However, when applied to critically ill patients with shock, its measurement may be affected by changes in FiO2 and PaO2 and potential associated O2 diffusion between urine and ureteric or bladder tissue. We aimed to investigate PuO2 measurements in septic shock patients with a fiberoptic luminescence optode inserted into the urinary catheter lumen in relation to episodes of FiO2 change. We also evaluated medullary and urinary oxygen tension values in Merino ewes at two different FiO2 levels.Continuous measurement of urinary PO2 decreases and 31 increases in FiO2. Median pre-decrease FiO2 was 0.36  and median post-decrease FiO2 was 0.30 , p = 0.006. PaO2 levels decreased from 83 mmHg  to 72  mmHg, p = 0.009. However, PuO2 was 23.2 mmHg  before and 24.2 mmHg  after the intervention (p = 0.56). The median pre-increase FiO2 was 0.30  and median post-increase FiO2 was 0.35 , p = 0.008. PaO2 levels increased from 64 mmHg  to 71 mmHg , p = 0.04. However, PuO2 was 25.0 mmHg  before and 24.3 mmHg  after the intervention (p = 0.65). A mixed linear regression model showed a weak correlation between the variation in PaO2 and the variation in PuO2 values. In 9 Merino ewes, when comparing oxygen tension levels between FiO2 of 0.21 and 0.40, medullary values did not differ  and this was similar to urinary oxygen values .In 10 human patients, there were 32 FiO2 and PaO2 within the context of usual care did not affect PuO2. Our findings were supported by experimental data and suggest that PuO2 can be used as biomarker of medullary oxygenation irrespective of FiO2.Changes in FiOThe online version contains supplementary material available at 10.1186/s40635-022-00479-y. There were 14 episodes of successive increase/decrease in the same patient. Such episodes were successive and conditional on the presence of a PaO2 measurement and the time difference between these episodes was 4.29  hours.We observed 63 episodes of changes in the FiO2 was decreased, PaO2 fell from 83  mmHg to 72  mmHg  while urinary oxygen tension values significantly increased .At higher FiO2 (from 0.21 to 0.60), medullary oxygen tension values were similar  but urinary oxygen tension values increased .For a larger change in FiO2 is affected by changes in systemic oxygenation during routine care of patients with septic shock. As expected, changes of FiO2 resulted in significant changes in PaO2. However, we found no significant differences between PuO2 measured before and after the interventions occurred. We supported our clinical findings with data from an experimental sheep experiment showing that medullary and urinary oxygen tension measurements did not differ within a similar range of FiO2 variation.We conducted an observational study in human septic patients to determine whether PuO2 and PuO2 during cardiopulmonary bypass [2 and PuO2, accounting for ≤ 6% of the variation of PuO2 [To our knowledge, there have been no previous investigations of the relationship between systemic and urinary oxygenation in human patients with septic shock. Ngo et al. addressed this issue in a group of patients undergoing cardiac surgery, finding no significant relationship between PaOy bypass . Importay bypass , 12, 13,2 in response to modest but clinically significant changes in FiO2 and thus PaO2 indicate that renal medullary tissue PO2 was not markedly affected by these clinical maneuvers. Experimental evidence supports the concept that extreme variations in FiO2 and/or PaO2 lead to corresponding changes in the oxygen tension of renal tissue. For example, in anesthetized rats a reduction in FiO2 from 1.0 to 0.1 resulted in a decline in cortical and medullary microvascular PO2 as assessed by dual-wavelength phosphorimetry [2 with variations in FiO2 [2 variation is associated with greater PuO2 response, in particular at 0.60 and 1.00 FiO2.The absence of detectable changes in PuOorimetry . Likewis in FiO2 , 15–17. 2 from 0.21 to 1.00 increased cortical and medullary tissue PO2 [2 is less responsive to changes in PaO2 than the renal cortical tissue PO2 [2 and/or PaO2 may not appreciably alter renal medullary tissue PO2. In support of this concept, no appreciable difference was observed in medullary tissue PO2 in our group’s preceding experiment when FiO2 was varied from 0.4 to 0.6 [2 did not vary significantly when FiO2 was varied from 0.21 and 0.30 [2 in patients and can only draw indirect inferences from measurement of PuO2 and consideration of available experimental evidence. However, the most parsimonious interpretation of our current findings is that modest changes in FiO2 and thus PaO2 neither markedly alter renal medullary tissue PO2 in patients with sepsis nor confounded the relationship between medullary tissue PO2 and PuO2.Our ovine study sample derived from a larger experiment comprising 18 healthy sheep undergoing abdominal surgery under total intravenous or volatile anesthesia. In this study, increasing FiOssue PO2 , 18, 19.ssue PO2 . Consequ2 settings in patients with sepsis do not result in significant changes in PuO2. In consonance of these findings, we observed that variations of FiO2 between 0.21 and 0.40 did not alter either medullary or urinary oxygen tension measurements in a sheep experiment. Thus, variations of systemic oxygenation seem unlikely to confound or affect the utility of urinary oxygenation as a biomarker for risk of AKI. Nevertheless, at higher FiO2 (0.60 and 1.00), significantly increased PuO2 values were obtained. One possible explanation is that in our septic patients, the FiO2 gap was far smaller in comparison to the experimental study. Also, one could argue that a type 2 error was present in the observational study which may have been controlled for during the experimental protocol.Our findings suggest that commonly performed adjustments to FiO2 variation in face of PaO2 changes derives from the presence of confounding factors affecting medullary oxygen values. Also, further studies in critically ill patients are needed to elucidate whether sustained differences in oxygen exposure [2, as a consequence of altered FiO2 in routine care of patients with septic shock, is unlikely to be a major confounder of the relationship between renal medullary tissue PO2 and PuO2. In the current study, PaO2 was used as a measure of systemic oxygenation because it reflects the balance between oxygen delivery and consumption. Had SpO2 been used, the accuracy would have been affected by peripheral tissue perfusion, use of vasoactive agents and altered cardiac output. Finally, continuous measurement of PuO2 might be useful for monitoring the impact on renal medullary oxygenation.Additional investigation is needed to explore whether the lack of PuOexposure , 23 infl2 and PuO2 is unlikely to be confounded by changes in FiO2 or PaO2 in the range commonly encountered in the ICU. As such, they provide further support for the use of PuO2 as a clinical surrogate of renal medullary PO2.We evaluated systemic and urinary oxygenation in human septic patients and assisted our proposition with experimental data. Our findings are consistent with previous observations in sepsis  and prov2 where the observed intervention (change of FiO2) was not protocolized. Moreover, controlling for variables such as creatinine or urine output was not feasible due to technical limitations and the limited number of patients. However, we aimed to undertake an exploratory analysis to generate a preliminary hypothesis to guide advanced studies. Moreover, we added data from a sheep experiment where FiO2 variation was protocolized. Also, due to the lower number of measurements in the experimental study, greater heterogeneity was observed. Second, the inclusion of septic patients in our clinical study did not occur in the early stage of resuscitation. On the other hand, the instances of FiO2 change we captured took place in a stable state with lower propensity for PuO2 to be affected by additional confounding effects of interventions intended to optimize oxygen delivery to the tissues. Furthermore, changes in FiO2 performed under stable conditions might have reduced the likelihood of reverse causation or provided mitigation of any potential effect of other interventions. Third, the observational nature of the study may have led to confounding by indication. For instance, the reasons motivating the clinician to change FiO2 settings could have affected the relationship between FiO2 and PuO2. However, a larger degree of FiO2 change would be expected if optimization measures capable of affecting such relationship were in place. Our patients were enrolled in the stabilization phase of sepsis, a time when, in general, only limited interventions are performed to achieve physiologic parameters aiming to prevent organ dysfunction. Finally, we addressed only the variation of systemic oxygenation within the normoxemic range. However, such a normoxemic range is typical in the care of patients in the ICU.Our study has several limitations. First, the clinical component was an observation designed to assess the effects on PuO2 and PaO2 within the context of usual care did not appreciably affect PuO2. Our findings suggest that, within the values reported, PuO2 measured in a clinical and experimental setting is not confounded by changes in inspired oxygen fraction or arterial oxygen tension and that PuO2 can be used as biomarker of medullary oxygenation irrespective of FiO2.Changes in FiOAdditional file 1. Relationship between PaO2 and PuO2 values in each individual study patient.Additional file 2. Box plot illustrating the hourly urinary output before and after a change in FiO2.


Acute Confusional Syndrome (ACS) is the most common neuropsychiatric complication in COVID-19 infection. Its management is still a challenge because the data and recommendations based on the evidence are limited.To describe the differential characteristics in the management of ACS in patients with COVID-19 pneumonia compared to ACS secondary to other causes.We present a descriptive study that is has been carried out in 62 patients with ACS (26 of them diagnosed with COVID 19 pneumonia), who have required assessment by the liaison psychiatry service of Hospital del Mar between February and April, 2020. The sample was divided in 2 groups (with and without COVID 19 pneumonia). Chi square and Fisher’s tests were used to comparisons.Dexmetomidine (26 vs 0) and olanzapine (13 vs 3) were significantly more frequently used in COVID-19 patients (p< 0 001). A greater number of different antipsychotic drugs were used in COVID 19 patients (2.40± 1 323 number of drugs), (p<0.0001). Further neuroimaging tests were requested in COVID 19 patients and they received less family support (4) compared to non COVID-19 (22), (p<0.005).ACS associated with COVID-19 pneumonia in the patients in our sample is more difficult to manage than ACS associated with other pathologies, similar to which described in other series. It is associated with a longer duration of confusional symptoms and difficulties for control it.No significant relationships.


To assess the uptake of cervical screening in patients under Birmingham and Solihull Assertive Outreach Teams; this included a re-audit of patients under the Central Birmingham Assertive Outreach Team.Patients with severe and enduring mental illness are known to have poorer physical health outcomes. In Birmingham and Solihull there are 6 Assertive Outreach Teams. These teams manage patients with a diagnosis of psychosis who have complex needs requiring intensive multidisciplinary input and often struggle to engage with health services. The national cervical screening programme aims to prevent cervical cancer by detecting and treating cervical abnormalities. Acceptable coverage is defined as screening at least 80% of people aged 25–49 years within the last 3.5 years and 80% of people aged 50–64 years within the last 5.5 years. In 2018 71.4% of women in England and 70.9% in the West Midlands were screened adequately. An audit of 15 patients under the Central Birmingham Assertive Outreach Team in 2014 showed 46.2% had taken up screening, measured in the last 5 years for those aged 50–64 years and the last 3 years for those aged 25–49 years.A list was obtained of all female patients under the Assertive Outreach Teams with patients excluded if they were under 25 years or over 64 years or if they were known to have undergone a total hysterectomy. All GP practices with eligible patients registered to them were written to requesting the date of the patient's most recent smear test. Cervical screening was classed as in date if carried out in the last 3.5 years for patients aged 25–49 years or 5.5 years for patients aged 50–64 years.Out of 127 eligible patients, 110 had correct GP details on their record. Responses were received regarding 101 patients, 48 of whom had in date cervical screening (47.5%). Of 58 patients aged 25–49 years, 26 had in date cervical screening (44.8%). Of 43 patients aged 50–64 years, 22 had in date cervical screening (51.2%).13.4% patients did not have a known GP practice, increasing the risk of multiple poor physical health outcomes. The rates of cervical screening among Assertive Outreach Team patients are similar to the original audit in 2014 and fall significantly below the national standards and averages. These findings, along with the importance of working together to address the need for physical health monitoring in this population, will be communicated with the local Assertive Outreach Teams and GP practices.


