P=0.015), T stage (P=0.006), lymph node metastasis (P=0.028) and the worst prognosis of PC patients (P=0.004). Meanwhile, a positive relationship between FAM172A and E-cadherin (E-cad)  was observed in clinical samples, which contributed to the better prognosis of PC patients (P=0.014). FAM172A silencing induced EMT in both AsPC-1 and BxPC-3 cells, including inducing the increase of Vimentin, MMP9 and pERK and the decrease of E-cad and β-catenin expression, stimulating EMT-like cell morphology and enhancing cell invasion and migration in PC cells. However, MEK1 inhibitor PD98059 reversed FAM172A silencing-enhanced EMT in PC cells. We conclude that FAM172A inhibits EMT of PC cells via ERK-MAPK signaling.FAM172A, as a newly discovered gene, is little known in cancer development, especially in pancreatic cancer (PC). We investigated the potential role and molecular mechanism of FAM172A in epithelial to mesenchymal transition (EMT) in both human clinical samples and PC cells. FAM172A was downregulated in human PC tissues compared with that in non-cancerous pancreas cells by immunohistochemistry and qRT-PCR. FAM172A expression was negatively associated with tumor size ( Summary: FAM172A inhibits EMT in pancreatic cancer via specifically regulating ERK-MAPK signaling. Pancreatic cancer (PC), as a deadly malignancy, ranks as the fourth leading cancer-related cause of death in Europe . It is aFamily with sequence similarity 172 member A (FAM172A), cloned from human aortic tissues, is subsequently observed in human endothelial and vascular smooth muscle cells, and macrophages . HoweverThe ERK-MAPK signaling plays a significant role in regulating cell proliferation, differentiation, survival, migration senescence and apoptosis via transmitting signals from cell surface receptors . ERK-MAPP<0.01) (P=0.002)  A. In mosP<0.01) B, and viP<0.01) C. SpearmP=0.002) .Fig. 1.P=0.015), T stage (P=0.006), lymph node metastasis (P=0.028), but had no relationship with age, gender, tumor location, differentiation, vascular invasion and preoperative CA199 level in PC patients (P>0.05) (P=0.003) (P=0.159) (P=0.014) (P=0.020) (Chi-squared test showed that FAM172A was negatively associated with tumor size ((P>0.05) . PatientP=0.003) D. ThoughP=0.159) E, patienP=0.014) F. In mulP=0.020) .Table 2.P=0.002)  A. Both FP=0.002) B,C. The in vitro.AsPC-1 and BxPC-3 cells with high FAM172A expression were used for FAM172A silencing experiments. FAM172A protein expression in the FAM172AsiRNA group was much lower than that in the siRNAcontrol group in both cell lines . FAM172APD98059 was identified as a highly selective inhibitor of MEK1 activation and the MAP kinase cascade . PD98059In addition, FAM172AsiRNA transfected AsPC-1 and BxPC-3 cells presented EMT-like cell morphology: most cells lost their epithelial properties of tight junction, and presented a spindle-shaped and fibroblast-like morphology . Howeverin vitro  was significantly reversed by MEK1 inhibitor PD98059. PD98059 also inhibited FAM172A silencing-induced EMT like cell morphology and cell invasiveness. Previous studies have shown that FAM172A promotes the proliferation of papillary thyroid carcinoma cells via p38/MAPK signaling , which were cultured in the recommended growth media with 10% FBS .IHC was performed as described previously ,b. BriefAs described previously , whole-cell lysates of PC cells were put into 12% SDS-polyacrylamide gels, transferred to PVDF membrane  and incubated with primary FAM172A , E-cadherin , β-catenin , N-cadherin , Vimentin , MMP9 , and α-Smooth muscle actin , pERK , ERK  and GAPDH  antibodies overnight. Then membranes were incubated with secondary antibodies (Proteintech) at room temperature and were visualized with the ECL machine . PC cells were pretreated with 20 µM of MEK1 inhibitor PD98059  for 2 h before western blotting. The experiment was repeated three times.As described previously , qRT-PCRThe FAM172AsiRNA sequences were: sense: 5′-GCCACTGAGAGTGAACCAAAG -3′, which were synthesized from GenePharma company . siRNA transfections (20 µM) were mixed with Oligofectamine 3000  for transient transfection following the manufacturer’s instructions.As described previously , FAM172ABriefly, FAM172AsiRNA and siRNAcontrol transfected AsPC-1 and BxPC-3 cells (pretreated with 20 µM of PD98059 for 2 h only once) was seeded onto 8.0-µM pore size membrane inserts  coated with matrigel  in 24-well plates with FBS-free growth media. Growth media plus 10% FBS was added to the bottom wells as a chemoattractant. 24 h later, cells that moved to the underside of the inserts were stained with Crystal Violet Hydrate . The migratory cells were counted in five random fields per well. Results were expressed as cells migrated per field and repeated three times. The transwell assay was performed in a similar way without matrigel.in vitro were described as means±s.e. (standard deviation) and were compared via t-test. P-value is presented as follow: *P<0.05; **P<0.01.Under SPSS software 20.0 , paired nonparametric test, chi-squared test and spearman correlation test were used for IHC assays, respectively. The log-rank test and Cox's regression was used to evaluate the postoperative survival time of PC patients. Western blotting, qRT-PCR and transwell assays


The present study evaluates the protective effects of myricitrin and its solid lipid nanoparticle (SLN) on diabetic nephropathy (DN) induced by streptozotocin-nicotinamide (STZ-NA) in mice. In this experimental study, 108 adult male NMRI mice were divided into 9 groups: control, vehicle, diabetes, diabetes + myricitrin 1, 3, and 10 mg/kg and, diabetes + SLN containing myricitrin 1, 3, and 10 mg/kg. After the experimental period, the plasma and tissue samples were collected for experimental, histopathological, real-time PCR and apoptosis assessments. Total antioxidant capacity, catalase, glomerular filtration rate, plasma level of albumin, urine (BUN) and, creatinine (Cr) levels decreased, and the kidney weight, intake/output, malondialdehyde, plasma level of BUN and Cr, urine level of sodium, potassium, albumin and glucose, fractional excretions of sodium and potassium, transforming growth factor-β (TGF-β) and nuclear factor kappa B (NF-κB) gene expression, red blood cell accumulation and infiltration of inflammatory cells, and kidney apoptosis increased in untreated diabetic mice compared to the control group, and administration of myricitrin and its SLN recovered all of these changes. Ultimately, myricitrin and its SLN administration improved DN changes by reducing oxidative stress and increasing antioxidant enzymes level, and these effects were more prominent in the SLN-administered mice. Myrica cerifera (Diabetic nephropathy (DN), known as a kidney progressive disease is characterized by persistent albuminuria, progressive decrease in glomerular filtration rate (GFR) and increases in plasma level of creatinine (Cr). This disease occurs in 45 % of diabetic patients . The rencerifera . Oral bicerifera . Solid lcerifera . Streptocerifera . Howevercerifera . So, basChemicals and experimental kitsUnited Kingdom) assay kits, blood urea nitrogen (BUN), Cr, albumin, glucose assay kits , RNeasy Mini Kit , cDNA Synthesis Kit, Sybergreen , TGF-β and NF-κb, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers , TUNEL assay kit , Proteinase K .Myricitrin (Purity 98 %) , Citrate buffer, Tween 80 , Streptozotocin , Nicotinamide, Hematoxylin, Eosin, , Ketamine/Xylazine , phosphate-buffered saline (PBS) , Iran), malondiadehyde (MDA), total oxidant capacity (TAC), catalase (CAT) , superoxide dismutase (SOD)  and encapsulation efficiency (EE %) were assessed in that study .Animalsad libitum. After one-week acclimatization of mice, T2DM induced by intraperitoneal injection of STZ (65 mg/kg) and NA (120 mg/kg) (dissolved in a citrate buffer (pH: 4.5) and normal saline, respectively) with 15 min interval. Mice with fasting blood glucose above 200 mg/dl were considered as diabetic and entered the study at 3 days after STZ-NA administration  mice (25-30 g) were obtained from the Ahvaz Jundishapur University of Medical Sciences (AJUMS) animal facility and, treated in accordance with the principles and guidelines on animal care of AJUMS as reviewed by an ethics committee (IR.AJUMS.REC.1395.136) and kept at a 20 °C ± 4 °C temperature with a 12 hr / 12 hr light and dark cycle. They received tap water and commercial chow Group I: ControlGroup II: Vehicle  solvents)Group III: DiabetesGroups IV, V and, VI: Diabetes in addition to administration of Myricitrin 1, 3, and 10 mg/kg, respectively.Groups VII, VIII and, IX: Diabetes in addition to administration of SLN containing myricitrin 1, 3, and 10 mg/kg, respectively  injected anesthetized mice. Then, plasma samples were prepared by blood samples centrifuging at 3500 rpm for 20 min. Following that, the kidney of all animals was separated for histological, gene expression, and apoptosis assessment. The plasma and kidney samples were maintained at −80 °C until the laboratory measurements were performed . Experimental measurementsThe kidney homogenate was prepared using Sharma and Singh method. In brief, the separated kidney slab on the dry ice pack, homogenized in 1/5 (w/v) PBS (pH: 7.4) with a Teflon homogenizer, and centrifuged at 2000 rpm for 10 min. Finally, the supernatant was used to measure the levels of lipid peroxidation (MDA) and antioxidant defense variables  by specific commercial kits .Plasma and urine level of the BUN, Cr, albumin, sodium (Na), potassium (K) and, urine level of glucose were measured by using an Autoanalyzer  electrolyte analyzer devices and biochemical assay kits . Also, GFR, and fractional excretion of sodium and potassium  were assessed by the following formula: GFR= Urine Cr × Urine volume / Plasma Cr; FE Na= (Urine Na × Plasma Cr / Plasma Na × Urine Cr) × 100; FE K= (Urine K × Plasma Cr / Plasma K × Urine Cr) × 100 .Gene expression assessment-ΔΔCT). The sequences of forward and reverse primer for TGF-β, NF-κb, and GAPDH genes are presented in Real-time PCR method was used for gene expression measurement in the present study. In brief, total RNA and cDNA were purified and synthesized from the kidney by the RNeasy mini kit and Reverse Transcriptase kit, respectively according to their instructions . Real-tiHistopathological and apoptosis assessmentsIn Situ Cell Death Detection Kit, POD, and the dark brown stained nucleus was included as dead cells. The index of apoptosis was measured in 3 randomly slides/animal and ten fields for each slide as a percentage of TUNEL-positive cells  and hematoxylin and eosin (H&E) staining method. Then, seven microscopic slides of tissue sections (5 to 7 µm) were assessed and read using blind method. Apoptosis assessment was carried out by TUNEL staining according to the labeling of DNA strand breaks by administration of ve cells .Statisticspost hoc least significant difference (LSD) tests. Moreover, the data were represented as the mean±standard error of the mean (SEM), and the statistically significant differences were considered at P<0.05.The obtained results were statistically analyzed by SPSS software (version 16) with one-way analysis of variance (ANOVA) and The role of myricitrin and its SLN on kidney weight and intake/outputP<0.01) compared to the control and decreased in the groups II (P<0.01), IX (P<0.05), and others (P<0.01) versus diabetes group. The water and food intake showed a significant increase in diabetic mice when compared to control . The water intake decreased in groups IV, V (P<0.05), and others (P<0.01) compared to the diabetes group. Food intake assessment showed a similar effect in the groups II, IV, V, VI (P<0.01), VII, VIII, and IX (P<0.001) compared to diabetes group. The urinary volume of untreated diabetic mice increased significantly (P<0.01). Further, the urine output decreased in groups IX (P<0.001) and others (P<0.01) versus diabetes group.As shown in Lipid peroxidation and antioxidant enzymes level in the kidneyP<0.01). This factor of lipid peroxidation reduced in the groups II (P<0.05), IV, V (P<0.01), VI, and VII (P<0.01) versus diabetes group (P<0.05) and group VI (P<0.01), and increased in groups V (P<0.01), VIII (P<0.05), and IX (P<0.001) in comparison with control. Further, this variable increased in groups II (P<0.05), IV (P<0.01), V (P<0.001), VII (P<0.01), VIII and IX (P<0.001) compared to untreated diabetic mice (P<0.001), groups IV (P<0.01), VI (P<0.001), VIII and IX (P<0.05) when compared to control, but this variable increased in groups II (P<0.001), IV (P<0.05), V (P<0.001), VII, VIII (P<0.01), and IX (P<0.01) versus diabetes group . This variable increased in the groups II (P<0.01), VIII (P<0.001), and others (P<0.05) versus diabetes group. Plasma level of BUN increased in diabetes (P<0.001) and groups IV (P<0.001), V (P<0.01), VI (P<0.05), and VII (P<0.01) compared to the control. Also, this factor decreased in the groups II (P<0.001), IV (P<0.01), and others (P<0.001) compared to untreated diabetic mice. Plasma Cr level showed a significant increase in diabetes and groups IV, V, VI, and IX in comparison with the control group (P<0.001). This variable decreased in the groups II (P<0.001), IV, V (P<0.05), VIII, and IX (P<0.001) versus diabetes group. Plasma level of albumin reduced in all experimental groups except group II versus the control (P<0.001). Moreover, this factor increased in the groups II (P<0.001), V, and VIII (P<0.05) when compared to the diabetes group (P<0.001). Further, this variable increased in the groups IV, VII (P<0.01), and others (P<0.001) versus diabetes group (P<0.01) and groups VI and IX (P<0.001) compared to the control group. Also, this factor increased in the groups II, IV, V, VII, and VIII (P<0.01) when compared to diabetes group (P<0.001) and groups IV, V (P<0.01), VI (P<0.001), VII (P<0.01), VIII (P<0.01), and IX (P<0.001) versus the control group. Moreover, urine albumin level decreased in the groups II (P<0.001), V, and VIII (P<0.01) when compared to untreated diabetic mice  compared to the control. Also, this variable decreased in all groups (P<0.01) except IV and VII versus the diabetes group. The urine K level increased in diabetes compared to control (P<0.01). Also, this factor decreased in the groups II (P<0.01), IV, V (P<0.05), VI (P<0.001), VII (P<0.05), VIII (P<0.01), and IX (P<0.001) when compared to diabetes group. FE Na showed a remarkable increase in diabetes (P<0.001) and groups IV (P<0.05), VI (P<0.001) and, IX (P<0.01) versus to the control group. Further, this variable reduced in the groups II (P<0.001), IV (P<0.01), V (P<0.001), VII, VIII (P < 0.001) and, IX (P<0.05) compared to the diabetes. The results of FE K indicated a significant increase in diabetes (P<0.001) and groups IV, V (P<0.05), VI (P<0.01), and IX (P<0.01) in comparison with the control group. Moreover, FE K decreased in the groups VI (P<0.01) and others (P<0.001) when compared to the diabetes group  and groups V (P<0.01), VI (P<0.001), VIII (P<0.05), and IX (P<0.001) compared to the control group. Also, TGF-β gene expression decreased in the groups II (P<0.001), IV (P<0.001), V (P<0.01), VII, and VIII (P<0.001), and increased in the group IX (P<0.001) when compared to the diabetes group . NF-κb gene expression showed a remarkable increase in diabetes (P<0.001) and groups VIII (P<0.01) and IX (P<0.001) versus the control group. This variable decreased in the groups II (P<0.001), IV (P<0.001), V (P<0.01), VII (P<0.001), and VIII (P<0.001) compared to the diabetes group .The gene expression of TGF-β increased in diabetes (Effect of myricitrin and its SLN on the kidney histopathology and apoptosisP<0.001) and groups IV (P<0.01), V (P<0.05), VI (P<0.001), VII (P<0.05), and IX (P<0.01) when compared to the control. Furthermore, this variable decreased in the groups II (P<0.001), IV, V (P<0.001), VI (P<0.05), VII, VIII, and IX (P<0.001) compared to the diabetes group  accumulation and infiltration of inflammatory cells in diabetes group compared to the control group. These alterations decreased in the groups II and others treated groups versus diabetes group, and this decreasing effect was more obvious in the groups VII and VIII . The reses group .et al. demonstrated an increase in kidney weight of type 2 diabetic rats due to renal enlargement, hypertrophy, and hyperfunctioning, and treatment with silymarin as a plant-derived antioxidant improved kidney weight (The present study showed that polyphagia, polydipsia, and polyuria occurred in untreated diabetic mice, and myricitrin and its SLN administration improved these disorders. The present results indicated that the kidney weight increased in diabetic mice and administration of myricitrin and its SLN recovered this alteration. Consists with this result Sheela y weight . When thy weight . TherefoT2DM increases ROS production and attenuates free radical scavenging molecules such as antioxidant enzymes. Free radicals induce basement membrane damage and lead to altering the membrane fluidity, ion transports and increased urinary albumin excretion . It seemMurraya paniculata ameliorated kidney dysfunctions of DN in diabetic rats through reducing oxidative stress and increasing antioxidant enzymes level (Urinary albumin excretion, hypoalbuminemia, elevated BUN, serum Cr, urine glucose, and decreased GFR levels occurred in DN, which may be related to T2DM and formation of free radicals . The enhes level . MoreoveGlomerular damage may accelerate tubulointerstitial injury by multiple pathways such as tubular chemokine expression that results in inflammatory cell infiltration in DN . AbnormaThe obtained results indicated that T2DM induced by STZ-NA causes to DN, kidney apoptosis and, inflammation via the increasing lipid peroxidation and decreasing antioxidant defense. Moreover, administration of myricitrin and its SLN in low and moderated doses improved all of the DN changes through reducing oxidative stress and increasing antioxidant enzymes level, and these effects were more potent in the SLN-administered diabetic mice.


Brachycephalus sulfuratus was recently described from southeastern and southern Brazil. In its description, the authors overlooked previous records of flea toads that had been identified as “Brachycephalus sp. nov.” and B. hermogenesi occurring in the same regions, which could suggest the possibility of up to three flea toads coexisting in southern Brazil. In addition, B. sulfuratus is characterized by substantial phenotypic variability, to an extent that compromises its current diagnosis with respect to its congener B. hermogenesi. Therefore, the current state-of-affairs regarding the geographical distribution of these two species and the identification of previously known populations is hitherto uncertain. Our goals are to reassess previous records of flea toads attributable to B. hermogenesi, B. sulfuratus and “Brachycephalus sp. nov.”, considering the description of B. sulfuratus, and to review the diagnosis of B. sulfuratus.The flea toad B. hermogenesi, B. sulfuratus, or to a potentially undescribed species from southeastern and southern Brazil was based either on the analysis of morphology or on their advertisement calls. These analyses include our independent examinations of specimens and, when not possible, examinations of published descriptions. To allow for a consistent comparison of advertisement calls between B. hermogenesi and B. sulfuratus, we made recordings of both species, including in the type locality of the former.A critical analysis of the species identity of flea toad specimens attributable to B. sulfuratus in relation to B. hermogenesi vary intraspecifically. Live individuals with ventral yellow spots correspond to B. sulfuratus; individuals without yellow spots can be either B. sulfuratus or B. hermogenesi. In preservative, they are indistinguishable. Previous records of Brachycephalus sp. nov. correspond to B. sulfuratus. We propose that the reduced number of notes per call and the presence of only isolated notes in the call of B. sulfuratus, as opposed to a high number of notes per call with isolated notes and note groups in the call of B. hermogenesi, as the only diagnostic characters between them. Regarding their distributions and based in our assessment, only B. sulfuratus occurs in southern Brazil, without any overlap with B. hermogenesi. There is a narrow gap between the distributions of these species around the southeast of the city of São Paulo. Our revision also revealed that some records previously attributed to B. hermogenesi in Rio de Janeiro and north São Paulo represent a distinct, unidentified flea toad that is not B. sulfuratus. Both species occur side by side in Corcovado, São Paulo, a locality from where five paratypes of B. hermogenesi were obtained. Biogeographic events that might have led to vicariance between B. hermogenesi and B. sulfuratus are discussed.We found that morphological and call characters originally proposed as diagnostic for  Brachycephalus Fitzinger, 1826 includes 36 small diurnal anuran species that live in the leaf litter across the Brazilian Atlantic Rainforest , municipality of Guaraqueçaba, in the northern coast of Paraná . Third, if the unidentified species of B. sulfuratus, there could be a single species of flea toad in southern Brazil (B. sulfuratus).The absence of a nomenclatural review of records of flea toads in southern Brazil can be evidenced by the fact that a single location in Santa Catarina, called Castelo dos Bugres, was recorded as harboring specimens identified as “ nov. 1” , or Bracs sp. 1.  and B. smogenesi , Brachycalus sp.  and B. slfuratus . Second, records  were assp. nov.” , leadingBrachycephalus sp. nov. 1” , Curitiba, Paraná, Brazil, Coleção Herpetológica do Departamento de Zoologia (DZUP), Universidade Federal do Paraná, Curitiba, Paraná, Brazil, and Museu de História Natural (ZUEC), Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. The sound collection analyzed include MHCNI, Xeno-Canto sound collection (www.xeno-canto.org), and Fonoteca Neotropical Jacques Vielliard .The critical analysis of the species identity of specimens attributable to B. sulfuratus .The analyses began by the assessment of the original diagnosis of lfuratus . We lookB. sulfuratus and B. hermogenesi, we noticed that the calls of B. hermogenesi described by B. hermogenesi  and digital  devices, with Sennheiser ME 66 and ME 67 microphones. Analogical recordings were digitized at 44.1 kHz and 16 bit using Raven Pro 1.4 . Digital recordings were made equally with sampling frequency rate of 44.1 kHz and 16-bit resolution. We analyzed calls under note-centered approach , as BornB. sulfuratus and B. hermogenesi, vouchered with specimens collected and deposited in the MHNCI. Collection permits were issued by ICMBIO . Geographical coordinates are based on the WGS84 datum. Elevations for literature records and author’s records were obtained from Google Earth, after plotting the location point  It “differs from… B. hermogenesi… by having (in life) yellow blotches on the ventral surfaces of the throat, chest, arms, and forearms” . The inverted v-shaped mark can be absent in individuals of B. sulfuratus ” .”orearms” : 43, 50;genesi…” : 43, 50;ogenesi” : 50. SpeCatarina  have revCatarina . Moreove compare . Additio pulex)” : 50. Als pulex)” : 46. Fin pulex)” : 50: “ThB. sulfuratus from B. hermogenesi. However, for identification purposes, we considered individuals with yellow spots on their ventral side as B. sulfuratus, whereas individuals without yellow spots could be either B. sulfuratus or B. hermogenesi. It is important to note that specimens with yellow spots of B. sulfuratus must be observed in life because in the preservative the change in color prevents separate them in relation to specimens of B. hermogenesi.Currently, there are no unique morphological character that could differentiate either live or preserved specimens  for B. sB. sulfuratus the following parameters of the advertisement call: “advertisement call long, composed of a set of 4–7 high-frequency notes (6.2–7.2 kHz) repeated regularly.” In the section “Comparisons with other species”, B. hermogenesi is the most similar to the new species (B. sulfuratus), being quite similar in frequency (dominant frequency = 6.8 kHz), which are the highest recorded for the genus. However, the advertisement call of B. hermogenesi can be simple or composed of 2–7 shorter notes with 1–3 pulses  is within the range of B. sulfuratus (6.2–7.2).In addition to morphological characters, 3 pulses .” In sumB. hermogenesi call can be simple or composed  and the presence of “attenuated notes”  with only isolated notes of B. sulfuratus, while in B. hermogenesi the advertisement call has a high number of notes (≥24) with the presence of isolated notes and note groups . One of these records involves “B. hermogenesi” from the municipality of Piedade, state of São Paulo, of HQ435682.1 and HQ435709.1; B. sulfuratus, which was placed on the tree together with a specimen from the Municipality of Barra do Turvo, in an early-diverging branch of the B. sulfuratus clade on the tree , also examined by us, collected in the state of Paraná, that was first identified as “ nov. 1” , “Brachys sp. 1” , and, fifuratus” ). There part.), two localities previously considered as occurrence of B. hermogenesi . The phylogenetic analysis revealed that the