diff --git "a/deduped/dedup_0138.jsonl" "b/deduped/dedup_0138.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0138.jsonl" @@ -0,0 +1,49 @@ +{"text": "Pagets disease of the nipple presents as an eczematous lesion, occurs in 1 \u2013 4% of all female breast carcinoma cases and is invariably associated with underlying malignancy either overt or occult. The majority of these cases are invasive disease although 40\u201345% are associated with DCIS.A 39 year old lady presented to our unit with a palpable lump in the right breast. Radiological and histological investigation proved this to be an extensive area of Ductal Carcinoma in Situ (DCIS) for which she underwent a simple mastectomy and immediate latissimus dorsi flap reconstruction. Histology revealed high grade DCIS with 2 small foci of invasive carcinoma. At 1 year the patient represented with a nodule adjacent to the reconstruction scar which was proved on biopsy to be consistent with Paget's disease. This was proved on formal excision.In the absence of underlying breast or apocrine tissue this case details a case of Paget's disease of uncertain origin. Pagets disease of the nipple accounts for between 1\u20134% of all cases of female breast carcinoma and presents as a chronic eczematous change of the nipple often with an underlying palpable lump. Classically the underlying carcinoma is invasive in nature although in 40\u201345% of cases the underlying pathology is DCIS. A prerequisite to the development of this condition is the presence of the nipple or at least underlying mammary tissue both of which were absent in this case. We present a case of a 39 year old lady who re-presented with Pagets disease following mastectomy and latissimus dorsi reconstruction, in the absence of a nipple or underlying breast tissue.A 39 year old lady was referred to our breast unit with a one week history of a lump in the lateral aspect of the right breast. On examination, a generalized hardness was felt in the right breast together with central retraction of the nipple.Mammography revealed malignant microcalcification affecting the whole breast with core biopsy proving high grade ductal carcinoma in situ (DCIS).Following discussion at the multi disciplinary meeting, she underwent a mastectomy and axillary sampling together with an immediate latissimus dorsi (LD) reconstruction flap. Post-operative recovery was unremarkable and the cosmetic result was good.Histology of the breast revealed 120 mm of high grade DCIS of mixed comedo and solid type. Two separate foci of invasive ductal carcinoma (Grade 1 and Grade 2) were identified. The margins were clear of tumour by 1.3 mm (in-situ disease) and 5.8 mm (invasive component). No evidence of Paget's disease was documented at this time. The axillary sample was negative for tumour.As the tumour demonstrated strong Estrogen/Progesterone positivity, Mrs K.S was started on postoperative Tamoxifen treatment but this was discontinued within 6 months due to adverse side effects. On her second post-operative visit, a small erythematous skin lesion was noted on the superio-medial aspect of the flap adjacent to the scar Fig .A punch biopsy was obtained which revealed foci of atypical cells within the epidermis with Pagetoid spread. Immunostaining showed positivity for CAM 5.2, CEA and EMA, consistent with. Paget's disease.A wide local excision of the lesion was performed and the specimen was sent for a histological second opinion. This showed acanthosis, hyperkeratosis with focal parakeratosis and extensive replacement of the epidermis by pleomorphic epithelial cells with marked pagetoid pattern Fig . Tumour The differential diagnosis was between that of Paget's disease of the breast and extra-mammary Paget's disease of the skin.Because of the past history of breast carcinoma, a diagnosis of Paget's disease of the breast was made.Paget's disease of the breast was first described by Sir James Paget in 1874 .Opinion is divided regarding the pathogenesis of this condition.erb 2 and EMA) [It is thought that malignant epithelial cells from intraductal carcinoma, extend into the overlying epidermis through mammary duct epithelium and proliferate in the epidermis causing thickening of the nipple and areolar skin. This is supported by the observation that Paget cells often share cell surface markers with the underlying breast carcinoma ,4. NormaOthers believe that Paget's cells are derived from clear nipple epithelium (Toker) cells and thatExtramammary Paget's disease was first described by Radcliffe Crocker in 1889. It is histologically identical but anatomically different to mammary Paget's disease affecting sites rich in apocrine glands such as the genetalia, axillae, perineum, and external auditory canal .In our case, following mastectomy and latissimus dorsi reconstruction, a focus of Paget's disease was found in the scar bridging the breast and transplanted skin. Neither of these areas are apocrine rich and therefore would be unlikely candidates for extramammary Paget's disease. In the absence of a a nipple, underlying ductal epithelium or breast tissue in general it is also very hard to explain as part of mammary Paget's disease.In this case, neoplastic cells from an axillary apocrine carcinoma may have migrated up the hair follicle and along the surgical scar but the exact pathogenesis still remains unclear."} +{"text": "Mammary Paget's disease and extramammary Paget's disease are neoplastic conditions, in which there is intraepithelial infiltration by neoplastic cells showing glandular differentiation. Mammary Paget's disease occurs exclusively on the nipple/areola complex from where it may spread to the surrounding skin.We here describe a case of Paget's disease occurring on the thoracic wall site of a previous simple mastectomy, and also briefly summarise the most important aspects leading to a diagnosis of mammary Paget's disease.To the best of our knowledge, this is the first reported case of mammary Paget's disease occurring after mastectomy. The absence of the nipple/areola complex obviously raised some questions concerning whether it was mammary or extra-mammary Paget's disease, and how it could occur in the absence of the nipple/areola complex. Mammary Paget's disease occurs exclusively on the nipple/areola complex from where it may spread on to surrounding skin. Extramammary Paget's disease occurs most commonly in the anogenital region but can arise in any area of skin and mucosa.Mammary Paget's disease accounts for 2\u20133% of neoplastic conditions of the breast and in most cases tumour cells have spread to the skin of the nipple and areola from underlying invasive carcinoma or ductal carcinoma in situ -3. The nMammary Paget's disease has been reported in the male breast with no evidence that the disease behaves differently, although the numbers of cases reported are small .This paper describes a case of Paget's disease occurred on the thoracic wall where simple mastectomy was performed many years before.A 57-year-old woman underwent simple mastectomy (according to Madden procedure) and axillary dissection on 7 December 1995. She reported the appearance of eczema of the nipple-areola complex some years before, and the presence of a palpable mass for about two months; no mention was made of nipple discharge or ulceration. Histological examination revealed unifocal Paget's disease of the nipple underlying an invasive mucinous ductal breast carcinoma (pT2 2.2 cm) with focal aspects of intraductal carcinoma; none of the 21 nodes obtained upon axillary dissection were involved in the disease. Immunohistochemically, the tumour presented high estrogen and low progesterone receptor positivity, and a low-intermediate proliferative rate (Ki67 14%). No secondary lung, liver or bone lesions were detected. On the basis of the disease stage and histology, and age at diagnosis, adjuvant hormonal treatment with tamoxifen was started on 9 January 1996.In February 1999, the patient complained of a 1 \u00d7 1.5 cm hyperpigmented eczematoid erythematous area at the site of ths previous mastectomy, whose size remained unchanged for four years.The beginning of its slow growth in 2003 led to a dermatological evaluation being advised, but this was delayed by the patient until December 2004, when a biopsy was performed. No scraping was possible as there was no crusted lesion Figure . HistoloTo the best of our knowledge, this is the first reported case of mammary Paget's disease occurring after mastectomy. The absence of the nipple/areola complex obviously raised some questions concerning whether it was mammary or extra-mammary Paget's disease, and how it could occur in the absence of the nipple/areola complex.The first question was answered by the immunohistochemical examination because hormonal receptors can only be detected in mammary Paget's disease. The second one could be explained by the high prevalence of multifocal disease in patients with Paget's mammary disease. However in this case the pathologist revealed unifocal Paget's disease of the nipple underlying an invasive mucinous ductal breast carcinoma with focal aspects of intraductal carcinoma and surgical negative margins in the histological specimen in 1995 when mastectomy was performed and unifocal Paget's disease of the thoracic skin with no microinvasion and negative margins at the time of the second resection.In order to understand this better, we here briefly summarise the most important aspects leading to the diagnosis of mammary Paget's disease, which was first described by James Paget in 1874 as a neoin situ or invasive ductal carcinoma in the underlying breast tissue [Mammary Paget's disease accounts for 2\u20133% of all neoplastic conditions of the breast and, in most cases (up to 90%), microscopic examination shows intraepithelial infiltration by neoplastic cells with glandular differentiation. Most cases originate from t tissue -3, but rt tissue ,5.in situ ductal carcinoma only [When a mass is palpable, invasive carcinoma is likely to be found, which is said to be more commonly multifocal with axillary node involvement, and to lead to worse survival than in patients whose tumour does not show an epidermal spread . Howeveroma only . The breThe surgical management of mammary Paget's disease associated with neoplastic disease consists of mastectomy, which is frequent in such patients because of the high prevalence of multifocal disease; breast-conserving surgery, which in these cases consists of a wide local excision of the tumour with the excision of the nipple/areola complex, is regarded as being more appropriate to an underlying unifocal breast tumour. Conservative surgery with adjuvant radiotherapy may be possible in selected cases ,8, althoin situ ductal carcinoma, and is more commonly present in high-grade (comedo and micropapillary) lesions. S100 is useful in differentiating Paget's disease from melanoma, but may be expressed in up to 25% of cases of mammary Paget's disease (a similar frequency to that observed in mammary carcinoma) [The presence of an associated invasive tumour obviously drives the therapeutic decision-making process. Immunohistochemistry can be very helpful in diagnosing mammary Paget's disease, particularly in cases like ours with an unusual clinical appearance, because pathologists can make some immunohistochemical analysis using particular markers, some of which are quite sensitive or specific. Among the cytokeratins, cytokeratin 7 and 20 are sensitive but not specific markers of mammary Paget's disease, and most cases are labelled by EMA antibody. Only approximately 35% of the cases of mammary Paget's disease show anti-CEA antibodies. The tumour cells in mammary Paget's disease generally have a similar immunohistochemical steroid receptor profile to that of the associated breast carcinoma; c-erbB2 is detected in most cases of mammary Paget's disease (90\u2013100%) and in the associated breast tumour in about 44% of cases of rcinoma) .The author(s) declare that they have no competing interests.MG as an oncologist observed for the first time the lesion on the thoracic wall and described the case, reviewing the existing literature on this topic.CD as a surgeon performed the surgical resection of the lesion and took the pictures before its removal.QP as a pathologist performed the histological examination of the lesion.AM as an oncologist worked as the supervisor of the breast team involved in this case.All authors read and approved the manuscript."} +{"text": "Mammary and extramammary Paget's diseases are rare neoplasms of epidermis and mucosal epithelium. Due to their nonspecific and variable clinical view, they have differential diagnosis with eczema, melanoma, Bowen's disease, etc. To the best of our knowledge, no such study has been performed in Iran regarding the prevalence, clinical aspects, underlying disease and pathological characteristics of these two diseases. In this study, we have evaluated the clinical and histopathological aspects of this disorder.In this retrospective study, all Paget's biopsied samples referred to the Pathology Department of Imam-Reza hospital, Mashhad, since 1984 till 2004 were evaluated. Collected data were analyzed by descriptive statistical methods.Among 98925 specimens, there were 29 cases of Paget's disease. All cases were married women suffering from mammary Paget. The mean age was 53 \u00b1 11 years. Left and right breast involvement was observed in 17 and 12 cases, all unilateral. The most common clinical view was ulcerated (27%) and then erythematosus exudative plaques. More than 50% of patients were symptomatic. Most common symptoms were itching, pain and burning. The exclusive underlying pathological diagnosis was ductal carcinoma (55%).In most cases, the clinical view of mammary Paget's disease was helpful. Unilateral ulcerated plaque was the most common clinical sign. Majority of the accompanying pathology was ductal carcinoma. We had no cases of extramammary Paget's disease in our study. Paget's disease is an uncommon and relatively rare intraepithelial adenocarcinoma that presents clinically as ulcerated or excoriated plaques.2Mammary Paget's disease (MPD) was discovered by James Paget in 1874 and extramammary type was found 15 years later by Radcliffe Crocker.3MPD manifests as an underlying breast carcinoma4Extramammary Paget's disease (EMPD) is most often observed in areas that are rich in apocrine glands. Patients usually present with the eczematous crusted or excoriated gray white plaques.4To the best of our knowledge, since there is no study performed in Iran regarding the prevalence, clinical aspects, underlying disease and pathological characteristics of these two diseases, we have evaluated the clinical and histopathological aspects of this disorder in this study.In this descriptive retrospective study, all Paget's biopsied samples referred to Pathology Department of Imam-Reza hospital, Mashhad, since 1984 till 2004 were evaluated. Data collection was performed using questionnaires that consisted of questions regarding age, sex, site of involvement, underlying disease, underlying breast cancer, clinical findings and pathology reports. Collected data were analyzed by statistical methods such as Qui square, Kruskal-Wallis and Mann-Whitney tests using SPSS software (version 15).In this 20-year study, all the 29 cases under this study were married women suffering from mammary Paget's disease. The mean patient age was 53 \u00b1 11 years (ranging from 23 to 80 years). All MPDs were unilateral and more common in the left. The most common symptoms were itching (24.1%), pain (20.7%) and burning (6.9%); Among these cases, 48.3% were asymptomatic.Ulcerated plaque (27.6%) and then exudative erythematosus plaque (24.3%), nodule on intact skin (24.2%), excoriated erythematosus plaque (10.3%), brown plaques (6.9%) and ulcerative erythematosus plaque (6.8%) were the main clinical manifestations.The most prevalent diagnosis of the specimens was Paget's disease and breast cancer. Other differential diagnosis includes eczema, lichen simplex, psoriasis, candidiasis and morphea.P = 0.001). All the nodular cases were associated with underlying ductal carcinoma.Among our cases, 55.2% had an underlying breast carcinoma. There was significant relationship between clinical manifestations and underlying disease -positive, diastase-resistant granules, indicating the presence of neutral mucopolysaccharides. Rarely, acid mucopolysaccharides may be identified by Alcian blue reaction at pH 2.5. Usually, basal keratinocytes lie between the Paget cells and the papillary dermis.510Positive MUC1 and low-molecular-weight cytokeratins (CK) in conjunction with immunonegativity for high-molecular-weight CKs are the most diagnostically useful immunohistochemistry markers for mammary Paget.5Although there are few articles in which MPD is reported in men, all of our 29 cases were women.1010Similar to literatures, majority of our patients were asymptomatic (48.3%). Among symptomatic cases, itching (24.1%), pain (20.7%) and burning (7%) were the most common symptoms.101213in situ or invasive ductal carcinoma mostly multifocal (73%) was found in 92% of cases even when no palpable mass was evident (85%).in situ malignant transformation theory of Paget cells.We found underlying carcinoma in 55.2% cases. This was 98% in Kao series and 82-92% in Lioyd cases.In this study no significant relationship between clinical symptoms and underlying disorder was detected. In MPD cases without underlying disease, no nodule was found and plaques were more common. However, in cases with underlying ductal carcinoma, nodule has a greater prevalence. Clinically, differential diagnosis for MPD includes nipple eczema, nipple erosive adenoma, Bowen's disease, superficial basal cell carcinoma, tinea, candidiasis and contact dermatitis.122214268All cases in our study were mammary Paget's cases and ductal carcinoma was the most prevalent accompanying disorder. Therefore, if unilateral ulcerated plaques on nipple-areola complex suggest MPD, breast examination, mammography and even biopsy are highly recommended for ruling out the possible underlying carcinoma."} +{"text": "In patients with melanoma, knowledge of regional lymph node status provides important information on outlook. Since lymph node status can influence treatment, surgery for sentinel lymph node (SLN) biopsy became a standard staging procedure for these patients. Current imaging modalities have a limited sensitivity for detection of micrometastases in lymph nodes and, therefore, there is a need for a better technique that can accurately identify occult SLN metastases.18F]-2\u2032-fluoro-2\u2032-deoxy-1-\u03b2-D-\u03b2-arabinofuranosyl-5-ethyluracil ([18F]FEAU) positron emission tomography (PET) and confirmed by histochemical assays. An animal foot pad model of melanoma lymph node metastasis was established. Mice received intratumoral injections of NV1023, and 48 hours later were imaged after i.v. injection of [18F]FEAU. NV1023 successfully infected and provided high levels of lacZ transgene expression in melanoma cells. Intratumoral injection of NV1023 resulted in viral trafficking to melanoma cells that had metastasized to popliteal and inguinal lymph nodes. Presence of virus-infected tumor cells was successfully imaged with [18F]FEAU-PET, that identified 8 out of 8 tumor-positive nodes. There was no overlap between radioactivity levels (lymph node to surrounding tissue ratio) of tumor-positive and tumor-negative lymph nodes.B16-F10 murine melanoma cells were infected with replication-competent herpes simplex virus (HSV) NV1023. The presence of tumor-targeting and reporter-expressing virus was assessed by [18F]FEAU-PET was successful in a murine model. Similar studies could be translated to the clinic and improve the staging and management of patients with melanoma.A new approach for imaging SLN metastases using NV1023 and [ Solid tumors including malignant melanoma, breast, head and neck, gastrointestinal and genitourinary carcinomas are prevalent cancers worldwide and are a primary cause of mortality in the Western world \u2013thymidine kinase (tk) gene product. As opposed to the mammalian tk-1, the HSV1-tk gene product has the ability to catalyze the phosphorylation of [18F]-2\u2032-fluoro-2\u2032-deoxy-1-\u03b2-D-\u03b2-arabinofuranosyl-5-ethyluracil ([18F]FEAU). Phosphorylated [18F]FEAU, is trapped inside HSV1-tk expressing cancer cells; it accumulates in proportion to the level of HSV1-tk expression and can be imaged using PET supplemented with high glucose (HG), 10% fetal calf serum (FCS), 1.5 mmol/L sodium bicarbonate, 100 \u00b5g/L penicillin, and streptomycin. Cells were maintained in 5% CO2 in a 37\u00b0C humidified incubator.The syngeneic malignant melanoma cell line B16-F10 was used for the US2-2 through US2-5) inserted in the UL/S junction. Attenuation was achieved by a 15-kB deletion in the inverted repeat region that extends from the 3\u2032 end of UL55 to the promoter for ICP4, deleting UL56 and 1 copy of the diploid genes ICP0 and ICP4 and the neurovirulence gene \u03b3134.5. The Escherichia coli \u03b2-galactosidase gene (lacZ) was inserted at the US10-12 locus as an infection marker.NV1023 is an attenuated, replication-competent oncolytic HSV whose construction was previously described 4 cells per well in 96-well plates in 100 \u00b5L medium. After incubation for 6 hours, NV1023 (500 \u00b5L) was added to each well at a multiplicity of infection of 0.1. Expression of the lacZ gene was measured at 24-hour intervals with an enhanced \u03b2-galactosidase assay kit using a spectrophotometer at 570 nm. All samples were measured in triplicate.Cancer cells were plated at 1\u00d7102 inhalation.Animal studies were performed in compliance with all applicable policies, procedures and regulatory requirements of the Institutional Animal Care and Use Committee (IACUC), the Research Animal Resource Center (RARC) of MSKCC and the National Institutes of Health (NIH) \u201cGuide for the Care and Use of Laboratory Animals\u201d. All animal procedures were performed by inhalation of 2% isoflurane. After the studies all animals were sacrificed by COC-2J/J albino mice were anesthetized with inhalational isoflurane for all procedures. The right hind limb foot pad was sterilized with alcohol, and then slowly injected with 50 \u00b5L of cell suspension at a concentration of 2\u00d7105 cells/\u00b5L over a 2-minute period. The mice were then awakened and their foot pad monitored for tumor size and signs of pain or ulceration twice a week. Three to 4 weeks after tumor injection we could monitor the presence of isolated black-colored sub centimeter lymph node at the popliteal area, i.e., the first echelon lymphatic region of the foot pad. In some animals there was also evidence for positive lymph nodes in the inguinal area, the secondary echelon of the foot pad. Only animals with primary tumors <5 mm that were confined to the foot pad area were used in the PET imaging study. NV1023 was injected to the foot pads bilaterally . Five of the animals with tumors were injected with saline before [18F]FEAU/PET imaging and served as a negative control. Another 5 animals without tumors were injected with NV1023 and the morphology of their lymph nodes was monitored.A foot pad syngeneic melanoma model was established as described previously by Harrell et al Histologic examination confirmed the presence of metastatic melanoma cells in cases of palpable/black-colored nodes, which were generally 0.8 mm in dimension at the time of diagnosis. Metastatic melanoma cells were deposited in the subcapsular sinus of the lymph node and infiltrated the nodal parenchyma.18F]FEAU PET imaging with NV1023 is based on the ability of the HSV-1-tk reporter gene, to catalyze the phosphorylation of the thymidine analog [18F]FEAU. tk transgene serves as a molecular marker for the detection of melanoma metastases.The concept of [18F]FEAU tracer was synthesized by coupling the radiolabeled fluoro sugar with the silylated pyrimidine derivative following a procedure previously reported by Serganova [18F]FEAU tracer 2 hours before PET imaging was performed. Mice were sacrificed with CO2, degutted, and immediately imaged with a dedicated small-animal PET scanner . The foot pad of the animals was amputated prior to imaging to avoid high background levels of radiotracer uptake in the tumor. In all animals the tumor was localized to the foot pad and did not infiltrate below the wrist. Images were acquired using a transaxial field of view of 10 cm and an axial field of view of 7.8 cm. An energy window of 350\u2013750 keV and a coincidence timing window of 6 nanoseconds were used. The resulting list-mode data were sorted into 2-dimensional histograms by Fourier rebinning, and transverse images were reconstructed by filtered backprojection into a 128\u00d7128\u00d763 (0.72\u00d70.72\u00d71.3 mm) matrix. Image analysis was performed using the ASIPro . After imaging, the popliteal and inguinal lymph nodes were excised bilaterally, weighed, and measured for radioactivity (%ID/g) using a gamma counter .Following virus injection, animals were intravenously injected with \u223c250 \u00b5Ci (9.25 MBq) of FEAU (250 \u00b5Ci).We randomly selected 5 of the tumor bearing animals for the test group. These mice were injected with 1\u00d71018F]FEAU PET scans in mice injected with NV1023 identified all tumor-positive lymph nodes (with melanoma metastases) (n\u200a=\u200a8). Conversely, all tumor-negative lymph nodes in mice injected with NV1023 (n\u200a=\u200a5) and mice treated with saline (n\u200a=\u200a5) had [18F]FEAU signal levels similar to background (P<0.001) for tumor-positive lymph nodes compared to tumor-negative nodes. Nucleoside analogs are not accumulated or trapped by mammalian tissue, as only virus-containing cells can induce phosphorylation of these tracers by the HSV1-tk reporter gene 18F]FEAU uptake in an animal with a lymph node metastasis injected with saline. The figure clearly demonstrates no radiotracer accumulation by mammalian tissue, aside from gallbladder uptake, where colonies of thymidine kinase forming bacteria may augment the background signal.Region of interest (ROI) measurements from FEAU, [124I]-2\u2032-fluoro-1-\u03b2-D-arabino-furanosyl-5-iodo-uracil ([124I]FIAU), and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) 124I]FIAU or [18F]FEAU 124I]FIAU administration, saline-treated tumors demonstrated a mean [124I]FIAU accumulation of 0.0021\u00b10.0002 percent dose/g tissue, whereas tumors treated with 1\u00d7107 p.f.u. of NV1020 demonstrated a mean accumulation of 0.0047\u00b10.0004 percent dose/g . Similarly, tumors treated with 5\u00d7107 p.f.u. of NV1020 demonstrated a mean accumulation of 0.0070\u00b10.0009 percent dose/g . The specificity and efficacy of [18F]FEAU imaging using NV1023/HSV1-tk reporter gene was recently demonstrated by our group for various other tumors Reporter gene imaging with HSV1-tk has been used extensively in animal models of cancer. The expression of HSV1-tk can be imaged and monitored using specific radiolabeled substrates that are selectively phosphorylated by HSV1-tk and trapped within transfected cells. Three radiotracers that are widely used by many investigators for imaging of HSV1-tk gene expression are [tk in cancer cells invading lymph nodes that can be selectively imaged with [18F]FEAU PET. This imaging system was sensitive and accurate enough to detect micrometastases in lymph nodes that measured <3 mm in diameter. In one tumor-positive lymph node, the lymph node-to-surrounding tissue radioactivity ratio was close to unit, 1.5. This low ratio was primarily due to the high level of background radioactivity that surrounded this lymph node. This illustrates the importance of imaging when background radioactivity is low 18F]FEAU was detected in all nodes invaded by cancer compared to non invaded normal nodes (>2 standard deviations).Here we describe a novel method for detection of occult SLN metastases using an oncolytic HSV/NV1023 and reporter gene imaging. Using NV1023 as the delivery vector, we demonstrate that a single injection of this attenuated, replication-competent virus into the primary tumor will induce expression of HSV1-Lymph node size may be correlated to immune infiltrates and not only to metastatic volume. We demonstrated that although tumor invaded lymph nodes were slightly heavier than normal nodes, the majority of lymph node with metastases (7 out of 8) were within the weight range of normal nodes. This suggests that lymph node weight alone can not predict the presence tumor micrometastases, and that NV1023-PET imaging system can identify tumor-positive lymph nodes within the range of \u2018normal\u2019 size lymph nodes, which is not the case with conventional imaging methods.Studies have implicated the ability of oncolytic viruses, including HSV-1, to be effectively transported into the lymphatic circulation 18F]fluoro-3-hydroxymethylbutyl) guanine ([18F]FHBG) Renilla luciferase reporter genes 18F]FEAU with HSV-1 oncolytic viruses was shown to enhance the detection of nerves infiltrated by cancer cells using noninvasive imaging Gene expression imaging of occult lymph node metastases was recently described by Burton et al. for direct PET visualization of prostate cancer Aotus owl monkeys, a primate exquisitely sensitive to herpes viral infections Aotus monkeys compared with HSV-1. A similar virus, NV1020, was recently studied in a phase I trial for patients with hepatic colorectal metastases. Doses of \u22641.3\u00d7109 PFU were administered by hepatic infusion pump without dose-limiting toxicity or significant adverse events attributable to the virus None of the NV1023-treated animals suffered from clinically apparent side effects attributable to viral administration. The parent virus from which NV1023 was derived, R7020, has a very favorable safety profile in One potential diagnostic application of this viral-imaging paradigm is the staging of disease and guiding future treatment. In melanoma, head and neck, and breast carcinomas, imaging the targets of replication-competent viruses could be performed following a single intratumoral injection. In mucosal tumors such as gastrointestinal and genitourinary carcinomas, NV1023 or similar vectors can be delivered through endoscopic injections to intraluminal primary tumors 18F]FEAU imaging tool for diagnostic staging. In several human PET studies using nucleoside analogs and in our laboratory it was found that intestinal background uptake does not play an important role in patients a more sensitive noninvasive method for the detection of lymph nodes invaded by cancer, (2) delineation of the extent and anatomic location of regional metastases (3), provide better pre-surgical assessment of disease extent in patients with suspected positive lymph nodes and (4) provide guidance for adjuvant radiotherapy, immunotherapy, or chemotherapy to patients with lymph node metastases.Our study shows that the NV1023 herpes virus expressing the HSV-1-Figure S1LacZ staining 48 hours after NV1023 infection in vitro revealed cytoplasmatic expression of beta-galactosidase.(0.42 MB TIF)Click here for additional data file."} +{"text": "For mice (n = 8 in each group), only 1\u2009mA was used, by which C fiber of the mice can be activated. Gastric antrum motility was measured by intrapyloric balloon. Gastric motility was facilitated by EAS at ST36 and inhibited by EAS at CV12. The half maximal facilitation intensity of EAS at ST36 was 2.1\u20132.3\u2009mA, and the half maximal inhibitory intensity of EAS at CV12 was 2.8\u2009mA. In comparison with C57BL/6 mice, the facilitatory effect of ST36 and inhibitive effect of CV12 in ASIC3\u2212/\u2212 mice decreased, but the difference was not statistically significant (P > 0.05). However, these effects in TRPV1\u2212/\u2212 mice decreased significantly (P < 0.001). The results indicated that there existed an \u201cintensity-response\u201d relationship between EAS and the effect of gastric motility. TRPV1 receptor was involved in the regulation of gastric motility of EAS.The aim of this study was to explore the \u201cintensity-response\u201d relationship between EAS and the effect of gastric motility of rats and its underlying peripheral neural mechanism by employing ASIC3 knockout (ASIC3\u2212/\u2212), TRPV1 knockout (TRPV1\u2212/\u2212), and C57BL/6 mice. For adult male Sprague-Dawley ( Acupuncture therapy, as a traditional Chinese medicinal treatment, has been widely used in clinical practice in oriental countries. And it has been more accepted by practitioners and patients worldwide after its therapeutic effects for the treatments of postoperative dental pain, nausea, and vomiting have been confirmed by NIH in 1997 .\u03b4) and IV (C) primary afferent fibers [During the last decades, a large number of studies have been performed to investigate the effects of acupuncture on gastrointestinal secretion, motility, and gastric myoelectrical activity \u20135. Some t fibers , EAS wit\u03b2 and A\u03b4 mechanical receptors, as well as C-polymodal receptors, played important roles in the acupuncture stimulation perception [\u03b2 primary afferent fibers innervating the skin and muscle [\u03b4 and C fibers. It can be activated by capsaicin, noxious heat, low PH, and voltage and closely related to noxious physical detection [Previous studies showed that Arception \u201314. But rception and locad muscle , 16. Traetection \u201319. In oetection .\u03b2 and A\u03b4/C afferent fiber mice models, respectively, in order to investigate the roles of A\u03b2 and A\u03b4/C fibers in the EAS-modulated gastric motility.In the present study, both ASIC3 knockout (ASIC3\u2212/\u2212) mice and TRPV1 knockout (TRPV1\u2212/\u2212) mice were employed to establish dysfunction of An = 18), weighing 250\u2013300\u2009g, were purchased from Institute of Animal, Academy of Chinese Medical Sciences. Male ASIC3\u2212/\u2212 mice (n = 8), TRPV1\u2212/\u2212 mice (n = 8), and C57BL/6J mice (n = 8), weighing 25\u201330\u2009g, were purchased from Jackson Lab (USA) and bred at the China Academy of Chinese Medical Science Animal Care Facility. The animals were housed under a 12\u2009h light/dark with free access to food and water. All animals were treated according to the Guide for Use and Care of Medical Laboratory Animals from Ministry of Public Health of People's Republic of China.Male Sprague-Dawley (SD) rats was administered. About 1\u2009h after the urethane administration, the animals were under deep anesthesia, and the trachea was cannulated but not immobilized to keep respiratory tract unobstructed. A catheter was inserted into one of the jugular veins for infusion. A small longitudinal incision was made in the duodenum about 1\u2009cm from the pylorus. A small balloon made of flexible condom rubber was inserted via incision of the duodenum into the pyloric area of rat and kept in position by tying the connecting catheter to the duodenum. And another catheter (inner diameter of 1\u2009mm) was also inserted into the same hole by incision in order to drain digestive juices secreted from stomach. The balloon was filled with about 0.2\u20130.3\u2009mL warm water to keep pressures at about 100\u2009mmHPressure in the balloon was measured by a transducer through a thin polyethylene tube (1.5\u2009mm in outer diameter) and then input into a polygraph amplifier . The signal was captured online and analyzed offline using a data acquisition system and Chart 5.2 software. Demifasting gastric motor activity was recorded as a control for at least 30\u2009min before any stimulation. The gastric motility induced by EAS was compared with the background activity in terms of average amplitude (the average difference between the cyclic maxima and minima in the selected cycles), integral , and frequency (per minute) of gastric contraction waves. Systemic blood pressure and heart rate were continuously monitored by using of BIOPAC data acquisition system , and rectal temperature kept constantly around 37\u00b0C by a feedback-controlled heating blanket .\u03b4A), 1\u2009mA (TC), 7\u2009mA (>TC), and 9\u2009mA (>TC), were applied at ST36 or CV12 in an ascending order. The latter stimulus can only be applied when the gastric motility recovered to control state. The background gastric activity and gastric activity during and after EAS were recorded continuously, 60\u2009s for each session. Gastric motility during and after EAS was compared with background activity. If the change rates of gastric motility during or after EAS were 15\u201320% of the basal activity, the response was then considered to have an excitatory or inhibitory effect. The first EA stimulus was applied when gastric motility wave maintained stable, usually at about 30 minutes after the surgical procedure. Different intensities of EAS, including 0.5\u2009mA ( 0.05) . However, 3\u2009mA, 5\u2009mA, 7\u2009mA, and 9\u2009mA EAS at ST36 elicited a significant enhancement on the amplitude and integral of gastric contraction compared with the background activities . The facilitation of EAS at ST36 appeared from a low intensity with an EC50 value of approximately 2.3\u2009mA for amplitude , while 3\u2009mA, 5\u2009mA, 7\u2009mA, and 9\u2009mA EAS at ST36 induced significant enhancement on the frequencies of gastric motility compared with the background activities . The maximal facilitatory response of the frequency appeared as the intensities reached to 5\u2009mA.P < 0.05; 1\u2009mA: \u221218.8 \u00b1 3.2%, 3\u2009mA: \u221242.0 \u00b1 5.5%, 5\u2009mA: \u221256.7 \u00b1 10%, 7\u2009mA: \u221256.3 \u00b1 10%, and 9\u2009mA: \u221257.3 \u00b1 7.2%, P < 0.01) . The inhibition of EAS at CV12 appeared from a low intensity (0.5\u2009mA), with IC50 value of approximately 2.8\u2009mA for both amplitude and integral . But 3\u2009mA, 5\u2009mA, 7\u2009mA, and 9\u2009mA EAS at CV12 induced a significant inhibition on the frequency of gastric motility compared with the background activities . The inhibitory response of the frequency was prone to be maximal when the intensity reached to 5\u2009mA.\u03b4 and C fiber played important roles for modulating gastric motility. According to the threshold of C fiber of mice [P < 0.001, Figures P < 0.001, Figures P < 0.05, P < 0.05, The previous data showed that there existed a possibility of \u201cintensity-response\u201d relationship between stimulation and effects of gastric motility. We speculated that the EAS with intensities of activation A of mice , 1\u2009mA wa50 of EAS at ST36, was 2.1\u20132.3\u2009mA, which was near the threshold of A\u03b4 fiber. EAS at CV12 displayed inhibitory effects which were also related to the intensities. The IC50 of EAS at CV12, was about 2.8\u2009mA, which was also near the threshold of A\u03b4 fiber. These data suggested that the activation of A\u03b4 fiber was important for EAS-modulated gastric motility. Further study in ASIC3 and TRPV1 knockout mice showed that both ASIC3 and TRPV1 receptors were involved in the effects of EAS on gastric motility, but there was a quantity difference in the changes of gastric motility between ASIC3 and TRPV1 knockout mice. TRPV1 played a more important role in the effects of EAS. In the present study, we investigated the \u201cintensity-response\u201d relationship between EAS and the effect of gastric motility in rats. And we firstly observed which afferent fibers were involved in the effect of EAS on gastric motility by using of knockout mice. Our findings strongly indicated the existence of\u2009\u2009\u201cintensity-response\u201d effects of EAS on gastric motility. EAS at ST36 induced facilitatory effects which were related to the intensities. After data fitting, the EC\u03b2 fibers. Relationship between TRPV1 and effects of acupuncture was further investigated recently. Our previous study suggested an involvement of TRPV1 receptors in acupuncture analgesia [\u03b4 and C fiber were more critical than A\u03b2 fiber in the effects of EA-modulated gastric motility. In another somatovisceral reflex study, Noguchi et al. revealed that to decrease duodenal motilities, EAS to the abdomen needed to be strong enough to excite group IV (C) fibers of intercostal nerves. To increase motilities, EAS to the hindpaw needs to be strong enough to excite the higher threshold group III (A\u03b4) fibers of tibial nerves. Their results also indicated the critical roles of A\u03b4 and C primary afferent fibers in effective regulation of EAS on visceral organ, which were quite similar to our results [Based on another experiment in our research group, 1\u2009mA was strong enough to activate the C primary afferent fiber in mice. The different gastric responses induced by 1\u2009mA EAS between ST36 and CV12 were mainly caused by diverse somatoautonomic reflexes; that is, the facilitatory effect of EA at ST36 was mediated via the parasympathetic pathway, whereas the inhibitory effect of EA at abdomen was reasoned to be attributed to the sympathetic pathway. The involvement of the opioidergic pathway has also been frequently reported , 22. EA nalgesia . Wang etnalgesia . Moreovenalgesia . Accordi results .It is generally believed that acupuncture at different acupoints produces different effects, and the site-specific inhibitory or facilitatory effects of acupuncture on gastric motility had already been proposed , 27, 28.\u03b4 fiber to get remarkable modulatory effects, and these effects tended to maximization when C fiber was activated.There existed \u201cintensity-response\u201d relationship between stimulation and effects on gastric motility. TRPV1 receptor was involved in the regulation process of EAS. It is necessary to activate A"} +{"text": "Chemotherapy-induced amenorrhea (CIA) is common in young breast cancer patients. The incidence of CIA associated with regimens involving epirubicin and taxane was not well known. Furthermore, previous studies suggested leucopenia and amenorrhea may reflect inter-individual variations in pharmacokinetics. The purpose of this study was to investigate the association between leucopenia after first cycle of chemotherapy and CIA in young breast cancer patients receiving epirubicin and taxane based chemotherapy. Furthermore, the incidence of CIA was also assessed.P<0.001) and previous childbearing were significantly associated with probability of CIA. Compared to patients treated without taxane, patients treated with taxane-contained regimens did not have a significantly higher rate of CIA (P>0.05). The rate of CIA in leucopenia group (52.56%) was significantly higher than that in normal leukocyte group (34.62%) (P\u200a=\u200a0.024). In patients treated with a FEC regimen , the rate of CIA in leucopenia group (59.57%) was significantly higher than that in normal leukocyte group (36.84%) (P\u200a=\u200a0.037).Between October 2008 and March 2010, 186 consecutive premenopausal patients, treated with epirubicin and taxane based chemotherapy, were recruited. Information about CIA was collected by telephone and out-patient clinic. Of these 186 patients, data from 165 patients were included and analyzed. Of all 165 patients, CIA occurred in 72 patients (43.64%). In multivariate analysis, age older than 40 y (OR: 16.10, 95% CI: 6.34\u201340.88, Age at diagnosis and previous childbearing were both found to significantly increase the risk of CIA, whereas additional taxane was not associated with increased rate of CIA. Importantly, leucopenia after first cycle of chemotherapy was associated with increased risk of CIA, which suggested that leucopenia may be an early predictor of chemotherapy-induced infertility. Breast cancer is a worldwide malignant disease. Adjuvant chemotherapy can significantly improve disease-free survival (DFS) and overall survival (OS) for early breast cancer patients Many factors, including patients' age, dosage of chemotherapy, and schedule of chemotherapy are associated with the risk of CIA. Usually, old patients had a high risk of CIA due to a reduced number of active ovarian follicles present with increasing age When the above factors were adjusted, CIA rates may be still different in different individuals. Inter-individual variations in pharmacokinetics, influence the degree of the toxicity, may be inner factors. Leucopenia, chemotherapy-induced bone marrow toxicity, is common after chemotherapy, and it may be positively related to the prognosis In this study, we aimed to investigate the association between leucopenia after first cycle of chemotherapy and CIA in young breast cancer patients receiving epirubicin and taxane based chemotherapy. A secondary aim was to evaluate the impact of epirubicin and taxane based regimens on the rate of CIA in Chinese patients. Furthermore, other potential risk factors of CIA were also assessed.The study was conducted according to ethical considerations for observational retrospective studies, and this study was in compliance with the Helsinki Declaration. All breast cancer patients provided written informed consent for their clinical data to be reviewed by us. Between October 2008 and March 2010, 186 consecutive premenopausal patients, treated with epirubicin and taxane based chemotherapy, were recruited at our hospital. Patient data were included in this retrospective study when they met the following criteria: (1) not receiving bilateral oophorectomy or luteinizing hormone releasing hormone (LHRH) agonists; (2) not receiving chemotherapy previously and (3) without recurrent disease in 12 months. Information about CIA was collected by telephone and out-patient clinic. The following information was collected: (1) after which cycle of chemotherapy did the patients experience amenorrhea; (2) when did the menstruation recover; (3) how many times did the menstruation occur after amenorrhea. Since 5 of the 186 patients had recurrent disease within 12 months, 11 were lost during follow-up, 5 had been treated with LHRH, data from 165 patients were included and analyzed at last. The median follow-up time from the initiation of chemotherapy was 26 months .2 on day 1, epirubicin 75 mg/m2 on day 1, and cyclophosphamide 500 mg/m2 on day 1) every 3 weeks for six cycles; (2) sequential-ECT: CE every 3 weeks for four cycles followed by T (docetaxel 75 mg/m2 on day 1 every 3 weeks or paclitaxel 175 mg/m2 on day 1 every 2 weeks) for four cycles; (3) FEC-T: FEC every 3 weeks for three cycles followed by docetaxel every 3 weeks for three cycles; and (4) concurrent-ECT (docetaxel 75 mg/m2) on day 1 every 3 weeks for six cycles. To reach 100% dose, patients treated with taxane were granulocyte colony stimulating factor (G-CSF) supported. All the patients were G-CSF supported in the case of leucopenia. White blood cell (WBC) count and neutrophilic granulocyte count were assessed on day 7 after chemotherapy. Tamoxifen was given as adjuvant endocrine therapy after chemotherapy when patients were positive for estrogen receptor (ER) and/or progesterone receptor (PR). Radiotherapy was administered to some patients according to NCCN guidelines. Other clinical information was collected for this study, including age, tumor size, nodal involvement, hormone receptor status, and pathology.The chemotherapy regimens were determined based on National Comprehensive Cancer Network (NCCN) guidelines and included: (1) FEC .In this study, percentiles, median, and range were analyzed for each continuous variable. Differences between subgroups were examined using the chi-square test. The candidate explanatory variables in the multivariate analysis of CIA onset were: age at diagnosis, chemotherapy regimen, childbearing, and use of tamoxifen. Logistic regression was used for multivariate analysis. In all, 165 patients were included in this study. Of these patients, 85 were treated with a FEC regimen, 52 with a sequential-ECT regimen, 19 with a FEC-T regimen, and 9 with a concurrent-ECT regimen. The patients' characteristics are shown in Almost all patients had amenorrhea, most of which came up after first three cycles of chemotherapy. Of all 165 patients, CIA occurred in 72 patients (43.64%). Most patients with temporary amenorrhea experienced resumption of menstruation in about 8 months after the end of chemotherapy.The incidences of CIA according to different variables were shown in P<0.001) and previous childbearing were significantly associated with probability of CIA. Patients treated with tamoxifen had a trend towards higher rate of CIA compared with patients not receiving tamoxifen. Compared to patients treated with a FEC regimen, patients treated with taxane-contained regimens did not have a significantly higher rate of CIA .In multivariate analysis , age oldThe patients treated with concurrent-ECT regimen were G-CSF supported after first cycle of chemotherapy, so these patients were excluded from the analysis of the relationship between leucopenia and CIA. In all, 156 patients were included for this analysis .P>0.05). The rate of CIA in leucopenia group (52.56%) was significantly higher than that in normal leukocyte group (34.62%) . In patients treated with a FEC regimen, the rate of CIA in leucopenia group (59.57%) was significantly higher than that in normal leukocyte group (36.84%) . Patients treated with a sequential ECT regimen in leucopenia group (50%) had a trend towards higher CIA rate compared with patients in normal leukocyte group (36.67%), but no significant difference was observed (P>0.05) due to small sample size.Of these 156 patients, 78 (50%) experienced leucopenia after first cycle of chemotherapy. In multivariate analysis, age at diagnosis, chemotherapy regimen, and previous childbearing were not associated with leucopenia after first cycle of chemotherapy (P\u200a=\u200a0.055), the association between leucopenia and CIA was also analyzed in patients older than 40 y or 40 y and younger treated with a FEC regimen. In patients older than 40 y treated with a FEC regimen, the rate of CIA in leucopenia group (25/32) was significantly higher than that in normal leukocyte group (12/23) . In patients 40 y and younger treated with a FEC regimen, the rate of CIA in leucopenia group (3/15) was not significantly higher than that in normal leukocyte group (2/15) (P>0.05). However, the association between leucopenia and CIA was not analyzed in patients older than 40 y or 40 y and younger treated with other two regimens due to small sample size.Because there was a trend toward more leucopenia in older patients (Chemotherapy-induced premature menopause and infertility influence patients' life quality seriously. It is important to investigate the risk factors of CIA. The CIA risk of epirubicin and taxane based chemotherapy is not well known. Our results demonstrated that the risk of CIA was significantly related to age at diagnosis and previous childbearing. Furthermore, compared with the FEC regimen, taxane based regimen did not show a higher rate of CIA. Importantly, leucopenia after first cycle of chemotherapy was associated with CIA in patients treated with epirubicin and taxane based chemotherapy.The definitions of CIA were different in previous studies. CIA was defined as the absence of menses for at least three consecutive months from the point of breast cancer diagnosis in some studies Previous studies reported discordant results in the incidences of taxane-induced amenorrhea This study showed that age was still the most important risk factor of CIA, which was consistent with previous studies. In addition, we found that previous childbearing was significant associated with CIA, which was the same as the previous study Previous studies demonstrated that chemotherapy-induced amenorrhea and leucopenia were both associated with improved survival On the other hand, several limitations were present in this study. First, this is a retrospective study with marginally significant differences found with regard to age and leucopenia, future studies would be required to investigate these. Second, only Chinese patients were included in this study. Future studies should be taken in other populations. Third, endocrinological data were not assessed in this study. Since oligo-amenorrhoea may be caused by tamoxifen, multivariate analysis was used to assess the risk factors of CIA in this study.Our study suggested age at diagnosis and previous childbearing were both found to significantly increase the risk of CIA. Compared to patients treated with the FEC regimen, additional taxane did not significantly increase the rate of CIA. Importantly, leucopenia after first cycle of chemotherapy was associated with increased risk of CIA. When patients want to maintain their fertility, our results should be considered. Methods of fertility preservation should be administrated as early as possible for patients with leucopenia."} +{"text": "In this article, we reveal a reciprocal interaction whereby circulating human plasmablasts are potent inducers of the Tfh cell\u2013differentiation program, including the expression of their key transcription factor Bcl-6. The markedly increased propensity of plasmablasts, compared with naive B cells, to induce Tfh cell differentiation was due to their increased production of IL-6. Specific targeting of IL-6 using tocilizumab therapy in patients with rheumatoid arthritis led to a significant reduction in circulating Tfh cell numbers and IL-21 production, which was correlated with reduced plasmablast formation. Our data uncover a positive-feedback loop between circulating plasmablasts and Tfh cells that could sustain autoimmunity and spread Ab-driven inflammation to unaffected sites; this represents an important therapeutic target, as well as reveals a novel mechanism of action for tocilizumab.B cells require CD4 This Tfter (GC) , 3. Murin values in the figure legends refer to independent donors.Blood was obtained from healthy individual volunteers and patients with rheumatoid arthritis (RA) before and 6 mo after treatment with tocilizumab. The mean disease activity score (DAS28) in the paired samples before and after tocilizumab treatment was 7.2 and 3.5, respectively. These patients were not taking any other disease-modifying antirheumatic drugs and were on <7.5 mg prednisolone/d. The University College London Hospital ethics committee approved the study. The +CD25\u2212CD127+ and CD4+CD45RA+CD27+, respectively. B cell subpopulations, including plasmablasts and naive and memory B cells, were sorted as CD19+CD38+CD27+, CD19+IgD+CD38\u2212/intCD27\u2212, and CD19+CD38\u2212CD27+ respectively.B cells were depleted from PBMCs by positive selection with magnetic beads (Miltenyi Biotec). Responder and naive T cells were sorted as CD4+ T cells were resorted after 4 d of culture, with or without plasmablasts, and cultured with autologous freshly sorted naive B cells in the presence of 2 \u03bcg/ml endotoxin-reduced Staphylococcal enterotoxin B (Sigma-Aldrich). Naive T cells were cultured with 10 ng/ml of IL-21 or IL-6 or a combination of both (PeproTech).PBMCs were stimulated with 2 \u03bcg/ml soluble anti-CD3 (HIT3a) and anti-CD28 (CD28.2) (eBioscience). A total of 50,000 cells each was used for cocultures of T and B cells. For Tfh functional assays, CD4The following Abs were used: CD4\u2013Alexa Fluor 700, CXCR5-Biotin, ICOS-PECy7, Bcl-6\u2013Alexa Fluor 647, CD27\u2013allophycocyanin\u2013H7, CD45RA-PE, CD19-allophycocyanin or V450, IgD-FITC, IL-21\u2013Alexa Fluor 647, IFN-\u03b3\u2013PECy7, IL-10\u2013PE, Streptavidin\u2013PE\u2013Texas Red (BD Biosciences), CD38\u2013PerCP\u2013eFluor 710, and IL-6\u2013FITC (BioLegend). Neutralizing Abs specific for human IL-6 and IL-21R and isotype controls were from R&D Systems.For analysis of intracellular cytokines, cells were stimulated for 4 h with 50 ng/ml PMA, 250 ng/ml ionomycin (Sigma-Aldrich), and GolgiPlug (BD Biosciences). Data were acquired on an LSR II (Becton Dickinson) and analyzed with FlowJo software (TreeStar).IL-6 (eBioscience) and Ig (IgM and IgG) production (Sigma-Aldrich) was measured in supernatants by ELISA, according to the manufacturers\u2019 instructions.t test using Prism . The Mann\u2013Whitney U test was used to compare healthy individuals and patients with RA. Correlation coefficients and their significance were analyzed by the Pearson correlation.Data were analyzed for significance by the paired +CXCR5+ICOS+. Only a small fraction of circulating CD4+ T cells in healthy donors possessed these characteristics were cultured with CD19+ B cells and stimulated with either anti-CD3 and IL-4 (+CD38highCD27high) (+IgD+CD38\u2212/lowCD27\u2212) or memory B cells (CD19+CD38\u2212CD27+) (This finding prompted us to address whether B cells directly support the expansion of Tfh cells. Responder T (Tresp) cells but not 8\u2212CD27+) .Given the close correlation between plasmablasts and Tfh cells, we hypothesized that this B cell subset was responsible for the increased frequency of Tfh cells. Therefore, plasmablasts were isolated from peripheral blood, according to their expression of CD38 and CD27 Supplem, and com+ T cells/plasmablasts and assessed the induction of Tfh cell differentiation. The ratio of plasmablasts/naive CD4+ T cells in these cultures was directly proportional to Tfh cell differentiation (We next assessed whether circulating plasmablasts possessed the capacity to induce the differentiation of Tfh cells from naive T cells. Thus, plasmablasts, naive and memory B cells, and monocytes were isolated and added to naive T cells in the presence of anti-CD3/CD28. Plasmablasts were the most potent inducers of Tfh cell differentiation . Tfh celntiation . To ascentiation . Plasmabntiation and IgG ntiation , respectntiation . This diIn our efforts to elucidate the mechanism by which plasmablasts induce Tfh cell differentiation, we discovered that plasmablasts produce large amounts of IL-6, more than either naive or memory B cells . PlasmabWe next investigated whether the production of IL-6 and IL-21 mediated the differentiation of Tfh cells by plasmablasts. In vitro blockade of IL-21R or IL-6 in cocultures of naive T cells and plasmablasts significantly decreased the acquisition of a Tfh cell phenotype . Blockad+ T cell IL-21 production was decreased significantly after treatment with tocilizumab (To glean some insight into the in vivo relevance of our findings in patients, we investigated the interrelationships between Tfh cells and plasmablasts in patients with RA before and after tocilizumab therapy. Tocilizumab targets IL-6R and is an effective treatment for patients with RA . In agreilizumab . Tocilizilizumab , 3J. Morilizumab . These rOur data implicate a previously unrecognized function of human circulating plasmablasts as potent inducers of Tfh cell differentiation via IL-6. One can envisage a feedback mechanism by which circulating plasmablasts traffic from one inflammatory site and create further foci of inflammation through the differentiation of Tfh cells, which, in turn, induces plasmablast formation. This feedback mechanism is tightly controlled under normal conditions; however, in infections and autoimmune diseases it could rapidly result in large numbers of Tfh cells and plasmablasts in several locations. This amplification loop represents an important therapeutic target in autoimmune diseases, such as RA, and it also could be boosted to improve responses to infection and to enhance vaccination strategies.Data Supplement"} +{"text": "However, the mechanism(s) of action remains to be elucidated. In the present study, we found that niclosamide induced cell toxicity in human glioblastoma cells corresponding with increased protein ubiquitination, ER stress and autophagy. In addition, niclosamide treatment led to down-regulation of Wnt/\u03b2-catenin, PI3K/AKT, MAPK/ERK, and STAT3 pro-survival signal transduction pathways to further reduce U-87 MG cell viability. Taken together, these results provide new insights into the glioblastoma suppressive capabilities of niclosamide, showing that niclosamide can target multiple major cell signaling pathways simultaneously to effectively promote cell death in U-87 MG cells. Niclosamide constitutes a new prospect for a therapeutic treatment against human glioblastoma.Glioblastoma is the most common and lethal malignant primary brain tumor for which the development of efficacious chemotherapeutic agents remains an urgent need. The anti-helminthic drug niclosamide, which has long been in use to treat tapeworm infections, has recently attracted renewed interest due to its apparent anticancer effects in a variety of Glioblastoma multiforme is the most common and aggressive brain tumor for which an effective pharmacotherapy remains unavailable. The current initial treatment combines surgery with chemotherapy, yet the overall survival rate for glioma has not significantly improved in the past three decades because these tumors have a high incidence of recurrence and commonly lead to death within less than a year from diagnosis \u20133. Extenin vitro and in vivo by triggering apoptosis and oxidative stress or by inhibiting several important signaling pathways including Wnt/\u03b2-catenin was performed in 96-well plates using a CellTiter 96 non-radioactive cell proliferation colorimetric assay kit according to manufacturers' instructions. Briefly, cells were plated in 96-well plates the day before the experiment. At the end of various treatments, 100 \u03bcl medium was removed from well, followed by the addition of 20 \u03bcl of MTS solution to each well and then incubated for 1 h at 37\u00b0C. Samples were read by a microplate reader at a wavelength of 490 nm. At least 6 replicates of each treatment were used.The autofluorescent agent MDC (Sigma-Aldrich) was used as a specific autophagolysosome marker to analyze the autophagic process . U-87 MGChanges in the amounts of protein expression were measured by Western blot analysis. Protein homogenates were prepared as follows: the cells were lysed in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitor cocktails . Clear lysates were obtained by centrifugation at 4\u00b0C for 20 min at 13,000 rpm in a refrigerated microcentrifuge. Protein concentrations were determined, according to the manufacturer\u2019s instruction, using the Pierce BCA Protein Assay Kit . Equal amounts of the protein samples (25\u201330 \u03bcg) were separated on a 10% or 4\u201320% gradient polyacrylamide gel , transferred to nitrocellulose membranes, and blocked for either 1 h at room temperature or overnight at 4\u00b0C with Tris buffer containing 0.1% Tween 20 and 5% (w/v) nonfat dried milk. The blotted membranes were incubated with specific primary antibodies for 1 h at room temperature or overnight at 4\u00b0C. Ubiquitin, cyclin D1, survivin, P-ERK and ERK antibodies were purchased from Santa Cruz Biotechnology while P-AKT, AKT, CHOP, LC3, Cleaved PARP, and cleaved caspase-3 antibodies were purchased from Cell Signaling . \u03b2-Actin loading control was obtained from Sigma-Aldrich. The membranes were washed and incubated for 1 h with appropriate horseradish peroxidase-conjugated secondary antibodies. The protein bands were detected using a chemiluminescent (ECL) method, according to the manufacturer\u2019s instructions. Band densities were analyzed by ImageJ software.p\u22640.05, ***p\u22640.001) between different treated cell cultures in comparison to untreated controls.Statistical analysis was undertaken using one-way analysis of variance and Tukey comparison test, using GraphPad Prism 4.0 (GraphPad Software). Data is presented as the mean \u00b1 SEM of at least three independent experiments. In the figures, asterisks indicate the degree of significance difference is another important cellular signal transduction pathway necessary for the degradation of intracellular proteins and it can play a role in the regulation of pathways necessary for tumor cell growth and survival. Studies have shown that ubiquitination also can play a role in the regulation of apoptosis \u201331. A pr pathway . Therefo pathway .Finally, cells were treated with 2.5 \u03bcM or 5 \u03bcM niclosamide for 24 hours and Western blot analysis was performed with ubiquitin antibody and PARP (Poly ADP ribose polymerase) antibody, a well-known and commonly used marker for apoptosis. As shown in It has been demonstrated that accumulation of misfolded or ubiquitinated proteins can function like proteasome inhibition and induce endoplasmic reticulum (ER) stress \u201336. ER sRecent studies have found that ER stress is linked to autophagy which can function as a protective mechanism during ER stress ,38. As nThe Wnt/\u03b2-catenin signaling pathway is involved in cell proliferation, differentiation, and migration and it is required for neural stem cell development. Many studies have demonstrated that Wnt/\u03b2-catenin signaling is up-regulated in glioblastoma and can promote glioblastoma growth and invasion given its function in stem cell development . TherefoThe PI3K/AKT pathway is an intracellular signaling cascade important in regulating the cell cycle and it is directly related to cellular proliferation and cancer development. The MAPK/ERK signaling pathway is an evolutionarily conserved kinase module that links extracellular signals to cellular responses that control fundamental cellular processes such as proliferation, differentiation, and apoptosis. Given the importance of these two signal transduction pathways, they can often interconnect . PreviouSTAT3 regulates major cellular processes such as cell growth, survival and differentiation and, as previously mentioned, it is dysregulated in a number of cancers and thereby is intimately linked to tumorigenesis. Studies have demonstrated that niclosamide is a potential anticancer drug against cancers with activated STAT3 given that it can disrupt STAT3 transcription ,27. AlsoRecent studies have shown that niclosamide is cytotoxic and, more importantly, it exhibits anticancer activity in different cancer cell lines. It has been demonstrated that, like many other anticancer reagents, niclosamide has an effect on various important cellular mechanisms involved in carcinogenesis. Niclosamide is able to inhibit signaling pathways such as Wnt/\u03b2-catenin, NF-\u03baB (a regulator of inflammation), STAT3, and mTORC1 (a negative regulator of autophagy), depending on cancer cell type. However, the effect of niclosamide on human glioblastoma has not been thoroughly studied. Our study aimed to investigate the effect of niclosamide in the human glioma U-87 MG cell line. In the current study, we sought to evaluate the toxicity of niclosamide in U-87 MG cells. Our results are consistent with other published reports on the mechanisms of niclosamide-induced apoptotic cell death in other model systems, suggesting that niclosamide may employ common cytotoxic signaling pathways in different cancer cell lines. Results presented in this study demonstrated that niclosamide dramatically inhibits U-87 MG cell growth and proliferation, which correlates with results from a previous study . SignifiPrevious studies have shown controversial results regarding the effect of niclosamide on protein ubiquitination. It was reported that niclosamide was able to prevent the formation of large ubiquitin-containing aggregates in human neuronal SH-SY5Y cells . HoweverAnother important outcome of this research was the demonstration, for the first time, that niclosamide induces endoplasmic reticulum (ER) stress in U-87 MG cells. We found that U-87 MG cells exposed to niclosamide showed increased expression of CHOP, a key marker of ER stress. Moreover, our results showed that niclosamide is able to induce ER stress in a time-dependent manner. Thus, the ability of niclosamide to trigger ER stress in U-87 MG cells may be one mechanism by which niclosamide can trigger cell death of cancer cells and thus function as an effective anticancer agent.Previous results have shown that niclosamide is able to induce autophagy in various cancer cells including human neuroblastoma SH-SY5Y cells. Balgi et al. demonstrated that niclosamide induces autophagosome formation and inhibits mTORC1 signaling in MCF-7 breast cancer cells . NiclosaThe Wnt signaling pathway plays a pivotal role in human cancer cell growth and carcinogenesis. Multiple publications by various groups have shown that niclosamide is a potent inhibitor of Wnt/\u03b2-catenin signaling. In particular, niclosamide has been reported to inhibit Wnt/\u03b2-catenin signaling in various cancers \u201325,48. OBoth PI3K/AKT and MAPK/ERK signaling pathways have been reported to play an important role in the tumorigenesis of glioma cells \u201351. The via phosphorylation [Niclosamide has also recently been reported as a potent STAT3 inhibitor that functions as a tumor suppressor in cancer cells . In agrerylation . Thereforylation ,56. Highrylation ,57. FurtThe present study demonstrated that niclosamide potently induced protein ubiquitination, endoplasmic reticulum stress and autophagy in human glioblastoma cells and considerably disrupted multiple signal transduction pathways that have been reported to be associated with glioblastoma development . Wnt/\u03b2-cThe present investigation demonstrates the tumor suppressive properties of niclosamide in human glioma U-87 MG cells. Niclosamide treatment simultaneously inhibited multiple key cell signaling pathways that are involved in cell proliferation and survival and it resulted in the initiation of ER stress, autophagy, and apoptosis. Even more interestingly, niclosamide acted like a proteasome inhibitor and markedly induced expression of abundant ubiquitinated proteins in U-87 MG cells, which mostly likely contributed to its effectiveness in suppressing cell survival and promoting cell death. It is rare to find a drug, especially one that has been established as safe for human use, which can target so many intracellular pathways involved in carcinogenesis. Overall, these results suggest that niclosamide may be a novel potential therapeutic candidate for the treatment of glioblastoma.S1 Fig2. Cell lines were treated with the indicated concentrations of niclosamide for 24 h and then total cell lysates were collected. Protein was resolved by SDS-PAGE and immunoblotted with antibody specific to ubiquitin. \u03b2-Actin was utilized as a loading control.The human U-118 MG cell line was purchased from ATCC and were cultured in DMEM medium containing 10% FBS at 37\u00b0C, 5% CO(TIF)Click here for additional data file.S2 Fig2. Cell lines were treated with the indicated concentrations of niclosamide for 24 h and then total cell lysates were collected. Protein was resolved by SDS-PAGE and immunoblotted with antibody specific to ubiquitin. \u03b2-Actin was utilized as a loading control.Cell lines were purchased from ATCC. BJ skin fibroblast cells (A) were cultured in EMEM containing 10% FBS; U-2 OS cells (B) were cultured in McCoy\u2019s 5A medium containing 10% FBS; and MCF-7 (C) and MDA-MB-231 (D) cells were cultured in DMEM medium containing 10% FBS at 37\u00b0C, 5% CO(TIF)Click here for additional data file.S3 FigU-87 MG cells were treated with the indicated concentrations of niclosamide for 24 h. 20S proteasome activity was quantified by a colorimetric assay read at 480 nm and normalized by cell number. Data represent the mean \u00b1 S.E.M of at least three independent experiments.(TIF)Click here for additional data file.S4 Fig(XLSX)Click here for additional data file.S5 Fig(XLSX)Click here for additional data file.S6 Fig(XLSX)Click here for additional data file."} +{"text": "Drosophila ( http://www.indian-drosophila.org/). The clinal data on ecologically relevant phenotypes of Indian drosophilids will be useful in addressing questions related to future challenges in biodiversity and ecosystems in this region.The unique geography of the Indian subcontinent has provided diverse natural environments for a variety of organisms. In this region, many ecological indices such as temperature and humidity vary predictably as a function of both latitude and altitude; these environmental parameters significantly affect fundamental dynamics of natural populations. Indian drosophilids are diverse in their geographic distribution and climate tolerance, possibly as a result of climatic adaptation. These associations with environmental parameters are further reflected in a large number of clines that have been reported for various fitness traits along these geographical ranges. This unique amalgamation of environmental variability and genetic diversity make the subcontinent an ecological laboratory for studying evolution in action. We assembled data collected over the last 20 years on the geographical clines for various phenotypic traits in several species of drosophilids and present a web-resource on Indian- Drosophilids as a broader taxonomic group are very diverse in subtropical India, with 25 genera and over 287 species identified to date4\u20136. As in several other taxonomic groups, the genus Drosophila is species-rich in India, presumably due to the extensive heterogeneity in ecological conditions. This environmental heterogeneity may have spurred diversification among populations and taxa, and many species in this genus are endemic to the Indian subcontinent6.Drosophilid flies are classical models for population genetic and evolutionary ecology studieset al.7 and Rajpurohit and Nedved8.The Indian subcontinent ranges from 8.4 to 37.6 \u00b0N in southern Asia. It is mostly situated on the Indian plate and extends southward into the Indian ocean from the Himalayas. The region is characterized by a diverse geography with an extensive altitudinal range, vast central plains, and large river valleys in the northern foothills of Himalayas. The coefficient of variance of temperature and relative humidity changes predictively from the subtropical lower Himalayas to the tropical southern peninsula. The cumulative effects of the cold northern Himalayas, inland hot plains, and surrounding water bodies are considered as major drivers of the climatic gradients. Further details on these climatic variables can be found in Rajpurohit 8. These studies have primarily examined latitudinal and/or altitudinal variation among populations in traits measured under common garden conditions. Some intriguing patterns have emerged, such as opposing clines for desiccation resistance and starvation resistance (reviewed in Rajpurohit and Nedved8). The environmental gradients present on the Indian subcontinent, in conjunction with the observed phenotypic and genetic clines, sets a platform for studies of local adaptation to natural variation in environmental conditions. These are also the ideal places to investigate adaptive dynamics of fitness-associated traits8. Natural selection acting along environmental gradients could result in the formation and maintenance of such clines8\u201310. Such patterns of association can also reflect aspects of demography11; if a clinal pattern is replicated in independent samples the inference of selection is strengthened12.Phenotypic and genetic clines in various drosophilid species on the Indian subcontinent have been well documentedDrosophila cline dataset contains data from ten Drosophila species from studies published between 1998 and 2013 and includes traits related to morphological variation, life-histories, stress resistance, behavioral differences and genetic markers. A majority of these studies used common garden experiments to test for latitudinal and/or altitudinal differentiation among populations, thus focusing on variation at the phenotypic level8. More recently, studies have also examined range shifts in species boundaries in the context of climate change scenarios14.However, studies of wild drosophilid populations in India are not as well-known as similar studies done on European, North American and Australian populations, presumably because of limited circulation or studies published in local journals. Studies from this region are not as well incorporated in the conceptual and empirical understanding of climatic adaptation. Therefore, we assembled data from previous studies on ecologically relevant fitness traits of the Indian drosophilids to generate a comprehensive, one-stop portal for the scientific community. The Indian The compilation of historical datasets and species distributions in a central database repository will also be useful for assessing climate change related studies in this region.Drosophila, and to track shifts in these clines over time, the \u2018Indian-Drosophila\u2019 portal (http://www.indian-drosophila.org/) was established. The \u2018Indian-Drosophila\u2019 web-resource contains: (1) latitudinal clinal data , (2) altitudinal cline data , and (3) details on species taxonomy and distribution. The web-resource will be continually updated and user-friendly interfaces will be incorporated as needed. In the present format of the resource a user-friendly application named DrosoCline has been incorporated for data visualization purposes.To summarize existing studies on phenotypic and genetic clines of the Indian populations of We began the development of this community resource with an exhaustive search of papers published between 1987 and 2015 that examined clinal variation in Indian populations of various drosophilid species. We selected papers and datasets which conformed to the following criteria: (1) at least three populations were included along the latitudinal/altitudinal transect; (2) traits were measured under common garden conditions, i.e., excluding studies where direct measurements were done on wild collected samples; and (3) if parallel studies of the same cline had been conducted, we selected the first, initial study only. With these criteria 16 papers were selected and incorporated to this web-resource. A point to note, most of the traits in this web-resource exhibited significant clines and were reported accordingly; there is the possibility of some bias, as work that failed to identify significant clines may not have been published. In the present clinal datasets five major categories of traits have been incorporated: (1) morphology, (2) reproduction and behavior, (3) metabolites, (4) stress resistance, and (5) allozymes see .Drosophila species populations are given in http://www.imdpune.gov.in/).A total of 61 datasets have been incorporated in this resource from which 32 are latitudinal and 29 are altitudinal (see Data Citation 1). These datasets span ten different species, representing the most comprehensive range of studies in drosophilids. This is the only collection of datasets that incorporates both latitudinal and altitudinal clinal data from a single geographical region (the Indian subcontinent). The geographical distribution of populations from which these 61 clinal datasets are derived is between 9\u201333\u00b0 N/ 75\u201383\u00b0 E see . This co15\u201318. Thorax length was measured from the posterior tip of the scutellum to the anterior margin of the thorax. Wing length measured from the point of attachment with thorax up to the tip of the third longitudinal vein. The thorax and wing measurements are expressed in mm\u00d7100. Trident pigmentation was scored by visual examination of dorsal side of the thorax using four phenotypic classes. Abdominal pigmentation was scored by the visual examination of lateral side of the abdominal tergites using discrete phenotypic classes. Body weight is expressed as mg\u00d7100.A total of 18 different traits under five major categories have been incorporated in this resource (Data Citation 1). The morphological traits include thorax length, wing length, sternopleural bristle number, body weight, trident pigmentation, and abdominal pigmentation20. Ovariole number was presented as average or sum of both ovary lobes. Fecundity was reported as the number of eggs laid per fly per day. Copulation duration was measured as the time from successful mounting to separation and is expressed in minutes.The category for reproduction and behavior included ovariole number, fecundity and copulation duration21. For lipid content, the estimation method as described by Marron et al.22 was followed. Individual flies were dried at 60\u2009\u00b0C for 48\u2009h in smaller tubes and weighed. Di-ethyl ether was subsequently added to the samples and agitated at 37\u2009\u00b0C for 24\u2009h. Flies were then removed from the solvent and dried again at 60\u2009\u00b0C for 24\u2009h and reweighed. Lipid content was calculated by subtracting lipid free mass from dry mass. Trehalose content was estimated using commercially available kits . Flies were homogenized in groups and heated in a water bath set at 95\u2009\u00b0C for 20\u2009min. Subsequently, samples were centrifuged at 12,000\u2009r.p.m. for 15\u2009min and aliquots of the supernatant were digested with trehalase at 37\u2009\u00b0C. The reaction absorbance was measured at 340\u2009nm.The metabolites category included total lipid content as well as trehalose content23\u201330. Desiccation assays were performed in narrow size Drosophila culture vials where no food and water was provided. Flies were kept individually or in groups of ten with silica gel desiccant; flies were separated from the silica gel desiccant by a foam plug. To measure starvation resistance, flies were kept in narrow Drosophila culture vials in the absence of food. Water was made available with a cotton ball soaked in water or with 1\u20132% agar media in the vial. For heat-knockdown time measurements, individual flies were placed in glass tubes submerged in a water bath set at 39\u2009\u00b0 C. To measure chill-coma recovery time, flies were placed in empty glass vials and buried in ice for a specific period of time. Flies were then brought to room temperature and the recovery time of each individual was recorded.Stress resistance traits included desiccation resistance, starvation resistance, heat-knockdown time, and chill-coma recovery timeAdh and Est-6 allozymes were examined for allele frequency variation among populations using standard starch gel electrophoresis and staining methods. Each population was characterized by the frequency of one of the common electrophoretic alleles . For all the traits described above, the original publications give further methodological details. The original data with the accompanying reference can be downloaded using the DrosoCline application.Drosophila web-resource (http://www.indian-drosophila.org/). The datasets can be accessed through the DrosoCline application (https://indian-drosophila.shinyapps.io/DrosoCline/). A direct link for this application is provided in the Indian-Drosophila web-resource. DrosoCline allows the user to choose the type of geographical range , species, sex, and phenotype. The user can also download the data for the selected combinations by clicking the associated download tab. The downloaded data file (in.csv format) also contains information regarding the published references. This resource currently archives data on latitudinal and altitudinal variations for 18 ecologically relevant traits in 10 species, and more datasets will be incorporated as they become available. We propose to update the data portal via biannual literature surveys and direct author submission.A total of 61 datasets are made available through the Indian-23\u201331. The data presented here in the paper and on the online resource have also been statistically analyzed in the associated published work. Data have been re-visualized and checked for possible typing errors in the various published papers. If any discrepancies were found they were corrected before placing them on the web-resource. Only the experiments that included control treatments and adequate replication were included in this analysis and the associated web-resource.The datasets reported in this collection are already published in peer-reviewed journals32. A temperature profile for the past 100 years (1900\u20132000) on the Indian subcontinent is shown elsewhere .Severe effects of global warming are projected for South Asia. The Regional Climatic Modeling System PRECIS developed by the Hadley Center predicts a 2\u20134\u2009\u00b0C temperature rise in India as the 21st century progresses, with the northern parts of the India being more sensitive to this change34. Studies on phenotypic differentiation across environmental gradients could be a valuable tool to understand fundamental aspects of the evolutionary response to climate change. In Drosophila species, genetic and phenotypic change across geographical transects could provide insight into the connectivity between climate change and evolutionary shifts. Along these lines, recent work on Indian altitudinal transects in the Western Himalayas has shed light on habitat shrinkage and expansion associated with climate change14. Ongoing studies from the Indian subcontinent will provide further opportunities for more precise studies merging patterns of phenotypic and genetic variation, species distributions, historical data, and climate change.The global temperature rise may result in increasing physiological challenges that put organisms, taxa, and entire ecosystems at risk. Recent studies have clearly shown range expansion for many populations, and that such shifts are accompanied by changes in phenology and rapid adaptation to environmental changeet al.35 proposed that information on species distribution and geographical variations over time could provide a basic framework to infer evolutionary responses associated with climate change35. The resource we develop here will help the scientific community track spatiotemporal changes in phenotype and associated genomic regions, and can also be used to examine associations between pattern shifts and temporal change in specific climatic variables.This unique amalgamation of taxonomic and genetic diversity, as well as the presence of robust clines for multiple traits across species, makes the Indian subcontinent an ecological laboratory for studying evolution in action. Pimm Drosophila Database also allow us to make comparisons across populations residing on different continents. A resource for Australian drosophilids already exists36. A cross geographical comparison could be used to explore fundamental and unaddressed questions in Drosophila ecology and evolution.Resources such as the Indian-How to cite this article: Rajpurohit, S. et al. A resource on latitudinal and altitudinal clines of ecologically relevant phenotypes of the Indian Drosophila. Sci. Data 4:170066 doi: 10.1038/sdata.2017.66 (2017).Publisher\u2019s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3+ T cells both efficiently induced AIP, while CD4+ and CD8+ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4+CD44high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (Tregs) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred Tregs on pancreatic histopathology. The results of our studies suggest a key role of T cell\u2010mediated processes in murine AIP. The effects of CD4+CD44high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow\u2010up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.The immunopathogenesis of autoimmune pancreatitis ( It is a differential diagnosis of pancreatic carcinoma, because clinical symptoms of both entities are very similar.Both, humoral and cellular immunity have been implicated into the pathogenesis of AIP.