The gender distribution of the study participants should have been reported as 227 girls (60.5%) and 148 boys (39.5%). The article has been corrected.In the Research Letter titled “Evaluation of Anxiety and Depression in a Community Sample of Transgender Youth,”


It is sensitive, with a lower limit of detection of 0.2 CCID50/mL , and generates reproducible results. This assay can be used for quality control and lot release of the nOPV2.To control circulating vaccine-derived type 2 poliovirus outbreaks, a more genetically stable novel Oral Poliovirus Vaccine type 2 (nOPV2) was developed by targeted modifications of Sabin 2 genome. Since the use of OPV2 made of Sabin 2 strain has been stopped, it is important to exclude the possibility that batches of nOPV2 are contaminated with Sabin 2 virus. Here, we report the development of a simple quantitative one-step reverse-transcription polymerase chain reaction assay for the detection and quantitation of Sabin 2 virus in the presence of overwhelming amounts of nOPV2 strain. The method is specific and linear within 8 log There are two prophylactic vaccines against poliomyelitis caused by the three serotypes of poliovirus: inactivated poliovirus vaccine (IPV) and live oral poliovirus vaccine (OPV). The use of these vaccines has dramatically reduced polio incidence worldwide. However, IPV does not stimulate adequate mucosal immunity to prevent transmission of circulating polioviruses ,2, and OThe Global Polio Eradication Initiative (GPEI) led to the eradication of wild polioviruses of types 2 and 3 , but wilSeveral molecular-based procedures have been developed for detection of OPV strains, including ELISA , reverseThe assay for the presence of Sabin 2 contamination in nOPV2 stocks is simple, rapid, reproducible, sensitive, and has large linearity range suitable for quality control of the nOPV2 vaccine.295K was provided by Dr. Andrew Macadam  and Dr. Raul Andino , and was used as negative control for the Sabin 2 contamination assay. Monovalent bulks of nOPV2 candidate 1 (nOPV2-c1) batches nPOL 2016 and nPOL 2018C, monovalent nOPV2 candidate 2 (nOPV2-c2) batches nPOL 2038C and nPOL 2056C, and drug product nOPV2-c1 batches 2060119C, 2060219C and 2060319C were provided by P.T. Bio Farma (Indonesia) and were tested for the presence of Sabin 2 virus.Batch of US neurovirulence poliovirus reference vaccine (Sabin 2 strain of OPV having GenBank accession number AY184220) and plasmid-containing Sabin 2 genome were used as positive control (Lab. method development inventory at US. FDA). Plasmid-containing nOPV2-c1 genome with mutation E295K by transfecting HEp-2C monolayers with T7 transcripts . A,4. A3,4]10 CCID50/mL was subjected to RNA extraction as described above and the RNA was serially diluted 10-times using 10-fold steps in a total volume of 100 μL. The original and diluted RNA samples were tested, and the resulting Ct values were averaged (mean) for each dilution and plotted against the respective Sabin 2 titers expressed as log10 CCID50/mL as shown in 50/mL  and had a very broad linearity range of at least 8 log10 (from 8.34 to 0.34 Log10 CCID50/mL). RNA from a sample with the lowest titer analyzed (0.034 Log10 CCID50/mL) was undetectable.To evaluate the linearity and sensitivity of the RT-PCR in absence of nOPV2 background, a Sabin 2 strain with titer of 8.34 Log10 CCID50/mL) and a plasmid containing Sabin 2 genome (0.1 ng/mL) were run as positive controls, while another plasmid containing the nOPV2-c1genome (0.1 ng/mL) and water were used as negative controls. The controls were run in quadruplet repeats, and the test and standard reference samples were tested in triplicates. The results of positive and negative controls are presented in In the same run, Sabin 2 RNA . Briefly, the Sabin 2 detection assay yielded consistent and reproducible results.Three Sabin RNA samples with equivalent viral titers of 2.16, 21.63 and 216.27 CCID10 CCID50/mL was serially diluted in 10-fold steps in nOPV2c1-295 virus suspension with the titer of 8.07 log10 CCID50/mL . All diluted samples were subjected to RNA extraction and then to the assay for detection Sabin 2 contamination in nOPV2c1-295 as separate samples in triplicate repeats. The results showed that the presence of nOPV2 virus with high titer did not interfere with the assay that was able to detect Sabin 2 virus at levels of 0.22 CCID50/mL which correspond to traces of Sabin 2 virus in nOPV2 (2 × 10−7% of Sabin 2 in nOPV2 suspension)  . A linea 8 log10 . All neg 8 log10 , confirm50 to those based on genome copy (GC) numbers we analyzed the spiked samples with qmosRT-PCR as described above and the resulting GC numbers were used to calculate the percentages of the Sabin 2 in the samples  and the percentages of Sabin 2 in samples were calculated as the average percentages of the 18 SNPs in domain V  that are mentioned above  and presented in Vaccines are the most efficient tools for preventing, controlling and even eradicating infectious diseases. However, contamination can occur at various stages of vaccine preparation, resulting from the unintentional introduction of extraneous agents present in raw materials or introduced during the manufacturer’s process.As stated above, the nOPV2 was created by introducing targeted genetic changes to the Sabin 2 strain that further stabilize the attenuated phenotype. Since the original Sabin 2 virus can easily convert to circulating vaccine-derived poliovirus type 2 (cVDPV2), it is important to ensure that batches of nOPV2 do not contain Sabin 2 virus if handled in the same manufacturing facility. Therefore, a sensitive and specific assay for detection of Sabin 2 strain is useful for quality control of the nOPV2 vaccine.The previous molecular methods were designed for specific detection of Sabin strains in clinical and environmental samples but cannot distinguish Sabin strains from their genetically modified nOPV derivatives ,23,24,256 to 101 of the relative CCID50/mL), blank (water) and nOPV2 plasmid DNA and/or nOPV2 RNA as negative controls, and Sabin 2 RNA and/or Sabin 2 plasmid DNA as positive controls. All the samples are tested in triplicates or quadruplicates (for controls) in the same 96-well plates by qosRT-PCR that specifically detects Sabin 2 virus and differentiates it from the nOPV2. The qosRT-PCR run is considered valid if all the following conditions are met: R2 of the standard curve is more or equal 0.95, for three out of four positive control repeats Ct values are less or equal to 40, and for at least three out of four negative control repeats have Ct values undetermined (or Ct ≥ 40). The test sample is positive if at least two out of three repeats have Ct values less than 40. In this work the Ct 40 was chosen as threshold. However, to establish a real Ct threshold needs further work to ensure that the assay is specific and works under different conditions, and in different hands and labs.The following provisional layout and acceptance criteria are proposed: the assay for detection of Sabin 2 contamination in nOPV2 RNA samples are tested in triplicate along with reference–Sabin 2 RNA serially diluted in 10-fold steps  of Sabin 2 virus diluted in nOPV2 suspension, and 198 GC/mL corresponds to 0.396 GC/reaction which is in line with having reached the maximum theoretical limit of one GC detected per reaction. The limit of detection may vary from virus to virus depending upon the GC:CCID50 ratio for each virus. No cross-amplification was observed with nOPV2 and a plasmid that contains nOPV2 genome . No Sabin 2 virus was detected in these lots and the results were confirmed by NGS . Based oIn conclusion, the assay for detection Sabin 2 contamination in nOPV2 stocks described in this communication offers a simple and rapid method for the detection and quantitation of Sabin 2 virus either individually or in the presence of nOPV2 virus. The assay for Sabin 2 contamination in nOPV2 stocks is designed specifically to be applied during manufacture of nOPV2 vaccine, for quality control to detect Sabin 2 virus as a potential contaminant in nOPV2 vaccine.