specimen from Municipality of Paraibuna is indeed B. hermogenesi  with distinct durations (= transliteration size) related to the number of notes in the call. This diagnosis between B. sulfuratus and B. hermogenesi is only feasible under the note-centered approach. Considering their calls under the call-centered approach, there would be no diagnosis to be proposed between them at this moment, because each note would represent a call , consolidate the benefit of the note-centered approach over the call-centered approach in describing calls of species of this genus , for example, from the B. ephippium group, which includes most species from the state of São Paulo to the north up to Espírito Santo and Minas Gerais.The advertisement calls of ix group , 2018a, B. hermogenesi in southern Brazil and the presence of B. sulfuratus as far north as the east of São Paulo city, only 25 km in straight line from the southernmost site of a confirmed record of B. hermogenesi  since 2009, focusing on their distribution, ecology and conservation , on the border between the municipalities of Jaraguá do Sul and Massaranduba, where we described B. albolineatus . In the studies . In the  studies  and snak studies .Brachycephalus toads and Scytalopus birds of São Paulo, Paraná, and Santa Catarina originated less than 2–5 million years ago  and Mesozoic, respectively  and Eleoscytalopus indigoticus , and calls of B. hermogenesi in Núcleo Santa Virgínea, Parque Estadual da Serra do Mar, municipality of São Luiz do Paraitinga, São Paulo, can be heard in a recording of E. indigoticus  at Corcovado, São Paulo, is the only confirmed case of sympatry between species of Brachycephalus in the same group. Other cases of sympatry include Brachycephalus from distinct groups . RIO DE JANEIRO: Trilha do Corisco, municipality of Paraty MHNCI 206–12. SÃO PAULO: Corcovado, municipality of Ubatuba MHNCI 193–205.10.7717/peerj.10983/supp-1Supplemental Information 1Abbreviation: NA = not available.Click here for additional data file.10.7717/peerj.10983/supp-2Supplemental Information 2MHNCI 124 Brachycephalus sulfuratus near Jurupara dam voucher MHNCI 10791 or MHNCI 10792 29 set 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-3Supplemental Information 3MHNCI 124 Brachycephalus sulfuratus near Jurupara dam voucher MHNCI 10791 or MHNCI 10792 29 set 2016 MRBornschein cutted.Click here for additional data file.10.7717/peerj.10983/supp-4Supplemental Information 4MHNCI 125 Brachycephalus sulfuratus near Jurupara dam not collected 29 set 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-5Supplemental Information 5MHNCI 126 Brachycephalus sulfuratus Nucleo Itutinga Piloes ind 1 not collected 08 out 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-6Supplemental Information 6MHNCI 127 Brachycephalus sulfuratus Nucleo Itutinga Piloes ind 2 not collected 08 out 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-7Supplemental Information 7MHNCI 128 Brachycephalus sulfuratus Biquinha not collected 18 set 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-8Supplemental Information 8MHNCI 129 Brachycephalus sulfuratus Serra Agua Limpa MHNCI 11583 26 out 2011 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-9Supplemental Information 9MHNCI 130 Brachycephalus sulfuratus Serra do Guarau not collected 22 out 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-10Supplemental Information 10MHNCI 131 Brachycephalus sulfuratus Caratuval near Parque Estadual Lauraceas MHNCI 11571 L Correa.Click here for additional data file.10.7717/peerj.10983/supp-11Supplemental Information 11MHNCI 132 Brachycephalus sulfuratus Caratuval Parque Estadual Lauraceas not collected 18 dez 2010 L Correa.Click here for additional data file.10.7717/peerj.10983/supp-12Supplemental Information 12MHNCI 133 Brachycephalus sulfuratus Reserva Particular do Patrimonio Natural Salto Morato not collected 10 marco 2016 MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-13Supplemental Information 13MHNCI 134 Brachycephalus sulfuratus Fazenda Thalia not collected MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-14Supplemental Information 14MHNCI 135 Brachycephalus sulfuratus Truticultura not collected MRBornschein.Click here for additional data file.10.7717/peerj.10983/supp-15Supplemental Information 15MHNCI 136 Brachycephalus sulfuratus Morro Garuva not collected 02 nov 2016 Andre Confetti.Click here for additional data file.10.7717/peerj.10983/supp-16Supplemental Information 16MHNCI 137 Brachycephalus sulfuratus Morro do Garrafão not collected LFRibeiro.Click here for additional data file.10.7717/peerj.10983/supp-17Supplemental Information 17MHNCI 165 000107 0122S4 Brachy hermogenesi Corcovado specimen one two starting notes lost without interruption.Click here for additional data file.10.7717/peerj.10983/supp-18Supplemental Information 18MHNCI 168 190330 05 Brachy hermogenesi ind 03 Boraceia 30 marco 2019 MRB.Click here for additional data file.10.7717/peerj.10983/supp-19Supplemental Information 19MHNCI 169 190330 06 Brachy hermogenesi ind 04 Boraceia 30 marco 2019 MRB.Click here for additional data file.10.7717/peerj.10983/supp-20Supplemental Information 20MHNCI 172 BLR00061 Brachy hemogenesi Picinguaba ind 01 ex 01 sem detalhes 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-21Supplemental Information 21MHNCI 173 BLR00062 B hermogenesi Picinguaba ind2 ex1 4 notas perdidas 13dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-22Supplemental Information 22MHNCI 174 BLR00064 B hermogenesi Picinguaba ind3 6 notas perdidas 13dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-23Supplemental Information 23MHNCI 175 BLR00065 B hermogenesi Picinguaba ind1ex2 tudo 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-24Supplemental Information 24MHNCI 176 BLR00067 B hermogenesi Picinguaba ind1ex3 tudo ind4ex1 3 perdidas 13dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-25Supplemental Information 25MHNCI 177 BLR00068 B hermogenesi_Picinguaba ind2 ex2 tudo 05cm 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-26Supplemental Information 26MHNCI 178 BLR00069 B hermogenesi Picinguaba ind3 ex2 5 notas perdidas 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-27Supplemental Information 27MHNCI 179 BLR00070 B hermogenesi Picinguaba ind 04 ex 02 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-28Supplemental Information 28MHNCI 180 BLR00071 B hermogenesi Picinguaba ind 03 ex 03 sem detalhes 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-29Supplemental Information 29MHNCI 181 BLR00072 B hermogenesi Picinguaba ind3 ex4 perdeu uma nota 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-30Supplemental Information 30MHNCI 182 BLR00073 B hermogenesi Picinguaba ind2 ex3 soh o final do canto 13 dez 2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-31Supplemental Information 31MHNCI 183 BLR00076 B hermogenesi Picinguaba ind4ex3 tudo 5-20cm distancia 13dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-32Supplemental Information 32MHNCI 184 000106 0115S4 Brachy hermogenesi Picinguaba specimen one several starting notes lost without interruption.Click here for additional data file.10.7717/peerj.10983/supp-33Supplemental Information 33MHNCI 185 000106 0117S4 Brachy hermogenesi Picinguaba specimen two two starting notes lost without interruption.Click here for additional data file.10.7717/peerj.10983/supp-34Supplemental Information 34MHNCI 186 000106 0119S4 Brachy hermogenesi Picinguaba specimen three one starting notes lost without interruption.Click here for additional data file.10.7717/peerj.10983/supp-35Supplemental Information 35MHNCI 187 000106 0120S4 Brachy hermogenesi Picinguaba specimen four several starting notes lost without interruption.Click here for additional data file.10.7717/peerj.10983/supp-36Supplemental Information 36MHNCI 213 080409 02 B hermogenesi ind1 muito perdido ruim Paranapiacaba MRB.Click here for additional data file.10.7717/peerj.10983/supp-37Supplemental Information 37MHNCI_188_000103_0135S4_Brachy hermogenesi Trilha do Ipiranga ind 1 e unknown starting notes with interruption.Click here for additional data file.10.7717/peerj.10983/supp-38Supplemental Information 38MHNCI 189 000103 0137S4 Brachy hermogenesi Trilha do Ipiranga ind 2 three starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-39Supplemental Information 39MHNCI 190 000103 0140S4 Brachy hermogenesi Trilha do Ipiranga ind 3 three starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-40Supplemental Information 40MHNCI 191 000103 0141S4 Brachy hermogenesi Trilha do Ipiranga ind 4 four starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-41Supplemental Information 41MHNCI 193 BLR00084 Brachy sp ind1 parte final Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-42Supplemental Information 42MHNCI 194 BLR00085 Brachy sp ind2 2m Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-43Supplemental Information 43MHNC 195 BLR00086 Brachy sp ind3 perdeu notas 20cm Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-44Supplemental Information 44MHNCI 196 BLR00087 Brachy sp ind4ex1 perdeu 3 notas 30-100cm Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-45Supplemental Information 45MHNCI 197 BLR00089 Brachy sp ind5 soh final Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-46Supplemental Information 46MHNCI 198 BLR00091 Brachy sp ind6ex1 4 notas perdidas 10-50cm Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-47Supplemental Information 47MHNCI 199 BLR00092 Brachy sp ind6ex2 soh final 50-80cm Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-48Supplemental Information 48MHNCI 200 BLR00093 Brachy sp ind4ex2 perdeu 7 notas 20cm Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-49Supplemental Information 49MHNCI 201 BLR00094 Brachy sp ind7 perdeu 4 notas 20-100cm Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-50Supplemental Information 50MHNCI 202 BLR00095 Brachy sp ind8 perdeu 6 notas Corcovado 18dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-51Supplemental Information 51MHNCI 203 000102 0129S4 Brachycephalus sp ind1 Corcovado two starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-52Supplemental Information 52MHNCI 204 000102 0130S4 Brachycephalus sp ind2 Corcovado two starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-53Supplemental Information 53MHNCI 205 000102 0132S4 Brachycephalus sp ind4 ind5 Corcovado two starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-54Supplemental Information 54MHNCI 211 000104 0113S4 Brachycephalus sp ind1 Trilha do Corisco several starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-55Supplemental Information 55MHNCI 212 000104 0114S4 Brachycephalus sp ind2 Trilha do Corisco several starting notes lost.Click here for additional data file.10.7717/peerj.10983/supp-56Supplemental Information 56MHNCI 206 BLR00053 Brachycephalus sp Trilha do Corisco ind1 perdeu inicio 40 cm 12dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-57Supplemental Information 57MHNCI 207 BLR00054 Brachycephalus sp Trilha do Corisco ind2 perdeu inicio 12dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-58Supplemental Information 58MHNCI 208 BLR00055 Brachycephalus sp Trilha do Corisco ind3 microfone flutuou 12dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-59Supplemental Information 59MHNCI 209 BLR00056 Brachycephalus sp Trilha do Corisco ind4 20-60 cm 12dez2017 MRB.Click here for additional data file.10.7717/peerj.10983/supp-60Supplemental Information 60MHNCI 217 190206 05 Brachycephalus sp mais B sulfuratus Serra do Pico MRB 6fev2019.Click here for additional data file.10.7717/peerj.10983/supp-61Supplemental Information 61MHNC 218 BLR00165 Brachy sp mais Brachy sulfuratus Torre Embratel MRB não coletado.Click here for additional data file.10.7717/peerj.10983/supp-62Supplemental Information 62MHNCI 219 200123 0448S4 Brachycephalus sulfuratus Entroncamento Teba 20 cm from microphone LFR JAN-2020.Click here for additional data file.10.7717/peerj.10983/supp-63Supplemental Information 63MHNCI 220 Brachycephalus sulfuratus Morro do Canal 150 cm from microphone LFRibeiro JAN-2017.Click here for additional data file.10.7717/peerj.10983/supp-64Supplemental Information 64MHNCI 221 161115 02 Brachycephalus sulfuratus Monte Crista 100 cm from microphone Luiz Fernando Ribeiro NOV-2016.Click here for additional data file.10.7717/peerj.10983/supp-65Supplemental Information 65Brachycephalus hermogenesi ind1 parte do canto Morro do Cantagalo-Caraguatatuba LC.MHNCI 222 180802 08 Click here for additional data file.10.7717/peerj.10983/supp-66Supplemental Information 66Brachycephalus hermogenesi ind2 parte do canto Morro do Cantagalo-Caraguatatuba LC.MHNCI 223 180802 09 Click here for additional data file.


Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant neoplasm. It is necessary to improve the understanding of the underlying molecular mechanisms and identify the key genes and signaling pathways involved in PDAC.GSE28735, GSE62165, and GSE91035 were downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified by integrated bioinformatics analysis, including protein–protein interaction (PPI) network, Gene Ontology (GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The PPI network was established using the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape software. GO functional annotation and KEGG pathway analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery. Hub genes were validated via the Gene Expression Profiling Interactive Analysis tool (GEPIA) and the Human Protein Atlas (HPA) website.The microarray datasets FN1, COL1A1, COL3A1, BGN, POSTN, FBN1, COL5A2, COL12A1, THBS2, COL6A3, VCAN, CDH11, MMP14, LTBP1, IGFBP5, ALB, CXCL12, FAP, MATN3, and COL8A1. These genes were validated using The Cancer Genome Atlas (TCGA) and Genotype–Tissue Expression (GTEx) databases, and the encoded proteins were subsequently validated using the HPA website. The GO analysis results showed that the most significantly enriched biological process, cellular component, and molecular function terms among the 20 hub genes were cell adhesion, proteinaceous extracellular matrix, and calcium ion binding, respectively. The KEGG pathway analysis showed that the 20 hub genes were mainly enriched in ECM–receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and protein digestion and absorption. These findings indicated that FBN1 and COL8A1 appear to be involved in the progression of PDAC. Moreover, patient survival analysis performed via the GEPIA using TCGA and GTEx databases demonstrated that the expression levels of COL12A1 and MMP14 were correlated with a poor prognosis in PDAC patients (p < 0.05).A total of 263 DEGs (167 upregulated and 96 downregulated) were common to the three datasets. We used STRING and Cytoscape software to establish the PPI network and then identified key modules. From the PPI network, 225 nodes and 803 edges were selected. The most significant module, which comprised 11 DEGs, was identified using the Molecular Complex Detection plugin. The top 20 hub genes, which were filtered by the CytoHubba plugin, comprised MMP14 and COL12A1 is associated with poor overall survival, and these might be a combination of prognostic biomarkers in PDAC.The results demonstrated that upregulation of  Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of the pancreas and is a lethal malignancy with poor prognosis, which is in part due to its rapid progression and the lack of diagnostic and therapeutic targets. In 2018, pancreatic cancer (PC) ranked 11th among the most common cancers, with 458,918 new cases and 432,242 deaths due to PC worldwide . Recent KRAS  and biological pathways linked to various malignant tumors. Therefore, microarray techniques are promising and efficient ways to identify candidate biomarkers involved in the pathogenesis of PDAC. The purpose of our study was to determine significant DEGs and pathways implicated in PDAC by integrated bioinformatics analysis and to provide novel insights into the progression, diagnosis, and therapeutic targets of PDAC.https://www.ncbi.nlm.nih.gov/geo/) is a public repository of high-throughput gene expression genomics datasets  is an online analysis tool that is based on the R programming language and can be used to identify DEGs that differentiate between cancer and normal samples in a GEO series  is an online application that can be used to assess DEG-encoded proteins and protein–protein interaction (PPI) networks  is a public online bioinformatics database (p < 0.05. We performed enrichment of the GO terms and KEGG pathways for the candidate DEGs using DAVID.The Gene Ontology (GO) is used to perform enrichment analysis, which covers the cellular component (CC), biological process (BP), and molecular function (MF), of the selected genes . The Kyodatabase  that conhttp://gepia.cancer-pku.cn/) is used to perform functions such as survival analysis, the detection of similar genes, and correlation analysis to clarify the relationships between diseases and DEGs  database and the Genotype–Tissue Expression (GTEx) database, the Gene Expression Profiling Interactive Analysis tool  website on the basis of spatial proteomics data and quantitative transcriptomics data (RNA-Seq) obtained from immunohistochemical analysis of tissue microarrays.The expression of proteins encoded by the PDAC candidate genes was validated using the Human Protein Atlas Functional and pathway enrichment analyses were accomplished using DAVID. GO analysis showed that the most significant module was mainly enriched in cell adhesion, extracellular matrix structural constituent, and proteinaceous extracellular matrix  Table 2Table 2. COL12A1 and MMP14 were correlated with an unfavorable prognosis in PDAC patients (p < 0.05) (p > 0.05).Patient survival analysis performed via the GEPIA using TCGA and GTEx databases demonstrated that the high expression levels of  < 0.05) . The oveFN1, COL1A1, COL3A1, BGN, POSTN, FBN1, COL5A2, COL12A1, THBS2, COL6A3, VCAN, CDH11, MMP14, LTBP1, IGFBP5, FAP, MATN3, and COL8A1 were significantly overexpressed in PDAC tissues in comparison with normal pancreatic tissues, whereas ALB was underexpressed in PDAC tissues (p < 0.05) .To ensure the reliability of the identification of the top 20 hub genes, we validated these via the GEPIA using TCGA and GTEx databases. Boxplots of the hub genes associated with PDAC were downloaded from the GEPIA. The results demonstrated that  < 0.05) . CXCL12 COL5A2, IGFBP5, and MATN3 are reported on the HPA website, and expression profiles of the other 17 genes in PDAC clinical specimens are shown in We obtained the expression levels of proteins encoded by the 20 hub genes associated with PDAC from the HPA website. No data for proteins encoded by FN1, MMP14, COL12A1, COL3A1, COL1A1, POSTN, VCAN, LTBP1, FBN1, and FAP were upregulated in PDAC tissues in comparison with normal tissues, with only ALB being downregulated in PDAC tissues. COL6A3, COL8A1, CDH11, and CXCL12 were not expressed in either PDAC tissues or normal tissues, and BGN and THBS2 were overexpressed in both cancer and normal tissues.The protein expressions of GSE28735, GSE62165, and GSE91035. The main findings deduced from the studies used to compile GSE28735 were that dipeptidase 1 and a unique set of free fatty acids played roles in the development, progression, and prognosis of PC and might be potential targets in PDAC (GSE62165 found that hepatocyte nuclear factor (HNF)-1 α and HNF-1 β seem to be good candidates as tumor suppressors in PDAC . Ca2+ is a ubiquitous and versatile second messenger involved in the regulation of numerous cellular functions, including gene transcription, vesicular trafficking, and cytoskeletal rearrangements  and enhances the production of type I collagen by increasing transforming growth factor- β signaling  are a family of calcium- and zinc-dependent membrane-anchored or secreted endopeptidases that are overexpressed in various diseases, including breast cancer . MMP14 i of PDAC . Moreoveasive PC , and MMPasive PC . Type I al cells , and COLignaling . Ottaviac tissue . These fCOL1A1, COL3A1, COL5A2, COL6A3, FN1, and THBS2, were significantly associated with ECM–receptor interactions, focal adhesion, and the phosphatidylinositol-3-kinase–protein kinase B (PI3K-Akt) signaling pathway. In addition, collagen-encoding genes, including COL1A1, COL3A1, and COL5A2, were also enriched in protein digestion and absorption and platelet activation.The KEGG pathway analysis showed that six hub genes, namely, COL1A1 and COL3A1 were significantly downregulated in PC (p <0.0001) after treatment with gemcitabine in combination with EC359   . As we hFBN1 and COL8A1 appear to be involved in the progression of PDAC. FBN1 encodes a structural component of the microfibrils of the ECM that have diameters of 10–12 nm, which impart both regulatory and structural properties to load-bearing connective tissues  and the related enriched functions or pathways, which regulate the progression and metastatic invasion of PDAC, as well as overall survival. The results demonstrate that the upregulation of MMP14 and COL12A1 in PDAC is closely associated with poor overall survival, that these might be a potential combination of prognostic biomarkers in patients with PDAC, and that FBN1 and COL8A1 might be biomarkers of PDAC. In brief, our study increases the understanding of the potential critical genes and related pathways that participate in the pathogenesis of PDAC.In conclusion, we screened the top 20 hub genes (10.7717/peerj.10419/supp-1File S1Click here for additional data file.10.7717/peerj.10419/supp-2Table S1Click here for additional data file.10.7717/peerj.10419/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10419/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.10419/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.10419/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.10419/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.10419/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.10419/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.10419/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.10419/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.10419/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.10419/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.10419/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.10419/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.10419/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.10419/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.10419/supp-18Supplemental Information 18Click here for additional data file.10.7717/peerj.10419/supp-19Supplemental Information 19Click here for additional data file.10.7717/peerj.10419/supp-20Supplemental Information 20Click here for additional data file.10.7717/peerj.10419/supp-21Supplemental Information 21Click here for additional data file.10.7717/peerj.10419/supp-22Supplemental Information 22Click here for additional data file.10.7717/peerj.10419/supp-23Supplemental Information 23Click here for additional data file.10.7717/peerj.10419/supp-24Supplemental Information 24Click here for additional data file.10.7717/peerj.10419/supp-25Supplemental Information 25Click here for additional data file.10.7717/peerj.10419/supp-26Supplemental Information 26Click here for additional data file.10.7717/peerj.10419/supp-27Supplemental Information 27Click here for additional data file.10.7717/peerj.10419/supp-28Supplemental Information 28Click here for additional data file.10.7717/peerj.10419/supp-29Supplemental Information 29Click here for additional data