+ and CD8+ T lymphocytes are present in pancreatic parenchyma of AIP patients,+ T cells predominate in tissue infiltrates. Increased production of interferon (IFN)\u2010\u03b3 by CD4+ Thelper\u2010cells type 1 (Th1) has been proposed to promote AIP,Less is known about cellular immune processes in AIP, although T cells are the prevalent type of infiltrating immune cells in affected tissue.The MRL/MpJ mouse model has proven useful to study the pathogenesis of AIP. These mice spontaneously develop an AIP at the age of about 6\u00a0months.+CD44high memory T cells in the pathogenesis of AIP.Tregs) in the mediation of the therapeutic effects of rapamycin through a suppression of the effector T cell response.Using this mouse model in previous studies, we gained genetic evidence for an involvement of CD4One characteristic of autoimmune diseases such as AIP is the possibility to adoptively transfer the disease from sick to healthy individuals in in\u00a0vivo experiments. In 1992, Kanno et\u00a0alHowever, splenocytes consist of a variety of different lymphocytes and the specific populations necessary for the transfer are still unknown. Thus, the aim of this work was to gain mechanistic insights into processes of cellular autoimmunity in murine AIP. We have used the MRL/MpJ mouse model to perform adoptive transfer experiments. Different T cell subpopulations were transferred to identify those that are able to induce AIP or to inhibit progress of the disease.+ T cells and CD4+CD44high memory T cells are sufficient to induce AIP in susceptible MRL/MpJ recipient mice, suggesting key roles of these specific T cell subpopulations in the immunopathogenesis of murine AIP. Interestingly, the transfer of immunosuppressive Tregs failed to inhibit progress of AIP in spontaneously diseased MRL/MpJ mice.Our data indicate that both adoptively transferred CD322.1MRL/MpJ mice were purchased from Charles River Laboratories . These mice spontaneously develop an AIP at an age of about 24\u00a0weeks.2.2g, 10\u00a0minutes, 4\u00b0C), resuspended in 1\u00a0mL phosphate\u2010buffered saline (PBS) and incubated for 3\u00a0minutes on ice with 4\u00a0mL of ice\u2010cold 0.25\u00a0M NH4Cl solution to lyse erythrocytes. Lysis was stopped by adding 10\u00a0mL of culture medium and centrifugation . The cells were sowed at a density of 1\u00a0\u00d7\u00a0106\u00a0cells/mL in cell culture flasks. Based on the protocol of Kanno et\u00a0al,2 humidified atmosphere.Splenocytes were obtained from female MRL/MpJ mice. Young and adult mice served as spleen donors. The animals were sacrificed by an overdose of ketamine/xylazine, serum was collected using clot activator containing tubes , and the pancreas, liver and kidneys were harvested and cryo\u2010 or paraffin\u2010embedded for further analyses. The spleen was kept on ice in splenocyte culture medium RPMI\u20101640 supplemented with 10% foetal calf serum , 1% penicillin/streptomycin (Biochrom) and 50\u00a0\u03bcmol/L \u03b2\u2010mercaptoethanol . Subsequently, the organ was forced through a 70\u00a0\u03bcm cell strainer and incubated in 10\u00a0mL splenocyte culture medium containing 50\u00a0\u03bcg/mL DNase I for 10\u00a0minutes. The cell suspension was centrifuged ; CD4+ Thelper\u2010cells: CD4+ T Cell Isolation Kit, mouse ; CD8+ cytotoxic T cells: CD8+a T Cell Isolation Kit, mouse ; CD4+CD44high memory T cells: Mouse Memory T Cell CD4+/CD62L\u2212/CD44high Column Kit . All kits were carried out following the manufacturer's instructions.On day 3 of splenocyte culture, cells were harvested and washed with PBS, pH 7.4. Unpurified splenocytes were either used directly for adoptive transfer or subjected to isolation of lymphocyte subpopulations. The following kits were used to isolate untouched cells by negative selection: CD36 cells or 2\u00a0\u00d7\u00a0106 cells (CD4+CD44high T cells and another group of unpurified splenocytes) in a volume of 200\u00a0\u03bcL were injected into the tail vain of recipient mice. Mice without cell transfer served as controls. Table\u00a0As recipients, 8.1\u00a0\u00b1\u00a00.2 to 9.5\u00a0\u00b1\u00a00.5\u2010week\u2010old MRL/MpJ mice of the indicated gender were employed. One week prior to cell transfer, recipient mice were treated once with 200\u00a0mg/kg bodyweight cyclophosphamide to facilitate engrafting following the manufacturer's instructions.For the transfer of +CD25+ T cells were cultured using the Treg Expansion Kit, mouse . Briefly, 2\u00a0\u00d7\u00a0106\u00a0cells/mL were dissolved in splenocyte culture medium supplemented with 2000\u00a0U/mL interleukin (IL)\u20102 and sowed into 24\u2010well plates applying 200\u00a0\u03bcL/well. Afterwards 200\u00a0\u03bcL of 6\u00a0\u00d7\u00a0106 CD3/CD28 MACSiBeads/mL in splenocyte culture medium plus 2000\u00a0U/mL IL\u20102 was added to each well. On day 1, 400\u00a0\u03bcL/well of splenocyte culture medium, supplemented with 2000\u00a0U/mL IL\u20102, was added to each well. Half of the medium was replaced with fresh medium and IL\u20102 on days 3 and 5. On day 7, the MACSiBeads were removed employing the MACSiMAG Seperator (Miltenyi Biotec) and cells were sowed out again with fresh beads. The expansion was repeated for another week.CD46 cells in a volume of 200\u00a0\u03bcL were injected into the tail vain of 30\u00a0\u00b1\u00a00.2\u2010week\u2010old female MRL/MpJ mice , and pathological changes were graded on a semi\u2010quantitative scale from 0 (none) to 4 (severe) as previously described (H&E\u2010score).AIP\u2010score for each animal represents the highest score that was obtained by the evaluation of H&E and CD3 stains, through the assessment of at least 8 pancreatic sections per mouse.As a complementary method, immunohistochemistry was performed on 6\u00a0\u03bcm thick cryosections of pancreatic tissue. Immunohistochemistry was carried out using an avidin\u2010biotin\u2010peroxidase (ABC) system . The slides were then counterstained with Mayer's hemalum solution, dehydrated by two short incubations in ethanol and xylene each and embedded in Pertex . To define the composition of the inflammatory regions, the following antibodies were used: rat anti\u2010mouse CD3 , rat anti\u2010mouse CD4 (Immunotools), rat anti\u2010mouse CD8\u2010\u03b2 , rat anti\u2010mouse CD44 and biotinylated goat anti\u2010rat IgG antibody (Vector Laboratories). Extension and density of the specifically stained lymphocytic infiltrates were assessed in a semi\u2010quantitative manner. Therefore, scores that resembled the H&E\u2010based scoring system were applied as follows (CD3\u2010score): 0, no infiltrate; 1, minimal infiltration of periductal tissue; 2, moderate periductal infiltration and beginning infiltration of parenchyma; 3, extensive and multifocal parenchymal and periductal infiltrates; 4, dense and confluent infiltrates throughout the entire section. The so\u2010termed 2.6Unpurified splenocytes or isolated subpopulations were subjected to flow cytometric analyses to validate the quality of the cells. Prior to staining, the Fc receptors were blocked by preincubation with anti\u2010CD16/CD32 antibodies for 10\u00a0minutes on ice. Surface staining was accomplished by incubating the cells with fluorochrome\u2010conjugated specific antibodies for 20\u00a0minutes in the dark on ice. The following antibodies were employed: anti\u2010CD3\u2010FITC (130\u2010102\u2010496), anti\u2010CD4\u2010FITC (130\u2010102\u2010541), anti\u2010CD4\u2010PE (130\u2010102\u2010619), anti\u2010CD8\u2010PE (130\u2010102\u2010595), anti\u2010CD19\u2010FITC (130\u2010092\u2010042), anti\u2010CD25\u2010APC (130\u2010102\u2010787), anti\u2010CD44\u2010APC (130\u2010102\u2010563), anti\u2010CD62L\u2010PE (130\u2010102\u2010907).Intracellular staining of FoxP3 was performed using an anti\u2010FoxP3\u2010PE antibody and the FoxP3 Staining Buffer Set following the given instructions.Flow cytometric analyses were run on a FACS Verse (BD Biosciences) or FACS Calibur (BD Biosciences). A total of 10\u00a0000 events per sample were acquired and data were evaluated using the FACS Suite or CellQuest Pro software (both BD Biosciences).2.7U\u2010test and Bonferroni's post hoc test. P\u00a0<\u00a0.05 (Bonferroni\u2010adjusted) was considered to be statistically significant.Data were analysed using the IBM SPSS Statistics 22.0. Values are expressed as mean\u00a0\u00b1\u00a0standard error of mean (SEM) for the AIP\u2010scores of the different mouse cohorts as well as for affected livers and kidneys and the age of the animals. Statistical significance was checked using the Kruskal\u2010Wallis test followed by the Mann\u2010Whitney 33.1Female MRL/MpJ mice spontaneously develop AIP at an age of at least 6\u00a0months.3.2+, CD4+ or CD8+ T cells were purified. All isolations lead to highly pure cell populations .As expected, both animals of the control group and male recipient mice exhibited autoimmune lesions at a low frequency only . In contrast, the transfer of unpurified splenocytes into female MRL/MpJ mice induced the development of inflammatory foci within the pancreatic tissue of recipient mice . The transfer of CD4+ and CD8+ T cells lead to average AIP\u2010scores of 1.6\u00a0\u00b1\u00a00.3 and 1.9\u00a0\u00b1\u00a00.3, respectively. When unpurified splenocytes from young mice without a pancreatic phenotype at the time of cell isolation were transferred, an average AIP\u2010score of 1.8\u00a0\u00b1\u00a00.4 was obtained. The three latter scores were not significantly higher than the score of untreated controls.Interestingly, CD33.3+ T cells, with CD4+ T cells being predominant over CD8+ T cells number of unpurified splenocytes, enabling a direct comparison of the findings.To gain further mechanistic insights, we employed CD4+CD44high memory T cells , expanded for 2\u00a0weeks . Recipient mice were at the advanced age of 30\u00a0\u00b1\u00a00.2\u00a0weeks, when spontaneous AIP occurs at a high frequency.Next, we asked if transferred Tregs reduced the average AIP\u2010score to 1.6\u00a0\u00b1\u00a00.4. This effect, however, was statistically not significant (P\u00a0=\u00a0.274 vs controls).The AIP\u2010scores for the injected mice and a control group (no cell transfer) are shown in Figure\u00a04The immunopathogenesis of AIP, a rare form of CP, is largely unknown. In particular, the role of cellular autoimmunity has not been studied in detail. The results of this study identify two lymphocyte subpopulations that efficiently trigger AIP in MRL/MpJ mice, an experimental model of the disease.Kanno et\u00a0al+ T cells induce an AIP as efficient as unfractionated immune cells when adoptively transferred from adult and sick MRL/MpJ mice into young and still healthy animals of the same strain. Consequently, cellular immunity mediated by T cell\u2010dependent processes is essential and sufficient for the transfer of the disease under the given experimental conditions. Evidence for T cell\u2010mediated processes has also been found in experimental AIP in rats before: Amylase\u2010specific CD4+ T cells could induce AIP in adoptive transfer experiments.Furthermore, our results revealed that CD3+CD44high memory T cells were sufficient to transfer murine AIP. Strikingly, the average AIP\u2010score for recipient mice treated with memory T cells was as high as the AIP\u2010score for recipient mice treated with CD3+ T cells, although the number of transferred cells was less than the half. The studies on the role of CD4+CD44high T cells were encouraged by the results of previous investigations of our group: Genotype\u2010phenotype correlations studies in AIP\u2010susceptible mice lead to the mapping of quantitative trait loci (QTLs) containing putative susceptibility genes for AIP.+CD44+ splenocytes and the development of AIP were found to be controlled by overlapping QTLs.+CD44high memory T cells in the spleen correlated with the severity of AIP.+CD44high memory T cells in murine AIP is underscored by the fact that they were the only type of immune cell that fulfilled both criteria (overlapping QTLs with AIP and correlation with disease severity). CD4+CD44highCD62Llow effector memory T cells (TEM) are, in contrast to na\u00efve T cells, antigen\u2010primed and thus deliver T cell memory.TEM are located in lymphoid and non\u2010lymphoid tissue and provide immediate local immune response.+CD44high memory T cells in the pathogenesis of autoimmune\u2010related experimental pancreatitis.Moreover, CD4+ and CD8+ T cells in AIP has been discussed before.+ nor CD8+ T lymphocytes alone were sufficient to induce AIP in healthy (but susceptible) recipients. Together, these findings suggest a contribution of both types of T cells to the exhibition of the disease phenotype.The role of CD4Tregs, which suppress the activity of effector T cells.Tregs on T cells could also lead to an inhibition of the progression of AIP in\u00a0vivo. However, under the given experimental conditions, the transfer of activated Tregs alone was not sufficient to attenuate AIP in MRL/MpJ mice, suggesting that the availability of these cells was not a limiting parameter in our model. Furthermore, the stage of the disease at the time of Treg application as well as the specific protocol of cell application needs to be considered as influencing factors.Regulatory T cells are immune cells with largely inhibitory characteristics.The MRL/MpJ model of spontaneous AIP that was used in this study mimics the human disease, specifically type 1, in some important regards. Thus, inflammation starts from the exocrine tissue, involves destruction of acinar architecture and increased deposition of extracellular matrix, and leaves the islets largely unaffected. Moreover, dense infiltrates of immune cells, such as T cells and activated B\u2010cells/plasma cells, are characteristic of both the human and the murine disease.While this study was dedicated to the analysis of the role of different T cell populations, previous experimental studies by us and others have implicated macrophages and neutrophils into the pathogenesis of the disease.+ and CD4+CD44high T cells effectively transfer AIP from sick MRL/MpJ mice to healthy, but AIP\u2010susceptible individuals. While our data clearly support a key role of T cells in experimental AIP, they do not address the involvement of autoantibodies to disease induction and progression. As both cellular and humoral immune reactions are likely to contribute to the pathogenesis of human AIP, the interplay between these processes is subject of follow\u2010up studies of our laboratory.In summary, the results of this study provide new insights into the immunopathogenesis of experimental AIP by showing that both CD3The authors confirm that there are no conflict of interests.LE performed the experiments and wrote the manuscript with the assistance of RJ. SR and RJ participated in the cell culture and animal experiments. All authors contributed to the design of the experiments, evaluated the data and approved the manuscript.\u00a0Click here for additional data file."} +{"text": "Pseudoaneurysm is one of the most serious complications of femoral trochanteric fracture surgery. Since the rupture of pseudoaneurysm may lead to death, early detection is important. We report the case of an 80-year-old male who developed pain in the proximal thigh and severe swelling after internal fixation of a femoral trochanteric fracture with a hip nail. Angiography revealed a pseudoaneurysm of a branch of the deep femoral artery near the interlocking screw. The vascular lesion was immediately treated by transcatheter embolization, and the vascular leakage was completely resolved with catheter embolization. After embolization, the patient's clinical state improved rapidly, and the laboratory values improved to normal after several weeks. The current case study reminds us that pseudoaneurysm can occur after intramedullary nail placement to treat a femoral trochanteric fracture. Femoral trochanteric fracture is a fracture that is often encountered in the clinical setting. One of the most serious complications of femoral trochanteric fracture repair surgery is pseudoaneurysm. Pseudoaneurysm occurring in a branch of the deep femoral artery is a relatively rare complication after surgical repair of a femoral trochanteric fracture. The diagnosis of pseudoaneurysm is usually delayed because of nonspecific clinical features like pain, subcutaneous bleeding, swelling, or unexplained anemia. However, rupture of the pseudoaneurysm may lead to hemorrhagic shock, so a pseudoaneurysm should be suspected early. Moreover, appropriate tests are necessary, such as CT angiography and angiography, for the early detection of pseudoaneurysm. Furthermore, appropriate early treatment intervention such as open surgical repair and coil embolization is necessary to prevent the rupture of the pseudoaneurysm. We present a case of a deep femoral arterial branch pseudoaneurysm diagnosed in an 80-year-old man after the placement of an intramedullary nail for a femoral neck fracture.An 80-year-old man in good health was admitted to our hospital for severe pain in the left hip associated with functional impairment after a fall at home. X-rays and computed tomography (CT) of the painful hip showed a femoral trochanteric fracture with an Evans classification of 1c . Two dayPseudoaneurysms arise from a disruption in arterial wall continuity resulting from inflammation, trauma or iatrogenic causes such as surgical procedures . PseudoaIn the current study, rising CRP levels were also observed with the progression of anemia without signs of infection of the surgical wounds or other organs. CRP levels for early detection are said to correlate with early postoperative complications such as infection, dislocation, and hematoma after proximal femoral fracture surgery . It shouIn conclusion, we described a case of effective embolization for a pseudoaneurysm of the deep femoral artery caused by an intramedullary nail used to treat a femoral trochanter fracture."} +{"text": "The incidence of in-hospital cardiovascular adverse events (AEs) in patients with ST-segment elevation myocardial infarction (STEMI) following primary percutaneous coronary intervention (PCI) is relatively high. Identification of metabolic markers could improve our understanding of the underlying pathological changes in these patients. We aimed to identify associations between concentrations of plasma metabolites on admission and development of in-hospital AEs in post-PCI patients with STEMI. We used targeted mass spectrometry to measure plasma concentrations of 26 amino acid metabolites on admission in 96 patients with STEMI who subsequently developed post-PCI AEs and in 96 age- and sex-matched patients without post-PCI cardiovascular AEs. Principal component analysis (PCA) revealed that PCA-derived factors, including branched chain amino acids (BCAAs), were associated with increased risks of all three pre-specified outcomes: cardiovascular mortality/acute heart failure (AHF), cardiovascular mortality, and AHF. Addition of BCAA to the Global Registry of Acute Coronary Events risk score increased the concordance C statistic from 0.702 to 0.814 (p\u2009<\u20090.001), and had a net reclassification index of 0.729 . These findings demonstrate that high circulating BCAA concentrations on admission are associated with subsequent in-hospital AEs after revascularization in patients with STEMI. However, these patients may develop in-hospital cardiovascular adverse events (AEs) such as cardiac rupture, malignant arrhythmias, and onset of heart failure (HF) after PCI as a result of myocardial ischemia and ischemia\u2013reperfusion injury2. The 30-day mortality rate and incidence of in-hospital acute heart failure (AHF) among patients with STEMI who undergo primary PCI are reportedly 7.9% and 28%, respectively4. An early and efficient interventional strategy could reduce the incidence of cardiovascular AEs and improve these patients\u2019 short- and long-term prognoses5.Primary percutaneous coronary intervention (PCI) is currently the recommended treatment for patients presenting with ST-segment elevation myocardial infarction (STEMI)6. However, the limited effectiveness of these strategies in preventing and treating post-MI cardiovascular AEs suggests that interruption of the pathways responsible for the AEs are needed to improve outcomes. Maladaptive molecular processes may be a significant cause of such AEs; thus, a metabolomics approach may help to address these problems.Current optimal secondary prevention therapies for patients with STEMI who have undergone PCI primarily involve drugs such as antiplatelet agents, statins, \u03b2-blockers, angiotensin-converting enzyme inhibitors, and aldosterone antagonists8. Amino acids are important metabolites of nutrients that are vital to survival and growth of cells and serve as substrates for intracellular biosynthesis and signalling molecules9. Previous studies have shown that ischemia and ischemia\u2013reperfusion injury lead to disordered amino acid catabolism, especially of the branched-chain amino acids (BCAAs) leucine, isoleucine, valine, and taurine12. Defective amino acid catabolism sensitizes the heart to ischemia\u2013reperfusion injury, leading to adverse myocardial remodelling and post-MI HF13.Following primary PCI, there are metabolic changes in the hearts of patients with STEMI and emerging evidence suggests that metabolic remodelling in the heart is the key to development and progression of heart failureIn this study, we aimed to assess abnormalities in circulating amino acid metabolites in admission blood samples of patients with STEMI who went on to undergo PCI and who developed in-hospital cardiovascular AEs and compare them with those in age- and sex-matched controls who did not develop AEs.14, history of hypertension, diabetes mellitus and smoking, and objective laboratory measures, including blood lipids, glucose, and creatine concentrations. Troponin I (TnI) and N-terminal B-type natriuretic peptide (NT-pro BNP) concentrations were significantly higher in the AE than the No-AE group . Furthermore, Global Registry of Acute Coronary Events (GRACE) risk scores were significantly higher in the AE than the No-AE group .Of the 96 patients with STEMI who developed the composite primary outcome of cardiovascular death or AHF during hospitalization, 20 had cardiovascular deaths and 76 AHF. The baseline clinical characteristics of patients who did and did not develop cardiovascular AEs are listed in Table\u00a0Differences in admission plasma concentrations of 26 amino acids between the AE and No-AE groups are shown in Supplementary Table\u00a0Six meaningful metabolomics factors were identified by principal component analysis (PCA) , Factors 1, 2, and 3 were found to be associated with an increased risk of the composite primary outcome of cardiovascular death or AHF analysis identified the same six metabolomics factors, Factor 2 having the highest value for distinguishing between patients with and without AEs Fig.\u00a0.Figure 1Patients in the highest tertile for Factor 2 were at a 9.45-fold greater risk of developing a primary outcome than those in the lowest tertile identified both NT-pro BNP and Factor 2 as predictors of primary outcomes . However, TnI was not identified as a predictor in the current study the receiver-operating characteristic (ROC) curves. According to this analysis, Factor 2 had a slightly higher predictive value than NT-pro BNP (AUC: 0.69 vs. 0.74 for the composite primary outcome) ; the net reclassification index (NRI) was 0.729 and the integrated discrimination improvement (IDI) 0.186 . Adding NT-pro BNP only moderately improved prediction by GRACE score, increasing the C statistic to 0.760 (p\u2009=\u20090.069). Importantly, adding a combination of Factor 2 and NT-pro BNP to the GRACE score further improved risk prediction, increasing the C statistic to 0.869 (p\u2009<\u20090.001). The NRI and IDI were also significantly higher , is activated by dephosphorylation with mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm)23. Transcriptomic and metabolomics studies have revealed that expression of PPM1K (the gene encoding PP2Cm) is suppressed in cardiomyopathy and that proteins involved in the rate-limiting step of BCAA degradation, including BCKDH E1 \u03b1/\u03b2 and E2 subunits, are downregulated during pathological stress, leading to marked accumulation of branched-chain alpha-keto acids (BCKAs) in stressed heart tissue24. Abundant BCAAs directly inhibit mitochondrial respiratory function, leading to superoxide accumulating in the mitochondria of cardiomyocytes24. Furthermore, plasma concentrations of BCAAs and BCKAs are significantly increased in PPM1K gene knockout mice, whereas in PPM1K-null mice cardiac function is compromised at a young age and deteriorates more quickly with mechanical overload25. Accumulation of BCAAs has been shown to suppress glucose metabolism and sensitize the heart to ischemic injury26. Oral BCAA administration activates mammalian target of rapamycin (mTOR) signalling and exacerbates cardiac dysfunction and remodelling in mice with surgically induced MI26. These lines of evidence support the hypothesis that abnormal BCAA catabolism in the ischemic or otherwise pathologically stressed myocardium may cause marked accumulation of BCAAs and BCKAs, which may in turn promote mitochondrial dysfunction, superoxide accumulation, and cardiomyocyte death9, resulting in heart failure or death post-MI.The association between high admission concentrations of BCAAs and cardiovascular AEs after STEMI may be linked to previously identified correlations between abnormal BCAA catabolism and MI and ischemia\u2013reperfusion injuryet al.27 have reported that serum concentrations of BCAAs are remarkably higher in patients with coronary artery disease than in healthy individuals, and are independent of diabetes, hypertension, dyslipidaemia, and BMI. Fan et al.28 demonstrated that plasma leucine concentrations are up-regulated in patients with acute MI compared with patients with unstable angina. A case-control study22 showed that plasma leucine and isoleucine concentrations are higher in patients with chronic heart failure than in healthy controls. Several recent studies have investigated the correlation between metabolic disorders of BCAAs and prognosis in patients with cardiovascular disease. Ruiz-Canela et al.29 reported that high plasma baseline BCAA concentrations are associated with higher cardiovascular disease risk during 1-year follow-up. Furthermore, another recent study8 demonstrated that the prognostic value of several metabolites, involving BCAAs, exceeded that of B-type natriuretic peptide in terms of AEs in patients with chronic heart failure.Several other patient-based studies have shown that disturbances of BCAA metabolism are associated with multiple cardiovascular diseases, including coronary artery disease, MI, and HF. Yang et al.30 reported finding remarkable impairment of BCAA catabolism in the myocardium in response to infarction in a murine MI model; oral administration of BCAA further increased BCAA concentrations, activated mTOR signalling, and exacerbated cardiac dysfunction and remodelling. Importantly, these researchers showed that inhibition of mTOR by rapamycin or pharmacological inhibition of branched chain keto acid dehydrogenase kinase, a down-regulator of myocardial BCAA catabolism, significantly improves cardiac BCAA catabolism, reduces amounts of myocardial BCAA, and ameliorates post-MI cardiac dysfunction and remodelling. Additional data, including data from experimental studies in larger animals, are needed to clarify whether BCAAs are a potential therapeutic target.In the current study, we found that high plasma BCAA concentrations on admission are associated with high risk of in-hospital cardiovascular AEs in patients treated with primary PCI for STEMI. These results suggest that increasing BCAA catabolism to lower plasma concentrations of BCAAs may improve the prognosis of patients with STEMI after PCI. Wang 31. Second, the study was also limited by the fact that blood samples were taken in a non-fasting state. Third, our findings are based on blood metabolite profiles, which are not necessarily representative of myocardial metabolism.The present study had several limitations. First, this was a small single centre study; our results should therefore be validated in larger cohorts. However, in light of the small samples sizes, we used RSF analysis to validate and increase confidence in our resultsIn conclusion, we found that high plasma BCAA concentrations on admission are associated with in-hospital cardiovascular AEs in post-PCI patients with STEMI and that addition of plasma BCAA concentrations and NT-pro BNP improves the predictive ability of GRACE risk scores. Of note, BCAAs have been shown to be involved in the pathogenesis of ischemia\u2013reperfusion injury and adverse cardiac remodelling in experimental studies, supporting the hypothesis that disordered BCAA metabolism may represent a novel insight into the pathological mechanisms underlying prognosis after MI. Further studies of BCAA metabolism aimed at improving the prognosis of patients with myocardial infarction and ischemic\u2013reperfusion injury are warranted.32. Briefly, patients >18 years of age referred for primary PCI were enrolled if they met the following criteria: (i) first STEMI; and (ii) onset of chest pain <12\u2009h before presentation. STEMI was defined as continuous chest pain for at least 20\u2009minutes, and new ST-segment elevation \u22650.1\u2009mV in at least two contiguous leads, or new-onset left bundle branch block. Patients with Killip class\u2009>\u2009I on admission, heart failure, serum creatinine >250 \u00b5mol/L, alanine aminotransferase >135\u2009U/L, severe fatty liver or liver cirrhosis, or malignant tumours were excluded, as were patients who had taken amino acid supplements in the preceding 3 months. Aspirin and clopidogrel (both 300\u2009mg) or ticagrelor (180\u2009mg) were administered before catheterization. The postoperative therapeutic strategy was performed in accordance with standard guidelines1. The Medical Ethics Committee of the First Hospital, Jilin University approved the study protocol and the study participants provided written informed consent. The primary clinical outcome was the composite variable of cardiovascular death and AHF after PCI during hospitalization. Secondary clinical outcomes included the individual components of the primary outcome separately were also investigated. Cardiovascular death included death from pump failure, arrhythmia, or mechanical complications including ventricular septal rupture and free wall rupture. AHF was defined as presence of dyspnoea, peripheral oedema and pulmonary rales on physical examination, radiological evidence of pulmonary congestion, need for administration of intravenous diuretic agents, or continuous positive airway pressure. A total of 96 patients with STEMI treated with primary PCI who developed in-hospital cardiovascular death or AHF comprised the AE group. We analysed selected metabolites in these 96 AE patients and 96 No-AE patients matched (1:1 ratio) for age group (\u00b13 years) and sex was conducted using an Ultimate 3000 UHPLC system coupled to a mass spectrometry system ,which provided quantitative assessment of concentrations of 26 amino acids. Details of methods can be found in the Data Supplement.Blood samples were collected from patients with STEMI on admission.33. In the current study, GRACE risk scores were calculated using age, heart rate, systolic blood pressure, creatinine, Killip class, presence or absence of cardiac arrest on admission, presence or absence of ST-segment elevation, and cardiac enzyme concentrations on admission.The GRACE score is widely used to evaluate the risk of death or recurrent MIU-tests for continuous variables and \u03c72 tests for categorical variables.Baseline characteristics were compared between the No-AE and AE groups using Mann\u2013Whitney 34 was used to identify significant factors: only factors with an eigenvalue of \u22651.0 were considered. Individual metabolites with an absolute value for factor load of \u22650.7 are reported as components of a given factor . A large cut-off was chosen to avoid false positives. The relationships between PCA-derived factors and clinical outcomes were evaluated using univariate and multivariate logistic regression models, adjusting for traditional STEMI risk factors . ORs and 95% CIs are given.PCA was used to reduce the large number of correlated metabolites to a smaller number of uncorrelated factors. Varimax rotation35, this being a powerful machine-learning statistical algorithm for validating results of studies with small sample sizes. PCA factors were used as covariates and events as the outcome. The results were validated by ranking the PCA factors on the basis of importance of the variable as determined by RSF.The PCA results were validated by RSF analysisP value of\u2009<0.05 was considered to denote statistical significance.Model performance was assessed by calculating the C statistic, NRI, and IDI. The 95% CIs were calculated for each variable. ROC curves were generated and the AUCs calculated to compare the accuracies of traditional biomarkers and the PCA factors. Statistical analyses were performed using commercial software and R version 3.3.3 . A two-tailed Supplementary Table1,Supplementary Table2"} +{"text": "Planning community resilience to sea level rise (SLR) requires information about where, when, and how SLR hazards will impact the coastal zone. We augment passive flood mapping by simulating physical processes posing recurrent threats to coastal infrastructure, communities, and ecosystems in Hawai\u2018i . We find that the \u201cbathtub\u201d approach, alone, ignores 35\u201354 percent of the total land area exposed to one or more of these hazards, depending on location and SLR scenario. We conclude that modeling dynamic processes, including waves and erosion, is essential to robust SLR vulnerability assessment. Results also indicate that as sea level rises, coastal lands are exposed to higher flood depths and water velocities. The prevalence of low-lying coastal plains leads to a rapid increase in land exposure to hazards when sea level exceeds a critical elevation of ~0.3 or 0.6\u2009m, depending on location. At ~1\u2009m of SLR, land that is roughly seven times the total modern beach area is exposed to one or more hazards. Projected increases in extent, magnitude, and rate of persistent SLR impacts suggest an urgency to engage in long-term planning immediately. Satellite altimetry from 1993 to 2015 indicates it rose nearly three times faster (~3\u2009mm/yr) than during the previous century (~1.2\u2009mm/yr)3. Depending on the trajectory of global greenhouse gas emissions, SLR may reach 1\u2009m or more by the end of the century4, especially if the contribution from Antarctic ice melt has been under-predicted5.Global mean sea level rise (SLR) is accelerating6). But local SLR could reach 8\u2009mm/yr during the second half of this century, which would exceed global mean SLR. This is due to gravitational redistribution of meltwater leading to higher sea levels in the equatorial Pacific (near Hawai\u2018i), relative to other oceanic locations7. Additionally, the 10 to 20\u2009cm of SLR expected no later than 2050, will more than double the frequency of extreme water-level events in the Tropics, where Hawai\u2018i is located8. The main Hawaiian Islands, a developed high island chain with low-lying coastal plains , might serve as a model for other Pacific islands and island nations for understanding and adapting to SLR in the Tropics.In Hawai\u2018i, local long-term SLR is presently mild combines with episodic rises in sea level caused by other processes such as storm surge20, tides21, eddies22, wave events23, and regional climate indices . For example, a recent high water anomaly April\u2013August, 2017, coupled with seasonal wave events, led to wave inundation, episodic erosion, groundwater flooding, and saltwater blockage of drainage infrastructure at vulnerable sites in Hawai\u2018i terrain flood model29. The passive flood model is a projection of a flood surface onto a digital elevation model (DEM). The flood surface, in its simplest form, is a horizontal plane of pre-determined height or elevation (e.g. SLR). It is easy to implement and provides the requisite spatial specificity.Before planning can begin, it is necessary to identify coastal areas that are vulnerable to SLR. This is challenging because sea level-related hazards vary in their intensity and spatial distribution across landscapes due to local topography, bathymetry, and wave exposure. To produce spatially-explicit estimates, localized modeling of complex coastal processes is needed. However, available models typically require large, detailed datasets and are computationally intensive34. Despite the documented need for more comprehensive impact modeling, only a limited number of studies have modeled multiple SLR-related processes at a spatial scale appropriate for local planning33.However, passive flood modeling does not provide a comprehensive picture of SLR hazards because it ignores dynamic processes such as seasonal waves, storm surge, and erosion, which have been shown to have a considerable impact on coastal communitiesTo address this gap and inform local planning, we model recurring coastal processes to comprehensively quantify the projected land area exposed to sea level-related hazards. We examine how land exposure changes, as a function of SLR, and investigate the relative importance of modeling each coastal process, in terms of the overall exposure. To identify hazards specific to each area, we take a place-based approach and use models that capture SLR impacts at the local level. We use a combination of numerical and empirical models to estimate the chronic impacts of SLR under the RCP 8.5 emissions pathway on all low-lying coasts of three of the most populated Hawaiian Islands: O\u2018ahu, Kaua\u2018i, and Maui.Modeling is performed under four higher sea level scenarios \u2013 corresponding to the upper limit of the likely range (83rd percentile) for years 2030, 2050, 2075, and 2100, as set forth in IPCC AR5 RCP8.5 \u2013 and a baseline scenario (2015) representing present-day conditions ; and seawater flowing directly across the shoreline into lands that lie below the water level. Our methodology does not distinguish between water table flooding and inland storm drain backflow; hence we refer to them collectively as \u201cgroundwater flooding/storm drain backflow.\u201d Direct flow of seawater across the shoreline we refer to as \u201cmarine overland flooding\u201d.Low elevation coastal plains and shallow groundwater tableset al.36 to map low-lying areas exposed to flooding under SLR. First, a temporally static yet spatially varying mean higher high water (MHHW) surface is created. Future tidal surfaces are then created by increasing the water level above the MHHW surface by the projected amount of SLR. Flooding during high tide is modeled by subtracting the tidal surface model from a DEM (the DEM is described in Methods) using ArcGIS37. If, from a map view, the boundary of a mapped flooded area is connected to the ocean, that area is identified as \u201cmarine overland flooding\u201d. If the boundary of a mapped flooded area is unconnected to the ocean (land-locked), we assume that it represents either groundwater flooding or inland storm drain backflow from the ocean.We follow the method in Cooper et al.38 found that three-dimensional groundwater modeling at high tide in Hawai\u2018i was well represented by the passive MHHW surface. Monitored well sites in Hawai\u2018i show that during high tide, the groundwater level near the coast is similar to the tidal elevation; while farther inland, the contribution from tides is reduced, but is compensated by an elevated mean groundwater level35, which results in a groundwater surface that mimics the high tidal elevation. We also assume that groundwater levels will increase at the same rate that SLR is occurring because we assume that the water table is perched on the salt water.In our method, we assume that the groundwater level is equal to the predicted tidal heights. Habel 40. As swells dissipate across reefs, they lead to wave set-up (increased water elevation) and an increase in infragravity waves (longer period waves), which contribute to wave run-up and overwash43. Since any rise in mean sea level will increase the mean water level over the reef, the response of wave inundation to SLR is non-linear. Accordingly, we use a numerical model to capture changes in wave dynamics as sea level rises, and model the dominant annual wave climate at all shores, even if the waves are shorter period wind waves.Storm surge associated with hurricanes can cause significant damage to island communities. However, it is rare in Hawai\u2018i. More common are swells, series of long-period waves that are generated from distant storms, which impact Hawaiian shores on a seasonal basis46, which is widely used in assessing vulnerability to SLR in both reef and non-reef environments47. The 1-dimensional (1D) model was selected because the more robust 2-dimensional (2D) model requires a large computation time in non-hydrostatic mode, making it infeasible for large-scale application. Modeling is performed along shore-normal profiles spaced 20\u2009m alongshore. Modeled parameters are interpolated to create 2D grids. Any parameters at grid locations with velocity <0.1\u2009m/s or depth <0.1\u2009m are omitted to ignore static, inland water that does not result from waves, and to remove thin veneers of water excursion. This last step also helps to distinguish between active wave-induced floodwater and standing water (passive marine overland flooding or groundwater/drainage flooding). The resulting wave inundation is the horizontal extent of the modeled wash of the waves. Data and boundary conditions such as the DEM, wave forcing, and bottom friction values are described in the Methods section.We model the seasonal high wave event for each location and for each SLR scenario with the non-hydrostatic XBeach model48. Larger SLR rates have been correlated with increased rates of shoreline retreat in Hawai\u2018i49, and future SLR is likely to exacerbate retreat rates17.Coastal erosion is prevalent in the Hawaiian Islands. A previous study estimates that roughly 70% of beaches on the islands of Kaua\u2018i, Maui, and O\u2018ahu are retreating, and 21.5\u2009km (11%) of those beaches have been completely lost to chronic erosion and shoreline hardening over the 80-year study periodet al.17, which combines the long-term historical shoreline change rates with a geometric Bruun-type model to adjust for future SLR . The equation for the projected vegetation line follows from the shoreline change equation in Anderson et al.17, and isyveg(t) is the vegetation position at future time t, yveg(t0) is the vegetation position at initial time t0, r is the historical shoreline change trend, (Sf\u2009\u2212\u2009Shist) is the difference between the IPCC projected sea level and the extrapolated Hawai\u2018i sea level at time t, and tan\u03b2 is the slope of the submerged portion of the active beach profile. In this equation, negative values indicate retreat. Erosion hazard areas are created in ArcGIS by connecting an erosion hazard line (determined from the projected vegetation) with the present-day shoreline contour), to represent land at risk of future erosion.To estimate future erosion hazards, we use the probabilistic method presented by Anderson It is important to note that the erosion model differs from the passive and wave models, in that it depends on the time it takes for sea level to reach a pre-determined elevation. By contrast, the passive and wave models can be applied to a higher sea level, regardless of when that higher sea level occurs.50 . Here, we present a subset of these map\u00a0areas to illustrate the spatial distribution of coastal lands exposed to coastal hazards, under several SLR scenarios, for one location on O\u2018ahu. We then present the quantitative results: initially, we show land area exposed to individual hazards and the cumulative exposure as a function of SLR; thereafter, we present rates of change in exposure area with respect to SLR; lastly, we display the contribution of each modeled physical process to the overall exposure.From the above-described modeling, TetraTech Inc. produced maps depicting the spatial extent of land exposed to modeled hazards. These maps, covering the islands of Kaua\u2018i, O\u2018ahu, and Maui, are available for viewing and download through the Pacific Islands Ocean Observing System51, and many homes are being renovated and expanded.To illustrate the spatial distribution of lands exposed to hazards, Fig.\u00a0Passive marine overland flooding is minimal at \u2018Ewa Beach, even in the 0.98\u2009m SLR scenario. By contrast, passive groundwater/storm drain flooding (green), representing both outcropping water table and salt-water intrusion of drainage infrastructure, is minimal, until the 0.98\u2009m scenario, in which large green areas indicate flooding of the presently-shallow water table. Wave inundation (dark blue) is the result of modeling the annual high wave event. The \u2018Ewa Beach area is most prone to long-period southern swells during the summer months (June\u2013August). Wave parameters for \u2018Ewa Beach, derived from an extreme value statistical analysis of offshore wave data (see Methods for details), are: 1.8\u2009m significant wave height, 16\u2009second peak period, and 181\u00b0N dominant direction (waves approaching from the south). Waves begin to overtop the limestone bench (left side of image) and a portion of the beach berm (middle of image) at 0.60\u2009m of SLR. A considerably larger area is exposed to wave inundation for the 0.98\u2009m SLR scenario. Coastal erosion (red) has the largest relative impact on land area during lesser amounts of SLR (0.17 and 0.32\u2009m SLR scenarios), but is milder than the other hazards at higher levels of SLR. However, due to the density of oceanfront homes, small amounts of erosion will have a substantial impact to the existing beach. Most erosion occurs at the left side of the beach, adjacent to the limestone bench, where sand dunes have the lowest elevations, due to the density of oceanfront homes. For all hazards, the spatial distribution shows great dependence on local topography and geology.To investigate the spatial extent to which sea level-related processes impact coastal lands, for each SLR scenario, we calculated the total land area exposed to each modeled hazard, as well as the cumulative land area exposed to one or more hazards on Kaua\u2018i (11.3\u2009\u00b1\u20093.3\u2009km2), O\u2018ahu (13.3\u2009\u00b1\u20092.4\u2009km2), and Maui (3.2\u2009\u00b1\u20090.5\u2009km2), combined . Normalized areas show that, at 0.98\u2009m SLR, land area exposed to passive flooding will be 4.2 times present-day exposure on Kaua\u2018i; 3.7 times on O\u2018ahu; and 2.6 times on Maui , O\u2018ahu (7.9\u2009\u00b1\u20092.0\u2009km2), and Maui (2.7\u2009\u00b1\u20090.8\u2009km2), combined , O\u2018ahu (2.7\u2009\u00b1\u20091.4\u2009km2), and Maui (2.4\u2009\u00b1\u20091.0\u2009km2), combined. Normalized exposure areas , O\u2018ahu (20.1\u2009\u00b1\u20095.7\u2009km2), and Maui (6.3\u2009\u00b1\u20092.9\u2009km2), combined. Normalized cumulative exposure areas ; when sea level is between 0.60\u20130.98\u2009m above present MSL, the area exposed to flooding expands at a rate of ~23\u2009km2 per m of SLR (or ~2\u2009km2 of newly flooded land for each 10\u2009cm of SLR). The only hazard that indicates a reduction in expansion rate at higher SLR intervals is erosion , to ~60\u2009km2 per m of SLR (for sea level between 0.60\u20130.98\u2009m above present MSL).To investigate how the extent of impacted lands changes, as a consequence of SLR, we calculate a simple rate at which lands become exposed to sea level-related hazards, with respect to SLR, by taking the difference in consecutive exposed areas and dividing that value by the difference in consecutive sea levels Fig.\u00a0. Aside fion Fig.\u00a0; note thion Fig.\u00a0 roughly To quantify the effect of augmenting the passive flood model with the wave and erosion models, we calculate the areas exposed to combinations of overlapping hazards and compare them to the cumulative exposure area. Figure\u00a0Our results indicate that there is considerably more land at risk of flooding or erosion, by SLR, than shown by simple passive flood mapping alone. In Hawai\u2018i, coastal planning responses must account for wave inundation and erosion to avoid missing 23.3\u2009km (38%) of threatened land over all three islands, under 0.98\u2009m of SLR; this is equivalent to nearly four times the land area of existing beaches. Such an oversight can have significant adverse consequences for coastal communities. And, while the percent contribution of erosion alone to cumulative SLR risk appears small under 0.98\u2009m SLR and two wetlands on Maui . The threshold between slow and fast phases is attributed to the prevalence of low-lying coastal plains that exist near the critical elevations. Similarly, the expansion rate of the land area exposed to wave inundation increases suddenly when SLR exceeds 0.60\u2009m on all islands : from the mean historical shoreline change rate of \u22120.08\u2009\u00b1\u20090.02\u2009m/yr to a mean rate of \u22120.32\u2009\u00b1\u20090.03\u2009m/yr by the year 2100 histograms of depth-velocity pairs , and lack the funds to conduct such modeling or collect the needed data. For those locations, data acquisition is the primary need. In the interim, we recommend using any combination of available forecasting methods in planning for SLR, as some guidance is still better than none, and will raise awareness that SLR is a threat, and that more data is needed.There are limitations to our approach and the models that we used. Projections for future SLR include large uncertainty values, mainly because of limited understanding of ice sheet contributions to the sea level budget, and unknowns related to curbing greenhouse gas emissions. Large uncertainty in the SLR projections contributes to large uncertainty in the erosion model. Concerning wave inundation, some physical systems might require at least a 2-dimensional (2D) model (as opposed to the 1D model we used) to capture alongshore wave transformation more accurately. Moreover, waves are run on a DEM which was not updated to reflect future changes in beach morphology. Thus, when creating adaptation strategies, it would be prudent to perform higher-resolution investigations that use our modeling results to focus the scope of such studies. For example, 2D wave modeling might provide required accuracy for planning where the reef is highly irregular. The need for additional study depends on individual sites and project objectives.We found that modeling the impacts of the annual high wave event and coastal erosion, in addition to the standard passive (bathtub) flood model, is essential to providing a more comprehensive vulnerability assessment of future SLR scenarios in Hawai\u2018i. Depending on the island and SLR scenario, our results show that relying solely on the passive model results in missing 35\u201354 percent of the total land area that is exposed to one or more hazards.2 per m of SLR (for scenarios between 0.17\u20130.32\u2009m) to ~60\u2009km2 per m of SLR (for scenarios between 0.60\u20130.98\u2009m). This increase is mainly a consequence of prevalent low-lying coastal plains. For Hawai\u2018i, we identified the following critical elevation thresholds, beyond which damage accumulates rapidly: 0.32 and 0.60\u2009m for passive flooding on Maui and O\u2018ahu, respectively; and 0.60\u2009m for wave flooding on all islands. Once lands are exposed to hazards, they are subsequently subjected to increasing flood depths and velocities as sea level rises, which leads to greater damage to structures and habitats that exist within threatened areas.The geographic extent and magnitude of SLR impacts are also expected to increase rapidly with SLR. The rate at which new land becomes exposed to one or more hazards, per unit SLR, nearly doubles from ~30\u2009kmBecause of the rapid increase in (1) land exposure to SLR hazards and (2) magnitude of SLR impacts, the time to engage in long-term planning is now. Taking this next step will allow for the creation and implementation of adaptation measures specifically-tailored to meet each locality\u2019s unique SLR vulnerability profile. With sufficient modeling to identify potential hazard vulnerabilities, individual locations become well informed to craft adequate SLR adaptation responses. As our study shows, it is crucial that SLR impact modeling is conducted at the local scale and include as many hazards as possible for the sake of building more resilient communities.Passive flood modeling is described in the main text, under the section: Approach and Models, Passive flooding at high tide. For validation, modeled flooded areas were compared against known elevations during present day conditions.In passive modeling, we assume that the amount of SLR is known. Thus, we estimate the error in the baseline scenario For higher SLR values, we assume that the error increases proportionally to projected exposure area; and, the constant of proportionality is the fractional uncertainty (error in area divided by estimated area) of the baseline area. This helps account for larger errors that correspond to larger increases in exposure area when, at higher SLR scenarios, ground slopes are generally milder than the baseline scenario. At milder slopes, the vertical error in the DEM leads to larger errors in exposed area projections. The resulting error for projected exposure under future SLR is:http://shoals.sam.usace.army.mil/). This DEM extends seaward to depths of approximately 35\u2009m, while the landward coverage extent is typically between 400 and 1000\u2009m inland from the shore. The 2013 DEM was augmented with the 3\u2009m resolution NOAA SLR Viewer DEM36 to fill in low elevations that extend far inland. Only the terrain portion of the DEM (not the bathymetric portion) was used for passive flood modeling.The DEM for passive flood modeling is a digital surface that represents the local terrain. It consists mainly of a 1\u2009m resolution bare-earth DEM that was derived from light detection and ranging (LiDAR) data collected in 2013 by the U.S. Army Corps of Engineers (USACE) XBeach simulation using maximum annually recurring wave conditions along shore-normal profiles spaced 20\u2009m apart. Then, we define the extreme water level as the average of the five highest water elevations at each grid point along each profile, and identify the corresponding velocity. Water depth is calculated as the difference between the annual extreme water level surface and the ground elevation. Depths and velocities are then interpolated to create 2D surfaces using ArcGIS, and resampled at 5\u2009m grid spacing. Modeled parameters at grid locations with velocity <0.1\u2009m/s or depth <0.1\u2009m are omitted to ignore static, inland water that does not result from waves, and to remove thin veneers of water excursion. The error in land area exposed to wave inundation is determined using the same method described in modeling passive flooding, except the 36. Data gaps in shallower water (<10\u2009m) are left unfilled because we find that these gaps only occur in small patches whose spatial footprints are much smaller than the resolution of the 1999\u20132003 10\u2009m DEM; and using the lower resolution DEM values to fill these gaps results in obvious elevation errors near the shoreline. Instead, we linearly interpolate across any small gaps in elevation after we have extracted the 1D profiles from the DEM.We augmented the 1\u2009m resolution 2013 USACE DEM with lower resolution (10\u201350\u2009m) bathymetric data collected by the Hawai\u2018i Mapping Research Group for water depths that exceed the maximum LiDAR depth range (roughly >35\u2009m depth). However, the lower resolution data is rarely used since most modeling is conducted over shallower water depths. To fill bathymetry gaps within nearshore areas, we use a 10\u2009m DEM that was created from all 1999\u20132003 USACE bathymetric and topographic surveys available from NOAA\u2019s Digital Coast57, which has been providing a 5-day hourly forecast that is calibrated using local wave buoys, for Hawaiian regions since 2009. This forecast is produced by using the WaveWatchIII and SWAN wave models to dynamically downscale atmospheric forcing from the NCEP Global Forecast System weather model57. From this forecast, we use the hourly time series of significant wave height, peak wave period, and dominant direction, for the years 2009\u20132015.Mean water levels are determined by increasing the water level above MHHW by the desired amount of SLR. To obtain wave information at the local level, we use the wave forecast available from PacIOOS39 and extract peak wave heights from the time series of significant wave height, and fit those to a generalized extreme value (GEV) distribution, from which the maximum annually occurring significant wave height and the associated peak period are found. To determine the dominant direction, we perform the GEV fit on subsets of wave series that are limited to incoming wave directions within 30-degree bins, and let the bin ranges shift by 15 degree increments . The direction from which waves will produce the highest run-up is typically shore-normal, and has the largest wave height and period. On the rare occasion that these three conditions do not align, we use expert knowledge and generally select the direction corresponding to longer period waves. See Supplementary Tables\u00a0We then follow the method presented by Vitousek and Fletcher58. From the wave parameters, we use XBeach routines to generate the 1-hour time series of incoming waves.We repeat this procedure on wave data at locations spaced 1.5\u20132.5\u2009km apart, in roughly 25\u201335\u2009m depth around each island. The annually-recurring maximum wave parameters are then interpolated along the offshore boundary to match profile locations. Since projections of future wave conditions due to climate change do not indicate significant changes in wave characteristics near Hawai\u2018i, we use the same wave parameters for all future SLR scenarios59; for submerged areas, we use benthic structural classification maps produced as part of the Mapping of Benthic Habitats for the Main Eight Hawaiian Islands project for NOAA\u2019s National Ocean Service Center for Coastal Monitoring and Assessment60. We then assign friction values to each geologic type and structural classification that classify the geology and structural type of land and sea topography. For land areas, we use the USGS geology classification map for Hawai\u2018iet al.17 (and introduced in equation (yveg(t) by numerically combining the pdfs for each term on the right hand side of equation is then projected back to map coordinates and exported as a GIS line shapefile. These calculations are done using Matlab. Some edits are manually performed in ArcGIS to smooth areas where transects overlap along coasts with high curvature. Erosion hazard areas are created in ArcGIS by connecting the erosion hazard line with the existing shoreline (MSL elevation contour).Sf and time t is determined as ith projected shoreline position, and \u0394x is the distance between transects (20\u2009m).The error in new land area exposed to erosion for future sea level 48 and augmented with newer digitized shorelines for the north Maui and west Maui regions49. The positional error in each digitized shoreline was estimated as the root mean square of up to seven sources of positional error. The reader is referred to Fletcher et al.48 for a detailed description of the historical shorelines.We use historical shorelines and vegetation lines that were digitized as part of the USGS National Assessment of Shoreline Changeth century, can influence the long-term shoreline change rate and violate the assumption that shoreline change remains fairly constant over the roughly 80 years of available data. To capture ongoing behavior of the beach, we calculate shoreline change rates only over the period of time following any significant beach alternation, such as sand mining. In the case where a beach is completely lost to erosion and a seawall has been built, we calculate the shoreline change rate up to and including the first shoreline where the wall was erected. Since we cannot predict if a wall will be built or removed, we use the preexisting rate to provide an estimate of exposure to erosion, in the beach\u2019s natural state.At some beaches, past human activity, such as sand mining in the early 2062. Rates are then smoothed in the alongshore direction using a weighted [1 3 5 3 1] moving average to reduce spurious variations between adjacent beach locations.A straight line is fit to the historical shoreline data using weighted least squares regression63, as well as additional surveys at 35 locations on O\u2018ahu and 27 locations on Kaua\u2018i over the period 2006\u2013200817. Following Anderson et al.17, we extract, for each repeated beach survey, the slope between two morphologic features: the beach toe64, which is located at base of the foreshore, and; either the point at which the repeated surveys converge (depth of closure) or the intersection of the sandy profile with the reef platform65. The final beach slope, tan\u03b2, is determined as the average of the individual slopes. We use the standard deviation of the slopes as our estimate for the error associated with tan\u03b2. If more than one profile location exists along a study area, slopes are interpolated using cubic splines.To determine beach slope, we use the collection of biannual cross-shore beach surveys (1994\u20131999) on beaches of O\u2018ahu and MauiSupplementary MaterialDataset 1Dataset 2"} +{"text": "The aim of this paper is to map the scientific landscape related to cancer research worldwide between 2012 and 2017. We use scientific publication data from Web of Science Core Collection and combine bibliometrics and social network analysis techniques to identify the most relevant journals, research areas, countries and research organizations in cancer scientific landscape. The results show: Oncotarget as the journal with most publications; a significant increase in China\u2019s publications, reaching United States\u2019 publications in 2017; MD Cancer Center, University of California and Harvard University as organizations with most publications; cell biology as the most frequent research area; breast, lung and colorectal cancer as the most frequent keywords; high density of co-authorship between organizations in the West, especially in the US, and low density between organizations in Asian and lower and medium income countries. Our findings can be used to guide a global knowledge platform guiding policy, planning and funding decisions as well as to establish new institutional collaborations. Cancer cases worldwide reached 17.5 million cases in 2015, along with 8.7 million deaths. The incidence rate increased by 33% between 2005 and 2015 partly due to population growth and aging. Currently, cancer is the second cause of death worldwide and is expected to hit 27.1 million people by 2030 - especially in rich countries [Bibliometrics in studies related to cancer is relatively common in the literature. For example, bibliometrics has already been used to inform cancer research policy and spending and pathFrom this perspective, the aim of this paper is to map the scientific landscape related to cancer research worldwide between 2012 and 2017, combining bibliometrics and Social Network Analysis (SNA) techniques. To map the scientific landscape related to cancer, we used data from scientific publications available in the Web of Science Core Collection (WoS), Thomson Reuters. The paper describes the comprehensive research status of the cancer field by analyzing the quantity of publications, main journals, frequent research areas and most scientifically productive countries and organizations. It also explores the global cooperation network of these countries and organizations, identifying most central players, and the association of different research topics, highlighting the one that is mostly associated with innovative efforts in the field. It aims to generate evidence that could ultimately inform managers, researchers and policy makers, supporting decision-making, R&D planning and financing strategies.Figure Much of the journals are focused entirely on cancer. Molecular Medicine Reports, whose scope includes different topics in molecular medicine was one exception . CA: TheFigure Figure Figure The network highlights the prominent role of the United States. This country is the one with the biggest node, which means it is the one with more partnerships. Japan and China are respectively second and third in the ranking, but the size of its nodes show a lower degree of partnership than other countries with fewer publications. For example, France and the United Kingdom are countries with significantly fewer publications than these two, but with higher degree of entry into the network. Data suggests that for China and Japan there are more partnerships between organizations within the country than with organizations in other countries.Figure Another highlight among organizations is the Karolinska Institutet, in Sweden. This organization is the thirteenth in this ranking, with far superior representation compared with Sweden amongst the total publications of the countries. The Karolinska Institutet is responsible for more than half of the cancer publications in Sweden and publishes slightly less than the main China\u2019s cancer organizations. Founded in 1810, this organization is one of the world\u2019s leading medical organizations with 80 percent of its revenue exclusively dedicated to research .Figure Again, the comparison of total publications and partnerships in China has different results. Although this country has four of the twenty organizations with more publications on cancer, the number of partnerships is significantly lower than some organizations with much less publications. One notes that the smaller scale of partnerships in Chinese organizations is not only for partnerships between different countries, but also within the country itself. The data suggest that the high volume of publications in this country is more often the result of intrainstitutional efforts.Another relevant category to map scientific publications on cancer is the research area. Thomson Reuters\u2019 team reads indexed articles in the Web of Science database and assigns one or more research areas to each of them. The database is currently divided into 151 research areas, which come from five major research areas: (1) Biomedicine and Life Sciences, (2) Physical Sciences, (3) Technology, (4) Arts and Humanities, and (5) Social Sciences . Figure The most prominent research areas can be divided into large areas of knowledge segmented by cancer sites , types of treatment and types of scientific knowledge . One can notice the prominence of research related \u200b\u200bto cellular biology in relation to the others, with more than 10 thousand publications indexed. The other research areas represents the main modalities of cancer diagnosis and treatment, except immunology, which is only in the sixteenth position. Although this modality of treatment is currently considered one of the most modern on cancer care, traditional approaches to treatment are still those that concentrate the largest amount of publications.Figure Research and experimental medicine is also highlighted. According to the Thomson Reuter classification, this category includes papers that analyze or promote the creation of techniques considered extremely innovative. Most of the work involves research at an early stage of development, that is, still distant from the market and with great uncertainty associated with their future. This category increased steadily in this period (86% increase). The search for innovative interventions on cancer care has been a trend in the industry and academia for some decades and the funding of these researches has been growing substantially . ResearcFigure Figure Figure The prominence of breast cancer among the keywords probably relates to its relevance in the global epidemiological profile. Breast cancer accounts for 6% of cancer deaths and 12% of new cases recorded in 2012. Although there is more up-to-date data for specific countries, the figures for the total number of cancer cases in the world, which is compiled by the Agency International Cancer Research (IARC) of the World Health Organization, are only updated to 2012. Although male breast cancer exists in a smaller proportion, if only cancers in women are considered, this is the one with the highest mortality rate and a higher incidence in 2012. Breast cancer is the most common cancer in women in 140 countries and the most frequent cause of cancer mortality in 101 countries .Besides breast cancer, lung and colorectal cancers also have prominence in the global epidemiological profile. Lung cancer is the cancer with the highest rates of mortality and incidence among all cancers in the year 2012, while colorectal cancer is the third in terms of incidence and the fourth in terms of mortality. An analysis of incidence and mortality numbers on cancer shows that the publications are much more consistent with disease incidence rates than with their mortality rates.The total amount invested in research in each of these areas may also be another factor to explain this distribution. A proxy for these investments can be found in the total investments made by the National Institutes of Health (NIH), the leading research funding organization in the United States. Breast cancer is the one with the highest investment among all other cancers, with almost double the funding compared to second-placed pediatric cancer ($ 656 million and $ 351 million in 2016). Lung and colorectal cancers are also cancers with a lot of funding in recent years .To map the scientific landscape related to cancer, we used data from scientific publications available in the Web of Science Core Collection (WoS), Thomson Reuters, and combined bibliometrics and social networks analysis techniques. In WoS, the following search strategy was used:((ti=(cancer* or neoplasia* or neoplasm* or tumor*) and su=(oncology))) AND DOCUMENT TYPES: (Article) Indexes=SCI-EXPANDED Timespan=2012-2017It was decided to restrict the search only to articles because these documents meet higher quality standards than other types of scientific dissemination materials . In ordeDuplicates were removed using the \u2018ISI Unique Article Identifier\u2019. After that, the number of documents was reduced to 89,067. The fields \u2018author affiliations (Organization and City and Country)\u2019 were normalized using the general fuzzy logic from Vantage Point\u2019s list cleanup tool as well as manual cleaning. The keywords were grouped according to the type of cancer, form of treatment or technology. The rankings were produced in VantagePoint and exported to Microsoft Excel for building the graphics. Bubble charts were the only ones produced in VantagePoint itself.The networks were produced using the software Gephi 0.9.1 from co-occurrence matrices generated in Vantage Point. We used the Fruchterman Reingold (FR) algorithm, which assumes the existence of groups or clusters within the network . For eacOur paper provides interesting insights to cancer scientific landscape. It provides a concise view of the scientific knowledge distribution through thousands of publications around the world and the key elements for understanding the dynamics of this knowledge. Although the timespan was only five years, it was enough to show trends among research areas, and specially China\u2019s rise as an important contributor in the generation of knowledge. Also, the networks showed how cancer is the subject of interinstitutional efforts worldwide - with a higher density among Western countries."