FOXC1 and PITX2, account for almost half of known cases, while the genetic lesions in the remaining cases remain unresolved. Given the genetic similarity between zebrafish and humans, as well as robust antisense inhibition and gene editing technologies available for use in these animals, loss of function zebrafish models for ARS have been created and shed light on the mechanism(s) whereby mutations in these two transcription factors cause such a wide array of developmental phenotypes. This review summarizes the published phenotypes in zebrafish foxc1 and pitx2 loss of function models and discusses possible mechanisms that may be used to target pharmaceutical development and therapeutic interventions.Axenfeld–Rieger syndrome (ARS) encompasses a group of developmental disorders that affect the anterior segment of the eye, as well as systemic developmental defects in some patients. Malformation of the ocular anterior segment often leads to secondary glaucoma, while some patients also present with cardiovascular malformations, craniofacial and dental abnormalities and additional periumbilical skin. Genes that encode two transcription factors,  Axenfeld–Rieger syndrome (ARS) is a clinically heterogeneous disorder characterized by ocular anomalies with systemic multi-organ system involvement in some patients. This relatively rare disorder, with a prevalence of 1 in 50,000–100,000 live births ,3, cerebFORKHEAD BOX C1 (FOXC1) and PAIRED-LIKE HOMOEDOMAIN (PITX2). DNA lesions involving PITX2 result in ARS type I, in which patients have fully penetrant ocular phenotypes often observed with craniofacial and dental anomalies. Mutations in FOXC1 result in ARS type III, defined by fully penetrant ocular phenotypes often observed with cardiovascular defects and sensorineural hearing loss. While linkage analysis supports an additional gene causing ARS type II on chromosome 13q14 [CYP1B1 gene have been found in a single family with ARS [CYP1B1 appear to be an extremely rare cause of ARS.ARS is inherited as an autosomal dominant disorder, with the genetic lesion defined in approximately 40% of cases. Mutations or copy number variation (CNVs) in two genes identified through a variety of family-based studies account for ARS with fully penetrant ocular manifestations; me 13q14 , no causme 13q14 . This cowith ARS , and whiwith ARS ,19, mutaDanio rerio) has provided valuable mechanistic insights into ARS disease etiology. The zebrafish genome contains two homologues of the FOXC1 gene denoted foxc1a and foxc1b, arising from an ancient duplication in the teleost lineage, and one homologue of PITX2 (pitx2). Gene expression studies using in situ hybridization have highlighted specific cell types that require the expression of foxc1 and pitx2 for normal ocular development. The use of morpholino based antisense inhibition and genome editing have produced zebrafish strains that mimic ARS phenotypes and have provided novel mechanistic data highlighting downstream genes and signaling pathways that are required for ocular and systemic manifestations of the syndrome. This review will focus on the role of foxc1 and pitx2 in the regulation of genes and signaling pathways that regulate formation of the eye, cardiovascular system, and craniofacial skeleton, as defined by loss of function zebrafish mutant strains or antisense inhibition data.The zebrafish ) as well as the lateral plate mesoderm (LPM) [foxc1a continues to be expressed in the adult zebrafish eye, including the anterior segment and retinal ganglion cell layer [At early stages of development, zebrafish express st cells ,20  . Neural rm (LPM) . Neural rm (LPM) ,24,25. Trm (LPM) ,26,27. All layer , possiblpitx2 gene that encodes two isoforms via alternative splicing that correspond to human PITX2A and PITX2C. The expression of pitx2a is found in partially overlapping domains with foxc1a and foxc1b in developing zebrafish embryos. As early as 24 h post fertilization (hpf), pitx2 expression is observed in the periocular mesenchyme [foxc1a and foxc1b, pitx2 is expressed in the neural crest derived tissues of the pharyngeal arches that contribute to the craniofacial skeleton [pitx2c isoform in the lateral plate mesoderm [pitx2a in the neural crest [The zebrafish genome contains a single senchyme   inhibition, a number of zebrafish models have been reported that recapitulate the phenotypes of foxc1a and foxc1b display underdeveloped or absent anterior segments beginning around 3 dpf [foxc1a/foxc1b zebrafish, which are typically not observed in ARS patients. This discrepancy may be the result of complete loss of Foxc1 function in double homozygotes, compared to heterozygous mutations or copy number variations that define ARS. Heterozygous mutations of both foxc1a and foxc1b in zebrafish do not alter early development of the anterior segment, however, published analyses only describe assays performed up to 6 dpf and thus additional analyses on older heterozygous larvae or adults when the anterior segment has matured, could potentially reveal anterior segment anomalies consistent with ARS. Loss of Foxc1 function in zebrafish also leads to endophenotypes of glaucoma, including reduced number of retinal ganglion cells and thinner optic nerves [The zebrafish anterior segment begins to develop as early as 26 hpf when the lens vesicle detaches from the surface ectoderm . By 3 dand 3 dpf . Additioc nerves  . However, many ARS patients have normal tension glaucoma (NTG), defined as having normal intraocular pressure, and thus other mechanisms are likely involved. For example, a number of studies have demonstrated that foxc1 regulates the formation of the zebrafish hyaloid and retinal vasculature [atonal homolog 7 (atoh7). Atoh7 is required for RCG differentiation during embryonic development [ATOH7 in human populations are associated with persistent hyperplasia of the primary vitreous [atoh7 expression via Foxc1 could account for ocular vascular defects in ARS patients and could influence the development of glaucoma independent of, or in combination with, potential increased IOP caused by the anterior segment dysgenesis. Variants in ATOH7 have also been associated with primary open angle glaucoma [atoh7 as a key contributor of RGC and optic nerve health.Certainly, the observed anterior segment defects in culature ,47. Defeelopment ,49, and vitreous  whereby glaucoma  and endoglaucoma ,53,54, iatoh7, other genetic targets regulated by Foxc1a/Foxc1b in zebrafish have been identified that may shed light on potential mechanisms of glaucoma development. Foxc1a/Foxc1b regulates the expression of FOXO1A/foxo1a, a gene expressed in the zebrafish POM that mediates the response to oxidative stress [foxc1a or foxo1a resulted in aberrant responses to increased oxidative stress and increased cell death in the eye [FOXC1. Foxc1a also regulates the expression of fgf19 [foxo1a and fgf19 have not been identified in ARS or glaucoma patients and thus the dysfunction of either gene alone likely cannot cause overt visual defects, however, their reduced expression due to loss of Foxc1 function, as well as other yet to be identified downstream targets, likely contribute to the complex etiology of RGC loss in FOXC1-attributable ARS patients.In addition to e stress . Manipul the eye , indicatof fgf19 ,56, anotof fgf19 ,58. Mutafoxc1a mutants, as well as foxc1a/foxc1b double mutants, have craniofacial defects consistent with abnormalities in the facial structure of ARS patients. In these patients, hypertelorism (increased space between the eyes) and a prominent forehead are often described. In zebrafish, foxc1a and foxc1b are expressed in the neural crest cells that populate the first and second pharyngeal arches that give rise to anterior jaw structures. Mutation of foxc1a alone results in craniofacial dysmorphism [foxc1a/foxc1b mutants. Phenotypes in double homozygous mutants included under-developed palatoquadrate and hyomandibula cartilages [foxc1a and foxc1b play critical roles in the development of anterior facial cartilages (foxc1a). Analysis of genetic targets downstream of foxc1 revealed both foxc1a and foxc1b regulate Sox9-dependent expression of cartilage specification genes, accounting for reduced jaw structures in these animals. While Foxc1 does not directly regulate sox9 expression, loss of foxc1 paralogs in zebrafish causes a decrease in chromatin accessibility for transcription factors such as Sox9 in chondrocytes [Zebrafish morphism ,37 includrocytes , and thufoxc1a mutants in conjunction with homozygous loss of foxc1b (which survive to adulthood) demonstrates craniofacial abnormalities in adults that include a misshapen head that closely mimics that of ARS patients, as well as mandibular retrognathia and dorsally positioned eyes [FOXC1.Analysis of heterozygous ned eyes . CombineFOXC1 mutations or CNVs that include hypoplastic ventricular outflow tract morphology, dysplastic arcade mitral valve, and atrial septal defect [FOXC1 plays an important role in early heart and cerebral vascular development, with such phenotypes recapitulated in zebrafish loss of foxc1 function models.Cardiac anomalies have been described in ARS patients with l defect ,59. Withl defect —all of wl defect . These cfoxc1a alone or in combination with foxc1b leads to cardiac phenotypes that include hypoplastic myocardium and ventricular outflow tract, as well as defects in cardiac valve formation that are similar to that observed in patients with FOXC1-attributible ARS [foxc1 in heart development, reports differ in their analysis of heart morphology, assay different time points, and study different combinations of mutations. For example, Yue et al. [foxc1a−/− embryos  formation has been proposed. Such studies show that Foxc1a directly binds to the promoter of nkx2.5 [nkx2.5 in zebrafish foxc1 homozygous mutants provides mechanistic insight into the hypoplastic myocardium observed in ARS patients. Furthermore, genes expressed specifically in the AVC are downregulated in foxc1 homozygous mutants, including notch1b, tbx2b and bmp4, providing additional genetic targets that downstream of foxc1 that may contribute to cardiac defects. While foxc1a is expressed in LPM, the heart also receives a contribution of cells from the neural crest, and thus foxc1 expression in neural crest cells could also influence the presence and severity of cardiac phenotypes observed in ARS patients and zebrafish foxc1 mutants.Utilizing f nkx2.5 , a gene foxc1 function in zebrafish has been undertaken. Combined morpholino inhibition of foxc1a and foxc1b causes cerebral hemorrhages [sox10 positive neural crest cell populations. Furthermore, defects in cerebral angiogenesis [As CSVD in ARS patients increases stroke risk, analysis of cerebral vasculature due to loss of orrhages   with early onset glaucoma (a diagnosis similar to ARS) identified a 748 kb deletion containing a conserved PITX2 regulatory element [pitx2 expression and like mutation of the gene itself, resulted in reduced or shallow anterior chambers. These data clearly demonstrate that mutation of pitx2, or genomic modifications that alter pitx2 expression result in anterior segment defects reminiscent of ARS. Although no direct quantification of optic nerve morphology or RGC number has been undertaken, these data demonstrate that mutant pitx2 zebrafish represent an ideal model for subsequent studies that focus on the mechanism by which glaucoma may result in patients with PITX2-attributable ARS.Like elopment ,33,43  [dkk2 in the anterior segment of the eye [pitx2 mutants. Wnt signaling plays a key role in the development of neural crest cells in part through regulation of foxd3 and sox10 [pitx2 expression in early neural crest cells, and its continued expression in the adult anterior segment [pitx2 is likely required for development and maintenance of the anterior segment in part through a Wnt dependent mechanism.Analysis of genetic targets in the eye demonstrates that Pitx2 deficiency reduces the expression of the Wnt ligands ( wnt10a)  as well  the eye . A role nd sox10 ,62 that  segment , pitx2 ipitx2 depletion demonstrate normal expression of pax6a that is required for anterior segment development [pax6a was observed, it has been shown that pitx2 expression is regulated by Pax6a/b in zebrafish [PAX6/pax6 mutations [foxc1, pitx2 may play an important role in early trophic support of the lens and anterior segment structures early in development.Analysis of additional ocular targets due to elopment . While nebrafish  which mautations . The expfoxc1, disruption of pitx2 using