file.10.7717/peerj.10419/supp-30Supplemental Information 30Click here for additional data file.10.7717/peerj.10419/supp-31Supplemental Information 31Click here for additional data file.10.7717/peerj.10419/supp-32Supplemental Information 32Click here for additional data file.10.7717/peerj.10419/supp-33Supplemental Information 33Click here for additional data file.10.7717/peerj.10419/supp-34Supplemental Information 34Click here for additional data file.10.7717/peerj.10419/supp-35Supplemental Information 35Click here for additional data file.10.7717/peerj.10419/supp-36Supplemental Information 36Click here for additional data file.10.7717/peerj.10419/supp-37Supplemental Information 37Click here for additional data file.10.7717/peerj.10419/supp-38Supplemental Information 38Click here for additional data file.10.7717/peerj.10419/supp-39Supplemental Information 39Click here for additional data file.10.7717/peerj.10419/supp-40Supplemental Information 40Click here for additional data file.10.7717/peerj.10419/supp-41Supplemental Information 41Click here for additional data file.10.7717/peerj.10419/supp-42Supplemental Information 42Click here for additional data file.10.7717/peerj.10419/supp-43Supplemental Information 43Click here for additional data file.10.7717/peerj.10419/supp-44Supplemental Information 44Click here for additional data file.10.7717/peerj.10419/supp-45Supplemental Information 45Click here for additional data file.10.7717/peerj.10419/supp-46Supplemental Information 46Click here for additional data file.10.7717/peerj.10419/supp-47Supplemental Information 47Click here for additional data file.10.7717/peerj.10419/supp-48Supplemental Information 48Click here for additional data file.10.7717/peerj.10419/supp-49Supplemental Information 49Click here for additional data file.10.7717/peerj.10419/supp-50Supplemental Information 50Click here for additional data file.10.7717/peerj.10419/supp-51Supplemental Information 51Click here for additional data file.10.7717/peerj.10419/supp-52Supplemental Information 52Click here for additional data file.10.7717/peerj.10419/supp-53Supplemental Information 53Click here for additional data file.10.7717/peerj.10419/supp-54Supplemental Information 54Click here for additional data file.10.7717/peerj.10419/supp-55Supplemental Information 55Click here for additional data file.10.7717/peerj.10419/supp-56Supplemental Information 56Click here for additional data file.10.7717/peerj.10419/supp-57Supplemental Information 57Click here for additional data file.10.7717/peerj.10419/supp-58Supplemental Information 58Click here for additional data file.10.7717/peerj.10419/supp-59Supplemental Information 59Click here for additional data file.10.7717/peerj.10419/supp-60Supplemental Information 60Click here for additional data file.10.7717/peerj.10419/supp-61Supplemental Information 61Click here for additional data file.10.7717/peerj.10419/supp-62Supplemental Information 62Click here for additional data file.10.7717/peerj.10419/supp-63Supplemental Information 63Click here for additional data file.10.7717/peerj.10419/supp-64Supplemental Information 64Click here for additional data file.10.7717/peerj.10419/supp-65Supplemental Information 65Click here for additional data file.10.7717/peerj.10419/supp-66Supplemental Information 66Click here for additional data file.10.7717/peerj.10419/supp-67Supplemental Information 67Click here for additional data file.10.7717/peerj.10419/supp-68Supplemental Information 68Click here for additional data file.10.7717/peerj.10419/supp-69Supplemental Information 69Click here for additional data file.10.7717/peerj.10419/supp-70Supplemental Information 70Click here for additional data file.10.7717/peerj.10419/supp-71Supplemental Information 71Click here for additional data file.10.7717/peerj.10419/supp-72Supplemental Information 72Click here for additional data file.10.7717/peerj.10419/supp-73Supplemental Information 73Click here for additional data file.10.7717/peerj.10419/supp-74Supplemental Information 74Click here for additional data file.10.7717/peerj.10419/supp-75Supplemental Information 75Click here for additional data file.10.7717/peerj.10419/supp-76Supplemental Information 76Click here for additional data file.10.7717/peerj.10419/supp-77Supplemental Information 77Click here for additional data file.10.7717/peerj.10419/supp-78Supplemental Information 78Click here for additional data file.10.7717/peerj.10419/supp-79Supplemental Information 79Click here for additional data file.10.7717/peerj.10419/supp-80Supplemental Information 80Click here for additional data file.10.7717/peerj.10419/supp-81Supplemental Information 81Click here for additional data file.10.7717/peerj.10419/supp-82Supplemental Information 82Click here for additional data file.


Fear of Falling (FOF) is common among community-dwelling older adults and is associated with increased fall-risk. In this cross-sectional study we examined the relationships between FOF and factors associated with fall-risk such as gait quality, cognition, and walking-confidence. Using baseline data from older adult participants in a randomized exercise trial , we quantified the following outcome measure for (1) gait quality: harmonic ratio (smoothness) and time-frequency spatiotemporal variables from triaxial accelerometry during 6 minute walk; gait speed, step-time CoV (variability) and walk-ratio (step-length/cadence) on an instrumented walkway; (2) cognition: Trails A and B (3) walking-confidence: Gait efficacy Scale. Mann Whitney U-tests indicated individuals without FOF had better gait quality (p<0.05): greater smoothness (2.38±.58 vs 1.14±.73), speed (1.10±.15 vs 1.04±.17 m/s) and walk-ratio (.56±.07 vs .53±.08 cm/steps/min), lower step-time CoV (3.72±1.24 vs 4.17±1.66), and greater walking-confidence (89±11 vs 79±13). A random forest classifier predicted FOF with 64% (gait only) and 70%  accuracy; Gini-index based ranking indicated gait quality  direction, walking speed) were consistently important variables. Linear Support Vector Machine learning yielded accuracies of 60% (only gait) and 68% : smoothness V, mediolateral frequency bandwidth, gait speed among top 4 ranked variables in both models, and walking-confidence in the additional measures model; smoothness-V the highest weighted coefficient (-0.52). Based on these findings, interventions targeted for gait quality and walking-confidence may be important to overcome FOF and reduce fall risks.


Staphylococcus aureus (MRSA), there have been few studies focused on the molecular characterization of methicillin-susceptible Staphylococcus aureus (MSSA). In this cross-sectional study, 85 MSSA isolates were characterized by antimicrobial susceptibility testing, virulence genes analysis, accessory gene regulator (agr) typing, and S. aureus protein A locus (spa) typing.Compared to methicillin-resistant agr types detected in tested strains were mainly type I (76.5%), II (12.9%), and III (10.6%). Of 85 MSSA examined isolates, 48 (56.5%) isolates were toxinogenic with 27 producing pvl (31.8%) and 21 tst (24.7%). The findings of the study show a high genetic diversity in MSSA strains warranting continued surveillance to provide critical insights into control and treatment of MSSA infections.In present study, 9 different clonal complexes namely CC8-MSSA-t037 (22.4%), CC8-MSSA-t008 (11.8%), CC7-MSSA-t091 and CC30-MSSA-t021 (each 9.4%), CC8-MSSA-t037 (8.3%), CC398-MSSA-t034 (7.1%), CC22-MSSA-t005 (5.9%), CC5-MSSA-t002 and CC15-MSSA-t084 (each 4.7%), CC22-MSSA-t790 and CC59-MSSA-t437 (each 3.5%), CC22-MSSA-t1869, CC5-MSSA-t045, and CC45-MSSA-t015 (each 2.3%), CC30-MSSA-t318 and CC15-MSSA-t491 (each 1.2%) were found.  Staphylococcus aureus is a common hospital- and community-acquired pathogen [S. aureus strain diversity appears to differ by geographic region, there has been a dramatic increase in the prevalence of S. aureus strains associated with human infections around the world and this appears to be especially true for methicillin-susceptible Staphylococcus aureus (MSSA) [S. aureus infections [pathogen . It is rpathogen . Althougs (MSSA) –5. MSSA s (MSSA) , 6. Comps (MSSA) . Antimicfections , 8. Knowfections , 10. AltS. aureus isolates phenotypically by using standard microbiological techniques. Polymerase chain reaction (PCR) assay targeting the S. aureus-specific nuc gene was applied to verify the isolates [S. aureus isolates susceptible to cefoxitin disc  in a disc diffusion assay using established methods (CLSI 2018) and negative for the presence of mecA gene by PCR were considered as MSSA strains [Eighty-five MSSA isolates were obtained from hospitalized patients at four hospitals affiliated to Shahid Beheshti University of Medical Sciences during an 9-month collection period from March 2019 to November 2019. This study protocol was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran (IR. SBMU. MSP.REC. 1398. 774). Furthermore, we confirmed isolates , 12. TheS. aureus ATCC 25923, ATCC 43300, and ATCC 29213 strains.In present study, Kirby–Bauer disk diffusion method was applied to determine the antimicrobial susceptibility of isolates based on the clinical and laboratory standards institute (CLSI) criteria (CLSI 2018). The antimicrobial agents included penicillin, teicoplanin, gentamicin, kanamycin, amikacin, tobramycin, clindamycin, erythromycin, tetracycline, linezolid, rifampicin, mupirocin, ciprofloxacin, quinupristin–dalfopristin, and trimethoprim–sulfamethoxazole . The minimal inhibitory concentrations (MIC) values of vancomycin was evaluated by broth microdilution method. Susceptibility test was quality controlled by using eta, and etb), Panton-Valentine leukotoxin gene (pvl), and toxic shock syndrome toxin (tsst-1) by PCR assay [S. aureus ATCC49775 and toxin positive S. aureus strains obtained from our previous were used as reference strains [S. aureus strain ATCC 25923 were also used as negative control.Genomic DNA was isolated using the phenol–chloroform extraction method. All of the isolates were screened for virulence encoding genes namely: exfoliative toxin genes (CR assay –14. The agr type detection using primer set comprising a common forward primer (Pan) and reverse primers  specific to each agr group [agr types were identified by comparing the banding patterns of isolates to RN6390 (agr type I), RN6607 (agr type II), RN8465 (agr type III), RN4550 (agr type IV), and RN6911 (negative control), as reference strains. PCR amplification was used for spa typing as described previously [spa gene amplified by PCR with forward (5′-AGACGATCCTTCGGTGAGC-3′) and reverse (5′-GCTTTTGCAATGTCATTTACTG-3′) primers. The purified PCR products were sequenced and then edited. The Ridom SpaServer database (http://www.spaserver.ridom.de) was applied to determine the spa type of each isolate. Each set of PCR reactions include a spa-type t008 isolate from our previous study as positive control sample (14), and