Frequency-potency analysis of IgG+ memory B cells delineates neutralizing antibody responses at single-cell resolution.

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.

Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method.

The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR Cq values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 103 to 108 CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products.

Development of a SNP-based strain-identified method for Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 using pan-genomics analysis.

The health benefits conferred by probiotics is specific to individual probiotic strains, highlighting the importance of identifying specific strains for research and production purposes. Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 are exceedingly valuable for commercial use with an excellent mixed-culture fermentation. To differentiate these 2 strains from other S. thermophilus and L. delbrueckii ssp. bulgaricus, a specific, sensitive, accurate, rapid, convenient, and cost-effective method is required. In this study, we conducted a pan-genome analysis of S. thermophilus and L. delbrueckii ssp. bulgaricus to identify species-specific core genes, along with strain-specific single-nucleotide polymorphisms (SNPs). These genes were used to develop suitable PCR primers, and the conformity of sequence length and unique SNPs was confirmed by sequencing for qualitative identification at the strain level. The results demonstrated that SNPs analysis of PCR products derived from these primers could distinguish CICC 6038 and CICC 6047 accurately and reproducibly from the other strains of S. thermophilus and L. delbrueckii ssp. bulgaricus, respectively. The strain-specific PCR method based on SNPs herein is universally applicable for probiotics identification. It offers valuable insights into identifying probiotics at the strain level that is fit-for-purpose in quality control and compliance assessment of commercial dairy products.

Rapid sand filtration for <10 µm-sized microplastic removal in tap water treatment: Efficiency and adsorption mechanisms.

The omnipresence of microplastics (MPs) in potable water has become a major concern due to their potential disruptive effect on human health. Therefore, the effective removal of MPs in drinking water is essential for life preservation. In this study, tap water containing microplastic <10 µm in size was treated using constructed pilot-scale rapid sand filtration (RSF) system to investigate the removal efficiency and the mechanisms involved. The results show that the RSF provides significant capacity for the removal and immobilization of MPs < 10 µm diameter (achieving 98 %). Results showed that silicate sand reacted with MPs through a cooperative assembly process, which mainly involved interception, trapping, entanglement, and adsorption. The MPs were quantified by Flow cytometry instrument. A kinetics study underlined the pivotal role of physio-chemisorption in the removal process. MP particles smaller than absorbents, saturation of adsorbents, and reactor hydrodynamics were identified as limiting factors, which were alleviated by backwashing. Backwashing promoted the desorption of up to 97 % MPs, conducive for adsorbent active site regeneration. These findings revealed the critical role of RSF and the importance of backwashing in removing MPs. Understanding the mechanisms involved in removing microplastics from drinking water is crucial in developing more efficient strategies to eliminate them.

Comparison of Nanotrap® Microbiome A Particles, membrane filtration, and skim milk workflows for SARS-CoV-2 concentration in wastewater.

Introduction: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for Coronavirus Disease 2019 (COVID-19) surveillance. Grab (quantitative) and passive samples (qualitative) are two distinct wastewater sampling methods. Although many viral concentration methods such as the usage of membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. Methods: The objectives of this study were to compare two workflows (Nanotrap® Microbiome A Particles coupled with MagMax kit and membrane filtration workflows coupled with RNeasy kit) for SARS-CoV-2 recovery in grab samples and two workflows (Nanotrap® Microbiome A Particles and skim milk workflows coupled with MagMax kit) for SARS-CoV-2 recovery in Moore swab samples. The Nanotrap particle workflow was initially evaluated with and without the addition of the enhancement reagent 1 (ER1) in 10 mL wastewater. RT-qPCR targeting the nucleocapsid protein was used for detecting SARS-CoV-2 RNA. Results: Adding ER1 to wastewater prior to viral concentration significantly improved viral concentration results (P < 0.0001) in 10 mL grab and swab samples processed by automated or manual Nanotrap workflows. SARS-CoV-2 concentrations in 10 mL grab and Moore swab samples with ER1 processed by the automated workflow as a whole showed significantly higher (P < 0.001) results than 150 mL grab samples using the membrane filtration workflow and 250 mL swab samples using the skim milk workflow, respectively. Spiking known genome copies (GC) of inactivated SARS-CoV-2 into 10 mL wastewater indicated that the limit of detection of the automated Nanotrap workflow was ~11.5 GC/mL using the RT-qPCR and 115 GC/mL using the digital PCR methods. Discussion: These results suggest that Nanotrap workflows could substitute the traditional membrane filtration and skim milk workflows for viral concentration without compromising the assay sensitivity. The manual workflow can be used in resource-limited areas, and the automated workflow is appropriate for large-scale COVID-19 wastewater-based surveillance.