} +{"text": "The recent arrival of CDK4/6 inhibitor agents, with an approximate doubling of progression-free survival (PFS) associated with their use in hormone receptor-positive, HER2-negative advanced breast cancer (BC), has radically changed the approach to managing this disease. However, resistance to CDK4/6 inhibitors is considered a near-inevitability in most patients. Mechanisms of resistance to these agents are multifactorial, and research in this field is still evolving. Biomarkers with the ability to identify early resistance, or to predict the likelihood of successful treatment using CDK4/6 inhibitors are yet to be identified, and represent an area of unmet clinical need. Here we present selected mechanisms of resistance to CDK4/6 inhibitors, largely focussing on roles of Rb, cyclin E1, and the PIK3CA pathway, with discussion of associated biomarkers which have been investigated and applied in recent pre-clinical and clinical studies. These biological drivers may furthermore influence clinical treatment strategies adopted beyond CDK4/6 resistance. RB1 complex. Once activated, CDK4/6 phosphorylates the retinoblastoma protein (Rb), an event which causes Rb to lose the ability to bind to the E2F family of transcription factors. E2F is therefore released, activating gene transcription and thus initiating progression of the cell cycle from G1 to S phase (resulting in DNA synthesis) , human epidermal growth factor receptor-2 (HER2) negative BC. Endocrine therapy (ET) as monotherapy, previously thought to be the gold-standard in first and often subsequent lines of management, has been augmented and displaced in the treatment hierarchy by the emergence of selective, small-molecule inhibitors of CDK4/6. Three such agents are currently in widespread clinical use, with all three, when given in combination with ET, resulting in an approximate doubling of progression-free survival (PFS) in patients with advanced HR+/HER2\u2013negative BC, compared to ET plus placebo. These consistent positive PFS results have been demonstrated in large phase III trials in the upfront setting [PALOMA-2 for ribosistance \u201314; convRB1, the gene encoding for Rb, and cyclin D1 (and lower levels of p16) are observed in human BC cell lines sensitive to palbociclib . RBsig, y (4.7%) , and hasy (4.7%) . Similary (4.7%) . Of notesistance .CCNE1, the gene that encodes cyclin E1, is upregulated in models with resistance to CDK4/6 inhibitors than those with high CCNE1 expression . The predictive power of CCNE1 mRNA was stronger in metastatic biopsies (interaction p < 0.001) than archived primary biopsy samples (interaction p = 0.09). Investigators provided further validation in an independent cohort (N = 61) drawn from the Preoperative Palbociclib (POP) Clinical Trial (NCT02008734), wherein high CCNE1 mRNA expression correlated with a significantly lower anti-proliferative response to palbociclib. Contrastingly, no such association with CCNE1 expression and PFS was found in the biomarker analyses of MONALEESA-2, with near-identical hazard ratios observed across expression groups . An earlbociclib .CCNE1 and down-regulation of Rb occurred at the time of palbociclib resistance. Subsequent in silico analyses, correlating the ratio between CCNE1 and RB1 expression levels (CCNE1/RB1) with palbociclib IC50 in a large dataset of cell lines, showed the ratio outperformed both CCNE1 and/or RB1 when they were utilized as sole markers. Furthermore, retrospective analyses showed CCNE1/RB1 to be an adverse prognostic factor, with the ability to differentiate between palbociclib sensitive and resistant patients enrolled in the neoadjuvant NeoPalAna trial catalytic subunit (PIK3CA) are found in approximately 40% estrogen receptor-positive BC . CirculaThymidine kinase-1 (TK1) is an enzyme in the pyrimidine salvage pathway that plays a critical role in the synthesis of DNA and in cell proliferation . High TKp = 0.012) (p = 0.0026).Previous studies have validated the use of DiviTum, both as a prognostic marker, and as one of response to ET. A pilot study of 31 women with advanced HR+/HER2 negative BC commencing on a new line of palliative endocrine therapy showed that those with low baseline levels of plasma TKa had a median PFS of 25.9 months, vs. 5.9 months in those with high baseline levels (= 0.012) . Furtherp < 0.001). Patients whose TKa increased from baseline after 3 months of treatment had a significantly shorter mTTP than those whose TKa did not increase (P = 0.0045). After adjusting for major prognostic factors, TKa remained an independent marker . Concurrently, cellular proliferation (as assessed by methylene blue assay) was observed to drop significantly after a minimum of 6 days, suggesting TKa may be an early marker of proliferative inhibition in response to palbociclib. This phenomenon was not observed in models with acquired resistance to palbociclib. The prognostic ability of TKa has been clinically validated in planned translational studies of plasma derived from the TREnd study (NCT02549430), a phase II trial which tested the activity and safety of single-agent palbociclib against palbociclib combined with the ET the patient had received (and progressed on) most recently before enrollment . Mutations in sistance . This inn = 341). After a median follow up of 20 months, the median PFS was almost double in mutation-positive patients receiving alpelisib compared to those receiving placebo . Further data, reporting the efficacy of alpelisib according to mutational status evaluated by ctDNA, suggested an even greater clinical benefit than tissue analysis (n = 186), there was a 45% risk reduction in PFS (HR 0.55 95% CI 0.39\u20130.79). In the small number of patients who had previously received CDK4/6 inhibition (n = 20), there was a 52% risk reduction in PFS in favor of alpelisib over placebo (HR 0.48 95% CI 0.17\u20131.36). Alpelisib is selective for the alpha isoform of PI3K, which has so far set it apart from pan-PI3K inhibitors, which have reported notably poor safety profiles (PI3K-dependent activation of non-canonical cyclin D1-CDK2 and resultant recovery of Rb phosphorylation and S phase entry has been implicated in early resistance to CDK4/6 inhibition, with combined PI3K and CDK4/6 inhibition demonstrating the ability to overcome resistance to CDK4/6 inhibitors in BC cell lines . Hence, analysis . In patiprofiles , 57. Nevprofiles .Whilst the number of patients with prior exposure to CDK4/6 inhibitors subsequently enrolled in SOLAR-1 was small, it is not unreasonable to consider\u2014in patients harboring an actionable mutation\u2014PIK3CA inhibition following disease progression on a CDK4/6 agent. This is particularly relevant, given the likelihood of emergence of driver mutations of PIK3CA secondary to previous ET and CDK4/6-targeted therapies . Whilst All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.AD has received honoraria from, and served as an advisory board member for, Pfizer, Lilly, AstraZeneca, and Novartis. AD has received research funding from Pfizer, AstraZeneca, and Novartis. LM has received consultation fees from Novartis and research support from Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Although pelvic incidence (PI) is a key morphologic parameter in assessing spinopelvic sagittal alignment, accurate measurements of PI become difficult in patients with severe hip dislocation or femoral head deformities. This study aimed to investigate the reliability of our novel morphologic parameters and the correlations with established sagittal spinopelvic parameters.One hundred healthy volunteers , with an average age of 38.9 years, were analysed. Whole-body alignment in the standing position was measured using a slot-scanning X-ray imager. We measured the established spinopelvic sagittal parameters and a novel parameter: the sacral incidence to pubis (SIP). The correlation coefficient of each parameter, regression equation of PI using SIP, and regression equation of lumbar lordosis (LL) using PI or SIP were obtained. The intraclass correlation coefficient (ICC) was calculated as an evaluation of the measurement reliability.R\u2009=\u20090.96) and pelvic tilt (PT) (R\u2009=\u20090.92). PI could be predicted according to the regression equation: PI = \u2212\u20099.92\u2009+\u20090.905 * SIP . The ideal LL could be predicted using the following equation using PI and age: ideal LL\u2009=\u200932.33\u2009+\u20090.623 * PI \u2013 0.280 * age and using SIP and age: ideal LL\u2009=\u200924.29\u2009+\u20090.609 * SIP \u2013 0.309 * age .Reliability analysis showed high intra- and inter-rater agreements in all the spinopelvic parameters, with ICCs\u2009>\u20090.9. The SIP and pelvic inclination angle (PIA) demonstrated strong correlation with PI .This study was retrospectively registered with the UMIN Clinical Trials Registry ( More than two decades ago, the concept of pelvic incidence (PI) was introduced, and it has become the significant unique parameter for assessment of standing spinal alignment . For exaMoreover, lumbar degenerative spondylolisthesis, a common degenerative disorder, which causes sagittal malalignment as well as disability of daily life, tends to exhibit a high PI , 15, andThere is a reciprocal relationship between the lumbar spine and hip joint characterised by \u2018hip-spine syndrome\u2019, which was reported by Offierski and MacNab . There aTherefore, alternative morphological pelvic parameters need to be established. The upper edge of the pubic symphysis has also been proposed as a key anatomic landmark of the anterior pelvic plane and used in assessing pelvic orientation , 22. We This prospective observational study was approved by the Clinical Research Ethics Committee of the hospital. All Niigata Spine Surgery Center staff were informed of the purpose of the study according to the Helsinki Declaration. After signing the informed consent form, anthropometric, clinical, and radiographic data were collected from those who agreed with the purpose of the study. One hundred and twenty-one volunteers with no history of treatment for spinal disease were enrolled. After whole spinal radiographic images were obtained, we excluded five cases with lumbarisation, three cases with sacralisation, one case with 11 thoracic vertebrae, two cases with scoliosis (Cobb angles\u2009>\u200920 degrees), three cases with lumbar spondylolisthesis with %slip of at least 5\u2009%, three cases with axial pelvic rotation\u2009>\u20095 degrees, two cases with total hip arthroplasty or postoperation of the ilium, and two cases that moved during scanning, so that accurate radiographic measurements could be obtained. Exclusion of the transitional vertebrae is important because transitional vertebrae affect spinal and pelvic parameter measurements. Finally, 100 patients, including 25 males and 75 females, with an average age of 38.9 years (20\u201370 years), were included for radiographic analysis. The following epidemiologic and morphologic characteristics of patients were obtained: age, sex, body weight, height, and body mass index (BMI).2 including the front and side surfaces.Whole-body alignment in the standing position was measured using a slot-scanning X-ray imager . Patients were instructed to stand in the centre of the imager and look straight ahead at a horizontal gazing mirror, while placing their hands on their cheeks. The patients were scanned within 30 s from the skull to the feet at a speed of 7.6\u00a0cm/s . The aveWe measured the PI, PT, SS, and LL L1-S1) as routine spinopelvic sagittal parameters estimates and their 95\u2009% confidence intervals were calculated based on a single-rating, absolute-agreement, 2-way mixed-effects model. The correlations between the investigated parameters were analysed by Pearson\u2019s correlation coefficients. Simple linear regression analysis was performed to establish a formula to predict the value of PI using the newly-established anatomical parameter. In addition, we defined the LL of healthy volunteers with no history of treatment for spinal disorders who participated in this study as the ideal LL. Stepwise multiple regression analyses were performed to establish formulas to predict the ideal LL value using age, sex, height, weight and PI or SIP. As SS, PT and PIA are dependent factors affecting spinal deformity, we did not include them as independent variables. JMP and SPSS were used for the analyses, and a p value less than 0.05 was considered statistically significant.Demographic and radiographic parameters are shown in Table\u00a0For the radiographic measurements, the reliability analysis showed high intra- and inter-rater agreements for all the spinopelvic parameters, with ICCs\u2009>\u20090.9 Table\u00a0.Table 2correlation coefficient (R)\u2009>\u20090.9] were found between PI and SIP, and PT and PIA. Moreover, the correlation coefficient between SIP and LL was similar to that between PI and LL. The same applied to the correlation coefficient between SS and PI, and that between SS and SIP .SIP was shown to be a predictor of PI according to the regression equation: PI = \u2212\u20099.92\u2009+\u20090.905 * SIP . Therefore, these two parameters could be alternative morphologic or positional parameters of the pelvis to assess whole body sagittal alignment.As mentioned previously, the evaluation of spinal sagittal alignment, as well as pelvic version according to pelvic morphology, especially PI, are crucial in hip or spine surgeries. Although use of HA, which is the centre of pelvic version, is optimal to assess pelvic morphology, an accurate assessment of PI is impossible on lateral plane radiographs in cases with deformed or dislocated femoral heads due to severe osteoarthritis or necrosis. Recently, alternative morphologic parameters have been proposed, without use of HA. Imai et al. reported a strong correlation between PI and anatomical-SS as a morphological parameter, which is the value of the SS measured from the pelvis adjusted in the anterior pelvic plane . Wang etPrevious studies have reported that deterioration of sagittal alignment, especially loss of LL due to common spinal disorders, decreases HRQOL \u201311. TherTwo limitations of our study should be acknowledged. Firstly, the proportion of female volunteers we recruited in this study was as high as 75\u2009%. However, a previous report showed that sex has no effect on spinopelvic parameters . SecondlOn the other hand, the present study has some strong points compared to the previous studies. Firstly, we investigated sagittal whole-body skeletal alignment and used a scanning X-ray imager with a biplanar upright scanning imaging modality to achieve reduced X-ray particle scatter, improved image quality and significantly reduced radiation to the patient . SecondlFrom the perspective of future possibilities, we need to investigate the accuracy or reliability of SIP and PIA measurements on conventional X-ray images compared to PI and PT. Moreover, further studies are needed on whether our novel concepts can predict the HRQOL status in patients with progressive hip arthritis with hip dislocation or femoral head deformity. We believe that use of our novel concepts can support future research in the elucidation of the various pathologic conditions on hip-spine syndrome.Since the surgical goal is to achieve optimal global sagittal alignment by restoring an optimal LL for spinal deformity patients to enhance quality of life status, our novel pelvic parameters accurately assessed spinopelvic sagittal alignment, and allowed us to create formulae for predicting PI and an ideal LL. Our new measurement parameters and formulae may assist clinicians in treatment decision-making, and lead to better quality of life for patients with ASD and those with progressive disorders of the hip joint."} +{"text": "In order to describe the physical properties of large time scale biological systems, coarse-grained models play an increasingly important role. In this paper we develop Coarse-Grained (CG) models for RNA nanotubes and then, by using Molecular Dynamics (MD) simulation, we study their physical properties. Our exemplifications include RNA nanotubes of 40 nm long, equivalent to 10 RNA nanorings connected in series. The developed methodology is based on a coarse-grained representation of RNA nanotubes, where each coarse bead represents a group of atoms. By decreasing computation cost, this allows us to make computations feasible for realistic structures of interest. In particular, for the developed coarse-grained models with three bead approximations, we calculate the histograms for the bond angles and the dihedral angles. From the dihedral angle histograms, we analyze the characteristics of the links used to build the nanotubes. Furthermore, we also calculate the bead distances along the chains of RNA strands in the nanoclusters. The variations in these features with the size of the nanotube are discussed in detail. Finally, we present the results on the calculation of the root mean square deviations for a developed RNA nanotube to demonstrate the equilibration of the systems for drug delivery and other biomedical applications such as medical imaging and tissue engineering. The accurate modeling of the complex biomolecules like ribonucleic acid (RNA) nanoclusters is important for many biological processes taking place in the human body, as well as for their applications such as drug delivery. The influence of the dynamic behavior of RNA on the cellular processes of modifying genetic circuits within functionally productive pathways is rather complex ,2,3. WitCurrent and potential applications of RNA nanotubes drive the research envelope in the development of novel CG models further. The long enough RNA nanotubes . Long enough RNA nanotubes, as considered in this paper, have attracted substantial interest at the practical level for drug delivery, along with other biomedical applications ,39. By uPerform the all-atom MD simulation.Convert the all-atom simulation trajectory into the corresponding coarse-grained model and then calculate the radial distribution function for this CG pseudoatom.T is the temperature and Using these pseudoatomic radial distribution functions, calculate the pairwise potential for each pair of the CG pseudoatoms using the following Equation :(2)uCG:Finally, the CG simulation is repeated as is done in step 4 until the value of In the CG modeling, when the system reaches its minimum energy equilibrium, a number of characteristics are responsible for the determination of thermodynamical properties. Among them, an important role is played by the sum of pseudo atoms, as well as the effective energy function, Equation . On the otential . The BolWe use the above procedure in the next section for parameter fitting to obtain the structures of RNA nanotubes for the subsequent CG modeling.All-atom molecular dynamics simulations have been performed by using the CHARMM force field implemented in the NAMD MD simulation package ,48,49. Tfollowed and addeTo model RNA nanotubes, we have used the RNAIi/RNAIIi as building blocks and self-assembled them to form RNA nanorings, which are later connected by links to form the nanotubes. This is done in a way analogous to references ,51 whereIn order to fit the CG parameters for the development of model structures, we use the radial distribution functions, histograms of several bonds, angles, and dihedral angles from the all-atom MD simulations. During the calculation, the total energy of the system is coming from three different contributions. Out of these three contributions, the first one is the contribution from the chain connectivity that includes bonds, angles, and dihedral angles. The second contribution is coming from the base-pairing which comes from the hydrogen bonding between electronegative atoms and the hydrogen (electropositive) atom of two adjacent nucleobases of the RNA. Specifically, the hydrogen bonding comes from the base pairs in such a way that there is an attraction between two kinds of the base pairs, such as the cytosine pairs with guanine and the adenine pairs with the thymine. For example, in the base pairing of guanine and cytosine, there is a hydrogen bond among O6 of guanine with the hydrogen at N2 of cytosine, hydrogen at N1 of guanine with the N3 of cytosine and hydrogen at N2 of guanine with the O2 of cytosine. The last and the third contributions in the energy of the CG model comes from non-bonding interactions such as van der Waals interactions. The effective potential of mean force is calculated from the probability distribution function and the parameters are fitted by using the Boltzmann inversion method as described in Using molecular dynamics simulations we modeled a range of different RNA nanotubes of various sizes. One of our main results, as shown in Our three-bead approximation is a development of our earlier model for the nanoring , where wIn In In in Ref. . The RNA in Ref. . In the In summary, we have developed new coarse-grained models for different sized RNA nanotubes. Based on the three-bead approximation approach, we have used the center of mass of each bead to be one atom in the sugar ring, phosphate backbone, and the nucleobase, respectively. Based on the developed coarse-grained models, for multiple-ring RNA nanotubes, we have calculated the radial distribution functions, root mean square deviations, histograms for the dihedral angles of the backbone, and the angles of three beads. We have shown that the intensity of the radial distribution functions decreases with the increasing number of RNA nanorings in the RNA nanotube. We have also shown that there are significant classes of RNA conformations and that the peaks in the two-dimensional map of the dihedral pair distributions would be corresponding to the kissing loops. Finally, a number of physical properties and characteristics of different size RNA nanotubes possess similar behaviors. This reveals that it is possible to efficiently manipulate such properties and characteristics for larger size RNA nanotubes, which are practically important for drug delivery and other biomedical applications of these structures. The developed models could be further generalized to account for more complex multiscale interactions, integrating our developed methodology with predictive, dynamic, and stochastic coarse-grained approaches ,60,61,62"} +{"text": "Effectiveness of prenatal iron supplementation program depends on utilization of the supplement by pregnant women. Nevertheless, in Ethiopia, regardless of increasing efforts, lower proportion of pregnant women use the supplementation for recommended 3 months and above but, the reason is not clear. This study aimed to identify the level and factors associated with utilization of prenatal iron supplementation.A community based cross-sectional quantitative study complemented with qualitative component was carried out in January, 2016. A total of 411 pregnant women who gave birth 6 months preceding data collection were selected using multi-stage cluster sampling technique. Qualitative data were collected by conducting four focus group discussions (FGDs) of local pregnant mothers and interviewing district maternal and child health (MCH) focal, 3MCH heads of health centers and four health extension workers. Factors associated with appropriate utilization of supplementation were assessed using multivariate logistic regression. The outputs of analyses were presented using Adjusted Odds Ratio (AOR) with 95% confidence intervals (CI).From the study participants, 11.5% (95% CI\u2009=\u20099.9\u201313.1%) took the supplement for the recommended duration of 3 months or above. Pregnant women who could not read and write had 77% reduced odds of using iron supplementation than their counterparts (AOR\u2009=\u20090.23(95% CI: 0.07\u20130.75)). As compared to women who had four or more antenatal care (ANC), women with 2 and 3 ANC visits had 78% (AOR\u2009=\u20090.22(0.07\u20130.63)) and 66% (AOR\u2009=\u20090.34(0.14\u20130.81)) reduced odds of using the supplementation, respectively. As compared to women who were aware of benefits of taking the supplement for maternal and child health, the counterparts had 90% (AOR\u2009=\u20090.10 (0.10\u20130.63)) reduced odds of using the supplement. Women who were not knowledgeable of anemia had 85% (AOR\u2009=\u20090.15(0.04\u20130.62) reduced odds of using the supplement than those who were knowledgeable. The qualitative study indicated that there was no problem in the supply and logistic system of iron supplement and leading reasons for not taking the supplement were late initiation of Antenatal Care, lack of awareness and occurrence of side effects; unpleasant taste, nausea, vomiting.In the study area utilization of prenatal iron supplementation is very low. Improving maternal education, ensuring early and frequent ANC, educating pregnant women about the benefits of service and ensuring comprehensive knowledge of anemia expected to improve the utilization of prenatal iron supplementation. Anemia affects more than 2 billion people of the world and majority of the burden is attributed to iron deficiency (ID) . ID reprEthiopian national nutrition strategy (NNS) targeted to increase proportion of pregnant women who take iron supplementation for 3 month or more to 50.0% by 2015 . HoweverThere is no conclusive and consistent evidence in Ethiopia. Moreover, there is no well documented information on the level and factors associated with utilization of iron supplementation. Thus, purpose of the current study was to determine the proportion and factors associated with utilization of prenatal iron supplementation in wolaita zone, south Ethiopia.Community based cross-sectional study using quantitative study design complemented with qualitative approach was conducted via home to home visit in January 2016.The study was carried out in Offa woreda which is found in Wolaita zone 362 Km to the South of Addis Ababa and 185 Km to the South West of Hawassa . According to 2007 Central Statistical Agency (CSA) report, the Woreda has estimated total population of 135,527; with an estimated male to female ratio of 1to1 and 4878 pregnant mothers. There were 4 health centers and 27 health posts that serve the population with potential health services coverage of 73.8%. According to health management information system (HMIS) report, about 97.6 and 81.5% of pregnant women got first and fourth ANC services, respectively and the annual health professional assisted delivery coverage was 59% in 2015 .The source and study populations of the study were women who gave birth 6 months preceding data collection and living in entire woreda and randomly selected kebeles respectively.The sample size for quantitative study was calculated by using EPI info version7 for windows using single population proportion formula using the following assumptions; The expected proportion of utilization for 91 or more days 20.4% , 95% sigRandomly 10 kebeles were selected by simple random sampling (SRS) technique. Total number of sample required from each randomly selected kebele was proportionally allocated. The participants were selected by SRS from list of women who gave birth in 6 months preceding data collection which was established prior to data collection by conducting listing through mobilization of health development army (HDA). Eight to ten pregnant mothers for each FGD were purposively selected from list of antenatal care attending women.A structured questionnaire was prepared in English focal person, MCH department heads of health centers and Health extension workers (HEW) and FGD among pregnant women. Interviews and FGDs on each topic facilitated using semi-structured guideline that included some questions from quantitative part that need further exploring. In-depth interview was undertaken in respective working places of the interviewees and ultimately eight interviews among district MCH focal person (1), health center MCH heads (3) and HEWs (4) were carried out. Participants for FGD were selected purposively from pregnant women who attended ANC at once, twice, thrice and four times during study period and four FGDs were undertaken. All the discussants had been informed on the purpose of the discussion, why we take the record, coached to be in line with discussion topic and encouraged to freely participate in discussion. Beside recording, note had been taken on some pertinent information that cannot be recorded like facial expression and gesture while discussion was proceeding. Information maturation (the point where no new thought was coming out) was considered during both interviews and FGDs.Knowledge of anemia: women who have heard of anemia and know at least one symptom, cause, prevention method and consequence of anemia.Late pregnancy: time period after second trimester of pregnancy.Utilization of iron supplementation: Proportion of pregnant women who took iron supplement for 3 month or more.P-value of less than 0.05 was taken as the statistical significance level.The raw data were checked for completeness and consistency and had been given individual unique identification number and then entered into Statistical Package for Social Science (SPSS) version 20. The wealth index (indicator of living standard of household) was constructed through principal component analysis (PCA). Attitude towards utilization of iron supplementation and anemia was assessed by using health belief model (HBM) constructs ; these wThe information recorded during qualitative data collection was first transcribed into hard copy document. Both the transcription and notes taken in the field had been read thoroughly and related ideas were summarized together to develop themes. Themes were arranged in their logical order and finally results had been narrated manually making the use of themes and quoting ideas that were strongly expressed during interview and FGD.Before collection of the actual data, 5% of total sample size was pretested and necessary corrections on questionnaires were made accordingly. Translating of questionnaire from English to Amharic and back to English to check consistency was made. At the end of each data collection day, data were checked for completeness and consistency and discussion with the research assistants was undertaken. Information saturation and homogeneity of FGD participants have got concern.Four hundred one women who gave birth 6 months preceding the data collection responded the questionnaire successfully and the response rate was 97.6%. Mean age (\u00b1SD) of the respondents was 27.4 (\u00b14.4) years. The median (IQR) number of children ever born to a woman was 3 (2) and about 15.5, 71.8 and 12.7% of respondents gave birth to one, two to four and five or more children, respectively. The majority of respondents were in the age range between 25 and 34\u2009years (Table\u00a0More than three fourth (75.6%) of participants attended ANC at least once during the recent pregnancy and. Mean (\u00b1SD) gestational age at first ANC visit among respondents who had at least one ANC was 5.1 (\u00b11.7) months. Forty four percent received ANC from the health post and 17% had the recommended four or more ANC visit during the index pregnancy Table\u00a0.Table 2Respondents who had ANC visit reflected on the quality of ANC services. They perceived service quality as poor 4.5%, neutral 7%, good 6.7% and very good 27.4%. More than half (51.4%) of respondents perceived that they can face life threatening conditions during pregnancy.Out of the entire respondents, 62.3% women had ever heard of anemia and they mentioned symptoms of anemia; tiredness 50.1%, pale appearance 35.4% and loss of appetite 32.9%. Concerning the causes of anemia during pregnancy, 59.9% mentioned failure to take iron supplementation, 42.6% identified inadequate dietary iron intake as a cause. Other causes reported were blood loss 14% and parasitic infection 13.7%. Most frequently mentioned anemia prevention methods were taking iron supplementation 59.4% and proper maternal nutrition 43.1% and other prevention mechanism mentioned was preventing parasitic infection 13.7%.Regarding the respondents knowledge towards consequences of anemia, 45.1% reported that anemia increases likelihood of maternal death, 41.1% anemic women become seriously ill and 9 and 4.7% mentioned that anemia can result in fetal death and LBW, respectively.Knowledge of anemia among respondents was assessed based on whether they heard of anemia or not and knowledge of symptoms, causes, prevention methods and consequences of anemia they had. Woman was categorized to be knowledgeable of anemia if she heard of anemia and knows at least one symptom, cause, prevention and consequences. Regarding this, 50.6% women were knowledgeable of anemia. From those heard of anemia, 96.8% could mention at least one symptom of anemia Table\u00a0.Table 3Among the respondents, 80.5% had ever heard of iron supplementation and 72.1% have been informed about it during their recent pregnancy. The sources of information were HEWs 38.2%, health professionals 34.4%, mass media 27.9% and HDA members 4.7%. About 27.9 and 4.5% of respondents have received home to home visit care including information about iron supplementation from HEWs and HDAs, respectively.From the respondents who had at least one ANC visit, 91.1% had been informed about the significance of iron supplementation. Type of information they get were iron supplementation prevents anemia 74.9%, all pregnant women need to take it regardless of their anemia status 34.0%, taking iron supplementation can cause side effects 19.1% and how to manage side effects if happened 2.6%.Attitude of the study participants regarding utilization of prenatal iron supplementation and anemia was assessed using health belief model (HBM) constructs . On thisOut of the entire respondents, 70.1% took iron supplement at least once during their recent pregnancy. The median number of days the respondents had taken iron supplementation was 50 (IQR\u2009=\u200945). The length of days which women have taken iron during their pregnancy period ranges from 4 to 140\u2009days. From all the study participants, only 11.5% (95% CI\u2009=\u20099.9\u201313.1%) took the supplementation for the recommended duration of 3 months or above [Among those who took iron supplementation at least once, nearly three fourth (76.5%) have been pre-informed about the total number of days iron supplements have to be taken. Of the respondents those have been taking the supplement, 68.3 and 11.2% got information on benefits and possible side effects, respectively. Amongst those who took a supplementation at least once, more than three fourth (86.8%) reported that they have been advised to take the supplementation on daily bases.Among women who had taken iron supplement at least once during the recent pregnancy, 13.9, 60.1 and 26.6% started taking iron supplementation within first, second and third trimesters respectively. Reasons for starting late in the pregnancy (after second trimester) were delayed initiation of ANC visit 43.2%, having no information about iron supplementation 36.5%, no specified reasons16.2% and others 4.1%.Regarding the source of iron supplements, 34.5% respondents took from health post, 34% from health center and 0.7% from hospital. About 8.2% reported that they encountered a moment when they got no or inadequate iron supplement during their visit to health facility. About 86.5% of respondents who have been prescribed iron supplementation during their recent pregnancy period were reminded about taking iron during their consecutive ANC visits. Utilization of recommended dose of IFA is determined by mother\u2019s adherence towards recommendation by the health worker. Among the respondents who had taken iron supplementation at least once, 55.2% reported that they have been taking the supplementation on daily bases while the remaining 44.8% reported otherwise. Frequently mentioned reasons for missing the supplement were forgetfulness (46.4%), side effects (26.4%), finding it difficult to take the supplement daily (16.8%), with no specific reason (8.0%) and others (2.4%). Of the respondents who started taking the supplement, 83.6% stopped taking iron supplementation at some point before reaching the recommended 91\u2009days plus. The main reasons for discontinuation were side effects (39.8%), birth of the baby (25.4%), fear of taking recommended number of tablets during pregnancy (22.9%), fear of fetal weight gain (4.7%), fear of other health problems (5.5%) and others (1.7%). Amongst the women who took the supplement 60.1% complained of side effects due to taking the supplementation; unpleasant taste (50.9%), nausea (47.3%), vomiting (29.6%), gastric irritation (24.3%), diarrhea (9.5%) and constipation (5.9%).From the entire respondents, 29.9% did not take iron folic acid supplementation during the index pregnancy. Reasons for not taking at all were; had no information regarding importance of iron (12.7%), fear of side effects (4.2%), don\u2019t know where to get the supplement (4.5%), dislike the taste (2.7%), due to religious reasons (2.2%), unavailability of iron tablet in the health facility during time of the visit (2.1%) and assume getting enough iron from food (1.5%).Maternal age, birth order, maternal education, spousal education and household income, two-way distances from the nearest health facility, health service utilization, perception on taking iron and knowledge related factors were considered as variables that affect utilization of prenatal iron supplementation. Maternal education status, spousal education status, birth order, household economic status, time taken for two way trip to the nearest health facility, ANC frequency, maternal knowledge towards anemia, perceived susceptibility to anemia and perceived benefit of taking the supplement were covariates included in adjusted analysis. Educational status of women, family economic status, Number of ANC visits, maternal knowledge on anemia and mother\u2019s perceived benefit of taking the supplement were significantly associated with utilization of prenatal iron supplementation; odds of using prenatal iron supplementation among illiterate women was 77% reduced than their counterparts (AOR\u2009=\u20090.23(95% CI\u2009=\u20090.07\u20130.75)). As compared to women in the richest wealth tertile, those in poorest tertile had 4.43 times increased odds of utilizing the supplementation (AOR\u2009=\u20094.43(95% CI\u2009=\u20091.74\u201311.28)). As compared to women who had four or more antenatal care (ANC), those who conducted 2 and 3 ANC visits had 78% (AOR\u2009=\u20090.22(0.07\u20130.63)) and 66% (AOR\u2009=\u20090.34(0.14\u20130.81)) reduced odds of using the supplementation, respectively. Women who lack comprehensive knowledge of anemia had 85% reduced odds of utilization than their counterparts (AOR\u2009=\u20090.15 (0.04\u20130.62)). As compared to women who perceived that taking iron supplements improves maternal and child health, the counterparts had 90% reduced odds of using the supplementation (AOR\u2009=\u20090.10(0.01\u20130.63) mentioned that ANC service components utilization is improving in the Woreda over time. The main underlining reason is successful promotion activity through HEWs and HDAs. HDA members are involved in identification and listing of pregnant women in their respective catchment areas so that pregnant women can book for the service. Conducting home to home visit by HEW and HDAs and encouraging pregnant women to undertake visit also facilitates ANC service utilization. All discussants agreed that ANC follow-up is necessary to every pregnant woman regardless of her health status as risk factors are unpredictable.Regarding time of initiating ANC visit, majority of the discussants claimed that they started follow-up late after 5 month of pregnancy. Health professionals and HEWs also explained that majority of the pregnant women in their respective catchment health facilities start ANC visit late. The reported reasons for doing so were lack of awareness, late recognition of their pregnancy by the women themselves, late identification of pregnancy by health care providers and lack of access to health institutions. When women start ANC visit late, the duration of taking the supplement is shortened because most of them stop taking after delivery of the baby.Most of the health care providers stated that they were providing iron folic acid supplement to pregnant women. Regarding information being provided to the beneficiaries during iron provision, benefit of taking iron and how many tablets to take once were frequently mentioned.\u201cwe also advice the beneficiaries to delay taking tea/coffee after taking the supplementation\u201d. The information had been provided focused on benefit that can be obtained from taking iron; no discussant had information on side effects that can occur due to taking the supplement, how to manage them and total tablets to be taken.A midwifery nurse in health center said that Sickness in general and pregnancy itself were most frequently mentioned cause of extreme tiredness and fatigue. Few of the FGD participants mentioned that anemia can cause extreme tiredness and fatigue.\u201cAnemia is a disease that results in extreme tiredness and fatigue; even it is not possible to anemic women to stand up and sit down\u201d (23\u2009years old pregnant woman who completed grade 10 at school). Anemia can increase risk of maternal death, can damage the fetus and result in severe illness among mother were frequently mentioned consequences among FGD members. Discussants had fairly good knowledge level on anemia, its consequences and many of them agreed the possibility of preventing anemia through taking iron supplementation.Interviewed health workers agreed that all pregnant women are in need of iron supplementation and mentioned that majority of pregnant women in their catchment were using the supplement. Similarly most of the FGD discussants knew iron tablet and why it is given for.Majority of the FGD participants discussed as they were using iron supplementation;\u201cWhatever the living standard of the pregnant women is, she is at risk of developing anemia; therefore, all pregnant women should take iron tablet\u201d (25\u2009years old pregnant woman who completed grade 9 at school). Pregnant discussants who were taking iron supplement told that they were happy to take the supplement.Some member of FGD talked that they were not taking the supplementation; because, lack of awareness, perceived side effects, having no ANC visits and negligence. Others used to miss the supplement and frequently mentioned reasons for doing so were side effects like nausea, gastric irritation and forgetfulness. No cultural and religious beliefs and negative community perceptions that hinder utilization had been mentioned. The discussants mentioned that some other pregnant do not consume iron at all because they think that there is enough iron in the food they eat. HEWs and health professionals reported that they have been checking adherence of pregnant women towards iron supplementation by randomly conducting pill count during home to home visit, take self report from attendants during subsequent visits on number of tablets they have in home and compare days between previous and current visit.HEWs and health workers reported that they did face no stock out of iron supply in the last year and they had adequate iron folic acid in their stock during interview. Health centers access iron tablet from woreda health office based on their consumption level that was managed by bin card and internal facility request and resupply form (IFRR). Health posts reported that they control their stock using bin card and access iron from their respective catchment health centers based on their need. Health workers monitor management of iron during their supportive supervision both in health posts and health centers.\u201cTo improve the future utilization of iron supplementation, it needs integrating activities at all level; HEW and health professionals should improve quality of counseling, empower HDAs, should early identify pregnancies, continuous follow-up and supervision from health center and district level\u201d (Woreda MCH coordinating person).The group members of respective FGD forwarded some recommendations to improve the future utilization of iron tablet among pregnant women. The majority of group members agreed on strengthening information and education provision to improve mother\u2019s knowledge and follow-up to women to improve their adherence to iron.Prevention of Iron deficiency anemia (IDA) during pregnancy relies on utilization of prenatal iron supplementation. Thus, current study tried to investigate the level and factors associated with utilization of prenatal iron supplementation. The study revealed that the utilization level (taking the supplement for recommended 91\u2009days or above) was still low and predicted by different variables including maternal education, economic status, frequency of ANC visits, knowledge of anemia and perception of women as taking iron supplement improves maternal and child health.The proportion of women who had appropriate utilization of iron supplementation was found to be 11.5%. Even though the figure is low, it is higher than finding from recent EDHS that was less than 1% and studDemographic and health surveys analysis in Indonesia and studNumber of studies revealed that maternal education is positively associated with utilization of prenatal iron supplementation. According to studies in Amhara region , EthiopiIt is common to report economically empowered women use increased number of iron supplementation than their counterparts. Studies undertaken in Indonesia , NortherAs witnessed by several studies, utilization of prenatal iron supplementation increases with increasing frequency of ANC visits. Studies undertaken in Rio Grande , TanzaniThough half (50.6%) of the study participants were knowledgeable of anemia; it became significantly associated with utilization of prenatal iron supplementation. Women who lack comprehensive knowledge had reduced utilization of iron supplementation. This is in consistence with studies undertaken in India , Kenya , Amhara The current study revealed that perceived benefit of taking iron supplementation during pregnancy for maternal and child health had been predictor of prenatal iron supplement utilization. This goes in line with study undertaken in Senegal . Study cSide effects played a major role for missing, discontinuing and failure to take the supplement. It has been reason for missing 26.2%) and discontinuing the supplement (23.4%). Forgetfulness also takes a large share for missing the supplement. About 44.8% had missed (did not taking daily) the supplement and 46.8% of these were due to forgetfulness. The qualitative study also found that side effects, lack of awareness and forgetfulness, missing and discontinuing as reasons for none use. Studies have been done in Nigeria .2% and d, SenegalAs strength, current study used relatively shorter reference period after delivery to identify utilization rate and it mixed both qualitative and quantitative research approaches to strengthen findings. The following limitations need to be considered when interpreting findings. Utilization rate presented was self reported by women that there may be over or under estimation. The sample sizes in some of cells are small and this may be affected statistical power. Study used 6 month reference period after delivery to identify level of iron supplement utilization; thus, still the recall bias might have been introduced.Utilization of prenatal iron supplementation in the study area was very low (11.5%) and left far behind target that intended to increase utilization for 91\u2009days or more days to 50% by 2016. Factors that predict under utilization among women were, maternal illiteracy, having no or less than four ANC visits, lack of knowledge of anemia and perceiving no benefit up on taking iron supplementation. Economically better off women had reduced utilization of prenatal iron supplementation than their counterparts. Lack of awareness towards iron supplement and perceived side effects contributed to non-use of the supplement. Forgetfulness and side effects facilitated missing to take the supplement on daily bases.Thus, I recommend health sector in the study area to ensure early and frequent ANC visits through effective mobilization of HDAs, ensure comprehensive knowledge of anemia among pregnant women, provide comprehensive counseling on benefit, possible side effects and how to reduce them; as result, missing or stopping to take the supplement can be reduced, use HDAs to encourage and confirm pregnant women\u2019s adherence to the supplement of iron recommended for betterment of utilization, users to adjust iron taking time with other usual events like meal time and bed time to reduce forgetfulness and education office in the study area to work hard on girls education and women empowerment as well.Additional file 1. Questionnaires (English version)."} +{"text": "Ovarian cancer is the eighth most commonly occurring cancer in women. Clinically, the limitation of conventional screening and monitoring approaches inhibits high throughput analysis of the tumor molecular markers toward prediction of treatment response. Recently, analysis of liquid biopsies including circulating tumor DNA (ctDNA) open new way toward cancer diagnosis and treatment in a personalized manner in various types of solid tumors. In the case of ovarian carcinoma, growing pre-clinical and clinical studies underscored promising application of ctDNA in diagnosis, prognosis, and prediction of treatment response. In this review, we accumulate and highlight recent molecular findings of ctDNA analysis and its associations with treatment response and patient outcome. Additionally, we discussed the potential application of ctDNA in the personalized treatment of ovarian carcinoma.ctDNA-monitoring usage during the ovarian cancer treatments procedures. Most ofgnancies . Clinicagnancies . Althouggnancies . Accordignancies or humangnancies . Genomicgnancies . Also, tgnancies . In recegnancies , 9, opengnancies . Of thesgnancies , 11. WhiUnderstanding the molecular mechanisms of ovarian cancer, as a heterogeneous disease and underlying treatment resistance, can lead to discovery of some new therapeutic agents . The staLack of an appropriate approach in chemotherapy response prediction in ovarian cancer is likely leading to poor patient\u2019s survival . CA125, HE4 (Human Epididymis Protein 4) is overexpressed in ovarian cancer. Also, the combination of CA-125 and HE4 have the highest sensitivity and specificity in patients sera and also helps in the prediction of malignancy . In addiOva1 measures five proteins named as CA-125, transthyretin, apolipoprotein A1, beta-2 microglobulin, and transferrin with the FDA approval . VasculaKallikreins (KLKs) with 15 family members are responsible in cell growth, angiogenesis, invasion, and metastasis . The decOsteopontin (OPN) is a secreted extracellular matrix glycoprotein, which is involved in wound healing, the immune response, inflammation, tumorigenesis, bone remodeling, and apoptosis inhibition .Mesothelin is a cell surface glycoprotein that are important in tumor metastasis, cancer cell survival, proliferation, and drug resistance . Also, MIn addition to these biomarkers, few prognostic biomarkers were introduced as valuable indicators including macrophage colony-stimulating factor (M-CSF), bikunin, EphA2, Transthyretin (TTR), Transferrin receptor 1, B7-H4, Prostasin, and soluble EGF receptor \u201351 and the increased risk of recurrence independently [TP53 was evaluated in both patient-derived tumor specimens and corresponding ctDNA, which resulted into the detection of similar hotspot mutation in TP53 in both sources of biopsy [TP53 mutations across 103 tumor tissues from 61 patients with a high grade ovarian cancer and also confirmed TP53 mutations in 41 patients. They concluded that, detection of TP53 mutation in ctDNA is a potential tumor-specific biomarker for the treatment response monitoring [CLDN4), RAS oncogene family (RAB25), and ATP binding cassette subfamily F member 2 (ABCF2) [Moreover, BRCA1 and BRCA2 play pivotal roles in DNA repair and germline mutations [BRCA1/2 reversion mutations could be detected by ctDNA sequencing analysis in the patients who received platinum and/or PARP inhibitors [TP53, KIT, KDR, KRAS, PIK3CA, and PTEN) were identified in tumors pre-NACT, while 59 variants out of 19 genes were detected in the ctDNA. In this study, targeted NGS determined the increased level of ctDNA variants with a minimal overlap between ctDNA and tumor DNA. Most of the mutations found in ctDNA were not present in tumor resulting from the amplifying of ctDNA. Besides, the heterogeneity in the tumor can be detected in ctDNA, in contrast with tumor tissue [Over the last few years, genome-wide analysis revealed numerous alterations in ovarian cancer genomes including the inactivation of mutations in tumor suppressor genes such as , ARID1A , 103. Otnd ERBB2 , 104\u2013106nd ERBB2 using tha et al. assessedendently . In a prf biopsy . Moreovef biopsy assessednitoring . Clinicanitoring includin (ABCF2) , 113. Ho (ABCF2) reported (ABCF2) . Moreoveutations . Additiohibitors . Rebeccahibitors comparedr tissue .NF1, RAD51C, PTEN, BRCA2, STAG2, FANCA, CDKN1B, ERBB2, ERBB4, and MAP\u00a02\u2009K1), and also alterations associated with the clinically available drugs were detected. One chemo resistant patient therapy has changed based on the detection of ERBB2 amplification and ctDNA-guided decision. These results can be considered as a proof of using ctDNA concept to guide the clinical decisions during the cycles of chemotherapy in ovarian cancers [TP53, KCAN5, and GJA8) decreased following NAC. Also, in two out of the four NAC-resistant cases, the VAF increased in the non-synonymous somatic mutations . The rate of TP53 mutation was significantly higher in the resistant cases compared with the sensitive cases. In addition, the blood tumor mutation burden significantly decreased after the treatment in the sensitive cases. These findings showed that, gene mutation can be profiled and then monitored using ctDNA in ovarian cancer patients during treatment. The Table To date, FDA has not approved any ctDNA-related test in ovarian cancer. In this regard, a clinical ctDNA workflow was recently designed in the management of the high-grade ovarian cancer, to investigate the clinically actionable alterations of 500 cancer-related genes, which was performed in 12 patients. In seven patients, a good concordance of mutations and copy number alterations in ctDNA and tumor samples ( cancers . In addi cancers comparedIn summary, ctDNA detection before treatment facilitates early detection and leads to appropriate treatment decision-making based on patient stratification. Monitoring of the residual disease helps in prevention of recurrence of the tumor. During the course of treatment, regular monitoring of ctDNA can elucidate drug resistance acquired from genetic alterations, which are always present but not detectable by conventional approaches. Therefore, genomic-based drug response prediction can open new horizons in oncology to enable better cancer patient\u2019s management. In addition, a considerable number of clinical trials, mentioned in Table"} +{"text": "Circulating tumor DNA (ctDNA) detection is a minimally invasive technique that offers dynamic molecular snapshots of genomic alterations in cancer. Although ctDNA markers can be used for early detection of cancers or for monitoring treatment efficacy, the value of ctDNA in guiding treatment decisions in solid cancers is controversial. Here, we monitored ctDNA to detect clinically actionable alterations during treatment of high-grade serous ovarian cancer, the most common and aggressive form of epithelial ovarian cancer with a 5-year survival rate of 43%.We implemented a clinical ctDNA workflow to detect clinically actionable alterations in more than 500 cancer-related genes. We applied the workflow to a prospective cohort consisting of 78 ctDNA samples from 12 patients with high-grade serous ovarian cancer before, during, and after treatment. These longitudinal data sets were analyzed using our open-access ctDNA-tailored bioinformatics analysis pipeline and in-house Translational Oncology Knowledgebase to detect clinically actionable genomic alterations. The alterations were ranked according to the European Society for Medical Oncology scale for clinical actionability of molecular targets.ERBB2 amplification, and this ctDNA-guided decision was followed by significant tumor shrinkage and complete normalization of the cancer antigen 125 tumor marker.Our results show good concordance of mutations and copy number alterations in ctDNA and tumor samples, and alterations associated with clinically available drugs were detected in seven patients (58%). Treatment of one chemoresistant patient was changed on the basis of detection of Our results demonstrate a proof of concept for using ctDNA to guide clinical decisions. Furthermore, our results show that longitudinal ctDNA samples can be used to identify poor-responding patients after first cycles of chemotherapy. We provide what we believe to be the first comprehensive, open-source ctDNA workflow for detecting clinically actionable alterations in solid cancers. Key ObjectiveThe aim of the study was to identify the clinical usefulness of circulating tumor DNA (ctDNA) in the treatment of high-grade serous ovarian cancer (HGSOC) and to provide a clinical workflow for reliable detection of ctDNA alterations.Knowledge GeneratedWe show that longitudinal ctDNA sampling can be used to detect response to platinum-taxane primary therapy after the first cycles of chemotherapy and to identify clinically applicable mutations and copy number alterations. In one patient with chemoresistant HGSOC, ctDNA-guided treatment including trastuzumab was administered during tumor progression and yielded tumor shrinkage.RelevanceThe early detection of poor-responding patients allows the design of alternative treatment strategies in HGSOC. Especially for platinum-resistant patients who have limited treatment options, rapid discovery of resistant cell populations can provide an early opportunity to interfere with the development of recurrence. The provided approach allows the selection of treatment options that target subclones that persist during therapy. This is a substantial improvement in the management of recurrent solid cancers, where tumors are not usually sampled either because of the risk of potential intervention complications or simply because the sample could be insufficient or not representative of the disease.1 Indeed, ctDNA sampling is a clinically attractive, minimally invasive technique that is based on the observation that tumor cells leak DNA to the bloodstream, where it can be captured by genomic assays. ctDNA can be used to monitor tumor evolution,2 detect cancer early,3 and monitor treatment efficacy.4 However, the value of ctDNA in guiding treatment decisions still remains controversial,1 especially in cancers lacking dominant oncogenic mutations.Effective treatment of metastatic solid cancers is hampered by intrapatient heterogeneity, tumor evolution, and the paucity of representative tissue samples to guide treatment decisions. Analysis of circulating tumor DNA (ctDNA) is an approach with the potential of overcoming all three obstacles.5 Translating genomic alterations in ctDNA data into a clinical decision requires multidisciplinary expertise in medical genetics, bioinformatics, and clinical oncology, as discussed in response to a recent study.6 Another reason is that most earlier studies focused on a few or tens of candidate genes, which significantly decreases the probability of finding an effective therapy on the basis of ctDNA data, whereas exome or whole-genome ctDNA sequencing is currently too expensive for clinical use.A major reason for controversy in the use of ctDNA to guide clinical decision making stems from the varying, and often poorly described, analysis approaches.7 The current standard-of-care therapy for advanced HGSOC consists of cytoreductive surgery and platinum-paclitaxel chemotherapy.8 Although the initial response to standard-of-care therapy is usually good, nearly all patients eventually relapse and have limited treatment options. Thus, there is an urgent need to find effective therapy targets for HGSOC in general and for relapsed, chemotherapy-resistant disease in particular.Here, we present a comprehensive ctDNA analysis approach that consists of longitudinal ctDNA sampling, a panel of more than 500 cancer-related genes, ctDNA analysis\u2013tailored bioinformatics pipelines, and a Translational Oncology Knowledgebase to identify clinically actionable mutations and copy number alterations (CNAs). We demonstrate the usefulness of this approach in high-grade serous ovarian cancer (HGSOC), which is the most common and aggressive form of epithelial ovarian cancer, with a 5-year survival rate of 43%.We prospectively collected 78 plasma samples from 12 patients treated for ovarian or primary peritoneal HGSOC at Turku University Hospital, Finland or partially platinum-sensitive disease were regarded as poor responders (50%), whereas patients with platinum-sensitive disease were considered good responders .All patients participating in the study gave their informed consent, and the study was approved by the Ethics Committee of the Hospital District of Southwest Finland.g. The samples were stored at \u221280\u00b0C as 1 to 2 mL aliquots frozen less than 2 hours after collection to avoid lysis of the white blood cells.1 Cell-free DNA (cfDNA) was extracted from plasma samples and subjected to 1000\u00d7 targeted Illumina Hi-Seq sequencing at BGI using the Oseq Solid Cancer Panel with more than 500 clinically actionable genes (Data Supplement).Venous whole blood (5 to 6 mL) was collected into an EDTA tube, shaken eight to 10 times, and centrifuged twice for 10 minutes at 2000 \u00d7 2 snap-frozen tumor tissue and ascites samples (Data Supplement), as well as from whole blood (germline) buffy coat samples, and was sequenced using the Oseq panel with coverage of 200\u00d7.DNA was isolated from 21 liquid LN10 using the ctDNA data analysis pipeline, which is based on best practices for sequencing data analysis (Data Supplement). A detailed description of the pipeline and validation data can be found in the Data Supplement. Briefly, somatic single nucleotide variants (SNVs) and indels were detected using Mutect2.11 We tracked mutations detected in at least one plasma or tumor tissue sample with at least 1% variant allele frequency (VAF) but not in the corresponding germline blood control (maximum two reads or VAF 0.001 accepted). Filtering steps included detection of likely spurious mutations by identifying clusters of variants shared between patients but not known in cancer,12 and functionally irrelevant variants by a Combined Annotation Dependent Depletion13 score of less than 15. CNAs were estimated using PanelDoC14 (Data Supplement) and CNVPanelizer.Mutation detection was performed in the computational ecosystem AndurilTP53 mutation4 VAF identified as truncal mutations from the pretreatment tissue and plasma samples . To estimate the change in mutational profiles, we identified the largest proportion of significantly decreased variants during treatment compared with the pretreatment sample . Because of varying numbers of mutations in the pretreatment samples, the proportion of significantly decreased variants was corrected for mutation counts.Detected somatic alterations were used to confirm the presence and quantity of ctDNA . The proportion of ctDNA in cfDNA was evaluated from P < .01, Fisher\u2019s exact test; Data Supplement) using the ConsensusPathDB15 algorithm. The ConsensusPathDB integrates data from 32 databases and allows comprehensive over-representation analysis for mutations.Pathway analysis was performed for genes with a nondecreasing mutation frequency (16 and shown to exist in patients\u2019 tumor tissue (Data Supplement).Potentially clinically actionable alterations were validated through immunohistochemistry (IHC) and in situ hybridization for alterations classified as most prominent We collected 78 longitudinal ctDNA samples and 21 tumor tissue samples from 12 unselected patients with HGSOC (Data Supplement). We applied a sequencing panel of more than 500 genes, followed by variant and CNA calling, filtering, and prioritization of clinically relevant aberrations as shown in TP53 gene are reported in nearly 100% of patients with HGSOC.4 In our cohort, TP53 mutations were observed in all patients. We noted, consistent with previous studies,4 a moderate positive correlation between ctDNA TP53 VAF and serum cancer antigen 125 levels . The most selectively enriched pathways in poor-responding patients were related to chromatin regulation and included 10 genes. We prioritized and interpreted all 265 mutations and 113 CNAs using the Translational Oncology Knowledgebase, which integrates information from 11 databases and 14 scientific articles (Data Supplement). We identified high-confidence, potentially clinically actionable mutations or CNAs in seven patients 58%; . The KnoPTEN (patient EOC49) and NF1 (patient EOC429), which cause overactivation of the mTOR pathway.22 Patients with the activated mTOR pathway have been shown to be responsive to phosphatidylinositol 3-kinase/mTOR inhibitors.22 We validated overactivity of the mTOR pathway in the two patients with IHC from tumor tissue samples and plasma matched the pattern of TP53 mutation, indicating that the PTEN mutation exists in all tumor cells and presents an early event in tumor evolution (Data Supplement). PTEN was detected in a follow-up sample at 8 months after primary treatment with VAF of 0.1%, confirming its persistence during therapy.The highest allele frequency mutations matching tumor content were detected in therapy . In patiBRCA2 and STAG2, which potentially confer homologous recombination (HR) DNA repair deficiency and sensitivity to poly-ADP ribose polymerase (PARP) inhibitors. However, the frequencies of these mutations were low in the tumor tissues (0.2% for BRCA2 and 3.1% for STAG2), suggesting subclonal events. In patient EOC429, we detected a germline deletion in RAD51C from plasma, which has been shown to confer HR deficiency and PARP inhibitor sensitivity,21 and, consistent with this, the patient had a prolonged response to platinum-based chemotherapy. We also detected a somatic deletion of FANCA in patient EOC1067. This patient also had concurrent deletions in CDKN1B and CDKN2B. These alterations are predicted to cause sensitivity to CDK2/425 and/or CDK4/6 inhibitors.26 Regardless of the presence of HR-deficiency\u2013conferring alterations, this patient, who had no residual tumor after surgery, progressed 8.9 months after the last platinum cycle.We observed mutations and CNAs in genes associated with DNA repair in three patients. In patient EOC677, we detected mutations in ERBB4 mutation, which is predicted to confer sensitivity to EGFR inhibitors.27 Patient EOC587 had a MAPK mutation and a simultaneous MAPK1 amplification, which are associated with resistance to the EGFR targeting therapies.29 We detected a high copy-count ERBB2 amplification in the ctDNA in patient EOC736. The ERBB2 locus is amplified in 3% to 11% of HGSOCs31 and is a predictive biomarker for trastuzumab. The ERBB2 amplification was validated in primary omentum tissue using SNP genotype\u2013based CNA detection and in the tumor tissue sample from interval debulking surgery with in situ hybridization and IHC pathway. Patient EOC568 had an and IHC .TP53 VAF remained detectable throughout the treatment, which, on the basis of our findings, indicates a poor response to platinum taxane (Data Supplement). Indeed, disease progression was detected only 5 months after the end of primary therapy. This patient also suffered from severe paclitaxel-induced neuropathy after primary treatment, and therefore, pegylated liposomal doxorubicin (PLD) was started at the first relapse instead of dose-dense paclitaxel therapy. PLD was discontinued after four cycles because of constantly rising CA-125 (from 193 to 769) and progression in lymph nodes as evidenced by a computed tomography scan.Patient EOC736 was treated with NACT, and she participated in a clinical trial after primary treatment, receiving maintenance bevacizumab and olaparib or placebo . AlthougERBB2 amplification detected in ctDNA, treatment of patient EOC736 was changed to trastuzumab 600 mg subcutaneously once every 3 weeks, combined with reduced doses of carboplatin (AUC4) and dose-dense paclitaxel (80 mg/m2 on days 1 and 8). The patient responded to this combination well, and a biochemical complete response was achieved after only three treatment cycles .The value of ctDNA has been demonstrated in early detection and disease monitoring, but evidence for using ctDNA to guide clinical decision making is weaker.1 Indeed, our filtering approach using longitudinal samples removed false-positive alterations in all patients. Our results further emphasize the possibility that tissue samples may not represent the progressive tumor in all cases. This highlights the potential of ctDNA as a clinically feasible, noninvasive method to uncover genetic alterations and drug targets during disease progression. Thus, the optimal discovery of clinically useful alterations requires multiple samples from one patient combined with careful cross-disciplinary interpretation conducted by geneticists, bioinformaticians, and clinicians.We detected high ctDNA\u2013tumor congruence, which, together with the absence of alterations from the blood cells used as germline samples and comprehensive filtering of mutations through clustering, maximizes the likelihood that the reported alterations originated from tumor and not from other sources, such as clonal hematopoiesis.4 The early hints of chemoresistance , may be used to predict prognosis and open a window of opportunity for therapeutic interventions before disease relapse and additional evolution of the tumor. The most informative comparisons for predicting treatment outcome are samples at the preoperative stage, after two cycles of chemotherapy, and after debulking surgery, because they provide information on treatment response and on persisting tumor subclonesLongitudinal ctDNA sampling can be used to monitor response to first-line therapy and to identify poor-responding patients in HGSOC.22 In patient EOC429, we identified alterations conferring both mTOR pathway activation and HR deficiency, which suggests potential benefit from combination treatment with, for example, mTOR inhibition and PARP inhibition. In addition, two patients had more than one concurrent, potentially targetable alteration. Patient EOC1067 progressed after 9 months of platinum-based therapy despite the FANCA deletion conferring HR deficiency, potentially because of the contribution of the CDK alterations on cell-cycle regulation and tumor progression.32 On the basis of the concurrent CKDN1B and CKDN2B deletions, this patient could potentially benefit from a combination targeting both pathways, for instance, PARP inhibitor and CDK4/6 inhibitor. These results indicate that longitudinal ctDNA sampling can detect and monitor a relapse at an early stage and identify potentially effective drug combinations.We identified potentially clinically applicable mutations or CNAs in more than one half of the patients with HGSOC. These included two patients with mTOR pathway\u2013activating mutations that were validated. Both patients had a good response to the platinum-based first-line therapy, but in the case of relapse they could most likely benefit from mTOR inhibitors alone or in combination with chemotherapy.ERBB2 amplification in the ctDNA of a patient with platinum-resistant HGSOC and used this information to change the patient\u2019s treatment to include trastuzumab with reduced doses of platinum and paclitaxel. This patient had relapsed 5 months after primary platinum-taxane\u2013based therapy and did not respond to PLD. Thus, rapid response to the ctDNA-guided treatment, detected by complete normalization of CA-125 values within 2 months after starting the combination treatment, was surprising. Earlier studies that have combined ERBB2 inhibitors with platinum and paclitaxel report similar encouraging responses for relapsed patients with advanced HGSOC.31 Although the number of patients in these studies and our study remains small because of the low number of patients with ERBB2-amplified HGSOC, these results warrant testing ERBB2 amplification from relapsed HGSOC patients with advanced disease.We identified a high-confidence ctDNA sampling and sequencing with a large panel offers several benefits to physicians. Response to therapy can be inferred using two or three consecutive ctDNA samples during therapy. Rapid discovery of resistant cell population expansion provides an early opportunity to interfere with the development of recurrence. This is particularly important in chemoresistant relapse in which larger tumor burden is associated with low therapy responses, with both conventional and targeted therapies. Moreover, large gene panels offer a higher probability of identifying clinically relevant mutations and CNAs, as compared with targeted approaches with a few targets. This is a substantial improvement in managing recurrent solid cancers in which tumors are not usually sampled either because of the risk of potential intervention complications or because the sample could be insufficient or not representative of the disease."