antisense morpholinos or with genome editing induced mutations disrupts pharyngeal arch cartilage and jaw formation. Specific defects were observed in the Meckel’s and ceratohyal cartilages, which were under-developed and positioned abnormally [sox10 positive neural crest cells that line the pharyngeal arches and jaw elements demonstrate reduced cell number [foxc1 and pitx2 depleted zebrafish likely involves overlapping mechanisms of decreased neural crest cell migration and survival.Patients with type I ARS typically have dysmorphic craniofacial features that include maxillary and dental hypoplasia, in addition to the anterior segment ocular phenotypes that define ARS. Similar to normally ,43  [pitx2a and pitx2c) [pitx2a is predominantly expressed in neural crest cells and thus mutations that affect this isoform may result in ocular, craniofacial, and cardiovascular anomalies associated with ARS, while pitx2c is predominately expressed in the lateral plate mesoderm and the heart, which could contribute to cardiac defects observed in some ARS patients. Patients with PITX2-attributable ARS may thus present with or without cardiac defects that may depend in part on the location of the disease-causing mutations [pitx2a) or both isoforms (pitx2a and pitx2c) in order to identify similar defects in fish, and to gain mechanistic understanding of such phenotypes in patients and animal models.Like humans, the zebrafish  PITX2d)  with two pitx2c) . During utations ,68. Mutapitx2c) did not result in cardiac defects consistent with PITX2-attributable ARS in one study, although asymmetric looping and overall morphology were the only phenotypes tested [pitx2c specific mutant strain and uncovered cardiomyopathy with fibrosis (PITX2 as a causative gene for atrial fibrillation [foxc1 regulates expression of nxk2.5 in the lateral plate mesoderm, nkx2.5 is required for maintenance of pitx2 expression in this area [foxc1 and pitx2 in regulating heart development.Mutations in zebrafish that affect both isoforms (or just s tested . A seconfibrosis E–H and aillation ,70. Whilhis area , highligfoxc1 and pitx2 loss of function models provides understanding of the mechanisms that lead to most ARS related phenotypes. Both genes when mutated in zebrafish result in defects consistent with mammalian ARS models and patient phenotypes. Zebrafish ARS mutants have ocular anterior chamber defects, and foxc1 mutants additionally display endophenotypes of glaucoma. Disruption of foxc1 or pitx2 in zebrafish display craniofacial anomalies consistent with ARS, as well as cardiovascular defects that are often observed in patients. pitx2 mutants additionally display tooth hypoplasia that is often observed in Type 1 ARS. Mechanisms involving downstream gene regulation are beginning to be uncovered using homozygous embryos and larvae, however, analysis of adult phenotypes in heterozygous mutants is somewhat lacking in the literature. The continued analysis of such mutants will reveal novel insights into disease mechanisms, and given the utility of zebrafish for translational research, pharmaceutical approaches using high-throughput drug screening for phenotypic rescue provides a path toward testing therapeutic interventions.Analysis of zebrafish


Cytokines were determined by multiplex assays and ELISA, and gene expression by qPCR. MALAT1 loss of function was performed in OA patient osteoblasts using locked nucleic acids. The osteoblast transcriptome was analysed by RNASeq and pathway analysis. Bone expression of MALAT1 positively correlated to serum DKK1 and galectin-1 concentrations, and in OA patient osteoblasts was induced in response to IL-1β stimulation. Osteoblasts depleted of MALAT1 exhibited differential expression (>1.5 fold change) of 155 genes, including PTGS2. Both basal and IL-1β-mediated PGE2 secretion was greater in MALAT1 depleted osteoblasts. The induction of MALAT1 in human OA osteoblasts upon inflammatory challenge and its modulation of PGE2 production suggests that MALAT1 may play a role in regulating inflammation in OA subchondral bone.Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1) is implicated in regulating the inflammatory response and in the pathology of several chronic inflammatory diseases, including osteoarthritis (OA). The purpose of this study was to examine the relationship between OA subchondral bone expression of MALAT1 with parameters of joint health and biomarkers of joint inflammation, and to determine its functional role in human OA osteoblasts. Subchondral bone and blood were collected from hip and knee OA patients ( Osteoarthritis (OA) is a degenerative joint disease and a leading cause of pain and disability for which there are currently no approved pharmacological disease modifying therapeutics . CurrentMALAT1) lncRNA modulates the inflammatory phenotype of synovial fibroblasts in the OA synovial joint lining by mediating the production of CXCL8 [MALAT1 lncRNA has now emerged as a central mediator of osteoblast function and bone homeostasis. Expression of MALAT1 is reported to be greater in the bone tissue of patients who exhibit aseptic loosening following a hip replacement [MALAT1 has been demonstrated to inhibit the proliferation of the human osteoblast cell line hFOB 1.19 [MALAT1 sponging of the microRNA miR-30 has been shown to promote the osteoblast differentiation of mesenchymal stem cells by inducing RUNX2 expression [MALAT1 is associated with abnormal osteogenic and adipogenic differentiation of BMSCs in the patients with osteonecrosis of the femoral head [Recently, long non-coding RNAs (lncRNAs) have emerged as novel epigenetic regulators of gene transcription ,14 and oof CXCL8 . Howeverlacement  and its lacement . In vitrFOB 1.19 , and MALpression . Furtherral head .MALAT1 could play a key role in the pathological changes that occur in OA diseased bone. However, currently the expression and functional role of MALAT1 in OA subchondral bone has not been reported. Therefore, the aim of this study was to profile the expression of MALAT1 in the subchondral bone tissue of patients with either knee or hip OA and its relationship to parameters of joint damage and to examine the functional role of MALAT1 in OA patient primary osteoblasts.These data suggest that n = 6), who were undergoing surgery at Russell’s Hall Hospital  or the Royal Orthopaedic Hospital (Birmingham UK). OA patient characteristics are detailed in Following ethical approval , subchondral bone tissue and blood was collected from a total of 17 patients  with end-stage OA comprising of 9 patients with hip OA and 8 patients with knee OA and from neck of femur fracture (NOF#) patients without OA  to remove excess fat. The bone chips were then placed in a T75 flasks with fresh culture media and incubated in at 37 °C in a humidified atmosphere containing 5% CO2. The media was changed every 3–4 days, and the chips were removed after 7–14 days when the osteoblasts outgrowth occurred.Primary OA osteoblasts were cultured from OA subchondral bone tissue. The bone was cut into small pieces approximately 2 mmThe serum concentration of 24 cytokines were determined using Luminex multiplex platform (Luminex R&D systems) according to the manufacturer’s instructions, having been diluted to 1:2 in assay buffer.MALAT1 or with a control (NC) LNA. To determine the effect of MALAT1 knockdown on the OA osteoblast transcriptome, total RNA was extracted  24 h following transfection with LNAs. RNA integrity (RIN) was evaluated  with a RIN of >7 deemed of sufficient quality for RNA sequencing analysis using the QuantSeq 3′ kit . The sequenced reads were mapped to the hg38 reference human genome using Star Aligner version 2.5.2b [In vitro loss of function studies were performed using lipofectamine 3000  to transfect primary osteoblasts with two different Locked nucleic acids (LNAs) targeting n 2.5.2b . Differen 2.5.2b .MALAT1 knockdown on the production of PGE2 and osteoprotegrin (OPG) was determined by ELISA according to the manufacturer’s instructions . Alkaline phosphatase (ALP) activity was quantified in osteoblast lysates prepared in RIPA buffer diluted 1:5 with 1 mM MgCl2. In brief, diluted osteoblast lysates were combined with ALP substrate  and incubated at 37 °C for 15 min. The reaction was stopped with the addition of 0.1 N NaOH and absorbance read at 405 nm on a microplate reader. The degree of osteoblast mineralisation was quantified by staining of mineralised nodules using an alizarin red solution . Following 10 min incubation at room temperature, cells were washed with PBS to remove excess stain, and then incubated in 10% cetyl pyridinium chloride  for 10 min. The supernatant was then collected, diluted 1:10 with the 10% cetyl pyridinium chloride and absorbance read at OD550 nm on a microplate reader.The effect of www.ingenuity.com, accessed on 31 January 2020). Differentially expressed genes  were analysed using a core functional analysis to identify significant canonical pathways and cellular processes. Pathway analysis was performed using the software Ingenuity Pathway Analysis . However, this did not reach significance and there was no significant difference in expression between patients with knee OA and those with hip OA (MALAT1 expression in OA patients with greater disease severity (KL4 and joint space < 1 mm), compared to those patients with KL grade 3 and joint space > 1 mm, there was no significant relationship between MALAT1 expression and disease severity  and galectin-1   . HoweverMALAT1 was induced upon an inflammatory challenge. To this end, primary osteoblasts from n = 3 OA patients were stimulated for either 6 h or 24 h with IL-1β (1 ng/mL), and the expression of IL-6 and MALAT1 determined by qPCR. As expected, stimulation of osteoblasts with IL-1β induced a significant increase in the expression of the pro-inflammatory cytokine IL-6 within 24 h , compared to non-stimulated control. Similarly, osteoblasts stimulated with IL-1β for 24 h exhibited significant increase in the expression of MALAT1 , compared to non-stimulated control  and examined the resulting osteoblast transcriptome by RNA sequencing. Primary OA osteoblasts (n = 3 OA patients) were transfected with either a non-targeting control LNA or one of two LNA duplexes targeting MALAT1. Following 24 h transfection, LNAs targeting MALAT1 induced knockdown of between 60–90% in the expression of MALAT1, compared to the control LNA  in osteoblasts transfected with the MALAT1 LNAs, compared to LNA control transfected cells. Of the upregulated transcripts, 80 were protein coding genes (including PTGS2) and 2 were antisense lncRNAs. Of the down-regulated transcripts, 66 were protein-coding genes and 7 were lncRNAs, which comprised of 2 lincRNAs, 3 antisense, 1 sense intronic lncRNA and 1 pseudogene  to identify significantly affected canonical pathways and cellular processes. The most significant canonical pathways affected included phosphatidylcholine biosynthesis, fMLP signalling in neutrophils, NAD biosynthesis, eicosanoid biosynthesis and prostanoid biosynthesis. The significantly affected cellular processes included cell–cell signalling, DNA replication, cellular growth and proliferation and cellular development  or left unstimulated and secretion of PGE2 quantified by ELISA. MALAT1 knockdown was confirmed by qPCR  (Given the finding that knockdown of  by qPCR A. Compar < 0.05) B.MALAT1 would affect the innate function of osteoblasts with regard to bone remodelling by determining OPG production, alkaline phosphatase activity and osteoblast mineralisation. To this end, OA osteoblasts were transfected with either a control LNA or a MALAT1 LNA twice per week for a period of 3 weeks. During the 3-week time course, supernatants were collected to analyse OPG production by ELISA and cells lysed to measure alkaline phosphatase activity. At the end of the 3 weeks, cells were stained with alizarin red to quantify mineralisation. Twice weekly transfection with LNA maintained the significant knockdown in MALAT1 expression of between 65–85%  in the subchondral bone tissue of both knee and hip OA patients irrespective of disease severity. On average MALAT1 expression was greater in the bone tissue from patients with greater OA severity, as measured by either KL grade or joint space narrowing. However, it should be noted that all the OA patients in this study had advanced end-stage disease, with radiographic severity determined to be either KL3 or KL4, and most patients having <1 mm joint space. Therefore, we cannot comment on whether expression of MALAT1 