When case reporting becomes untenable: Can sewer networks tell us where COVID-19 transmission occurs?

Monitoring SARS-CoV-2 in wastewater is a valuable approach to track COVID-19 transmission. Designing wastewater surveillance (WWS) with representative sampling sites and quantifiable results requires knowledge of the sewerage system and virus fate and transport. We developed a multi-level WWS system to track COVID-19 in Atlanta using an adaptive nested sampling strategy. From March 2021 to April 2022, 868 wastewater samples were collected from influent lines to wastewater treatment facilities and upstream community manholes. Variations in SARS-CoV-2 concentrations in influent line samples preceded similar variations in numbers of reported COVID-19 cases in the corresponding catchment areas. Community sites under nested sampling represented mutually-exclusive catchment areas. Community sites with high SARS-CoV-2 detection rates in wastewater covered high COVID-19 incidence areas, and adaptive sampling enabled identification and tracing of COVID-19 hotspots. This study demonstrates how a well-designed WWS provides actionable information including early warning of surges in cases and identification of disease hotspots.

Induction of bacteria in biofilm into a VBNC state by chlorine and monitoring of biofilm structure changes by means of OCT.

The occurrence of viable but non-culturable (VBNC) bacteria in drinking water may result in significant underestimation of viable cell counts detected by culture-based method, thus raising microbiological safety concern. Chlorine disinfection has been widely used in drinking water treatment to ensure microbiological safety. However, the effect of residual chlorine on inducing bacteria in biofilms into a VBNC state remains unclear. We determined cell numbers of Pseudomonas fluorescence in different physiological states (culturable, viable, dead) by heterotrophic plate count method and flow cytometer in a flow cell system under 0, 0.1, 0.5, 1.0 mg/L chlorine treatment. Numbers of culturable cells were 4.66 ± 0.47 Log10, 2.82 ± 0.76 Log10, 2.30 ± 1.23 Log10 (CFU/112.5 mm3) in each chlorine treatment group. However, viable cell numbers remained at 6.32 ± 0.05 Log10, 6.11 ± 0.24 Log10, 5.08 ± 0.81 Log10 (cells/112.5 mm3). Significant difference between numbers of viable and culturable cells demonstrated chlorine could induce bacteria in biofilms into a VBNC state. In this study, flow cells combination with Optical Coherence Tomography (OCT) were applied to construct an Automated experimental Platform for replicate Biofilm cultivation and structural Monitoring (APBM) system. The OCT imaging results demonstrated that changes of biofilm structure under chlorine treatment were closely related to their inherent characteristics. Biofilms with low thickness and high roughness coefficient or porosity were easier to be removed from the substratum. Biofilm with high rigid properties were more resistant to chlorine treatment. Even though >95 % bacteria in biofilms entered a VBNC state, the biofilm physical structure was still remained. This study revealed the possibility of bacteria to enter a VBNC state in drinking water biofilms and changes of biofilm structure with different characteristics under chlorine treatment, which provide reference for biofilms control in drinking water distribution systems.

A novel qPCR-based method to quantify seven phyla of common algae in freshwater and its application in water sources.

The light microscope is widely used to count algae, however, there are some disadvantages associated with this method, such as time consuming and laborious. In this study, a qPCR-based method was established for quantifying seven phyla of common algae in freshwater, including Cyanophyta, Chlorophyta, Euglenophyta, Bacillariophyta, Dinophyta, Cryptophyta, and Chrysophyta. The accuracy of qPCR in estimating algal cells was confirmed by comparing it with the microscopic counting. The qPCR was used to detect the cell concentration of seven phyla of algae in Longhu Reservoir, showing that green algal blooms occurred during the monitoring period. The intensity of algal blooms was further evaluated according to the classification standard, which suggested that the grade of this bloom was mild. An early warning system was proposed to early warn the occurrence of algal blooms in two water sources, Longhu Reservoir and Dongzhang Reservoir. The qPCR method developed in this study could be a useful tool in the monitoring of algae. The early warning system reported here will have important implications for the effective warning of algal blooms.

Grassland ecology system: A critical reservoir and dissemination medium of antibiotic resistance in Xilingol Pasture, Inner Mongolia.

