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Abstract Background High-density short oligonucleotide microarrays are a primary research tool for assessing global gene expression. Background noise on microarrays comprises a significant portion of the measured raw data, which can have serious implications for the interpretation of the generated data if not estimated correctly. Results We introduce an approach to calculate probe affinity based on sequence composition, incorporating nearest-neighbor (NN) information. Our model uses position-specific dinucleotide information, instead of the original single nucleotide approach, and adds up to 10% to the total variance explained (R2) when compared to the previously published model. We demonstrate that correcting for background noise using this approach enhances the performance of the GCRMA preprocessing algorithm when applied to control datasets, especially for detecting low intensity targets. Conclusion Modifying the previously published position-dependent affinity model to incorporate dinucleotide information significantly improves the performance of the model. The dinucleotide affinity model enhances the detection of differentially expressed genes when implemented as a background correction procedure in GeneChip preprocessing algorithms. This is conceptually consistent with physical models of binding affinity, which depend on the nearest-neighbor stacking interactions in addition to base-pairing.

Gestational diabetes mellitus (GDM) is a maternal metabolic disorder that perturbs placental development and increases the risk of offspring short- and long-term metabolic disorders. The mechanisms by which GDM impairs placental development remain poorly understood. Here, we defined the DNA methylome of GDM placentas and determined whether GDM perturbs methylation at genes important for placental development. We conducted an epigenome-wide association study of 42 placentas from pregnancies in the South African Soweto First 1000 days cohort (S1000). Using genome-wide bisulfite sequencing, we compared non-GDM placentas to GDM placentas with similar proportions from obese and non-obese mothers. Compared to non-GDM, GDM placentas exhibited a distinct methylation profile consisting of 12,210 differentially methylated CpGs (DMCs) that mapped to 3,875 genes. Epigenetically altered genes were enriched in Wnt and cadherin signalling pathways, both critical in placentation and embryogenesis. We also defined regional DNA methylation perturbation in GDM placentas at 11 placental development genes. These findings reveal extensive changes to the placental epigenome of GDM pregnancies and highlight perturbation enriched at important placental development genes. These molecular changes represent potential mechanisms for GDM-induced placental effects that may serve as candidate biomarkers for placental, maternal, and foetal health. Using a study design that used similar proportions of obese and non-obese mothers in our case and control pregnancies, we minimized the detection of changes due to obesity alone. Further work will be necessary to investigate the extent of the influence of obesity on these GDM-related placental epigenetic changes.

The human microbiome has been strongly correlated with disease pathology and outcomes, yet remains relatively underexplored in patients with malignant endometrial disease. In this study, vaginal microbiome samples were prospectively collected at the time of hysterectomy from 61 racially and ethnically diverse patients from three disease conditions: 1) benign gynecologic disease (controls, n=11), 2) low-grade endometrial carcinoma (n=30), and 3) high-grade endometrial carcinoma (n=20). Extracted DNA underwent shotgun metagenomics sequencing, and microbial α and β diversities were calculated. Hierarchical clustering was used to describe community state types (CST), which were then compared by microbial diversity and grade. Differential abundance was calculated, and machine learning utilized to assess the predictive value of bacterial abundance to distinguish grade and histology. Both α- and β-diversity were associated with patient tumor grade. Four vaginal CST were identified that associated with grade of disease. Different histologies also demonstrated variation in CST within tumor grades. Using supervised clustering algorithms, critical microbiome markers at the species level were used to build models that predicted benign vs carcinoma, high-grade carcinoma versus benign, and high-grade versus low-grade carcinoma with high accuracy. These results confirm that the vaginal microbiome segregates not just benign disease from endometrial cancer, but is predictive of histology and grade. Further characterization of these findings in large, prospective studies is needed to elucidate their potential clinical applications., Competing Interests: Disclosure/Conflict of Interest: The authors declare no potential conflicts of interest.