} +{"text": "The mixture of isopropanol, polyethyleneimine, and benzoic acid proved to be an optimized dispersing agent for EPD. The kinetics of EPD for oxidants (Al) and reductants (MoO3) were systematically investigated, which contributed to adjusting the equivalence ratio of targeted energetic chips after changing the EPD dynamic behaviors of Al and MoO3 in suspension. In addition, the obtained nano-Al/MoO3 MIC energetic chips showed excellent heat-release performance with a high heat release of ca. 3340 J/g, and were successfully ignited with a dazzling flame recorded by a high-speed camera. Moreover, the fabrication method here is fully compatible with a micro-electromechanical system (MEMS), which suggests promising potential in designing and developing other MIC energetic chips or devices for micro-ignition/propulsion applications.Film-forming techniques and the control of heat release in micro-energetic chips or devices create challenges and bottlenecks for the utilization of energy. In this study, promising nano-Al/MoO Compar3 [2O3 ,12,13. T3 MIC, as a desirable energetic system, has continuously aroused great interest, owing to its high heat of reaction (4698 J/g) and adiabatic flame temperature . Recently, different fabrication methods have been explored to prepare Al/MoO3 MICs or nanothermites for developing their exothermic performances, including the thermal co-evaporation method [3 MICs with different multilayer internal structures via the magnetron sputtering method on a semiconductor bridge as a promising micro-energy storage device, and analyzed the condensed state reaction process in the obtained nano-multilayered films [3 nanocomposite powders fabricated by the arrested reactive milling technique [3 MICs fabricated by the traditional mechanical mixing technique were shown to fuel a dramatic combustion exothermic process with a high burning rate of 100 \u00b1 4 m/s and a high pressurization rate of 35 kPa/\u03bcs [2O3 MIC system [3) chips via the EPD technique. The Al/MoOn method , magnetrn method ,16, sonin method , and arrn method . For exaed films . E.L. Drechnique . In addi5 kPa/\u03bcs . Neverth5 kPa/\u03bcs ,23, or p5 kPa/\u03bcs ,25 in a 5 kPa/\u03bcs and the 5 kPa/\u03bcs , respectC system . MoreoveC system and oxidizer (MoO3). Thus, further exploration of this aspect was analyzed and verified theoretically. Finally, the heat-release properties and ignition test of the product were investigated.Thus, for these reasons, a novel nano-Al/MoOPolyethyleneimine, PEG-2000, benzoic acid, and nano-Al powders (99.9%) were purchased from Aladdin Inc. Corporation. . Isopropyl alcohol was purchased from Kelong Industrial Inc., . The other reagents (including hydrogen peroxide and ethanol) from Sinopharm Chemical Reagent Co., Ltd. were used as analytical grade purity without further purification. Deionized water (18.2 \u03a9) was used in all experiments.3 MIC, the EPD technique was exploited, and the corresponding detailed schematic diagram is displayed in 3 powders. To be specific, 0.25 M Mo powders were added into 200 mL deionized water with trace PEG2000 marked as mixture A, then H2O2 (30 wt%) was dripped into mixture A slowly, until the yellow molybdenum peroxide sol appeared. After ultrasonic treatment for 0.25 h, the obtained sol was moved into a hydrothermal reactor at 110 \u00b1 2 \u00b0C for 4 h, and the MoO3 powders were fabricated after repeated centrifugal cleaning at a rotation speed of 10,000 r/min and vacuum drying.In the fabrication of the nano-Al/MoO3 powders with different mass ratios were added into the optimized dispersant of isopropanol, polyethyleneimine, and benzoic acid with a volume ratio of 50:1:1 to obtain a stable suspension after ultrasonic treatment for 20 min at 25 \u00b0C. During EPD, a micro-ignition bridge was the working electrode, and the copper sheet with the same area was used as the counter electrode; the detailed size of electrodes is shown in 3 MIC chip was finally obtained after cooling to room temperature for the subsequent ignition experiments. The deposited efficiency (deposit weight per area (mg cm\u22122)) of the deposits was calculated by dividing the increased weight of the working electrode after EPD by the deposition area. Each experiment was repeated five times, and the average value of five parallel experiments was used as the final valid result.Then, the nano-Al and MoO3 MIC were measured with a field emission scanning electron microscope equipped with energy dispersive X-ray spectroscopy (EDX), and X-ray diffractometer with a scanning rate of 5\u00b0/min, respectively. Atomic absorption spectroscopy was used to determine the mass or mole ratio of Al and MoO3 in deposited energetic film. The heat-release (Q) of the energetic chip was analyzed by differential scanning calorimetry measured in a temperature range from 25 to 1000 \u00b0C at a low heating rate of 10 K/min under a 99.999% argon flow. Ignition of the product was studied using home-made capacitor charge/discharge initiating equipment, and video recordings of the deflagration were recorded by a high-speed camera at an imaging speed of 104 f/s.The morphology, element distribution, and crystalline structures of the nano-Al/MoO3 MIC by EPD at normal tempearture and pressure. For verifying the EPD controllability, dynamic behaviours of particles in optimized suspension were studied in detail. Shown in 2) as a function of deposited time under applied electric field strengths ranging from 6 to 12 V/mm during EPD of Al/MoO3 MIC films. It was obviously observed that the deposition efficiency increased with the EPD time when the field strength was fixed at 6 V/mm, and a similar trend was also seen in a higher field strength of 9 or 12 V/mm. The higher field strength provides a higher EPD efficiency at a certain deposited time . Moreover, the deposited efficiency increased linearly with deposited time increasing from 0 to Tc ) in cT decreased with an increase of applied field strength; that is, cT for 6 V/mm was larger than cT for 9 and 12 V/mm, which was primarily due to the more severe precipitation and collision of particles under higher field strengths. Thus, the EPD process of Al/MoO3 MIC can be more precisely controlled in the linear control region (ct < T) in this study, which is due to the relatively complex relationship of deposited efficiency and EPD time in the nonlinear variation region for all field strengths. Furthermore, the effect of the distance of electrodes on the deposited efficiency of the nano-Al/MoO3 MIC is analyzed in A successful EPD generally largely depends on various dispersing agents, and a large number of experimental studies have been carried out to compare the dispersion systems for EPD of specific sorts of particles ,30,31. A3). Generally, in MIC energetic reactions, the equivalence ratio (\u03a6) is defined as the actual ratio of fuel to oxidizer divided by the stoichiometric ratio of fuel to oxidizer in an energetic reaction, that is \u03a6 = (F/O)actual/(F/O)stoich [3 particles, the equivalence ratio in the starting suspension (\u03a6s) was adjusted accurately in weighed samples, and the equivalence ratio in the deposited product (\u03a6d) was determined by EDX and AAS techniques. d of Al and MoO3 particles in the Al/MoO3 MIC chip as a function of \u03a6s of nano-Al and MoO3 particles in the starting suspension. Clearly, it can be seen that \u03a6d increased linearly with \u03a6s by EDX and AAS analysis, and the fitted equations were similar . Thus, \u03a6d in the nano-Al/MoO3 MIC could be adjusted by changing \u03a6s in suspension, which contributes to optimizing the proportion of components in product, and further developing the exothermic performance of the product.In addition, the exothermicity of MICs is a key indicator that largely depends on the mass or mole ratio of fuel and oxidizer stoich . For the3 MIC in 3 ) on the product, demonstrating the successful co-EPD of the Al and MoO3 particles. In addition, the fact that there are no other clear peaks of Al2O3 and Mo in XRD analysis was used to investigate the crystal structures of the nano-Al/MoO3 MIC films via EPD are displayed in 3 MIC appears to be uniformly distributed, without rare unmixed zones. The higher-resolution images in 3, which significantly helps to enlarge the contact areas of Al and MoO3, and shorten the mass transportation length (MSL) during the thermite exothermic reaction of nano-Al/MoO3 MIC. Moreover, there were numerous gaps among the particles [3 MIC obtained by EPD.The as-obtained nano-Al/MoOarticles b,c, cont698 J/g) ,33. In a3 MIC with the \u03a6d of ~1.0 and a low heating rate of 10 K/min. In addition to an unobservable exothermic peak at ~400 \u00b0C, probably due to the reaction between Al nano-particles with much smaller-sized MoO3 particles, there are several observable exothermic peaks in 3 particles [3 particles, which is consistent with the results from the Zhu group [3 MIC was as high as ~3340 J/g, which was >70% of the theoretical value, indicating the relatively sufficient thermite reaction. Furthermore, the effect of the deposited time on the heat-release of the product is analyzed in 3 particles in suspension. The heat-release values as functions of \u03a6d and \u03a6s of Al and MoO3 are clearly shown in the 3D histogram and target energetic films or initiators (\u03a6d).The thermite reaction deflagration processes of an electric explosion for the nano-Al/MoO quickly ,40,41,423 MIC chip initiator was firstly fabricated by a high-efficiency EPD technique in an optimized mixture dispersant of isopropanol, polyethyleneimine, and benzoic acid at normal temperature and pressure. The microstructures and chemical compositions of the product were demonstrated by FESEM, EDX, and XRD. The deposited energetic films exhibited even mixing between the oxidizer (Al) and reductant (MoO3), contributing to enhancing their exothermic performance. The EPD dynamic behaviors of nano-Al and MoO3 particles were studied, which can act as a theoretical bridge for connecting the \u03a6s in starting suspension and \u03a6d in energetic chips. DSC results showed the apparent exothermic peaks of nano-Al/MoO3 MIC chips, due to the thermite reaction between Al and MoO3, and the corresponding total heat-release was as high as ca. 3340 J/g when \u03a6d of Al and MoO3 was close to 1.0. In addition, the Al/MoO3 MIC chip initiator can be successfully ignited with a typical capacitor charge/discharge ignition device, exhibiting outstanding detonation performance with a short burst time and a dazzling flame. In short, the design of the Al/MoO3 MIC chip initiator in this study will provide a universal approach for fabricating other thermite energetic chips with wide civilian and military applications, especially in micro-initiation or micro-propulsion systems.In this study, a novel Al/MoO"} +{"text": "High-sensitivity C-reactive protein (hs-CRP) elevation frequently occurs in acute myocardial infarction (AMI) and is associated with adverse outcomes. Since diabetes mellitus (DM) is characterized by an underlying chronic inflammation, hs-CRP may have a different prognostic power in AMI patients with and without DM.We prospectively included 2064 AMI patients; hs-CRP was measured at hospital admission. Patients were grouped according to hs-CRP quartiles and DM status. The primary endpoint was a composite of in-hospital mortality, cardiogenic shock, and acute pulmonary edema. Two-year all-cause mortality was the secondary endpoint.Twenty-six percent (n\u2009=\u2009548) of patients had DM and they had higher hs-CRP levels than non-DM patients . The primary endpoint incidence in the overall population , in DM , and non-DM patients increased in parallel with hs-CRP quartiles. The adjusted risk of the primary endpoint increased in parallel with hs-CRP quartiles in DM and non-DM patients but this relationship was less evident in DM patients. In the overall population, the adjusted OR of the primary endpoint associated with an hs-CRP value\u2009\u2265\u20092\u00a0mg/L was 2.10 (95% CI 1.46-3.00). For the same risk, hs-CRP was 7 and 2\u00a0mg/L in patients with and without DM. A similar behavior was observed for the secondary endpoint when the HR associated with an hs-CRP value\u2009\u2265\u20092\u00a0mg/L found in the overall population was 2.25 (95% CI 1.57-3.22). For the same risk, hs-CRP was 8 and 1.5\u00a0mg/L in DM and non-DM patients.This study shows that hs-CRP predicts in-hospital outcome and two-year mortality in AMI patients with and without DM. However, in DM patients, the same risk of developing events as in non-DM patients is associated to higher hs-CRP levels. Type 2 diabetes mellitus (DM) is a common comorbidity in acute myocardial infarction (AMI), and it is associated with two-fold higher in-hospital and long-term mortality rates and with a higher risk of recurrent cardiovascular events \u20133. Both C-reactive protein (CRP), an acute phase protein secreted by the liver, is the most widely used biomarker for detecting inflammatory conditions . The eleElevated hs-CRP levels in AMI patients may reflect a variable combination of chronic and acute (due to the ongoing cardiac event) inflammation. Since DM is more frequently associated with some degree of chronic inflammation, it is possible that, in AMI patients with DM, hs-CRP has a different prognostic relevance as compared to their non-DM counterpart.The purpose of this study was to investigate the association between hs-CRP levels, measured at hospital admission, and in-hospital outcome and two-year mortality in a cohort of AMI patients according to DM status.This was a prospective, observational study. We enrolled all consecutive patients with AMI (n\u2009=\u20092178), both ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI), admitted to the Intensive Cardiac Care Unit of Centro Cardiologico Monzino in Milan between June 1, 2012 and October 1, 2017. Patients experiencing AMI as a complication of elective percutaneous coronary intervention (PCI) (Type 4a AMI) and those with concomitant systemic inflammatory conditions, including active infections (n\u2009=\u200988) or malignancies (n\u2009=\u200926) were excluded. The study complied with the Declaration of Helsinki, and the Ethics Committee of our center approved the research protocol (n. R520-CCM549). Written informed consent was obtained from all participants.Patients were considered as suffering from DM if one of the following conditions were present: personal history of DM reported in clinical record, treatment with glucose lowering drugs, or a glycated hemoglobin value\u2009\u2265\u20096.5% (48\u00a0mmol/mol). Glycated hemoglobin was measured at hospital admission in all patients as a part of our routine laboratory package using a method NGSP certified and standardized to the DCCT assay .\u00ae assay (particle-enhanced immunoturbidimetric assay) on Cobas c501 (Roche) and 0.67 [95% CI 0.61\u20130.72]) than in DM patients (0.63 [95% CI 0.58\u20130.68] and 0.61 [95% CI 0.54\u20130.67]).The adjusted OR and HR of the primary and secondary endpoint, respectively, associated with an hs-CRP value\u2009\u2265\u20092\u00a0mg/L found in the overall population Fig.\u00a0 correspoThis study supports previous evidence showing that hs-CRP measured at hospital admission in AMI patients is a predictor of in-hospital outcome and long-term mortality. This seems to be true for both DM and non-DM patients. However, we demonstrated that the relationship between the outcomes considered in our study and hs-CRP levels is downshifted in DM patients, who show for each hs-CRP value a lower risk than that of non-DM patients. In other words, in DM patients, the hs-CRP values associated to each event risk were higher than those of non-DM patients.The involvement of inflammation in atherosclerosis and, consequently, in AMI is well established \u20137, as weIn our study, we confirmed the presence of a close association between inflammation and DM status in AMI. Indeed, DM patients were more likely to have admission hs-CRP levels\u2009\u2265\u20092\u00a0mg/L and had a higher median hs-CRP value than non-DM patients. Moreover, both inflammation and DM status, considered separately, were predictors of in-hospital outcome and two-year mortality, even after adjustment for major confounders. However, when we investigated the relationship between inflammation and outcomes, hs-CRP showed a different behavior in DM and in non-DM patients. In particular, the adjusted risk of the primary and secondary endpoints increased in parallel with hs-CRP quartiles in both groups, but with a more evident trend in non-DM patients. Notably, in the overall population, an hs-CRP value\u2009\u2265\u20092\u00a0mg/L was associated with an almost two-fold higher risk of both endpoints. This same risk corresponded to higher hs-CRP values in DM patients, when compared to non-DM patients, thus suggesting that the prognostic relevance of inflammation is maintained also in DM patients but it is shifted towards higher hs-CRP levels. To the best of our knowledge, this is a novel finding, which, if confirmed in future studies, could pave the way for prognostic stratification and intervention strategies tailored according to DM status.The mechanisms underlying the different prognostic behavior of hs-CRP in DM and non-DM patients are beyond the purpose of the present analysis. However, the following hypothesis can be proposed. In AMI patients, admission hs-CRP level may be considered the result of a variable combination of chronic and acute inflammation. Thus, high hs-CRP levels at hospital admission may not necessarily represent only the inflammatory response associated with AMI severity. Given the well-established association between DM and inflammation, the contribution of chronic inflammation to hs-CRP levels in AMI patients is possibly more relevant in DM than in non-DM patients. Consistently with this theory, a similar hs-TnI peak value, an estimate of myocardial infarct size, was observed in our study in DM and non-DM patients, although the median hs-CRP level was significantly higher in the former group. Moreover, the correlation between hs-CRP levels and hs-TnI peak value was closer in non-DM than in DM patients.Another intriguing issue is represented by the mechanisms underlying the association between hs-CRP and in-hospital outcome in AMI. In this regard, there is growing evidence that inflammation in AMI is not only a marker of AMI severity but it may directly exacerbate the cardiac dysfunction \u201333. IndeOur study may have some potential clinical implications. Firstly, in AMI patients, hs-CRP allows physicians to identify high-risk patients. This is true also for DM patients, in whom, however, a higher hs-CRP threshold than that usually considered (2\u00a0mg/L) should be identified to improve risk stratification. This concept is further suggested by the fact that, in our study population, a significant interaction was found between DM status and hs-CRP when long-term mortality was considered. Moreover, the RR of two-year mortality was constantly lower in patients with DM than in those without DM at each given hs-CRP level. For instance, the two-year mortality RR of a non-DM patient with hs-CRP level of 2\u00a0mg/L was similar to that of a DM patient with hs-CRP level of 14\u00a0mg/L Fig.\u00a0. SecondlThe strengths of our study include its prospective design, a well-characterized population, and a special focus on the relationship between inflammation and DM status in AMI. However, some limitations warrant mention. Firstly, we evaluated an AMI population admitted to a single center and treated, in most cases, with PCI. As this therapeutic strategy may have influenced the results of our study, the overall applicability of our findings to AMI patients not undergoing coronary revascularization needs to be clarified. Moreover, the promptness, extent, and efficacy of myocardial revascularization was not assessed as a confounder event. Secondly, because of the observational nature of the study, a cause-effect relationship between hs-CRP and outcomes cannot be established. Moreover, we did not evaluate the effectiveness of and the adherence to pharmacological treatment, in particular of lipid- and glucose-lowering therapies, during follow-up. Indeed, previous studies reported that the prognostic effect of admission hs-CRP is attenuated in high-intensity statin users and in pThe results of this study show that hs-CRP level measured at hospital admission predicts in-hospital outcome and two-year mortality in AMI patients with and without DM. However, in patients with DM, the same risk of developing events as in non-DM patients is associated to higher hs-CRP levels."} +{"text": "Background: High sensitivity CRP (hs-CRP) has attracted intense interest in risk assessment. We aimed to explore its prognostic value in patients with acute myocardial infarction (AMI).Methods and Results: We enrolled 4,504 consecutive AMI patients in this prospective cohort study. The associations between hs-CRP levels with the incidence of in-hospital HF was evaluated by logistic regression analysis. The association between hs-CRP levels and the cumulative incidence of HF after hospitalization were evaluated by Fine-Gray proportional sub-distribution hazards models, accounting for death without HF as competing risk. Cox proportional hazards regression models were constructed to estimate the association between hs-CRP levels and the risk of all-cause mortality. Over a median follow-up of 1 year, 1,112 (24.7%) patients developed in-hospital HF, 571 (18.9%) patients developed HF post-discharge and 262 (8.2%) patients died. In the fully adjusted model, the risk of in-hospital heart failure (HF) [95% confidence intervals (CI)] among those patients with hs-CRP values in quartile 3 (Q3) and Q4 were 1.36 (1.05\u20131.77) and 1.41 (1.07\u20131.85) times as high as the risk among patients in Q1 (p trend < 0.001). Patients with hs-CRP values in Q3 and Q4 had 1.33 (1.00\u20131.76) and 1.80 times (1.37\u20132.36) as high as the risk of HF post-discharge compared with patients in Q1 respectively (p trend < 0.001). Patients with hs-CRP values in Q3 and Q4 had 1.74 (1.08\u20132.82) and 2.42 times (1.52\u20133.87) as high as the risk of death compared with patients in Q1 respectively (p trend < 0.001).Conclusions: Hs-CRP was found to be associated with the incidence of in-hospital HF, HF post-discharge and all-cause mortality in patients with AMI. C-reactive protein (CRP), an acute-phase protein reflecting the early inflammation response, has emerged as a simple tool for risk assessment for coronary events in general population \u20133. In thHowever, most of the recent studies were focused on mortality, recurrent myocardial infarction or late development of HF and most of them were limited by relatively small sample sizes or only conducted in stable myocardial infarction survivors, ST-segment elevation myocardial infarction (STEMI) or non\u2013ST-segment elevation myocardial infarction patients (NSTEMI) patients alone. Few of the above studies focused on in-hospital HF and what is particularly noteworthy is that high sensitivity CRP (hs-CRP) detected by an ultrasensitive automated enzyme immunoassay has been shown to be a better indicator of outcomes than CRP levels measured through traditional assays .In the present study, we aimed to assess the prognostic value of hs-CRP on clinical outcomes including in-hospital HF and HF post-discharge as well as all-cause mortality and recurrent myocardial infarction in patients with acute myocardial infarction (AMI).n = 537): patients who had prior HF, liver disease, uremia, malignancy or infectious disease before admission, patients who had received thrombolysis therapy or deep venous thrombosis and who had no hs-CRP assessments as well as other key clinical variables on admission. As 537 patients were excluded and 4,504 AMI patients with complete data were enrolled in this cohort in the final count. Patients alive at discharge consented to an additional 1\u201324 months follow-up owing to a non-normal distribution and analyzed by Kruskal-Wallis H test. Categorical variables were presented as number (percentages) and analyzed by chi-square test or Fisher exact test.To visually assess non-linear relationships between hs-CRP levels and HF during and after hospitalization as well as mortality risk, restricted cubic spline curves were performed. The cumulative incidence of HF post-AMI after hospitalization or all-cause mortality were estimated according to hs-CRP quartile using Kaplan-Meier method and compared with the log-rank test.The associations between hs-CRP levels with the incidence of in-hospital HF was evaluated by logistic regression analysis. The association between hs-CRP levels and the cumulative incidence of HF after hospitalization were evaluated by Fine-Gray proportional sub-distribution hazards models, accounting for death without HF as competing risk. Cox proportional hazards regression models were constructed to estimate the association between hs-CRP levels and the risk of all-cause mortality. In addition to the unadjusted model, we constructed another five adjusted models , 15, modp-value of < 0.05 was considered statistically significant in this study.Stratified analysis was presented with a fully adjusted model (except for subgroup factors). Interaction effects between hs-CRP and subgroup variables were assessed using the log likelihood ratio test. A 2-tailed All the analyses were performed with the statistical software R version 3.6.1 .A total of 4,504 AMI patients with complete data were enrolled in this cohort in the final count, including 3,070 STEMI and 1,434 NSTEMI. Four thousand four hundred thirty-two patients alive at discharge consented to an additional follow-up. The median length of follow-up was 1 year (1\u201324 months). The median age was 62 years (53\u201369). Baseline characteristics of the study population according to hs-CRP quartile concentrations were listed in p trend = 0.012). Similar observations were acquired when treating hs-CRP as continuous variable patients developed HF . The restricted cubic spline curves showed that hs-CRP had a L-shaped relationship with in-hospital HF . Hs-CRP variable .Figure 4A).We also performed several sensitivity analyses to evaluate the robustness of the association between hs-CRP levels and in-hospital HF. Stratified analysis showed that the prognostic effect of hs-CRP levels was similar across all the various sub-populations of the 4,432 patients alive at discharge experienced a HF event after hospitalization. The restricted cubic spline curves showed that hs-CRP also had a L-shaped relationship with HF post-discharge . The cump trend < 0.001). Moreover, stratified analysis showed that the prognostic effect of hs-CRP levels was robust across all the various sub-populations patients died. Hs-CRP also had a L-shaped relationship with death in the restricted cubic spline curves . Elevatep trend < 0.001). When analyzing hs-CRP as a continuous variable, this association remained with a 52% increased risk of death for each SD increase in hs-CRP and 2.42 times (1.52\u20133.87) as high as the risk of death compared with patients in Q1, respectively (26\u20131.83) . In the 26\u20131.83) .Our study demonstrated the prognostic effect of hs-CRP assessed on admission on the incidence of in-hospital HF, the cumulative incidence of HF post-discharge and all-cause mortality in patients with AMI, which were independent of other relevant covariables.Some previous studies also found that CRP was predictive for the occurrence of HF in stable myocardial infarction survivors or in patients with STEMI or AMI \u201312. In aAfter AMI, CRP dramatically increases, peaking in 2\u20134 days and returning to baseline in 3\u20134 weeks . In thisOn the other hand, CRP can amplify the immune response through complement activation, which has the effect of expanding the infarct size , 25. InfWith respect to mortality, some previous epidemiologic studies in initially healthy populations have also shown that CRP levels may be associated with cardiovascular morbidity and death , 30. SomHowever, the prognostic value in our study was significant. There are two main factors may explain the reason for this prognostic effect of hs-CRP for death post-AMI. First, CRP is related to the dysfunction of endothelial cells and the progression of atherosclerosis, it has a direct proinflammatory effect by inducing a significant increment of adhesion molecule expression in human endothelial cells and decreasing the synthesis of nitric oxide as well as release of prostacyclin produced by endothelial cells which will facilitate the development of diverse cardiovascular diseases \u201335. FurtOur study has several limitations. First, this is a single-center study and was performed in a Northeast Chinese population, therefore the findings should be extrapolated cautiously to other populations with different genetic backgrounds. Second, we did not conduct serial measurements for hs-CRP levels at other time points, particularly the peak levels after the occurrence of AMI, however the hs-CRP levels on admission also gave us strong information for the prognosis of AMI patients at the early phase. Third, this is an observational study and the observed differences in clinical outcomes and hs-CRP levels may be subject to possible confounders which we were unable to control for. However, our findings were strongly statistically significant and clinically meaningful, and relevant results were shown by viewing the numerical data. Above all, when observing during a short time, it is indeed beneficial to investigate the potential correlation between hs-CRP and the incidence of HF, with less unknown affecting factors, because HF is characterized by highly heterogeneous manifestations and mechanisms.Our study demonstrated that there was a significant association between hs-CRP and the incidence of in-hospital HF and HF post-discharge and it could also be a prognostic marker of all-cause mortality in AMI patients, independent of established conventional risk factors. This association could improve clinical decision making for post-AMI management.dryu_hmu@163.com.The data analyzed in this study is subject to the following licenses/restrictions: The dataset is still in privacy of our team. Requests to access these datasets should be directed to The studies involving human participants were reviewed and approved by the Ethics Committee of Harbin Medical University (Reference Number: KY2017-249). The patients/participants provided their written informed consent to participate in this study.XZ and SW contributed to conceptualization and formal analysis. XZ contributed to the writing of the manuscript. BY and SF contributed to project administration and supervision. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "OsHMA2, a gene controlling root-to-shoot Cd/Zn translocation, was not influenced by Cd exposure in Capataz and progressively increased in Beirao with the external Cd concentration, suggesting that activity of the OsHMA2 transporter may differentially limit root-to-shoot Cd/Zn translocation in Capataz and Beirao.Two rice accessions, Capataz and Beirao, contrasting for cadmium (Cd) tolerance and root retention, were exposed to a broad range of Cd concentrations and analyzed for their potential capacity to chelate, compartmentalize, and translocate Cd to gain information about the relative contribution of these processes in determining the different pathways of Cd distribution along the plants. In Capataz, Cd root retention increased with the external Cd concentration, while in Beirao it resulted independent of Cd availability and significantly higher than in Capataz at the lowest Cd concentrations analyzed. Analysis of thiol accumulation in the roots revealed that the different amounts of these compounds in Capataz and Beirao, as well as the expression levels of genes involved in phytochelatin biosynthesis and direct Cd sequestration into the vacuoles of the root cells, were not related to the capacity of the accessions to trap the metal into the roots. Interestingly, the relative transcript abundance of Cadmium (Cd) is a toxic heavy metal for most living organisms and represents one of the principal dangerous substances released into soil, water, and atmosphere by anthropogenic activities ,2,3,4. AOryza sativa L.), an important staple food for more than half of the world\u2019s population, has been recognized as the major source of Cd intake in Japan and other Asian countries, where a high prevalence of renal proximal tubular dysfunctions has been documented [\u22121 established by the Codex Alimentarius Commission of FAO/WHO [Rice for accumulation in the shoots, as they probably use the same transport systems to be loaded into the xylem . A promilocation ,33,34. Ld grains .Selection of low-Cd accumulator rice accessions is still the most hopeful approach to minimize the dietary intake of Cd, in terms of both effectiveness and economic value ,25. In tjaponica rice panel.This paper describes and compares two rice accessions, Capataz and Beirao, selected as the most contrasting accessions following a preliminary screening for different traits related to Cd accumulation and tolerance performed on a Rice plants were grown hydroponically and exposed for 10 days to a broad range of Cd concentrations (from 0.01 to 1 \u03bcM) in the growing medium.Results showed that Cd exposure significantly inhibited the growth of Capataz. Growth inhibition, calculated with respect to the control (absence of Cd), progressively increased from 14.7 to 56.9% and from 4.7 to 63.1%, in roots and shoots, respectively, as Cd external concentration did. On the other hand, in the same conditions, Cd did not produce significant effects on the growth of Beirao, except at the highest concentration analyzed (1 \u03bcM), where a significant inhibition (26.8%) of shoot growth was observed A,B.The total amount of Cd absorbed by plants increased by enhancing the external concentration of the metal. Except for the lowest Cd concentration analyzed, the amount of the metal in the whole plants was significantly higher in Beirao than in Capataz C. Cd conFrom these data, we can easily evince that the two accessions were characterized by two contrasting behaviors concerning their root capacity to retain Cd : in Capataz, Cd root retention was not constant and rose from 29.8 to 82.2%, while in Beirao, Cd root retention was significantly higher than in Capataz at the lowest Cd concentrations analyzed (0.01 and 0.1 \u03bcM) and resulted independent of the amount of Cd in the growing medium F.Since thiol metabolism may be directly involved in Cd sequestration inside the vacuoles of the root cells, we analyzed the effects of increasing Cd exposure on the accumulation of non-protein thiols (NPTs) in the roots. As expected, NPT levels of the roots increased as Cd concentration in the external medium did and resulted significantly higher in Beirao than in Capataz under all the Cd concentrations analyzed A. The ovOsPCS1 and OsPCS2, the two rice genes encoding PC synthases expressed in the roots, was not significantly affected by Cd exposure in both Capataz and Beirao , but also on the activity of transport systems, like OsHMA2, which continuously removing free Cd ions from the root cells, determine the \u201cpassive\u201d capacity of the roots to retain the metal ions within them [OsHMA3 gene expression. In such a context, it would be expected that the amount of Cd ions escaping the active components of the root firewall progressively increases with the Cd concentration in the root tissues, according to the pictures previously described by fractioning Cd ions accumulated in rice roots [OsHMA2 transcripts in the roots, we can reasonably suppose that the activity of the OsHMA2 transporter may differentially limit root-to-shoot Cd translocation in Capataz and Beirao, determining a different Cd root retention. Such a hypothesis seems to be supported by the observation that in Capataz, Cd xylem loading early approached saturation at 0.1 \u03bcM Cd2+, while in Beirao, it linearly increased with the external Cd concentration , the concentration of Zn in the shoots was significantly lower in Beirao than in Capataz. Thus, the Cd-induced accumulation of the OsHMA2 transcripts observed in Beirao could be the consequence of its lower capacity to maintain Zn homeostasis in the shoots under Cd stress.Several works indicate OsHMA2\u2014a P1B-type ATPase\u2014as the main transporter controlling Zn and Cd translocation in rice ,33,34. Od Beirao D, suggeshin them ,25. Datace roots . Considentration A. Moreovntration B. Such at system B. Finallolerance . Under cOsHMA2 observed in Beirao under Cd exposure could be the result of a complex interaction among a plethora of transporters involved in maintaining Zn homeostasis, rather than the molecular phenotype of an accession-specific OsHMA2 allele.Taken as a whole data presented in this work confirm that the ability of rice to retain much of the Cd absorbed within the roots results from an equilibrium among the physiological and biochemical processes acting in Cd chelation, sequestration, and translocation. Although phenotypic differences among the rice accessions may mainly depend on specific differences in their capability to compartmentalize Cd into vacuoles of the root cells ,43,44,45Oryza sativa L. ssp. japonica) accessions, Capataz and Beirao, belonging to a japonica rice panel maintained at the CREA-Research Centre for Cereal and Industrial Crops in Vercelli, Italy [All the experiments were performed using two rice 2, 500 \u03bcM MgSO4, 250 \u03bcM NH4H2PO4, 25 \u03bcM Fe-tartrate, 46 \u03bcM H3BO3, 9 \u03bcM MnCl2, 0.8 \u03bcM ZnSO4, 0.3 \u03bcM CuSO4, 0.1 \u03bcM (NH4)6Mo7O24, 30 \u03bcM Na2O3Si (pH 6.5). Seedlings were kept for 12 days in a growth chamber maintained at 26 \u00b0C and 80% relative humidity during the 16 h light period and at 22 \u00b0C and 70% relative humidity during the 8 h dark period. Photosynthetic photon flux density was 400 \u00b5mol m\u22122 s\u22121. At the end of this period, plants were treated with Cd by supplementing the nutrient solutions with different amounts of CdCl2 to reach the final concentrations of 0.01, 0.1, and 1 \u03bcM. The treatment period was 10 day long. All hydroponic solutions were renewed daily to minimize nutrient depletion.Rice caryopses were placed on filter paper saturated with distilled water and incubated in the dark at 26 \u00b0C. Seven days later, seedlings were transplanted into 3 L plastic tanks (21 seedlings per tank) containing the following complete nutrient solution: 1.5 mM KNO2 solution to displace extracellular Cd [2 and stored at \u221280 \u00b0C or analyzed immediately.Plants were harvested and roots were washed for 10 min in ice-cold 5 mM CaCllular Cd , rinsed 3, clarified with 1.5 mL 33% (w:v) H2O2, and finally dried at 80 \u00b0C. The mineralized material was dissolved in 5 mL 0.1 M HNO3 and filtered on a 0.45 \u03bcm nylon membrane. Cd and Zn content was measured by inductively coupled plasma-mass spectrometry .Samples of c.a. 400 mg fresh weight (FW) of both roots and shoots were mineralized at 120 \u00b0C in 5 mL 14.4 M HNOTotal non-protein thiols of roots were determined as described by Nocito et al. . ResultsAt the end of the exposure period, shoots were cut 2 cm above the roots with a microtome blade. Xylem sap exuded from the lower cut surface was collected by trapping into a 1.5 mL plastic vial filled with a small piece of cotton for 30 min. The amount of collected sap was determined by weighing, and the Cd and Zn concentrations were measured by ICP-MS after complete mineralization.\u00ae Reagent and then purified using PureLink\u00ae RNA Mini Kit (Life Technologies), according to the manufacturer\u2019s instructions. Contaminant DNA was removed on-column using PureLink\u00ae DNase (Life Technologies). First-strand cDNA synthesis was carried out using the SuperScriptTM III First-Strand Synthesis SuperMix for qRT-PCR , according to the manufacturer\u2019s instructions.Total RNA was extracted from roots using TRIzolOsPCS1 , OsPCS2 , OsHMA3 , and OsHMA2 was performed on first-strand cDNA in a 20 \u03bcL reaction mixture containing GoTaq\u00ae qPCR Master Mix and the specific primers, using an ABI 7300 Real-Time PCR system . The relative transcript level of each gene was calculated by the 2\u2212\u0394\u0394Ct method [S16 coding for the 40S ribosomal protein S16 (LOC_Os11g03400) gene as reference. Primers for qRT-PCR are listed in qRT-PCR analysis of t method using thn = 6). In each independent experiment, three distinct 3 L tanks were used for each control (0 \u03bcM Cd2+) and Cd treatment . ANOVA was carried out using SigmaPlot for Windows version 11.0 . Significance values were adjusted for multiple comparisons using the Bonferroni correction. Student\u2019s t-test was used to assess the significance of the observed differences between Capataz and Beirao. In both cases, statistical significance was at p < 0.05.Quantitative values are presented as mean \u00b1 standard error of the mean (SE) of two independent experiments run in triplicate ("} +{"text": "Triticum aestivum L.) has a higher Cd absorption capacity than other cereal crops causing an excess daily Cd intake and a huge threat for public health. Therefore, the reduction of Cd accumulation in wheat from the soil is a crucial food-security issue. A pot trial was performed on Cd-stressed wheat seedlings to evaluate the morphological and physio-biochemical responses via foliage spray of two different bio-stimulants, i.e., ascorbic acid (AsA) and moringa leaf extract (MLE). Two wheat cultivars (Fsd-08 and Glxy-13) were exposed to cadmium (CdCl2.5H2O) stress , along with foliar spray of AsA (0 and 50 mM) and MLE (0 and 3%). The most observable growth reduction was documented in plants that are exposed to a higher Cd concentration , followed by the lower Cd level (500 \u03bcM). The wheat growth attributes, such as number of leaves per plant, number of tillers per plant, biomass yield, shoot/root length, and leaf area, were greatly depressed under the Cd stress, irrespective of the cultivar. Under the increasing Cd stress, a significant diminution was observed in maximum photochemical efficiency (Fv/Fm), photochemical quenching (qP), and electron transport rate (ETR) accompanied with reduced gas exchange attributes. However, Cd-induced phytotoxicity enhanced the non-photochemical quenching (NPQ) and internal carbon dioxide concentration (Ci), which was confirmed by their significant positive correlation with Cd contents in shoot and root tissues of both cultivars. The contents of proline, AsA, glycine betaine (GB), tocopherol, total free amino acid (TFAA), and total soluble sugar (TSS) were greatly decreased with Cd stress , while MLE and AsA significantly enhanced the osmolytes accumulation under both Cd levels . The Cd accumulation was predominantly found in the root as compared to shoots in both cultivars, which has declined after the application of MLE and AsA. Conclusively, MLE was found to be more effective to mitigate Cd-induced phytotoxicity up to 500 \u03bcM Cd concentration, compared with the AsA amendment.It has been established that wheat ( Zea mays L. has a higher Cd absorption capacity than other cereal crops leaf extract (MLE) has been categorized as one of the novel and natural biostimulants that are enriched with higher nutrient contents, improved growth, and Cd tolerance in Phaseolus vulgaris L. (Zea mays L. (Lepidium sativum L. (Triticum aestivum L. (Hordeum vulgare L.) (Triticum aestivum L.) and wheaivum L.) grown univum L.) .The beneficial role of the combined application of MLE and AsA to mitigate Cd stress in the wheat plant is scarcely available. Therefore, the current study was based on the hypothesis that MLE and AsA will enhance the tolerance of wheat seedlings by maintaining morpho-physiological responses and accumulation of secondary metabolites to reduce the impact of Cd. Our findings will provide an insight to guide further studies of the Cd resistance with MLE in wheat at the molecular level, as well as tolerance against other heavy metals in other plant species. The objectives of the current study were to: (i) investigate the impact of Cd on wheat growth attributes, chlorophyll fluorescence, antioxidant, and osmolyte performance, and (ii) explore valuable physiological and biochemical traits as a marker to induce Cd tolerance with exogenously applied MLE and AsA. The findings would further highlight the mechanisms involved to ensure tolerance against Cd in wheat seedlings, along with the provision of useful, innovative, and cheap techniques in the form of moringa to mitigate other abiotic stresses.A pot investigation was performed in the wirehouse of the old botanical garden, University of Agricultural, Faisalabad, Pakistan . The growing conditions in the wirehouse remained at 27/20\u00b0C day/night temperature, a 12-h illumination per day, and 65\u201370% moisture contents. The seeds of two wheat cultivars, i.e., Faisalabad-08 (Fsd-08) and Galaxy-13 (Glxy-13), were provided by Ayyub Agriculture Research Institute (AARI), Faisalabad, Pakistan. Healthy seeds were surface-sterilized by soaking in the solution of sodium hypochlorite for 5 min and washed with distilled water repeatedly. The plastic pots (32 \u00d7 22 cm) were filled with 5.5 kg dried and sieved sand. Later, the dried seeds were sown in pots for germination. All the pots were watered with a half-strength Hoagland nutrient medium once a week.2.5H2O in a full-strength Hoagland nutrient medium . The concentrated extract was further diluted to 3% with distilled water before foliar application. To maintain the moisture level of plants up to 75\u201385%, pots were watered regularly, while Cd and foliar application were given once a week (6 weeks). To avoid nutrient deficiency, the nutrient solution was irrigated once a week as well. All the below-mentioned attributes were recorded 45 days after sowing (DAS), with and without treatment. All the measurements were performed according to the set criterion to reduce the difference caused by determination time.Both wheat cultivars were allowed to grow up to the three-leaf stage, thinning was done to ensure 5 uniform seedlings/pot before application of Cd, ascorbic acid (50 mM), and moringa leaf extract (3%). The Cd was saturated in the rhizosphere by dissolving CdClt medium , while A2) was calculated by taking the length and width of three randomly selected leaves/plant/replicate with a measuring tape. The number of tillers and the number of leaves were recorded by counting the respective trait.The shoot and root length and the fresh biomass were measured separately with the measuring tape (cm) after harvesting and subsequent weighing, respectively. Leaf area (cmA), stomatal conductance (Gs), internal CO2 concentration , and transpiration rate (E), were assessed using fully turgid uppermost leaves with a portable photosynthesis system, Infra-Red Gas Analyzer at a light-saturating intensity between 9:00 am and 12:00 pm. On the other hand, water-use efficiency was calculated by adopting the formula (A/E).The gaseous attributes, such as carbon dioxide assimilation rate (\u20131 FW) were examined by following the method devised by a + b), and carotenoids by applying the respective formula.The concentrations of chlorophyll a or b, total chlorophyll, and carotenoid contents (mg g0) was determined by a weak red light (< 0.1 \u03bcmol m\u20132 s\u20131). Then, the maximum fluorescence (Fm) was determined using a saturating pulse of.8 -s duration. The activity of photosystem II (PSII) was determined by V/FMF, when the leaves had been dark-adapted for 30-min, using the dark leaf clip. Quantum efficiency of photochemical transports used for photosynthesis (\u03a6PSII), photochemical quenching (qP), quantum efficiency of thermal dissipation promoted by the photoprotective non-photochemical quenching via the xanthophyll cycle (\u03a6NPQ), and electron transport rate (\u03a6ETR) was calculated using 1,500 \u03bcmol m\u20132 s\u20131 actinic light.The chlorophyll fluorescence (CF) parameters from the upper leaf surfaces of wheat seedlings were determined with an OS5p Modulator Fluorometer . Three 2SO4. From the mixture,0.5 ml was further mixed with.5 ml of KI3. The mixture was cold at 90 min, then, was added a pre-chilled distilled water and 1,2-dichloroethane. It will form two layers, the upper layer will be discarded, and absorbance was taken from the spectrophotometer at 365 nm. Endogenous ascorbic acid was determined according to the standard protocol was taken with a spectrophotometer (IRMECO 2020) at 750 nm. Total soluble sugar (TSS) contents were extracted by using fresh leaves (0.1 g) of both cultivars by cutting and boiling them in the distilled water (5 ml) for 1 h. The extract was filtered and the volume was up to 25 ml. The TSS was analyzed by using a reaction of 1 ml of an extract with 5 ml fresh anthrone reagent (Flavonoid contents were determined by following the method of % H2SO4) . Thereaf3) and kept at room temperature for 3 h. The samples were transferred to a heating block and digested at 300\u00b0C until samples turned black to tinted yellow. The samples were taken from the heating block, cooled, diluted with distilled water up to 25 ml, and were filtered. The acid extract of the samples was used to determine Cd concentration by atomic absorption spectrum .The root and leaf samples were dried in a hot-air oven at 72\u00b0C for six days. Shoot and root dry samples (0.25 g) were separately placed in a 50 ml digestion flask and digested in 5 ml concentrated nitric acid (HNOp < 0.05).All the attributes discussed above were measured by 45 DAS and subjected to statistical analysis using the SPSS . For each treatment, the mean values with SE (\u00b1 SE) are described in the figures and tables. The parameters were analyzed by multivariate ANOVA (MANOVA). The significant difference among the means was calculated by Tukey\u2019s test , transpiration rate (E), water use efficiency (WUE), and stomatal conductance (Gs) in both wheat cultivars were deprived of the increasing levels of Cd stress, except internal CO2 concentration (Ci) (2 assimilation rate (A) was prominently increased with the application of both foliar stimulants, with and without Cd stress (2 concentration (Ci) was increased under stress, while slightly decreased by MLE and AsA foliar treatments (The results exhibited that carbon dioxide assimilation rate (ion (Ci) . Howeverd stress . Both wheatments .A reduction in biomass accumulation and photosynthetic attributes governed by Cd stress was concentration-dependent; therefore, higher levels of Cd resulted in a higher loss in biomass accumulation and chlorophyll contents in both wheat cultivars . The resFv/Fm), and electron transport rate have significantly declined with the increasing Cd stress in the rhizosphere, with one exception of non-photochemical quenching (NPQ), which increased significantly under the elevated level of Cd concentration in both wheat cultivars , with and without a foliar spray of MLE and AsA, in both cultivars exposed to Cd stress (p \u2264 0.05) escalation with the exposure to Cd stress. However, the effect of MLE and AsA on alpha-tocopherol contents was more obvious at 500 \u03bcM Cd stress compared to the 1,000 \u03bcM Cd level. After the supply of MLE and AsA, an increment in tocopherol contents was noticed by 52 and 20% in Fsd-08, while 37 and 26% in Glxy-13 cultivar exposed to 500 \u03bcM Cd stress, respectively, compared with the related treatments without a foliar spray of biostimulants.According to the obtained results, ascorbic acid contents have decreased, while tocopherol, glycine betaine, and proline contents have increased under the Cd toxicity, particularly with 500 \u03bcM Cd concentration. Meanwhile, exogenous application of biostimulants modulated the innate mechanisms to boost the non-enzymatic antioxidant defense system of both wheat cultivars . Brieflyd stress . The alpp \u2264 0.05) reduction for flavonoid pool and total free amino acids at all levels of Cd stress. However, flavonoid contents displayed a positive response in Fsd-08 and Glxy-13 cultivars after the foliar spray of MLE (38% and 12%), in reaction with 500 \u03bcM Cd stress, respectively, relative to the similar treatments without MLE application. However, the mitigation of 1,000 \u03bcM Cd concentration was not obvious with the spraying of biostimulants and of flavonoid contents that are sharply diminished , total phenolics, and TSS contents, decreased with the increasing Cd stress . Both whminished . The seeminished . Similarminished . Likewisminished .p < 0.05) reduced Cd accumulation in the shoot in root tissues of both wheat cultivars in comparison to the no-spray treatment. Cd concentration in the shoot reduced by 58\u201361% in Fsd-08 and 33\u201340% in Glxy-13 with the foliar spray of AsA followed by MLE under the lower Cd stress (500 \u03bcM).The Cd concentrations in the different wheat tissues (shoot and root) under the foliar applications of the two biostimulants on two wheat cultivars are presented in 2 concentration (Ci) and non-photochemical quenching (NPQ), while a negative interaction was observed with other attributes of both wheat cultivars.A correlation analysis was performed to evaluate the linkage between the accumulated Cd concentration and various physio-biochemical attributes of Cd-stressed wheat cultivars after the amendment of MLE and AsA . There ivia foliage has already been proven to boost plant growth , total soluble protein, and ascorbic acid in the rocket plant (Erusa vesicaria) (It was previously exhibited that Cd stress downregulated the gas exchange features in wheat seedlings . Moreoveeristics . Accordieristics . The higeristics . Moreovea leaves . Similarsicaria) .2O2), ionic leakage, and lipid peroxidation resulting in an escalation in the activities of non-enzymatic antioxidants, antioxidative enzymes, and enzyme gene expression have been reported earlier in participating in osmotic balance and maintenance of macromolecules concentration, hence, improving the tolerance to abiotic conditions could be a useful strategy to enhance Cd tolerance in wheat plants by improving growth, reducing Cd uptake, and accumulation of metabolites in various tissues (In this study, Cd caused a significant reduction in growth, photosynthetic apparatus, and osmoprotectant activities in two wheat cultivars, tissues .The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.FF: data curation, formal analysis, investigation, and writing \u2013 original draft. MA: formal analysis, investigation, and writing \u2013 original draft. XW, RI, and HT: data curation and writing \u2013 review and editing. AT: supervision, writing \u2013 original draft, and writing \u2013 review and editing. MK: data curation, investigation, resources, writing \u2013 original draft, and writing \u2013 review and editing. IT: investigation and writing \u2013 review and editing. FM-P: data curation, investigation, and writing \u2013 review and editing. AE-S: resources and writing \u2013 review and editing. HE: resources, data curation, and writing \u2013 review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The aggregated vehicle, in particular the \u03b3-CD-added system, showed a remarkable sustained effect because of slow dynamics. Furthermore, a prominent cytoprotective effect by the \u03b3-CD\u2013Toc vehicle under the oxidative stress condition was confirmed. Thus, the novel vitamin E vehicle motif using \u03b3-CD as a stabilizer was proposed, widening the usability of Toc for biological applications.Development of a novel antioxidant-delivery vehicle exerting biosafety has been attracting a great deal of interest. In this study, a vehicle comprising a natural composite consisting of vitamin E and cyclodextrin (CD) additives was developed, directed toward aqua-related biological applications. Not only Vitamin Es are representative lipophilic antioxidants, consisting of a chromanol head and a phytyl tail ,6,7,8,9.The low solubility and dispersibility of vitamin E in water media have been typically shown ,7,8,9. T\u03b1-CD, \u03b2-CD, and \u03b3-CD are the representative natural CDs that consist of six, seven, or eight glucose residues, respectively, and are used in various medical applications greatly enhanced the solubility of Toc, whereas the natural CDs, such as \u03b2-CD and \u03b3-CD, which are also water-soluble CDs, formed opaque systems by mixing with Toc. From the preliminary study, we deemed that the dispersion systems can be used as antioxidant-delivery vehicles in biological applications. Both components, Toc and CD, have been used as food ingredients and pharmaceutical excipients [Recently, we have studied the solubility enhancement of Toc by CDs . As a recipients , and the\u03b3-CD\u2013Toc systems, were investigated in detail. Furthermore, the radical scavenging ability in water systems was evaluated, together with an assessment of the cytoprotective effect on cells under oxidative stress using a lipid oxidation inducer. These studies were performed in the absence of an organic solvent. Throughout the study, 2,2,5,7,8-pentamethyl-6-chromanol . 2,2\u2032-Azinobis (ABTS) was purchased from TCI Co. Ltd. .Toc (MW: 430.71), and PMC (MW: 220.31) were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. . 2. Cells were then cultured in the presence or absence of \u03b3-CD (60 \u00b5M) or \u03b3-CD (60 \u00b5M)-Toc (10 \u00b5M) for 30 min under identical incubation conditions, before exposure to 0, 50, 100, or 200 \u00b5M tert-butyl hydroperoxide (TBHP) [The African green monkey kidney cell line COS-7 (JCRB9127) was cultured in Dulbecco\u2019s Modified Eagle\u2019s Medium supplemented with 10% fetal bovine serum , penicillin , streptomycin , and amphotericin B . Cells were seeded into wells of 96-well or 24-well plates at a concentration of approximately 0.3 cells/mL, in 100 or 500 \u00b5L per well, respectively, and plates were incubated for 24 h at 37 \u00b0C in an atmosphere containing 5% COTo prepare the CD-Toc mixture system, an excess amount of Toc or the Toc analogue PMC was added to 4 mL of an aqueous CD solution at different concentrations. Stirring at ambient atmosphere was conducted for 16 h. It was found that some amount of added Toc adhered to the Teflon magnetic bar or the glass wall during stirring , and thu2 gas atmosphere in the dark. The dispersion was prepared by the addition of water and subsequent mixing with vortex and ultrasonication within 1 min, respectively.II-1:4: CD-Toc dispersion systems were prepared using the CD-Toc mixing solids ,27. Brie\u03b3-CD-added system.II-1:2: Toc (0.46 mmol) in 5 mL EtOH and CDs in 10 mL pure water were also used for the preparation of dispersion II-1:2 to demonstrate the effectiveness of the The Toc concentration of each sample was determined spectrophotometrically at 291 nm using a V-650 spectrophotometer, JASCO Corp. ,23,24,25The analysis of the compositions in the solid samples, used for the preparation of II, was performed by NMR spectroscopy using a JEOL ECA 500 instrument . The physicochemical properties of the solids were analyzed by Fourier transform infrared (FTIR) using a Nicolet iS50+iN10 . To obtain novel insight into the physical properties, emission quantum yields in solid states were evaluated using a combination of an integrating sphere , a monochromated xenon light source, and a cooled CCD spectrometer.For the radical scavenging test, an ABTS biradical was used similar to previous works ,28. Name2. Optical density at 450 nm was measured using a Food Mark microplate absorbance reader . Cell morphology was assessed using a DM IL LED microscope .Cell viability was assessed using a Cell Counting Kit-8 , according to the manufacturer\u2019s protocol. Briefly, CCK-8 reagent was added to cells, which were then incubated for 2 h at 37 \u00b0C in an atmosphere containing 5% COt-test. Differences were considered statistically significant at * p < 0.05. Levels of statistical significance for each comparison are indicated in each figure.All statistical analyses were conducted using Excel 2016. All data are presented as the mean \u00b1 standard deviation (SD) of four independent experiments. Between-group differences were compared using Student\u2019s \u03b2-CD system with a vortex mixing afforded a distinct agglomeration within 10 min). The results indicated the high stability of the dispersion formed in \u03b3-CD-added systems.When stirring a water-soluble CD solution at 10 mM concentration with a water-insoluble vitamin E (Toc), an increase in turbidity was observed for each CD-added system, especially for the D system . The disto 10 mM , right, \u03b2-CD-, and \u03b3-CD-added systems became trace. This indicates the formation of large aggregates containing Toc for the \u03b2-CD- and \u03b3-CD-added systems, which were insoluble in water.Noteworthy, when transparent solutions were obtained by the filtration of these dispersions through a 0.2 \u03bcm nanopore membrane filter, the Toc contents in the \u03b3-CD. The amount of Toc suddenly increased above 20 mM \u03b3-CD concentrations for I, suggesting that the concentration can be regarded as the critical aggregation concentration (CAC) to afford \u03b3-CD/Toc-dispersions. As far as we know, the observation of CAC for CD/Toc systems was unprecedented. Furthermore, as shown in \u03b3-CD-added system, whereas those of the \u03b1-CD-added and \u03b2-CD-added systems were unstable of Toc in mol was expressed as the scavenging ability of PMC in mol, which was evaluated in the water system in the absence of both organic solvent and CD and used as a reference (unity). It should be noted that the addition of CDs does not affect the RSA of PMC at the concentrations used for the study. The representative results of the RSA test are shown in \u03b2-CD and 30 mM \u03b3-CD aq showed effective RSA under diluted conditions. Noteworthy, while the 2,6-DMCD-added system showed no sustained effect in the previous study [\u03b2-CD-added and \u03b3-CD-added systems, respectively. This means that the dispersed Toc stabilized by \u03b2-CD and \u03b3-CD, especially in the latter case, showed limited access owing to the hindered molecular diffusion of the self-assembled aggregates. Similar behavior was observed for sample II prepared using an inclusion solid consisting of \u03b3-CD and Toc , as demonstrated by improved cell viability and preserved cell morphology (\u03b3-CD\u2013Toc complex exhibited more potent cytoprotective activity than \u03b3-CD alone. These data suggest that the \u03b3-CD\u2013Toc complex enters COS-7 cells and scavenges intracellular lipid radicals in a cytoprotective manner. Thus, this complex may provide utility not only for molecular biological research, but also perhaps as a novel therapeutic agent for clinical use in the context of oxidative stress-associated diseases.Exposure of COS-7 cells to TBHP induced loss of viability in a dose-dependent manner a and resrphology . The \u03b3-C\u03b3-CD and Toc formed a highly stable dispersion when the initial CD concentration was above 20 mM or when it was prepared using inclusion complex solids obtained via the co-precipitation method. On the other hand, the mixture of \u03b2-CD and Toc also formed a stable dispersion when the initial CD concentration was 10 mM, but the system lost the dispersing state under dilution unlike the dispersion formed in the \u03b3-CD-added system, meaning that the dispersion formed in the \u03b3-CD-added system must be more stable and useful.The formation of CD\u2013Toc inclusion dispersion and the effects on the radical scavenging and the cytoprotection were demonstrated. The mixture of Several studies including the phase solubility profile of CD\u2013PMC systems and the spectroscopic analysis on the solid consisting of CD and Toc indicated the correlations between the characteristic dispersing behavior of the CD\u2013Toc systems and the self-assembly of inclusion complex formed between CD and Toc. Detail investigations on the formation of mechanisms at the molecular level were required as future researches.\u03b3-CD\u2013Toc system exhibited a distinct packaging effect, and a sustained effect was obtained. Moreover, the effectiveness of \u03b3-CD\u2013Toc inclusion assembly was confirmed for the cytoprotective activity in vitro. These results showed unique fascinating features of \u03b3-CD\u2013Toc inclusion assembly for novel antioxidant-delivery-vehicles for biological applications, in which water-soluble analogues, PMC and Trolox, cannot be used. The use as a therapeutic agent for clinical use in the context of oxidative stress-associated diseases was expected.Noteworthy, most of the inclusion complexes were not solubilized, but the CD\u2013Toc self-assembly dispersion systems showed a radical scavenging effect comparable to that of PMC. In particular, the"} +{"text": "Greenhouse vegetable production (GVP) has grown rapidly and has become a major force for cucumber production in China. In highly intensive GVP systems, excessive fertilization results in soil acidification, increasing Cd accumulation and oxidative stress damage in vegetables as well as increasing health risk of vegetable consumers. Therefore, enhancing antioxidant capacity and activating the expression level of Cd transporter genes seem to be feasible solutions to promote plant resistance to Cd stress and to reduce accumulated Cd concentration. Here, we used transcriptomics to identify five cucumber transporter genes in response to cadmium stress, which were involved in Cd transport activity in yeast. Ionomics, gene expression, and REDOX reaction level association analyses have shown that the transcript of CsNRAMP4 was positively correlated with Cd accumulation and antioxidant capacity of cucumber roots. The expression level of CsHMA1 was negatively correlated with Cd-induced antioxidant capacity. The overexpression of CsHMA1 significantly relieved Cd stress-induced antioxidant activities. In addition, shoots with high CsHMA2 expression remarkably presented Cd bioaccumulation. Grafting experiments confirmed that CsHMA1 contributed to the high antioxidant capacity of cucumber, while CsHMA2 was responsible for the transport of Cd from the roots to the shoots. Our study elucidated a novel regulatory mechanism for Cd transport and oxidative damage removal in horticultural melons and provided a perspective to regulate Cd transport artificially by modulating Cd accumulation and resistance in plants. Greenhouse vegetable production (GVP), a main part of vegetable production in China, has grown rapidly. Chinese GVP areas reached 4.1 million ha in 2021 ,4. AnecdCucumber is the dominant staple vegetable plant in many countries of the world. China produces 187 million tons of cucumber annually, accounting for approximately 35% of global production . The qua2+ uptake activities. For instance, AtIRT1, a ZIP family transporter for Fe2+, Zn2+, and Mn2+, mediates Cd uptake in the roots of Arabidopsis thaliana [2+ into the xylem and transportation from the root to shoot are mediated by ATPases HMA2 and HMA4, respectively [Cd transduction is controlled by a group of metal transporters . Given tthaliana ,9. OsNrathaliana . Cd mustthaliana . P1B-typthaliana ,13. Furtthaliana . CsHMA1 thaliana . Loadingectively ,16. Simiectively . The Cd ectively . The aboectively , OsCAL1 ectively OsZIP1 [ectively , and OsCectively . Furtherectively ,23,24. A2O2 acts as a signaling molecule in response to heavy metals and other stresses [2O2 levels increase in response to Cu and Cd treatment in A. thaliana upon Hg exposure in tomato [ROS is a toxic and signal material caused by the stress of heavy metals. Heavy metals (like Cd) can produce ROS via the Fenton and Haber\u2013Weiss reactions and indirectly inhibit the activities of antioxidant enzymes . In partstresses ,28,29,30n tomato and in rn tomato . Modificn tomato ,23. Plann tomato ,33. Relen tomato ,27,28,34n tomato ,23, suggIn this study, we adopted RNA sequencing (RNA-Seq) to identify five Cd responsive metal transporters in Cd-exposed cucumber seedlings. Yeast metal transport experiments have shown that the genes can transport Cd. Ionomics, gene expression profile, and oxidative stress correlation analysis showed that CsNramp4 in cucumber was correlated with Cd uptake in roots and high ROS oxidation level in seedlings. Meanwhile, CsHMA3 was correlated with low ROS oxidation level in seedlings. In addition, CsHMA2 was involved in the transport of Cd from the roots to the shoots of cucumber and had affected the accumulation of Cd in the fruits. This work highlights the importance of metal transporters in plant responses to oxidative stresses from Cd. These transporters may be used to reduce Cd accumulation and the damage of oxidative stress in cucumber, thereby preventing the environmental risks of Cd to human health through the food chain.4 for 48 h to 20 days, and the nutrient solution was renewed every 2 days. Three independent biological replicates were performed for each treatment. The treated seedlings were prepared for primary root length, fresh weight, Cd content, oxidative stress level, and gene expression.Cucumber inbred line \u2018R1461\u2032 was provided by Prof. Zhang Xiaolan from China Agricultural University . Twenty-two cucumber cultivars were purchased from Kerun Cucumber Research Institute in Tianjin. Plump cucumber seeds were selected and placed into a sterilized Petri dish with filter paper to germination. After germinating, the seedlings were cultured in a Petri dish for 3 days. The seedlings with the same growth rates were selected, and the hypocotyls were fixed with a sponge and placed into a 1 L black beaker containing Yamazaki nutrient solution for hydroponics for 10 days. The seedlings were treated with 0, 1, 10, 50, and 150 \u03bcM CdSOThe treated seedlings and their control counterpart were used to isolate total RNA by TRIzol reagent . Approximately 1 \u03bcg of the RNA was treated with DNaseI. EasyScript One-Step gDNA removal was followed by the synthesis of cDNA by using SuperMix based on the manufacturer\u2019s protocol. qRT-PCR analysis was performed in 18 \u03bcL reaction solution containing cDNA (2 \u03bcL), 2 \u00d7 SYBR Premix Ex Taq (10 \u03bcL), and 200 nM primers. The reaction was carried out under denaturation at 95 \u00b0C for 30 s for one cycle, followed by 40 cycles of denaturation at 85 \u00b0C for 5 s and 50 \u00b0C for 34 s for extension. Actin1 and Ubiquitin10 were used as internal controls with specific primers.p values. In this study, FDR \u2264 0.05 was used as the threshold to determine the significance of gene expression variation.Ten-day-old cucumber seedlings were exposed to 0 and 50 \u03bcM Cd for 4 d and were sampled. Total RNA from Cd-treated (+Cd) and Cd-free cucumber seedlings was isolated using the TRIzol Reagent and pooled for RNA sequencing. The extracted RNA was treated with DNaseI at 25 \u00b0C for 30 min. mRNA was purified with oligo (dT)-rich magnetic beads and broken into short fragments. First- and second-strand cDNAs were synthesized. cDNAs were end-repaired and phosphorylated using T4 DNA polymerase and Klenow DNA polymerase. The Illumina paired-end solexa adaptors were ligated to cDNA fragments. The ligated products were purified on a 2% agarose gel. Six libraries were sequenced using Illumina hiseq2500. The original image data generated by the sequence providers were transferred into nucleotide sequences by base calling and defined as raw reads. All subsequent analyses were performed on high-quality clean read datasets according to bioinformatics analysis approach summarized in http://cucurbitgenomics.org/, accessed on 12 July 2021), rice , and Arabidopsis Genome Database. Sequence comparison and evolutionary tree analysis were carried out using MEGA6.0 software . The neighbor-joining phylogeny of these sequences was constructed with 1000 bootstrap replicates [The structures of the transporter sequence on the protein sequences were downloaded from the cucumber [Cucumber and MA, USA) .Arabidopsis were grown hydroponically for 10 d and transferred to the same culture solution containing 0, 10, or 50 \u03bcM Cd for 48 h. H2O2 content was quantified as described previously [Arabidopsis seedlings was extracted in 1 mL of 80% ethanol. In brief, 100 \u03bcL of the plant extracts were incubated for 30 min with 1 mL of solution containing 90% methanol (v/v), 25 mM H2SO4 (v/v), 250 \u03bcM ferrous ammonium sulfatehexahydrate, and 100 \u03bcM xylenol orange. The absorbance of the homogenate was recorded at 560 nm. Standard curves ranging from 0 to 200 \u03bcM were established and used to calculate H2O2 concentration. MDA content was measured using the method described by Heath and Packer (1968) with slight modification. The cucumber and Arabidopsis seedlings (0.1 g) were ground in 1 mL of 10% (w/v) trichloroacetic acid. After centrifugation at 12,000\u00d7 g for 10 min at 4 \u00b0C, the supernatant was collected, and 2 mL of the supernatant fraction was mixed with 2 mL of 0.6% TBA solution. The mixtures were heated at 95 \u00b0C for 30 min and then cooled quickly in an ice bath. The resulting mixtures were centrifuged at 10,000\u00d7 g for 10 min, and the absorbance of the supernatants was recorded at 450, 532, and 600 nm.The seedlings of 22 cucumber cultivars and transgenic eviously . One graTo explore the association between Cd accumulation in the natural populations of cucumber and the transporters mentioned above, we selected 23 cucumber cultivars collected throughout China and cultured them in soils with simulated Cd pollution conditions (0.5 \u03bcM) in facilities. Cd content was measured in both shoots and roots.Jinyou12, Jinyou49, Jinmei3, and R1461). Ten-day-old cucumber plants were selected for grafting. After 7 days of growth in Yamazaki nutrient solution, the live seedlings were supplemented with 0 and 0.5 \u03bcM Cd for 48 h (determination of Cd content) and 4 days (gene expression detection).To investigate the physiological role of CsNramp4 and CsHMA2 in plants, we designed a series of grafting experiments to verify their function. We selected four widely cultivated cucumber varieties and 4 days (gene expression detection).To study the direct correlation between oxidative stress level and Agrobacterium tumefaciens strain GV3101 and then into Arabidopsis by floral dip method [\u22121 basta. Ten independent UBI10:CsNramp4 and 15 UBI10:Cs CsHMA1 transgenic lines (single copy and homozygous line) were obtained. Five lines were randomly selected for transcription analysis by qRT-PCR, and two of them were used for functional characterization.pSY06 was used as an expression vector with ubiquitin 10 promoter . CsNrampp method . PositivArabidopsis leaf mesophyll protoplasts [The coding sequences of CsNramp1 and CsNramp4 were amplified by RT-PCR and inserted into pCAMBIA1300-GFP vectors driven by the 35S promoter. The CsNramp1 and CsNramp4-GFP fusion vector was transformed into toplasts . Fluoresycf1 (Cd-sensitive strain [ycf1 complementation by drop-spotting assays was performed on synthetic defined (SD)-Ura medium containing 2% galactose, 0.67% yeast nitrogen base (Sigma), 2% agar [The cDNA fragments containing an entire open reading frame of CsNramp1, CsNramp4, CsZIP1, and CsZIP8 were amplified. The fragments were cloned into a pYES2 vector. The resulting plasmids were transformed into the mutant yeast strain e strain ). ycf1 c 2% agar , and supn = 3), and each replicate contained at least 10 plants. Data between different treatments were statistically analyzed by ANOVA, followed by means separation by least significant difference (LSD) test if the ANOVA result is significant at p < 0.05. Data were analyzed using SPSS 22 .Results were presented by means of three independent replicates were employed to test the growth responses of lication A\u2013D. The lication B. Cd ovein roots ,42. SimiCsNramp1 and CsNramp4), two ZIP family genes (CsZIP1 and CsZIP8), and an HMA family gene (CsHMA5) were significantly upregulated at the transcriptional level and Cd-free cucumber seedlings. We generated 252.47 Gb clean reads with average base quality (Q30) of 94.17% and sampal level G. CsHMA2eedlings G. These Arabidopsis, and rice, we respectively collected a data set of 17 putative Nramp protein sequences, 39 putative ZIP protein sequences, and 25 putative HMA protein sequences including 27 from Arabidopsis, 31 from rice, and 23 from cucumber for phylogenetic analysis , we predicted that CsNramp1 and CsNramp4 are membrane proteins with 12 transmembrane domains , respectively. The inhibition of growth was more serious in yeast expressing CsNramp1, CsNramp4, CsZIP1, and CsZIP8 than that expressing pYES2 empty vector C\u2013E. The CsNramp1, CsNramp4, CsZIP1, CsZIP8, and CsHMA2) and HMA1 [CsNramp1, CsZIP1, CsZIP8, and CsHMA1 were not significantly correlated with Cd content in the roots or shoots, indicating that the four genes were not the major regulatory genes involved in Cd distribution in the roots or shoots of these varieties.To explore the association in cucumber between Cd accumulation and the transporters mentioned above, we selected 23 cucumber cultivars collected throughout China and cultured them in soils with simulated Cd pollution conditions (0.5 \u03bcM) in facilities. We measured the Cd content in shoots and roots. The Cd accumulation in the roots of all varieties was significantly higher than that in the shoots A,B. At tand HMA1 in the r2O2 were significantly enhanced in 23 varieties accumulated more Cd in shoots, while roots with high level of CsNramp4 (Jinyou12 and R1461) had positive correlation with Cd content in the roots. In rice, the CsNramp4 homologous gene OsNramp5 is localized to the plasma membrane of root cells and functions as a high-affinity transporter for Cd and Mn uptake [Arabidopsis displayed a similar Cd efflux function, which are involved in the loading of Cd into the xylem from root-to-shoot transport of Cd [Up to now, transgenetic cucumber and mutants were obtained difficultly. To investigate the physiological role of CsNramp4 and CsHMA2 in plants, we designed a series of grafting experiments to verify their function A. We seln uptake ; it actin uptake . These rn uptake . The mosrt of Cd ,46. MeanCsHMA1 and CsNramp4 in cucumber. Concretely, varieties Jinyou 1 and Jinyuan 11 were low in expression of CsHMA1 in the roots, while Jindong F6 and Jinyou 315 were high in expression of CsHMA1 as rootstock and scion. In addition, the expression of CsNramp4 in the roots of Jinyou 315 and Jinyuan 11 was lower, while the expression of CsNramp4 in the roots of Jindong F6 and Jinyou 1 was higher. Then, these four cucumber varieties were applied to graft experiments. When the varieties Jindong F6 and Jinyou 315 with high CsHMA1 expression were used as rootstocks, the ROS content in the grafted seedlings was at a relatively low level (Jinyou 1 and Jinyuan 11 with lower CsHMA1 expression were used as rootstocks, the plants of each combination accumulated more MDA and H2O2 (CsHMA1 expression level in cucumber roots directly determined Cd-induced ROS content. Although previous results showed that the expression of CsNramp4 was positively correlated with the accumulation of Cd-induced ROS in the plant, when CsHMA1 was at high expression level, the increased level of CsNramp4 could not increase the accumulation of ROS, indicating that CsHMA1 had a stronger regulation ability of Cd-induced ROS than CsNramp4. This reason may be that Cd absorbed by CsNramp4 in the root is rapidly chelated into vacuoles by CsHMA1, thereby reducing oxidative damages (We further used four cucumber variety to study whether a direct correlation exists between oxidative stress level and expression of ow level . Howeverand H2O2 . These r damages . In concA. thaliana overexpressing CsHMA1 and CsNramp4, respectively. The homozygous OE lines showed 10- to 35-fold higher CsHMA1 and CsNramp4 transcript levels than the wild type. Seedlings grown on 1/2MS medium for 7 days were treated with 0, 10, and 80 \u03bcM Cd for 10 d. The growth in OE lines of CsHMA1 exhibited more tolerance to Cd stress than the wild type. The primary root length of the OE lines increased by 12.6\u201319.2% (CsNramp4 was more sensitive to Cd toxicity, with the primary root length decreased by 19.9\u201334.6% compared with the wild type (CsHMA1 and CsNramp4 had higher Cd concentration than the wild type under Cd stress (2O2 detection in Cd-exposed seedlings. Compared with WT, UBI10:CsHMA1 plants exposure to Cd showed a lower level of MDA and H2O2, while in UBI10:CsNramp4 roots, the levels of MDA and H2O2 were relatively higher in Cd stress (To investigate the effects of CsHMA1 and CsNramp4 on plant REDOX response under Cd stress, we constructed transgenic .6\u201319.2% C. By conild type B. Followd stress B. To exad stress D,E. Thes2O2 and MDA production during short-term exposure of cucumber cell cultures to Cd2+ has been reported previously [2O2 and MDA were gradually elevated with increasing Cd content in cucumber. The control of Cd accumulation is maintained by a group of metal transporters [Cadmium pollution has attracted worldwide attention . Cd toxieviously . Many steviously . The releviously . Howeversporters . Accumulsporters ,20. ThisNRAMPs, ZIPs, and HMAs family trans porters in response to cadmium exposure. Our results of gene expression profiles in cucumber tissues showed that CsNramp4 was highly expressed mainly in cucumber roots, was located in the plasma membrane of cucumber cells, and had similar expression pattern to the main Cd absorption transporter OsNramp5 in rice [A. thaliana, which is also involved in the absorption of Fe2+, Zn2+, and Mn2+. OsIRT1 is also involved in the absorption of Cd in rice. We also identified CsZIP1 and CsZIP8 as homologous genes of OsIRT1, complementing CsNramp1, CsNramp4, CsZIP1, and CsZIP8 through yeast experiments. It is worth exploring which one above is mainly responsible for the accumulation of Cd ions in cucumber cultivarsWe used transcriptome analysis to screen seven in rice ,44. ThisCsNramp4, while Cd accumulation in cucumber aboveground parts was positively correlated with the expression of CsHMA2, suggesting the role of CsNramp4 and CsHMA2 in the absorption and transduction of Cd into cucumber shoots. Grafting experiments further showed that the expression of CsNramp4 contributed to the accumulation of Cd in cucumber roots, while CsHMA2 was involved in the transport of Cd from the root to shoot. Our work represents the transporter pathway for Cd from soil to the fruit of cucumber.In addition, we found that the pattern about CsHMA2 gene in response to Cd stress is the most closely related to HMA2 and HMA4 in plant root transport of Cd to shoot ,16, suggCsHMA1 was negatively correlated with oxidative stress indicators in the roots, suggesting that CsNramp4 and CsHMA1 may maintain the REDOX balance at cytoplasmic and vacuolar levels in cucumber. We further selected cucumber seedlings with different expression levels of CsHMA1 and CsNramp4 as scions and grafting stock to study whether a direct correlation exists between oxidative stress level and expression of CsHMA1 and CsNramp4 in cucumber. The results indicated that the high expression of CsNramp4 aggravated Cd-induced oxidative damage in cucumber roots. The CsHMA1 expression level in cucumber roots directly determined the Cd-induced ROS content. Overexpressed CsNramp4 and CsHMA1 in Arabidopsis showed the same ROS change level with cucumber when the seedlings were exposed to Cd. However, when CsHMA1 was at high expression level, the increased level of CsNramp4 could not increase the accumulation of ROS, indicating that CsHMA1 had a stronger regulation of Cd-induced ROS than CsNramp4. This finding may be because Cd absorbed by CsNramp4 in the root is rapidly chelated into vacuoles by CsHMA1, thereby reducing oxidative damage. In Arabidopsis, seedlings employ a two-step mechanism to detoxify toxic ions. First, phytochelatins, such as GSH and PCs, bind to the toxic ion. Then, the metal\u2013phytochelatin complex is sequestered by two ABCC-type transporters, AtABCC1 and AtABCC2, in the vacuole [A large number of Cd accumulation and detoxification transporters have been identified in plants, but the direct relationship between these transporters and Cd-induced oxidative stress is rarely reported ,54,55,56 vacuole . EffectiOur study is a good example of a molecular approach for mining of Cd accumulation transporters in greenhouse vegetables. Root uptake and chelating, xylem loading, and phloem transportation are important transport processes that determine Cd accumulation in the edible parts of crops ,44. In tCsHMA2 in stocks was found to be associated with low Cd accumulation in the fruits, which contributed to the cucumber accumulation of less Cd in plants. Our work not only helps to understand the transport and regulatory mechanisms underlying Cd detoxification and accumulation in greenhouse vegetables, but it also addresses the environmental issues of Cd contamination in the greenhouse.This study demonstrated that five Cd-responsive transporters transcriptionally responded to Cd stress. Functional identification revealed that CsNramp4 and CsHMA2 led to high accumulation of Cd in the cucumber roots and shoots, respectively. Importantly, high CsHMA1 expression resulted in cucumber tolerance to Cd stress by strengthening antioxidant capacity. Grafting evidence showed that the reduced expression of"} +{"text": "Among various models for dose\u2013response relationships, a probit model was adopted as the best fitting model. The obtained BMDLs of cumulative Cd-A were 272.3 yrs \u00b5g/m3 (BMR5%) and 707.5 yrs \u00b5g/m3 (BMR10%). The BMDLs of mean cumulative Cd-B were 3967.2 months nmol/L (BMR5%) and 7798.1 months nmol/L (BMR10%). The BMDLs of least-squares cumulative Cd-B were 3588.6 months nmol/L (BMR5%) and 8616.3 months nmol/L (BMR10%). Assuming a working period of 40 years, the BMDLs for BMR10% corresponded to 17.7 \u00b5g/m3 (Cd-A) and 1.8~2.0 \u00b5g/L (Cd-B). Discussion: This study provides new valuable information to enhance the reliability of limit values and thereby make a significant contribution to preventing the health effects of Cd in exposed workers.Objectives: The aim of this study was to determine the no observed adverse effect level (NOAEL), the lowest observed adverse effect level (LOAEL) and the benchmark dose low (BMDL) of cadmium exposure by re-evaluation of the dose\u2013response relationship between cumulative cadmium exposure and renal tubular damage reported previously. Methods: The participants were workers (326 men and 114 women) employed for at least three months between 1931 and 1982. Blood cadmium (Cd-B) and air cadmium (Cd-A) were collected at regular intervals with urinary \u03b22-microglobulin as the tubular effect marker. Cumulative Cd-A and Cd-B were estimated by multiplying concentration and working period. The BMDL was calculated using Benchmark Dose Software (version 3.1.2). The benchmark response (BMR) was set at 5% or 10%. Results: By logistic regression, the NOAEL of mean cumulative Cd-B was 7122 months nmol/L. The LOAEL of cumulative Cd-A and least-squares cumulative Cd-B was 691 yrs \u03bcg/m Chronic respiratory exposure to cadmium (Cd) causing emphysema, renal and bone damage, and Cd-induced adverse renal effects starts with proximal tubular damage followed by glomerular damage. Bone effects are characterized by osteomalacia and osteoporosis ,2. Itai-3), calculated as air Cd concentration \u00d7 years of work, and Cd concentrations in liver, kidney, blood, and urine [Exposure to cadmium through the inhalation of fumes or dust in occupational settings has long been known as the main cause of adverse health effects on lungs and kidneys . Therefond urine . Furthernd urine .In terms of blood Cd level (Cd-B), it is well-known that there are fast and slow components for the reduction of blood cadmium, reflecting recent exposure and long-term exposure, respectively . After l3, 2 \u00b5g/m3 (respirable particulate fraction)) [3 ) and the Biological Exposure Values ) of the Scientific Committee on Occupational Exposure Limits (SCOEL) of the European Union [Based on this information, the biological tolerable limits for occupational Cd exposure have been proposed as the Threshold Limit Value ) and Biolaction)) of the A10) in laboratory animals; however, in the case of human epidemiological data, it may be necessary to adopt a lower BMR of 5% (BMDL05) or 1% [The concept of the benchmark dose (BMD), published by Crump et al., has been widely applied to the risk assessment of environmental chemicals ,12. This5) or 1% . HoweverThis prompted us to determine the NOAEL, LOAEL and BMDL by re-evaluation of the fine dose\u2013response relationship between cumulative Cd exposure and renal tubular damage in previously reported reliable data .i = years of employment at a particular level of Cd-A, Ci = average Cd-A during the corresponding period. Cd-B values have been collected since 1967 at regular intervals together with other laboratory data, such as urinary \u03b22-MG, which was chosen as the tubular effect marker in this study. The mean number of blood tests per individual was 7 . Three workers were excluded because of missing Cd-B data, and thus 437 workers were included for the cumulative blood-cadmium analyses. Initially, average Cd-B was computed from the available Cd-B values for each worker. This average value was then multiplied by the number of months employed at the factory according to Equation (2):i = number of months employed, Bi = average Cd-B.As noted in the previous report , the pari = number of months employed, D0 = k Y0 + I, D1 = k Y1 + I, k = slope, I = intercept for the fitted line, Y0 = year of first employment and Y1 = last year of employment or end of study, whichever comes first [Considering the very high exposure in the 1940s or 1950s, this mean Cd-B may underestimate the whole cumulative exposure [As the renal tubular marker, creatinine-adjusted urinary \u03b22-MG was measured using a radioimmunoassay method from 1972 to 1982. \u03b22-MG uria was defined as \u03b22-MG > 35 \u00b5g/mmol cre 309.4 \u00b5g/g cre). This urinary \u03b22-MG level was based on the upper 2.5 percentile for the urinary \u03b22-MG among persons without tubular dysfunction \u00b5g/g cre or the iOdds ratios of the categorized cum Cd-A, mean cum Cd-B and leasq cum Cd-B level for the \u03b22-MG uria were evaluated using logistic regression by IBM SPSS 19J . Lowest categories were adopted as the reference one.Furthermore, we calculated the BMD and BMDL of the cum Cd-A, mean cum Cd-B and leasq cum Cd-B for \u03b22-MG uria using 5% and 10% of the benchmark response (BMR). Values of cumulative Cd exposures were log-transformed according to the previous study . To findThis study is based on previously published data and does3, which corresponds to 17.3 or 23.0 \u03bcg/m3, assuming the whole working period to be 40 or 30 yrs. In terms of Cd-B, NOAEL corresponded to 14.8 or 19.8 nmol/L (1.7 or 2.2 \u03bcg/L), and LOAEL was 17.9 or 23.9 nmol/L (2.0 or 2.7 \u03bcg/L).The responses with cum Cd-A, mean cum Cd-B and leasq cum Cd-B as the dose parameters are shown in With BMDS software, the probit model was the best-fit model for all data for cum Cd-A, mean cum Cd-B and leasq cum Cd-B, based on Akaike\u2019s information criteria. 