Antibiotic resistance is a major threat to human health. It is necessary to explore all the potential sources and comprehend the pathways that antibiotic resistance genes (ARGs) are transmitted. In this study, by applying high-throughput quantitative PCR and high-throughput sequencing, ARGs and microbial community structure were determined, to understand the reservoirs and spread of ARGs in the Xilingol grassland system. A total of 151,140 and 138 different ARGs were observed in manure, soil, and water samples, respectively. Only 12 ARGs were shared in all environmental and animal manure samples. Multidrug defense system, such as efflux pump, was the most dominant factor in manure and soil samples, followed by antibiotic deactivation processes. These genes coffering resistance to major classes of antibiotics including ß_Lactamase (blaSFO, fox5, blaCTX-M-04, blaOXY), vancomycin (vanC-03, vanXD), MLSB (vatE-01, mphA-01), aminoglycoside (aadA2-01), Multidrug (oprJ) and others (oprD, qacEdelta1-02), except sulfonamide and tetracycline. The 12 ARGs were significantly enriched in water samples compared to manure and soil samples (p < 0.01) and demonstrated that the water environment was an important transmission source of ARGs in the grassland. The highest enrichment was up to 324.5-fold. Moreover, the 12 shared ARGs were positively correlated with the mobile genetic elements (p < 0.01). The nonrandom co-occurrence network patterns between ARGs and microbial community suggested that a total of three bacterial phyla were viewed as the potential ARGs hosts. These findings indicate that ARGs were highly enriched in water samples, demonstrating that the water environment was a critical source and sink of ARGs in the grassland system. It may illuminate the mechanism stressing the effects of human activity on the occurrence and transmission of ARGs in the grassland system.

A sensitive, simple, and low-cost method for COVID-19 wastewater surveillance at an institutional level.

SARS-CoV-2 is a respiratory virus, but it is also detected in a significant proportion of fecal samples from COVID-19 cases. Recent studies have shown that wastewater surveillance can be a low-cost tool compared to massive diagnostic testing for tracking COVID-19 outbreaks in communities, but most studies have focused on sampling from wastewater treatment plants. Institutional level wastewater surveillance may serve well for early warning purposes because specific geographic areas/populations with emerging cases can be tracked and immediate action can be executed in the event of a positive wastewater signal. In this study, a novel Moore swab method was developed and used for wastewater surveillance of COVID-19 at an institutional level. Of the 442 swab samples tested, 148 (33.5%) swabs collected from the three campuses and two buildings were positive for SARS-CoV-2 RNA. Further study of the quarantine building with a known number of cases indicated that this method was sensitive enough to detect few cases in the building. In addition, comparison between grab samples and Moore swab samples from the hospital sewage line indicated that Moore swabs were more sensitive than grab samples and offer a simple, inexpensive method for obtaining a composite sample of virus in wastewater over a 24-48 h period. These results suggest that collection and analyses of Moore swabs for SARS-CoV-2 RNA detection is a sensitive, low-cost, and easy to use tool for COVID-19 surveillance that is useful for institutional settings and could be deployed in low-resource settings to identify emerging COVID-19 clusters in communities.

A Defect Detection Method for Rail Surface and Fasteners Based on Deep Convolutional Neural Network.

As a result of long-term pressure from train operations and direct exposure to the natural environment, rails, fasteners, and other components of railway track lines inevitably produce defects, which have a direct impact on the safety of train operations. In this study, a multiobject detection method based on deep convolutional neural network that can achieve nondestructive detection of rail surface and fastener defects is proposed. First, rails and fasteners on the railway track image are localized by the improved YOLOv5 framework. Then, the defect detection model based on Mask R-CNN is utilized to detect the surface defects of the rail and segment the defect area. Finally, the model based on ResNet framework is used to classify the state of the fasteners. To verify the robustness and effectiveness of our proposed method, we conduct experimental tests using the ballast and ballastless railway track images collected from Shijiazhuang-Taiyuan high-speed railway line. Through a variety of evaluation indexes to compare with other methods using deep learning algorithms, experimental results show that our method outperforms others in all stages and enables effective detection of rail surface and fasteners.

Assessment of the UV/Chlorine Process in the Disinfection of Pseudomonas aeruginosa: Efficiency and Mechanism.