would differ in the subchondral bone of patients with early OA. This paper reports the expression and functional role of the long non-coding RNA MALAT1 in OA synovial fibroblasts [MALAT1 expression in OA osteoblasts was induced during the IL-1β inflammatory response. This suggests MALAT1 may play an important regulatory role in bone homeostasis under the inflammatory conditions exhibited in OA patients. Of note, although the subchondral bone expression of MALAT1 was not related to the serum concentration of IL-1β we did observe positive correlations between MALAT1 expression and the serum concentration of both DKK1 and galectin 1. DKK1 is an endogenous inhibitor of the Wnt/beta-catenin signalling pathway and is implicated in bone development and in the pathological remodelling of bone in both OA and osteoporosis and mediating inflammation-induced bone loss by inhibiting osteoblast differentiation [Similar to previous findings on the role of roblasts , MALAT1 ntiation ,31. In ontiation . In contntiation , and in ntiation . MALAT1 in primary human OA osteoblasts profoundly affected the transcriptomic phenotype with pathway analysis revealing significant activation of pathways that promote the production of inflammatory prostacyclins and eicosanoids. Amongst the most differentially expressed upregulated genes were TNFSF12 (>11-fold upregulated), which encodes TWEAK (TNF-related weak inducer of apoptosis). TWEAK is a known mediator of inflammatory bone remodelling [MALAT1 exhibited a significant >10-fold upregulation in the expression of PTGS2, the gene which encodes for the enzyme COX2 that mediates the production of inflammatory prostaglandins including the putative OA pain mediator PGE2 [MALAT1 compared to control cells. PGE2 acting through the E prostanoid receptors EP2 and EP4 sensitizes nociceptors, possibly by acting synergistically with IL1β to induce IL-6 and iNOS expression [MALAT1 expression in OA osteoblasts regulates PTGS2 expression as well as both basal and IL-1β induced PGE2 production suggests that MALAT1 may play an important role in regulating inflammatory pain in the bone.LNA-mediated knockdown of odelling , and tarodelling . In additor PGE2 ,38. Indepression  and selepression . The subpression  and bonepression . TherefoMALAT1 knockdown on the osteoblast transcriptome and the acute effect on PGE2 production we did not observe any chronic effect of MALAT1 knockdown on several key osteoblast functions. During a time-period of 3 weeks, we induced sustained MALAT1 knockdown but did not observe any significant difference in the secretion of OPG or in the activity of ALP and ability of the osteoblast to form mineralised bone nodules. This contrasts with previous publications which have implicated MALAT1 in mediating both OPG production in osteoblasts [Despite the effect of eoblasts  as well eoblasts .MALAT1 have been reported to exert a pro-osteogenic function by acting as miRNA sponges [MALAT1 in mediating osteoblast differentiation has been linked to the sponging of several miRNAs including miR-204 [MALAT1 depletion, we did not identify any miRNAs which were altered upon MALAT1 KD. However, the isolation of total RNA using columns would likely have excluded many miRNAs from our sequencing analysis.The underlining molecular mechanisms by which lncRNAs mediate their function is complex and for the majority of lncRNAs remains to be determined. Several lncRNAs, including  sponges . Indeed, miR-204 , miR-30  miR-204  and miR- miR-204 . In our MALTA1 in OA subchondral bone, its induction in osteoblasts upon inflammatory challenge and its functional role in modulating the production of the prostaglandin PGE2 suggest that MALAT1 may play an important role in the development of OA bone pain and inflammation.In conclusion, the expression of


Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal  Porphyromonas gingivalis, induces chronic infection of gingiva and tooth loss, and also enhances the risk of cardiovascular diseases such as heart attack [Approximately 10–15% of adults suffer from periodontal disease with dental bone loss leading to tooth loss in patients and affecting their quality of life . Publisht attack . Additiot attack . Other st attack ,8. ThereThere have been several reported causes of dental bone loss in periodontal disease which increase the risk of congenital disease. For example, hormone or endocrine disorders might inhibit the repair of collagen fiber in the gingival tissue and cause alveolar bone loss . UnhealtPorphyromonas gingivalis, were injected to create inflammation. In addition, alcohol administration through oral exposure of 10% alcohol in drinking water was provided to observe the effects of dental bony defects in mice. In this study, we demonstrated that under LPS-induced toxicity and alcohol exposure, deficiency in the ALDH2 enzyme played a significant role in dental bone loss. The results from this study may provide important clinical information and a potential therapeutic target for the treatment or prevention of dental bone loss in periodontal disease, especially for the large population of East Asians carrying the vulnerable ALDH2*2 genotype.The role of genetic factors in alcohol-induced dental bone loss has not been explored thus far. The polymorphism of a single amino acid substitution which leads to the loss of enzyme function in the mitochondrial aldehyde dehydrogenase 2 (ALDH2) gene is well known . FollowiThe shRNA sequence of ALDH2 was obtained from the RNAi database . The ALDH2 shRNA vector was purchased from Academia Sinica. Osteoblasts expressing silenced human ALDH2 were developed by transfecting cells with lentivirus carrying the ALDH2 shRNA. The targeted ALDH2 sequence was GCAGATCATTCCGTGGAATTT.2. Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS), 100 unit/mL penicillin +100 μg/mL streptomycin , and 2 mM L-glutamine  were used for culture. Cells were grown to 80% confluence, and then passaged. Osteoblasts expressing silenced human ALDH2 were selected by transfecting cells with lentivirus  carrying the ALDH2 shRNA (multiplicity of infection (MOI) = 10) vector. The MOI is defined as the ratio of infectious agents to infection targets. It is also the ratio of viral particles to the number of target cells in a defined space, such as in a cell culture well. The cell line, designated as ALDH2 knockdown osteoblasts were confirmed with low ALDH2 expression by Western blot analysis using the ALDH2 antibody , Abcam, Cambridge, MA, USA). Osteoblasts transfected with an empty lentivirus were used as the control. Cells at 3–4 passages were used for the MTT assay.The human osteoblast (transfected with SV40 large T antigen) cell line (hFOB 1.19) was obtained from Bioresource Collection and Research Center . Cells were cultured at 37 °C in a humidified incubator with 5% CO4 cells per well in a 96-well microplate (n = 8) in the DMEM culture medium for 7 and 14 days. On Day 7 and Day 14, MTT was added to each culture well at a final concentration of 2 mg/mL followed by an incubation at 37 °C for 4 h. Then, the medium was harvested and centrifuged at 900 rpm for 5 min and aspirated. The pelleted formazan reaction products were dissolved in 200 μL of dimehtyl sulfoxide  solution and shaken for 15 min. The optical density of the formazan solution was recorded using an enzyme-linked immunosorbent assay (ELISA) plate reader  at 570 nm. The absorbance was proportional to the metabolic activities and viabilities of the cells.Cell viabilities of both control and ALDH2 knockdown transfected osteoblasts were analyzed by using the assay of 3--2,5-diphenyl tetrazolium bromide  . Both pa3 cells/mL and high, 1 × 105 cells/mL). The degree of mineralization was observed by phase contrast microscopy from the independent culture wells of the control and ALDH2 knockdown groups.In this assay , both coPorphyromonas gingivalis  under anesthesia and manipulated under a microscopy. The injection was performed twice a week and continued for 6 weeks. The corresponding right side of the upper 1st molar and 2nd molar interdental alveolar bone areas and between the 2nd and 3rd molar interdental areas of all mice were injected with 2 μL vehicle (endotoxin-free water), twice a week, and continued for 6 weeks as the control. Drinking water with 10% alcohol and standard dry food were administered to all mice. After 6 weeks, all mice were sacrificed for evaluation by micro-CT and histological observation with hematoxylin and eosin staining.The animal use was approved and followed the guidelines of the Laboratory Animal Center at the National Taiwan University College of Medicine, Taiwan. The ALDH2*2 knockin mice representing the ALDH2*2 single amino acid substitution of human ALDH2*2 were generated and obtained from Prof. Daria Mochly-Rosen’s lab, Stanford University. The construction, characterization, and phenotype of the ALDH2*2 mice were as described previously . Four C5-, d1 site) was designated as 100% for comparisons with other distance measurements of bone loss. The percentage of bone loss in the wild-type group of the vehicle injected into the right side of the upper 2nd and 3rd molar interdental areas  was designated as 100% for comparisons with other distance measurements. In addition, 2 μL (20 μg) of purified lipopolysaccharide from Porphyromonas gingivalis was injected into the corresponding left side of the upper 1st and 2nd molar interdental areas  and the 2nd and 3rd molar interdental areas  as the treatment groups.Micro-CT imaging protocol was as described in our previous study . To obta-, d1 site. The percentage of bone loss of the right upper jaws between the 2nd and 3rd molar interdental areas of the vehicle-injected ALDH2*2 knockin mice was designated as the ALDH2 LPS-, d2 site group. In addition, the percentage of bone loss of the left upper jaws between the 1st and 2nd molars, and between the 2nd and 3rd molar interdental alveolar areas of the ALDH2*2 knockin mice injected with LPS were designated as the ALDH2 LPS+, d1 site group and ALDH2 LPS+, d2 site group, respectively. Three-dimensional (3D) reconstruction of micro-CT image was used to compare bone loss in the upper jaw of the areas defined above in each animal.Similarly, the percentage of bone loss of the right upper jaws between the 1st molar and 2nd molar interdental alveolar areas of vehicle-injected ALDH2*2 knockin mice was designated as the ALDH2 LPSAfter the examination of micro-CT, all samples were demineralized using 15% EDTA for 4 weeks and embedded with paraffin. The samples were sectioned horizontally (5 μm thickness), perpendicular to the long axis of the teeth, and stained with hematoxylin and eosin for histological observation. The sections were screened from the cemental enamel junction to the apical area vertically and the top first section with bony structure. The expression of osteoblasts in the area of interest of each mouse was analyzed and the total number of osteoblasts was counted.+, WT LPS-, ALDH2 LPS+, and ALDH2 LPS-), were counted three times under a light microscope  and quantified. Comparison of the average total number of osteoblasts in the ROI areas were calculated by using ImageJ software and presented as the mean ± standard deviation (SD). Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by post hoc procedure  (p < 0.05 was considered to be significant)The interdental areas between 1st and 2nd molar and interdental areas between the 2nd and 3rd molar in the right upper jaws and left upper jaws were defined as the region of interest (ROI) areas of each animal. The average total number of osteoblasts in the top section with bony structure in the ROI areas of each mice of the four different groups . Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by post hoc procedure  (p < 0.05 was considered to be significant).For in vitro measurement of ALDH2 expression, the results were expressed as means ± standard error from at least three separate experiments. The statistical analyses were determined by Student’s t-test, To evaluate the effect of human osteoblasts that were transfected by lentivirus carrying the human ALDH2-silencing shRNA, ALDH2 protein expression was analyzed by Western blot using the ALDH2 antibody. Compared with the control osteoblasts transfected with an empty lentivirus, ALDH2 protein expression in the ALDH2 knockdown transfected osteoblasts was reduced  staining was used to quantify the mineralization abilities of the osteoblasts on Day 7 and on Day 14 after transfection using the matrix calcification index, as described by Ratisoontorn et al. . On Day Porphyromonas gingivalis LPS-induced dental bone loss by localized injection of purified LPS (20 ug in 2 uL of endotoxin-free water) to mimic the bacterial infection and inflammation of the upper jaw interdental alveolar bone area on the left sides of the region of interest (ROI) areas in all mice. In addition, drinking water with 10% alcohol was provided every day continuously to mimic the effects of chronic alcohol drinking. We compared the percentage of bone loss in the left side of the upper jaws between the 1st and 2nd, and between the 2nd and 3rd molar interdental areas between the wild-type mice and the ALDH2*2 mice injected with 20 μg (2 μL in endotoxin-free water) of purified lipopolysaccharide from Porphyromonas gingivalis and the corresponding animal groups injected with the vehicle control (2 μL of endotoxin-free water). Three-dimensional (3D) reconstruction of micro-computed tomography (micro-CT) image was used to compare bone loss in the upper jaw of the animals. +), the water-injected wild-type group (WT LPS-), the ALDH2*2 knockin LPS-injected group (ALDH2 LPS+), and the ALDH2*2 knockin water-injected control group (ALDH2 LPS-). There are three upper molar teeth on each side of the upper jaw of each mouse. In -, d1 site) was calculated as 100%. The distances were measured and calculated in all other groups and compared with the wild-type control group, as shown in +) or without LPS injection (WT LPS-) (-) showed similar bone loss at the d1 and d2 sites as the wild-type mice with LPS (WT LPS+) or without LPS injection (WT LPS-) (+) showed 30% higher bony loss at the d1 site and 30% higher bony loss at the d2 site than the corresponding sites of the wild-type animals (WT LPS- and WT LPS+) and those of the ALDH2*2 knockin mice without LPS injection (ALDH2 LPS-) (- and WT LPS+) and those of the ALDH2*2 knockin mice without LPS injection (ALDH2 LPS-) (+) > (ALDH2 LPS-) = (WT LPS+) = (WT LPS-).We used a genetic model of ALDH2*2 knockin mice to evaluate the effect of periodontal bone defect in vivo with age-matched wild-type mice as a control. The ALDH2*2 knockin mice mimics the ALDH2*2 single amino acid substitution of ALDH2*2 mutation in humans . We alsoWT LPS-) . The bonWT LPS-) . HoweverH2 LPS-) . Since dH2 LPS-) . In summTo support our findings, a histological analysis of hematoxylin and eosin (H&E) stained paraffin sections of the upper jaws of all mice was also carried out. We counted the total numbers of osteoblasts in the region of interest (ROI) areas of all specimens from both sides of the upper jaws of the four different animal groups.- and WT LPS+, - and ALDH2 LPS+, +) was significantly diminished as compared with the ALDH2*2 mice without LPS injection  was also reduced as compared with the group without LPS injection  was reduced by 17% as compared with the non-treated group . In the ALDH2*2 knockin mice, the total number of the osteoblasts in the LPS treated group (ALDH2 LPS+) was reduced by 21% as compared with the untreated group . Comparing the effects of LPS injection in the wild-type mice and ALDH2*2 knockin mice, the total number of osteoblasts in the ALDH2*2 knockin mice (ALDH2 LPS+) was only about 80% of that of the wild-type mice  indicating that the ALDH2*2 mutation rendered the mice more susceptible to the Porphyromonas gingivalis LPS toxin. Comparing the wild-type mice and the ALDH2*2 knockin mice without LPS injection, we found that the total number of osteoblasts in the ALDH2*2 knockin mice (ALDH2 LPS-) was only about 72% as compared with that of the wild-type mice (WT LPS-). This indicated that alcohol intake alone, without LPS injection, was able to inhibit bone regeneration potential with the ALDH2*2 genotype, although we did not observe this difference using the technique of micro-CT imaging (+). In this group, the number of osteoblasts was reduced by 33.3% as compared with the wild-type mice that did not receive the treatment of LPS toxin . These findings indicated that under alcohol intake, the ALDH2 mutation by itself may reduce the growth of the osteoblasts and injection of Porphyromonas gingivalis LPS toxin further exacerbated the growth of osteoblasts in the ALDH2*2 knockin mice. imaging . As expeA previous study has revealed a significant association between the ALDH2 Glu504Lys (or ALDH2*2) polymorphism and osteoporosis . The prePorphyromonas gingivalis lipopolysaccharides for 6 weeks under the exposure of 10% alcohol in drinking water. Excess alcohol drinking has been shown to directly cause systemic diseases. Many studies have also indicated that alcohol consumption is a risk factor for periodontitis [Porphyromonas gingivalis [Porphyromonas gingivalis is a well-known periodontal pathogen and is one of the predominant species in the gingival pockets of patients with advanced and severe periodontal disease [Porphyromonas gingivalis has been demonstrated to be able to cause bone resorption and inhibit bone formation as a culprit of chronic periodontitis [Porphyromonas gingivalis to create periodontal inflammation and bony defects. Previous studies have shown that LPS from Porphyromonas gingivalis prevented apoptosis of HL60-derived neutrophils and the signaling of P. gingivalis through toll-like receptor 2 (TLR2) and may account for the inhibitory effect of P. gingivalis LPS on apoptosis, thus, provided a mechanism for the development of destructive periodontal disease [P. gingivalis LPS caused a more severe periodontitis in mice [P. gingivalis LPS could activate the p38 MAPK and NF-κB signaling pathways and induce periodontitis and gingival bone resorption [P. gingivalis LPS is a strong inducer of oral bone resorption and periodontal diseases. In our study, we found that P. gingivalis LPS-injected ALDH2*2 knockin mice had a significantly higher percentage of bone loss as compared with the LPS-injected wild-type mice and both ALDH2*2 knockin and wild-type mice without LPS injection based on micro-CT scan  > (ALDH2 LPS-) > (WT LPS+) > (WT LPS-)  . We noteWe believe that these results are important for periodontitis and warrant confirmation by epidemiological and clinical studies in human populations where ALDH2*2 mutation, alcohol drinking, and poor oral hygiene are common. Such confirmation would benefit future development of precision dental care and treatment or prevention of dental bone loss among East Asians with the ALDH2*2 genotype.Porphyromonas gingivalis LPS-induced toxicity in dental bone loss. The ALDH2 function was suppressed by silencing its gene activity in human osteoblasts. After transfecting cells with a lentivirus vector expressing ALDH2 shRNA, ALDH2 protein level was reduced by 80% as compared with the non-transfected osteoblasts. The ALDH2 knockdown osteoblasts lost their proliferation capability and, concomitantly, had reduced mineralization potential. Using a genetic animal model of ALDH2, we compared dental bone loss in wild-type and ALDH2*2 knockin mice with the injection of purified Porphyromonas gingivalis LPS and the administration of 10% alcohol in drinking water for 6 weeks. The micro-CT scan results indicated that dental bone loss was significantly more serious in the ALDH2*2 knockin mice injected with LPS. We also found that alcohol alone is a risk factor for bony defects in animals carrying the ALDH2*2 mutation based on the histology of stained bony samples, and the ALDH2 mutation together with LPS injection exacerbated the extend bony defects. These results suggest that the genetic factor of ALDH2 mutation interacts with lifestyle factors, such as alcohol consumption and poor oral hygiene such as the oral toxicity of bacterial-induced Porphyromonas gingivalis LPS, and therefore plays a crucial role in the pathology of dental bone loss in periodontal disease. Our findings may have important clinical implications for the dental health of 560 million East Asians carrying the ALDH2*2 mutation.In this study, we investigated the roles of ALDH2, alcohol, and


The graph labels for placebo and hydroxychloroquine were mistakenly transposed, the hazard ratio for lopinavir and ritonavir should have been 1.16 instead of 1.17, and the Population section should not have mentioned expected hospital stays. This article has been corrected.1In the Original Investigation titled “Effect of Early Treatment With Hydroxychloroquine or Lopinavir and Ritonavir on Risk of Hospitalization Among Patients With COVID-19: The TOGETHER Randomized Clinical Trial,”


The World Health Organization has declared SARS-CoV-2 COVID-19) a pandemic in March 11, 2020 9 a pande during tFourteen children who were victims of sexual abuse consulted the pediatric medical emergency department; this number is 2.3 times the number who consulted during the same period in 2019. Although the rate is increased by 2.3, it certainly remains underestimated due to the difficulties by families of accessing hospitals and social structures [


Triglycerides (TGs) are transported in the bloodstream by TG-rich lipoproteins in the form of chylomicrons and VLDLs. The hydrolysis of circulating TGs is rate-limiting for their uptake into tissues and is catalyzed by the enzyme lipoprotein lipase (LPL) . The actet al. (APOAI/CIII/AIV gene cluster, while van der Vliet identified APOA5 as the most highly induced gene after partial hepatectomy (APOA5 variants are associated with elevated plasma TG levels (Of this list of proteins, the most elusive LPL regulator is probably APOA5. APOA5 was discovered independently by two groups in 2001. Pennacchio atectomy . Targeteatectomy . The rolHow APOA5 lowers plasma TGs has remained controversial. A report suggested that APOA5 reduces VLDL-TG production . Alternaet al. (Here, inspiration may be taken from the angiopoietin-like proteins ANGPTL3, ANGPTL4, and ANGPTL8. These proteins function as inhibitors of LPL and are present in plasma at concentrations that resemble the plasma APOA5 concentration. Although ANGPTLs were initially believed to operate individually, recent findings indicate that ANGPTL3 and ANGPTL4 form complexes with ANGPTL8 , 11, 12.et al.  now findet al. stumbled upon APOA5 when trying to identify proteins in human serum that associate with immobilized ANGPTL3/8. They also succeeded in immunoprecipitating APOA5 from human serum when using an antibody directed against the ANGPTL3/ANGPTL8 complex. Subsequent biolayer interferometry experiments showed that recombinant APOA5 is able to efficiently bind to the ANGPTL3/8 complex. At the functional level, recombinant APOA5 impaired the ability of the ANGPTL3/ANGPTL8 complex to inhibit LPL. By contrast, APOA5 did not influence LPL inhibition by ANGPTL3 and ANGPTL4 alone or in the absence of any angiopoietin-like proteins. Answering to the original conundrum about the low plasma APOA5 concentration, it was found that the suppression of ANGPTL3/8-mediated LPL inhibition by APOA5 occurred at a molar ratio consistent with the plasma concentrations of APOA5 and ANGPTL3/8.Chen After 15 years of uncertainty, these new biochemical data represent a potential breakthrough in our molecular understanding of how APOA5 lowers plasma TGs. Whether APOA5 also interferes with the function of ANGPTL3/ANGPTL8 in vivo is still unknown. Accordingly, it would be of interest to investigate the effect of overexpression of APOA5 or injection of recombinant APOA5 on postprandial plasma TGs or on plasma TG clearance in mice deficient in ANGPTL3 or ANGPTL8.et al., another therapeutic approach could be to mimic the binding of APOA5 to ANGPTL3/ANGPTL8, thereby relieving LPL inhibition by ANGPTL3/ANGPTL8. To enable the design of such a strategy, better insight is needed into the specific domains in APOA5 and ANGPTL3/ANGPTL8 involved in their mutual interaction.This exhilarating new discovery might create potential novel opportunities for the therapeutic targeting of hyperlipidemia, an important risk factor for atherosclerotic cardiovascular disease. Currently, ANGPTL3 is a hot target for the treatment of atherosclerotic cardiovascular disease. Inactivation of ANGPTL3 via antisense oligonucleotides and monoclonal antibodies effectively lowers plasma TGs and LDL-C in mouse models and in human volunteers , 16. BasANGPTLs and APOA5 have followed a very different path since they were first cloned about twenty years. Although the importance of APOA5 in regulating plasma TG levels quickly gained ground, the recognition of the role of ANGPTLs in controlling human plasma lipoproteins took a much more gradual course. Their mutual interaction adds an exciting new twist to a now rapidly evolving field.