3 (BMR5%) and 707.5 yrs \u00b5g/m3 (BMR10%). Assuming the whole working period to be 40 or 30 yrs, Cd-A would correspond to 6.8 or 9.1 \u00b5g/m3 (BMR5%) and 17.7 or 23.6 \u00b5g/m3 (BMR10%). The BMDLs of mean cum Cd-B were 3967.2 months nmol/L (BMR5%) and 7798.1 months nmol/L (BMR10%). Assuming the whole working period to be 40 or 30 yrs, Cd-B was estimated to be 8.3 or 11.0 nmol/L (BMR5%) and 16.2 or 21.7 nmol/L (BMR10%), corresponding to 0.9 or 1.2 \u00b5g/L (BMR5%) and 1.8 or 2.4 (BMR10%). The BMDLs of leasq mean Cd-B were 3588.6 months nmol/L (BMR5%) and 8616.3 months nmol/L (BMR10%). Assuming the whole working period as above, Cd-B was estimated to be 7.5 or 10.0 nmol/L (BMR5%) and 18.0 or 23.9 nmol/L (BMR10%), corresponding to 0.8 or 1.1 \u00b5g/L (BMR5%) and 2.0 or 2.7 \u00b5g/L (BMR10%).05/BMDL05 of urinary Cd for abnormal urinary RBP and \u00df2-MG were estimated to be 5.1/3.0 and 9.6/5.9 \u00b5g/g cre in all workers, 12.6/6.6 and 12.2/5.5 \u00b5g/g cre in non-smokers, and 6.3/4.9 and 4.3/3.5 \u00b5g/g cre in smokers, respectively. In the present study, we estimated the NOAEL and LOAEL of Cd-A and Cd-B, which had not been determined in the previous publication [3 for Cd-A and 2.0 \u00b5g/L for Cd-B.In the present study, we re-examined the previous data and estimated the BMD/BMDL of Cd for renal tubular damage. The concept of BMD/BMDL has been widely adopted for health risk assessment of various compounds. However, to our knowledge, the BMD/BMDL of Cd-A or Cd-B has not been estimated in terms of occupational exposure. Furthermore, it is also worth mentioning that we could compare several models for dose\u2013response relationships between Cd exposure and renal effects by using BMDS software. The probit model adopted in the previous study was the As noted above, blood cadmium is a good indicator of internal dose after chronic exposure, with its biological half-life of 7\u201316 years [In recent years, the background Cd exposure is considered to have decreased. Therefore, we need to pay attention to the obtained data in view of the possibility that the renal outcome represents occupational and background exposure simultaneously about the earlier data. However, as there are limited data on Cd-A, we believe that the present data are still valuable for the occupational setting. On the other hand, Cd-B reflects the whole body burden of Cd. In view of the low background exposure in recent years, higher Cd-B depends on the amount of occupational exposure, emphasizing the importance of countermeasures against it. In terms of outcome, we adopted tubular damage as reflecting the early change of Cd exposure. For a further long-term health outcome, increased mortality was associated with cadmium exposure in longitudinal studies in the general population ,18. Ther3, there was evidence of renal abnormalities . The percentage of workers with renal abnormalities was found to increase as the cumulative Cd exposure index increased. Mason et al. [3. The 95% lower limit of inflection points was 509 yrs \u00b5g/m3 for urinary \u03b22-MG and 636 yrs \u00b5g/m3 for RBP. Simple dose\u2013response analysis of the exposed group showed a greatly increased incidence of tubular proteinuria when the cumulative Cd exposure was greater than 1000 yrs \u00b5g/m3. Thus, these results indicated that the threshold level of cumulative Cd-A was 510\u20131100 yrs \u00b5g/m3, corresponding to a 40-year exposure of 12.8\u201327.5 \u00b5g/m3. Thus, we believe that the results of the present study and those of previous studies complement each other and add important findings on the health effects of Cd-A for occupational exposure.Several studies have previously investigated the dose\u2013response relationship between cumulative Cd-A and renal damage in the working environment. Ellis et al. evaluaten et al. investigWith respect to Cd-B, several studies have shown a higher incidence of renal dysfunction in exposed workers. Chia et al. studied 3 , 2 \u00b5g/m3 (respirable particulate fraction) (Cd-A), 5 \u00b5g/L (Cd-B) and 5 \u00b5g/g cre (urinary Cd). In particular, for Cd-B, ACGIH decided to retain the BEI of Cd-B because there was no new information after the previous study [3 and the BEV of urinary Cd at 2 \u03bcg/g cre, without setting the BEV of Cd-B due to the limited information [05, and BMDL10 for Cd-A in this study were generally in agreement with the guideline of ACGIH, but were higher than the EU guideline level. On the other hand, the results for Cd-B were lower than the guideline of ACGIH, suggesting the need for reconsideration of the guidelines in the future.In terms of the allowable limit for occupational exposure, ACGIH reviewed the TLV of Cd-A and BEIsus study which waormation . AssuminOne limitation of the present study was the lack of information on other factors, which thus could not be corrected for in this study, especially smoking. The analysis of publicly available biomonitoring data showed that B-Cd and urinary Cd are higher in cigarette smokers . TherefoThe present study revealed essential information for health risk assessment, such as NOAEL and BMDL for Cd exposure. In addition, it was suggested that the reference concentration of Cd-B, for which less information was available, was lower than the previously assumed one. In the ongoing discussion on the exposure limits for Cd, this study provides new valuable information to enhance the reliability of limit values, thereby making a significant contribution to preventing adverse health effects of Cd in exposed workers."} +{"text": "However, it has very low solubility in water. The formulation of this compound requires high amounts of solubilizers, which limits its clinical application. In addition, its low solubility in water is a barrier to the distribution of this drug, thus affecting its potency. Cyclodextrin (CD) is widely used as a solubility enhancer of poorly soluble drugs. This study aimed to increase the solubility of \u03b1-M in water through complex formation with CD. The complex of \u03b1-Mangostin and \u03b3-Cyclodextrin (\u03b1-M/\u03b3-CD CX) was prepared by the solubilization method, resulting in a solubility improvement of \u03b1-M in water. Characterization of \u03b1-M/\u03b3-CD CX by using FTIR-Spectrometry, XRD, H-, C-, and HMBC-NMR showed that \u03b1-M was able to form an inclusion complex with \u03b3-CD. The complex yielded an entrapment efficiency of 84.25 and the thermodynamic study showed that the \u03b1-M/\u03b3-CD CX was formed spontaneously, based on the negative values of Gibbs energy and \u0394H. Interestingly, the solubility of \u03b1-M/\u03b3-CD CX significantly increased by 31.74-fold compared with \u03b1-M. These results suggest that \u03b1-M/\u03b3-CD CX has the potential in the formulation of water-based preparation for clinical applications. Garcinia mangostana pericarp extract -9H-xanthen-9-one] (\u03b1-M) is a major compound in extract [1]. \u03b1-MCyclodextrin (CD) is an oligosaccharide molecule that has a unique cavity structure: it has a hydrophobic property in the inner cavity but is hydrophilic in the outer cavity. The conical truncated structure of CDs proposed to include the hydrophobic molecule in the cavity, resulting in their improved water solubility. CD molecules are formed by glucopyranose units via \u03b1-1,4-glycosidic linkages. The type of CDs correlated with the number of glucopyranose units. \u03b1-, \u03b2-, and \u03b3-CD contain six, seven, and eight units, respectively . Some neIn this study, the inclusion complexes of \u03b1-M and \u03b3-CD were prepared by solubilization method to improve the solubility of \u03b1-M in water. The phase solubility study was implemented to display the solubility enhancement of \u03b1-M in several concentrations of \u03b3-CD and a Job plot predict the molar ratio of the complex formation. Several characterizations such as FTIR-Spectrometry, XRD, SEM, H-, C-, and HMBC-NMR were performed to study the configuration of the inclusion complex. The termodynamic studies were implemented to study the complex formation process. \u03b1-M could be more widely used both in formulation and several pharmacological applications due to the increased solubility of \u03b1-M in the inclusion complex.\u03b1-Mangostin was purchased from Chengdu Pharmaceutical Industries, China. \u03b3-CD was a kind gift from Kumamoto University, Kumamoto, Japan. Ethanol 95% and aqua deionized to a high purity were purchased from Sigma Aldrich, St. Louis, MO, USA. All the reagents were of analytical grade and were used without any further purification.\u03b1-M and \u03b3-cyclodextrin were weighed in a 1:2 molar ratio and mixed for 1 min using a vortex mixer ,21.The stoichiometry of the \u03b1-M/\u03b3-CD complex (\u03b1-M/\u03b3-CD CX) formation was determined using the continuous variation method (Job plot). Each concentration (0\u20131 mM) of the \u03b1-M and \u03b3-CD solution was mixed in a constant molar fraction. All the mixtures were stored at room temperature for 24 h. After equilibration, the solution was filtered using a 0.45 \u03bcm filter membrane ,22. The This study was performed according to The Higuchi and Connors Method . An exceThe solubility improvement of \u03b1-M/\u03b3-CD CX in water was investigated by dispersing 0.1\u20130.3 g of \u03b1-M, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX in 10 mL of distilled water. The mixtures were shaken for 24 h at 25 \u00b0C. The mixtures were then filtered with 0.45 \u03bcm filter membrane. The concentration of \u03b1-M was measured using an Analytic Jena Specord 200 at 316 nm .The complex was prepared by the solubility method. A total amount of 1 mM of \u03b1-M solution in ethanol 95% and 2 mM of \u03b3-CD solution in water was made in a separated volumetric flask. \u03b1-M solution was gradually added into \u03b3-CD solution and mixed for about 24 h at room temperature. Then, the solution was evaporated at 60 \u00b0C to yield the complex powder. The entrapment efficiency of the complex was measured by an Analytic Jena Specord 200 at 316 nm ,9,22,26.\u22121. About 2 mg of sample was blended with 200 mg KBr powder. The mixture was pressed into a disc and analyzed at 400\u20134000 cm\u22121 [The samples of the complex were analyzed using IR Prestige-21 Spectrometry at 400\u20134000 cm000 cm\u22121 ,26.The XRD pattern of \u03b1-M, \u03b3-CD, the physical mixture (\u03b1-M/\u03b3-CD PM), and the inclusion complex (\u03b1-M/\u03b3-CD CX) were analyzed using a XRD Bruker D8 Advance 3 kW with LynxEye XE-T detector and radiation source CuK alfa. Each sample was scanned at 2\u03b8 from 5\u201350 ,19,26.The morphology surface of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX were assessed by scanning electron microscopy (SEM) JEOL JSM 6510 LA. The sample was placed in a sample holder, sprayed to remove impurities, and coated with gold. The analyses were carried out at an acceleration voltage of 15 kV, and each sample was observed at 250 and 3000 magnification ,26.The structure of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM and \u03b1-M/\u03b3-CD CX were analyzed using NMR spectrometry 500 Hz JEOL, JNM ECA 500 ,27.sK) was calculated by using the slope value from the solubility phase diagram, utilizing the following equation:An excess of \u03b1-M (\u00b125 mg) was added into 5 mL \u03b3-CD solution . Each mixture was shaken continuously in an incubator shaker at 24 \u00b0C for 96 h until equilibrium was reached ,21,24. TS0 is the solubility of \u03b1-M without the presence of cyclodextrin, and slope is the gradient of the solubility phase diagram [ diagram ,24.H), and Gibbs energy (\u2206G) of \u03b1-M/\u03b3-CD CX formation were determined using the stability constant (Ks) vs. temperature, following the Van\u2019t Hoff equation:The change of enthalpy , R is the gas constant (8.314 J/mL/K), and \u2206H is calculated using the slope value from the sK vs. 1/T graph [/T graph ,24.G was calculated by the following equation:Furthermore, the value of \u2206p-Value \u2264 0.05 is considered as statistically significant between-group population.Statistical comparison was performed by using Scheffe\u2019s test. A phase solubility study was undertaken to analyze the molar ratio of \u03b1-M and \u03b3-CD in the complex \u03b1-M/\u03b3-CD CX was prepared by the solubilization method: \u03b1-M was dissolved in ethanol, while \u03b3-CD was dissolved in water. Both of the solutions were mixed for 24 h and evaporated at around 60 \u00b0C. The entrapment efficiency of this complex was 84.25 \u00b1 6.80%.L type of solubility diagram, indicated that \u03b1-M solubility was reached linearly with \u03b3-CD [The phase solubility study can be seen in ith \u03b3-CD ,28. At tith \u03b3-CD .The number of \u03b1-M to form the complex results in the improvement of its solubility in water. The high percentage of artemisinin complexed with \u03b3-CD was higher than \u03b1- and \u03b2-CD . MoleculThe stoichiometry of complex formation was established using a Job plot. A gradient concentration of \u03b1-M was mixed with a gradient concentration of \u03b3-CD in a constant mole fraction, and the mixture was allowed to stand overnight. The mixture was filtered by 0.45 \u03bcm membrane, and the \u03b1-M in the filtrate was measured at 316 nm. In max value at 0.4, corresponding with the stoichiometry ratio of the complex formation at 1:1. This ratio implies the inclusion of a single guest molecule entrapped with one molecule of the host [The toroidal structure of \u03b3-CD can entrap \u03b1-M in its cavity . The stothe host ,33.\u22121. The FTIR spectra of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX are shown in \u22121 corresponds to the hydrogen bond of the hydroxyl groups, and the weak absorption near 2900 cm\u22121 is related with sp3 hybridization of aliphatic carbon. The bend absorption at 1300\u20131000 cm\u22121 is the absorption of C-O groups, and the slight absorption at wavenumber 1600\u20131700 cm\u22121 is the vibration of C=C groups.The functional group analysis of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX was carried out using FTIR spectrophotometry at wavenumber 4500\u2013500 cm\u22121. For interpretation, in the first graph (\u03b1-M), the stretching vibration at 3410 and 3252 cm\u22121 corresponds with the \u2013OH in \u03b1-M. The band at 2970 and 2931 cm\u22121 is given by the asymmetric stretching vibration of sp3 hybridization of the aliphatic carbons of the \u03b1-M methyl (CH3) and methylene (CH2) groups. The absorption bands at 1580 cm\u22121, 1645 cm\u22121, and 1630 cm\u22121 are the stretching vibrations of unconjugated C=C in \u03b1-M. The band at 1470 cm\u22121 is given by the unconjugated carbonyl group (C=O) on the B ring of \u03b1-M; 1362 cm\u22121 is given by CH3 bending; 1083 cm\u22121 and 1051 cm\u22121 correspond with the stretching vibration of C-O; and 1011 cm\u22121, 862 cm\u22121, 804 cm\u22121, 772 cm\u22121, and 598 cm\u22121 are given by C-OH stretching, \u2013CH=CH- (trans), \u2013CH=CH- (cis), CH2, and \u2013CH=CH- bending, respectively [The FTIR spectra of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX are shown in \u22121 with a maximum absorption at 3395 cm\u22121. This band is related to the stretching vibration of the \u2013OH groups in the glucose ring. The band at 2930 cm\u22121 comes from the stretching vibrations of the C-H bonds in the CH2. The high-intensity bands at 1030 cm\u22121 and 999 cm\u22121 are related to the C-H out-of-plane bending vibrations and the C-O stretching vibration modes, respectively [The second graph (\u03b3-CD) shows a broad absorption around 3000\u20133600 cmectively .\u22121 corresponds to the hydrogen bond of the hydroxyl groups. The weak absorption 2930 cm\u22121 corresponds with the methylene (CH2) groups, 1640 cm\u22121 is given by CH3 bending, 998 cm\u22121 is the bend absorption of C-OH stretching vibration, and 598 cm\u22121 indicates \u2013CH=CH- bending [The same absorption-type can be seen for the \u03b1-M/\u03b3-CD PM (the third graph). The strong band at 3000\u20133500 cm bending ,35.\u22121 with the maximum absorption at 3346 cm\u22121 is given by the stretching vibration of the \u2013OH groups in the glucose ring of \u03b3-CD. The maximum band at 2930 cm\u22121 comes from the stretching vibrations of the C-H bonds in the CH2. A high-intensity doublet, with maximum absorption at 1030 cm\u22121 and 1000 cm\u22121, is assigned to the C-H out-of-plane bending vibrations and the C-OH stretching vibration modes, respectively. The band at 598 cm\u22121 is given by \u2013CH=CH- bending [The last graph is the spectra for \u03b1-M/\u03b3-CD CX. The broad band around 3000\u20133600 cm bending .The crystallinity of intact \u03b1-M was 91.1% with the specific peak at 5.890. The crystallinity of intact \u03b3-CD was 62.6% with the specific peak at 5.19. These peaks still remain in \u03b1-M/\u03b3-CD PM at 5.14 and 5.95. In contrast, both of the peaks disappeared, and the crystallinity of \u03b1-M was decreased into 28.2% in \u03b1-M/\u03b3-CD CX; the peak was seen around 16\u201318 with an intensity of about 1000. This indicates that \u03b1-M formed a complex with \u03b3-CD .The crystallinity of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX was examined using PXRD . Intact SEM was performed to analyze the morphology of each sample . The surThe surface of \u03b1-M was rough, and the particle size was around 150 \u03bcm . The surThe NMR spectra of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX were obtained using NMR Spectrometry 500 Hz JEOL, JNM ECA 500. The analysis was performed for proton (H-NMR), C-NMR, and HMBC.1H NMR spectra of \u03b1-M, \u03b3-CD, \u03b1-M/\u03b3-CD PM, and \u03b1-M/\u03b3-CD CX are shown in The signal of a,b,c,d and e, given by the proton in glucopyranose monomer of \u03b3-CD at a,b,c,d and e position. The integration of the overlapped peaks was used to calculate the molar ratio of \u03b1-M and \u03b3-CD in the complex. The result emphasized the Job plot, which showed the molar ratio of this complex is 1:1. The chemical shift of \u03b1-M/\u03b3-CD CX was influenced by the guest molecule (\u03b1-M) entering into the hydrophobic cavity. This phenomenon indicates that the complex of \u03b1-M/\u03b3-CD CX was formed ,17,18.The study continued by using C-NMR and HMBC to investigate the molecular interaction of \u03b1-M and \u03b3-CD. The HMBC spectrum of \u03b1-M/\u03b3-CD CX is given in A hydrogen bond was found at the hydroxyl group of \u03b1-M, which interacted with the hydrogen ion of the \u03b3-CD molecule. The signal of a, b, c, d and e, given by the proton in glucopyranose monomer of \u03b3-CD at a, b, c, d and e position. The signal 1, 3, 4a, 6 and 7 given by the carbon of \u03b1-M molecule at the same postion. The interaction of \u03b1-M and \u03b3-CD shows the interaction between the proton (H) from the hydroxyl groups with the C of the methoxy groups. The hydrogen bond was found at the hydroxy group at the A and C ring of \u03b1-M, which interacted with the hydrogen ion at a, b, c, and d of the \u03b3-CD molecule. The interaction occurred at C 4a of \u03b1-M with a hydrogen atom at the d position of \u03b3-CD. Two other signals appear at C7 of \u03b1-M, with hydrogen at the a and d position of \u03b3-CD. This interaction describes the successful complex formation of \u03b1-M and \u03b3-CD ,25.sK) in each temperature was calculated by using Equation (1) by using the slope of the graph in each temperature and \u2206H is the slope of the diagram in Thermodynamic studies of \u03b1-M/\u03b3-CD CX formation were undertaken to study the complex formation process. This procedure was carried out at temperatures of 25 \u00b0C, 31 \u00b0C, and 37 \u00b0C. The stability constant yielded an energy of complexation of \u22125.68 kcal/mol, while the parameterization method 7 (PM7) gave an energy of complexation of 5.29 kcal/mmol [The thermodynamic study of \u03b1-M/\u03b3-CD CX formation gave an energy of complexation of about \u22125.019 \u00b1 0.224 kcal/mol and \u0394H of \u22125.520 kcal/mol. The in silico study using PM6 yielded an energy of complexation of \u22125.68 kcal/mol, while the PM7 method gave an energy of complexation of 5.29 kcal/mmol [The in silico study showed that \u03b1-M molecules were included in the inclusion complex with \u03b3-CD. In the latest study, PM6 molecular docking indicated that the O4 of \u03b3-CD attached with the hydroxyl groups at C3 and C6 in \u03b1-M, while PM7 resulted in the interaction of hydroxyl groups in \u03b3-CD with the methoxy groups at the C ring and the carbonyl group at the B ring of \u03b1-M .\u03b1-M is able to form an inclusion complex with \u03b1-, \u03b2-, and \u03b3-CD. The in silico study using PM7 found that the inclusion complex of \u03b1-M with \u03b3-CD is the most favorable, because this complex has the lowest negative value of free binding energy (\u0394G), \u22125.68 kcal/mol, while a higher energy was needed for \u03b1- and \u03b2-cyclodextrin inclusion complexes . The calThe Job plot diagram and H-NMR revealed that \u03b1-M/\u03b3-CD CX has formed as an inclusion complex at a stoichiometry ratio of 1:1. Analysis by FTIR, XRD, and NMR corroborates that the complex was formed by the molecular interaction of \u03b1-M and \u03b3-CD, resulting in an amorphous complex with the solubility improvement and different physical properties compared with \u03b1-M. Furthermore, the thermodynamic study exhibited that the Gibbs energy of the complex formation was negative, indicating that this complex was formed spontaneously."} +{"text": "Arabidopsis halleri ssp. gemmifera hyper-accumulates Cd and Zn, and may be used to remediate Cd-contaminated sites. However, few studies have focused on Cd accumulation by A. halleri ssp. gemmifera. Herein, we demonstrate the accumulation of Cd by A. halleri ssp. gemmifera. The biomass, Cd, and Zn concentration of the plant increased in the 103 days of experimentation. Cd concentration of soil significantly decreased compared to its initial concentration (\u224810%). The material balance of Cd uptake by plant and Cd decrement from soil ranged from 63.3% to 83.7% in each growth stage. Analysis indicated that the water-eluted and exchangeable forms of Cd were stable during the experiment. However, Cd concentration extracted with 0.1 M HCl decreased , and this fraction was not bioavailable. The study exhibits the mass balance of Cd between plant uptake and decrement from the soil and the changes in the chemical form of Cd during stages of A. halleri ssp. gemmifera cultivation.Cadmium is a hazardous heavy metal and causes contamination globally. Phytoremediation can potentially become a low-cost and eco-friendly technique for mitigating Cd contamination. Cadmium (Cd) is present in trace amounts in zinc (Zn), lead (Pb), and copper (Cu) ores. During mining and beneficiation of these ores, Cd is discharged into rivers and accumulates in downstream soil through seepage water from mine shafts, drainage, and waste rock deposits . Cd poll2. Generally, for remediation of such contaminated sites, excavation is believed to be a swift and certain remediation technique. However, it is time-consuming, and moving the contaminated soil is expensive. Hence, a low-cost novel technology with a low environmental load is required.As Cd accumulates in rice, the regions with high rice consumption, such as Japan and other Asian countries, are concerned about the effects of Cd on human health. Although Cd in nature does not cause acute poisoning, chronic poisoning may occur due to long-term Cd exposure at relatively low levels. A serious example of this is Itai-Itai disease, which occurred in the Jinzu River basin in Toyama Prefecture . Itai-ItArabidopsis halleri ssp. gemmifera is a plant native to East Asia [Phytoremediation is an environmental remediation technology that utilizes the biological functions of plants, and the cost and environmental impact are lower than other remediation technologies such as excavation and soil washing . Heavy mast Asia that hypast Asia ,12.Heavy metals exist in various chemical forms in the soil; water-soluble form is easily eluted by water as ions and is available for plant uptake . The exchangeable form is bound to layers of clay or adsorbed on the soil surface, and this form can be exchanged for another cation. Therefore, it is considered potentially bioavailable. Carbonate form is bound to carbonates, and oxide form is bound to oxide compounds of Fe and Mn . These hA. halleri ssp. gemmifera, the total Cd concentration of soil decreased from 5.0 mg/kg to 1.0 mg/kg [A. halleri ssp. gemmifera [A. halleri ssp. gemmifera from soil where only a fraction of metal is bioavailable [A. halleri ssp. gemmifera, its material balance between soil and plants and Cd chemical form.In the earlier studies with .0 mg/kg . Additio.0 mg/kg . In anotemmifera . Howevervailable . Hence, A. halleri ssp. gemmifera, the chemical behavior of Cd resembles that of Zn which is essential nutrient, and it has been reported that Cd and Zn pass through a similar transportation pathway [Generally, plants change their nutrient requirements and tran pathway . TherefoA. halleri ssp. gemmifera and the consequent mass balance between soil and plants in a pot experiment. Additionally, the relationship between the chemical form of Cd and its accumulation by plant uptake was explored.The present study investigated Cd accumulation by A. halleri ssp. gemmifera were grown from the seed, and plants with heights of 3 to 5 cm were used for the experiment. The soil was filled in pots of two sizes up to 80% of the total volume and A. halleri ssp. gemmifera was planted in the center of each pot. The dry weight of the soil in the small pots was 0.61 \u00b1 0.03 kg, and that in the large pots was 3.06 \u00b1 0.19 kg. To ensure that plant growth was not limited by the previous study [Soil containing Cd and Zn was collected from a farmland located downstream of the Hosokura Mine, Tome city, Miyagi Prefecture. The initial concentrations of Cd and Zn in the soil were 4.94 mg/kg and 601.3 mg/kg, respectively. us study , the amo3 was added and heated at 130 \u00b0C by a heat block for 2 h. The samples were then digested and diluted by Milli-Q water so that the concentration of the target element was approximately 100 \u03bcg/L. The obtained sample was analyzed via inductively coupled plasma mass spectrometry , just as in Sugawara et al. [Plant materials were harvested in a manner to avoid separation of shoots and roots, and then washed with Milli-Q water. Thereafter, plants were divided into shoots and roots and were measured for fresh weight before oven-drying at 60 \u00b0C for 48 h, following which the dry-weight was measured. Plant samples weighing 0.01 g were placed in a glass test tube to which 2 mL of HNOa et al. .4)2SO4, and HCl [4)2SO4 solution and was also bioavailable because it is assumed that the exudation of the plant dissolves this fraction. HCl-soluble fraction was extracted by HCl solution. The details of extraction are shown in After harvesting, soil samples were sieved through a pore diameter of 2 mm and homogenized. Different fractions of the soil sample were extracted by Milli-Q water, (NH and HCl . The watg) for 20 min and the supernatant liquid was diluted by Milli-Q water for ICP-MS analysis. For finding total concentration of Cd and Zn, soil samples were oven-dried at 60 \u00b0C until the weights stabilized. The dried soil sample was ground . The powdered samples were made into pellet using a press machine and then analyzed via X-ray fluorescence spectrometer , as also described in Sugawara et al. [Extracted samples were centrifuged at 3300 rpm , which represents the mass balance of Cd between soil and plant, was calculated from the results as shown in p/s, TF, BCF, and AF are shown in The above results show a significant accumulation of Cd by the plants and its consequent reduction from the soil. The ratio of removal (Rp/s) ranged from 63.3 \u00b1 16.7% to 83.7 \u00b1 24.3%. This indicated that the most of Cd in soil was absorbed by A. halleri ssp. gemmifera, rather than drained from the pot. This study highlights the relationship between Cd in plants and in soil, which has not been reported previously.After the 75th day, the mass balance [Cd TF values ranged from 2.03 \u00b1 0.75 to 7.49 \u00b1 1.56. Meanwhile, BCF and AF values rapidly increased from the 75th to the 94th day, and ranged from 31.2 \u00b1 18.1 to 424 \u00b1 80.3 and 28.0 \u00b1 15.3 to 336 \u00b1 59.6, respectively. The earlier study of ge = 88) . The AF A. halleri ssp. gemmifera using radioactive Zn and Cd . Therefore, the same trend was confirmed in the present study.Additionally, it appeared that the translocation speed of heavy metals from roots to shoots was different between the early growth stage and late growth stage in this study . The TF Extraction experiments were conducted to verify the Cd fraction absorbed mainly by the plant. Cd concentrations of water-soluble fraction, exchangeable fraction, and HCl-soluble fraction in the soil are shown in A. halleri. Generally, Cd contaminated soil contains Zn, as Cd is derived from zinc ore. A. halleri ssp. gemmifera is also a hyperaccumulator of Zn. Therefore, phytoextraction of Cd should be performed without interference by Zn. If the Cd pathway is the same as Zn, then Cd uptake by the plant could be obstructed by Zn uptake. However, A. halleri ssp. gemmifera absorbed Cd adequately (maximum concentration in shoot: 1.89 \u00d7 103 Cd mg/kg) without Zn obstruction in this experiment. Hence, A. halleri ssp. gemmifera can be applied to Cd phytoremediation of soil containing Zn. Furthermore, Cd uptake pathway may be different from that of the Zn uptake. Zn concentration in soil was 100 times that of Cd, and the concentration in shoots was approximately 10 times that of Cd. Although heavy metal concentration in soil was drastically different, the difference in concentration in shoots was smaller than in soil. This result indicated different pathways for Cd and Zn uptake. In cases of iron (Fe) deficiency, the root of Arabidopsis induces the expression of the divalent cation transporter such as IRT1, which is essential for the uptake of Fe from the soil and responsible for the uptake of heavy metals such as Cd [Fukuda et al. suggestech as Cd .3 pot, the maximum Cd concentration in the plant was 753 mg/kg at the flowering phase [A. halleri ssp. gemmifera. Contrastingly, soil properties affect plant biomass owing to the presence of nutrients and water retention potential. As the phytoextraction potential is determined by the production of biomass [In a previous pot experiment conducted for 3\u20134 months of plant cultivation using 5.4 mg/kg Cd contaminated soil and 3000 cmng phase . The resThe concentrations of exchangeable fraction and HCl-soluble fraction decreased through the experimental period of the study. Hence, to confirm that the exchangeable fraction is the dominant form absorbed by plants, the difference in the decrement in concentrations between three fractions, namely total Cd, exchangeable, and HCl-soluble, is shown in A. halleri ssp. gemmifera cultivation. Degryse et al. [Although it is assumed that plants absorb the exchangeable fraction of Cd, decrement in the exchangeable fraction was less than total Cd after 75 days. In contrast, the decrement in the HCl-soluble fraction of Cd was the same as the total Cd on the 75th and 103rd days. Nevertheless, the HCl fraction was not bioavailable. Kubota et al. reportede et al. suggestee et al. reportede et al. .The extraction method may give useful information on heavy metals, although the chemical form of Cd in soil is affected by several factors. Some previous reports attempted to upregulate the change in heavy metal chemical forms from stable to bioavailable form . This stA. halleri ssp. gemmifera reached to maximum Cd concentration in shoot in a short period (3 weeks) regardless of biomass in the previous study using hydroponic culture [Nicotiana tabacum L., which is one of the Cd hyperaccumulators, enhances the mobility of Cd in soil [Thlaspi caerulescens known for Cd and Zn hyperaccumulation did not enhance Cd mobility [A. halleri ssp. gemmifera enhances Cd mobility in soil, the relationship between plant uptake and form of Cd must be demonstrated to understand rhizospheric interaction. To summarize, the study revealed Cd accumulation by A. halleri ssp. gemmifera and its mass balance between Cd chemical form in soil and subsequent shifting in form by plant uptake in pot experiments. This report may contribute to the improvement in the phytoremediation technique and understanding the relationship between heavy metals and plants in the environment. For future research, the relationship between root exudates and Cd fraction in the soil must be investigated.Generally, plants acquire nutrients including metal form soil by releasing root exudates and micr culture . This re culture . Additio culture reported culture reported culture , maize [ culture , and soy culture also rel culture . Root ex in soil . As orgamobility . Thus, tA. halleri ssp. gemmifera. The biomass, Cd, and Zn concentration of the plant increased in the 103 days of our experiment. Cd concentration of soil significantly decreased compared to its initial (\u224810%). Mass balance of Cd uptake by plant and Cd decrement from soil ranged from 63.3% to 83.7% in each growth stage. Analysis indicated that the water-eluted and exchangeable forms of Cd were stable during the experiment. However, Cd concentration extracted with 0.1 M HCl decreased , and this fraction was not bioavailable. The study showed the mass balance of Cd between plant uptake and decrement from the soil and the changes in the chemical form of Cd during different stages of A. halleri ssp. gemmifera cultivation. However, influence of A. halleri ssp. gemmifera on the Cd chemical form change are not investigated adequately. Therefore, in future study, the effect of root exudates of each growth stage on soil needs to be investigated. This study may contribute molecular biological analysis and other methods to facilitate the understanding of the mechanism of hyperaccumulation and tolerance to heavy metals, which is not exhibited by ordinary plants.In the present study, we investigated the effect of growth stage and chemical form of heavy metal to the accumulation of Cd and Zn by"} +{"text": "Oryza sativa L.) is one of the most important food crops, feeding half of the world\u2019s population. However, rice production is affected by cadmium (Cd) toxicity. Due to an increase in Cd-contaminated soil and rice grains, and the serious harm to human health from Cd, research on Cd uptake, transport and resistance in rice has been widely conducted, and many important advances have been made. Rice plants absorb Cd mainly from soil through roots, which is mediated by Cd absorption-related transporters, including OsNramp5, OsNramp1, OsCd1, OsZIP3, OsHIR1,OsIRT1 and OsIRT2. Cd uptake is affected by soil\u2019s environmental factors, such as the concentrations of Cd and some other ions in soil, soil properties, and other factors can affect the bioavailability of Cd in soil. Then, Cd is transported within rice plants mediated by OsZIP6, OsZIP7, OsLCD, OsHMA2, CAL1, OsCCX2, OsLCT1 and OsMTP1, from roots to shoots and from shoots to grains. To resist Cd toxicity, rice has evolved many resistance strategies, including the deposition of Cd in cell walls, vacuolar Cd sequestration, Cd chelation, antioxidation and Cd efflux. In addition, some unresolved scientific questions surrounding Cd uptake, transport and resistance in rice are proposed for further study.Rice ( In recent years, the Cd content of farmland soils has continuously increased due to increasing discharges of industrial wastewater, waste gas and other residues, excessive use of metal-containing pesticides and fertilizers. The total concentration of Cd in agricultural soils is higher than that in non-agricultural soils . CompareRice is the main source of Cd in Chinese diet, and 55% of Cd intake by the Chinese population is from rice . Cd enteTaken together, it is important to investigate the mechanisms of Cd uptake, transport and resistance in rice. In recent decades, scientists have made many important advances in understanding Cd uptake, transport and resistance in rice. Here, we introduce and discuss the research progress in this field. We also explore unresolved scientific questions regarding Cd uptake, transport and resistance in rice that demand further study in the future. The research progress in this field can be used to reduce the Cd-caused yield loss by enhancing rice resistance to Cd toxicity, produce safe rice grains by reducing Cd accumulation in rice grains and repair the Cd-contaminated paddy fields by cultivating and planting rice varieties with strong Cd enrichment.Rice plants primarily absorb heavy metal ions through their roots. Roots are the first organ to contact Cd in the soil and also the first barrier used by rice to resist Cd toxicity. Cd concentration in rice roots is significantly higher than that in aerial organs . Cd is aOsNramp5, a member of the rice natural resistance-associated macrophage protein (Nramp) family, is a membrane transporter involved in Cd and manganese (Mn) uptake by rice roots. This transporter is polarly localized on the distal sides of both exodermis and endodermis cells and is the main pathway through which rice roots absorb Cd and Mn , Table 1OsNramp1, a Cd transporter that can be up-regulated in response to Cd, participates in the absorption and transport of Cd in rice , Table 1As the physical and chemical properties of Cd ions are similar to zinc (Zn) and iron (Fe) ions, Cd ions can be transported into rice cells through Zn and Fe transporters. The Zn and Fe transporters OsIRT1 and OsIRT2, which transport Zn and Fe across the membrane, have high affinities for Cd . These tArabidopsis reduces Cd accumulation in roots and buds, thereby enhancing Cd tolerance \u2013Cd chelates from the cytoplasm to the vacuoles, and a knockout of these genes causes a hypersensitive response of Arabidopsis to Cd . In. In38]. is to Cd ,83. AtABvacuoles . HoweverThe chelation of Cd plays an important role in plant tolerance of Cd toxicity. Chelating agents in plants can prevent contact between Cd and organelles through Cd chelation and impede biochemical reactions between Cd and other substances, thereby reducing Cd toxicity ,84,85,862) is a tripeptide that can form an active transport complex of bis (glutathionato) cadmium (Cd\u00b7GS2) . Yeast co plants . PC is ao plants . Moreovetoxicity 41]..2) gene and thus enhance the tolerance of rice to Cd via the chelation between MT and Cd (Escherichia coli (E. coli) enhances the Cd resistance of transgenic E. coli , such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals. Excessive ROS disrupt the redox balance of rice plants, leading to a dysfunction of the antioxidant system, which in turn causes membrane lipid peroxidation and other types of oxidative damage, including programed cell death .gshII gene encoding GS from E. coli in Brassica juncea promotes glutathione synthesis and enhances Cd tolerance of transgenic Brassica juncea [Arabidopsis resistance to arsenic [GSH plays important roles during the antioxidation process of rice exposed to Cd. It has been reported that glutathione synthetase (GS) is an important enzyme involved in catalyzing GSH biosynthesis, and the overexpression of the gene encoding \u03b3-glutamylcysteine synthetase (\u03b3-ECS) and the a juncea ,101. Pre arsenic . GSH can arsenic . Moreove arsenic . In addi arsenic . In addi arsenic .2O2, regulates antioxidant activity in rice plants by inducing the expression of OsMSRMK3 and OsWJUMK1 are signal molecules involved in the regulation of rice growth and stress responses . Methyl OsHMA9, which encodes a heavy metal efflux transporter that is located in the plasma membrane, has Cd, Cu, Zn and Pb efflux activities , Table 1Cd pollution of farmland is a serious problem. In recent decades, scientists have made many important advances in understanding Cd uptake, transport and resistance in rice, but some of the molecular mechanisms underlying these processes are still unknown. Previous reports indicated that the expression of OsCd1 in the root cortex may promote the absorption of Cd by rice roots, but it is unclear whether the expression of OsCd1 in the root stele can facilitate the transport of Cd from the root to the shoot by promoting Cd loading. OsCDT1 is located in the cytoplasmic membrane and can restrict the entry of Cd into cells by chelating Cd on the cell surface, thereby enhancing the tolerance of plants to Cd. However, the chelation site for Cd on OsCDT1 has not yet been reported, and the chelation mechanism has not been well studied. Although PC plays an important role in the process of Cd chelation and the mitigating of Cd toxicity, overexpression of PC synthase genes leads to increased Cd tolerance in some plants and decreased Cd tolerance in others, and the different mechanisms for these opposite effects need further confirmation. Previous studies have shown that loss-of-function mutation of OsAUX1 results in increased Cd sensitivity due to the severe oxidative damage in the rice mutant under Cd exposure, but the regulatory mechanism of OsAUX1 in the antioxidation process under Cd exposure remains unclear. OsABCG36 promotes Cd efflux from rice roots, thereby improving resistance to Cd. However, the form (ionic or chelated) of Cd that is excreted due to OsABCG36 remains unknown, and the molecular mechanism through which OsABCG36 promotes Cd efflux has not yet been elucidated.indica rice grains is generally higher than that in japonica rice grains [indica rice varieties that carry the japonica rice allele OsCd1v449 exhibit reduced Cd accumulation in grains [japonica rice varieties is the main reason for the accumulation of Cd in the grains of some japonica rice varieties [indica and japonica rice varieties remains obscure. Moreover, the transport of Cd and Cd complexes into vacuoles is also an important detoxification method. However, the transporters that can transport Cd complexes such as PC\u2013Cd and Cd\u00b7GS2 into the vacuoles of rice cells have not been identified. In future research, the unresolved scientific questions concerning Cd uptake, transport and resistance in rice may be revealed through the construction of mutants and use of molecular, genetic, physiological and multi-omics techniques, and bioinformatics. In addition, it is of great significance to identify unknown genes, which are related to Cd uptake, transport and resistance in rice, and to investigate the functional mechanisms of these genes. Additionally, with the intensification of climate change, the decomposition of soil organic matter is also strengthening [The Cd content in e grains , and indn grains . Loss-ofarieties ,82. Howegthening , which m"} +{"text": "Cyclodextrin (CD) derivatives are the most efficient and frequently used chiral selectors (CSs) in capillary electrophoresis (CE). There are situations when the use of a single CD as CS is not enough to obtain efficient chiral discrimination of the enantiomers; in these cases, sometimes this problem can be resolved using a dual CD system. The use of dual CD systems can often dramatically enhance enantioseparation selectivity and can be applied for the separation of many analytes of pharmaceutical interest for which enantioseparation by CE with another CS systems can be problematic. Usually in a dual CD system an anionic CD is used together with a neutral one, but there are situations when the use of a cationic CD with a neutral one or the use of two neutral CDs or even two ionized CDs can be an efficient solution. In the current review we present general aspects of the use of dual CD systems in the analysis of pharmaceutical substances. Several examples of applications of the use of dual CD systems in the analysis of pharmaceuticals are selected and discussed. Theoretical aspects regarding the separation of enantiomers through simultaneous interaction with the two CSs are also explained. Finally, advantages, disadvantages, potential and new direction in this chiral analysis field are highlighted. More than half of the pharmaceutical substances currently used in therapy are chiral, however only about 25% are used in the form of a pure enantiomer. It is known that usually the desired pharmacological effect is restricted to only one of the enantiomers, called eutomer, while the other, called distomer, can be less potent, exhibit different pharmacological activity and sometimes can even be responsible for the adverse effects reported after racemic mixture administration .In the past 25 years the number of pure enantiomers introduced in therapy increased constantly, after the publication in 1992 of FDA regulations regarding the development of new stereoisomeric drugs. Currently the properties of individual enantiomers of a racemic mixture should be studied and characterized individually; and the stereoisomeric composition of an optically active drug must be verified from pharmacokinetic, pharmacologic, and toxicologic point of view is the CD concentration.When separation occurs because of the difference in the complexation constants, enantiomeric separation can be achieved when the mobilities of the complexed and free form of the analyte differ. This situation in the presence of a single CS system is described in the equation below :(1)\u0394\u03bc= and the analyte: CD complexation ratio is 1:1. The model can be applied for both neutral and charged analytes and CDs .f is the electrophoretic mobility of the free form, \u03bcc1 and \u03bcc2 are the electrophoretic mobilities of the analyte-CD complexes, cK1 and cK2 are the complexation constants of the enantiomer with CD1 and CD2 and [CD1] and [CD2] are the concentration for the two CDs.The following equation for the apparent electrophoretic mobility of the enantiomers in dual CD systems is more appropriate :(2)\u03bc=-\u03b2-CD (DM-\u03b2-CD) and sulfobutyl ether \u03b2-CD (SBE-\u03b2-CD) proved to be useful for the simultaneous enantioseparation of illicit drugs using a Tris-phosphate BGE at pH 2.45. When using only a single CD some coelutions happened, however using a dual CD all primary amines were separated. This first application has already shown one of the huge benefits of dual CD systems when the goal is the simultaneous separation of enantiomers of several substances. Chiral resolution and migration times of the enantiomers were controlled by adjusting the ratio of the two CDs; the anionic CD playing the role of counter migrating complexing reagent, while the neutral CD ensured enantiorecognition. An initial theoretical model was developed to characterize the impact of neural and anionic CDs on the chiral resolution of cationic analytes. However, the addition of SBE-\u03b2-CD resulted in increased tailing and migration times when compared with the use of single DM-\u03b2-CD, probably due to electrodispersion .In another study by Anigbogu et al. the enantiomers of aminoglutethimide were separated in a basic BGE at pH 9.0 using a mixture of neutral \u03b2-CD and anionic carboxymethyl-\u03b2-CD (CM-\u03b2-CD). At this pH, aminoglutethimide is neutral and no chiral interactions were observed when using the CDs individually. Apparently the neutral \u03b2-CD provided enantioselectivity, while the anionic CM-\u03b2-CD provided a differential migration rate. The basic pH of the optimized selected method resulted in a strong electroosmotic flow (EOF), which was accompanied by a short analysis time. The method was compared with a micellar electrokinetic chromatography method (MEKC) at the same pH using only \u03b2-CD as CS which resulted only in partial resolution of the enantiomers and a capillary zone electrophoresis (CZE) technique at acidic pH (3.0) where small chiral interactions were observed with neutral \u03b1- and \u03b3-CD .2-\u03b2-CD) and a neutral one, trimethyl-\u03b2-CD (TM-\u03b2-CD) in an acidic BGE at pH 2.3. In this case, interactions with NH2-\u03b2-CD, are not stereoselective, however provide the differential migration rates of the enantiomers, while TM-\u03b2 -CD allows the chiral recognition. Also, a theoretical model regarding selectivity was developed for dual CDs separation systems, based on the concept of the apparent constant of complex formation [Leli\u00e8vre et al. developed a CE chiral separation method for arylpropionic acid non-steroidal anti-inflammatory drug (NSAID) derivatives using a CD mixture composed of a cationic CD, amino-\u03b2-cyclodextrin Fillet et al. obtained the baseline enantioseparation by CE using a dual CD selector system containing neutral TM-\u03b2-CD and the anionic SBE-\u03b2-CD at pH 3.0. In an acidic BGE these analytes are almost neutral, consequently, the use of uncharged CDs alone is inefficient, while the use of charged CDs gave some chiral resolution, which was significantly improved when the dual CD system was applied. In this system the charged CD played the role of the carrier giving the analytes an apparent electrophoretic mobility, while the uncharged CD provided enantioselectivity. Triethanolamine was used as BGE additive to provide a weak EOF; as the anionic CD moved with EOF; remarkably high resolution and relatively short analysis time (less than 15 min) for a chiral separation were obtained .Based on the comparison of the two previously mentioned studies we can observe that aryl propionic acid derivative NSAIDs in a mixture can be separated at acidic pH using a dual CD system. One of the CD should be charged, as both cationic and anionic derivatives were applied to play the carrier role, and the other CD is neutral and is used to improve selectivity.In a follow-up study, the utility of the previously developed CD system was verified for the enantioseparation of weak acids and neutral compounds . Systems containing DM-\u03b2-CD and TM-\u03b2-CD as neutral CDs and CM- \u03b2-CD and SBE-\u03b2-CD as anionic ones were tested at different pH values. All the analytes were baseline enantioseparated at pH 3.0 except for the anxiolytic drug chlormezanone, which was resolved at pH 5.0 using a combination between TM-\u03b2-CD and CM-\u03b2-CD. Higher resolutions were obtained at pH 5.0 for all the analytes; at this pH, the results obtained when using CM-\u03b2-CD as anionic CD were better than in the case of SBE- \u03b2-CD, due to the substantial increase in the negative charge of CM-\u03b2-CD, because of pH increase from 3.0 to 5.0 .In another study by Fillet et al. the enantioseparation selectivity in dual and single CD CS systems was compared using basic drugs as model substances. Different CD combinations were tested: CDs with opposite chiral recognition pattern but with the same effect on analyte mobility (CM-\u03b2-CD and SBE-\u03b2-CD) and CDs with the same chiral recognition pattern but with opposite effect on analyte mobility (DM-\u03b2-CD and TM \u03b2-CD). The use of a CD system in which the CSs have the same effect on the mobility of the analytes, decreased separation efficiency, while the use of a CD system in which the CSs have opposite effect on the mobility of the analytes seem to be beneficial for the separation .It can be seen, that as well as in the case of acidic character compounds, basic drugs can be separated using a dual anionic-neutral CD system. However, in contrast to acidic drugs, basic drugs have their own mobility at acidic pH. Moreover, combination of methylated CD with CM- \u03b2-CD or SBE- \u03b2-CD seem to be the most effective dual system.S-naproxen in tablets. Several single and dual CD systems were applied in a phosphoric acid-triethanolamine BGE at pH 3.0, the best results were obtained when using a mixture of TM-\u03b2-CD and SBE-\u03b2-CD, similar with the one used previously by the same research group for the enantioseparation of other NSAIDs [R-naproxen, in a 0.1\u20132% range. This method proves the applicability of dual CD systems for the verification of enantiomeric purity of analytes used as pure enantiomers [CE with a dual CD chiral system was applied by Fillet et al. for the enantioselective determination of r NSAIDs . The metntiomers .R- and S-thalidomide [Meyrig et al. developed a CE method for the enantioseparation of thalidomide and three of its hydroxylated metabolites using a dual CD system composed of native \u03b2-CD and anionic SBE-\u03b2-CD and a polyacrylamide-coated capillary. The separation of thalidomide and its hydroxylated metabolites by CE was verified in a previous study by the same research group using four charged CDs, and their combinations with \u03b2-CD, the best results being obtained when using CM-\u03b2-CD, however the simultaneous baseline separation of all the enantiomers was not achieved . In the lidomide . The uselidomide .In a study published by Abushoffa et al. a single isomer anionic heptakis-6-sulfato-\u03b2-cyclodextrin (HS-\u03b2-CD) was used in combination with neutral TM-\u03b2-CD for the CE enantioseparation of arylpropionic acid NSAIDs using a pH 2.5 phosphoric acid\u2013triethanolamine BGE in the reversed polarity mode. The model compounds are acidic and are not ionized at pH 2.5, the use of HS-\u03b2-CD as a single CS gave poor resolution, however in dual CD system HS-\u03b2-CD provided the analytes with the suitable mobility while TM-\u03b2-CD provided enantioselectivity. Affinity constants for the enantiomers with the both CDs were determined, using linear regression in a two-step approach; for HS-\u03b2-CD were calculated in single CD systems while with TM-\u03b2-CD were calculated in dual systems. A mathematical model was developed to predict the best analytical conditions for the CE separation of aryl propionic derivatives in HS-\u03b2-CD\u2014TM-\u03b2-CD dual systems .The same research group applied the use of a mixture of charged CDs for the enantioseparation of the four analytes from the previous study. A combination of two oppositely charged CD derivatives were used; a single isomer cationic CD, permethyl-6-monoamino-6-monodeoxy-\u03b2-CD (PMMA-\u03b2-CD) and an anionic CD, HS-\u03b2-CD in a phosphoric acid-triethanolamine BGE at pH 2.5. Significant improvement in selectivity and resolution was observed, as the two CDs lead independent enantioselective complexation with the analyte enantiomers and exhibit not only opposite effects on the electrophoretic mobility of the analytes but also opposite affinity patterns towards the enantiomers. Binding constants were determined using linear regression method, to predict conditions, giving rise to high selectivity and resolution using the previously developed mathematical models. The use of single isomer CDs allows prediction of the analytical performance of the method. However, it should also be noted, that these derivatives are much more expensive, and they separation capacity does not exceed the one of randomly substituted CD derivatives .S-enantiomer (E-6232), while the R-enantiomer (E-6231) was considered a chiral impurity; other five degradation impurities were alsoanalyzed. A mixture of DM-\u03b2-CD and SBE-\u03b2-CD was used in the chiral separation, the limit of detection (LOD) and limit of quantitation (LOQ) was 0.03% and 0.1% of distomer. The use of single SBE-\u03b2-CD proved to be also efficient, however the dual CD system offered advantages in terms of higher enantioresolution and sensitivity [P\u00e9rez-Maseda et al. developed a CE enantioseparation method for a potential novel NSAID cyclooxygenase-2 (COX-2) inhibitor. The eutomer of this compound is the sitivity .N-deprenyl oxide. The enantioresolution of all analytes was achieved by using a dual CD system containing DM-\u03b2-CD in combination with CM-\u03b2-CD in a Tris-phosphate BGE at pH 2.7. The method was applied for the determination of deprenyl metabolites in rat urine; stereoselective metabolism of deprenyl was confirmed. This method proves that CE with dual CD selector system could be an attractive tool for detect and separate chiral metabolites in biological fluids [T\u00e1bi et al. developed a CE enantioseparation method for deprenyl, an irreversible monoamine oxidase enzyme (MAO) inhibitor and eight of its metabolites, including the active metabolite, l fluids .O-phenylcarbamoyl-\u03b2-CD (MPC-\u03b2-CD) and \u03b2-CD as CS. The separation resolution significantly increased using the dual CD system when compared with the individual CDs alone [Lin et al. developed a MEKC enantioseparation method for miconazole, an antifungal agent with an imidazole structure, using a borate-sodium dodecyl sulphate (SDS) BGE at pH 9.5 and a mixture of mono-3-Ds alone .R,S-enantiomer, while the R,R-, S,S- and S,R-forms are considered to be chiral impurities. The combination of hydroxypropyl-\u03b3-CD (HP-\u03b3-CD) and CM-\u03b2-CD was used in the separation added in a phosphate BGE at pH 6.4 [An enantioselective method for the verification of chiral purity of a potential antianginal agent with a benzoaxathiepin structure was developed by Beaufor et al. The substance has two chiral centers, consequently four optical isomers, however the eutomer is considered to be the t pH 6.4 .S-efaroxan at 0.05% level [The enantiomeric purity of a basic drug efaroxan, a selective \u03b1-2-adrenoreceptor antagonist, was verified by CE using a dual CD system. The method published by Lorin et al. uses a mixture of DM-\u03b2-CD and CM-\u03b2-CD added in a phosphoric acid\u2013triethanolamine BGE at pH 3.0. The method was validated for the enantiomeric impurity determination of the distomer 5% level .Chu et al. developed a CE enantioselective analysis method for chiral separation of the antiparkinsonian drug rotigotone, a non-ergolinic dopamine receptor agonist and two related chiral impurities. A mixture of methyl-\u03b2-CD (M-\u03b2-CD) and sulfated-\u03b2-CD (S-\u03b2-CD) was added in a phosphate buffer at pH 2.5. The LOD and LOQ were 0.003 and 0.01 mM for each enantiomer, respectively .S-citalopram, a selective serotonin reuptake inhibitor (SSRI) antidepressant. Citalopram is used in therapy both as racemic mixture and in the form of its eutomer, S-citalopram. A phosphate BGE at pH 2.5 containing a mixture of native \u03b2-CD and S-\u03b2-CD was applied in the enantioseparation. For method optimization a central composite face-centered factorial design was used. During the optimization, the concentration of \u03b2-CD was kept constant at 0.5 mg/mL as preliminary experiments revealed not significant effect in the range of 0.5\u20132 mg/mL; and the effect buffer concentration, applied voltage, temperature and S \u03b2-CD concentration were studied. The method was validated for the determination of the distomer, R-CIT and the enantiomers of an impurity, citadiol. LOD was 0.02%, while LOQ 0.05% for all compounds reported to a 5 mg/mL concentration of S-CIT. The method was applied for the determination of chiral purity of S-CIT in bulk substance and tablets. A representative electropherogram for the determination of S-CIT chiral impurities is presented in Sungthong et al. developed a stereospecific CE method for the simultaneous determination of optical purity and other related compounds of 1H-NMR studies were performed to characterize the CD-analyte interactions; the results showed that SBE-\u03b2-CD interacts primarily with the basic part of glitazone derivatives while DM-\u03b2-CD interacts with the phenyl ring [A CE chiral separation method for the enantioseparation of three glitazone derivatives was developed by Jamali et al. Initially enantiorecognition of DM-\u03b2-CD and SBE-\u03b2-CD was investigated in single CD systems at different pH values. The baseline separation of all analytes was achieved when using a dual CD system composed of the two CDs added to a sodium tetraborate BGE ay pH 9.70. Optimization of analytical parameters was made using factorial design experiment. nyl ring .S,2S)-norpseudoephedrine, -norephedrine, phenylacetone and phenylacetone oxime. The method developed by Wongwan et al. used a phosphate BGE at pH 3.0 and a combination of SBE-\u03b2-CD and S-\u03b2-CD as a dual CD selector system. The effect of the degree of substitution of SBE-\u03b2-CD was investigated. The LOD values were between 0.01\u20130.02%. This study proves the applicability of dual CD systems containing two anionic CD in the enantioseparation by CE [A CE method was used for the simultaneous determination of impurities of dexamphetamine, including levoamphetamine and related substances (1on by CE .N-acetyl-5-methoxytryptamin) binding site. The baseline separation was successful using polyethylene oxide dynamically coated capillaries and a dual CD selector system. The composition and concentration of the CD system were optimized systematically. The best results were obtained with a mixture of \u03b3-CD and highly sulfated-\u03b2-CD (HS-\u03b2-CD) in a phosphate buffer at pH 2.5 [Lipka et al. worked on the CE enantioseparation of six agonists and antagonists with tetrahydronaphthalenic structures for the melatonin -propylamino-\u03b2-cyclodextrin (HPA-\u03b2-CD) and DM-\u03b2-CD was used by Wagner et al. for the determination of excitatory amino acids in brain samples by CE using laser induced fluorescence (LIF) detection. The method was used to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-aspartate and D-glucose in the central nervous system (CNS). 4-Fluoro-7-nitro-2,1,3-benzoxadiazole was used as fluorescent derivatization agent. The method was applied to analyze brain samples of 1-day-old chickens .Imperata cylindrica using CDs as CS. A complex CD screening involving 27 CDs was performed to establish the optimum CS. The best result was obtained using a dual CD system containing 6-monodeoxy-6-mono-(3-hydroxy)-propylamino-\u03b2 CD (MPA-\u03b2-CD) and sulfobutyl-ether-\u03b3-CD (SBE-\u03b3-CD) added to a borate BGE at pH 9.0. The average stoichiometry of the complex was determined with Job\u2019s method using NMR-titration, the results showed a 1:1 complex for both enantiomers. One of the curiosities of this study is that the best result was obtained using a dual system containing CDs with opposite charge based on a complex CD screening [Sohajda et al. developed a CE method for the enantioseparation of imperanene, a polyphenolic compound extracted from the plant creening .Wan Ibrahim et al. developed a MEKC method for the simultaneous determination of three imidazole antifungal drugs in a mixture. The baseline separation was obtained using a mixture of two neutral CD, HP-\u03b3-CD and DM-\u03b2-CD added to a phosphate- SDS BGE at pH 7.0. The use of single CDs gave an incomplete separation, however the mixture of the two neutral CD derivatives was able two separate the six stereoisomers. LOD values for the three analytes ranged from 2.70\u20137.70 mg/L. The method was applied for the determination of the three drugs in spiked human urine and from pharmaceutical preparations .Separation of five agomelatine analogues, with potential antidepressant effect, was achieved by Lipka et al. using dynamically coated capillary with polyethylene oxide and a dual CD system as CS. The best results were obtained using a phosphate BGE at pH 2.5 and a mixture of 6-monodeoxy-6-monoamino-\u03b2-CD (MMA-\u03b2-CD) and sulfated-\u03b3-CD (S-\u03b3-CD) . Like inYu et al. verified the applicability of a newly synthetized cationic CD, single isomer mono-6-deoxy-6-piperdine-\u03b2-cyclodextrin (PIP-\u03b2-CD), for the enantioseparation by CE of meptanizol, an opioid-type analgesic with mixed agonist\u2013antagonist effects and three of its intermediates. To enhance chiral resolution a dual CD system containing \u03b2-CD and PIP-\u03b2-CD was applied. This example shows the advantage of using dual CD system in the separation of molecules with more chiral centers .S,R- and R,S-enantiomers, HP-\u03b3-CD resolved the separation of the S,S- and R,R-enantiomers; while the use of the dual CD system separated all four stereoisomers. The influence of single and dual CD on the enantioseparation of the tapentadol four stereoisomers is presented in The chiral separation of the four stereoisomers of tapentadol, a centrally acting analgesic agent, was described by Znaleziona et al. The best results were obtained when using a mixture of two neutral CDs, hydroxypropyl-\u03b2-CD (HP-\u03b2-CD) and HP-\u03b3-CD added to a borate BGE at pH 9.5. HP-\u03b2-CD resolved the separation of the S-sulpiride) by CE using a dual CD system composed of M-\u03b2-CD and S-\u03b2-CD added in a Britton-Robinson BGE at pH 3.45 [Melani et al. developed a CE method for the chiral separation of sulpiride, an atypical antipsychotic drug with benzamide structure. Previously the use of a Quality by Design (QbD) strategy was reported by Orlandini et al. for the chiral purity study of the eutomer levosulpiride . After an initial CD screening the use of a dual CD system composed of \u03b2-CD and SBE-\u03b2-CD added in a phosphate BGE at pH 7.0 was selected as the optimum solution. The two selected CDs showed synergic effect on enantioseparation; resulting in the simultaneous baseline separation of all 8 enantiomers in a short analysis time (less than 8 min) with good chiral resolution. Even if chemically related drugs from the same therapeutic class are usually not administered together, the development of generic methods for their simultaneous determination became a tendency in recent years, as it can be applied for the determination of any of these analytes without the need to develop separate methods for each analyte. The method was validated and used for the determination of levocetirizine from tablets .Nov\u00e1kov\u00e1 et al. reported a CE method for the enantioseparation of leucine and phenylalanine benzothiazole derivatives, with potential antimicrobial effects. Dual CD system consisting of two neutral CDs; \u03b2-CD with HP-\u03b2-CD for the leucine derivative and \u03b2-CD with HP-\u03b3-CD for the phenylalanine derivative, added in a phosphate BGE at pH 3.5, proved to be the best solution for the enatioseparation .Delplanques et al. developed a CE method using single and dual CD systems for the enantiomeric and diasteromeric separation of dihydropyridine analogues, with one or two chiral centres. Ten dual CD systems proved to be successful in the separation, among the tested CDs, HS-\u03b3-CD and SBE-\u03b2-CD proved to be the most efficient selectors. This work underlines the challenges analysts must overcome in developing chiral separations as there is a large diversity of CDs used as CSs which can be mixed in multiple combinations .The chiral separation method for ketamine metabolites, norketamine, hydroxynorketamine and 5,6-dehydronorketamine by CE was published by Sandbaumh\u00fcter et al. The best results were obtained using a mixture of CDs composed of S-\u03b2-CD and HS-\u03b3-CD added to a phosphate BGE at pH 3.0. A liquid-liquid extraction (LLE) was used for sample preparation. The method was applied to analyze plasma samples from dogs and horses after i.v. ketamine administration and in an in vitro study in which ketamine was incubated with equine liver microsome .S-lansoprazole, S-rabeprazole) as chiral impurities in dexlansoprazole and dexrabeprazole samples [Papp et al. developed a CE method for the enantioseparation of two proton pump inhibitors (PPI) . Single and dual CD systems were screened in a preliminary analysis. A mixture containing \u03b3-CD and SBE-\u03b2-CD was selected and added in a phosphate BGE at pH 7.0. An experimental design approach was used for method optimization, a fractional factorial screening design followed by a central composite design. The optimum ratio of the two CDs was different for lansoprazole and rabeprazole, respectively. The method was validated and applied for the determination of distomers , regarding the distomer R-dapoxetine and structurally related impurities. For the optimization of the analytical method an initial fractional factorial design was applied followed by face centered central composite design. The optimum CD CS system contained DM-\u03b2-CD and S-\u03b3-CD added to a phosphate BGE at pH 6.3. The method was validated and applied for the analysis of dapoxetine in tablets [Harnisch et al. published a CE method for the simultaneous determination of chiral and achiral purity of tablets .The same methodology was applied by Krait et al. for the determination of chiral purity of dextromethorphan, a centrally active antitussive drug, regarding its distomer, levometorphan. A dual CD system containing M-\u03b2-CD and S-\u03b2-CD added in a phosphate BGE at pH 6.5 was used. An initial fractional factorial design followed by a face centered central composite design was used for method optimization. The method was validated and applied for the analysis of dextromethorphan in capsules .Casado et al. applied CE method with a dual CD system for the simultaneous enantioseparation of six phenoxy acid herbicides from a complex mixture. The CD CS system contained two neutral derivatives HP-\u03b2 CD and TM-\u03b2-CD added in a phosphate BGE at pH 7.0. The method development was based on a previously published theoretical model proposed by Dubsk\u00fd et al., which permits prediction of what happens in a dual CD system by performing individual measurements with each CD individually at different concentration levels . The resThe previously described methods analytical conditions are summarized in To choose an efficient dual CD system, and explain chiral separation, analysts must consider both chiral interactions which occurs at molecular level and chiral recognition which leads to chiral discrimination. Consequently, both mobility and affinity effects must be taken into consideration and an explanation about affinity patterns that generates increased enantioseparation may be drawn depending on the effect of both CSs on the electrophoretic mobility of the analyte enantiomers.In some situations, an increase in the selectivity of enantioresolution can be observed when changing from a single CD CS system to a dual CD system. Also, sometimes, the intrinsic chiral separation mechanism can be better understood when a dual CD system is applied as it is possible to define selectivity whether complexing agents are considered separately or all together.Several combinations of CDs can be used in practice: combinations between a neutral CD with a charged one, between two charged CDs with the same charge or between two neutral CDs ,14,16.Analyte-CD complexation is the main trigger that leads to enantiomer separation by CE when this leads to differences in mobility between the free and complexed formed of the analytes and stability differences between the complexes formed by the CD with the enantiomers of the analyte. In the case of charged CDs, they have their own electrophoretic mobility, and can be used as carriers for the separation of neutral and ionizable analytes in uncharged forms ,19.In some dual CD systems, the chiral discrimination capacity of one CD can lead to enantioresolution only in the presence of the second CD. For example, if we have an uncharged chiral analyte, its enantiomers can exhibit chiral interaction with a neutral CD without significant enantioresolution because of the small mobility differences between the free and the complexed forms. When a charged CD is added to the BGE, it can provide an apparent electrophoretic mobility to the analyte, which increases enantioresolution; even if the interactions with the charged CD are not enantioselective ,19,62.In other dual CD systems, the interactions with the two CDs can be both enantioselective, and have opposite effects on the migration of enantiomers, one CD accelerates while the other decelerates them, in this case enantioselectivity is obtained if the affinity patterns of the enantiomers towards the CDs are different. The same condition must be met, if one of the CDs has little effect on the electrophoretic mobility of the analyte, as in the case of an uncharged analyte and a neutral CD. A mixture of two CDs exhibiting the same effect on analyte mobility, as both CDs accelerate or decelerate migration of the enantiomers, seem to be inefficient in the separation ,19.But in another hand, the variations in chiral selectivity observed when using dual CD systems are not always explainable; in some situations, the two CDs can have synergistic or antagonistic effects on the chiral resolution, which makes the overall process unpredictable.To obtain information about complex stoichiometry, binding constants, stereoselectivity or intermolecular analyte-CD interactions other instrumental techniques such as NMR, MS or X-ray crystallography can be used. Molecular modelling can also be a valuable tool in establishing analyte-CD complex structure and mechanism of enantiorecognition ,64.Initially a trial-error strategy was applied in choosing the optimum CD for the separation, however in the past 20 years the theory enantioseparation in dual CD mixtures has been explained and understood. Theoretical mathematical models have been developed and can be used as feasible practical approaches in chiral method development ,60,65.Also, the use of QbD principles and design of experiments (DoE) represents a new strategy for the development of CE separation methods suitable for chiral analysis ,66It is essential that choice of the optimum CD mixture as CSs, should be complemented using the right ratio between the two CD and DoE is an efficient tool to establish this with a minimum number of experiments ,66,67.It should be noted that even if in CE enantioseparations usually dual CD systems are used, CD may be combined sometimes also with other CSs or rarely combined CS system without CDs can be applied ,68.There is only one review published in literature regarding the specific use of CD dual systems in CE for the analysis of pharmaceuticals, this review was published in 2000 by Fillet et al. . HoweverOf course, there are a series of reviews published in the last ten years regarding the application of CDs in general as CSs in CE, reviews in which CD dual system are mentioned as an interesting and useful option. We can mention here the reviews published by Rezanka et al. and Saz It is interesting to mention that initial works involving the use of CD dual system in CE enantioseparations focused mainly on the analysis of model compounds with particular structural and electrophoretic properties in order to develop theoretical models used to characterize the influence of chiral discrimination ability and electrophoretic mobility of the two CDs on the overall selectivity of the enantiomeric separation. As information about separation mechanism in dual CD system was clarified, research direction changed, and studies focused mainly on the application of dual CD systems on the enantiomer separation of specific chiral analytes; the use of dual CD systems for the verification of enantiomeric purity, quantitative analysis from pharmaceutical formulations or pharmacokinetic studies have been reported.In CE, the right selection of the optimum CD to be used to achieve chiral resolution is the essential step in method development. When the use of a single CD does not provide enantioresolution, the addition of a second CD may generate separation. In the case of dual CD systems usually a combination of an anionic CD with a neutral CD is used. It is a fact that many analytes have basic character at an acidic pH, consequently, are positively charged and will have a higher affinity towards negatively charged CDs, increasing enantiorecognition capability. The use of dual CD systems is extremely useful in the case of analytes in uncharged form; in this situation it is preferable that the charged CDs should have no enantioselectivity towards the analyte or exhibit a chiral recognition pattern opposite to the neutral CD.Based on the described separation mechanism, the two enantiomers of a chiral analyte under interaction with a dual CD system are likely to differ in their mobilities, compared with single CD systems where the mobilities are likely to be the same. Although generally different distribution constants between the two enantiomers and CDs in single CD system dictates enantioseparation, in dual CD systems additional electrophoretic enantioselective mechanisms resulting from different electrophoretic mobilities of the enantiomer-CD complexes play an important role in separation. Dual CD systems are more selective than single CD systems due to this additional separation mechanism.The use of dual CDs system can be a useful tool in the enantioseparation of pharmaceuticals, offering good perspectives and high potential, as novel dual systems are continuously developed and applied in diverse analytical fields."