Introduction: Proton pump inhibitor (PPI) use was recently reported to be associated with increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and worse clinical outcomes. The underlying mechanism(s) for this association are unclear., Methods: We performed a prospective study of hospitalized coronavirus disease 2019 (COVID-19) patients and COVID-negative controls to understand how PPI use may affect angiotensin-converting enzyme 2 (ACE2) expression and stool SARS-CoV-2 RNA. Analysis of a retrospective cohort of hospitalized patients with COVID-19 from March 15, 2020 to August 15, 2020 in 6 hospitals was performed to evaluate the association of PPI use and mortality. Covariates with clinical relevance to COVID-19 outcomes were included to determine predictors of in-hospital mortality., Results: Control PPI users had higher salivary ACE2 mRNA levels than nonusers, 2.39 ± 1.15 vs 1.22 ± 0.92 (P = 0.02), respectively. Salivary ACE2 levels and stool SARS-CoV-2 RNA detection rates were comparable between users and nonusers of PPI. In 694 hospitalized patients with COVID-19 (age = 58 years, 46% men, and 65% black), mortality rate in PPI users and nonusers was 30% (68/227) vs 12.1% (53/439), respectively. Predictors of mortality by logistic regression were PPI use (adjusted odds ratio [aOR] = 2.72, P < 0.001), age (aOR = 1.66 per decade, P < 0.001), race (aOR = 3.03, P = 0.002), cancer (aOR = 2.22, P = 0.008), and diabetes (aOR = 1.95, P = 0.003). The PPI-associated mortality risk was higher in black patients (aOR = 4.16, 95% confidence interval: 2.28-7.59) than others (aOR = 1.62, 95% confidence interval: 0.82-3.19, P = 0.04 for interaction)., Discussion: COVID-negative PPI users had higher salivary ACE2 expression. PPI use was associated with increased mortality risk in patients with COVID-19, particularly African Americans., (Copyright © 2021 by The American College of Gastroenterology.)

Background: The gut microbiome protects the host from infection by promoting epithelial integrity and providing basal immunologic stimulation. Disruption of this delicate ecosystem is linked to morbidity and mortality among critically ill patients, but the impact of traumatic injury on the gut microbiome is poorly understood. This study sought to identify alterations in gut microbiota following trauma and persistent stress in rodents without confounding antibiotics., Methods: Male Sprague-Dawley rats aged 9 weeks to 11 weeks were randomized to naive, lung contusion with hemorrhagic shock (LCHS), and LCHS plus either 7 (LCHS/CS 7/7) or 14 days (LCHS/CS 14) of restraint cylinder stress for 2 hours daily. Stool was collected on Days 0, 3, 7, and 14 for bacterial whole genome DNA isolation. Alpha diversity, or the number and relative abundance of unique bacterial species within each cohort, was assessed using Chao1 indices. Beta diversity, or the measure of differences in biodiversity across cohorts, was assessed by principle coordinate analysis. False discovery rate correction was applied to all statistical analyses and corrected for cohousing effects., Results: Rodent groups subject to restraint stress demonstrated a progressive increase in alpha diversity over time. These microbiota changes resolved after cessation of stress (LCHS/CS 7/7) but continued to increase among rats subjected to ongoing stress (LCHS/CS 14). The LCHS/CS 7/7 also demonstrated reductions in class Actinobacteria and increased abundance of the genus Bacteroides by Day 7, which resolved by Day 14. Increased abundance of Bacteroides was also noted in the LCHS/CS 14 cohort, suggesting the role of chronic stress in its destabilization., Conclusion: This study points to persistent stress as a potential source of the destabilization of microbial diversity seen after trauma. This lack of microbiota stability could be associated with worse long-term outcomes in critically ill trauma patients. Further studies are warranted to elucidate mechanistic pathways and potential therapeutic modalities., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Progression of prostate cancer (PC) to terminal castration-resistant PC (CRPC) involves a diverse set of intermediates, and androgen receptor (AR) is the key mediator of PC initiation and progression to CRPC. Hence, identification of factors involved in the regulation of AR expression and function is a necessary first-step to improve disease outcome. In this study, we identified ubiquitous βArrestin 1 (βArr1) as a regulator of AR function in CRPC. Unbiased gene expression analysis of public datasets revealed increased levels of ARRB1 (the gene encoding βArr1) in CRPC when compared to normal tissue. Further, βArr1 expression correlated with enhanced AR transcriptional function in these datasets. The βArr1 partitions to both nucleus and cytosol and mechanistic studies showed that nuclear, and not cytosolic, βArr1 formed a complex with AR and AR-coregulator βCatenin and that the heterotrimeric protein complex was recruited to androgen-response elements of AR-regulated genes. Functionally, we demonstrate that depletion of βArr1 attenuates PC cell and tumor growth and metastasis, and rescued expression of nuclear, but not cytosolic, βArr1 restores the PC colony growth and invasion of Matrigel in vitro and tumor growth and metastasis in mice. The targeting of βArr1-regulated AR transcriptional function may be used in the development of new drugs to treat lethal CRPC.

In the originally published version of this Article, the affiliation details for Dorina Avram incorrectly included "Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, 2015 SW 16th Ave, Gainesville, FL, 32608, USA", instead of "UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA". This has now been corrected in both the PDF and HTML versions of the Article.