a reaction mixture containing no template DNA as a control for possible false positive results.Multiplex PCR was performed for gr group . agr typeviously . In thispvl (31.8%) and 21 tst (24.7%).The sources of isolates included: skin and soft tissue wounds (44.7%), purulent exudates from wounds or abscesses 17.7%), urine (14.1%), blood (11.8%), sputum (8.2%), and other body fluids (3.5%). Out of 85 MSSA isolates, 29 isolates were obtained from hospital H1 (34.1%), 25 isolates from hospital H2 (29.4%), 20 isolates from hospital H3 (23.5%), and 11 isolates from hospital H4 (13%). Among 85 MSSA isolates tested, the highest rate of resistance was detected for penicillin (74.1%), and gentamicin (54.1%). , II (12.9%), and III (10.6%). spa results showed 16 types corresponding to nine clonal complexes (CCs), namely CC8 (42.3%), CC22 (11.8%), CC30 (10.6%), CC7 (9.4%), CC5 (7.1%), CC398 (7.1%), CC15 (5.9%), CC59 (3.5%), and CC45 (2.3%). spa type t037 was the most common spa type identified among 85 MSSA isolates, with a frequency of 22.3%, followed by t008 (11.8%), t021 and t091 (each 9.4%), t030 (8.2%), t034 (7%), t005 (5.9%), t084 and t002 (each 4.7%), t790 and t437 (each 3.5%), t1869, t045, and t015 (each 2.4%), t318 and t491 (each 1.2%) (Table 5%), II 1.9%, and Inducible clindamycin resistance was observed in CC8-MSSA-t037 (n = 5), CC30-MSSA-t021 (n = 4) CC8-MSSA-t008 (n = 3) isolates; while constitutive resistance phenotype was observed in CC8-MSSA-t037 (n = 8), CC8-MSSA-t030 (n = 3), CC-MSSA-t034 (n = 4), CC-MSSA-t091 (n = 5), CC-MSSA-t045 (n = 4), CC-MSSA-t005 (n = 4), and CC-MSSA-t790 (n = 1) isolates. Detailed results of the genotype distribution are presented in Table spa types among MSSA isolates. We detected CC8 in 42.3% of isolates. Although multi-resistant CC8 was previously reported as one of the main international CCs of MRSA, the predominance of the CC8-MSSA clone has previously been reported in Europe [Infection with MSSA as the most common pathogen in hospitalized patients is becoming increasingly problematic globally and requires special attention . As presn Europe  and Afrin Europe , 19. We n Europe , Irelandn Europe , and Nign Europe . We confn Europe , 21. As n Europe .We noted a relatively low prevalence of CC7 (9.4%), CC5 (7.1%), CC398 (7.1%), CC15 (5.9%), CC59 (3.5%), and CC45 (2.3%) in our study. A study conducted in China during the 4-year period indicated that CC22-t309 26.0%), CC188-t189 (5.1%), CC796-t796 (4.8%), CC121-t435 (4.8%), and CC398-t571 (3.6%) were the most dominant clones among MSSA isolates . They de.0%, CC18pvl encoding gene. Prevalence PVL positive MSSA strains has varied among studies from different geographic regions including China (34.4%) [Present results demonstrated that out of the 85 tested MSSA, 27 isolates 31.8%) carried 1.8% carr 34.4%) , Ireland (34.4%) , Colombi.4% , Ire (34.4%) , Russia  (34.4%) . In a re (34.4%) . However (34.4%) , 19. In  (34.4%) , 26, 28, (34.4%) .tst positive. This reported rate was different from the earlier studies in Africa (7%) [tst gene were associated with CC8-MSSA-t037 (11.8%), CC8-MSSA-t0307 (5.9%), CC22-MSSA-t790 (3.5%), and CC59-MSSA-t437 (3.5%) clones. A multicenter study from china reported that 4.0% of MSSA isolates carried tst gene which belonged to CC5 clone [tst-positive MSSA was distributed into four STs and the majority of them belonged to CC30 clone [tst encoding gene among S. aureus clinical isolates (21.3%) [tst were significantly higher in MSSA isolates in comparison to MRSA isolates (18.1% vs. 11.6%) [tst-positive MSSA clones seems geographically different and CC8 may represent a newly emerging clone in Iran.In our strain collection, 24.7% of isolates were ica (7%) , China (ica (7%)  and Turkica (7%) . Contrarica (7%) , 31 whicC5 clone . A recenSSA-t037 .8%, CC8- (21.3%) . In a st. 11.6%) . Overallagr genotypes are strictly associated with the clonal lineages [agr type I, as the most predominant type (76.4%), was associated with CC8, CC22, CC7, CC45, CC398, and CC59 isolates. In line with our results, Croes et al. demonstrated that CC7, CC8, CC22, CC25 and CC45 clonal lineages harbored agr I [agr type I (68.8%), followed by agr III (18.7%), and agr IV (12.5%) among MSSA isolates [According to the evidence, the lineages , 35. In ant type 6.4%, wasOur research had some limitations. Firstly, present work lacks detailed clinical information about the patients, Secondly, our samples were not collected consecutively. Thirdly, whole genome sequencing technique was not applied in the present work due to some of technical limitations.


The results showed that a HSD, salt gene overexpression and dFOXO knockdown significantly reduced climbing endurance, climbing index, survival, dFOXO expression and SOD activity level, and increased malondialdehyde level in aging flies. Inversely, in a HSD aging flies, endurance exercise and dFOXO overexpression significantly increased their climbing ability, lifespan and antioxidant capacity, but they did not significantly change the salt gene expression. Overall, current results indicated that a HSD accelerated the age-related decline of climbing capacity and mortality via upregulating salt expression and inhibiting the dFOXO/SOD pathway. Increased dFOXO/SOD pathway activity played a key role in mediating endurance exercise resistance to the low salt tolerance-induced impairment of climbing capacity and longevity in aging Drosophila.A high-salt diet (HSD) is a major cause of many chronic and age-related defects such as myocardial hypertrophy, locomotor impairment and mortality. Exercise training can efficiently prevent and treat many chronic and age-related diseases. However, it remains unclear whether endurance exercise can resist HSD-induced impairment of climbing capacity and longevity in aging individuals. In our study, flies were given exercise training and fed a HSD from 1-week old to 5-weeks old. Overexpression or knockdown of This article has an associated First Person interview with the first author of the paper. Summary: Increased dFOXO/SOD pathway activity played a key role in mediating endurance exercise resistance to the salt tolerance-induced impairment of climbing capacity and longevity in aging Drosophila. Sodium chloride from dietary salt supplies essential electrolytes to the human body, and it plays a vital role in maintaining the stability of the intracellular and extracellular environments . DespiteExercise training is an efficient strategy for the prevention and treatment of many chronic diseases caused by diet or aging such as myocardial hypertrophy, hypertension and obesity . For exaDrosophila melanogaster provides significant practical advantages over other model systems to study the molecular mechanisms of exercise training, nutrition and aging, which include developed exercise and diet programs, a short lifespan (2–3 months), low genetic redundancy compared with mammals, and a plethora of tools available to easily manipulate gene expression      , but a 2%-SD had no remarkable influence on CI (P>0.05)   , a 4%-SD and an 8%-SD significantly reduced the climbing fatigue time (CFT) (ctively) B. In 3-wctively) C. In 5-wctively) D. In addctively) E–H. Nextctively) I. In 1-w(P>0.05) J. In 3-wctively) J. In 5-wctively) J. These Drosophila. In this experiment, the results showed that a 2%-SD significantly reduced the lifespan of Drosophila (P<0.05), and a 4%-SD and an 8%-SD acutely reduced lifespan of flies   K. These Drosophila  (P>0.05)  (P>0.05)  (P>0.05) , and senility significantly decreased their CI (P<0.01) (P<0.05), and senility significantly decreased their CI (P<0.01) (P<0.05), but the CI of 4%-SD and 5-week-old flies did not change significantly after exercise training (P>0.05), and senility significantly decreased their CI (P<0.01) (P>0.05), and senility significantly decreased their CI (P<0.01) (P<0.05) (P>0.05)  A–C, but (P>0.05) D. Furthectively) E–G, but (P>0.05) H. Moreovctively) I,J, and (P>0.05) K,L. The (P<0.01) M. In 2%-(P<0.01) N. In 2%-(P<0.01) O. The CI(P<0.01) P. What i(P<0.05) Q,U, but (P>0.05) V,W. ThesD. melanogaster , and the expression of Salt gene of 2%-SD flies, 4%-SD flies and 8%-SD flies did not change significantly after exercise training (P>0.05) (dFOXO gene (P<0.01), and exercise training significantly increased the dFOXO gene expression in the 0%-SD flies, 2%-SD flies and 4%-SD flies , but dFOXO gene expression of the 8%-SD flies did not change significantly after exercise training (P>0.05) (P<0.01), and exercise training significantly increased the SOD level of the 0%-SD flies, 2%-SD flies and 4%-SD flies , but the SOD level of the 8%-SD flies did not change significantly after exercise training (P>0.05) , and exercise training significantly decreased the MDA level of the 0%-SD flies, 2%-SD flies and 4%-SD flies (P<0.05), but the MDA level of the 8%-SD flies did not change significantly after exercise training (P>0.05)  A. In add(P>0.05) B. Moreov(P>0.05) C. Finall(P>0.05) D. These FOXO is associated with an increase in the expression of SOD and SOD activity , exercise training significantly increased the CFT (P<0.05) , exercise training significantly increased the CFT (P<0.05) , exercise training could not be said to significantly increase the CFT (P>0.05)  , but exercise training significantly increased the CI (P<0.05) , but exercise training significantly increased the CI (P<0.01) , but exercise training significantly increased the CI (P<0.01) , but exercise training significantly increased lifespan (P<0.05)  A. In 3-w(P<0.05) B. Howeve(P>0.05) C. Next, (P<0.01) D. In 1-w(P<0.05) E. In 3-w(P<0.01) E. In 5-w(P<0.01) E. Moreov(P<0.05) F,G. ThesSalt gene (P<0.01), but exercise training did not significantly decrease the expression of the Salt gene (P>0.05) , but exercise training significantly increased the expression of dFOXO gene (P<0.05) , but exercise training significantly increased the SOD level (P<0.01) , but exercise training significantly increased the MDA level (P<0.01)  H. In add(P<0.05) I. Moreov(P<0.01) J. Finall(P<0.01) K. These salt gene knockdown can improve salt tolerance, and it can increase the survival of a HSD fly , and a HSD also did not significantly reduce the CFT (P>0.05)  , and a HSD also did not significantly reduce the CI (P>0.05) , and a HSD also did not significantly reduce the lifespan (P>0.05) , but a HSD did not significantly increased the salt gene expression (P>0.05)   A–C. In s(P<0.01) D. Howeve(P>0.05) E. What i(P>0.05) F,G, whic(P>0.05) . Next, t(P>0.05) H. Finall(P>0.05) I–K. ThesSalt gene function takes part in regulating salt tolerance, and downstream it regulates antioxidant function, climbing capacity and aging.Two principal classes of manipulation are usually employed to study gene function. Loss-of-function (LOF) approaches attempt to eliminate gene function partially or completely. Gain-of-function (GOF) approaches