} +{"text": "Intraperitoneal (IP) chemotherapy has emerged as a promising method for the treatment of peritoneal malignancies (PMs). However, microenvironmental barriers in the tumor limit the delivery of drug particles and their deep penetration into the tumor, leading to reduced efficiency of treatment. Therefore, new drug delivery systems should be developed to overcome these microenvironmental barriers. One promising technique is magnetically controlled drug targeting (MCDT) in which an external magnetic field is utilized to concentrate drug-coated magnetic nanoparticles (MNPs) to the desired area. In this work, a mathematical model is developed to investigate the efficacy of MCDT in IP chemotherapy. In this model, considering the mechanism of drug binding and internalization into cancer cells, the efficacy of drug delivery using MNPs is evaluated and compared with conventional IP chemotherapy. The results indicate that over 60 min of treatment with MNPs, drug penetration depth increased more than 13 times compared to conventional IPC. Moreover, the drug penetration area (DPA) increased more than 1.4 times compared to the conventional IP injection. The fraction of killed cells in the tumor in magnetic drug delivery was 6.5%, which shows an increase of more than 2.5 times compared to that of the conventional method (2.54%). Furthermore, the effects of magnetic strength, the distance of the magnet to the tumor, and the magnetic nanoparticles\u2019 size were evaluated. The results show that MDT can be used as an effective technique to increase the efficiency of IP chemotherapy. Cancers that occur in organs associated with the peritoneal cavity are prone to metastasize to the intraperitoneal space. Patients with these malignancies experience low quality of life due to the fact of complications such as urinary blockage, ascites, pain, and inability to eat and drink. The five-year survival rate is 49% in these types of cancer. The average survival rate in peritoneal carcinomatosis of gastric origin is 1\u20133 months ,3,4. AltWhile some studies have shown that magnetic drug targeting in biological fluids is strongly hampered , IP chemIn this work, we used a numerical model to study the IP injection of drug-coated MNPs for the treatment of peritoneal malignancies. Size dependency was seen as a determining parameter, taking into account the impacts of the relative size of MNPs compared to the vessel wall pore size and ECM pore size in the model. Moreover, the drug transport process was examined by considering the mechanisms of drug binding and internalization into cancer cells. The spatiotemporal distribution of drug concentration in the forms of free, bound, and internalized cancer cells are presented. Finally, the fraction of killed cells (FK) was calculated as a quantitative treatment efficacy parameter.In clinical IP chemotherapy, chemotherapeutic agents are transferred to the desired location within a 1\u20132 h cycle. A schematic of drug delivery by IP injection of MNPs is shown in Considering the intercapillary distance (33\u201398 \u03bcm), which is usually 2\u20133 orders of magnitude smaller than the length scale of drug transfer ,27, tumo\u03ba is the interstitium\u2019s hydraulic conductivity. In addition, iP and Here, PL and B\u03c0 indicate the average osmotic reflection coefficient of plasma proteins and plasma osmotic pressure. Likewise, i\u03c0 is the interstitial fluid osmotic pressure. Lymphatic drainage ). Moreover, Gauss\u2019s law (Equation (6)) was used to describe the magnetic flux density ,30:(5)\u2207\u00d7H is the magnetic field, J is the current intensity, and B is the magnetic flux density. As we are using a permanent magnet, the current density (J) was considered 0. For the air and tissue domain, the constitutive equation B = \u03bc0H was used, while for the magnet domain, the equation B = \u03bc0r\u03bcH + remB was applied [\u03bc0 = 4 \u03c0 \u00d7 10\u22127 NA\u22122, the relative magnetic permeability r\u03bc = 1000, and the remnant magnetic flux was defined as remB. In a magnetic field (H), the magnetic force exerted on an MNP is determined as [Here, applied . In thesmined as :(7)Fm=VMHere, turation , [\u2202H\u2192\u2202x\u2192particle ,34,35. F with H\u2192 ,37. The FC is the free drug concentration, FD is the diffusion coefficient of free drug in the porous medium, and recC and \u03c6 are the concentration of cell surface receptors and the tumor volume fraction available for the drugs, respectively. ONK is the constants of the drug association rate to the cancer cells. In addition, v and mnpD are the velocity and effective diffusion coefficient of MNPs in tissue. The velocity (v) was calculated as the sum of the local velocity of interstitial fluid (iv) and the equilibrium velocity (ev). ev corresponds to when the Stokes drag force, Fs = (6\u03c0a\u03b7)ve, is equal to the magnetic force (Fm) [The free drug\u2019s concentration in the interstitial fluid is calculated using the convection\u2013diffusion equation as follows:rce (Fm) ,18:(9)ve\u03b7 = 1.12 \u00d7 10\u22123 Pa.s is the dynamic viscosity of the interstitial fluid.Here, \u03a6 in Equation (8), is the net rate of free drug exchange from blood and lymphatic vessels and is calculated as follows:B\u03a6 and L\u03a6 are expressed as follows:P is the permeability of tumor microvessels, Cp is the concentration of MNPs in the blood plasma, and f\u03c3 is the osmotic reflection coefficient for the MNPs. Pe is the Peclet number defined as follows:Using the pore model ,39 for tBC is the drug concentration bound to the cancer cell, and INTK is the constant rate of drug internalization into the cancer cells. Moreover, the equation internalized drug concentration is expressed as:IC is the concentration of the drug that is internalized into cancer cells.The equation for the bound drug concentration is as follows:The fraction of killed cells was calculated as ion (16) , which iion (16) for the In Equation (16), l, and width, h, lies at a distance, d, from the tumor. A circular tumor with radius, R, and a necrotic core with radius, Rn, were considered. Doxorubicin has been used as one of the most widely used drugs in the model.A solid tumor may have a necrotic area that lacks functional blood or lymph vessels; thus, there is no exchange of fluid with the interstitium in this region. In contrast, the regions of the tumor that are outside of the necrotic core contain rapidly dividing cells and blood vessels. In order to consider this non-uniform perfusion rate in the tumor, a biologically related structure, including an area with a necrotic core and a surrounding with leaky vasculature, is defined as a tumor . A repreThe boundary conditions for the present study are listed in To create the initial conditions for time-dependent numerical simulations, fluid flow equations were primarily solved to achieve a steady-state solution in the computational domain. Then, the values of velocity and pressure obtained at time zero were used to simulate drug delivery. The solution strategy for this section is also shown in FC), bound drug (BC), and drugs internalized into cancer cells (IC) were examined in the tumor during one hour of chemotherapy. Two main criteria were considered for the evaluation of the performance of chemotherapy with this method:FK) was utilized as the major parameter for quantitatively evaluating drug delivery efficiency. Since drug particles do not reach all tumor parts in chemotherapy, this fraction was first studied as the fraction of killed cells in the drug penetration area (PAFK). Then, in order to evaluate the treatment of the whole tumor, this parameter was calculated and examined as the effective fraction of killed cells (effFK), which represents the fraction of killed cells by considering the whole tumor;Fraction of killed cells and then plateaued. The same applied to the concentration of bound drug, but the difference was that the concentration of bound drug reached its maximum more gradually. Unlike free and bound drug concentrations, the concentration of internalized drug was constantly increasing. This increasing internalized drug concentration cell as well as the non-decreasing trend of the free and bound drugs were due to the constant concentration of the drug at the outer boundary of the tumor within one hour of administration. In other words, with the continuous injection of free drug, the concentration of free and bound drug reached a steady-state during treatment. Obviously, with the end of the drug injection into the peritoneal cavity, the mentioned concentrations will decrease. However, the main focus in this study was a one-hour time span in which a continuous concentration of the drug was injected into the peritoneal cavity.w1/2 was examined to evaluate the drug\u2019s penetration into the tumor during treatment. The value obtained for w1/2 in this section was equal to 60 \u03bcm and, given that the tumor\u2019s radius was 10 mm, the relative penetration depth of the tumor w1/2% was obtained as 0.6%. In addition, calculating the relative drug penetration area (relPA) revealed that 87.3% of the tumor area remained untreated (FK) in the drug penetration area is presented in PAFK values increased over 60 min of treatment and reached 28.3% at the end of treatment.In order to acquire a higher concentration of the drug internalized into the cancer cells, more extracellular drugs should be available. Moreover, the drug must penetrate the tumor at the proper depth so that the tumor is homogeneously exposed to the drug. For this reason, the ntreated c. These w1/2, the relative drug penetration area (relPA), fraction of killed cells in the area of drug penetration (PAFK), and the effective fraction of killed cells (effFK) after 60 min of treatment. According to this table, relPA and effFK are 8.97% and 2.54%, respectively.The results of this section generally indicate the low efficacy of the conventional IPC. Even though the IP injection has been conspicuous because of its potential in the regional treatment and direct transfer of drugs to the tumor, it is necessary to provide strategies that can improve treatment using IP injection. The results presented in this section provide a general insight into drug delivery by conventional IP injection and review it from the perspective of fluid and mass transfer, which can help design a treatment strategy that can overcome obstacles of drug delivery to the tumor.In this section, the results of the MNP delivery are presented. To evaluate the improvement of treatment using this method. The results of this section were compared to the results of conventional IPC. Firstly, a baseline model is presented in order to compare magnetic IP drug delivery with the conventional IPC. Next, the impact of the MNPs\u2019 size, the magnetic strength, and the distance of the magnet from the tumor were investigated.In the baseline model, a permanent magnet of 20 by 10 cm in dimension, which was placed at a distance of 5 cm from the tumor, was used to assist in drug delivery to the tumor. The radius of the MNPs used in the baseline model was 100 nm, and the magnetic strength was considered as 1.5 T. As larger tumors are more dangerous due to the possibility of disease recurrence after treatment [FC, B,C and IC were calculated to be 0.0054, 0.0022, and 0.00025, respectively. Compared to the corresponding values for conventional IPC , an increase of 4.5, 4.4, and 2.77 times in the mean concentration of the free, bound, and internalized drug were achieved, respectively. Contours of FC, B,C and IC are shown in In FC, BC, and IC distributions along the vertical line passing through the center of the tumor are shown 60 min after the start of injection. The drug had a relatively good penetration into the tumor at the lower area of the tumor (r = 2 cm), the concentration of the drug decreased in all of its three forms as we moved to the center of the tumor. PAFK) over time. PAFK gradually increased from zero at the starting point to 51.2% at the end of the treatment. For more accurate evaluation and comparing the efficacy of treatment using MNPs, w1/2, relPA, PAFK, and effFK were calculated and presented in w1/2 was equal to 0.08 cm using magnetic nanoparticles with a radius of 100 nm, which shows the significant effect of the proposed technique in increasing the penetration depth of the drug by more than 13 times. As mentioned above, the penetration of the drug in the magnetic nanoparticles\u2019 drug delivery occurred only in the lower area of the tumor. Moreover, the value of the drug penetration area in the magnetic drug delivery was equal to 0.40 cm2, which, compared to conventional IPC, showed an increase of more than 1.4 times. The value of efFKf in the tumor using the magnetic drug delivery was also calculated to be 6.5%, which shows a more than 2.5 times increase than the obtained value for the conventional method (2.54%). Therefore, considering all these criteria for the treatment\u2019s evaluation, it can be said that magnetically controlled IP drug targeting has improved drug delivery to the tumor compared to the conventional IPC using doxorubicin. The performance of the proposed method was also compared with the results of two other drugs including paclitaxel and cisplatin , and 300 nm nanoparticles with 55.02% had the largest relPA among the studied MNPs. effFK values for different sizes of MNPs after 60 min of treatment are presented in effFK was calculated as the lowest value for 25 nm nanoparticles, which was equal to 0.11%, whereas it was the highest for 300 nm particles with 24.1%.Efficacy parameters .The results of this section show that the size of nanoparticles can affect the efficiency of treatment using this method. By selecting an appropriate MNP size, the effective killed cells fraction can increase by 9.5 times compared with conventional IPC. Whereas an improper selection of nanoparticle size (in the range of 25\u2013100 nm) leads to a reduction in the treatment efficacy even lower than the conventional IPC. Additionally, it should be noted that experimental results demonstrremB) in the range of 0.5\u20132.5 T on the treatment output is investigated. In FC distribution in the tumor are presented for the three magnetic strengths of 0.5, 1.5, and 2.5 T. As can be seen, an increase in the magnetic strength will increase the drug\u2019s penetration into the tumor. At the lowest magnetic strength (0.5 T), the drug penetration into the tumor was negligible, whereas at the highest magnetic strength (2.5 T), the drug particles significantly penetrated the tumor.In this section, the effect of magnetic strength by changing the remnant magnetic flux , this parameter\u2019s effect on the treatment using MNPs was studied.According to Equation (7), the magnetic force exerted on the MNPs was commensurate with FC in the tumor for three different distances of the permanent magnet from the tumor including 3, 5, and 10 cm. The comparison of the contours shows that by increasing the distance, a smaller part of the tumor was affected by the drug. This reduction in penetration occurred dramatically, especially by increasing the distance of the magnet from the tumor to 10 cm. According to FC values 60 min after the injection at a distance of 3 cm was 0.0069 mol/m3, while increasing the distance to 10 cm decreased the FC value to 0.0019 mol/m3.w1/2, relPA, PA,FK and effFK for three distances between the magnet and the tumor after 60 min of treatment. Reducing the distance of the magnet from the tumor from 10 to 3 cm increased the w1/2 and relPA values by 4 and 4.4 times, respectively. effFK values for different magnetic distances after 60 min of treatment. effFK increased by 3.88 times by reducing the distance of the magnet from the tumor from 10 to 3 cm.According to the results of this section, changing the distance between the tumor and the magnet significantly affected the efficiency of the magnetically controlled IP chemotherapy. Considering that tumors may be located in different sites of the peritoneal cavity, treatment using MNPs will be more difficult for tumors in deeper parts of the body. Although, using optimal values for other parameters, such as magnetic nanoparticle size and magnetic strength, can help to improve drug delivery to these tumors.In this section, the validation of the numerical solution is presented. The IFP and IFV distributions were calculated using the Darcy law and, subsequently, the concentration distribution of the free, bound, and internalized drugs could be obtained by solving the mass transfer equations. Hence, each of these physics must be validated separately.Finding the IFP and IFV is one of the exigent steps of this modeling, which was obtained by solving Equation (1). To validate the distribution of IFP in the tumor, a comparison between the radial distribution of IFP with an experimental work by Boucher et al. was condAu et al. evaluateUsing a magnetically controlled IP drug targeting system can overcome the microenvironmental barriers of the tumor against the transfer of drug particles and significantly increase the treatment efficiency compared to conventional IP chemotherapy;There was an optimal size for MNPs: larger nanoparticles exerted a stronger magnetic force. On the other hand, tumor microenvironmental barriers further hinder the movement of large nanoparticles in the tissue compared to smaller ones. Therefore, the competition between these two factors determines the extent of penetration of different sizes of nanoparticles;Using a permanent magnet with more magnetic strength and reducing the magnet\u2019s distance from the tumor by increasing the magnetic force exerted on the MNPs improved IP delivery of doxorubicin-coated MNPs.In the present study, a mathematical model was developed to study a magnetically controlled IP drug targeting system as a solution to improve the drug penetration into the tumor. Considering the binding and internalization of the drug into the cancer cells, the mathematical model analyzed the drug distribution inside the tumor in three forms: free, bound, and internalized. The drug penetration area and the fraction of killed cells in the tumor were used to estimate the efficiency of the proposed drug delivery system. The main conclusions of the present study are as follows:There are a number of assumptions and limitations in our model. With the lack of appropriate experimental data associated with the drug concentration at the outer edge of the tumor, this value was assumed constant during the simulation. By sampling the intraperitoneal fluid during treatment, a more accurate boundary condition for concentration outside the tumor can be obtained. In addition, the tissue poroelasticity effect was not considered in this study. This effect should be considered in the future developments of the model, given that studies have shown the effect of these phenomena on drug binding and internalization . The par"} +{"text": "Hg concentrations in lichens ranged from 142 ng g\u22121 at sampling sites located north of the chlor-alkali plant to 624 ng g\u22121 in lichens collected south of the chlor-alkali plant. A north-south gradient of Hg accumulation in lichens along the Ossola Valley channel was observed, highlighting that the area located south of the chlor-alkali plant is more exposed to the dispersion of Hg emitted into the atmosphere from the industrial site. Long-term studies on Hg emission and dispersion in the Ossola Valley are needed to better assess potential impact on ecosystems and human health.We present the first assessment of atmospheric pollution by mercury (Hg) in an industrialized area located in the Ossola Valley , in close proximity to the Toce River. The study area suffers from a level of Hg contamination due to a Hg cell chlor-alkali plant operating from 1915 to the end of 2017. We measured gaseous elemental Hg (GEM) levels by means of a portable Hg analyzer during car surveys between autumn 2018 and summer 2020. Moreover, we assessed the long-term dispersion pattern of atmospheric Hg by analyzing the total Hg concentration in samples of lichens collected in the Ossola Valley. High values of GEM concentrations (1112 ng m As was recognized in 2013 by 127 world countries with the signing of the UNEP Minamata Convention, further scientific efforts are needed to improve atmospheric Hg monitoring and characterization of Hg sources in order to reduce risks associated with Hg contamination 38].With the aim of assessing the impact of Hg pollution on air quality in the contaminated area around the chemical plant located along the Toce River, in the present study we investigated spatial and temporal changes in Hg distribution coupling short-term measurements of GEM concentrations in air to the measurement of Hg concentration in epiphytic lichens collected in the Ossola Valley at selected sampling points around the chemical plant.Results were related to wind pattern along the Ossola Valley channel.This research was carried out in the Ossola Valley . The Osshttps://prtr.eea.europa.eu/#/facilitydetails?FacilityID=162&ReportingYear=2017; accessed on 26 September 2020).The valley hosts about 66,500 inhabitants and the main town is Domodossola, with about 18,000 inhabitants. Most industrial activities are localized close to this area. However, the biggest industrial plant is located close to Pieve Vergonte town (about 2493 inhabitants), and was used in the last century mainly for Dichlorodiphenyltrichloroethane (DDT), sulfuric acid and cloro-soda production. This latter product was obtained using a Hg-cell chlor-alkali plant, which was discontinued at the end of 2017 for conversion to non-Hg technology. The chlor-alkali facility reported total annual releases of Hg to air and water in the European Pollutant Release and Transfer Register (E-PRTR) managed by the European Environment Agency .The climate of the Ossola Valley was defined through long-term (2002\u20132020) data collected at the Fomarco (hamlet of Pieve Vergonte) and Domodossola meteorological stations operated by Agenzia Regionale per la Protezione Ambientale del Piemonte and DL = 0.3 ng m\u22123 (average time being 30 s) for Hg determination in air. The dynamic range covers four orders of magnitude \u22121 at incerved by , who fou\u22121) but at the Bosco Tenso site . The spatial gradient of Hg contamination can be explained considering the topography of the Ossola Valley. In fact, our finding is in agreement with literature data showing that in industrial areas where metallic Hg is handled, prevailing wind direction and topography are accountable for the dispersion pattern in the surrounding areas [In our study we observed a specific spatial gradient of Hg in lichens along the valley channel, and the highest concentration of total Hg was not observed near the industrial area , is wider with respect to the lower valley at the southern lichen sampling site (Bosco Tenso) surrounded by 1000\u20131500 m high mountains. The peculiar morphology of the Ossola Valley plays a fundamental role in modifying the atmospheric boundary-layer flow that undergoes a channeling along the valley, determining the observed spatial pattern of lichen contamination. In addition, it is necessary to take into account that another factor may have influenced the higher concentrations of total Hg measured in lichens collected at the Bosco Tenso site. In fact, in wooded areas the uptake capacity of lichen is higher than in open areas . MoreoveData presented in this paper provide the first evidence of atmospheric GEM concentration and its deposition in areas surrounding a chlor-alkali plant located near the Toce River in the Ossola Valley .Determining or predicting Hg concentration in the atmosphere is of crucial importance in identifying sensitive areas requiring ecosystem and health protection. We showed how an integrated investigation of airborne Hg analytical measurements and biomonitoring can be employed to locate Hg emission sources and to identify areas where Hg inhalation exposure may be of concern.Our study highlights that the wind pattern in the Ossola Valley and its morphology imply that the area located south of the chlor-alkali plant is more affected by dispersion of Hg emitted into the atmosphere from the industrial site. The ongoing reclamation at Pieve Vergonte industrial site will likely decrease the mercury emissions, but contaminated soils around the facility will remain a long-term potential source. Therefore, further studies are necessary to avoid or minimize risks for ecosystems and human health deriving from the dispersion of this airborne Hg. In this sense, greater effort should be directed towards continuous GEM measurements, together with an in-depth study of how meteorological factors could help understand Hg emission/deposition cycles, Hg dispersion and potential future contamination trends. Moreover, a better characterization of soil chemico-physical properties should be planned, together with measurements of Hg emission from soil, to improve our knowledge on Hg cycling and soil potential for Hg volatilization."} +{"text": "Croton lechleri, commonly known as Dragon\u2019s blood, is a tree cultivated in the northwest Amazon rainforest of Ecuador and Peru. This tree produces a deep red latex which is composed of different natural products such as phenolic compounds, alkaloids, and others. The chemical structures of these natural products found in C. lechleri latex are promising corrosion inhibitors of admiralty brass (AB), due to the number of heteroatoms and \u03c0 structures. In this work, three different extracts of C. lechleri latex were obtained, characterized phytochemically, and employed as novel green corrosion inhibitors of AB. The corrosion inhibition efficiency (IE%) was determined in an aqueous 0.5 M HCl solution by potentiodynamic polarization (Tafel plots) and electrochemical impedance spectroscopy, measuring current density and charge transfer resistance, respectively. In addition, surface characterization of AB was performed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy techniques. Chloroform alkaloid-rich extracts resulted in IE% of 57% at 50 ppm, attributed to the formation of a layer of organic compounds on the AB surface that hindered the dezincification process. The formulation of corrosion inhibitors from C. lechleri latex allows for the valorization of non-edible natural sources and the diversification of the offer of green corrosion inhibitors for the chemical treatment of heat exchangers. S. Ss is tcurs see , where aC. lechleri [Comparative XPS survey spectra for a polished metal sample (black line), AB immersed in 0.5 M HCl (red line), and AB immersed in 0.5 M HCl with 50 ppm CL2 (blue line) are shown in lechleri . The N1slechleri ,54,55. TThe mechanism of brass corrosion in acidic media has been reported as a two-step dezincification process . Initial2+ that prevents the formation of soluble metallic chlorides, limiting the overall oxidation process [C. lechleri latex offers two opportunities\u2014the valorization of a non-edible natural source and the diversification of the availability of corrosion inhibitors for the chemical treatment of equipment in different industrial sectors. These green corrosion inhibitors might provide an option for the corrosion mitigation in the cleaning process of heat exchangers increasing their lifecycle.Our experimental results agree with this process, as evidenced by the difference in the surface composition of Cu (70.48%) and Zn (29.42%) for a polished metal sample see compared process . A compa2 antisolvent extraction techniques were successfully employed to obtain novel green C. lechleri solid corrosion inhibitors of AB in 0.5 M HCl media. CL1, which is the dry C. lechleri latex, showed the presence of alkaloid and phenolic compounds with an IE% of 30.48%. Microparticles of CL3 enriched in phenolic content provided an IE% of 48.93%, while CL2 was enriched in alkaloid compounds showing an IE% of 57%. Adsorption onto the surface of AB of the alkaloid molecules in CL2 was proposed as the mechanism of corrosion by slowing down the dezincification process of the metal sample. Further purification and processing of CL2 should improve its solubility and corrosion-inhibition efficiency. Studies at various concentrations and temperatures as well as computational simulations are performing by our group to obtain information about the adsorption process and thermodynamics.In summary, lyophilization, chloroform Soxhlet extraction, and supercritical CO"} +{"text": "In prostate cancer, extraprostatic extension (EPE) is an unfavorable prognostic factor, and the grade of EPE is correlated with the prognosis. This study aims to evaluate the utility of length of capsular contact (LCC ) in predicting the grade of EPE by correlating the measurements from MRI images and the measurements performed from radical prostatectomy specimens.MR images and specimens of 110 tumors are analyzed retrospectively. The specimens are used as reference to validate the presence of EPE and to measure the ground truth LCC. MR images are evaluated by two radiologists to identify the presence of EPE and to predict the LCC indirectly. Reliability, accuracy, sensitivity, and specificity of the evaluations are analyzed in comparison with the findings obtained from the specimens.In detection of EPE existence, the radiologists achieve almost the same performance with optimal cut-off values lead to moderate sensitivity and specificity pairs . In distinguishing high-grade EPE from low-grade EPE, the radiologists accomplish very similar performances (AUCs = 0.73 and 0.72) Optimal thresholds of 20.0 mm and 18.5 mm for the readers retrospectively reveal medium sensitivity and specificity pairs .Consistent LCC estimates can be obtained from MR images providing a beneficial metric for detecting the existence of EPE and for discriminating the grades of EPE. Accurate local staging of prostate cancer is crucial for determining the prognosis and establishing the best treatment plan [1\u20133]. The staging is highly influenced by the status of extraprostatic extension (EPE). Moreover, a greater EPE is associated with a significant prognosis of the disease. It has also an impact on surgical strategy by modifying the surgical technique, i.e. performing a wider margin of excision versus narrower margin of excision that depends on the amount of EPE. In the case of a high amount of EPE, the patient can be informed about the increased risk of positive surgical margin, and neoadjuvant therapy can be considered. Besides, such a patient can be treated with radiation therapy or hormone therapy before surgery or instead of surgery. In detecting EPE and determining the amount of EPE from pathology specimens, several sub-classification methods are proposed [4\u20137]. However, there has been no common consensus for the optimal method and sub-classification categories are exempted from the 2010 tumor node metastasis (TNM) staging system [8]. In addition to this, there is a great need for a less complicated and easy to use technique to detect EPE presence and to predict the amount of EPE.Multi-parametric magnetic resonance (MR) imaging is the most favorable imaging technique for local staging of prostate cancer [9\u201311] and also offers many imaging findings linked to EPE. When compared to the findings from clinical examination, the findings from the images are demonstrated to be more beneficial in expressing EPE . Nevertheless, a recent meta-analysis shows that MR imaging-based local staging of prostate cancer shows high specificity with low sensitivity [14]. \u2018A tumor-capsule interface of greater than 1.0 cm\u2019 is an MR imaging finding introduced in the prostate imaging-reporting and data system version 2.1 (PIRADS V2.1) guideline that linked to EPE [15]. The tumor-capsule interface, also named as the length of capsular contact, measured as the tumor contact length with prostate capsule on the images establishes a good agreement and performance [16\u201322]. However, the relationship between the length of capsular contact and the amount of EPE has not been understood fully yet. The current study aims to figure out the utility of length of capsular contact (LCC) from MR images in detecting and grading the extraprostatic extension (EPE) for prostate tumors in comparison with the measurements performed on radical prostatectomy specimens.Institutional review board approval and informed consent are secured for this retrospective study. A search on the electronic databases at our institution explored a total of 121 prostate tumors from 121 patients who underwent MR imaging before radical prostatectomy intervention. Unreachable pathology records were of concern for four tumors. The time interval between the imaging and the RP was longer than six months for five cases. MR images of the two tumors were with severe artefacts. These tumors were excluded from the study and the remaining 110 tumors were taken into consideration . In addition, a portion of the study population (approximately 70%) was used to evaluate whether the International Society of Urological Pathology (ISUP) grade group of the tumors influenced the relationship between LCC and EPE presence (ref); however, for the first time, we analyzed the role of LCC in assessment of the amount of EPE with a larger study group in our current study.MR imaging of the prostate is conducted with a 3T MRI scanner and a sixteen-channel phased-array surface coil in a multi-parametric manner. To reduce motion artifacts associated with bowel peristalsis, imaging is performed after intramuscular injection of 20 mg of butylscopolamine . The imaging protocol respectively consists of T2-weighted imaging (T2WI), fat-suppressed dynamic contrast-enhanced imaging (DCE), free-breathing diffusion-weighted imaging (DWI), and apparent diffusion coefficient (ADC) mapping with the imaging parameters listed in Table 1. Following imaging, radical prostatectomy interventions are performed. All of the specimens gathered are fixed with 10% buffered neutral formalin, then surgical margins are painted with ink. The entire prostate gland and seminal vesicles are step sectioned from apex to base at 3\u20134 mm intervals in the plane perpendicular to the long axis of the prostate gland, and hematoxylin and eosin are used to stein these sections. An index lesion is marked according to the following benchmarks; 1: the prostate tumor foci that show EPE, 2: the prostate tumor foci that have the highest ISUP grading score, 3: the tumor foci that have the largest dimension. For an index lesion identified, the absence or presence EPE and the pathologic length of tumor capsule contact on the RP specimen (p-LCC) are determined by an experienced uropathologist. In the presence of EPE, the pathological radial distance of EPE (p-RD), defined as the length of tumor protrusion perpendicular to the outer margin of the prostatic stroma, is measured additionally . The index lesion is matched with a histopathological diagram as reference standard with the consensus of the uropathologist and two radiologists by taking into account of alterations on the shape and size of the prostate caused by the preservation of specimens. The two radiologists have 12 and 5 years of experience in genitourinary radiology (B.B and A.O). They independently explore all MR images using DynaCAD prostate software . Each radiologist identifies the dominant prostate tumor foci with low signal intensity on ADC maps and high signal intensity on high b-value DWI images with or without early contrast enhancement on DCE images. Next, the radiologist measures the length of the tumor capsule interface (MR-LCC) on the axial T2W image according to the method described by Baco et al [18] using the curved measurement tool offered by the software. If the radiologist cannot manage to identify any contact between the tumor and the capsule on the images, MR-LCC is considered to be zero. During measurements, the radiologists are aware that the patients have prostate cancer verified by radical prostatectomy, but they are blinded to the demographical, clinical, and final pathology findings.p-value of <0.05 is considered for statistical significance. All analyses are performed using IBM SPSS for Windows .Mann\u2013Whitney U test or independent-samples t-test is conducted to detect significant differences in the LCC estimates for the tumors with and without EPE and for the tumors having high and low grades of EPE. Spearman Rho (\u03c1) is used to assess the correlation between the p-RD and the LCC estimates and between p-LCC and the LCC estimated from MR images. The inter-observer agreement for the MR-LCC estimates across the radiologists is determined using the intraclass correlation coefficient (ICC). Performances of the LCC estimates in the diagnosis of EPE and in distinguishing the low-grade EPE from high-grade EPE are obtained by performing receiver operator characteristic curve analyses and by calculating the area under the curves (AUC). Youden analysis is implemented to obtain the optimal threshold for the LCC, and the sensitivity (Se) and specificity (Sp) are reported for that threshold. A Radical prostatectomy specimens and multi-parametric MR images of 110 prostate tumors diagnosed with prostate cancer are evaluated retrospectively. The mean time interval between the imaging and the intervention is 73.1 days (range: 11\u2013192 days). Organ-confined disease is acknowledged for 84 tumors, and EPE is detected for the remaining 26 tumors from the specimens. The radial distance of the extension (p-RD) measured from the specimens of the EPE positive tumors gives a median value of 1.0 mm that is later used as a cut-off to categorize the high-grade and low-grade EPE positive tumors. Consequently, among the 26 EPE positive tumors, 15 tumors are figured out to be with low-grade EPE and 11 tumors are with high-grade EPE. Figures 1a\u20131c and Figures 2a\u20132c demonstrate the measurements for the two representative tumors from the study dataset. Tumor localizations in the RP specimens of the same cases are shown in Figure 1d and Figure 2d.1 and MR-LCC2 is lower for the EPE negative tumors than the EPE positive ones and an increase in both p-LCC, MR-LCC1 and MR-LCC2 is a precursor for high-grade EPE positive tumors . Significant differences are present for both p-LCC and MR-LCC between EPE negative and EPE positive tumors and also between low-grade and high-grade EPE positive tumors . Table 3 shows the correlations between MR-LCC and p-LCC from the tumors. For EPE negative tumors, moderate correlations are observable between p-LCC and MR-LCC estimates by the radiologists while slightly better correlations are noticeable between p-LCC and MR-LCCs for the EPE positive tumors (\u03c1 = 0.72 and 0.67). Moderate correlations present between p-LCC and MR-LCCs both for low-grade EPE positive tumors (\u03c1 = 0.67 and 0.62), but good correlations exist for high-grade EPE positive tumors (\u03c1 = 0.82 and 0.74). Very strong correlations are noted between MR-LCC estimates by the radiologists for all EPE cases (\u03c1 = 0.92\u20130.98). Overall, very similar MR-LCC estimates are obtained by the radiologists . On the other hand, p-RD shows a fair correlation with p-LCC (\u03c1 = 0.39); however, moderate correlations are present between p-RD and MR-LCC estimates by the radiologists for the EPE positive tumors. All correlations are significant (p < 0.05). Average p-LCC and MR-LCC estimates from all the tumors taken into analyses are listed in Table 2, and conforming boxplots are presented in Figures 3a\u20133f). Both p-LCC, MR-LCCThe performances of LCC in detecting the EPE positive tumors and in distinguishing the low-grade from high-grade EPE positive tumors are presented in Table 4, and the ROC plots obtained during analyses are as seen in Figure 4a and Figure 4b. In the detection of the EPE positive tumors, p-LCC performs fair (AUC = 0.74), and almost the same performance is accomplished by MR-LCC for both of the radiologists as shown in Figure 4a. For the optimal cut-off of 16.5mm, p-LCC reveals fair sensitivity and moderate specificity (Se/Sp = 0.58/0.77). For the optimal cut-off values of 14.5 mm and 15.8 mm for the radiologists, MR-LCC provides moderate sensitivity and specificity pairs (Se/Sp = 0.77/0.62 and Se/Sp = 0.69/0.68). Higher optimal cut-off is of concern for p-LCC achieving higher specificity but lower sensitivity when compared to the ones for MR-LCC. In distinguishing the low-grade from high-grade EPE positive tumors, almost the same fair performance is delivered by p-LCC and MR-LCC (AUCs= 0.71\u201373) as shown in Figure 4b. For the optical cut-off of 21.0 mm, p-LCC gives moderate sensitivity and specificity (Se/Sp= 0.64/0.73). The optimal cut-offs of 20.0 mm and 18.5 mm for MR-LCC reveal the same moderate sensitivity and moderate specificity . Higher optimal cut-off is of concern for p-LCC achieving higher specificity when compared to the ones for MR-LCC.Detection and grading of extraprostatic extension (EPE) of prostate tumors are remarkably important for precise local staging of prostate cancer and management of the patients suffering from prostate cancer [4\u20137]. Multi-parametric MR images offer several metrics to improve the accuracy of prostate cancer staging. In the current study, the utility and the reproducibility of the length of capsular contact estimated from the multi-parametric MR images of the prostate tumors have been assessed for the purpose.The length of capsular contact estimated from the multi-parametric MR images of the prostate tumors (MR-LCC) is reported to be the most prevalent and relatively objective imaging measure satisfying fair to good performances with good inter-reader agreements in detection of EPE existence . Every 1 mm increase in the measure is thought to be linked to a 4% increase in the risk of EPE [19]. The optimal threshold for detection is associated with the balance between the sensitivity and the specificity and takes values from 6 mm to 20 mm [18\u201322]. In the current study, LCC measurements performed independently by two radiologists reveal moderate performances and very similar optimal cut-off values (i.e. 14.5 mm and 15.8 mm) that lead to moderate sensitivity and specificity in the detection of EPE positive tumors. LCC estimates from MR images offer good inter-observer agreements. The results of the current study is in consistent with the previous studies that reported fair performance with good interobserver agreement rates [18\u201322]. We recomended the LCC cut off value of 14.5 mm for detecting EPE in prostate cancer.For grading EPE for an EPE positive prostate tumor, the radial distance of EPE, described as the length of tumor protrusion perpendicular to the outer margin of the prostatic stroma, has been voted as a beneficial metric. However, several studies demonstrate that if used as a continuous metric, the radial distance determined from the radical prostatectomy specimens is insignificantly correlated with prognosis. Besides, an increase in the metric is shown to be signi\ufb01cantly associated with an increase in the risk of biochemical recurrence . Significant correlations can be obtained when the metric is converted into its categorical form by performing thresholding, and the median of the radial distance from a large population of prostate is recommended as an optimal threshold . In agreement with these studies, an optimal threshold of 1.0 mm is determined in the current study and later used to categorize the high-grade and low-grade EPE positive prostate tumors. In the current study, LCC estimate from multi-parametric MR images provides fair diagnostic performance and reveals moderate sensitivity and specificity in discriminating high-grade from low-grade EPE positive tumors with the optimal LCC cut-offs of values of 20.0 mm and 18.5 mm. The performance of the MR-estimated LCC in discriminating the EPE-positive patients according to the amount of EPE has not been previously studied. Nevertheless, the distinct cut-off values for diagnosing any EPE and established-high grade EPE has been published only in one previous study that reports the optimal thresholds of MR estimated LCC as 6 mm for detecting EPE and 10 mm for diagnosing established-high grade EPE [20]. When compared to that work, higher optimal LCC thresholds are reported for the detection and the diagnosis in the current work. This difference can be explained by two different perspectives. Firstly, LCC is obtained for an index lesion localized within the entire prostate gland in the current study, while it is measured for the dominant lesion localized within each of the lobes of the prostate gland in [20]. Secondly, while the cut-off value of pathological RD was taken as 0.5 mm for classifying patients as focal versus established in [20]., the current study reports the cut-off value of 1.0 mm for pathological RD to discriminate the high-grade EPE from the low-grade EPE. The median value of the pathological RD obtained from a patient population is recommended for use as a cut-off for discriminating the high-grade from the low-grade EPE positive tumors. A threshold value of 1.0 mm is obtained in the current study that is in accordance with some previous studies one of which enrolls the largest study cohort of EPE positive tumors and utilizes 1.0 mm cut-off as the optimal threshold for RD that is signi\ufb01cantly associated with the increase in BCR risk [24\u201326]. We recommend the LCC cut off value of 18.5 mm for distinguishing low grade EPE from high grade EPE.The results of the current study note a moderate correlation between pathological RD and the MR estimated LCC, while a poor correlation is demonstrated between pathological RD of EPE and pathological LCC. This is an unexpected finding that required an explanation on whether there is a significant difference between pathological LCC and MR determined LCC measurements according to the EPE status. We observed that pathological LCC and MR determined LCC measurements are highly correlated in all groups when patients are classified according to EPE status. However, this correlation is much stronger in patients with a high amount of EPE than in patients with a low amount of EPE (Table 3 ). Bak\u0131r et al. reports that, in the low ISUP grading group, the pathological LCC and MR estimated LCC measurements are less concordant, and statistical results involving the LCC and EPE relationship for pathological LLC and MR estimated LCC measurements are diverging [23]. Considering more aggressive tumors with higher ISUP grading group shows a higher tendency to extension beyond the prostatic capsule; the findings of the current study is in accordance with the literature. LCC measurements from MR images may be overestimated or underestimated for less aggressive prostate tumors, and this might cause lower correlations with LCC estimates from radical prostatectomy specimens. Tumor aggressiveness may play a role in establishing the relationship between LCC and EPE. There are some limitations of the current study. The study has a retrospective design, and this may lead to some selection bias for the prostate tumors taken into analysis. The study dataset covers a large number of prostate tumors, but the number of EPE positive tumors in the dataset is limited. Consequently, the results reported may not be generalized well for the EPE positive tumors. Although MR images of the prostate tumors are with high quality, the length of capsular contact may be under- or over-estimated especially for less aggressive tumors due to resolution margins of the recent MR imaging technology. In conclusion, multi-parametric MR images deliver reliable estimates of the length of capsular contact for prostate tumors that can be used in detecting and grading extraprostatic extension for the tumors in local staging of cancer and selection of appropriate surgical plan. We suggested further prospective studies with larger study cohorts to clarify potential benefits, and computational tools are needed to be developed to promote the use of MR-derived length of capsular contact in clinical practice. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u00a0Institutional review board approval and informed consent are secured for this retrospective study ."} +{"text": "Glucocorticoids are essential endogenous steroid hormones secreted in response to stress, and treatment with synthetic forms is widespread for numerous inflammatory conditions including asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, and arthritis . EmerginAs such, the recent prospective cohort study by Cairat et al. is an imUnlike prior studies, Cairat et al. were able assess whether the associations of glucocorticoids and breast cancer differed according to hormone receptor status, histological subtype, tumor grade, and stage at diagnosis. This is particularly important given the various pharmacological properties of glucocorticoids and evidence for cross-talk between estrogen receptors and glucocorticoid receptors in mammary epithelial cells , which sThis study is informative in teasing apart heterogeneity in glucocorticoid-breast cancer associations by hormone receptor status to isolate the potential impact of estrogen pathways. As authors of this study note, estrogen inhibition induced by glucocorticoids may explain the lower risk of ER-positive tumors observed in this study. Moreover, given that ER-positive tumors tend to be less aggressive than ER-negative tumors, the inverse associations observed for early stage disease may reflect the preponderance of ER-positive tumors in this group. Systemic glucocorticoid use also affects immune system function and insulin sensitivity, which could induce adipose aromatase activity and estrogen production and also directly stimulate breast cancer cell growth and invasion , therebyThe potential impact of these findings are substantial, given the widespread and increasing use of glucocorticoids over the past several decades , 9, and In conclusion, this study\u2019s findings are intriguing and merit follow-up to further characterize the potential role of glucocorticoids and other anti-inflammatory therapies in breast cancer etiology. Specifically, future studies recognizing the complex interplay between inflammation and adaptive immune response in breast cancer are needed to inform our understanding of etiology and the development of personalized medicine approaches in breast cancer."} +{"text": "In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-of-concept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken.We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Re-challenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. Mesothelioma, a rare aggressive cancer that develops in mesothelial cells lining the pleura and peritoneum \u20133 is chaTMEM173 is an endoplasmic reticulum (ER) signaling molecule that plays a key role in host defense by responding to pathogen or self-derived DNA, reviewed by (Stimulator of interferon genes (STING) encoded by iewed by . Activatiewed by . STING-eiewed by , 15\u201318.DMXAA , a flavone-acetic acid-based drug, directly binds murine STING , 20 has We have shown that chemotherapy-induced tumor cell death expands the T cell response to dominant and subdominant epitopes using murine AE17sOVA that expresses ovalbumin (OVA) as a neo tumor antigen in which SIINFEKL is the dominant MHC class I epitope of OVA, whilst KVVRFDK is a subdominant epitope . RevealiThis study aimed to identify a DMXAA treatment regimen that: modulates mesothelioma tumor vessels; enhances tumor antigen presentation and promotes functional dominant and subdominant tumor antigen-specific CTL that infiltrate mesothelioma to mediate tumor regression. We further hypothesized that DMXAA could render the tumor microenvironment more permissive and responsive to biological molecules such as agonist anti-CD40 antibody or IL-2 to further enhance the immune response.DMXAA, kindly provided by Professor Lai Ming Ching as the sodium salt, was freshly dissolved in PBS for each experiment.2 humidified atmosphere.AE17 and AB1 are murine mesothelioma cell lines derived from the peritoneal cavities of C57BL/6J and Balb/c mice respectively after injection with asbestos fibres . Injecti3 cells/well for 24 hours at 37\u02daC in 5% CO2. DMXAA was diluted 1mg/ml and 50\u00b5l added to 150\u00b5l RPMI for serial dilution in RPMI without serum; 100\u00b5l of DMXAA was added to each well in duplicate for 24-hour incubation. Each well was treated with 50\u00b5l of 2mg/ml 3--2,5-diphenyltetrazolium bromide in PBS and incubated for 4 hours at 37\u00b0C in the dark. After 5-minute centrifugation at 1000 rpm at 25\u02daC, cell pellets were treated with 100\u00b5l DMSO, and incubated in the dark for 30 min at RT with continuous shaking. Wells were read at 595nm using a Biorad 3550 microplate reader .MTT -2,5-diphenyltetrazolium bromide) tetrazolium reduction assays assess cellular metabolic activity due to the enzymatic action of mitochondrial dehydrogenase present in viable cells providing an indicator of cell viability and proliferation. Cells were seeded into 96 well plates at 5 x 105 cells/ml in media and incubated at 37\u00b0C and 5% CO2 for 24 hours were treated with different DMXAA concentrations and incubated under the same conditions for a further 24 hours. Adherent cells removed using trypsin were centrifuged at 400g for 10 minutes when the pellet was re-suspended in sample buffer (1g glucose in 1L PBS) and centrifuged at 400g for 10 minutes; this step was repeated once more. Cells were then vortexed in 70% ice-cold ethanol, fixed overnight at 4\u00b0C, vortexed and centrifuged at 3,500g for 5 minutes. The ethanol was discarded and the cell pellet vortexed to re-suspend cells in residual ethanol and 300ul PI staining solution added to each tube for 30 minutes in the dark at RT. Samples were analyzed using flow cytometry .PI integrates with DNA indicating the amount of DNA present. PI staining intensity is directly proportional to the amount of DNA present in a cell, representing the cell cycle status of the cell . Six-well plates seeded with 1.5 \u00d7 10Female C57BL/6J mice aged 6-8 weeks obtained from the Animal Resources Centre were maintained under standard animal housing conditions at Curtin University\u2019s animal holding facility. The OT-1 (H-2b) TCR transgenic mouse line, expressing a TCR recognizing the dominant H-2b OVA epitope (SIINFEKL) was kindly supplied by Professor I. Frazer and Dr. R. Steptoe and bred in house at the University of Western Australia (UWA) animal holding facility. All mice were used in accordance with institutional guidelines and UWA and Curtin University\u2019s Animal Ethics Committee approval .5 tumor cells per mouse in 100 \u03bcl of PBS. Mice were monitored daily and tumors measured using microcallipers. Mice were sacrificed using methoxyflurane in accordance with specific AEC approval requirements.Eighty percent confluent AE17 and AB1 tumor cells were harvested using trypsin and prepared for injection by washing twice in PBS. Viability was always greater than 90%. Mice were injected subcutaneously (s.c.) in the left flank with 5 x 10After removing tumors and organs, single cell suspensions were obtained by gently mashing between two frosted glass slides, removing debris by filtration through 30\u03bcm nylon mesh , centrifuging cells at 1200 rpm for 6 minutes and re-suspending the cell pellet in PBS/2% TBS. Where relevant, lysis buffer was used to lyse red blood cells.6/ml and 200 \u03bcl/well transferred into 96 well plates for staining with 50\u03bcl of antibodies: CD3-PE ), CD8-PerCP-Cy5-5 and CD4-APC-Cy7 or their isotype controls (BioLegend). Cells were incubated for one hour at 4\u00b0C, washed in PBS and fixed in 2% paraformaldehyde for 15 minutes at 4\u00b0C, all in the dark. Fixed cells were washed in PBS/2% TBS and data acquired on a FACSCanto II (BD) using Diva software and analyzed using FlowJo v10 software (BD).Cells were washed in PBS/2% TBS, adjusted to 10in-vivo using adoptively transferred T cells from OT-I TCR transgenic mice, which express a TCR recognizing the MHC class I H-2b-restricted dominant OVA epitope, SIINFEKL (7cells/ml and incubated with 2.5\u03bcM CFSE (stock solution 5mM in DMSO) for 10 min at RT. Cells were washed through an FCS underlay twice and PBS alone twice, and 107 cells injected i.v. into each recipient mouse. CFSE-labeled cells were recovered from lymphoid organs 3 days post adoptive transfer and OT-1 CD8+ cells analyzed by FACS analysis.The AE17-sOVA model was used to assess the ability of antigen presenting cells (APCs) such as DCs to present the neo tumor antigen (OVA) SIINFEKL , 33. OT-SIINFEKL , 32, 33.2O2) solution , avidin blocking solution A, followed by biotin blocking solution B, for 10-15 minutes . After one PBS wash, sections were incubated at RT for 45 mins with a rat anti-mouse Platelet Endothelial Cell Adhesion Molecule-1 (PECAM or CD31) IgG2a antibody or the rat IgG2 isotype control, washed three times followed by sequential incubations with a biotinylated anti-rat antibody for 45 minutes and streptavidin-horse-radish peroxidase for 30 minutes. To detect color, Sigma FAST\u2122 (D-4168) DAB tablets were used as a peroxidase substrate, as per manufacturer\u2019s instructions. Slides were rinsed in PBS, counterstained with Mayer\u2019s hematoxylin for 2 mins, washed with tap water, mounted in aqueous mounting media with cover slips .Cryocut sections (10\u00b5m) fixed in fresh 4% paraformaldehyde for 20 minutes were washed in PBS and sequentially blocked with 1% hydrogen peroxide (H257-264 (SIINFEKL) and subdominant peptide OVA55-62 (KVVRFDKL) were manufactured by the Centre for Cell and Molecular Biology at a purity of >89%.The dominant peptide OVAin-vivo CTL assay or low (0.05\u00b5M) CFSE concentrations respectively. Control uncoated target cells (the 3rd population) were labeled with an intermediate concentration of CFSE (0.5\u00b5M). For i.v. injection, 1 x 107 cells of each population were mixed in 200\u00b5l PBS per recipient mouse. Specific in-vivo cytotoxicity was determined by collecting lymphoid organs and tumors 18 hours later, and CFSE+ cells detected by flow cytometry.We used our modified \u20183 peak\u2019 TL assay , 34, 35.Int/CFSEhigh) was calculated to obtain a numerical value of cytotoxicity for each mouse. Further controls included na\u00efve (no-tumor) mice, mice bearing parental AE17 tumors that do not express OVA and tumor-bearing PBS-treated recipient mice. To normalize data, allowing inter-experimental comparisons, the data was expressed relative to the no tumor control mice that were included in every experiment, as previously described . Lyophilized PROLEUKIN\u00ae was reconstituted in sterile PBS (Sigma) and routinely assayed for its bioactivity using a murine IL-2-dependent cytotoxic T cell line (CTLL), as previously described (Agonist rat anti-murine CD40 antibody (Ab) (FGK45) kindly provided by Professor C. Melief was expanded by the Monoclonal Antibody Facility and diluted in PBS to 0.4mg/ml so that 40\u03bcg/100 \u03bcl/injection could be delivered intra-tumorally (i.t.) escribed . Six i.tescribed , 36.Statistical analyses were performed using GraphPad Prism 8 . One-way Analysis of variance (ANOVA) was used to compare differences between multiple groups followed by a multiple comparisons test to compare two selected groups. The Mann-Whitney U test was used to examine differences between two groups; p values of less than 0.05 were considered significant.The first experiments examined whether DMXAA exerted toxic effects on two murine mesothelioma cell lines (AE17 and AB1) using the MTT assay. At 20 and 40 hours the MTT data suggest low doses between 0.1 and 100 \u00b5g/ml increased metabolic activity, and by 60 hours metabolic activity was higher than the controls at the same doses (Flavones such as DMXAA exert their cytotoxic effect by interrupting the cell cycle at the G2/M phase . TherefoDMXAA has been reported to induce tumor regression following intravenous (i.v.) (refs). We believe that larger tumors better represent human mesothelioma, as they are mostly associated with a longer time to develop. Therefore, we propose that DMXAA needs to overcome the greater suppressive tumor microenvironment seen in larger tumors before it could be considered a potentially translatable therapeutic. We have also shown that local delivery of immunotherapy is more effective and less toxic than systemic delivery were treated i.p. with one dose ranging from 6.25mg/kg to 30mg/kg DMXAA. The lower doses demonstrated minimal anti-tumor or toxic effects (Others have shown that the maximum tolerated dose (MTD) for DMXAA in mice is between 25mg/kg or 30mg/ effects Table\u00a01.A second experiment consisted of four groups of AE17-tumor-bearing mice. Two groups commenced treatment when their tumors were small, the other two groups had large tumors. The DMXAA dose and treatment regimen for all groups was 25mg/kg, followed by two maintenance doses of 5mg/kg, with 3-day intervals given i.v. Tumor reduction was seen in small, but not large, tumors maintenance doses of 5mg/kg, all given i.p. induced short-term tumor regression in 2/4 mice with mice given two thirds of the effective dose to ensure sufficient tumor remained for analysis, i.e. AE17sOVA-bearing mice were given two i.t. injections of PBS or 25mg/kg DMXAA 9 days apart and left for four days before adoptive transfer of CFSE-labeled OT-1 cells, followed by organ collection 24 hours later. Proliferation of CFSE-labeled, SIINFEKL-specific CD8+ T cells was used as a real-time, in-vivo indicator of antigen presentation. Disaggregated cells from draining lymph nodes (DLN), non-draining LN, spleen, and tumors were stained for, and gated on, CD8+ cells and proliferating CD8+ OT-1 cells identified by CFSE expression, as previously described was calculated to obtain a numerical value of cytotoxicity for each mouse. To normalize data, allowing inter-experimental comparisons, the data was expressed relative to the no tumor control mice that were included in every experiment. That is, relative killing was calculated by determining the ratios between the percentages of peptide-coated targets in no tumor control mice versus tumor-bearing mice and multiplying by 100 to obtain a percentage value.The ratio between the percentages of uncoated (the Ref peak) versus peptide-coated targets in PBS-treated mice, with lower response levels to the subdominant (KVVRFDKL) epitope in DLNs, spleens, non-draining LN and tumor , 35 and + tumor vessels gene expression and cell death pathways enhanced antitumor CD8+ T responses that controlled murine melanoma and colon tumors of DMXAA directly induced AE17 tumor cell death; this was more effective over longer periods of time (40 and 60 hours). Lower doses (100\u00b5g or less) did not affect tumor cell viability and pushed cells into the G2/M phase of the cell cycle. We did not look at other cell types such as DCs, although others have shown that DMXAA activates DCs targeting the programmed death protein-1 (PD1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) pathways with a STING agonist. However, ICIs on their own or in combination, as first, second or third line treatment have yet to be fully evaluated before a consensus can be reached \u201358 makinIn summary, we show that DMXAA enhanced mesothelioma-associated tumor antigen presentation and that tumor-specific T cells more readily infiltrated tumors. However, tumor-specific CTL function was blunted. Our data also shows that STING treatment regimens need to be carefully designed, as there is a fine line between achieving tumor resolution and maintaining immune control, particularly T cell control, without unwanted toxicity. Collectively, the data show that local delivery is the optimal approach that enhances tumor antigen presentation. However, there is a risk of compromising CTL function. Nonetheless, mesothelioma resolution was achieved, supporting further research involving the use of STING agonists for this devastating disease. The role of STING agonist in combination with contemporary clinically used checkpoint blockade targeting the PD1 and CTLA-4 pathways remains unclear.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.The animal study was reviewed and approved by Curtin University Animal Ethics Committee and the University of Western Australia\u2019s Animal Ethics Committee.DN and AN conceived the ideas and concepts, successfully applied for funding and oversaw all aspects of the project including data analysis and publication preparation. PG, SC and IL performed the experiments and analyzed the data. All authors contributed to the article and approved the submitted version.Funding from the Cancer Council of Western Australia and the New South Wales Workers\u2019 Compensation Dust Diseases Board equally funded this project.Author IL was employed by Becton Dickinson Pty Limited.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Background and Objectives: In the presence of a persistent endodontic lesion or endodontic failure, the alternative for the recovery of the dental element is endodontic retreatment or endodontic surgery, which consists in the surgical removal of the root apices with retrograde closure of the endodontium. The objective of this systematic review and meta-analysis was to provide an updated value of the Risk Ratio between the two types of treatment in order to offer to clinicians who propose a non-surgical endodontic retreatment or an endodontic surgery a direct comparison. Materials and Methods: The revision was performed according to PRISMA indications: three databases were consulted through the use of keywords relevant to the revision topic: surgical endodontic retreatment, endodontic retreatment, apicoectomy. This search produced 7568 records which, after eliminating duplicates and applying the inclusion and exclusion criteria, resulted in a total of seven included articles. The meta-analyses were conducted by applying fixed-effects models, given the low percentage of heterogeneity. In addition, trial sequency analysis (TSA) was performed for the analysis of the statistical power of the results and GRADE for the quality of the evidence. Results: The results of the meta-analyses\u2019 data report an aggregate risk ratio (RR) between non-surgical endodontic retreatment and surgical endodontic retreatment of: 1.05 at one year of follow-up; RR 2.22 at two years of follow-up; an RR 1.08 [0.73 1.62] for a follow-up period of 3\u20134 years; and an RR 0.92 for a follow-up period of 8\u201310 years. Conclusions: The results of the present meta-analysis show that in the long term, the risk of failure is identical for the two groups, and there is only a slightly higher risk of failure for non-surgical endodontic retreatments, when only two years of follow-up are considered. The primary purpose of endodontic treatment is the complete disinfection and cleansing of endodontic canals from the presence of microorganisms and from potentially infected pulp endodontic tissue, with the aim of obtaining a completely shaped and disinfected space that can accommodate the canal filling material and obtain closure three-dimensional endodontium with a reduction in the possibility of recurrence of the endodontic pathology.The primary treatment of the canal does not always achieve its objectives, and after years, there may be endodontic failure, the prevalence of which could reach about 30% of endodontically treated teeth, as reported in large cross-sectional studies .Enterococcus faecalis, resistant to the action of irrigants, especially if not used for an appropriate time, can survive in the dentinal tubules, aggregating itself in structures such as the biofilm [The causes of a failure are to be found in the persistence of bacteria inside the endodontium, due to the presence of spaces not adequately left empty and/or cleaned and disinfected. in this way, the bacteria have the possibility, if in contact with the external biological fluids, to survive and proliferate giving a secondary lesion . Moreove biofilm .Actinomycetes spp. (Propionibacterium and Actinomyces), affecting the dental element external root surface and leading in this way to a persistent extra root lesion [The biofilm can also consist in t lesion .In the presence of a persistent endodontic lesion or endodontic failure, the options for the recovery of the dental element are non-surgical endodontic retreatment or surgical endodontic retreatment, which consists in the surgical removal of the root apices with retrograde closure of the endodontium ,6.I.Evaluation of the coronal seal: the presence of an adequate coronal restoration or a still functional prosthetic crown that seals the endodontium coronally or whose removal involves the non-restorability can lead to the choice towards surgical endodontic retreatment. On the contrary, a restoration no longer adequate, with good access to the endodontium, may lead to the choice of non-surgical endodontic retreatment .II.Radiographic evaluation of root canal obturation: the presence of untreated canals and a coarse filling of the canals may lead to the choice of non-surgical endodontic retreatment. On the contrary, a surgical approach could be recommended in the presence of an apparent adequate apical seal or in the presence of a root canal obturation that is far from the radiographic apex of only 1 mm , and a surgical approach should be evaluated ,10,11.III.The presence of clinical complications such as the finding of fractured instruments , the presence of zipping of internal resorptions and the presence of root perforations or ledge formations; in these cases, the surgical approach may be a suitable choice .IV.In the presence of recurrent infections in which a root canal retreatment has already been carried out, a surgical endodontic retreatment is absolutely to be considered as a therapeutic choice .In accordance with Karabucak and Setzer , the criThe survival rate of teeth that received non-surgical endodontic retreatment was 85% after 72 months, 86.8% after 48 months and 90% after 24 months. The survival rate of teeth that received surgical endodontic retreatment was 88% after 72 months, 90.5% after 48 months and 93.7% after 24 months . These dThere is only a previous systematic review conducted by Del Fabbro et al. in 2010 , which dHowever, two other reviews by Torabinejad et al. and KangTaking into account these premises, the aim of this systematic review and meta-analysis was to provide, in the light of new studies and the advent of new endodontic biomaterials such as bioceramics ,22,23, aThe writing of the review was carried out following the indications of the PRISMA and was conducted based on the indications of the Cochrane handbook ,25; the All retrospective prospective studies and RCTs evaluating the number of failures of teeth undergoing non-surgical endodontic retreatment and those undergoing surgical endodontic retreatment with retrograde filling were considered. In addition, the PICO question was formulated, and the following points were identified: (P)articipants , (I)ntervention , (C)ontrol and (O)utcome . The PICO question, therefore, was the following: \u201cWhat is the risk ratio between teeth undergoing non-surgical endodontic retreatment and those undergoing surgical endodontic retreatment?\u201d.The inclusion criteria were: (1) studies that clearly indicated the number of teeth undergoing non-surgical endodontic retreatment and (2) the number of teeth undergoing surgical endodontic retreatment with related failures. The studies to be included must necessarily have investigated both methods of retreatment in the same patient court.The exclusion criteria were: (1) studies published in a language other than English, (2) those that did not present data on retreatment failures and successes and (3) those at high risk of bias.Exclusion criteria were not considered studies for surgical and endodontic techniques that involved the use of microscopy, ultrasonic instruments and biomaterials such as bioceramics.The studies were identified through bibliographic searches in electronic databases by two investigators (M.D. and D.S.). Restrictions on the language of publication have been applied, and articles in a language other than English have been excluded. The search was performed on 3 different databases: Scopus, PubMed, and Cochrane library registry. The latest literature search was conducted on 11 March 2022. In addition, Google Scholar and Open Gray literature search were also consulted, for sources not otherwise identifiable, and systematic reviews were investigated in search of further records.We used the following database search terms: surgical endodontic retreatment, endodontic retreatment, apicoectomy. Duplicate results were removed using the EndNote x8 software , and the overlaps of articles that could not be uploaded to EndNote were manually removed after the screening phase. The records identified were assessed and examined by 2 reviewers (MD and DS) separately, the screening included the analysis of the title and abstract, and the studies potentially eligible to be included in the systematic review were read full text; in addition, a third reviewer (A.B.) was tasked with resolving doubtful situations.The data to be extracted were previously established by the 2 authors responsible for screening the articles and were reported independently in 2 tables to be compared later to reduce the risk of errors. The data extracted from the studies concerned the first author, the type of study, the year of publication, the country that conducted the study, the number of teeth, the number of patients, the endodontic filling used, the type of retreatment performed, the causes of the retreatment failure, the number of failures and the follow-up period.I2).The risk of bias in the individual studies was assessed by one author (M.D.), with a second author in charge of verifying the correct assessment (D.S.) For the risk of bias, a different scale was adopted from that proposed by the Cochrane handbook, because the latter is studied for RCTS, as the current systematic review, also includes prospective or retrospective non-randomize studies. It was considered to adopt a different tool evaluation checklist used by Mcfarlane (2001) ,27. StudI2. An I2 value greater than 75% was considered high, and an analysis of the effects was applied, in specific cases and through a funnel plot: the meta-analysis presented high heterogeneity indices, and a sensitivity analysis was performed, excluding only studies that had low confidence interval overlap or that emerged graphically from the funnel plot.The risk of bias between studies was evaluated graphically through the analysis of the overlaps of the confidence intervals, through the For the meta-analysis, and in particular for the calculation of the aggregate RR, the software Reviewer Manager 5.4 was used. We used the online software GRADE pro-Guideline Development Tool , to assess the quality of the evidence. The trial sequency analysis (TSA) was performed using Stata 13 , with the implementation of the R 4.2 software and by installing the idbounds and metacumbounds commands .The search in the Cochrane library databases, Scopus and PubMed provided in total 7568 bibliographic citations, removing the overlaps manually and using software (Endnote) and excluding the articles that did not meet the legibility criteria when reading the abstract, resulting in 54 potentially admissible articles. Following the full text reading, the number was reduced to only 7 studies that met the inclusion and exclusion criteria and were included in the meta-analysis. Furthermore, the gray literature analysis and the previous systematic reviews did not allowed to identify additional studies to be included in the meta-analysis . FinallyArticles included in the meta-analysis are as follows: Danin et al. 1996) , Curtis 96 , CurtOnly 7 studies were included in the meta-analysis; the articles included Retrospective Clinical Studies and RCTs, whose data were published between 1989 and 2018, and included a follow-up period from 1 to 10 years. The total number of patients investigated was 1833 (including those lost during follow-up and those excluded from retreatment), with 579 teeth retreated non-surgically and 454 retreated surgically, and most of the studies performed an orthograde and retrograde filling with gutta-percha: Curtis et al. (2018) reports Data on treatment failure have been grouped by years of follow-up, in order to simplify the execution of meta-analysis; the periods considered were 1 year, 2 years, 3\u20134 years and 8\u201310 years of follow-up.Risk of bias was assessed using the checklist used by Mcfarlane (2001) , which m(1)Non-response rate: Is the participation on/follow-up rate stated? Do the authors describe the effort to increase the participant/follow-up rate?(2)Representativeness of sample to target population: Were the subjects asked to participate in the study representative of the entire population from which they were recruited?(3)Validity and reliability of outcome measurement: Were the main outcome measures used accurate ?(4)Amount of loss to follow-up: Are the non-participants/subjects lost to follow-up described? Do the authors describe the effort to increase the participation/follow-up rate?