The present study investigated the joint impact of adolescent sport experience and dopamine‐related genes  on sport participation in adulthood.Using the National Longitudinal Study of Adolescent Health  data, the hierarchical multivariable logistic regression models for predicting sport participation in wave 3 (around 20 years of age) and wave 4 (around 30 years of age) were conducted separately by gender  and gene (DRD2 and COMT genes).Adolescent sport experience significantly interacted with the number of DRD2 A1 alleles and COMT Met alleles in affecting wave 3 sport participation among male adults. The interaction between adolescent sport experience and DRD2 gene significantly affected wave 4 sport participation in opposite direction to that affected wave 3 sport participation among male participants. Among female participants, there were no significant interaction effects between dopamine‐related genes and adolescent sport experience on sport participation in both wave 3 and 4.Since adult sport participation is most likely to be influenced by the joint impact of environmental and genetic factors, it is important to consider gene‐by‐environment interactions when designing policies or programs to promote adult sport participation. The significant effects of the number of DRD2 A1 alleles on wave 3 sport participation became insignificant if male participants actively participated in sports in adolescence, indicating that adolescent sport experience may overpower the effect of DRD2 genes on sport participation in adults aged around 20 years. Sports participation invariably involves physical activity and inherently includes various enjoyable aspects, such as personal challenge, social interaction, goal achievement, and competition. The International Society for Physical Activity and Health also suggests that sports participation is “an investment that works” to increase physical activity and improve health . The wave 2 follow‐up survey was conducted 1 year after wave 1 (89% response rate). The wave 3 (77% response rate) and wave 4 (80% response rate) follow‐up surveys were conducted 6 and 13 years after wave 1, respectively. More information about the survey design of Add Health can be found elsewhere  or strenuous team sports  in waves 3 and 4. Respondents who participated in individual or team sports more than 4 days per week in each wave were categorized as active participators.Using a salivary DNA collection device , participants provided 2 ml of saliva in wave 4. In order to extract DNA, saliva was packed in the container  and shipped to the University of Colorado, Institute for Behavioural Genetics. Nine (0.06%) were empty and 24 (0.15%) were damaged or leaking during shipping. Further information about Add Health wave 4 candidate gene data is reported in more detail elsewhere  were genotyped on either an Applied Biosystems TaqMan® OpenArray® (archived samples) or an Illumina BeadXpress® GoldenGate® (non‐archived samples) platform for both DRD2 Taq1A SNP rs1800497 in the 30 UTR and COMT val158met SNP rs4680. The DRD2 Taq1A (rs1800497) assay and COMT val158met (rs4680) assay were carried out using a fluorogenic 5′nuclease  method , education . Since addictive behaviors have been shown to be associated with DRD2 and COMT genes, smoking (current smoker), binge drinking (5 or more drinks in a row), and marijuana use (1 or more times) were also used as a covariate when predicting sport participation in adulthood.2.3p‐values lower than .05 were considered as significant. Analysis of variance (ANOVA) and Chi‐square test were used to compare between active and inactive participators in sports. All the analyses were performed using SAS version 9.4 .The hierarchical multivariable logistic regression models for predicting sport participation in wave 3 (around 20 years of age) and wave 4 (around 30 years of age) were conducted separately by gender  and gene (DRD2 and COMT genes). In model 1, the effect of gene on adult sport participation was examined after controlling for age, education, and addictive behaviors. In model 2, adolescent sport experience was included in model 1 to see if there is any change in the effect of gene on sport participation. In model 3, the interaction between adolescent sport experience and gene was included in model 2 to examine the joint impact of adolescent sport experience and gene on adult sport participation. Wave 3 Sport participation was included as a covariate in all models predicting wave 4 sport participation. Other covariates  were assessed at the same wave as dependent variables. Deviance statistics (‐2 log likelihood) were used to compare nested models. The 33.1Table The characteristics of male and female participants by sport participation in wave 4 are presented in Table 3.2The results of multivariable logistic regression for predicting sport participation in wave 3 (around 20 years of age) are presented in Table Table 4To our knowledge, the present study is the first study that investigated the joint impact of adolescent sports experience and dopamine‐related genes  on sport participation in adulthood. The results of the present study showed that adolescent sport experience is strongly associated with sport participation not only among adults aged around 20 years but also among adults around 30 years of age. This result is in line with previous findings showing that participating in sports during adolescence increases the probability of sports participation in adulthood (Batista et al., The present study found out that adolescent sport experience interacts with the number of DRD2 A1 alleles and COMT Met alleles in affecting sport participation among male adults around 20 years of age. As shown in Figures Another interesting finding in this study was that the interaction between adolescent sport experience and DRD2 gene affected wave 4 sport participation in the opposite direction to that affected wave 3 sport participation among male participants. The number of DRD2 A1 alleles did not affect wave 4 sport participation among male participants who were not active participators in sports in adolescence (see Figure Among female participants, there were no significant interaction effects between dopamine‐related genes and adolescent sport experience on sport participation in both waves 3 and 4. Previous studies have shown that women are more susceptible to socio‐cultural norms and perceptions compared to men because their social relationships are characterized by emotional support, self‐disclosure, and intimacy Maccoby, . Their hThe present study has several limitations. First, more detailed information on sport participation is needed because there exist many different types of sporting activity. In order to understand the influence of dopamine‐related genes on sport participation in more detail, future studies should at least distinguish team sport participation from individual sport participation. Second, since this study used self‐reported sport participation, there is a risk of recall bias. Third, since Add Health is a prospective longitudinal study that followed up a nationally representative sample of middle and high school students in the United States, the results of this study may not be generalizable to other countries. Despite these limitations, the present study may contribute to the literature by providing new information on the joint impact of adolescent sport experience and dopamine‐related genes on adult sport participation. Our results suggest that adolescent sport experience may overpower the effect of DRD2 and COMT genes on sport participation in male adults aged around 20 years. However, our results also indicate that adolescent sport experience may act as a trigger for increasing sport participation in male adults around 30 years of age, who carry the DRD2 A1 allele. Since adult sport participation is most likely to be influenced by the joint impact of environmental and genetic factors, it is important to consider gene‐by‐environment interactions when designing policies or programs to promote adult sport participation.https://publons.com/publon/10.1002/brb3.2459The peer review history for this article is available at


Glioblastoma (GBM) is an extremely heterogeneous tumor and its different regions are populated with phenotypically distinct types of cancer cells. However, it is still unclear how multiple GBM populations arise from the originally homogenous group of tumor precursor cells. Here we showed that GBM cells from the core and edge of the tumor have different composition of ribosomes due to the alternative RNA splicing of multiple ribosomal genes with highest differences observed for RPL22L1. We found that cells at the edge of the tumor express classical isoform of RPL22L1 (RPL22L1a) while core cells have a novel RPL22L1b isoform. RPL22L1b appears due to low pH condition at the core of the tumor. It allows cells to survive during acidosis, promotes more aggressive phenotype in vivo and correlate with worse patient outcome. Mechanistically, RPL22L1b binds to lncRNA MALAT1 in the nucleus and induces its degradation enhancing stemness of GBM cells. On the other hand, RPL22L1a interacts with ribosomes in cytoplasm and upregulates p53 translation favoring less aggressive edge phenotype of GBM. The splicing switch between RPL22L1 isoforms is regulated by SRSF4 proteins. We identified a small molecule compound that inhibits SRSF4 and impairs splicing of RPL22L1, inducing apoptosis of GBM cells and decreasing tumor growth in vivo. Altogether, our data unraveled the mechanism by which less aggressive edge-like GBM cells acquire more malignant core-like phenotype during tumor growth. It may also explain discrepancies between proteome and transcriptome of GBM cell populations. Targeting this pathway may help to decrease tumor heterogeneity and eliminate therapy resistant cells at the tumor core.Work was supported by the Russian Science Foundation grant 19-44-02027.