attempt to obtain functional information by creating conditions where the gene is excessively or ectopically expressed or its function exaggerated . In thisdFOXO gene expression was also changed by UAS/Gal4 system in flies, and their CFT, CI and lifespan were measured.In animals, it has been identified that a HSD increases the oxidative stress and causes cell damage . In fliedFOXO gene overexpression significantly increased the CFT (P<0.05), and it significantly increased the CFT in HSD 1-week-old flies (P<0.001) (dFOXO gene overexpression significantly increased the CFT (P<0.05), and it also significantly increased the CFT in HSD 3-week-old flies (P<0.001) (dFOXO gene overexpression significantly increased the CFT (P<0.05), and it also significantly increased the CFT in HSD 5-week-old flies (P<0.01)  (dFOXO gene significantly increased the CI (P<0.05), and it also significantly increased the CI in HSD 1-week-old flies (P<0.05) (dFOXO gene significantly increased the CI (P<0.05), and it also significantly increased the CI in HSD 3-week-old flies (P<0.01) (dFOXO gene significantly increased the CI (P<0.01), and it also significantly increased the CI in HSD 5-week-old flies (P<0.01) (dFOXO gene significantly increased the lifespan of flies (P<0.05), and it also significantly increased the lifespan of HSD flies (P<0.01)  A. In 3-wP<0.001) B. In 5-w(P<0.01) C. Moreov(P<0.01) D. In 1-w(P<0.05) E. In 3-w(P<0.01) E. In 5-w(P<0.01) E. Furthe(P<0.01) F,G. ThesdFOXO gene (P<0.01), it also significantly increase the dFOXO gene expression in HSD flies (P<0.01) , and it also significantly did not change the salt gene expression in HSD flies (P>0.05) (dFOXO gene significantly increased the SOD level (P<0.01), and it also significantly increased the SOD level in HSD flies (P<0.01) (dFOXO gene significantly decreased the MDA level (P<0.01), and it also significantly increased the MDA level in HSD flies (P<0.01)  I. In add(P>0.05) H. Howeve(P<0.01) J. Finall(P<0.01) K. These dFOXO gene knockdown significantly decreased the CFT and CI in flies   , and a HSD aggravated the lifespan reduction in dFOXO gene knockdown flies (P<0.01) , but a HSD significantly increased the salt gene expression in dFOXO gene knockdown flies (P<0.01) (dFOXO gene RNAi significantly decreased the dFOXO gene expression (P<0.01), and a HSD aggravated dFOXO gene expression reduction in dFOXO gene knockdown flies (P<0.01) (dFOXO gene knockdown significantly decreased the SOD activity level (P<0.01), and a HSD aggravated SOD activity level reduction in dFOXO gene knockdown flies (P<0.01) (dFOXO gene knockdown significantly increased the MDA level (P<0.01), and a HSD aggravated MDA level increase in dFOXO gene knockdown flies (P<0.01)  A–E, and P<0.001) A–E. More(P<0.01) F,G. Howe(P<0.01) H. Next, (P<0.01) I. Simila(P<0.01) J. Finall(P<0.01) K. TherefdFOXO gene function is closely to lifespan in flies. For instance, the dFOXO overexpression decreased mortality and increased lifespan of flies via reducing oxidative stress, and the dFOXO is required both for transcriptional changes that mark the fly's dietary history and for nutritional programming of lifespan by excess dietary sugar . The UAS-salt-overexpression flies , the UAS-dFOXO-overexpression flies , and the arm-gal4  flies were obtained from the Bloomington Stock Center. The UAS-salt-knockdown flies  and the UAS-dFOXO-knockdown flies were obtained from the Vienna Drosophila Resource Center .The KO saltmir-1014-KO’ and ‘arm-gal4>w*; TI /total dead numbers. Sample sizes were 200–210 flies per group  method. Ten fresh-frozen flies were converted to homogenates in a homogenizer filled with 1 ml PBS (pH 7.2–7.4). The homogenates were centrifuged at 4°C for 15 min with a speed of 2000 r/min. The supernatant was mixed with the reagents supplied in an MDA Assay Kit  and incubated at 95°C for 40 min. After cooling at room temperature, the mixture was centrifuged at 4000 The total SOD activity in flies was determined using the hydroxylamine method. Ten fresh-frozen flies were converted to homogenates in a homogenizer filled with 1 ml PBS (PH7.2–7.4). The homogenates were centrifuged at 4°C for 15 min with a speed of 2000 r/min. The supernatant was mixed with the reagents supplied in a SOD Assay Kit . The mixture was incubated at room temperature for 10 min, and the absorbance of the compound was then measured at 550 nm. All operations were according to the manufacturer's instructions. Each preparation was tested in triplicate. SOD activity was expressed as U/ml. All assays were repeated three times.salt were as follows: F: 5′-TTAATCGCAGGCGCGTCAGTG-3′; R: 5′-GGACGAGACCACCGTGTTAATCAG-3′. Primer sequences of dFOXO were as follows: F: 5′-AACAACAGCAGCATCAGCAG-3′; R: 5′-CTGAACCCGAGCATTCAGAT-3′. Primer sequences of Rp49 were as follows: F:5′-CTAAGCTGTCGCACAAATGG-3′; R:5′-AACTTCTTGAATCCGGTGGG-3′.About ten flies were homogenized in Trizol. First, 10 μg of the total RNA was purified by organic solvent extraction from the Trizol . The purified RNA was treated with DNase I  and used to produce oligo dT-primed cDNAs , which were then used as templates for quantitative real-time PCR. The rp49 gene was used as an internal reference for normalizing the quantity of total RNAs. Real-time PCR was performed with SYBR green using an ABI7300 Real time PCR Instrument (Applied Biosystems), with three biological replicates. Expression of the various genes was determined by the comparative CT method . Primer sequences of dFOXO overexpression on flies. Using a non-parametric followed by a log-rank test for analyzing survival and time to fatigue. Analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 16.0 for Windows , with statistical significance set at P<0.05. Data are represented as means±s.e.m.The one-way ANOVA with LSD tests were used to identify differences among the relevant groups. A two-way ANOVA was used to analyze the effects of a HSD and


Correction to: Glob Health Res Policy (2020) 5:54https://doi.org/10.1186/s41256-020-00183-yAfter publication of this article , it is nThe correct reference 7 is below:Wang X, Yu Y, Hu Y, Yu C. COVID-19 analysis and forecast based on exponential smoothing model in Hubei Province. J Public Health Prev Med. 2020;31(1):1–4. (in Chinese)The original article has been updated.


Correction to: Mol Cancer (2019) 18:126https://doi.org/10.1186/s12943-019-1054-7After publication of the article , the autThe correct figures are updated below.Fig.3i and jFig.4hAdditional file 3: Figure S2d


Candidatus Dehalogenimonas etheniformans” strain GP couples growth with the reductive dechlorination of vinyl chloride and several polychlorinated ethenes. The genome sequence comprises a circular 2.07-Mb chromosome with a G+C content of 51.9% and harbors 50 putative reductive dehalogenase genes.“ Candidatus Dehalogenimonas etheniformans” strain GP couples growth with the reductive dechlorination of vinyl chloride and several polychlorinated ethenes. The genome sequence comprises a circular 2.07-Mb chromosome with a G+C content of 51.9% and harbors 50 putative reductive dehalogenase genes.“ Dehalogenimonas are obligate organohalide-respiring bacteria implicated in the turnover of naturally occurring and anthropogenic chlorinated compounds in anoxic environments . Paired-end sequencing (2 × 150 bp) was performed using a NovaSeq PE150 flow cell . The sequence reads were trimmed and filtered (https://github.com/lh3/readfq), resulting in 4,073,240 high-quality reads. Assembly using NanoPore raw long reads  and Illumina short reads  was performed using Unicycler version 0.4.7 (Genomic DNA from strain GP biomass was extracted using the cetyltrimethylammonium bromide method . For Nanon 0.4.7 . Unicyclon 0.4.7 .rdh) subunit A genes, including cerA (locus tag number HX448_10020), which encodes a VC rdh  of D. formicexedens strain NSZ-14T (D. formicexedens strain NSZ-14T (GenBank accession number CP018258.1), D. alkenigignens strain IP3-3T (shotgun sequencing project accession number LFDV00000000.1), Dehalogenimonas sp. strain WBC-2 (GenBank accession number CP011392.1), and D. lykanthroporepellens strain BL-DC-9T (GenBank accession number CP002084.1), respectively. The new data expand the Dehalogenimonas pangenome and rdh sequence diversity.The genome comprises one circular 2,068,322-bp chromosome with a G+C content of 51.9%. The genome contains 2,029 coding sequences, 47 tRNA genes, and single copies of the 5S, 16S, and 23S rRNA genes. The GP genome harbors 50 nonidentical reductive dehalogenase (CP058566.1). The BioSample and BioProject accession numbers are SAMN15398252 and PRJNA258024, respectively. The raw reads were deposited in the Sequence Read Archive under accession numbers SRR12774736 (Nanopore) and SRR12774735 (Illumina).The genome has been deposited at the DNA Data Bank of Japan, the European Nucleotide Archive, and GenBank (accession number


Solanum tuberosum L., is the No. 1 vegetable crop and a critical food security crop. The genome sequence of DM1–3 516 R44, a doubled monoploid clone of S. tuberosum Group Phureja, was published in 2011 using a whole-genome shotgun sequencing approach with short-read sequence data. Current advanced sequencing technologies now permit generation of near-complete, high-quality chromosome-scale genome assemblies at minimal cost.Worldwide, the cultivated potato, Here, we present an updated version of the DM1–3 516 R44 genome sequence (v6.1) using Oxford Nanopore Technologies long reads coupled with proximity-by-ligation scaffolding (Hi-C), yielding a chromosome-scale assembly. The new (v6.1) assembly represents 741.6 Mb of sequence (87.8%) of the estimated 844 Mb genome, of which 741.5 Mb is non-gapped with 731.2 Mb anchored to the 12 chromosomes. Use of Oxford Nanopore Technologies full-length complementary DNA sequencing enabled annotation of 32,917 high-confidence protein-coding genes encoding 44,851 gene models that had a significantly improved representation of conserved orthologs compared with the previous annotation. The new assembly has improved contiguity with a 595-fold increase in N50 contig size, 99% reduction in the number of contigs, a 44-fold increase in N50 scaffold size, and an LTR Assembly Index score of 13.56, placing it in the category of reference genome quality. The improved assembly also permitted annotation of the centromeres via alignment to sequencing reads derived from CENH3 nucleosomes.Access to advanced sequencing technologies and improved software permitted generation of a high-quality, long-read, chromosome-scale assembly and improved annotation dataset for the reference genotype of potato that will facilitate research aimed at improving agronomic traits and understanding genome evolution. Solanum tuberosum L., NCBI:txid4113) was published in 2011 by the Potato Genome Sequencing Consortium (PGSC) using a whole-genome shotgun sequencing approach [The genome of the vegetable