(5)Appropriate statistical tests: Are the statistical methods described?To each category was assigned a value from one to 5 (where one = low and five = high). The questions that the review answered by assigning the score were the following:Studies and reports presenting a high risk of bias were not included in the analysis quantitative. Articles with a high risk of bias were excluded from the table and eliminated during the inclusion phase. The assessment of the risk of bias of the 7 included studies was conducted by the first Author (M.D.).The meta-analysis was performed using the Rev manager 5.4 software: in total 4 meta-analyses were performed by calculating the aggregated RR as a function of the follow-up periods of 1-year, 2-years, 3\u20134-years and 8\u201310-years follow-up, in order to reduce the bias between the data in the different studies and consequently to reduce the bias and heterogeneity between the studies. The aggregate RR was calculated for each analysis by applying a fixed effects model.2 = 3.40, df = 2 (p = 0.18), and the Higgins index (I2) is 41%.For the calculation of the aggregate RR for a one-year follow-up period, we have data from only 3 studies: Danin et al. (1996) , Allen ep = 0.80). The forest plot shows a neutral RR between the 2 groups . All the three studies intercept the line representing the confidence intervals, the central line of no effect , and I2 was 39% , Kvist a was 39% .I2 = 0 , with the integration of the R 4.2 software via the Metacumbounds commands as described by Miladinovic et al. .The O\u2019Brien\u2013Fleminge expense function was used by applying fixed effects. The AIS (accrued information size) and subsequently APIS (a priori information size) commands were used by the Dialog BOX to determine the optimal sample size and for the power of the results, assuming a RRR (relative risk reduction) of 30%, an Alpha value equal to 5% (type 1 error) and a beta value at 20% (type 2 error) .The TSA of the failure data included in the first meta-analysis highlights how the \u201cZ\u201d curve does not cross (Z = 1.96) indicating no evidence B. The meInstead, from the TSA of the failure data included in the second meta-analysis, it was noted how the \u201cZ\u201d curve crosses (Z = 1.96), providing a significant result but a spurious effect because the \u201cZ\u201d curve does not exceed the monitoring limit D.If in the first TSA, there was no evidence, with the possibility of having a false negative result. As an alternative, in the second TSA, we have a statistically significant result, but with the possibility of a false positive result. Furthermore, from the APIS graph, it is clear how the optimal number of patients to be included considering a RRR of 30% is 2578.The authors also performed a GRADE pro-GDT, to evaluate the quality of the results . The resIn this work, we conducted a systematic review with meta-analysis of data published in the literature on endodontic retreatments, trying to provide a complete picture of what was the least risk of failure treatment between surgical versus non-surgical endodontic retreatment, providing the most up-to-date data to the endodontist practitioner. In addition, to our knowledge, this is the first meta-analysis with TSA that included only studies that investigated the two therapeutic choices, with a direct comparison.The previous 2010 systematic review conducted by Del Fabbro et al. shows onAnalyzing the failures between the various follow-up periods, it emerges that, for the follow-up periods of 1 year, 3\u20134 years and 8\u201310 years, there is no systematic difference in the failure rates between surgical endodontic retreatments and non-surgical endodontic retreatments, while for the period a period of 2 years, the meta-analysis reports an aggregate RR of 2.2 in favor of surgical endodontic retreatments. Numerous studies agree that a follow-up period of 2 years is a suitable period of time to obtain valid results on success in retreatments . Indeed,In fact, the TSA conducted on the RR data on failures at one year shows that for the three included studies, there is no adequate statistical power of the results, indicating the absence of evidence, with a possibility of a false negative result; instead, if we consider a follow-up period of 2 years, the condition changes, and the TSA shows statistically significant data, but with the possibility of incurring a false positive result for the presence of a spurious effect determined by confounding factors, indicating how, in order to be confident of a true positive result, the optimal number of the patients included in the studies is 2578.The majority of the systematic reviews with meta-analyses data, including the Cochrane studies, present a number of studies with randomized participants which is too low to hold the adequate statistical power that would allow an adequate evaluation of the intervention effect size. The results deriving from a meta-analysis with few studies, and consequently with few participants, could be not very credible, with the risk of overestimation or of being underestimated, as they do not possess sufficient statistical power to accept or reject interventions even with large effect sizes .The results shown by the TSA are also confirmed by the evaluation of the quality of evidence (Grade), where moderate certainty is obtained for the aggregate RR for a follow-up period of 2 years.However, the individual studies agree that there is not a big difference between the failure rates between the two therapeutic choices.In 2018, Riss et al. reportedAlthough Cali\u015fkan et al. reports The main risk incurred by reading the resulting data of this meta-analysis is that of over-interpreting the data at 2 years: the analysis we conducted did not show statistically significant differences compared to 1, 3\u20134 and 8\u201310 years. Suggesting one treatment or the other, the 2-year period could be considered suitable in many studies, but the success of an endodontic therapy should be evaluated in reasonably longer follow-up periods, consistent with the planned duration of an endodontic therapy.Surgical endodontic retreatment has disadvantages that could nullify the improvement obtained in the short term. In fact, Riis et al. reports Furthermore, it is increasingly clear from the data in the literature that vertical fracture is among the main causes of failure in teeth with surgical endodontic retreatment. In fact, this issue emerges from a study on the incidence of vertical root fractures between conventional versus surgical endodontic retreatment conducted by Karygianni et al. , in whicIn this review, we also focused on the type of biomaterials used to obtain the apical seal, in order to understand if it could represent a cause of a different failure rate.According to a recent literature review conducted on root canal filling materials performed by Komabayashi et al. , the TriThe limitations of this systematic review are that the results, even if in part significant, show a small number of participants included, as also highlighted by the TSA.The results of this study indicate that clinicians can choose either non-surgical retreatment or root-end surgery after failed primary root canal therapy. Finally, only in the short-term period (2 years), with balanced RR values, data regarding surgical endodontic retreatments seem more in favor in terms of healing.As shown in the results , Curtis 2 : they reported 14 endodontic failures , of which in total, 10 causes were due to an error in filling the canal , 2 were due to broken instrumentation and, finally, 2 were due to loss of the coronal seal.Ercam et al. performeIn the other included studies, bioceramics or more generally, Tricalcium silicate or Salycilati, were not used as root-end fillings.Indeed, the studies by Kvist and Reit , Allen eThe data of the failures extrapolated and presented in The analysis of possible biases within the present systematic review was addressed and planned on three fundamental points. The first point is the presence of a publication bias; the real issue was the small number of articles included and the analysis of the overlap of the confidence intervals, and in order to reduce the heterogeneity, the data meta-analyzed were collected according to the follow-up period, in order to have data that could be as homogeneous and comparable as possible. In addition, an analysis of the sources of heterogeneity through funnel plots was also performed and tThe execution of a subgroup analysis was also considered in relation to the type of endodontic sealer used but it has not been performed for too few studies within each single meta-analysis .The third point is the risk of bias within the studies, and it was addressed through the evaluation of a scale, whose checklist is used and widely described in the results Based on the results of the systematic review and the data from the TSA, some objectives are recommended on which retreatment and surgical endodontic retreatments studies should focus.Primarily, to increase the level of scientific evidence, studies of good quality with a low risk of bias and which foresee the identification of univocal prognostic predictors of failure or success must be put in place. Secondly, the data from the studies could be compared much more easily if they were represented in a standardized way through the use of a format or tool that clearly presents success and failure criteria; thirdly, the data from the TSA indicate that further studies on surgical endodontic retreatments are needed compared to non-surgical endodontic retreatments with long-term outcomes.On the basis of this study, surgical endodontic retreatments after 2 years of follow-up represent a predictable treatment choice with guarantee of initial successful outcome with a lower risk of failure compared to non-surgical endodontic retreatments. However, future long-term clinical studies on surgical endodontic retreatments versus non-surgical endodontic retreatments, which consider longer observation periods, are needed."} +{"text": "In the Delta swab samples, amitriptyline and amantadine inhibited the channel activity of viral proteins, with IC50 values of 0.73 mM and 1.11 mM, respectively. In the Omicron swab samples, amitriptyline inhibited the channel activity, with an IC50 of 0.76 mM. Conclusions: We developed an efficient method to screen FDA-approved ion channel modulators that could be repurposed to detect and inhibit SARS-CoV-2 viral replication, independent of variants.Background: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants. Hypothesis: We hypothesized that in SARS-CoV-2, two highly conserved Orf3a and E channels directly related to the virus replication were a target for the detection and inhibition of the viral replication, independent of the variant, using FDA-approved ion channel modulators. Methods: A combination of a fluorescence potassium ion assay with channel modulators was developed to detect SARS-CoV-2 Orf3a/E channel activity. Two FDA-approved drugs, amantadine and amitriptyline (an antidepressant), which are ion channel blockers, were tested as to whether they inhibited Orf3a/E channel activity in isolated virus variants and in nasal swab samples from COVID-19 patients. The variants were confirmed by PCR sequencing. Results: In isolated SARS-CoV-2 Alpha, Beta, and Delta variants, the channel activity of Orf3a/E was detected and inhibited by emodin and gliclazide (IC SARS-CoV-2 has undergone continuous mutations throughout the pandemic, yielding clinically relevant variants such as Delta and Omicron. At the time of writing, the dominant variant was BA.5; this accounted for nearly 90% of all Omicron variants (including BA.2.12.1 and BA.4) in the US, according to the Centers for Disease Control and Prevention (CDC) ,4,5.Currently, the antiviral drug Paxlovid (Pfizer) is the standard therapy to treat mild\u2013moderate COVID-19 symptoms in patients with a confirmed viral infection . The eff+ and K+ cation permeable: the Orf3a channel is more selective to K+ [+ (10 times higher than K+) ions [2+ ions [+ ion assay, a rapid method, the Orf3a/E channel activity could be detected, a strong indicator of live SARS-CoV-2 in the blood of COVID-19 patients [There are two viroporins within the SARS-CoV-2 genome, Orf3a and E; both of which can form non-voltage-gated cation channels ,11,12. Tve to K+ ,14 whereK+) ions . The E c[2+ ions and its [2+ ions . We prevpatients .In this work, we report that clinically relevant mutations occurring in several variants of concern (VOC) did not affect Orf3a/E channel activity. Combined with our previous detection methods, we show that FDA-approved ion channel modulators can be screened for their ability to inhibit Orf3a/E channel activity. As Orf3a/E channel activity is directly associated with viral replication, we believe that repurposing ion channel modulators that are already clinically used for other diseases represents an effective alternative therapy for COVID-19 patients, independent of the causative variants.The use of blood and nasal swab samples obtained from COVID-19 patients was approved by the West Virginia University Institutional Review Board. The research was discussed with each patient and if the patient consented to the research project, whole blood was drawn by a standard venipuncture into one tube containing EDTA for anticoagulation. A total of 12 blood samples from 12 patients were included. A total of 58 nasal swab samples were collected from COVID-19 patients. Twenty-one (21) Delta and thirty-seven (37) Omicron nasal swab samples were used. The SARS-CoV-2 variant status (Delta or Omicron) was verified by sequencing.Isolated SARS-CoV-2 variants were obtained from the CDC. All purified viruses were UV-inactivated in a Biosafety Level 3 facility at the Health Sciences Center of West Virginia University. UV-inactivated viruses were studied in a Biosafety Level 2 facility.+) to permeate K+ channels [+ channels are open, Tl+ in the extracellular solution flows down its concentration gradient into the cells via K+ channels. Inside the cells, Tl+ binds to and activates a fluorogenic indicator dye preloaded into the cells, resulting in a dramatic increase in the fluorescence signal. This technique allows the rapid determination of K+ channel activity in a high-throughput manner [A fluorescence-based potassium ion channel assay utilizes the ability of thallium .Emodin, gliclazide, and 4-aminopyridine (4-AP) were purchased from Sigma. A stock solution (50 mM) was prepared in DMSO. Approximately 0.5 mM of emodin and gliclazide were used in ~100 \u03bcL solution. DMSO at ~1\u20132% of the test solution had no effect on the test results.2O.Amantadine (ATD) and amitriptyline (ATT) were purchased from Thermo Fisher Scientific (Cat # 18-600-501) and VWR (Cat # TCA0908-025G) , respectively. Stock solutions (50 mM) for both ATD and ATT were prepared in HAntigen testing was performed in a clinic laboratory using the CareStart Rapid Diagnostic Test for the detection of the SARS-CoV-2 antigen . Briefly, a nasopharyngeal swab was removed from a pouch and the swab was introduced into the nasal passage until it reached the posterior nasopharynx. The swab was rotated 3\u20135 times over the posterior nasopharynx, then removed from the nostril with a rotation to sample the anterior nares. In the laboratory, the seal was removed from an extraction vial containing the extraction buffer. The swab was placed in the extraction vial and vigorously rotated 5 times. The extraction vial was then squeezed whilst the swab was removed by rotating against the sides of the extraction vial to remove any excess fluid from the swab and a cap was placed on the extraction vial. The sample was mixed by tapping the bottom of the extraction tube whilst inverted and 3 drops were squeezed into the sample well. The results were read after ten minutes.A red control line appeared at the top of the well next to the letter \u201cC\u201d. If the test was positive, a blue line appeared below the red line and across from the letter \u201cT\u201d. Positive results produced both red and blue lines. Negative results revealed only a single red line. A result with a blue line without a red line was invalid and test was repeated.Fluorescence data were collected using Gen5 2.0 microplate reader software (from BioTek), processed in Excel, and analyzed and plotted using GraphPad Prism 8.50 calculation, four different concentrations of one drug were studied. The IC50 was calculated using a non-linear fit for an inhibitor versus a normalized response model; specifically, Y = 100/(1 + X/IC50), where X was the concentration of the inhibitor and Y was the normalized fluorescence signal.For the ICMost mutations occur in the spike proteins, but a few occur in other proteins, including Orf3a and envelope proteins. We first verified the methodology we used to screen the drugs in the blood samples. We then tested the inhibitory effects of emodin and gliclazide in the isolated SARS-CoV-2 variants. We then used nasal swabs to test the inhibitory effects of emodin and gliclazide on the Orf3a/E channel activities. To determine which FDA-approved ion channel blockers might inhibit the Orf3a/E channel activities, we used swab samples that were relatively easy to obtain. As shown in 50 of 0.76 mM.Finally, we found, using blood samples, that the Orf3a/E channel activity was much weaker for Omicron compared with the parental (prior to Delta) and Delta variants , suggestSARS-CoV-2 variants that are associated with increased transmission rates most often express changes in spike proteins . Whilst The FDA has recently emphasized the impact of further viral mutations (mostly by BA.4/BA.5) on SARS-CoV-2 antigen-based and molecular tests ,32 becauThe NIH COVID-19 treatment guidelines recommenAdditionally, the FDA has authorized Evusheld (AstraZeneca) for the preventive treatment of COVID-19 in patients with an immune compromise or a severe allergy . EvushelIn this work, we targeted virally-encoded viroporins in SARS-CoV-2 genomes due to their roles in inflammation, viral replication, and pathogenesis ,43,44. O2+ homeostasis in cardiomyocytes, increasing the risk of arrhythmias [The E protein deletion of SARS-CoV-2 causes a significant attenuation of viral virulence, suggesting an important role of E protein in the viral replication and disease pathogenesis . SARS-Cohythmias .In a pilot study, we tested two FDA-approved drugs associated with ion channel activity, amantadine and amitriptyline. Amantadine is used to treat movement difficulties in Parkinson patients ,52. It i+ current [Amitriptyline is a tricyclic antidepressant used to treat major depressive disorders and to a current . This maIt is worth noting that in the early exploratory experiments, we used high concentrations of drugs to shorten the experimental time and focus on testing the central idea, which was whether FDA-approved ion channel modulators used in a clinic could be repurposed to inhibit SARS-CoV-2 viral activity, directly controlled by the 3a/E channel activity.+ and K+ channel modulators have been used in clinics to treat a variety of diseases such as cardiac arrhythmias and neuropathic pain [Many Nahic pain ,63,64,65The sample size was not large in this work. The samples were collected when the disease prevalence was high (approximately up to 50%) at an outpatient clinic. A larger sample size is needed to test our method for detecting functional viral proteins in the blood during periods of lower disease prevalence. Additionally, we did not test the recent Omicron variants (BA.2\u20135).We developed an efficient method to screen FDA-approved ion channel modulators that could be repurposed to inhibit SARS-CoV-2 viral replication, independent of the variants."} +{"text": "Background and Objectives: Acetylsalicylic acid (ASA) is widely used for preventing cerebrovascular and cardiovascular diseases. Gastrointestinal (GI) tract injury is one of the major complications of aspirin use, potentially leading to severe GI bleeding. However, no drugs for preventing aspirin-induced small intestinal injury have been developed. The aim of this study was to establish a human experimental model for investigating aspirin-induced small intestinal mucosal injury. In addition, we evaluated the protective effect of Irsogladine against aspirin-induced small intestinal mucosal injury using human induced pluripotent stem cell-derived 2D monolayer crypt-villus structural small intestine (2D-hiPSC-SI). Materials and Methods: Human iPS cell-derived intestinal organoids were seeded and cultured in Air-liquid interface. The permeability of 2D-hiPSC-SI was evaluated using Lucifer yellow. Changes in structure and mucosal permeability of 2D-hiPSC-SI after addition of aspirin were confirmed over time, and changes in intestinal epithelium-related markers were evaluated by real-time qPCR and Immunofluorescence staining. The effect of Irsogladine on prevention of aspirin mucosal injury was examined by adding Irsogladine to the culture medium. Results: Cultured 2D-hiPSC-SI showed multi-lineage differentiation into small intestinal epithelium comprised of absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells, which express CD10, MUC2, chromogranin A, and lysozyme, respectively. RNA in situ hybridization revealed intestinal stem cells that express Lgr5. ASA administration induced an increase in the mucosal permeability of 2D-hiPSC-SI. ASA-injured 2D-hiPSC-SI showed decreased mRNA expression of multi-lineage small intestinal cell markers as well as intestinal stem cell marker Lgr5. Administration of Irsogladine on the basal side of the 2D-hiPSC-SI resulted in significant increases in Mki67 and Muc2 mRNA expression by 2D-hiPSCs at 48 h compared with the control group. Administration of 400 \u00b5g/mL Irsogladine to the ASA-induced small intestinal injury model resulting in significantly decreased mucosal permeability of 2D-hiPSC-SI. In immunofluorescence staining, Irsogladine significantly increased the fluorescence intensity of MUC2 under normal conditions and administration of 400 \u00b5g/mL ASA. Conclusions: we established a novel ASA-induced small intestinal injury model using human iPSC-derived small intestine. Irsogladine maintains mucosal permeability and goblet cell differentiation against ASA-induced small intestinal injury. Acetylsalicylic acid (ASA), also known as aspirin, is used as an anti-inflammatory drug. Low-dose-aspirin (LDA), defined as less than 325 mg per day, prevents cerebrovascular and cardiovascular diseases and is therefore widely used worldwide ,2,3. LDAVonoprazan and proton pump inhibitors (PPIs) reduce the incidence of LDA-induced gastric and duodenal ulcers, and therefore, these drugs are recommended for preventing upper GI injury related to LDA ,10,11. HCYP3A4 and ABCB1/MDR1 [Mouse models have been developed as research tools to reproduce NSAID- and LDA-induced small intestinal mucosal injury . HoweverCB1/MDR1 . TherefoCB1/MDR1 . Cells sHere, we report a novel ASA-induced small intestinal epithelial injury model using human iPSC-derived monolayer crypt-villus structural small intestine (2D-hiPSC-SI) cultured at the air-liquid interface (ALI). Using this model, we also show that Irsogladine prevents ASA-induced small intestinal damage.g for 5 min, washed once with phosphate-buffered saline (PBS), collected into a tube, and incubated in PBS with 0.5 mM EDTA for 5 min at room temperature. Following a centrifugation of the organoids, a pellet was created that was suspended and incubated in TrypLETM Select for 15 min at 37 \u00b0C. The cells were suspended in ADF containing 10% FBS, centrifuged at 200\u00d7 g for 5 min at room temperature, and the suspension was seeded onto 24-well cell culture inserts with a pore size of 0.4 \u00b5m. The cells were cultured in ADF containing 2% FBS, 1% GlutaMAX, 15 mM HEPES, 1% N2 supplement, 2% B27 serum-free supplement, 100 U/mL penicillin, and 100 \u00b5g/mL streptomycin (conditioned medium) with additional supplements for the first 3 days, as shown in Human iPS cells (Windy) were cultured as reported previously ,25. A moOn the eighth day of 2D-hiPSC-SI culture, ASA was dissolved in basal medium at concentrations of 400 \u00b5g/mL, 1200 \u00b5g/mL, and 2400 \u00b5g/mL and added to the basal side of the 2D-hiPSC-SI and incubated for 48 h. The aspirin concentrations were set considering the intestinal drug concentration as calculated using the following formula: clinical dose (mg)/250 mL.On the seventh day of 2D-hiPSC-SI culture, Irsogladine maleate was administered in basal medium at a concentration of 16 \u00b5g/mL and incubated for 48 h. On the eighth day of culture, ASA was administered in basal medium at concentrations of 400 \u00b5g/mL and 1200 \u00b5g/mL. Samples were collected 24 h after ASA administration for further analysis. The Irsogladine concentration was set considering the intestinal drug concentration as calculated using the following formula: clinical dose (mg)/250 mL.Lgr5, Mki67, Muc2, Vil1, Chga, Lyz, Cdh1, and Hprt genes in 2D-hiPSC-SI were measured by real-time reverse transcription PCR (RT-PCR). Hprt was used as an endogenous control for data standardization. Total RNA was isolated from 2D-hiPSC-SI using an Agencourt RNAdvance Tissue kit . In order to create single-stranded cDNAs, 0.5 \u00b5g of total RNA was used. ReverTra Ace qPCR RT Master Mix was used for the reverse transcription process in accordance with the manufacturer\u2019s instructions. Real-time PCR analysis was carried out on an Eco Real-Time PCR system using Eco Real-Time PCR System software, version 5.0 . PCR was performed with the primer pairs indicated in The mRNA expression levels of the Cultured 2D-hiPSC-SI on cell culture inserts was fixed in 10% formaldehyde, coated with collagen gel, processed routinely, and embedded in paraffin. The samples were then sectioned and stained with hematoxylin and eosin (HE). As in vivo human small intestine samples, we used a formalin-fixed and paraffin-embedded terminal ileum specimen in colectomy. The study was performed in accordance with the Declaration of Helsinki, and the protocol was approved by the institutional review board of Nagoya City University Hospital.2O2/methanol solution to inhibit endogenous peroxidase activity, the sections were treated with the appropriate primary antibodies, carefully rinsed with PBS, and then incubated with biotinylated secondary antibodies followed by avidin-biotinylated horseradish peroxidase complex to block endogenous peroxidase activity. Immune complexes were clarified by incubation in 0.01% H2O2 and 0.05% 3,3\u2032-diaminobenzidine tetrachloride (DAB). Nuclei were counterstained using Mayer\u2019s hematoxylin [Immunohistochemical (IHC) staining was performed using antibodies against the following antigens: MUC2 , CD10 , chromogranin A , and lysozyme . For immunohistochemical staining, paraffin sections on slides were deparaffinized using xylene and a graded ethanol series. After soaking in 0.3% Hatoxylin .Immunofluorescence staining was performed using antibodies against the following antigens: MUC2 . 2D-hiPSC-SI grown on a cell culture insert was fixed with methanol, followed by blocking for 20 min in PBS containing 5% FBS. The samples were then reacted with primary antibodies at room temperature for 1 h, washed with PBS, and then incubated with secondary antibodies at room temperature for 1 h. Nuclei were counterstained using 4\u2032,6-diamidino-2-phenylindole (DAPI). Furthermore, small intestine samples were washed and cut with the membrane. Finally, specimens were attached to glass slides and mounted using SlowFade Diamond Antifade Mountant. Confocal images were captured using a BZ-X810 microscope .Lgr5 was performed using an RNAscope FFPE assay kit according to the manufacturer\u2019s instructions. Tissue slices (4-\u03bcm thick) that had been formalin-fixed and paraffin-embedded were immediately processed with heat and protease digestion. The sections were then hybridized with a target probe specific for Lgr5, followed by horseradish peroxidase\u2013based amplification. DAB was added to evaluate the target RNA. Positive staining was judged visually as the presence of brown dots.RNA in situ hybridization (RNA-ISH) of Lucifer yellow CH dilithium salt (LY) was dissolved in dimethyl sulfoxide to prepare a 20 mM stock solution. Next, 600 \u00b5L of medium on the basal (plate) side of the 2D-hiPSC-SI was replaced with HBSS and incubated at 37 \u00b0C for 30 min. Medium on the apical (cell culture insert) side of the 2D-hiPSC-SI was replaced with 100 \u00b5L of HBSS containing 110 \u00b5M LY, and 50 \u00b5L of HBSS (liquid A) was immediately collected and incubated at 37 \u00b0C for 30 min. After incubation, the HBSS on the basal side was stirred thoroughly, and then 50 \u00b5L (liquid B) was collected. Finally, the fluorescence of liquid A and liquid B was measured using a multi-plate reader to examine the permeability of the 2D-hiPSC-SI.t-tests were used as appropriate. The number of samples is indicated by \u201cn\u201d. At least two independent replicates were performed for all experiments. Data are represented as the mean \u00b1 standard deviation (SD). A p value < 0.05 was considered statistically significant.Statistical analysis was carried out using GraphPad Prism software, version 9.2.0 for Windows . Two-tailed Dunnett\u2019s tests or two-tailed Student\u2019s Lgr5 of the 2D-iPSC-SI on culture day 8, when morphological examination of the 2D-iPSC-SI revealed a mature villus structure under ALI culture conditions A. Time-cp < 0.01). According to the high degree of detachment of cultured 2D-hiPSC-SI, we could not evaluate at 24 h and 48 h in the ASA 2400 \u00b5g/mL group. At 24 h, LY permeability was significantly greater in the ASA 400 \u00b5g/mL and 1200 \u00b5g/mL groups than in the control group . At 48 h, LY permeability was significantly greater in the ASA 1200 \u00b5g/mL group than the control group (p < 0.05). Thus, ASA induced an increase in the permeability of the small intestine.The barrier function of the 2D-hiPSC-SI was evaluated based on LY permeability B. A signLgr5 in the ASA 400 \u00b5g/mL, 1200 \u00b5g/mL, and 2400 \u00b5g/mL groups was significantly decreased at 6 h compared with the control group (p < 0.001). At 24 and 48 h, Lgr5 expression in the ASA 1200 \u00b5g/mL group was significantly lower than that of the control. Expression of the proliferative cell marker Mki67 in the ASA 1200 \u00b5g/mL group at 6 h and the 400 \u00b5g/mL group at 48 h was significantly decreased compared with the control group (p < 0.05). Expression of the goblet cell marker Muc2 in the ASA 1200 \u00b5g/mL was significantly decreased at 6 and 48 h compared with the control group (p < 0.05). Expression of the absorptive cell marker Vil1 in the ASA 1200 \u00b5g/mL group was significantly lower than that of the control group at 6, 24, and 48 h . In the ASA 400 \u00b5g/mL group, Vil1 expression at 48 h was also significantly lower than that of the control group (p < 0.001). Expression of the enteroendocrine cell marker Chga in the ASA 400 \u00b5g/mL, 1200 \u00b5g/mL, and 2400 \u00b5g/mL groups at 6 h was significantly lower than that of the control group . Significantly lower expression of Chga compared with the control was also observed in the ASA 1200 \u00b5g/mL group at 24 h and the ASA 400 \u00b5g/mL and 1200 \u00b5g/mL groups at 48 h . Expression of the Paneth cell marker Lyz in the ASA 1200 \u00b5g/mL group at 6 h and the ASA 400 \u00b5g/mL and 1200 \u00b5g/mL groups at 48 h was significantly decreased compared with the control group . Expression of the tight junction marker Cdh1 in the ASA 400 \u00b5g/mL and 1200 \u00b5g/mL groups at 48 h was significantly lower than that of the control group (p < 0.001 each). Thus, small intestine tissue injured by ASA exhibited decreased expression of multi-lineage differentiated cell and ISC markers.mRNA expression was compared in control and ASA-induced injury 2D-hiPSC-SI . ExpressMki67 and Muc2 was significantly increased in the Irsogladine group compared with the control group at 48 h (p < 0.05 each) C.p < 0.01) C.Mki67 and Muc2 expression under both normal conditions (ASA 0 \u00b5g/mL) and in the presence of 400 \u00b5g/mL ASA D.p < 0.01 and p < 0.05, respectively) (MUC2 protein expression in 2D-hiPSC-SI cultured with different ASA concentrations was examined by immunofluorescence staining A. Irsoglctively) B,C.Lgr5 , Vil1 (absorptive cell marker), Chga (enteroendocrine cell marker), and Lyz (Paneth cell marker), as well as the ISC marker Lgr5. These results suggest that ASA-induced small intestinal injury is associated with ISC dysfunction that leads to a reduction in multi-lineage small intestinal epithelial cell differentiation. Furthermore, we showed that ASA increases LY permeability in 2D-hiPSC-SI, which is consistent with previous reports of NSAID-induced small intestinal injury [p < 0.01 and p < 0.001, respectively). In ASA 400 \u00b5g/mL group, the significant difference was resolved at 48 h after the addition of ASA. Mucosal permeability is attributed to intercellular junctions and thickness of the mucosal layer [Muc2 (goblet cell marker), the crypt-villus structure of 2D-hiPSC-SI was sustained as shown in Previous studies suggested that non-ASA NSAID-induced small intestinal injury is correlated with increased epithelial permeability ,37,38. Hl injury ,36. LY pal layer ,40. As sMki67 and Muc2 expression in 2D-hiPSC-SI. These results suggest that Irsogladine promotes turnover and goblet cell differentiation in the small intestinal epithelium. Although Irsogladine did not affect small intestinal mucosal permeability under normal conditions, Irsogladine suppressed the increased mucosal permeability induced by ASA (400 \u00b5g/mL) in the model of ASA-induced small intestinal injury. Irsogladine also increased Mki67 and Muc2 expression, which had been reduced in ASA-induced small intestinal injury. The small intestinal epithelium is coated by a secreted mucus layer. MUC2 expressed by goblet cells is a major component of the gel-like secreted mucus layer that coats small intestinal epithelium [We also examined the effect of Irsogladine on normal small intestine and ASA-induced small intestinal injury . Under nithelium ,45,46. Tithelium ,48,49.There are some limitations in this study. First, our 2D-hiPSC-SI model does not include immune cells or microbiota. Microbiota organisms can be added to the apical surface of the 2D-hiPSC-SI, and immune cells can be co-cultured within the basal medium. In the future, we will conduct in vitro experiments to reconstitute the whole in vivo environment, including immune cells and microbiota. Second, the effect of Irsogladine in vivo remains unclear. Prospective and randomized controlled studies are necessary to elucidate the preventative effect of Irsogladine in ASA-induced small intestinal injury.In conclusion, we have established a novel ASA-induced small intestinal injury model using human iPSC-derived small intestine. Irsogladine maintains mucosal permeability and goblet cell differentiation against ASA-induced small intestinal injury."} +{"text": "The presence of an extra (third) kidney is an unusual congenital anomaly of the urinary system (US), having less than a hundred cases reported globally. Owing to the rare occurrence of this complex anomaly, the fused supernumerary kidney and horseshoe portion is very scarcely reported with unknown incidence. This paper presents a rare renal anomaly case of a fused supernumerary kidney with a horseshoe portion in a 41-year-old male who presented with fever, abdominal pain, and burning micturition. CT of the kidney urinary bladder showed non-rotation of the right kidney with a supernumerary malrotated horseshoe-shaped kidney and malrotated left kidney with features of acute pyelonephritis. The patient was managed with double J stenting and appropriate antibiotics till discharge. An additional reniform kidney with its specific diverse vasculature, collection system, and encapsulated parenchyma is known as a supernumerary kidney (SK). The presence of an extra (third) kidney is an unusual congenital condition of the urinary system (US), having less than a hundred cases reported globally -3. As thA 41-year-old male presented with right lumbar abdominal pain, burning with micturition, and high-grade fever ongoing for five days. He also had a complaint\u00a0of high-grade fever for the last five days. Abdominal pain was localized to the right lumbar region. There was no blood in the urine but was turbid. Vital signs were stable on general examination with a pulse of 90/min, BP was 100/70mmHg, oxygen saturation was 95% on room air, and respiratory rate was 18/min. Abdominal examination was soft with tenderness present in the right lumbar region. No organomegaly, guarding, or rigidity was present. The rest of the systemic examination was within normal limits. The provisional diagnosis was considered to be a case of complicated urinary tract infection.\u00a0On investigation, his hemoglobin was 9.7 g/dl, total leucocyte counts (TLCs) were raised: 16400/ cu mm, serum calcium was 6.8 g/dl, urea was slightly raised: 93 mg/dl with a creatinine of 2.4 mg/dl. All other investigations were normal. Urine routine and microscopy were s/o 4-5 pus cells/HPF and urine culture revealed growth of Klebsiella pneumonia.Ultrasonography of the abdomen and pelvis\u00a0suggested the presence of three kidneys with adjoined lower pole of all kidneys and malrotation of bilateral lumbar region kidneys: horseshoe kidney. Calculus of 11 * 11 mm (right side middle pole of the right kidney) was present.CT of kidney urinary bladderNon-rotation of right kidney with supernumerary malrotated horseshoe-shaped kidney was present. Right renal calculus was present. Malrotated left kidney with features of acute pyelonephritis with distal ureter calculus causing upstream hydroureteronephrosis was present as shown in Figure ManagementThe patient investigation report was suggestive of raised WBC count and pus cells in urinary microscopy. Ultrasonography and CT of the kidney urinary bladder were done. After considering all investigations, the patient was diagnosed with a supernumerary kidney with horseshoe malformation with bilateral renal calculi with pyelonephritis with acute kidney injury. The patient started with antibiotics like injection piperacillin and tazobactum 2.25gm IV three times a day and injection Levoflox 500mg IV once a day on alternate days and other supportive management like injection acetylcysteine 600mg IV three times a day and adequate hydration was done. Initially, creatinine was deranged and TLCs were raised. After 48 hours, he improved with the escalation of antibiotic therapy. After which left-sided double J stenting was done and the removal of the stone was planned for later once the infection cleared.\u00a0Temperature, pulse, and blood pressure monitoring was done regularly. The patient was vitally stable with no active complaints and then discharged with a creatinine of 1.3. The patient was advised\u00a0for follow-up after one month for double J stent removal and right renal calculi management.SK may arise from irregular and abnormal division of the nephrogenic cord, which gets divided into two metanephric blastemas, which ultimately develop\u00a0into two kidneys with incomplete or double ureteral bud -5. ArounSK is an unusual urinary system congenital anomaly, with just around a hundred cases recorded in past research. It might be completely separated from the usual kidney or attached by a loose areolar tissue and it is normally smaller and less functioning as compared to normal kidneys . To our SK is a rare congenital anomaly of the urogenital system. An SK is an accessory/extra or third present along with two normally located kidneys that can be presented as horseshoe kidneys.Patient presents with complaints such as fever and abdominal pain, abdominal mass in cases and is associated with complications like hydronephrosis, pyelonephritis, pyonephrosis, renal and ureteric calculi, carcinoma, papillary cystadenoma, and Wilm\u2019s tumor . VentricDiagnostic modalities employed in evaluation are intravenous pyelography, ultrasound, nuclear scintigraphy, CT, and MRI. Here we used only non-contrast CT kidney urinary bladder as the patient\u2019s creatinine was raised up to 2.4. So contrast-enhanced CT of the kidney urinary bladder was avoided\u00a0to prevent further\u00a0kidney damage.Management option depends on symptoms and complications in such a patient. Surgical interventions are performed on symptomatic cases with complications. A correct diagnosis is required for effective and successful treatment, which should eventually include uretro-nephrectomy. This drastic treatment is only recommended if acute kidney injury is there, and asymptomatic patients are closely monitored for early identification of any disease or problem inside the kidney. To summarise, there are no universally accepted treatment suggestions for the most successful therapy, save that each therapeutic option should be adapted to the particular circumstances.SK with horseshoe anomaly of the kidney even though rare, can be a cause of acute pyelonephritis. Diagnostics modalities employed in evaluation are intravenous pyelography, ultrasound, nuclear scintigraphy, CT, and MRI. Management options depend upon clinical features and complications present. Surgical interventions such as uretero-nephrectomy and double J stenting can be performed.The embryological basis and clinical significance of this\u00a0congenital anomaly are very important for the radiologist and the surgeon to know the anatomical variations in the blood supply of the horseshoe kidney as the surgery could be complicated in the presence of anomalous blood supply."} +{"text": "The phase of the VMPO circadian oscillations was not influenced by light. However, the VMPO clocks were reset by temperature changes within the physiological internal temperature range. This thermal-sensitivity of the VMPO circadian clock did not require functional Opn5 expression or a functional circadian clock within the Opn5-expressing cells. The presence of temperature-sensitive circadian clocks in the VMPO provides an advancement in the understanding of mechanisms involved in the dynamic regulation of core body temperature.Mammals maintain their internal body temperature within a physiologically optimal range. This involves the regulation of core body temperature in response to changing environmental temperatures and a natural circadian oscillation of internal temperatures. The preoptic area (POA) of the hypothalamus coordinates body temperature by responding to both external temperature cues and internal brain temperature. Here we describe an autonomous circadian clock system in the murine ventromedial POA (VMPO) in close proximity to cells which express the atypical violet-light sensitive opsin, Opn5. We analyzed the light-sensitivity and thermal-sensitivity of the VMPO circadian clocks Increasus (PVN) .Opn5-null mice do not regulate their body temperature in response to violet light as described in as described in and were euthanized with CO2 asphyxiation in room light for ex vivo luminescence experiments.Mice were weaned at 3 weeks of age and transferred from standard laboratory housing with white fluorescent lighting to housing cabinets with LED lighting which included violet (415-nm), blue (475-nm) and green (525-nm) light to activate known mouse retinal photoreceptors at a total intensity of 5 W/m2 asphyxiation at ZT18 and brains were rapidly dissected in ice cold HBSS. Brains were sliced either coronally or sagittally with a vibroslicer (World Precision Instruments) and imaged on an Olympus IX81 microscope.For fresh slices , 2A, mic2 asphyxiation in dim red light and brains were rapidly removed into 4% paraformaldehyde. The brains were postfixed for 24\u00a0h, and cryoprotected in 30% sucrose in PBS. Brains were sectioned in the coronal plane at 20\u00a0\u03bcm sections in a cryostat at \u221220\u00b0C. Slices were incubated for 1\u00a0h in blocking buffer and then incubated for 1\u00a0h in 5% goat serum in PB + 0.3 % Triton X-100. They were then incubated in rabbit anti-Per2 primary antibody diluted in PB + 0.3% Triton X-100 for 24\u00a0h. After incubation, samples were rinsed in PBS, incubated with secondary antibody goat anti-rabbit\u2013Alexa 633 for 1\u00a0h at room temperature, and mounted with ProLong Gold Antifade Mountant with 4\u2032, 6-diamidino-2-phenylindole (DAPI) (Invitrogen). Immunofluorescent images were analyzed using a Leica DM6000 microscope with a Leica SP8 confocal system and processed with FIJI/ImageJ . A separate researcher collected the tissues than analyzed the images who was blinded to the groups.For immunohistochemistry, mice were exposed to 12-h light:12-h dark cycles of violet (415-nm), blue (475-nm) and green (525-nm) light for 2\u00a0weeks. At the indicated ZT phases three mice per time point were euthanized by CO3, and 0.1\u00a0mM D-Luciferin potassuim salt (Biosynth). Cultures were maintained in 1.2\u00a0ml of media in 35\u00a0mm petri dishes which were sealed with sterile vacuum grease. Sealed dishes were maintained at 36\u00b0C with a microscope stage warmer and imaged with a Retiga Lumo camera (Teledyne Photometrics). Images of bioluminescence were collected over 1\u00a0h exposures and 3 1-h images were averaged into 1 using FIJI/ImageJ software. This procedure was repeated 4 times for each orientation of imaging.Fresh 300\u00a0\u03bcm brain slices were placed on cell culture membranes (Millipore #PICMORG50) in DMEM (Cellgro) supplemented with B-27 Plus (Life Technologies), 2\u00a0mM Glutamax (Gibco), 10\u00a0mM HEPES buffer (Life Technologies), 25\u00a0U/ml penicillin; 25\u00a0\u03bcg/ml streptomycin (Sigma Millipore), 352.5\u00a0\u03bcg/ml NaHCOn = 8. POA slices were approximately 600\u00a0\u03bcm anterior to the SCN. Raw data was used to measure periods using LM-Fit which uses a best-fit sine harmonic regression which also accounts for natural damping of the trace. Fourier power spectrum transforms with a time window between 22 and 40\u00a0h were performed using a Blackman-Harris filter within Lumicycle Analysis software (Actimetrics). Both period and Fourier amplitude was determined on days 2\u20137 in culture. Initial culture phase was determined using background-subtracted luminescence traces fit with a 2-degree polynomial fit line using Lumicycle Analysis and was determined as the time of the peak of the first full oscillation. For phase shifts, a best-fit sine wave was fit to data before or after a pulse. Phase shifts were measured as observed phase subtracted from expected phase. Amplitude was restored in brain slices by replacing culture media in Fresh 300\u00a0\u03bcm brain slices were cultured as above. Dishes were maintained in a Lumicycle PMT apparatus (Actimetrics) contained within an air-jacketed incubator (Sanyo MIR-262) at 36\u00b0C. POA and SCN were isolated from each mouse for 14 photons cm\u22122\u00a0s\u22121 or a heating block set to warm a sealed dish to a stable 38\u00b0C. POA-containing dishes remained either exposed to violet light or to a temperature bock (in darkness) for 1.5\u00a0h before being returned to the Lumicycle in darkness.Cultured POA slices were transferred using an insulated shuttle box to a separate incubator which either included violet LEDs (peak output 415\u00a0nm) at 2 \u00d7 1014 photons cm\u22122\u00a0s\u22121. Following the light: dark cycles the tissues were returned to constant darkness inside a Lumicycle apparatus. Time of the first peak after return to darkness was measured as the phase of traces after an ex vivo light:dark cycle.For light cycles a custom light: dark apparatus was used as described in . BrieflyBmal1flox experiments. No data was excluded.Statistics were run using SigmaPlot 11.0. Two-tailed paired t-tests were used to compare the initial phase and periods of POA to SCN from the same animals. One-way ANOVA tests were used to test for differences beyond the 95% confidence intervals of multiple groups in phase shifts and of periods/amplitudes of more than two groups as in analyses of Opn5Cre/+; Ai14 mice using tdTomato fluorescence as a marker. There is a pyramidal nucleus of Opn5-cells, approximately 500\u00a0\u03bcm in width at its base, which converges dorsally to its termination location just above the anterior commissure . The initial phase of POA was advanced compared with SCN, with the peak of the first full cycle in culture occurring approximately 6\u00a0h earlier than the SCN. This indicates that there is an endogenous clock within the POA that is capable of oscillating independently of the SCN.In previous work, we have observed a correlation between tissues which express the atypical opsin Opn5 and sustained clock gene expression . We wishmmissure . Althougs region . Using aame mice and Per2Luciferase bioluminescence and saw that the PER2 reporter was at the midline near the third ventricle, roughly within the bounds of the Opn5-cells is that the precise location of the luminescence cannot be assessed. We next imaged the POA of n5-cells . By slic 1200\u00a0\u03bcm . The VMPOpn5Cre/+; Ai14 mice in a 12\u00a0h: 12\u00a0h light: dark cycle at four time points and antibody labelled for PER2. Of the times tested, the highest expression of PER2 in the VMPO was observed at ZT 6, and PER2 expression was medial to Opn5 expressing cells. Using an arbitrary threshold of PER2 expression in individual cells, 44 \u00b1 8% of the Opn5 cells stained with the PER2 antibody at ZT 6 or lacking a functional clock within the Opn5-expressing cells . NeitherPer2Luciferase bioluminescence, a temperature pulse caused a 3.0 \u00b1 1.1\u00a0h phase advance, and during the descending phase caused a \u22122.2 \u00b1 0.9\u00a0h phase delay . These results demonstrate that although Opn5 is expressed near to the circadian clocks of the VMPO, the presence of Opn5 itself and a functional clock within the Opn5-cells are not necessary for the free-running rhythms or the thermal responses of the VMPO circadian clock.Extra-ocular opsins have been implicated in thermal responses in mammalian spermatazoa . To testPer2Luciferase circadian reporter and can maintain oscillations for at least 1\u00a0month with fresh tissue culture media. These cells are near the third ventricle, but do not border it. Because this brain region also hosts the brain\u2019s most concentrated expression of the atypical opsin, Opn5, we were interested in the direct photosensitivity of local VMPO circadian clocks , arcuate nucleus, and the paraventricular nucleus (PVN), which support the VMPO\u2019s role in autonomic modulation of body temperature . The facOpn5Cre; Ai14 cells co-expressed PER2 at peak expression, because PER2 is expressed as a gradient over time, this required use of an arbitrary threshold. It is possible that an underlying PER2 rhythm is present in Opn5-cells but is too low to be measured in the presence of the strongly rhythmic cells near the third ventricle (Bmal1-based rhythms within the Opn5 cells are not directly related to the circadian function of the primary VMPO clock. The autonomous circadian clocks within the VMPO appear to be functionally isolated from the violet-light sensitive Opn5-cells which surround them.Finally, while this work demonstrates that these Opn5-expressing cells are not necessary for the circadian function or thermal entrainment of the VMPO clock, it was limited in determining if the Opn5-cells themselves express functional circadian clocks. While we did observe that \u223c44% of entricle . In the"} +{"text": "A simple quantitative reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for assay determination of cannabidiol and tetrahydrocannabinol in hemp oil infused products. The RP-HPLC method was developed and optimized for the mobile phase composition, flow rate, column selection and detector wavelength. An isocratic elution of samples were performed on SOLAS 100\u00a0\u00c5 C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm, 5\u00a0\u03bcm column with a mobile phase containing 75/25 acetonitrile/water v/v, with a flow rate of 1.5\u00a0mL/min by using an ultraviolet\u2013visible (UV/Vis) detector operating at 214\u00a0nm. The RP-HPLC method was validated to meet regulatory requirements which covers specificity, accuracy, range, linearity, precision, system suitability and robustness. The validated assay test method was applied successfully to quantify cannabidiol and tetrahydrocannabinol in commercial hemp oil infused products such as tablets, soft gel capsules, plant extract oils, oral drops, tincture, and beverage enhancers. All the test results were found acceptable as per ICH guidelines, and this confirmed the feasibility of this method for its intended use in regular quality control and assay of cannabidiol and tetrahydrocannabinol in hemp oil infused products. Cannabaceae and contains various natural constituents. A total of 565 compounds have been isolated and identified from the C. sativa species where 120 belong to the category of cannabinoids2. Cannabinoids belongs to a group of terpenophenolic compounds with 21 carbon atoms and are further divided into 11 subgroups according to their chemical structure. This constitutes 7 compounds of Cannabidiol (CBD), 16 compounds of Cannabigerol (CBG), 23 compounds of \u22069-tetrahydrocannabinol (\u22069-THC), 5 compound of \u22068-tetrahydrocannabinol (\u22068-THC), 11 compounds of Cannabinol (CBN), 9 compounds of Cannabichromene (CBC), 5 compounds of Cannabielsoin (CBE), 2 compounds of Cannabinodiol (CBND), 3 compounds of Cannabicyclol (CBL), 9 compounds of Cannabitriol (CBT) types and remaining 30 compounds belongs to miscellaneous type3. CBD, THC, CBG, CBN, and CBC are neutral cannabinoids and are synthesised by decarboxylation reaction of their respective naturally occurring acid forms . Out of 120 cannabinoids, CBD and THC are the two major biomarkers in commercial hemp oil samples4. CBD 16, thin layer chromatography (TLC)17, HPLC coupled with mass spectrometry (HPLC/MS)18, ultra-high performance supercritical fluid chromatography (UHPSFC)19, liquid chromatography coupled with MS (LC\u2013MS)23, and centrifugal partition chromatographic techniques24 have been reported. Methods showing quantitative determination of CBD in pure isolate was also published earlier25. Out of all the methods, the HPLC methods were identified as the simplest as it allows the determination of both acidic and neutral forms and even the system does not require expensive MS detectors. Several methods were published that utilises HPLC31 and most of these methods employ\u00a0buffer mobile phases and gradient elution techniques33. Moreover, these methods demonstrate the separation of cannabinoid mixture where it includes several cannabinoids apart from CBD and THC37. The simplest HPLC methods makes use of isocratic elution techniques where the mobile phase consists of only organic modifiers were also reported39. The hemp industry demands the development of a simple, robust, accurate and efficient analytical methodologies for the quantification of the main constituents such as CBD and THC, in order to keep up with the rapidly changing regulatory requirements of this industry. Hemp oil is currently used to formulate several novel consumer goods and food supplements. Examples of such products include beverage enhancers, chocolates, capsules, topicals, baked goods, and also as non-prescription medicines in the form of tablets, tinctures, capsules, oral drops, and sublingual drops.An extensive literature survey revealed that there have been several analytical methods to quantify cannabinoids in cannabis plant extracts and most commonly used methods utilise gas chromatography (GC) coupled with mass spectrometry (MS) detectors40.Here, we demonstrate the feasibility of reverse phase HPLC (RP-HPLC) method for reliable and fast quantitative determination of CBD and THC in hemp oil products. This method uses isocratic elution that makes the method feasible to perform in any standard HPLC system. Also, the method performs well in ambient temperature conditions and uses a mobile phase of organic modifier only. Specificity, linearity, accuracy, range, precision, and robustness of the analytical method were determined according to assay method validation requirements specified in the International council for harmonization (ICH) quality guideline Q2(R1), \u201cValidation of Analytical Procedures: Test and Methodology\u201d for quantitation of CBD and THC by liquid chromatographyRP-HPLC analysis was performed on a Jasco Extrema connected with an in built autosampler (AS-4150), quaternary pump (PU-4180) with an on-line degasser, column oven (CO-4061), UV/Vis detector (UV-4075) and an interface box LC-NetII/ADC. Chromatographic data was collected and analysed using ChromNAV Ver.2 software. A second HPLC system (Agilent 1100 series) equipped with variable wavelength detector (G1314A VWD), autosampler (G1313A) and isocratic pump system (G1310A). This system automated with a Chemstation (Agilent technologies) software and which was used for intermediate precision (repeatability) testing and data collection. Chromatographic separations were achieved by using SOLAS 100\u00a0\u00c5 C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm 5\u00a0\u00b5m column . Different HPLC columns such as SOLAS ODS C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm 5\u00a0\u00b5m, SOLAS ODS C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm 3\u00a0\u00b5m, SOLAS BDS C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm 5\u00a0\u00b5m, SOLAS BDS\u00a0C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm 3\u00a0\u00b5m and EIROSHELL C18 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm 2.6\u00a0\u00b5m (Glantreo limited) were used for this method development purpose. Each Chemical and test samples were accurately weighed using an analytical balance .9-Tetrahydrocannabinol (\u03949-THC or THC) reference standards were commercially purchased from Cerilliant . Phytocannabinoid mixture 10 was purchased from Cayman chemical . Prism Science LLC, 30403 Kings Valley Dr., Conifer, CO 80433 kindly provided CBD tablets (Santeer LUV 5\u00a0mg CBD Tablets-Medium) and placebo tablets for testing. Millex-LCR PTFE 0.45um membrane filters were purchased from Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, Ireland.All reagents and chemicals of HPLC or analytical grade were used, including acetonitrile from Alfa Aesar and distilled water from in-house facility. Cannabidiol (CBD) and \u0394A mobile phase of acetonitrile and water 75/25 v/v with isocratic flow rate of 1.5\u00a0mL/min were used. Column temperature was maintained at 25\u00a0\u00b0C and detection was made at 214\u00a0nm. 10\u00a0\u00b5L of standard and sample solutions were injected in the HPLC system, where a run time of 10\u00a0min for CBD standard and 20\u00a0min for system suitability and assay samples were used. For method development, 20\u00a0\u00b5L of phytocannabinoid mixture 10 working standard solution was injected in the HPLC system.Stock standard solutions of 100\u00a0\u00b5g/mL CBD and 100\u00a0\u00b5g/mL THC were prepared by dissolving 1.0\u00a0mL of CBD and THC reference standards to a 10\u00a0mL of acetonitrile in volumetric flasks separately. 1.0\u00a0mL of THC stock standard solution was diluted by a 10\u00a0mL of acetonitrile and was used as final standard stock containing 10\u00a0\u00b5g/mL of THC.To optimise the chromatographic method parameters, working standard solution of 10\u00a0\u00b5g/mL CBD, prepared by diluting 1.0\u00a0mL of CBD stock standard to 10\u00a0mL with acetonitrile. A 25\u00a0\u00b5g/mL phytocannabinoid mixture-10 working standard solution, prepared by diluting 1.0\u00a0mL of 250\u00a0\u00b5g/mL standard solution to a 10\u00a0mL with acetonitrile were used. Mobile phase varying from 60/40 v/v to 80/20 v/v acetonitrile/water were considered and flow rate of 1.5\u00a0mL/min and 2.0\u00a0mL/min were studied. After optimising the mobile phase and flow rate parameters, testing was conducted in different wavelengths\u2014. Chromatographic columns of 150\u2009\u00d7\u20094.6\u00a0mm with different particle size and column chemistry were tested. The columns considered were SOLAS C18 5\u00a0\u00b5m, SOLAS ODS 5\u00a0\u00b5m and 3\u00a0\u00b5m, SOLAS BDS 5\u00a0\u00b5m and 3\u00a0\u00b5m, and EIROSHELL C18 2.6\u00a0\u00b5m. The chromatographic column which showed better separation efficiency was chosen for final testing.A 1.0\u00a0mL of CBD and THC stock standard solutions were diluted by 10\u00a0mL of acetonitrile in a volumetric flask (standard mixture of concentration 10\u00a0\u00b5g/mL CBD and 1\u00a0\u00b5g/mL THC each) and used for assay method. This solution was used for system suitability testing where six injections were made in HPLC and determined the plate count, tailing factor, resolution, and reproducibility .Assay linearity was demonstrated by preparing five standard solutions within the range of 50\u2013150% of the nominal sample concentration (0.1\u00a0mg/mL). Each solution was prepared by serial dilution from a single stock (5\u201315\u00a0\u00b5g/mL for CBD and 0.5\u20131.5\u00a0\u00b5g/mL for THC) and was injected in triplicate. Linear regression analysis was performed without considering the origin as a data point.For specificity, prepared un-spiked placebo solutions by weighing placebo equivalent to one placebo blend tablet weight and transferred into a 50\u00a0mL volumetric flask, added 35\u00a0mL of acetonitrile, and sonicated for 20\u00a0min. Diluted to volume with acetonitrile and mixed well (un-spiked placebo stock). Diluted 1.0\u00a0mL of this solution to 10\u00a0mL with acetonitrile, mixed well, filtered into auto sampler vial after discarding 2.0\u00a0mL of filtrate using 0.45\u00a0\u00b5m Millex PTFE filter unit. The placebo preparation was injected in duplicate. Prepared two separate spiked solutions containing the active at 100% by preparing un-spiked placebo stock separately and diluted 1.0\u201310\u00a0mL with acetonitrile, added 1.0\u00a0mL of CBD stock standard and 1.0\u00a0mL of THC final stock standard solutions, mixed well, filtered into auto sampler vial after discarding 2.0\u00a0mL of filtrate using 0.45\u00a0\u00b5m Millex PTFE filter unit. (Spiked concentration: CBD\u201410\u00a0\u00b5g/mL and THC\u20141\u00a0\u00b5g/mL each). Injected the spiked samples twice to confirm specificity.Range for the assay method was demonstrated by analysing placebo solutions spiked in a range between 50 and 150% of the nominal method sample concentration of CBD (10\u00a0\u00b5g/mL) and THC (1\u00a0\u00b5g/mL). Three weights were prepared at each of five concentration levels and each solution was analysed in triplicate. Linear regression analysis was performed without considering the origin as a data point.Accuracy and recovery of the method for assay was validated by analysing data obtained from spiked placebo solutions during the range portion of the validation. The percent recovery of each individual sample weight and the average at each concentration level was determined.For precision, assay sample solutions are prepared as follows:Five tablets (minimum) were accurately weighed, the average tablet weight was calculated and then the tablets were grinded into fine powder. A weighed sample equivalent to 5\u00a0mg of CBD transferred into a 50\u00a0mL volumetric flask, Approximately 35\u00a0mL of acetonitrile was added and the resulting solution was sonicated for 15\u00a0min with intermitted shaking. The samples were then diluted to volume with acetonitrile and mixed well. 1.0\u00a0mL of this solution was diluted to 10\u00a0mL with acetonitrile, mixed well and filtered a portion through Millex PTFE 0.45\u00a0\u00b5m sample filtration kit into HPLC vials after discarding initial 2\u00a0mL of sample solution. Following this procedure, the sample was calculated to contain 10\u00a0\u00b5g/mL CBD. 10\u00a0\u00b5L of the sample solutions were subsequently injected on to HPLC system.For method precision (repeatability), six assay samples prepared and percent label claim for CBD and THC were determined for each sample. Intermediate precision of the method was demonstrated by performing the repeatability experiment again with a second analyst. This analyst used different solution preparations, reagents, operating conditions, column, and HPLC systems and tested on different days from the first analyst.Method robustness was established by variation of chromatographic parameters to indicate the reliability of the proposed analytical method during normal usage. The chromatographic parameters considered were the variation in mobile phase composition, column oven temperature and mobile phase flow rate. In mobile phase composition increased the major component by 5% and 10%, column oven temperature was increased and reduced by 5\u00a0\u00b0C and flow rate increased and decreased by 10% and 25% from the proposed method condition. System suitability standard solution was injected in duplicate with each parameter change.The assay method was developed and validated according to ICH guidelines with respect to system suitability, linearity, specificity, accuracy, range, precision (repeatability and intermediate precision) and robustness.The focus of our research was to develop a new analytical method for the simultaneous determination of CBD and THC in hemp oil infused products. Moreover, the method proposed here can be utilized in commercial drug testing laboratories, pharmaceutical industries, and research laboratories as a standard quality control procedure. Optimization of chromatographic conditions plays a major role in the accurate detection and quantification of analytes in HPLC techniques. Mobile phases containing different percentages of acetonitrile were examined and the elution time for CBD and resolution between CBD and CBG were noted. During the testing for different mobile phase compositions, a flow rate of 2.0\u00a0mL/min and detection wavelength of 214\u00a0nm was employed. A detection wavelength of 305\u00a0nm was used to identify the CBN in the phytocannabinoid mixture because CBN generates a\u00a0strong peak at 305\u00a0nm. The test results lead us to commend a mobile phase concentration of 75/25 v/v acetonitrile/water. Flow rate of 1.5\u00a0mL/min and 2.0\u00a0mL/min were compared and finalised the flow rate as 1.5\u00a0mL/min where CBD eluted with better efficiency. Different wavelengths were checked and the peak intensity for CBD was noted. Different chromatographic columns with 150\u00a0mm\u2009\u00d7\u20094.6\u00a0mm column dimensions were tested for their ability to resolve CBD and THC from other cannabinoids. All the columns tested were manufactured by Glantreo limited using their in-house silica technology platform and it includes SOLAS 100\u00a0\u00c5 C18 5\u00a0\u00b5m, SOLAS ODS 5\u00a0\u00b5m and 3\u00a0\u00b5m, SOLAS BDS 5\u00a0\u00b5m and 3\u00a0\u00b5m, and EIROSHELL C18 2.6\u00a0\u00b5m. All the test results were tabulated and shown in Table From Table A system suitability test was performed for standard mixture containing 10\u00a0\u00b5g/mL CBD and 1\u00a0\u00b5g/mL THC and six replicate injections were performed in both HPLC systems (Agilent 1100 and Jasco Extrema HPLC\u2019s). The observed RSD values for retention time, peak area, and peak height, are well within the set limits of acceptance criteria of less than 1%. Theoretical plates (N), USP tailing factor (T), Capacity factor, USP resolution (Rs) between CBD and THC, and relative retention time (RRT) of THC were determined for assay. The results are summarized in Table 2\u2009>\u20090.999 . Linear regression analysis was performed without considering the origin as a data point. For both CBD and THC, percentage of RSD for the peak area for the triplicate injections were found to be less than 1.0% and exhibits linear responses with rPlacebo samples were prepared by weighing an appropriate amount of placebo (the placebo remains 100% of method concentration), with respect to the concentration specified in the method being validated. The placebo preparation was injected in duplicate. Prepared two separate spiked solutions containing the active at 100%. The spiked samples were injected twice to confirm specificity. Example chromatograms of diluent blank, un-spiked placebo, and spiked placebo preparations are presented in the Fig.\u00a02\u2009>\u20090.999 for the average of each set of three weights. Each individual sample recovery also found within the range of 90\u2013110%. The recovery data shows the high extraction efficiency for CBD and THC of this method.Percentage recovery at each individual spiked placebo samples and the average at each concentration level was calculated. The results were tabulated in Table Method precision (repeatability) was performed by preparing six sample solutions from the tablets for a concentration of 10\u00a0\u00b5g/mL CBD where equivalent of 5\u00a0mg CBD was weighed. Each preparation was injected twice, and the assay values calculated. Results including the percentage RSD values of CBD and THC for both repeatability and intermediate precision are presented in Table Method robustness was proved by variation of the mobile phase composition, temperature, and flow rate, from the parameters specified in proposed method. A system suitability standard (10\u00a0\u00b5g/mL CBD and 1.0\u00a0\u00b5g/mL THC) was prepared and injected in duplicate with each parameter change. This solution was used to perform robustness for assay and each parameter change was tested one factor at a time. The retention time (RT), theoretical plates (N), Tailing factor (T), Capacity factor (k), resolution between CBD and THC (Rs), and the relative retention time (RRT) for THC are reported in Table Additionally, solution stability was established for 48\u00a0h by testing system suitability of standard solutions twice by using a freshly prepared standard, the solutions kept in room temperature for 24\u00a0h, the solutions kept in 0\u20138\u00a0\u00b0C for 24\u00a0h and the solution kept in room temperature for 48\u00a0h. The results show negligibly small variation in peak area absorbance values and proved the standard solution stability of 48\u00a0h in room temperature. Also, an old column where\u2009>\u2009500 injections made was tested and found a higher tailing and backpressure as compared to new column indicating approximate lifetime of this HPLC column is less than 500 injections.41. The samples tested consisted of tablets, capsules, tincture, oral drops, beverage-enhancers, and plant extract oils. Furthermore, this method was found to be suitable to quantitate CBD and THC in complex matrices such as in hemp oil infused chocolates where the cannabinoids were extracted using QuEChERS extraction method43. Furthermore, Glantreo limited presently employs this assay method for testing commercial hemp oil infused samples as a quality control procedure.The suitability of this validated method was studied using a wide range of hemp oil infused tablets provided by Prism Science LLC, 30403 Kings Valley Dr., Conifer, CO 80433. The samples included Santeer LUV 2.5\u00a0mg, 5\u00a0mg, and 10\u00a0mg PET tablets, Santeer FOCUS and RENEW 5\u00a0mg CBD tablets, and Santeer DREAM 10\u00a0mg CBD tablets. Then, this method was applied to quantitate CBD and THC in various hemp oil infused products and presented in a paper published by Analakkattillam et al.37 and for the remaining 7 methods showed isocratic elution, four methods use an acid or base or salt containing mobile phase34. One isocratic elution method utilizes a column oven temperature of 40\u00a0\u00b0C and was originally an Ultra high performance liquid chromatographic method25 whereas in the other isocratic method, a column oven temperature of 55\u00a0\u00b0C and an autosampler thermostat of 4\u00a0\u00b0C was employed39. The only method that found to be simple used 85% methanol as mobile phase and with reduced runtime of 10\u00a0min, but the method showed CBD recovery of 97.1% and THC recovery of 99.3% whereas our method showed a 105.2% recovery for CBD and 100.2% recovery for THC41. Moreover, while performing the assay testing for the samples mentioned in the published paper41, initially acetonitrile was used as extraction solvent. For the samples where the CBD content was found to be very low as compared to its label claim, conducted the analysis using methanol as extraction solvent. For the samples where the CBD was not detected, further performed extraction using QuEChERS extraction method43 in order to confirm the extraction efficiency of acetonitrile and/or methanol. The assay test results were comparable for both acetonitrile and methanol extractions where acetonitrile found to be more suitable.Suitability of this method\u2019s efficiency to quantitate CBD and THC was compared by considering the recovery data for CBD and THC reported in different published papers where HPLC technique employed (Supplementary Table The precise quantification of compounds like cannabinoids where the products contain psychotic molecules such as THC, demands that laboratories have affordable and easily applicable validated procedures. The amount of THC within a product determines the legal restriction for the said product. The assay test method validated and presented here allows the quantitative and simultaneous determination of CBD and THC in Hemp oil infused products in 20\u00a0min run time. The method uses single wavelength of 214\u00a0nm, and the testing can be carried out using any traditional standard RP-HPLC and UV\u2013visible detectors. Moreover, the method utilizes only organic solvents for standard and preparation, and for mobile phase single organic modifier was involved with no pH adjustment and the testing follows an isocratic elution. Due to simplicity and consistency this method can be employed by any analyst with basic HPLC knowledge. The method was shown to be selective, precise, linear, and accurate within the range of 50% to 150% of the nominal concentration for tablet strengths. The method was shown to be robust with the established as critical parameters such as mobile phase concentration changes and flow rate.Supplementary Information."} +{"text": "We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection While reverse transcription occurs, the viral core undergoes uncoating, which is the dissociation of monomeric capsids proteins from the core resulting in the opening of the structure that houses the viral genome. Concomitant with these early replication events, the nuclear cleavage and\u00a0polyadenylation specific factor 6 (CPSF6) is recruited from nuclear paraspeckles to nuclear speckles (NS)5. NS are dynamic borderless structures that are distinct entities in the nucleus.Infection of human cells by HIV-1 requires fusion of the viral membrane with the cellular membrane, which delivers the viral core into the cytoplasm of the cell. The viral core, composed of 1500 monomers of the capsid protein (p24), houses the viral RNA genome. This viral core travels to the nucleus, where it has been suggested that the RNA is converted into viral DNA by reverse transcription2. Translocation of CPSF6 to NS induced by HIV-1 infection changes the immunofluorescence microscopy pattern of CPSF6 from diffuse nuclear staining to large CPSF6 condensates or puncta-like structures that are easily recognizable2. Although the translocation of CPSF6 to NS is widely accepted, the role of this translocation in HIV-1 replication, if any, is not understood.The early recruitment of CPSF6 to NS induced by HIV-1 infection requires an intact capsid protein. Viruses with capsid mutations N74D or N57S fail to induce the translocation of CPSF6 to NS8. NS are also known as SC35-rich domains9 as they are easily visualized by antibodies directed against the spliceosome assembly factor SC3510. One possibility is that the recruitment of CPSF6 to nuclear speckles plays an important role in replication. The addition of the small pharmacological molecules PF74, GS-CA-1, or lenacapavir during infection inhibits translocation of CPSF6 to NS11, and PF74 disassembles CPSF6 condensates preformed by HIV-1 infection. In contrast, neither GS-CA-1 nor lenacapavir triggers CPSF6 disassembly, indicating that they act by a different mechanism than PF7411. Contrary to protein aggregates in the cell, which are poorly dynamic, the ability of small molecules to destabilize the CPSF6 condensates suggests that they are dynamic structures.NS, which are important for the expression of highly active genes, comprise protein and RNA that coordinate transcription and splicing11. However, the absence of CPSF6 does affect integration site selection for HIV-1, suggesting that, like LEDGF/p75, CPSF6 is involved in the selection of integration sites in the host genome13. Furthermore, infection of CPSF6 knockout cells by HIV-1 results in an integration pattern similar to the integration pattern observed for HIV-1-N74D viruses13; these results imply that in the absence of CPSF6 expression, HIV-1 wild type virus behaves like HIV-1-N74D viruses. Therefore, knocking out CPSF6 expression may not be the best approach to understand its contribution to wild type HIV-1 infection. Clearly, it has been shown that the absence of CPSF6 hinders the reactivation of latent proviruses14.The contribution of HIV-1-induced CPSF6 condensates to infection is not understood. Although CPSF6 interacts with the viral capsid, the lack of CPSF6 has no effect on HIV-1 infection of stimulated primary human T cells 2. We initially investigated whether reverse transcription and/or integration are required for the formation of CPSF6 condensates. We found that neither reverse transcription nor integration were required for the formation of CPSF6 condensates. Similarly, the HIV-1 genome was not required for the formation of CPSF6 condensates. Because CPSF6 forms condensate-like structures in the nucleus upon HIV-1 infection, we determined whether these puncta-like structures were biomolecular condensates. To this end, we tested whether osmotic stress and the drug 1,6-hexanediol affected the nature and stability of these condensates. We found that hypertonic stress resulted in the disassembly of CPSF6 condensates, and reestablishing isotonic conditions resulted in the reassembly of CPSF6 condensates in the nucleus. By contrast, 1,6-hexanediol resulted in the disassembly of CPSF6 condensates, and the removal of the drug allowed reassembly of CPSF6 condensates in the cytoplasm. HIV-1-infected human cells transferred from an hypotonic to an isotonic medium reassembled CPSF6 condensates in the cytoplasm. Thus, unlike protein aggregates, CPSF6 condensates are dynamic structures that behave like biomolecular condensates. The HIV-1 inhibitor GS-CA-1 prevented the disassembly of nuclear CPSF6 condensates triggered by hypotonic stress; thus, GS-CA-1 stabilizes CPSF6 condensates under these conditions. To determine whether CPSF6 condensates play a role in infection, we challenged human cells with HIV-1 in a hypertonic medium that prevents the assembly of CPSF6 condensates during infection. Remarkably, the hypertonic medium dramatically inhibited wild-type HIV-1 infection, but poorly affected viruses bearing the capsid changes N74D and A77V, which are viruses that do not induce the formation of CPSF6 condensates2. Thus, the CPSF6 condensates induced by wild-type HIV-1 are important for infection. To further characterize the protein content of these structures, colocalization experiments identified the protein CPSF5 but not CPSF7 as part of the condensate. Our studies revealed that the accumulation of CPSF6 biomolecular condensates in NS is important for wild-type HIV-1 infection.The\u00a0translocation of CPSF6 to NS induced by HIV-1 infection requires an intact capsid since viruses containing capsid mutations such as N74D and A77V fail to induce condensates containing CPSF65. CPSF6 in uninfected human cells shows semi-diffuse nuclear labeling, whereas HIV-1 infection triggers the formation of nuclear condensates or puncta structures that contain CPSF6, which colocalize with the NS marker SC35 1\u20132 (infecting\u2009>\u200990% of HT1080 cells) for CPSF6 condensates. After infecting human cells THP-1 (monocytes), U937 (monocytes), HT1080 , HeLa , and A549 cells, we found that 80%\u201385% of A549 cells showed CPSF6 condensates in NS, defined as SC35 positive compartments. For HeLa and HT1080 cells, 30\u201340% of the cells contained CPSF6 condensates. In contrast, less than 1% of phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 and U937 cells had CPSF6 condensates. These results indicated that A549 cells provide a reliable model for studying the formation and function of CPSF6 condensates in NS after HIV-1 infection.We and others have previously demonstrated that HIV-1 infection induces the recruitment of CPSF6 to NSAs a proof of principle, we infected A549 cells by an HIV-1 virus expressing luciferase as a reporter of infection (HIV-1-Luc) at an MOI\u2009=\u2009\u2009~\u20091\u20132 and showed that infection triggers the translocation of CPSF6 to NS did not affect the formation of CPSF6 condensates in NS. Overall, these results demonstrated that neither integration nor reverse transcription is required for the recruitment of CPSF6 to NS.We studied the role of HIV-1 determinants in the formation of CPSF6 condensates in NS using viral mutations and inhibitors. To this end, we simultaneously measure formation of CPSF6 condensates and infectivity. First, we found that the HIV-1 reverse transcriptase mutant D185N produced the same percentage of cells containing CPSF6 condensates when compared to the wild-type virus and owl monkey TRIMCyp prevent the entry of viral capsid into the nucleus2; therefore, we determined whether cells expressing these factors showed differences in the HIV-1-induced recruitment of CPSF6 to NS. Consistent with the requirement for the nuclear capsid in the recruitment of CPSF6 to NS2, expression of the restriction factors TRIM5\u03b1rh and TRIMCyp prevented the formation of CPSF6 condensates in NS (data not shown). These experiments suggested that the CPSF6 condensates formed upon infection that serve an unknown function are subsequently disassembled.The formation of CPSF6 condensates is likely to be important for viral infection; therefore, we sought to determine whether the CPSF6 condensates are transient or stable structures since some viruses require the assembly and disassembly of condensates for the completion of their viral life cycle18. In cells, biomolecular condensates increase the local concentration of proteins to improve (1) enzymatic activity by bringing enzyme and substrate together, (2) avidity in low-affinity protein interactions, (3) nucleation for polymers such as actin and tubulin, and (4) cell signaling amplification19. Although cells use condensates to support their physiology, many viruses exploit biomolecular condensates for replication20. Because biomolecular condensates are sensitive to osmotic stress21, we determined whether hypotonic stress affected the stability of CPSF6 condensates. A549 cells were infected with HIV-1-GFP at an MOI of\u2009~\u20092 for 24\u00a0h to induce CPSF6 condensates to induce condensates in cells that had been treated with a hypertonic medium at 10\u00a0h post-infection to prevent the assembly of CPSF6 condensates is a protein complex that regulates mRNA 3\u2032 processing and polyadenylation site selection27. CFIm is a tetramer composed of two 25-kDa CPSF5 subunits and two proteins of either 59 or 68\u00a0kDa 28. To further characterize the condensates induced by HIV-1 infection, we used colocalization experiments to assess the presence of CPSF5 and/or CPSF7 in CPSF6 condensates. CPSF6 condensates induced by HIV-1 infection colocalized with CPSF5 . To further explore the capsid protein as a determinant for the formation of condensates, we tested mutants in other regions of the capsid, such as the cyclophilin A binding loop (capsid mutant P90A) and found this region was not important for the formation of CPSF6 condensates.We explored the role of CPSF6 condensates in HIV-1 infection. Because our previous findings suggested that the formation of CPSF6 condensates upon HIV-1 infection requires an intact capsid protein in the nuclear compartmentWe also determined the presence of these condensates over time. We found the highest percentage of cells containing condensates at\u2009~\u200924\u00a0h post-infection, which then declined over time in the next four days, indicating that these structures may be transient. Remarkably, as the percentage of cells containing CPSF6 condensates declined, the size of the remaining condensates increased, which may indicate that fusion of condensates is occurring during cell division.31. Our work combined with the studies of Scoca et. al., which showed fluorescent recovery after photobleaching of CPSF6 proteins fused to neon-green fluorescent protein26, provide strong evidence to suggest that HIV-1-induced CPSF6 puncta are in fact biomolecular condensates, since these structures are: (1) sensitive to osmotic stress, (2) sensitive to 1,6-hexanediol, (3) growing overtime due to fusion events, and (4) recoverable upon photobleaching.Our data showing that CPSF6 condensates were affected by osmotic stress and the drug 1,6-hexanediol indicate that HIV-1-induced CPSF6 condensates behave as biomolecular condensates. Either hypertonic stress or the drug 1,6-hexanediol triggered the rapid disassembly of CPSF6 condensates, consistent with the dynamic nature of biomolecular condensates2-CPSF62 tetramer. However, we found no accumulation of CPSF7 in condensates suggesting that HIV-1 infection does not induce accumulation of the tetramer CPSF52-CPSF72.To further characterize the protein content of these condensates, we determined whether proteins that function in a complex with CPSF6 were also present in the condensates. We found that CPSF5, which forms a complex with CPSF6, was present in the condensates suggesting that HIV-1 infection induces the formation of condensates that contain the CPSF5We found that the epithelial cell line A549 is a very good model to study HIV-1 induced biomolecular condensates since condensates are formed in almost 80\u201385% of infected cells. However, these cells are not the natural target of HIV-1. For this reason, we investigated whether HIV-1 infection of natural targets cells, such as human T cells and primary macrophages, induce the formation of condensates. Our investigations revealed that HIV-1 infection of human T cells and macrophages induces the formation of condensates containing CPSF5 and CPSF6.2, and do not require CPSF6 condensates for infection. In addition, the integration pattern of HIV-1 capsid mutant N74D is very similar to the pattern shown by wild-type HIV-1 infection of CPSF6 knockout cells13. Our data showed that CPSF6 condensates are important for wild-type HIV-1 infection. It is important to point out that due to the condensing nature of some nuclear pore complex proteins, such as nucleoporins, hypertonic treatment could also have an effect on them and thus affect HIV-1 infection32.Because hypertonic stress induces the disassembly of HIV-1-induced CPSF6 condensates, we determined whether the formation of condensates is important for infection. When we prevented the formation of CPSF6 condensates using a hypertonic medium during infection, wild-type HIV-1 infection was inhibited, but HIV-1 capsid mutants N74D and A77V were much less affected by the hypertonic medium. These results are consistent with the observation that HIV-1 capsid mutants N74D and A77V do not form condensatesInvestigations to understand whether other nuclear proteins are in proximity or part of the HIV-1-induced condensates revealed that the integration cofactor LEDGF/p75 tri-dimensionally surrounds the condensate that contains CPSF6 and CPSF5. This observation suggested a potential functional link between the condensate and LEDGF/p75. Although this requires further exploration, the fact that LEDGF/p75 gets in proximity of the condensate forming a surrounding layer raises the possibility of a second condensate or environment that will facilitate infection.2 at 37\u00a0\u00b0C. Human THP-1 and U937 monocytes and human Jurkat T cells were obtained from ATCC and maintained in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% FBS and 1% penicillin/ streptomycin/ L-glutamine in a humidified incubator with 5% CO2 at 37\u00a0\u00b0C. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donor using the Pan Monocytes Isolation kit from Miltenyi Biotec (130-096-537). Monocytes were differentiated into macrophages by culturing in DMEM supplemented with 10% human serum for 7\u00a0days. The A549 cell lines that stably express the restriction factors TRIM5\u03b1rh or TRIMCyp were previously generated . HeLa cell line stably expressing CPSF6 fused at C-terminal to the enhanced green fluorescent protein (eGFP) was generated by retroviral transduction.A549 (human lung carcinoma), HT1080 (human fibrosarcoma), and HeLa (human epithelium) cells were obtained from the American Type Culture Collection , and were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100\u00a0\u03bcg/ml streptomycin, 29.2\u00a0mg/ml L-glutamine (Life Sciences), and 5\u00a0\u03bcg/ml plasmocin , in a humidified incubator with 5% COWe used the following mouse monoclonal antibodies: clone SC-35 against SC35 , clone 3F8 against CPSF5 , clone A-9 against CPSF7 and clone F3 against CPSF6 . We used rabbit polyclonal antibodies against the following proteins: CPSF6 and LEDGF/p75 . The fluorescent nuclear stain 4\u2019,6-diamidino-2-phenylindole (DAPI) and the following fluorescently labeled antibodies were from Life Technologies: Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated donkey anti-rabbit IgG and anti-mouse IgG. The inhibitors nevirapine (Nev) (Cat# ARP-4666), zidovudine (AZT) (Cat# ARP-3485), and raltegravir (Cat# ARP-11680) were from NIH AIDS Reagent Program. Dimethyl sulfoxide (DMSO) (Cat# D2438), cyclosporin A (CsA) (Cat# 30024), and PF74 (Cat#SML0835) were from Sigma Aldrich. GS-CA-1 was kindly provided by Stephen Yant .33. Viruses were collected 48\u00a0h post-transfection, filtered, titered, aliquoted, and stored at\u2009\u2212\u200980\u00a0\u00b0C.Wild-type and mutant HIV-1 expressing green fluorescent protein (GFP) or luciferase (Luc) as a reporter gene were produced by cotransfecting HIV-1-gag-pol, LTR-GFP-LTR/LTR-Luc-LTR, tat, rev and VSV-G in HEK293T/17 cells as described34. We infected human A549 cells and determined the percentage of GFP-positive cells using a flow cytometer (BD Celesta). For mutant viruses that affect the infectivity of HIV-1, we normalized the amount of virus against p24 by ELISA and/or western blotting using antibodies against p24 (NIH AIDS repository). Virus titer calculation was performed according to the following equation: Infectious units (IU)/ml\u2009=\u2009(cell number)\u2009\u00d7\u2009(% of GFP-positive cells)\u2009\u00d7\u2009(dilution factor), where the dilution factor\u2009=\u20091000\u00a0\u03bcl/viral input (\u03bcl). To calculate the volume of virus used at a specific MOI, use the following equation: MOI\u2009=\u2009[(virus stock IU/ml)\u2009\u00d7\u2009(volume of virus used)]/(number of cells in infection).The infectivity and titer of HIV-1-Luc viruses were measured using TZM-bl GFP-reporter cells in which HIV-1 infection induces GFP as described2O) was DMEM supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine. For hypotonic stress (30\u00a0mOsm/H2O), tenfold diluted DMEM (supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine) with ultrapure H2O was used25. For hypertonic stress (~\u2009500\u00a0mOsm/H2O), DMEM was supplemented with 10% FBS, 1% penicillin/streptomycin/L-glutamine and 200\u00a0mM NaCl. For 1,6-hexanediol treatment, DMEM was supplemented with 10% FBS, 1% penicillin/streptomycin/L-glutamine and 3% 1,6-hexanediol.For osmotic stress treatments, the isotonic medium . Fluorescence microscopy images were acquired with AxioObserver.Z1 microscope equipped with a PlanApo 63\u00d7\u2009oil immersion objective (NA 1.4) and an AxioCam MRm digital camera (Carl Zeiss). Image acquisition was carried out using a Zeiss Z1 Observer inverted microscope using the ZEN 3.3 (blue edition) software. Image deconvolution was performed with the ZEN 3.3 software using an acquired point spread function. Images for figures were processed with Adobe Photoshop CS5 software .Samples for immunofluorescence analysis were prepared as described previously with some modificationsThe mean and standard deviation values were calculated using GraphPad Prism 8. Statistical analysis was performed using unpaired t tests.Supplementary Figure S1.Supplementary Figure S2."} +{"text": "Estimating effective coverage of childbirth care requires linking population based data sources to health facility data. For effective coverage to gain widespread adoption there is a need to focus on the feasibility of constructing these measures using data typically available to decision makers in low resource settings. We estimated effective coverage of childbirth care in Gombe State, northeast Nigeria, using two different combinations of facility data sources and examined their strengths and limitations for decision makers. Effective coverage captures information on four steps: access, facility inputs, receipt of interventions and process quality. We linked data from the 2018 Nigerian Demographic and Health Survey (NDHS) to two sources of health facility data: (1) comprehensive health facility survey data generated by a research project; and (2) District Health Information Software 2 (DHIS2). For each combination of data sources, we examined which steps were feasible to calculate, the size of the drop in coverage between steps and the resulting estimate of effective coverage. Analysis included 822 women with a recent live birth, 30% of whom attended a facility for childbirth. Effective coverage was low: 2% based on the project data and less than 1% using the DHIS2. Linking project data with NDHS, it was feasible to measure all four steps; using DHIS2 it was possible to estimate three steps: no data was available to measure process quality. The provision of high quality care is suboptimal in this high mortality setting where access and facility readiness to provide care, crucial foundations to the provision of high quality of care, have not yet been met. This study demonstrates that partial effective coverage measures can be constructed from routine data combined with nationally representative surveys. Advocacy to include process of care indicators in facility summary reports could optimise this data source for decision making. Ensuring access to high quality maternal and newborn care is a global priority in efforts to reduce preventable mortality and morbidity \u20135. MeasuThere is emerging consensus that effective coverage of maternal, newborn and child health (MNCH) is best conceptualised using a care cascade, which outlines six sequential steps that the target population is anticipated to have to move through to achieve the intended health benefit: 1) service contact; 2) input-adjusted coverage; 3) intervention-adjusted coverage; 4) process quality-adjusted coverage; 5) user-adherence adjusted coverage; and 6) outcome-adjusted coverage ,10. For Despite consensus on the concept, research on how to best operationalise the cascade, including which data sets and indicators to use, is limited \u201314. A reThis study aimed to address this issue by demonstrating whether and how the effective coverage of childbirth care can be generated using two different health facility data sources to adjust population-level data on contact coverage in Gombe State, northeast Nigeria. First, using comprehensive health facility survey data generated by a research project and second we examined the feasibility of replicating that measure using only routine data sources typically available to decisions makers.Ethical approval for this study was obtained from the Nigerian National Health Research Ethics Committee (NHREC/01/01/2007), the State Ministry of Health Gombe State (ADM/S/658/Vol. II/66) and the London School of Hygiene & Tropical Medicine (22330). For the health facility project specific data, all potential participants were provided with a study information sheet and a consent form in English and Hausa. The in-charge of each facility gave written informed consent for the facility survey; written consent was also obtained from the birth attendant interviewed and women observed. Participation was voluntary and participants were free to withdraw at any time.Gombe State is a predominantly rural (80%) and sparsely populated state in northeast Nigeria . It is mThe northeast region of Nigeria has some of the highest maternal and newborn death rates globally, estimated at 1,549 per 100,000 live births in 2015 and 33 per 1,000 live births in 2017, respectively ,19. HealBetween 2016 and 2019, the GSPHCDA led a maternal and newborn health partnership designed to implement a package of evidence-based interventions to improve access, use and quality of maternal and newborn health services, across the 11 LGAs of Gombe State \u201324. ThroGenerating effective coverage of childbirth requires linking care seeking data collected through population based data sources with information from health facilities on the quality of the interventions provided . Two souThe NDHS is conducted every five years using a two-stage stratified cluster sample, designed to be representative at the national and state level . The houWe used health facility survey data from August 2019. Data collection methods are reported in detail elsewhere . BrieflyDuring the facility survey, observations of childbirth were completed in the 10 PHCs with the highest number of births recorded in the maternity register ,35. ObseDHIS2 is an open source software platform used in more than 70 countries . In GombWe computed both effective coverage measures based on the coverage cascade steps for facility based childbirth care proposed by the Effective Coverage Think Tank Group\u2013a group of experts convened by WHO and UNICEF . ConsistInput-adjusted measures were estimated in the respective health facility dataset (project data or DHIS2) as a binary score calculated for each facility based on: 1) all items available and functioning on the day of the survey in the project data, and 2) not experiencing stock outs of any items in the previous six months in DHIS2. Mean input-adjusted score was calculated, by facility type , as the percentage of facilities with inputs available. For the project data mean intervention-adjusted and process quality-adjusted measures were estimated in the observation dataset, as the percentage of women observed in a PHC who received all components of care. In the data typically available to decision makers, intervention-adjusted coverage was calculated based on women\u2019s self-reports in the NDHS, as the percentage of women who reported they gave birth in a facility that received all interventions. All items contributed equally to each score and missing data was treated as the item not being present.Similar to previous examples, effective coverage was calculated at the State level using ecological linking methods \u201314,31,42From the linked datasets, we calculated each step of the cascade. The first step in the cascade, service contact coverage, was calculated as the percentage of women who reported giving birth in a facility across the State. Subsequent steps were calculated as the product of the prevalence of the step and the prevalence of the proceeding step. The analyses adjusted for the survey design using the svyset and svy commands in STATA version 15.1 and uncertainty of the estimates of effective coverage was assessed using the delta method ,43. MissThe analysis included 822 women who reported a live birth in Gombe State in the five years preceding in the NDHS (2013\u20132018) . The proIn the rest of the results section we first describe the composition of the four steps of the cascade in turn and then present the two effective coverage measures estimated using the different data sources.In the NDHS for Gombe State, representing births between 2013\u201318, the coverage of facility based childbirth was 30%: 19% at PHCs and 11% at a referral facility. We checked for evidence of changes in facility delivery over the period of the NDHS, and found the coverage of women seeking childbirth care at a health facility to be relatively stable over the five-year period: 37% among women who delivered five years preceding the survey, 31% in the three to four years preceding, 32% in two years preceding and 27% among women who delivered in the 12 months preceding the survey.The availability of inputs from the two facility data sources by facility type is presented in The number of input measures it was possible to estimate in the DHIS2 was limited; no information was available on facility infrastructure and data was only captured on three of the 10 supply and commodity items included in the project-based estimate. Less than a fifth of facilities had all inputs available: 18% of PHCs and 22% of referral facilities. No items were universally available. The item most frequently available in PHCs was skilled birth attendant (73%) and uterotonic in referral (83%), while the item least frequently available in PHCs was anticonvulsant (34%) and in referral facilities skilled birth attendant (35%).Over three-quarters of women were observed to receive all three interventions in the project data see ; rangingProcess of care data was available in the project data but not the data typically available to decision makers see . OverallFor the second effective coverage measure using data typically available to decision makers in this setting we were able to calculate effective coverage up to cascade step 3. We observed that less than 0.5% of women were estimated to receive high quality care during childbirth. Again the largest bottleneck was in access to a health facility with only 30% of women attending a health facility for childbirth, and there was a large reduction from service contact to input-adjusted coverage, 30% to 6%.Effective coverage measures are recommended as best practice for estimating population-level access to high quality maternal and newborn health care but there has been limited progress to operationalise measures. To maximise utility, there are increasing calls to make better use of routine data systems to generate estimates of effective coverage . In thisOur first approach linking NDHS to health facility survey data collected through a research project represents the most comprehensive health facility data available in this setting. It included a health facility survey and observations of birth, allowing linking of these different data sources with NDHS to calculate all four recommended cascade steps to estimate process quality-adjusted coverage of childbirth. The analysis revealed that less than 2% of women received effective coverage of childbirth care in Gombe state. Substantial gaps in the provision of high quality care were highlighted; coverage dropped from 30% of women who attended a facility for childbirth to 10% after accounting for the necessary inputs to provide high quality care during childbirth, dropping again to 7% after adjusting for intervention delivery and 2% after finally adjusting for processes of care. This finding adds to the wealth of evidence demonstrating large drops in coverage once some measurement of quality is accounted for Reviewers' comments:Reviewer's Responses to Questions Comments to the Authorpublication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented. 1. Does this manuscript meet PLOS Global Public Health\u2019s Reviewer #1:\u00a0YesReviewer #2:\u00a0PartlyReviewer #3:\u00a0Yes********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes********** 3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. The Reviewer #1:\u00a0YesReviewer #2:\u00a0NoReviewer #3:\u00a0Yes********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0NoReviewer #3:\u00a0Yes********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0To the best of what has been presented; the methods and materials used are appropriate to the research objectives. The data analysis techniques used are vigorous and the conclusions have been based on the findings . Therefore, the article meets APLOS publication criteria and is very informative for health practice.Reviewer #2:\u00a0Thank you for inviting me to review this manuscript, \"Operationalising effective coverage of facility based childbirth in Gombe State; a comparison of data sources\". Please find below comments from my review.(a) Title: The title is not clear. Whereas the study reports findings related to optimising metrics of coverage of maternal health services, this is not accurately represented in the title. I suggest that you include the word(s) \"indicator\", \"metric\", \"measurement\" or whichever you may deem appropriate to clarify that the manuscript content regards metrics and not implementation per se. The term \"operationalising\" in the title contributes to ambiguity.(b) Abstract: Whereas the abstract is concise, you need to revise to ensure consistence of tense. Since this is not a study protocol, I suggest using past tense consistently through the manuscript.(c) Introduction: Lines 54-57. I suggest that you limit this section to clearly stating the study aims. The study design descriptions, such as, \".... using comprehensive facility assessment project data....\", can be detailed in the methods section.(d) Conclusions: Please revise this section to reflect concordance with study aims. Whereas it is true that effective coverage of childbirth care is low in Gombe, this had already been demonstrated from the NDHS, albeit sub-optimally. What then should the reader draw from your work? How did the triangulation of data sources improve utility of routinely available data?(e) Study data: please make the study data available or otherwise state justification for not doing so.Reviewer #3:\u00a0This study used a combination of facility data to estimate the effective coverage of the childbirth care system in Gombe State, Nigeria. The study found effective coverage estimates for different data combinations and discussed the strength and weaknesses of their method. This adds to the current body of evidence in Gombe State as regards the effective coverage for maternal, newborn, child, and adolescent health and nutrition (MNCAHN). The result is well presented and discussed.(1.) The authors should consider discussing more on how they managed to adjust for the sample design for multiple data, given that the design could be different for different survey data.(2.)The authors should explicitly write out the governing formulas/ equations to calculate these measures, and if possible, share the code used in the computation for readers who wish to reproduce the result.(3.) The authors should proofread again for minor grammatical errors.The article has sufficient contribution, and I recommend consideration for publication.********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.Do you want your identity to be public for this peer review? If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Privacy Policy. For information about this choice, including consent withdrawal, please see our Reviewer #1:\u00a0NoReviewer #2:\u00a0NoYes:\u00a0Bayowa Teniola BabalolaReviewer #3:\u00a0**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, 17 Feb 2022AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 22 Mar 2022Operationalising effective coverage measurement of facility based childbirth in Gombe State; a comparison of data sourcesPGPH-D-21-00996R1Dear Ms Exley,We are pleased to inform you that your manuscript 'Operationalising effective coverage measurement of facility based childbirth in Gombe State; a comparison of data sources' has been provisionally accepted for publication in PLOS Global Public Health.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.globalpubhealth@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.Best regards,Victor Adagi AleganaAcademic EditorPLOS Global Public Health***********************************************************Reviewer Comments :"} +{"text": "Postoperative complications (POCs) following resection of colorectal liver metastases (CRLM) are common. The objective of this study was to evaluate risk factors for developing complications and their impact on survival considering prognostic factors of the primary tumor, metastatic pattern and treatment in a well-defined national cohort.Patients treated with resection for CRLM that was also radically resected for their primary colorectal cancer (diagnosed in 2009\u20132013) were identified in Swedish national registers. Liver resections were categorized according to extent of surgery (Category I\u2013IV). Risk factors for developing POCs as well as prognostic impact of POCs were evaluated in multivariable analyses. A subgroup analysis of minor resections was performed to evaluate POCs after laparoscopic surgery.P\u2009=\u20090.001). Comparing laparoscopic and open resections in the subgroup analysis of small resections, 6% (4/68) in the laparoscopic group developed POCs compared to 18% (51/289) after open resection . POCs were associated with a 27% increased excess mortality rate . However, primary tumor characteristics, tumor burden in the liver, extrahepatic spread, extent of liver resection and radicality had higher impact on survival.POCs were registered for 24% (276/1144) of all patients after CRLM resection. Major resection was a risk factor for POCs in multivariable analysis (IRR 1.76; Minimal invasive resections were associated with a decreased risk of POCs following resection of CRLM which should be considered in surgical strategy. Postoperative complications were associated with a moderate risk for inferior survival.The online version contains supplementary material available at 10.1007/s00268-023-07043-z. Approximately 25% of colorectal cancer patients develop liver metastases (CRLM) within three years of diagnosis and surgThe randomized Oslo-COMET trial showed a decreased complication rate and faster recovery following laparoscopic resections . ConsensTo date, knowledge on risk factors for complications following CRLM resection are scarce, but POCs have been associated with subsequent impaired long-term results , 12. An Data were collected in February 2018 from two national registries with prospectively registered data, i.e. the Swedish Colorectal Cancer Registry (SCRCR) and the National Quality Registry for Liver Cancer (SweLiv). Both registries have reported good conformity with source data throughout the study period and high coverage when compared to the Swedish Cancer Registry , 14\u201316. , heart infarction and cerebral infarction, ascites or pleural fluid demanding intervention, deep vein thrombosis or pulmonary embolism, portal vein thrombosis, postoperative intensive care more than 24\u00a0h, intra-abdominal infections demanding intervention, sepsis and other infections demanding intervention or reoperation. Special importance of infectious POCs has been proposed [All POCs classified as Clavien\u2013Dindo IIIa or worse were included but POCs were not registered according to the Clavien\u2013Dindo classification before 2013. The following registered complications were therefore included for the entire study period (2009\u20132016): bile leakage, wound dehiscence, postoperative bleeding and other surgical complications demanding intervention or reoperation, single organ failure including liver failure, renal failure demanding dialysisproposed , 18 and , anatomical resection of one segment or 1\u20132 wedge resections (Category I); anatomical resection of two segments or 3\u20134 wedge resections (Category II); anatomical resections including more than 2 segments but not including hemihepatectomies or extended resections, or in case of wedge resections more than 4 wedge resections (Category III); and all hemihepatectomies including extended hemihepatectomies and two-step resections (Category IV). Two-step resections were defined as two surgical events within 6\u00a0weeks.Based on SweLiv data of resected segments validated with surgical procedure codes, liver resections were categorized into four groups depending on the extent of surgery; i.e.P-value of\u2009<\u20090.05 was considered statistically significant. Risk factors for POCs were analyzed using Poisson regression and presented as incidence rate ratios (IRR) with 95% confidence intervals. Survival was calculated from the date of liver surgery to date of death or date of last follow-up (21st March 2019). Overall survival (OS) was computed using the Kaplan\u2013Meier method and RS was calculated using the Ederer II method [P\u2009<\u20090.10 in univariable analyses together with previously established statistically significant prognostic factors were tested in multivariable analyses through manual elimination to assess their confounding effect or independent effect on POCs and excess mortality.All statistical analyses were performed using Stata version 16.1 . A The following prognostic factors, previously found to influence patient survival , were inOut of the 20,853 colorectal cancer patients treated with radical resection between 2009 and 2013, a total of 1200 (5.8%) patients were also registered in SweLiv for resection of liver metastases. Of these, complication data after liver resection were registered for 1166 (97.3%) patients. Twenty-two patients treated for CRLM within 30\u00a0days of the primary surgery were excluded due to registered postoperative complications after colorectal surgery in SCRCR. Hence, 1144 patients constitute the study cohort, of which 733 (64.1%) patients were treated with chemotherapy before liver surgery. Open resection was performed in 957 (83.7%) patients, 91 (8.0%) patients were treated with laparoscopic resection and 96 (8.4%) patients with resection (open or laparoscopic) combined with ablation therapy. Major resection (Category IV) including 18 (1.6%) two-stage procedures was performed in 444 (38.8%) patients. Details on treatment are presented in Table P\u2009<\u20090.01), whereas infectious complications were not of the patients and patients with POCs had significantly longer median hospital stays (11\u00a0days) compared to patients with no POCs .Postoperative complications corresponding to CD IIIa or more were registered for 276 24.1%) whereof five patients died within 30\u00a0days of surgery (0.44%). Surgical complications, including bile leakage, wound dehiscence, and postoperative bleeding, were registered for 114 (10.0%) patients. Medical complications including single organ failure, ascites or pleural fluid demanding intervention and venous thromboembolism were registered in 124 (10.8%) patients and infectious complications in 99 (8.7%) patients. Surgical and medical complications were increasingly common when more extensive resections were performed (% whereofP\u2009=\u20090.48). In the laparoscopic group, 5.9% (4/68) of the patients were registered with POCs compared to 17.7% (51/289) in the open resection group. This corresponded to a 68% decreased incidence rate ratio in multivariable analysis adjusted for sex, preoperative oncological treatment, and anatomical or non-anatomical resection. Additional adjustment for anterolateral or posterosuperior resections did not affect the model and was not included in multivariable analysis of the subgroup . No postoperative bile leakage was registered after laparoscopic resection, while bile leakage was registered for 3.1% (9/289) of the patients after open resection (P\u2009=\u20090.22). Postoperative hospital stay was significantly shorter for patients treated laparoscopically (5\u00a0days) compared to open resection .The subgroup analysis designed to include cases suitable for laparoscopic resection included 68 patients treated with laparoscopy and 289 with open resection. There was no significant difference in median tumor size between the laparoscopic (20\u00a0mm) and open resection (20\u00a0mm) groups . In patients with postoperative complications the 5-year RS was 48.8% (95% CI 42.1\u201358.1) compared to 58.1% (95% CI 54.1\u201362.3) in cases without complications, corresponding to an increased relative risk of 27% in multivariable analyses . All risk factors including primary tumor characteristics and metastatic patterns are presented in Table The 5-year OS rate was 51.3% 95% CI 48.3\u201354.3) following resection of CRLM and the 5-year age-standardized RS rate was 56.1% to reduce complications and increase survival.Postoperative complications following liver surgery continue to be a concern. Enhanced Recovery After Surgery (ERAS) seems to be efficient in decreasing hospital stays, but not in preventing complications . In our By stratifying the liver resections into four groups, where only the fourth would be considered major, we aimed to give a clinical description of our cohort where single and small tumors are common. The definition of major liver resections as three or more liver segments is widelR1-resections were seen in 16% (40/276) of Category II resections and 29% (21/73) of Category III-resections, possibly indicating increased complexity in precision as the number of tumors rises. Since complications increase and radicality decreases in Category III, one can infer that multiple minor resections should not be regarded as minor surgery, and that further efforts to allow better precision and thus radicality are needed. Poor survival after R1-resections could indeed be related to unfavorable tumor biology or complications, but the close to 60% increased risk of excess mortality shown here underlines the importance of aiming for radical resections when feasible.Intraoperative bleeding is still a relevant clinical problem, although low-pressure anesthesia and surgical techniques including Pringle have decreased bleeding volumes , 26. BloOthers have reported that laparoscopy has the potential to reduce POCs , 30, 31.In our study, EMRR is increased after POCs. However, in multivariable analysis, ablation in combination with resection, R1-resection and category II\u2013IV resections were stronger risk factors associated with over 50% higher EMRR. The negative impact of complications on both long-term survival and tumor-free survival is in line with other studies , 12, 17.The strength of this study is the size of the study (n\u2009=\u20091144), and the fact that the impact of complications is studied in the context of other clinical factors known to have a prognostic impact. The most important limitation of this study is that it is retrospective, although data have been prospectively reported in national registries with high coverage. The validity of complication registration between different centers during a long period of time (2009\u20132016) will be inferior to prospective registrations by trained personnel. However, we have focused this paper on serious complications where the validity is better .In summary, almost one-quarter of all patients undergoing CRLM resection developed severe POCs within 30\u00a0days. Postoperative complications were significantly more common after major resections and were associated with both inferior long-term survival and prolonged postoperative hospital stays. Although the noted lower risk of complications in laparoscopic surgery was not translated into a benefit for long-term survival, our results support further development and the increased use of minimally invasive liver surgery for CRLM.Supplementary file1 (DOCX 15 kb)Below is the link to the electronic supplementary material."} +{"text": "\u0394H\u00b0) and indicates disorderness (+\u0394S\u00b0) at the solid-liquid interface owing to the strong affinity of metal ions with adsorbent. The heavy metal uptake selectivity followed the following decreasing order; Cr(VI) > Cu(II) > Ni(II) by both nanocomposites, with adsorption capacities falling in the range of 204.68\u2013343.05 mg g-1. Several adsorption kinetic and isotherm models were applied to experimentally calculated data, which suggest favorable adsorption of Cr(VI), Ni(II) and Cu(II) by TK-NC and TV-NC from the system while obeying general-order kinetics and R-P adsorption model, conferring the transition in adsorption kinetics order and involvement of multiple adsorption process.Heavy metal contamination of water has become a global environmental burden, which has stirred up agitation worldwide. Fabrication of adsorbents utilizing either low cost, environment friendly materials or waste products can be helpful in remediating environmental pollution. The current study evolved around the synthesis of nanocomposites derived from such raw precursors like spent tea waste biochar, hydroxyapatite, and clays. In this context, two nanocomposites, namely manganese ferrite doped hydroxyapatite/kaolinite/biochar (TK-NC) and manganese ferrite doped hydroxyapatite/vermiculite/biochar (TV-NC), were synthesized followed by their employment for decontamination of heavy metals from aqueous media. TK-NC and TV-NC exhibited the crystallite sizes in the range of 2.55\u20135.94 nm as obtained by Debye Scherrer Equation and Williamsons\u2013Hall equation The fabricated nanocomposites were characterized using FT-IR, SEM-EDX, and powder XRD. Batch adsorption studies were performed, and influence of different adsorption parameters on metal adsorption was examined. Thermodynamic studies revealed that the adsorption of Cr(VI), Ni(II) and Cu(II) on TK-NC and TV-NC was endothermic (+ The global shortage of freshwater resources, coupled with incessant accelerating human demands for safe and clean water, poor water management practices, and inequitable sharing of water, has urged the requisite to preserve current water resources for socioeconomic growth and environmental sustainability . The mod-1, but consumed prolonged time (2\u201372 hours) to reach equilibrium stage [Removal of contaminants, particularly heavy metals, from wastewater is an extremely challenging task. A multitude of water purification technologies, including chemical precipitation, coagulation and flocculation, ion exchange, ozonation, membrane filtration, electro-dialysis, biological treatment, and adsorption, have been tested for abatement of these pollutants from aqueous solutions . Though um stage \u201313 Lahore, Pakistan. Kaolinite and vermiculite clays were purchased from a local vendor in Akbari market, Lahore, Pakistan. All the chemicals were used as received without further purification.et al. [Spent black tea biochar was prepared by method described by Butt et al. with litet al. [2HPO4 (0.54g/500ml) solution, 13mM anhydrous CaCl2 (0.72g/500ml) solution was added dropwise while keeping temperature at 65\u2070C under vigorous stirring. During the reaction, pH of the solution was maintained at 10.5 by using 0.1M NaOH. After completion of reaction, the contents of reaction flask were allowed to rest overnight for aging of precipitates. Subsequently, the precipitates were filtered, washed with distilled water, and oven dried at 110\u00b0C overnight.HA nanoparticles were synthesized by chemical precipitation method described by Le et al. with litIn this study, two types of clays and vermiculite (VC)) were alternatively assimilated with activated tea waste biochar (TBC), calcium hydroxyapatite (HA), and transition metals precursors (manganese sulphate and ferrous sulphate) to fabricate manganese ferrite doped hydroxyapatite/kaolinite/biochar (TK-NC) and manganese ferrite doped hydroxyapatite/vermiculite/biochar (TV-NC) nanocomposites.et al. [2HPO4 solution (500ml) at 65\u00b0C under constant agitation. After half an hour, 1.8mM FeSO4.7H2O and 0.9mM MnSO4.H2O were added into the flask, and the reaction mixture was vigorously stirred for 30 minutes for homogenous mixing. To this suspension, 13mM CaCl2 solution (500ml) was added dropwise while maintaining pH of reaction 10.5 using 0.1M NaOH solution. After completion of reaction, the contents in the reaction flask were stand overnight for aging. Subsequently, the resulting greyish-brown precipitates were filtered, washed with distilled water, and dried in oven at 110\u00b0C. The Both nanocomposites were fabricated by method described by Wang et al. with mod-1 by using Tensor-27 performed to determine the surface functional groups of raw counterparts and fabricated nanocomposites. The crystalline nature and phase of samples were investigated by using X-ray Diffractometer . Zeta potential of nanocomposites was determined by using the Malvern Zetasizer Nano ZSP.Characterization of fabricated nanocomposites (TK-NC and TV-NC) and their pristine components was carried out by using different techniques. Scanning electron microscopy and energy dispersive X-ray (SEM-EDX) analysis were done using Nova NanoSEM 450 field-emission scanning electron microscope (FE-SEM) for examining the surface morphology and chemical composition of fabricated biochar supported metallo-inorganic nanocomposites and their pristine components. Fourier transform infrared spectroscopy (FTIR) was studied over a range 4000\u2013600 cmMBV was estimated to screen out potentially efficient adsorbents for metal ions adsorption studies. MBV is defined as the value of methylene blue standard solution in (ml) that is decolorized with 0.1 g of sample used. The test was performed using the standard protocol described by Cefic (1986) .-1 methylene blue solution in diluted acetic acid was prepared from methylene blue aqueous stock solution (1000 mg L-1), and its absorbance was recorded at 620 nm (0.84 \u00b1 0.01). 0.1 g of fabricated nanocomposites and their pristine counterparts were contacted with 5 ml of methylene blue experimental solution at room temperature under constant stirring. The samples were shaken until discoloration was observed, which was done by spreading few drops of treated methylene blue solution on a white surface and observing its hue. Then, another 1 ml of methylene blue solution was added and stirred to discolor. The inclusion of methylene blue experimental solution in 1 ml portions was repeated as long as discoloration occurred as noticed by a clear or almost colorless film on the surface. After appearance of color in treated sample, the methylene blue solution was added to the reaction vessel until the same absorbance was achieved as of the untreated methylene blue solution. The volume (ml) of methylene blue solution decolorized until the endpoint achieved was noted and recorded as MBV.To estimate the MBV, 5 mg L-1) of Cr(VI), Cu(II) and Ni(II) were separately prepared from potassium dichromate (K2Cr2O7), copper sulphate pentahydrate (CuSO4. 5H2O), and nickel chloride hexahydrate (NiCl2. 6H2O), respectively. The working solutions of preferred concentrations of respective metals were made by diluting the stock solutions.Batch experiments were performed to investigate the adsorption of target metal ions (Cr(VI), Ni(II) and Cu(II)) onto synthesized nanocomposites under varying conditions of different experimental parameters . To perform this aqueous stock solution (1000 mg L-1) taken in a conical flask with a known dose of adsorbent (0.1\u20130.5 g) was stirred at 200 rpm for a given time period (2\u201390 min) and temperature (30\u201380\u00b0C). The pH (1.0\u20138.0) of the solution was adjusted with the help of 0.1M HCl and 0.1 M NaOH. Afterwards, the sample suspension was filtered using cannula filtration to separate out solid and liquid phases. The residual amount of metal in filtrate was analyzed with the help of Flame atomic absorption spectrometer . The percentage removal efficiency (RE%) and the concentration of adsorbate sorbed on adsorbent (adsorption capacity (mg g-1), qexp) were calculated using Eqs (Ci (mg L-1) and Cf (mg L-1) are the initial and final concentrations of adsorbate in solution, respectively; V (L) is the volume of solution; and W (g) is the mass of adsorbent.For a series of experiments to be carried out to optimize the process parameters required for maximum metal adsorption, 50 ml of known concentration of respective metal ion solution and \u03b2 is known as full width at half maximum (FWHM). The average crystallite size obtained for HA is 5.64 nm. The Williamsons\u2013Hall equation (WH) as given in Where, crystallite size (D) and lattice strain (\u03f5) were measured through intercept and slope by linear plot between \u03b2cos\u03b8 vs 4sin \u03b8 by the Eq which isThe SEM micrograph of HA nanoparticles depicts The EDX analysis confirme\u22121 are attributed to stretching vibrations of\u2013OH group due to adsorbed water molecules in KC lattice. The appearance of these multi-characteristic bands denotes ordered structure of the KC [\u22121 correspond to Si-O group, and the ones appearing at 938 and 912 cm\u22121 are ascribed to Al-OH group [-1, while the band at 654 cm\u22121 reflects Al-OH functional group [\u22121 corresponds to stretching and bending vibrations of\u2013OH group, respectively, while the ones appearing at 972 and 664 cm\u22121 attributed to SiO2 band and Al-OH stretching, respectively [The FTIR spectra of procured well-ground clays (kaolinite (KC) and vermiculite (Vc)) are given in f the KC . The peaOH group . The peaal group . Similarectively , 27.The XRD diffractogram of KC refers tIn addition to that, SEM images of both the clays were also acquired , which dEDX spectra of KC and VC along with their elemental composition is presented in \u22121) corresponds to\u2013OH stretching of alcohols and phenolic compounds present in biochar [\u22121 are related to C\u2261C stretching [\u22121 ascribe to C = O group of carboxylic acids, ketones, and lactones [\u22121 correspond to stretching vibrations of C = C or C = O and C-O-C groups, respectively [\u22121 [The FTIR spectrum of TBC is shown in biochar . The pearetching . The siglactones . The peaectively , 35. Thevely [\u22121 .The powder XRD diffractogram of TBC illustraSEM image of TBC exhibite\u22121 [\u22121 in nanocomposite is evidenced to be little shifted from pristine TBC (\u22121 (C\u2261C stretching) in TBC spectrum was not appeared in the case of TK-NC. Furthermore, TBC contributed to bands at 1996 and 1876 cm\u22121 ascribing to C = O group of carboxylic acids, ketones and lactones. The peak evidenced at 1623 cm\u22121 corresponds to stretching vibrations of C = C or C = O groups with red shift from 1573 cm\u22121 representing interaction of TBC with other components in the nanocomposite [-1 correspond to asymmetrical bending vibrations of PO43- of HA and Si-O groups of KC, respectively [\u22121 are related to Al\u2013OH stretching. The Si-O-Al bond was detected at 796 and 750 cm\u22121, while the peak at 673 cm\u22121 attributed to Al\u2013OH functional group [For affirmation of nanocomposites formation, FT-IR, XRD, SEM and EDX analysis were performed. FT-IR spectra of nanocomposites (TK-NC and TV-NC) are presented in \u22121 . Similar85 cm\u22121) , while tomposite , 34. Theectively . The sigal group , 37.\u22121 is ascribed to O-H stretching vibrations of adsorbed water molecules in the nanocomposite [\u22121 [\u22121 accredited the appearance of C = O and C = C groups, respectively. The appearance of PO43- group asymmetrical vibrations at 1057 cm\u22121 affirmed the presence of HA in nanocomposite [\u22121 corresponds to Si-O group emanating from VC. The Mg/Al-OH vibrations are observed at 682 cm\u22121 [-1 in both nanocomposites are attributed to M-O bond present in Manganese ferrite. The FT-IR analysis of metal-laden TK-NC and TV-NC is also performed to explore the interaction between adsorbent and adsorbate, which is displayed in In the case of TV-NC , the peaomposite , 34. Thesite [\u22121 , 34. Theomposite . The pea682 cm\u22121 , 27. In , which conforms to JCPDS card no. 00-005-0143 [, which matches well with JCPDS card no. 09\u20130432 [2O4 (JCPDS card no. 10\u20130319) [2O4 and hydroxyapatite ascribes to their poor crystalline nature owing to smaller crystallite size suggesting the formation of nanocomposite [2O4 corresponding to hkl values of (220), (311), (400), (422), (440), and (620), which is in good agreement with JCPDS card no. 10\u20130319 [2O4 and hydroxyapatite with peak broadening, in this case as well, indicates poor crystallinity owing to smaller crystallite size suggesting the formation of nanocomposite [005-0143 . The pea 09\u20130432 . The dif10\u20130319) . The lowomposite , 41. XRDomposite . TV-NC e, (311), 0, (422), 10\u20130319 . The suclues of 2, (311), omposite . The cry2O4 nanoparticles over the clay bed surface due to the high propensity of nanoparticles towards agglomeration. These nanoparticles exhibit fluffy texture and have been observed to be evenly distributed on the surface of clay. These findings are in agreement with earlier studies [2O4 nanoparticles possessing a spongy nature are clumped together in micro aggregates, with fairly even localization on the clay surface. These findings are in agreement with the earlier studies [The morphology of the prepared nanocomposites was evaluated by acquiring SEM images . In the studies , 42. Sim studies , 42.2O4, while the remaining elements of Al and Si in %weight of 5.43 and 5.27%, respectively, represent the presence of KC in the nanocomposites. The EDX spectrum of TV-NC is presented in 2O4 is also confirmed by the occurrence of Ca/P and Mn: Fe in 1.64 and 1:2 ratios, respectively. A small quantity of S (1.39%) was also observed in the spectrum, appearing due to MnSO4 used in the nanocomposite synthesis. Hence, the EDX analysis in both cases confirmed the doping of TBC, HA, and MnFe2O4 on to clay structures.The elemental composition of TK-NC and TV-NC is confirmed from EDX analysis . The EDXMethylene blue adsorption test is extensively employed as an indicator for evaluating the adsorption efficiency of any adsorbent, particularly carbon-based matrix, as this dye is often taken into account as a model contaminant due to difficulty of its degradation. It measures the adsorption performance of test adsorbent in terms of methylene blue value, which is the volume of standard methylene blue solution discolored with 0.1 g of adsorbent. The TK-NC, TV-NC, and their pristine components were subjected to methylene blue test to explore their adsorptive potential. The methylene blue values for all the test adsorbents are displayed in TK-NC and TV-NC were examined for their efficacy against the removal of target heavy metals (Cu(II), Cr(VI) and Ni(II)) from aqueous solutions in batch mode as a function of varying experimental conditions .Contact time is an imperative function to be studied during the adsorption experiments . The infThe metal ions adsorption on TK-NC and TV-NC increased with an increase in contact time, which was followed by a gradual decline in removal efficiency until equilibrium was achieved, and afterwards an insignificant rise in sorption capacities for both nanocomposites was noticed. The adsorption of metal ions onto TK-NC achieved equilibrium at 40 minutes for Cu(II), 50 minutes for Ni(II), and 60 minutes for Cr(VI) . Though,qt (mg g-1) is the concentration of adsorbent at time t (min), qe (mg g-1) is the concentration at equilibrium, k1 (min-1) and k2 (g mg-1 min-1) and k3 (min\u22121 (g mg\u22121) n\u22121) are the rate constants for pseudo-first, pseudo-second, and general orders, respectively, a (mg g-1 min-1) is the initial rate of adsorption, b (g mg-1) is the desorption constant, and n is the kinetic sorption order .Adsorption kinetics describes the adsorption rate of solute onto the adsorbent and is very prominent function for evaluating the removal efficiency of an adsorbent. Different kinetics models and calculated adsorption capacities, comes out to be general order kinetics model for all six cases. The fitting parameters are presented in The best fitting of model, as assumed from highest 0.5) is the intra-particle diffusion rate constant, qt (mg g-1) is the adsorption capacity at time (t), and Ci represents the thickness of boundary layer of stage i. A higher value of Ci indicates a greater boundary layer effect. If the intra-particle diffusion is the sole limiting factor, the plotted lines will pass through the origin (Ci = 0). However, if there is some involvement of external mass transfer, the lines will deviate from origin. The obtained fitting parameters and plots for intra-particle diffusion model are presented in i \u2260 0), suggesting a multi-step adsorption process [The adsorption kinetics were further investigated to identify the rate-controlling steps occurring during the adsorption process by using the intra-particle diffusion model , which i process .Macadamia nutshell biochar and montmorillonite modified peanut shell biochar [Zea mays based biochar [To investigate the influence of temperature, a set of batch experiments were performed by changing temperature from 30 to 80\u00b0C . The exp biochar , 51. Sim biochar , 52. In biochar .G\u00b0), standard enthalpy change (\u0394H\u00b0) and standard entropy change (\u0394S\u00b0), are calculated using the following equations:KC, Cs and Ce are equilibrium constant, equilibrium concentration of the adsorbate sorbed on the adsorbent in mg L-1, and equilibrium concentration of adsorbate in the solution in mg L-1. R is ideal gas constant which value is 8.314 J mol-1 K-1 and T is the adsorption temperature in Kelvin (K).Thermodynamic parameters, i.e. standard free energy change (\u0394KC vs T-1 for the adsorption of Cu(II), Ni(II), and Cr(VI) on TK-NC and TV-NC but points to disorderness around the nanocomposite surface owing to the strong affinity of metal ions with adsorbent as reflected by +\u0394S\u00b0 value [\u0394G\u00b0 for Cu(II), Ni(II), and Cr(VI) by TK-NC and TV-NC suggest that the adsorption of all metal ions on both nanocomposites is non-spontaneous. These findings are in accordance with the previous work in the support of non-spontaneity of adsorption of metal ions (Cu(II), Pb(II), Z(II), ad Cd(II)) on olive branches derived activated carbon at the temperature interval (303\u2013323 K) [The plot of lnnd TV-NC is used ectively . The valS\u00b0 value . The pos3\u2013323 K) .-1) was agitated for predetermined optimum time period and temperature with 0.1 g of adsorbent at altering pH levels. The data is presented in The pH of solution is a significant experimental parameter that is essential to be studied in adsorption experiments as it controls the uptake of metal ions onto the adsorbent surface. It influences the adsorbents\u2019 surface charge and speciation of adsorbent and adsorbate molecules in aqueous solution , 56. In 4-) ions are the most predominant form of Cr(VI), and when the pH moves towards basic medium, chromate ions (CrO42-) are the most prevalent species to occur in the aqueous system [-1 were observed at pH 3.0 for TK-NC and TV-NC, respectively. If pH < pH(PZC), the charge on the nanocomposite surface would be positive, and if pH > pH(PZC), it would be negative [(PZC) as HCrO4-. With an increase in pH (above pH(PZC)) the positive charge density gradually reduces due to an increase in the density of OH- ions, and the nanocomposites\u2019 surface get an anionic charge, resulting in electrostatic repulsion between negatively charged adsorbents\u2019 surface and Cr(VI) anionic species, which leads to poor metal adsorption performance [In acidic medium, hydrogen chromate (HCrOs system . The adsnegative . In thisr TV-NC) , the surformance .-1 for Cr(VI) using magnetic modified Astragulus membranaceus derived biochar was achieved at pH 2.0 [3O4 doped hydroxyapatite encapsulated alginate beads, with maximum removal capacity recorded as 29.32 mg g-1 [2+ and CuOH+ ions are the most prevalent species of Cu(II), whereas at pH (>5.0), Cu(II) exists as insoluble precipitates of Cu(OH)2 [-1) onto TK-NC and TV-NC, respectively, were recorded at pH 5.0. At pH \u2265 5.0, Ni2+ is the most prevalent form of Ni(II) in aqueous media [2 precipitates. At pH 7.0, the highest uptake of Ni(II) onto TK-NC and TV-NC occurred, with removal capacities of 176.81 and 166.62 mg g-1, respectively. At low pH RL> 0 (favorable); and 1< RL (unfavorable).Here, CKF ((mg g-1) (L mg-1)1/n) represents the intensity of sorption and nF is the Freundlich dimensionless constants, which measures the deviation of sorption process from linearity. If the value of 1/nF is approaching 0, then it is indicative of the heterogeneous nature of the system, and if it is very close to 1, it specifies homogeneity of the system. The value of 1/nF ranging between 0 to 1 also implies the favorability of adsorption process.The Freundlich isotherm addresseThe Redlich-Peterson (R-P) isotherm is a 3 pKR is the R-P constant (L g-1), a is R-P isotherm constant (L mg-1), and g is an exponent that has a value in the range of 0 to 1. If the value of g is approaching 0, then it is reduced to Freundlich isotherm equation, and if its value is approaching 1, then it is inclined to Langmuir isotherm. Ce is the concentration of adsorbate at equilibrium (mg L-1), and qe is the adsorption capacity at equilibrium (mg g-1).2 and reduced chi-square. The Kl values in Langmuir isotherm model ranged between 0\u20131, which indicates a good interaction between the adsorbent and metal ions. In the case of both nanocomposites, the values of RL ranged between 0.233\u20130.487, which are less than unity, which affirms the favorability of sorption of metal ions on the nanocomposites surface. The favorability of adsorption of all metal ions on TK-NC and TV-NC was also confirmed by nF values (2.223\u20134.663) obtained from Freundlich isotherm model, which are in the range of 1\u201310. The 1/ nF values ranging between (0.215\u20130.450) showed the heterogeneous nature of TK-NC and TV-NC surfaces. For all metal ions investigated, the R-P exponent g values (0.916\u20131.00) have approached unity, indicating that R-P has reduced to Langmuir isotherm. This inferred that the adsorption of all metal ions took place via monolayer coverage on heterogeneous surfaces of both nanocomposites with a relatively uniform distribution of binding sites [-1) > Cu (252.65 mg g-1) > Ni (204.68 mg g-1) and Cr (281.82 mg g-1) > Cu (261.87 mg g-1) > Ni (230.34 mg g-1), respectively. For Ni(II) and Cu(II), the removal capacity of TV-NC is better than that of TK-NC, while for Cr(VI), V-NC performed better. The current study demonstrated that the prepared nanocomposites (TK-NC and TV-NC) showed enhanced adsorption capacity for all three heavy metals (Cu(II), Cr(VI), and Ni(II)), which is much greater than already reported adsorbents for TK-NC and TV-NC after metal adsorption was noticed might be chelated with\u2013OH and\u2013COOH functionalities from biochar present on the surface of both nanocomposites, as represented in Eqs 6(OH)2), and the release of proton in aqueous media from \u2261PO-H functional site of HA and its subsequent compensation by metal ion to form \u2261PO-M+ is given in Eqs of HA along with dissolution of HA, followed by chemical precipitation of metal ions as corresponding metal phosphate 6(OH)2 formation;M2O4 nanoparticles as proposed by Tatarchuk et al. [2O4 nanoparticles gets protonated under acidic pH and attained an anionic charge in alkaline conditions, which encouraged the binding of metal ions on its surface via electrostatic attraction > Cu (252.65 mg g-1) > Ni (204.68 mg g-1) for TK-NC, and Cr (281.82 mg g-1) > Cu (261.87 mg g-1) > Ni (230.34 mg g-1) for TV-NC. TV-NC exhibited better adsorption activity for Cu(II) and Ni(II) as compared to TK-NC, while for Cr(VI), TK-NC surpassed TV-NC. The adsorption of metal ions by TK-NC and TV-NC was predominantly accredited to ion exchange, electrostatic interaction, surface complexation, and chemical precipitation. The employment of TK-NC and TV-NC in wastewater purification signifies an economically viable and environment-friendly alternative since it provides an innovate and green perspective of reutilizing tea waste and natural clays of low economic value to fabricate plausibly efficient nanocomposites, which have registered profound Cu(II), Cr(VI), and Ni(II) removal from aqueous systems in addition to being less time consuming and requiring less stringent conditions for adsorption. Thus, TK-NC and TV-NC can be used as potential nanocomposites for abatement of heavy metals from aqueous environments.The kaolinite and vermiculite-based nanocomposites and manganese ferrite doped hydroxyapatite/vermiculite/biochar (TV-NC)) with crystallite sizes ranging from 2.55\u20135.94 nm were synthesized using a facile one pot synthesis approach for abatement of multiple heavy metals from water. The results of powder XRD and EDX analysis confirmed the successful integration of clay, hydroxyapatite, tea waste biochar, and manganese ferrite into the nanocomposite matrix. The SEM analysis exhibited that all the components of nanocomposites retained their structural integrity and were evenly localized on the surface of nanocomposite. TK-NC and TV-NC exhibited efficient removal of selected metal ions within the contact time of 30\u201360 min at 0.1 g adsorbent dose, temperature (50\u201360\u00b0C), and pH (3.0 (Cr(VI)), 5.0 (Cu(II)), and 7.0 (Ni(II))). The adsorption of metal ions, as obtained from the Langmuir isotherm, followed the following order: Cr and TV-NC (d-f) after adsorption of metal ions.(TIF)Click here for additional data file.S2 Figt vs t0.5) to adsorption of Cu(II), Ni(II), and Cr(VI) on to TK-NC (a-c) and TV-NC (d-f), respectively.Fitting of intra-particle diffusion model (q(JPG)Click here for additional data file.S3 FigFitting of thermodynamic model to adsorption of Cu(II), Ni(II), and Cr(VI) on to TK-NC (a-c) and TV-NC (d-f), respectively.(JPG)Click here for additional data file.S4 Fig(PZC) for TK-NC (a) and TV-NC (b).The zeta potential under different pH and pH(JPG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file."} +{"text": "Are concerning computed tomography findings among patients seen in the emergency department with acute, low-risk pulmonary embolism (PE) associated with differences in treatment and/or clinical outcomes?In this cohort study of 817 patients, concerning computed tomography findings were associated with increased hospitalization and resource utilization but not short-term adverse clinical outcomes.These findings suggest that concerning computed tomography imaging findings may be a significant barrier to outpatient treatment among patients with otherwise low-risk acute PE. This cohort study evaluates the association of concerning computed tomography (CT) findings with outcomes and treatment of patients in the emergency department with acute, low-risk pulmonary embolism (PE). Most patients presenting to US emergency departments (EDs) with acute pulmonary embolism (PE) are hospitalized, despite evidence from multiple society-based guidelines recommending consideration of outpatient treatment for those with low risk stratification scores. One barrier to outpatient treatment may be clinician concern regarding findings on PE-protocol computed tomography (CTPE), which are perceived as high risk but not incorporated into commonly used risk stratification tools.To evaluate the association of concerning CTPE findings with outcomes and treatment of patients in the ED with acute, low-risk PE.This cohort study used a registry of all acute PEs diagnosed in the adult ED of an academic medical center from October 10, 2016, to December 31, 2019. Acute PE cases were divided into high- and low-risk groups based on PE Severity Index (PESI) class alone or using a combination of PESI class and biomarker results. The low-risk group was further divided based on the presence of concerning CTPE findings: (1) bilateral central embolus, (2) right ventricle\u2013to\u2013left ventricle ratio greater than 1.0, (3) right ventricle enlargement, (4) septal abnormality, or (5) pulmonary infarction. Data analysis was conducted from June to October 2022.The primary outcome was all-cause mortality at 7 and 30 days. Secondary outcomes included hospitalization, length of stay, need for intensive care, use of echocardiography and/or bedside ultrasonography, and activation of the PE response team (PERT) .P\u2009<\u2009.001). Low-risk patients with concerning CTPE findings were less frequently discharged from the ED than those without concerning CTPE findings and had more frequent echocardiography and PERT activation for consideration of advanced therapies .Of 817 patients female patients; 129 [15.8%] Black and 645 [78.9%] White patients) with acute PEs, 331 (40.5%) were low risk and 486 (59.5%) were high risk by PESI score. Clinical outcomes were similar for all low-risk patients, with no 30-day deaths in the low-risk group with concerning CTPE findings (0 of 151 patients) vs 4 of 180 (2.2%) in the low-risk group without concerning CTPE findings and 88 (18.1%) in the high-risk group (In this single-center study, CTPE findings widely believed to confer high risk were associated with increased hospitalization and resource utilization in patients with low-risk PE but not short-term adverse clinical outcomes. Indeed, of the major strategies for identification of low-risk patients\u2014the PE Severity Index (PESI) score,10 the simplified PESI (sPESI) score,11 the Hestia criteria,12 and the European Society of Cardiology (ESC) guidelines7\u2014only the ESC guidelines include imaging findings, and these are limited to signs of RV enlargement or RV dysfunction (RVD).Of the approximately 250\u2009000 patients diagnosed with acute pulmonary embolism (PE) in US emergency departments (EDs) each year, most are hospitalized, despite evidence from multiple studies and society-backed guidelines recommending consideration of discharge for the 25% to 50% with low risk stratification scores.14 Later studies, which directly examined the prognostic value of CTPE findings, focused on their potential to predict decompensation in hemodynamically stable patients, rather than those with low risk stratification scores.16 Most recently, 2 meta-analyses18 produced conflicting results, with one finding higher rates of early adverse outcome in patients with low-risk PE and RV dysfunction on echocardiography or CTPE and the other finding no association between abnormal findings on CTPE and in-hospital or 30-day mortality.In reality, little evidence is available regarding the prognostic value of CTPE imaging in patients with acute PE and low risk stratification scores. Early studies focused on echocardiographic markers, rather than CTPE findings, and were performed prior to the validation and widespread use of risk stratification tools.These results aside, there remains concern among many emergency medicine (EM) clinicians that certain CTPE findings confer higher risk of adverse outcome, irrespective of risk stratification score. These include bilateral and centrally located emboli, extensive clot burden, pulmonary infarction, and radiographic evidence of RV strain . In this study, we sought to understand the association of these so-called concerning CTPE findings with hospitalization, resource utilization, and patient outcomes, taking advantage of a large and well-curated registry of patients with acute PE diagnosed in the ED.19 including use of a standardized data abstraction form (STROBE) guidelines.20This study was granted exemption from review and the requirement for informed consent by the institutional review board of the University of Michigan (UM). Construction of the UM registry of acute PEs diagnosed in the ED is described in detail in eMethods in ion form , which wRace and ethnicity were self-reported with the following categories: American Indian and Alaska Native, Asian, Black or African American, Native Hawaiian or other Pacific Islander, White, unknown, other, and patient refused. Race and ethnicity were included in our analysis to help describe the diversity of the population studied so that future studies can compare and assess how population differences may impact their resultsAbstractors recorded any PESI score or class documented in the patient medical record and also calculated the PESI score using abstracted variables and peak vital signs during the ED stay. Interrater reliability was determined for the calculated PESI class, and in the event of disagreement, an adjudicating review was performed.Troponin I and high-sensitivity troponin T were included in the registry when measured as part of the ED evaluation. For the subset of acute ED-PEs for which biomarkers were available, we performed a parallel analysis in which patients with abnormal troponin levels were classified as high-risk irrespective of PESI class.CTPE findings were abstracted from the radiology reading, including laterality , largest vessel involved , RV-to\u2013left ventricle (LV) ratio, and presence of RV enlargement, septal abnormality , or pulmonary infarct. Missing data were obtained by fresh review of CTPE images by 2 board-certified radiologists (A.L.L. and M.S.K.). Patients were classified as having concerning CT imaging findings if 1 or more were present: (1) bilateral embolus described as saddle or main pulmonary arteries, (2) RV-LV ratio greater than 1, (3) RV enlargement, (4) septal abnormality consistent with RV pressure overload , or (5) pulmonary infarction.Bivariate linear and logistic regression analyses were used to test hypotheses about risk-group differences in continuous and categorical outcomes, respectively. For continuous outcomes, effect sizes were calculated as unstandardized between-group differences. For categorical outcomes, effect sizes were calculated as unstandardized marginal effects . An \u03b1 level of .05 was used for all analyses, and all hypothesis tests were 2-sided. Analyses were conducted with the Stata statistical software package version 15 (StataCorp). Statistical analysis was performed in June to October 2022.Construction of the acute ED-PE registry is shown in flow sheet form in eFigure 1 in P\u2009=\u2009.36). However, more high-risk cases were saddle PEs or involved the main pulmonary arteries vs low-risk cases . Likewise, high-risk cases had statistically higher mean (IQR) RV-LV ratios . There was no statistically significant difference between groups for RV enlargement or septal abnormalities, but more patients in the high-risk group vs the low-risk group had these findings . Pulmonary infarction was less common in the high-risk group vs the low-risk group .We next examined the frequency of various CTPE findings in low- and high-risk groups . There wP\u2009<\u2009.001). Even low-risk acute ED-PEs with the most concerning appearing CTPEs, eg, 41 cases with 3 concerning findings of bilateral, central emboli with an RV-LV ratio greater than 1 and septal abnormalities, had no deaths at 30 days.As shown in P\u2009=\u2009.86). Both low-risk groups were significantly different from the high-risk group, in which 80 patients (16.5%) were admitted from ED directly to ICU . The pattern remained unchanged when considering requirement for ICU-level care at any time during hospitalization: 4 (2.6%) and 5 (2.8%) for low-risk groups with and without concerning CTPE findings, respectively and 103 (21.1%) for the high-risk group . Even multiple concerning CTPE findings for an individual patient did not have a significant association with ICU utilization in low-risk patients, with only 2 of 55 patients (3.6%) with 2 or more concerning CTPE findings requiring ICU-level care.Very few patients treated in the ED from either low-risk group required admission to the intensive care unit (ICU) , with no significant difference between the low-risk group with concerning CTPE findings and the high-risk group . The pattern was reversed for hospital LOS, which was a mean (SD) of 2.3 (1.9) and 2.6 (3.5) days for the low-risk groups with and without concerning CTPE findings, respectively , and 5.8 (5.9) days for the high-risk group .P\u2009<\u2009.001) and 87 (57.6%) vs 49 (27.2%) for TTE . Similarly, the multidisciplinary PE response team (PERT), which advises ED clinicians in cases in which they believe advanced therapies might be beneficial, was activated in 34 (22.5%) low-risk cases with concerning CTPE findings vs 11 (6.1%) without . The frequency of POCUS (130 [26.8%]), TTE (262 [53.9%]), and activation of the PERT (138 [28.4%]) in the high-risk group were not statistically different compared with the low-risk group with concerning CTPE findings (Both cardiac point-of-care ultrasonography (POCUS) and formal transthoracic echocardiography (TTE) were performed more frequently in low-risk cases with than without concerning CTPE findings: 35 (23.2%) vs 15 (8.3%) for POCUS had at least 1 troponin level measured as part of the ED evaluation. We performed a parallel analysis had no obvious association with short-term mortality or need for intensive care in otherwise low-risk patients.18 published in the past several years that concluded that RVD is associated with short-term mortality in low-risk PE. Both reports, however, pooled data from studies using different measures of RVD, and their primary conclusions were based on combinations of CTPE, echocardiography, and biomarker testing. In the one case in which imaging modalities and biomarkers could be separated\u2014due to an individual patient-data meta-analysis (IPDMA) method\u2014RVD assessed via CTPE was not found to be associated with in-hospital or 30-day mortality. Of note, this pooled IPDMA population also matched our cohort fairly well in terms of the age, comorbidities, rate of concerning CTPE findings, and overall 30-day mortality among low-risk patients.18At first glance, our results seem to conflict with recent reports in the field. Specifically, there have been 2 meta-analyses23 A number of implementation studies have been conducted, focusing on creation of tools for electronic clinical decision support and protocols to facilitate identification, appropriate management, and timely follow-up of low-risk patients.24 These studies, however, have either explicitly excluded patients with concerning CTPE findings or simply not addressed the potential association between perceived risk and physician decision-making. Our findings suggest not only that outpatient management may be considered for these patients, but that concerning CTPE findings represent an important barrier to discharge. Compelling evidence, including the results of the current study, will likely need to be paired with effective implementation strategies to overcome their perceived risk. Indeed, as clinicians and health systems become more comfortable and facile with other aspects of home treatment, we expect a widening of the gap in discharge of low-risk patients with and without concerning CTPE findings.While our findings require confirmation, it is worth considering their potential impact on EM practice. Over the past 2 decades, a strong evidence base has been established to support safe outpatient management of PE, but adoption by EM clinicians has been slow and remains limited in most settings.Beyond clinical implications, our study has interesting aspects from the stand point of PE research and quality improvement. In particular, construction of the acute ED-PE registry required a multimodal approach, combining manual medical record abstraction (by the MEDIC collaborative), query of the EMR, and a substantial amount of physician medical record review to identify and exclude inappropriate cases. Nonacute and nonsignificant PEs were often difficult to distinguish from acute ED-PEs without physician review of the documented medical decision-making. For example, patients who presented to our facility for second opinion after recent diagnosis and treatment at another hospital were treated quite differently by EM clinicians, as evidenced by the greater than 10-fold higher rate of discharge, but typically had acute-appearing emboli on imaging and final clinical impressions of acute PE. Similarly, identification and separation of insignificant subsegmental PEs, radiographic mimics , and chronic PEs are likely to remain a major challenge for automated EMR queries or non\u2013medically trained abstractors. Our approach with repeated physician reviews resulted in a high-fidelity registry with essentially no missing data and high confidence in the accuracy and acuity of each PE diagnosis. At the same time, the experience raises concerns about the accuracy of PE data acquired from national databases or unfiltered EMR queries. These observations may be particularly important as health systems and researchers create interventions to evaluate and increase outpatient PE treatment.Our study does have some limitations. The primary outcome was uncommon, so our study may be underpowered to detect modest differences between groups. The study was also retrospective and limited to a single center. These limitations are mitigated, to some extent, by the relatively large number of cases in the registry and the numerous steps taken to minimize issues typically associated with retrospective medical review, eg, repeated physician review of each medical record, radiology overread of CTPE scans with missing measurements, and so on. These measures aside, retrospective design limits the ability to discern what factors influenced physician behavior at the time of patient care and introduces the possibility of confounding variables that may not have been accounted for, eg, social and economic factors requiring admission. Similarly, our practice setting\u2014an academic, tertiary health care center with a large population of patients with advanced cancer and cardiopulmonary disease\u2014does not reflect all hospitals, and our cohort had limited racial and ethnic diversity. To be truly generalizable, our findings will need to be reproduced in prospective, multicenter studies encompassing the full spectrum of EM practice. Our study is also somewhat limited in its focus on concerning CTPE findings as opposed to ultrasonographic findings. While many patients in our registry underwent formal TTE, it was infrequently completed during ED evaluation, and POCUS was limited by inconsistency in the training of operators and quality of documentation. Our analysis was also limited to short-term clinical outcomes, and it is possible that CTPE findings could be associated with other clinically significant endpoints, eg, post-PE syndrome, recurrent venous thromboembolism, or chronic thromboembolic pulmonary hypertension. Furthermore, our study does not address risk of bleeding, although other studies have consistently shown low rates of major bleeding in low-risk patients with acute ED-PE.In summary, ED patients with acute, low-risk PE had similar short-term outcomes irrespective of CTPE results. Nonetheless, specific CTPE findings were associated with increased resource utilization and hospitalization of these patients. Future implementation studies aimed at maximizing guideline-recommended outpatient management of acute low-risk PE should account for and address EM clinician concerns regarding CTPE imaging." \ No newline at end of file