UV irradiation and chlorination have been widely used for water disinfection. However, there are some limitations, such as the risk of generating viable but nonculturable bacteria and bacteria reactivation when using UV irradiation or chlorination alone. This study comprehensively evaluated the feasibility of the UV/chlorine process in drinking water disinfection, and Pseudomonas aeruginosa was selected as the target microorganism. The number of culturable cells was effectively reduced by more than 5 orders of magnitude (5-log10) after UV, chlorine, and UV/chlorine treatments. However, intact and VBNC cells were detected at 103 to 104 cells/mL after UV and chlorine treatments, whereas they were undetectable after UV/chlorine treatment due to the primary contribution of reactive chlorine species (Cl•, Cl2•-, and ClO•). After UV/chlorine treatment, the metabolic activity determined using single cell Raman spectroscopy was much lower than that after UV. The level of toxic opr gene in P. aeruginosa decreased by more than 99% after UV/chlorine treatment. Importantly, bacterial dark reactivation was completely suppressed by UV/chlorine treatment but not UV or chlorination. This study suggests that the UV/chlorine treatment can completely damage bacteria and is promising for pathogen inactivation to overcome the limitations of UV and chlorine treatments alone.

Accumulation of antibiotic resistance genes in full-scale drinking water biological activated carbon (BAC) filters during backwash cycles.

Biological activated carbon (BAC) filtration, a process widely used in drinking water treatment, was recently reported to harbor antibiotic resistance genes (ARGs). This emerging contamination is poorly understood. This study was conducted to investigate the occurrence of ARGs and bacterial community in full-scale BAC filters during the backwash cycle using high-throughput qPCR and high-throughput sequencing. A total of 178 ARGs were detected in all biofilm samples, with relative abundance ranging from 0.1 to 1.37 copies per 16S rRNA and absolute abundance ranging from 4.48 × 107 to 3.09 × 109 copies/g carbon. Biofilms sampled from different filters shared most detected ARGs and dominant genera including Bryobacter, Pedomicrobium, Reyranella, and Terrimonas, though their bacterial community structure differed significantly. After backwashing, the relative ARGs abundance increased by 1.5- to 3.8-folds and the absolute ARGs abundance increased by 0.90- to 1.12-logs in all biofilm samples during filter ripening, indicating that ARGs accumulated in filters during this period. Redundancy analysis suggested that such ARGs accumulation was mainly driven by horizontal gene transfer in winter, but highly correlated with the increasing relative abundance of genera Bryobacter and Acidibacter in summer. It was observed that 80.6 %-89.3% of the detected ARGs persisted in the filters despite of the backwashing. Given the high richness and relative abundance of ARGs in BAC filter and the ineffectiveness of backwashing in ARG removal, more stringent downstream disinfection strategies are deserved and more research is necessary to assess potential human health risks due to the persistence of ARGs in drinking water.

Detection and distribution of vbnc/viable pathogenic bacteria in full-scale drinking water treatment plants.

Viable but non-culturable (VBNC) bacteria have attracted widespread attention since they are inherently undetected by traditional culture-dependent methods. Importantly, VBNC bacteria could resuscitate under favorable conditions leading to significant public health concerns. Although the total number of viable bacteria has been theorized to be far greater than those that can be cultured, there have been no reports quantifying VBNC pathogenic bacteria in full-scale drinking water treatment plants (DWTPs). In this work, we used both culture-dependent and quantitative PCR combination with propidium monoazide (PMA) dye approaches to characterize cellular viability. Further, we established a method to quantify viable pathogens by relating specific gene copies to viable cell numbers. Ratios of culturable bacteria to viable 16S rRNA gene copies in water and biological activated carbon (BAC) biofilms were 0-4.75% and 0.04-56.24%, respectively. The VBNC E. coli, E. faecalis, P. aeruginosa, Salmonella sp., and Shigella sp. were detected at levels of 0-103 cells/100 mL in source water, 0-102 cells/100 mL in chlorinated water, and 0-103 cells/g in BAC biofilms. In addition, differences between the total and viable community structures after ozonation and chlorination were investigated. The relative abundance of opportunistic pathogens such as Mycobacterium, Sphingomonas, etc. increased in final water, likely due to their chlorine resistance. In summary, we detected significant quantities of viable/VBNC opportunistic pathogens in full-scale DWTPs, confirming that traditional, culture-dependent methods are inadequate for detecting VBNC bacteria. These findings suggest a need to develop and implement rapid, accurate methods for the detection of VBNC pathogenic bacteria in DWTPs to ensure the safety of drinking water.

Analysis of microbial contamination of household water purifiers.