crop potato . Howeveg; a total of 6.2 g of shoot tissue was split across 6 separate nuclei isolations. Modifications to the protocol include squeezing the homogenate through 5 layers of Miracloth instead of gravity filtering alone and 2 washes with nuclear isolation buffer. DNA was isolated from nuclei using the Nanobind Plant Nuclei Big DNA—Alpha Version kit  following the Nanobind Plant Nuclei Big DNA Kit Handbook v0.17 (May 2018). DNA libraries were prepared using the ONT SQK-LSK109 Ligation Sequencing kit . Six libraries were prepared and sequenced on 6 separate R9 ONT flow cells . DNA repair and end preparation  were performed with an input of 1 μg of DNA. The repair and end preparation reaction were incubated for 5–45 minutes at 20°C and 5–45 minutes at 60°C. The reaction was cleaned using Agencourt AMPure XP beads  with an incubation time of 5–10 minutes on a rotator mixer and eluted for 2–5 minutes. Ligation of adapters to the prepared DNA was performed at room temperature for 10–60 minutes. The ligation reaction was cleaned using Agencourt AMPure XP beads on a rotator mixer with an incubation time of 5–10 minutes with an elution time of 10 minutes. Sequencing was performed on an ONT MinION   with the current release of MinKNOW (version 1.15.0). Sequencing was run for 48–92 hours (RRID:SCR_016383) in paired-end mode, generating 150 nt reads (DM plants were grown in Murashige and Skoog (MS) medium , shoots harvested, and flash frozen in liquid nitrogen. Nuclei were isolated following the Workman et al.  protocol92 hours . DNA wasnt reads . Hi-C lint reads .RRID:SCR_018927) [RRID:SCR_017016) [RRID:SCR_017642) [RRID:SCR_018550) [RRID:SCR_016157) [RRID:SCR_002105) [RRID:SCR_014731) [RRID:SCR_011841) [RRID:SCR_010910) [RRID:SCR_006525) [RRID:SCR_006525), all using default parameters. Pilon was run using the “–fix bases” option. The polished contigs are composed of 1,382 contigs with a total size of 745.6 Mb with an N50 contig size of 17.3 Mb and a maximum contig length of 42.1 Mb  on an A_018927)  to remov_017016)  with the_017642) . For eac_018550)  with the_016157) . Reads w_016157)  with the_002105) . An upda_014731) . The Ill_011841)  to remov_006525) , and theRRID:SCR_017226) [RRID:SCR_017227) [RRID:SCR_016665) [S. tuberosum mitochondrion genome  using blastn v2.9.0  [To construct chromosome-scale pseudomolecules, the Hi-C library was first processed using the juicer.sh pipeline from the Juicer package (git commit 6403a27)  . The pse_017227)  and the _017227)  (Supplem_017227)  database_016665)  with the_001598) . FifteenRRID:SCR_011841) [RRID:SCR_010910) [RRID:SCR_015008) [k-mers (k = 21) from the cleaned Illumina whole-genome shotgun library (PEP_AA_01) using Jellyfish2 v2.2.10  [k-mer count histogram was analyzed by the online version of GenomeScope  [To assess completeness and accuracy of the v6.1 assembly, ∼458 million paired-end reads from a whole-genome Illumina sequencing library   was used_010910) . Alignme_015008)  software_015008) . Of 1,61_005491) . The k-m_017014)  and the RRID:SCR_018970) [RRID:SCR_018969) [RRID:SCR_017623) [Arabidopsis thaliana TAIR10 (LAI = 14.9), Fragaria vesca v4.1 (LAI = 16.9), and Solanum pennellii (LAI = 14.8) [The Long Terminal Repeat (LTR) Assembly Index (LAI)  metric w_018970) , LTR_FIN_018969) , and LTR_017623) . LTR seq = 14.8) . The LAIin situ hybridization (oligo-FISH) probes, which mark 26 regions on the 12 chromosomes, have been used to characterize potato karyotypic variation [Solanum species. We aligned the oligo-FISH probes to v6.1 using BWA-MEM v0.7.12-r1039  [Two “barcode” oligonucleotide fluorescent ariation , as well_010910)  to confiRRID:SCR_018968) [RRID:SCR_010910) [RRID:SCR_006646) [Cen12 is properly positioned on the short arm of v6.1 chromosome 12   with 200_018968) . ChIP-se_010910)  using de 12 Fig. . The imp6.1 Fig. . In v4.0nce Fig. . The siznce Fig. . These cRRID:SCR_018967) [To better depict the improved contiguity and accuracy of v6.1 relative to v4.04, D-GENIES   was usedRRID:SCR_015027) [RRID:SCR_001653) [RRID:SCR_012954) [A custom repeat library (CRL) was generated using RepeatModeler2 v2.0.1   with the_015027)  using bl_001653)  with an Cen4, Cen6, Cen10, Cen11, and Cen12) lack typical centromere-specific satellite repeats and, instead, are composed of single- or low-copy sequences resembling neocentromeres [Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) that contain megabase arrays of satellite repeats. Interestingly, the satellite repeats for these 6 centromeres are unique to individual chromosomes, some of which are derived from retrotransposons. Centromeric repeat sequences from Gong et al. [RRID:SCR_004870) [Cen2, Cen5, and Cen7 in v.6.1 but not in v4.04   with aliRRID:SCR_018966) [RRID:SCR_018927) [RRID:SCR_018550) with the parameters -a -x splice -uf -G 5000; 5,783,924 (99.98%) of the 5,784,833 filtered reads aligned to the DM assembly. The cDNA alignments were assembled using Stringtie2 v2.1.2  [RRID:SCR_018965) [RRID:SCR_011841) [RRID:SCR_015530) [RRID:SCR_016323) [RRID:SCR_018965) [To facilitate annotation of gene models, ONT complementary DNA (cDNA) sequences were generated from DM. DM was grown under a 16-hour day length in tissue culture and RNA was isolated from whole tissue-culture plants using a modified hot borate method . DNA con_018966)  to ident_018927)  to remov_016323)  (-L -m 5_018965) . Illumin_011841)  with the_015530)  with the_016323)  with the_018965) . Both thRRID:SCR_018964) [RRID:SCR_008417) [RRID:SCR_011930) [ab initio gene predictions. The BRAKER2 pipeline was run using the command line: braker.pl –species = DM_v6_1 –gff3 –softmasking –UTR = off –bam {RNA-seq.alns.bam}. Ab initio gene predictions were refined using PASA2 v2.4.1  [RRID:SCR_004726) [RRID:SCR_005305) [RRID:SCR_016582) [The BRAKER2 (git commit 6219573)   gene pre_011930)  and the _005305)  with a c_016582) .Arabidopsis proteome  [RRID:SCR_004726) [RRID:SCR_002380). Search results were processed in the same order, and the function of the first hit encountered was assigned to the gene model. The quality of the annotation was evaluated using BUSCO [High-confidence gene models were defined as having a TPM value > 0 in ≥1 RNA-Seq library and/or having a PFAM domain match. Gene models that were partial or had matches to transposable element–related PFAM domains were excluded from the high-confidence model set. A total of 32,917 loci encoding 44,851 gene models are contained within the high-confidence set . To assi_004618) , the PFA_004726) , and theng BUSCO , and botUsing improved sequencing technologies, the genome sequence of the reference potato genotype DM was vastly improved in contiguity relative to the previous release, DM v4.04. Version 6.1 of the DM genome assembly represents 87.8% of the estimated genome, with 595-fold increase in N50 contig size, 99% reduction in number of contigs, and a 44-fold increase in N50 scaffold size. Importantly, 731.2 Mb of the 741.6-Mb assembly is non-gapped and anchored to the 12 chromosomes, indicating a high degree of contiguity that was reflected in a “reference quality” LAI score, demonstrating the ability of advanced sequencing methods to assemble large contiguous regions of a medium-sized plant genome. With access to full-length cDNA sequences, 32,917 high-confidence protein-coding genes encoding 44,851 gene models were annotated, which provided a substantial improvement in representation of conserved orthologs compared with the previous annotation that will facilitate future studies in potato biology, genetics, and genomics.GigaScience GigaDB [The clone, DM1–3516 R44, is available through the United States Department of Agriculture Potato Genebank via PI GS 233 . The rawe GigaDB , Dryad De GigaDB , and on e GigaDB , 73 via Supplementary Figure S1. Hi-C contact map showing the inter- and intra-chromosomal chromatin interactions in DM v6.1. Inter-chromosomal chromatin interactions are off the diagonal axis and intra-chromosomal chromatin interactions are within the blue boxes. Each pixel represents the degree of interaction between each 1-Mb locus, with a dark red color indicating a greater number of reads involved in the interaction. The blue boxes represent the boundaries of each pseudomolecule, and individual scaffold boundaries are represented by the green boxes.k-mer of 21.Supplementary Figure S2. Estimation of heterozygosity of the DM genome as determined by GenomeScope. The DM genome has an estimated heterozygosity rate of 0.0383% using a RRID:SCR_018968) [Supplementary Figure S3. Mapping of the DM × RH F1 population markers to the (a) DM v4.04 and the (b) DM v6.1 assembly. Flanking sequence (200 nt) of the markers was used for sequence alignments to the assembly using Vmatch  . The y-aSupplementary Table S1. Sequence datasets used in this study. Total reads for Oxford Nanopore Technologies sequencing are passed reads after base-calling.Supplementary Table S2. Oxford Nanopore Technologies whole-genome shotgun sequence reads used in the DM v6.1 assembly.Supplementary Table S3. Illumina whole-genome shotgun sequence read mapping statistics.Supplementary Table S4. BUSCO  results Supplementary Table S5. Centromere positions in the DM v6.1 assembly.Supplementary Table S6. Repetitive sequence content in v4.04 and v6.1 DM 1–3516 R44 genome assemblies.Supplementary Table S7. DM v6.1 gene annotation summary.giaa100_GIGA-D-20-00167_Original_SubmissionClick here for additional data file.giaa100_GIGA-D-20-00167_Revision_1Click here for additional data file.giaa100_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giaa100_Reviewer_1_Report_Original_SubmissionJian-Feng Mao, Ph.D. -- 7/7/2020 ReviewedClick here for additional data file.giaa100_Reviewer_2_Report_Original_SubmissionAlexis Sullivan -- 8/10/2020 ReviewedClick here for additional data file.giaa100_Supplemental_FilesClick here for additional data file.in situ hybridization; ONT: Oxford Nanopore Technologies; PASA: Program to Assemble Spliced Alignments; PGSC: Potato Genome Sequencing Consortium; RNA-Seq: RNA-sequencing; SRA: Sequence Read Archive; TPM: transcripts per million.BLAST: Basic Local Alignment Search Tool; bp: base pairs; BUSCO: Benchmarking Universal Single-Copy Orthologs; cDNA: complementary DNA; ChIP-Seq: chromatin immunoprecipation sequencing; CRL: custom repeat library; Gb: gigabase pairs; kb: kilobase pairs; LAI: LTR Assembly Index; LTR: long terminal repeat; Mb: megabase pairs; mRNA: messenger RNA; NCBI: National Center for Biotechnology Information; nt: nucleotide; oligo-FISH: oligonucleotide fluorescent The authors declare that they have no competing interests.C.R.B. conceived the study. G.M.P., J.P.H., B.V., and J.C.W. performed the experiments. J.T.B., J.P.H., J.J., G.M.P., S.O., B.V., J.C.W., and H.Z. analyzed data. C.R.B., J.P.H., J.J., G.M.P., B.V., J.C.W., and H.Z. wrote the manuscript. All authors approved the final manuscript.