Understanding Rejection Mechanisms of Trace Organic Contaminants by Polyamide Membranes via Data-Knowledge Codriven Machine Learning.

Data-driven machine learning (ML) provides a promising approach to understanding and predicting the rejection of trace organic contaminants (TrOCs) by polyamide (PA). However, various confounding variables, coupled with data scarcity, restrict the direct application of data-driven ML. In this study, we developed a data-knowledge codriven ML model via domain-knowledge embedding and explored its application in comprehending TrOC rejection by PA membranes. Domain-knowledge embedding enhanced both the predictive performance and the interpretability of the ML model. The contribution of key mechanisms, including size exclusion, charge effect, hydrophobic interaction, etc., that dominate the rejections of the three TrOC categories (neutral hydrophilic, neutral hydrophobic, and charged TrOCs) was quantified. Log D and molecular charge emerge as key factors contributing to the discernible variations in the rejection among the three TrOC categories. Furthermore, we quantitatively compared the TrOC rejection mechanisms between nanofiltration (NF) and reverse osmosis (RO) PA membranes. The charge effect and hydrophobic interactions possessed higher weights for NF to reject TrOCs, while the size exclusion in RO played a more important role. This study demonstrated the effectiveness of the data-knowledge codriven ML method in understanding TrOC rejection by PA membranes, providing a methodology to formulate a strategy for targeted TrOC removal.

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

Molecular Surveillance of MRSA in Raw Milk Provides Insight into MRSA Cross Species Evolution.

Methicillin-resistant Staphylococcus aureus (MRSA) in foods has been associated with severe infections in humans and animals worldwide. In the present study, the molecular characteristics of livestock-associated MRSA (LA-MRSA) and human-associated MRSA (hMRSA) isolates obtained in China, as well as MRSA isolates obtained from raw milk in 2018, were investigated. In total, 343 (20.38%; 343/1,683) S. aureus isolates were obtained from 1,683 raw milk samples from 100 dairy farms in 11 provinces across China. Among these, 49 (2.91%; 49/1,683) were mecA-positive MRSA. All LA-MRSA isolates were resistant to penicillin and highly resistant to erythromycin, sulfisoxazole, and clindamycin. Bioinformatic analysis the 49 genomes of LA-MRSA and 71 previously published hMRSA genomes isolated from Chinese individuals in 2018 indicated that blaZ, erm, ant(6)-Ia, aph(3')-III, tet(K), cat, and aph(2″)-Ia were more prevalent in MRSA from raw milk (P < 0.05) compared to hMRSA. Additionally, hMRSA isolates were more significantly associated with ST5 (P < 0.01) compared to LA-MRSA; in contrast, ST338 was more prevalent among LA-MRSA isolates (P < 0.01). Likewise, the SCCmec type II was only detected in hMRSA isolates, whereas SCCmec type V and IV were more prevalent among LA-MRSA (P < 0.01). Furthermore, core-genome phylogenetic analysis showed the endemic characteristics of LA-MRSA in local provinces, as well as the close evolutionary relationships between MRSA from cattle and humans. Finally, homology analysis of mecA and blaZ genetic contexts revealed a high possibility of horizontal transmission of MRSA resistance genes among raw milk-associated and hMRSA strains, which increases the risk for public health. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is considered a public health concern as it is resistant to multiple antibiotics, thus being in zoonotic transmission of antibiotic resistance genes. MRSA causes serious public health issues and leads to hard-to-treat infections in humans and animals; therefore, it was meaningful to determine the prevalence of MRSA in raw milk samples and investigate phenotype and genotype of antimicrobial resistance and molecular characteristics in livestock-associated MRSA (LA-MRSA) and human-associated MRSA (hMRSA) in China, which could provide a theoretical basis for preventing and controlling the spread of MRSA between livestock and humans.

Lipid Nanoparticle Delivery of Small Proteins for Potent In Vivo RAS Inhibition.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAS-targeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30─a charge-modified version of the protein─intracellularly in vitro and in vivo. This system efficiently encapsulates charge-modified proteins, facilitates delivery in up to 90% of cells in vitro, and maintains potency after at least 45 days of storage. In vivo, these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types─beyond RNA─for diverse therapeutic applications.

Development of a Real-Time Recombinase-Aided Amplification Method to Rapidly Detect Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant staphylococcus aureus (MRSA) is a major pathogen responsible for human hospital and community-onset diseases and severe invasive livestock infections. Rapid detection of MRSA is essential to control the spread of MRSA. Conventional identification methods and antibacterial susceptibility tests of MRSA are time-consuming. The commonly used qPCR assay also has the disadvantages of being complicated and expensive, restricting its application in resource-limited clinical laboratories. Here, a real-time fluorescent recombinase-assisted amplification (RAA) assay targeting the most conserved regions within the mecA gene of MRSA was developed and evaluated to detect MRSA. The detection limit of this assay was determined to be 10 copies/reaction of positive plasmids. The established RAA assay showed high specificity for MRSA detection without cross-reactivities with other clinically relevant bacteria. The diagnostic performance of real-time RAA was evaluated using 67 clinical S. aureus isolates from dairy farms, which were detected in parallel using the TaqMan probe qPCR assay. The results showed that 56 and 54 samples tested positive for MRSA by RAA and qPCR, respectively. The overall agreement between both assays was 97.01% (65/67), with a kappa value of 0.9517 (p < 0.001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R2 = 0.9012, p < 0.0001), indicating that this RAA assay possesses similar detection performance to the qPCR assay. In conclusion, our newly established RAA assay is a time-saving and convenient diagnostic tool suitable for MRSA detection and screening.