Household water purifiers are increasingly used to treat drinking water at the household level, but their influence on the microbiological safety of drinking water has rarely been assessed. In this study, representative purifiers, based on different filtering processes, were analyzed for their impact on effluent water quality. The results showed that purifiers reduced chemical qualities such as turbidity and free chlorine. However, a high level of bacteria (102-106 CFU/g) was detected at each stage of filtration using a traditional culture-dependent method, whereas quantitative PCR with propidium monoazide (PMA) treatment showed 106-108 copies/L of total viable bacteria in effluent water, indicating elevated microbial contaminants after purifiers. In addition, high-throughput sequencing revealed a diverse microbial community in effluents and membranes. Proteobacteria (22.06-97.42%) was the dominant phylum found in all samples, except for purifier B, in which Melainabacteria was most abundant (65.79%). For waterborne pathogens, Escherichia coli (100-106 copies/g) and Pseudomonas aeruginosa (100-105 copies/g) were frequently detected by qPCR. Sequencing also demonstrated the presence of E. coli (0-6.26%), Mycobacterium mucogenicum (0.01-3.46%), and P. aeruginosa (0-0.16%) in purifiers. These finding suggest that water from commonly used household purifiers still impose microbial risks to human health.

Profiling the microbial contamination in aviation fuel from an airport.

Microbial contamination during fuel storage can cause fuel system fouling and corrosion. Characterizing microbial contamination is critical for preventing and solving these problems. In this study, culture-based combing with the culture-independent methods, were used to profile the microbial contamination in aviation fuel. High-throughput sequencing (HTS) modified by propidium monoazide (PMA) revealed a higher diversity of contaminating microorganisms in samples than the culture method. Proteobacteria (47%), Actinobacteria (21%) and Ascomycota (>99%, fungi) were the most abundant phyla, and the neglected archaea was also detected. Additionally, qPCR-based methods revealed all samples contained a heavy level of microbial contamination, which was more accurate than its culturable counterparts, and fungal contamination was still a problem in aviation fuel. The application of a PCR-based method gives deeper insight into microbial contamination in aviation fuel than the conventional culture method, thus using it for regular detection and accurate description of fuel contamination is strongly recommended in the case of explosive microbial growth.

Population and single cell metabolic activity of UV-induced VBNC bacteria determined by CTC-FCM and D2O-labeled Raman spectroscopy.

The occurrence of viable but non-culturable (VBNC) bacteria will result in significant underestimation of viable bacterial counts in drinking water. Whereas, much is unknown in characterizing their viability. In this study, two environmental isolates (Aeromonas sp. and Pseudomonas sp.) and two model strains (E. coli and S. aureus) were induced into VBNC state by UV irradiation. Then, their metabolic activity was determined by 5-cyano-2,3-ditolyl tetrazolium chloride combination flow cytometry (CTC-FCM) and D2O-labeled Raman spectroscopy, respectively, at both population and single cell levels. The results showed that almost all strains could enter VBNC state irradiated by ≥ 5 mJ/cm2 UV. When determined by CTC-FCM, the population metabolic activity for each strain did not vary significantly (p > 0.05) unless the UV dose reached 200 mJ/cm2. Their single cell activity spectrum narrowed slightly, as indicated by changes in the standard deviation of the logarithmic normal distribution (σ) of 0.015-0.033. This minute difference suggested the CTC-FCM method was suitable for assessing the essential viability of VBNC bacteria. With respect to Raman method, an obvious dose-response effect was recorded. With the UV dosages increased from 10 to 200 mJ/cm2, the CD/(CD + CH) for the four strains were reduced to between 95.7% and 47.9% of unirradiated controls, depending on strain and UV dose. Meanwhile, the single cellular Raman spectrum showed much more heterogeneously metabolic activity distribution, with some cells even entering metabolic "silence". Considering the ubiquitous participation of water in biochemical processes, the Raman method was more appropriate in assessing the overall metabolic activity. The above findings can not only be a reference for VBNC mechanism studies, but also have the potential in optimizing disinfection and other bacterial removal processes.

Organic carbon: An overlooked factor that determines the antibiotic resistome in drinking water sand filter biofilm.