During helminth infection and allergic asthma, naive CD4 + T-cells differentiate into cytokine-producing Type-2 helper (Th2) cells that resolve the infection or induce asthma-associated pathology. Mechanisms regulating the Th2 differentiation in vivo remain poorly understood. Here we report that mice lacking Bcl11b in mature T-cells have a diminished capacity to mount Th2 responses during helminth infection and allergic asthma, showing reduced Th2 cytokines and Gata3, and elevated Runx3. We provide evidence that Bcl11b is required to maintain chromatin accessibility at Th2-cytokine promoters and locus-control regions, and binds the Il4 HS IV silencer, reducing its accessibility. Bcl11b also binds Gata3-intronic and downstream-noncoding sites, sustaining the Gata3 expression. In addition, Bcl11b binds and deactivates upstream enhancers at Runx3 locus, restricting the Runx3 expression and its availability to act at the Il4 HS IV silencer. Thus, our results establish novel roles for Bcl11b in the regulatory loop that licenses Th2 program in vivo.

Introduction: Ventilator-associated pneumonia (VAP) caused by multidrug-resistant Acinetobacter baumannii (MDR-AB) is common in hospitals and impacts patient survival. We determined the incidence of MDR-AB VAP in critical care units and examined the predictors of 14-day mortality in these patients., Methodology: A retrospective case series study was conducted at a tertiary referral teaching hospital in north Jordan. A list of patients with a positive culture of A. baumannii between January 2007 and June 2013 was retrieved using computerized hospital databases. Medical records of all these patients were reviewed, and cases of VAP infected with MDR-AB were identified. Predictors of 14-day mortality were determined using multivariable logistic regression adjusted for possible confounders., Results: Out of 121 A. baumannii-VAP cases, 119 (98.3%) were caused by MDR-AB. The incidence rate of MDR-AB VAP was 1.59 cases per 100 critical care unit admissions. The mortality of A. baumannii-VAP cases in critical care units was 42% (50/119). Being prescribed two or more definitive antibiotics (prescribed based on susceptibility data) (OR = 0.075, 95% CI = 0.017-0.340, p = 0.001) and ipratropium/salbutamol during mechanical ventilation (OR = 0.140, 95% CI = 0.028-0.705, p = 0.017) were independently associated with lower hospital mortality., Conclusions: Our results suggest incidence of MDR-AB VAP in critical care units is high and that prescription of antibiotics based on antibiotic susceptibility and use of bronchodilators is associated with lower mortality in this population. Larger prospective studies are needed to explore whether these findings can be replicated in different clinical settings.

Background: Molecular genetic studies of floral development have concentrated on several core eudicots and grasses (monocots), which have canalized floral forms. Basal eudicots possess a wider range of floral morphologies than the core eudicots and grasses and can serve as an evolutionary link between core eudicots and monocots, and provide a reference for studies of other basal angiosperms. Recent advances in genomics have enabled researchers to profile gene activities during floral development, primarily in the eudicot Arabidopsis thaliana and the monocots rice and maize. However, our understanding of floral developmental processes among the basal eudicots remains limited., Results: Using a recently generated expressed sequence tag (EST) set, we have designed an oligonucleotide microarray for the basal eudicot Eschscholzia californica (California poppy). We performed microarray experiments with an interwoven-loop design in order to characterize the E. californica floral transcriptome and to identify differentially expressed genes in flower buds with pre-meiotic and meiotic cells, four floral organs at preanthesis stages (sepals, petals, stamens and carpels), developing fruits, and leaves., Conclusions: Our results provide a foundation for comparative gene expression studies between eudicots and basal angiosperms. We identified whorl-specific gene expression patterns in E. californica and examined the floral expression of several gene families. Interestingly, most E. californica homologs of Arabidopsis genes important for flower development, except for genes encoding MADS-box transcription factors, show different expression patterns between the two species. Our comparative transcriptomics study highlights the unique evolutionary position of E. californica compared with basal angiosperms and core eudicots.

In this study, we revealed rapid detection of streptomycin-producing Streptomyces spp. by extraction of total soil DNA from 14 soil samples using a modified lysis method followed by PCR amplification ofa genus-specific sequence in the Streptomyces' 16S rDNA gene. DNA band of the expected size (438 bp) was seen with all the samples. Additionally, specific amplification of the streptomycin-coding gene (strb1) directly from soil revealed the presence of a single DNA band of 940 bp. These results indicate that PCR-amplification of Streptomyces specific genes could be used for direct detection of streptomycin-producing Streptomyces species from soil.