Saccharomyces cerevisiae have utilized haploid cells. In this study, diploid cells were used in the hope of identifying the distinct genetic mechanisms used by diploid cells to acquire drug resistance. While haploid cells acquire resistance to 2-deoxyglucose primarily through recessive alleles in specific genes, diploid cells acquire resistance through dominant alleles, haploinsufficiency, gene duplication and aneuploidy. Dominant-acting, missense alleles in all three subunits of yeast AMP-activated protein kinase confer resistance to 2-deoxyglucose. Dominant-acting, nonsense alleles in the REG1 gene, which encodes a negative regulator of AMP-activated protein kinase, confer 2-deoxyglucose resistance through haploinsufficiency. Most of the resistant strains isolated in this study achieved resistance through aneuploidy. Cells with a monosomy of chromosome 4 are resistant to 2-deoxyglucose. While this genetic strategy comes with a severe fitness cost, it has the advantage of being readily reversible when 2-deoxyglucose selection is lifted. Increased expression of the two DOG phosphatase genes on chromosome 8 confers resistance and was achieved through trisomies and tetrasomies of that chromosome. Finally, resistance was also mediated by increased expression of hexose transporters, achieved by duplication of a 117 kb region of chromosome 4 that included the HXT3, HXT6 and HXT7 genes. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts.Previous studies of adaptation to the glucose analog, 2-deoxyglucose, by  Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. In an earlier study, we examined the genetic mechanism used by haploid yeast cells to acquire resistance to 2-deoxyglucose. Here, we conducted the same genetic selection on diploid cells. Diploid cells use dominant alleles, haploinsufficiency, gene duplication and aneuploidy to achieve drug resistance. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts. Saccharomyces cerevisiae has been used as a model eukaryotic organism for genetic studies due to its many properties that are advantageous to laboratory research. These advantages include its short generation time, a small and well-annotated genome, robust recombination systems and its ability to stably grow in either haploid or diploid states [The yeast d states . Most geREG1 and duplication of a large segment on chromosome 4 that increased expression of three hexose transporters genes. While many of the genetic mechanisms for the acquisition of 2DG-resistance were utilized by both haploid and diploid cells, the use of a monosomy and haploinsufficiency are options only possible in diploid cells. As has been suggested for other traits in yeast [Genetic studies of the yeast cell’s adaptation and response to the metabolic inhibitor 2-deoxyglucose (2DG) have identified many of the genes required for the maintenance of and release from glucose repression . All of in yeast –9, these7 cells were spread onto agar plates containing synthetic complete media with 2% glucose and supplemented with 0.1% 2DG. Plates were incubated for 4–6 days until colonies appeared. The frequency at which spontaneous 2DG-resistant colonies appeared was approximately 4–6 colonies per 107 cells. These 2DG-resistant candidates were propagated in synthetic complete medium, and cultures were preserved in glycerol stocks at -80°C. Initial characterization of the 2DG-resistant isolates was conducted by examining growth properties of the candidates on agar plates in the absence or presence of 0.1% 2DG. Candidates which failed to retain significant resistance to 2DG were discarded. Nine of the isolates exhibited normal growth properties and stable resistance to 2DG. Seven of the 2DG-resistant diploid isolates formed small colonies when grown on agar plates lacking 2DG  of yeast AMP-activated protein kinase . One strain (DS9) contained both an aneuploid state (monosomy of chromosome 4 and trisomy of chromosome 2) as well as a 117 kb duplication of a region of the chromosome 4  in DS21 and in the beta subunit gene (GAL83) in DS16  and previously as the glycogen binding domain (GBD). This domain is conserved among all AMPK beta subunits from yeast to mammals [SIP1 and SIP2 genes  demonstrate even greater 2DG resistance but this comes with a heavy fitness cost [REG1 locus confers 2DG resistance.Two of the 2DG resistant diploid strains were heterozygous at the one copy . In DS15 alleles . To testn assays  and 2DG n assays . Cells h p<0.01; . HomozygSeven of the 2DG-resistant diploid strains contained a monosomy of chromosome 4 . These iExamination of the diploid strains exhibiting aneuploidy revealed that six of the 2DG resistant strains  shared the trait of having extra copies of chromosome 8 . In an eDOG1 and DOG2 genes are adjacent to each other and are located on chromosome 8. We next analyzed the diploid strain DS14 which contained a chromosome 1 monosomy and chromosome 8 tetrasomy. DS14 was subjected to sporulation and the viable progeny (haploid spores lacking chromosome 1 are not viable) were sequenced and used to generate diploid strains with 2, 3 or 4 copies of chromosome 8. Chromosome copy number was confirmed by whole genome sequencing and comparison of the normalized median read depth for each chromosome (2n) and the strain with chromosome 8 tetrasomy. The dose dependence of DOG gene copy number and 2DG resistance was further confirmed with haploid strains with 0, 1, 2 or more copies of the DOG1 and DOG2 genes  also located on this chromosome. Increased expression of HXT1 and plasma membrane retention [HXT4, HXT1 and HXT5 genes on one copy of the disomic chromosome 8 with the HIS3 gene to generate the hxt415Δ::HIS3 allele. Finally, as a negative control we replaced the SIP2 gene on chromosome 7 (sip2Δ::HIS3) since we knew from earlier studies that this gene deletion did not confer 2DG resistance or sensitivity [1n) reference strain. Deletion of the DOG loci from one of the disomic chromosomes significantly reduced the 2DG resistance while deletion of the HXT cluster and the SIP2 gene had no effect on 2DG resistance. Thus, the extra copies of the DOG1 and DOG2 genes are necessary for the 2DG resistance conferred by the disomy of chromosome 8.To confirm that the etention  had beensitivity . The disREG1 gene, which can by itself confer 2DG resistance via haploinsufficiency (DOG1/2 genes (p<0.001) 2DG resistance  exhibited significant 2DG resistance (HXT7 gene did not by itself confer any significant 2DG resistance (plasmid 4F6). However, cells transformed with plasmid 4G6 containing HXT3 and HXT6 were more resistant to 2DG than were cells transformed with plasmid 4H6 that contains only HXT3.While most of our 2DG resistant strains contained a single alteration that was sufficient to confer 2DG resistance, two strains contained more than one genetic change, each capable of contributing to a 2DG-resistant phenotype. For instance, DS7 contains both a stop codon in the ficiency , and a t/2 genes . Closer /2 genes . Sporulasistance . The genpe cells . Wild type cells  whose insistance . IncreasREG1 gene on chromosome 4  lacking the ability to phosphorylate 2DG are completely resistant to 2DG [Two-deoxyglucose is a potent inhibitor of glycolysis in both yeast and mammalian cells growing on glucose ,25. Yeast to 2DG . Also, gt to 2DG .HXK2 gene that reduce the enzyme’s catalytic activity confer resistance to 2DG [HXK2 gene confer 2DG resistance and not mutations in the other two hexokinase genes in yeast is a puzzle that remains to be solved. Both Hxk1 and Hxk2 enzymes are able to phosphorylate 2DG in vitro [HXK2 gene confer 2DG resistance. Loss of function alleles in the HXK2 gene is a genetic adaptation that has only been observed in haploid cells [HXK2 locus do not have a growth advantage in 2DG. In order to increase the rate of 2DG-6P degradation, both haploid and diploid cells increase the expression of the DOG1 and DOG2 genes by increasing the copy number of chromosome 8. 2DG resistance exhibits a strong positive correlation with DOG gene copy number  are hypersensitive to 2DG whereas cells with an activated allele of SNF1 are resistant [SNF1-G53R allele [GAL83-S224R and SNF4-N177Y alleles. Thus dominant, missense alleles in the genes coding all three subunits of the AMPK heterotrimer can confer 2DG resistance  exhibit modest 2DG resistance without the slow growth phenotype in the absence of 2DG observed in diploid cells homozygous for the reg1Δ allele.A second mechanism to activate AMPK signaling is to reduce activity in the PP1 phosphatase, an inhibitor of AMPK activity. The PP1 isoform that dephosphorylates and inactivates yeast AMPK is composed of the catalytic subunit, Glc7, bound to the regulatory subunit, Reg1 ,29. The ss costs . Haploidficiency . DiploidREG1 gene is on chromosome 4 and we suspect that it is the reduced expression of REG1 (3) for disomic states responsible for the phenotypic switching of yeast colony phenotype [Diploid cells with a chromosome 4 monosomy are resistant to 2DG  and show of REG1  that con of REG1 . The rathenotype . We havehenotype ,31.REG1, a PP1 phosphatase subunit, is a key driver of the chromosome 4 monosomy. The great advantage of this genetic strategy is that the chromosome 4 monosomy is freely reversible. Diploid cells can reap the benefits of a chromosome 4 monosomy when 2DG is present and rapidly return to normal ploidy when the 2DG selection pressure is removed . This mechanism for increasing gene expression using homologous retrotransposon sequences has been documented in other studies [DOG genes since the retrotransposons flanking the DOG genes would encompass the centromere on chromosome 8. If this region were duplicated, a dicentric and unstable chromosome would have been generated.Downstream targets of AMPK signaling that are important for 2DG resistance are the glucose transporters. Cells challenged with 2DG increase the endocytosis of the glucose transporters in a process that is promoted by the α-arrestins Rod1 and Rog3 . AMPK si studies  but woulSaccharomyces cells grown in the laboratory and clinical isolates of Candida from human patients utilize aneuploidy as a means for acquisition of drug resistance [Using selection for 2DG resistance as a model for the selection of drug resistance, our studies show that numerous genetic adaptations are utilized to impact a relatively simple pathway of drug resistance . Both Sasistance . Human csistance . Our stuSaccharomyces Genome Deletion Project [The yeast strains used in this study were all derivatives of S288C. Yeast strains with specific gene deletions were generated in our laboratory or by the  Project  and purc Project .7 cells were spread on agar plates containing synthetic complete media with 2% glucose (g/100ml) and 0.1% 2-deoxyglucose. Plates were incubated at 30°C for 4–6 days, and 2DG-resistant colonies were isolated for further study.Spontaneous mutations that conferred 2DG resistance were selected in the diploid strain MSY1527 . ApproxiThe genomes of the 2DG-resistant strains were analyzed by whole genome sequencing. Genomic DNA was prepared using a glass bead phenol extraction method . SequencPfu polymerase, followed by digestion of the starting plasmid template with the restriction enzyme DpnI [DOG1/2 plasmids were made by inserting the 4.1 kb BamHI—EcoRI fragment  into pRS316 (low copy number) and pRS426 (high copy number) plasmids [Oligonucleotide-directed mutagenesis of plasmids was performed with yme DpnI . Mutagenplasmids .2DG resistance was measured in synthetic complete media with 2% glucose (g/100ml) as previously described in McCartney, Chandrashekarappa (11) using either a titration of increasing concentration of 2DG or multiple replicates of cultures grown from independent colonies with and without 0.1% 2DG.S. cerevisiae mRNA using the kallisto software package [RNA samples were prepared from multiple independent yeast cultures grown on synthetic complete medium using the RNeasy Mini Kit (Qiagen). Sequencing libraries were prepared using the TruSeq Stranded mRNA library method (Illumina). RNA sequences were mapped to  package . Each RN package ,40,42. Rp values are indicated as follows: * p<0.05; ** p<0.01; *** p<0.001.Unless otherwise stated, mean values were calculated from a minimum of three independent measurements, and the error bars represent 1 standard deviation. Statistical significance was determined using the Student t test for unpaired variables with equal variance. In all cases, S1 Fig(A) Multiple sequence alignment of the yeast and human (Hs) beta subunit CBM domains. Position of the seven beta sheets (S1-S7) are indicated. The serine residue corresponding to Gal83-S224, Sip2-S226 and Sip1-S541 is indicated by the asterisks. (B) Three dimensional structure of the Sip2 CBM showing the seven beta sheets and the position of S226 (red mesh) is shown using the structure coordinates in 2QLV.pdb . (C and (TIF)Click here for additional data file.S2 FigHaploid strains RS17, RS18 and RS19 with multiple disomic chromosomes were mated with wild type haploid of opposite mating type (MSY188). After sporulation, the haploid progeny were analyzed for 2DG resistance by replica plating and scored as resistant (R) or sensitive (s). Four tetrads from each cross that showed 2:2 segregation of 2DG resistance were selected and the pooled DNA from resistant and sensitive strains was sequenced. The normalized median read depth for each chromosome of the parents  is plotted along with the resistant and sensitive pools.(TIF)Click here for additional data file.S3 Fig(A) Distribution of whole genome sequence read depth for each chromosome is plotted for four haploid strains with median read depth for each chromosome indicated by the yellow bar. (B) Read depth is plotted as a function of position along chromosome 8 near the DOG1/2 locus for MSY1553 (black) and MSY1560 (blue). The gene map for this region of chromosome 8 is shown above. (C) Distribution of read depth 10 kb before, 10 kb after and across the DOG1/2 locus is plotted for MSY1553 (black) and MSY1560 (blue). The median value is indicated by the yellow bar in the plot and numerically below. (D) Read depth is plotted as a function of position along chromosome 8 near the HXT415 locus for MSY1553 (black) and MSY1562 (blue). The gene map for this region of chromosome 8 is shown above. (E) Distribution of read depth 10 kb before, 10 kb after and across the HXT415 locus is plotted for MSY1553 (black) and MSY1562 (blue). The median value is indicated by the yellow bar in the plot and numerically below.(TIF)Click here for additional data file.S1 TableTranscripts per million mapped reads (tpm) is provided for 5917 yeast open reading frames. Samples S72-S83 are triplicate datasets from wild type diploid cells (MSY1527) and DS10 cells (chromosome 4 monosomy) washed off plates and grown for two hours in SC media with 2% glucose or in the same media with 0.1% 2DG. Samples S132-S147 are quadruplicate datasets from wild type diploid cells (MSY1527) and DS14 cells (chromosome 1 monosomy and chromosome 8 tetrasomy) grown to mid-log in SC media with 2% glucose and two hours after addition of 2DG to 0.1%.(XLSX)Click here for additional data file.