Biofilter, an essential water treatment process, is reported to be the harbor of bacterial antibiotic resistance genes (ARGs). Due to the oligotrophic characteristic of source water, filter biofilm is largely influenced by the concentration of organic carbon. The objective of this study was to investigate the effect of organic carbon concentration on shaping bacterial antibiotic resistome in filter biofilm. Our study was based on pilot-scale sand filters, and we investigated the antibiotic resistome using high-throughput qPCR. A total of 180 resistance genes from eight categories of antibiotics were detected in 15 biofilm samples of three sand filters. The results indicated that higher concentration of influent organic carbon led to lower diversity of bacterial community and richness of antibiotic resistance genes (ARGs) in biofilm. We discovered a negative correlation (p ≤ 0.01) between the richness of ARGs and the corresponding TOC level. Moreover, the absolute abundance of ARGs was positively correlated (p ≤ 0.05) with the abundance of 16S rRNA gene and was determined by the organic carbon concentration. Sand filters with gradient influent organic carbon concentration led to the formation of different antibiotic resistomes and canonical correspondence analysis (CCA) indicated that difference in bacterial community composition was likely the main reason behind this difference. We also observed a similar trend in the relative abundance of ARGs, which increased with the depth of sand filters. However, this trend was more pronounced in filters with low organic carbon concentrations. Overall, this study revealed that the organic carbon concentration determined the absolute abundance of ARGs and also shaped the diversity and relative abundance of ARGs in drinking water sand filters. These results may provide new insights into the mechanism of persistent bacterial antibiotic resistance in drinking water treatment.

Sox7 negatively regulates prostate-specific membrane antigen (PSMA) expression through PSMA-enhancer.

BACKGROUND: PSMA expression in the prostate epithelium is controlled by a cis-element, PSMA enhancer (PSME). PSME contains multiple binding sites for Sox proteins, and in this study, we identified Sox7 protein as a negative regulator of PSMA expression through its interaction with PSME. METHODS: The statistical correlation between Sox7 and PSMA mRNA expression was evaluated using five prostate cancer studies from cBioportal. In vitro and in vivo interaction between Sox7 and PSME was evaluated by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase reporter assay. Synthetic oligonucleotides were generated to define the sites in PSME that interact with Sox7 protein. Sox7 mutants were generated to identify the region of this protein required to regulate PSMA expression. Sox7 was also stably expressed in LNCaP/C4-2 and 22Rv1 cells to validate the regulation of PSMA expression by Sox7 in vivo. RESULTS: Sox7 mRNA expression negatively correlated with PSMA/FOLH1 and PSMAL/FOLH1B mRNA expression in Broad/Cornell, TCGA and MSKCC studies, but not in two studies containing only metastatic prostate tumors. PC-3 cells mostly expressed the 48.5 KDa isoform 2 of Sox7, and the depletion of this isoform did not restore PSMA expression. Ectopic expression of canonical, wild-type Sox7 in C4-2 and 22Rv1 cells suppressed PSMA protein expression. ChIP assay revealed that canonical Sox7 protein preferentially interacts with PSME in vivo, and EMSA identified the SOX box sites #2 and #4 in PSME as required for its interaction. Sox7 was capable of directly binding to PSME and suppressed PSME-mediated transcription. The NLS regions of Sox7, but not its ß-catenin interacting motif, are essential for this suppressing activity. Furthermore, restoration of wild-type Sox7 expression but not Sox7-NLS mutant in Sox7-null prostate cancer cell lines suppressed PSMA expression. CONCLUSIONS: The inactivation of canonical Sox7 is responsible for the upregulated expression of PSMA in non-metastatic prostate cancer.

Evolution and rapid spread of a reassortant A(H3N2) virus that predominated the 2017-2018 influenza season.

The 2017-2018 North American influenza season caused more hospitalizations and deaths than any year since the 2009 H1N1 pandemic. The majority of recorded influenza infections were caused by A(H3N2) viruses, with most of the virus's North American diversity falling into the A2 clade. Within A2, we observe a subclade which we call A2/re that rose to comprise almost 70 per cent of A(H3N2) viruses circulating in North America by early 2018. Unlike most fast-growing clades, however, A2/re contains no amino acid substitutions in the hemagglutinin (HA) segment. Moreover, hemagglutination inhibition assays did not suggest substantial antigenic differences between A2/re viruses and viruses sampled during the 2016-2017 season. Rather, we observe that the A2/re clade was the result of a reassortment event that occurred in late 2016 or early 2017 and involved the combination of the HA and PB1 segments of an A2 virus with neuraminidase (NA) and other segments a virus from the clade A1b. The success of this clade shows the need for antigenic analysis that targets NA in addition to HA. Our results illustrate the potential for non-HA drivers of viral success and necessitate the need for more thorough tracking of full viral genomes to better understand the dynamics of influenza epidemics.