In this study, two designed primers were evaluated to identify soil Streptomyces and to detect streptomycin production by strb1 targeted PCR. Potential Streptomyces-specific signatures were identified in their 16S rDNA sequences in regions located around nucleotide positions 576 and 995. Primer pair RI7/RI8 derived from these regions was investigated for its specificity in detecting and identifying Streptomyces isolates by PCR assays using DNA from pure cultures. The constructed primer pair showed high specificity in detecting and identifying Streptomyces type strains as well as soil isolates. Streptomycin-producers were detected by PCR assays through the selective amplification of streptomycin biosynthetic gene (strb1). Results suggest that PCR assay facilitates the differential identification of Streptomyces-specific antibiotic producers and a resident population of Streptomyces in Jordan with the capacity of streptomycin-production is present.

Three sets of known Salmonella enterica-specific primers were used collectively, for the first time, to evaluate the use of multiplex polymerase chain reaction (m-PCR) as a diagnostic tool to detect Salmonella enterica in naturally contaminated meat and poultry products. For this purpose a total of 300 samples representing the most frequently used fresh and frozen meat (beef and lamb) and poultry (chicken) products (whole, cut, ground, and processed) were collected from eight locations within Irbid city (Jordan). After an enrichment step, DNA was extracted directly from each food sample and amplified using six Salmonella enterica specific primers. Samples were also analyzed using conventional microbiological methods for the confirmation of Salmonella enterica presence. Out of 300 samples, 93 samples were positive by m-PCR. On the other hand, 67 samples were positive by conventional microbiological methods and only 26 samples were positive by m-PCR alone. The primer pairs, used here, proved to be highly specific for Salmonella enterica. Using m-PCR, Salmonella enterica detection could be achieved easily and the results had been confirmed effortlessly within a short period of time (24-36 hours) compared to 3-8 days for the conventional microbiological methods. Our findings emphasize the value of using a combination of specific primer pairs instead of a single primer pair in the detection process to minimize any chance for any inherent experimental or natural error.

Random amplified polymorphic DNA (RAPD) analysis has been used to determine the relatedness of 73 antibiotic-producing soil Streptomyces isolates that were recovered from different soil habitats in Jordan based on their RAPD-PCR fingerprints. Genetic polymorphisms between these isolates showed three common bands of 2777, 800 and 250 bp shared by approximately (95%) of them. Some specific bands were also observed. Further analysis of RAPD patterns with the UPGMA resulted in clustering the tested isolates into two main super clusters. Super cluster I was more homogenous than super cluster II and contained all the reference strains. However, super cluster II consists of unrelated isolates within five small groups. As RAPD fingerprints of the tested isolates linked to their phenotypes, differentiation between isolates with different cultural properties was observed.

A cDNA and two genomic clones comprising highly similar genes that encode a protein with a Myb-related DNA-binding domain were isolated from the resurrection plant Craterostigma plantagineum. The structure of cpm5 and cpm10 (Craterostigma plantagineum myb) genes consists of three putative exons encoding a protein of 36.6 kDa. The cDNA of cpm7 encodes a closely related protein of 36.8 kDa. The canonical Myb domain present in transcriptional activators of yeast, animals and plants was localized in the amino terminus of deduced Cpm5, Cpm7 and Cpm10 proteins and corresponds to the two Myb repeats found in plants. The Myb domain of Cpm deduced proteins and a short stretch of amino acids adjacent to this region are closely related to a myb gene from Arabidopsis thaliana which is expressed in response to osmotic stress and ABA. The rest of the deduced protein has no similarity to other reported sequences. The myb-related genes in the Craterostigma genome comprise a small gene family of 6-8 members as estimated by hybridization with a bona fide Myb domain probe. Northern blot experiments showed specific expression of cpm10 in undifferentiated callus tissue up-modulated by ABA and expression of cpm7 mRNA in roots up-regulated by dehydration.

We show here data suggesting that Entamoeba histolytica, the protozoan responsible for human amebiasis, presents DNA amplification in a fashion similar to that described for transformed mammalian cells. By transmission electron microscopy (TEM), we found linear, circular and concentric circular DNA molecules exhibiting the main events of the unscheduled DNA amplification process. Loops were formed after the recombination of two nonadjacent DNA regions, and bubbles appeared from the recombinant strands without involving the looped-out sequences. Bubbles grew up and underwent further replication rounds to produce a nested set of partially replicated circles. Multicircle complexes were also formed from putative replication origin without recombination of distant DNA regions. Clones derived from the strain HM1:IMSS exhibited different DNA contents, suggesting DNA amplification. The parental clone A and its daughter clone C2 differed in rDNA gene copy numbers, but this was observed only when total DNA was separated by pulse field gel electrophoresis, and no significant differences were detected in nuclear DNA. The dissection of the events observed by TEM led us to propose an onion skin model for gene amplification in E. histolytica.