New clues about the global MRSA ST398: Emergence of MRSA ST398 from pigs in Qinghai, China.

This study, a part of the China national surveillance program on antimicrobial resistance in zoonotic bacteria, was to determine the phenotypic and genomic characteristics of endemic pig-associated Staphylococcus aureus ST398 strains in China. A total of 68 (48.9 %) S. aureus strains were recovered from 139 samples collected from two pig farms and one slaughterhouse in Qinghai province. Genomic characterization of All S. aureus strains was performed by WGS and their evolutionary relationships were assessed by phylogenetic analysis. Their susceptibilities to antimicrobials were determined using the broth dilution method. All S. aureus strains consisted of 41 ST398-t571 MSSA, 26 ST398-t011 MRSA and 1 ST5-t002 MRSA. Among these, ST398 was frequently identified in 67 S. aureus strains, suggesting that ST398 was a frequent source of MRSA and MSSA infections in Qinghai province and its possibility of transmission between individuals in pigs from farms and slaughterhouse. Meanwhile, Livestock-associated-MRSA ST398 in our study was establishing closely evolutionary relationships with MRSA ST 398 in Europe and Australia. The clues about closely relatedness of the global S. aureus ST398 underscore the potential public health risk of S. aureus ST398 in the pork supply chain and offer significant guidance for veterinary and human health.

Development of an organic acid compound disinfectant to control food-borne pathogens and its application in chicken slaughterhouses.

During poultry slaughter, cross-contamination of chicken carcasses with microorganisms (including drug-resistant bacteria) can occur because of incomplete disinfection during the pre-cooling process, and surface contact with contaminated tools and equipment. The use of disinfectants is the most common way to reduce the risk of cross-contamination and bacterial spread, as they can effectively reduce the number of bacteria. We developed a disinfectant consisting of organic acids and sodium dodecyl sulfate (SDS) and tested its bactericidal effects at different concentrations against Salmonella and Campylobacter. The main effective components in the disinfectant were citric acid, lactic acid, and SDS, and together they exerted a synergistic bactericidal effect. The bactericidal efficacy of the disinfectant increased with increasing concentrations of the 3 active ingredients. To reach a 100% reduction rate during a 15-s treatment in vitro, for Salmonella, the lowest concentrations of citric acid, lactic acid, and SDS were 0.06, 0.08, and 0.02%, respectively; and for Campylobacter, the lowest concentrations were 0.02, 0.025, and 0.0125%, respectively. The disinfectant remained effective in presence of interfering substances (e.g., 15% fetal bovine serum). Further experiments showed that the disinfectant inactivated sensitive bacteria as well as 23 drug-resistant strains of Salmonella and Campylobacter. Treatment with the disinfectant for 15 s decreased the concentrations of all tested strains by more than 4.7 log colony forming units per mL, and the reduction rate was as high as 100%. In on-site disinfection tests in chicken slaughterhouses, the disinfectant significantly reduced the number of pathogenic bacteria on carcasses during the pre-cooling process, and on tools (such as knives and gloves) during the segmentation process. Thus, this disinfectant has potential uses in preventing cross-contamination of food-borne pathogens (including resistant bacteria) in slaughterhouses.

Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc.

The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered "undruggable" with current small molecule drugs and biologics.

Emergence of livestock-associated MRSA ST398 from bulk tank milk, China.

OBJECTIVES: To detect livestock-associated MRSA (LA-MRSA) ST398 from bulk tank milk in China and to determine the phenotypic and genomic characteristics of the strains. METHODS: LA-MRSA ST398 strains were isolated from bulk tank milk samples in Shanghai and their susceptibilities to antimicrobials were determined using the broth dilution method. Genomic characterization of MRSA ST398 strains was performed by WGS and their evolutionary relationships were assessed by phylogenetic analysis. RESULTS: Two LA-MRSA ST398 isolates were recovered from bulk tank milk samples in two geographically distant farms in China. Whole-genome analysis strongly suggested that the LA-MRSA ST398 strains were closely related to the highly virulent hospital-associated MRSA (HA-MRSA) ST398 strains in China. CONCLUSIONS: The presence of LA-MRSA ST398 in bulk tank milk might be a serious threat to public health, highlighting the need for active surveillance of LA-MRSA in healthy cattle in China.

Genomic epidemiology of animal-derived tigecycline-resistant Escherichia coli across China reveals recent endemic plasmid-encoded tet(X4) gene.

Public health interventions to control the recent emergence of plasmid-mediated tigecycline resistance genes rely on a comprehensive understanding of its epidemiology and distribution over a wide range of geographical scales. Here we analysed an Escherichia coli collection isolated from pigs and chickens in China in 2018, and ascertained that the tet(X4) gene was not present at high prevalence across China, but was highly endemic in northwestern China. Genomic analysis of tet(X4)-positive E. coli demonstrated a recent and regional dissemination of tet(X4) among various clonal backgrounds and plasmids in northwestern China, whereas a parallel epidemic coincided with the independent acquisition of tet(X4) in E. coli from the remaining provinces. The high genetic similarity of tet(X4)-positive E. coli and human commensal E. coli suggests the possibility of its spreading into humans. Our study provides a systematic analysis of the current epidemiology of tet(X4) and identifies priorities for optimising timely intervention strategies.

Overlooked Role of Fe(IV) and Fe(V) in Organic Contaminant Oxidation by Fe(VI).

Fe(VI) has received increasing attention since it can decompose a wide range of trace organic contaminants (TrOCs) in water treatment. However, the role of short-lived Fe(IV) and Fe(V) in TrOC decomposition by Fe(VI) has been overlooked. Using methyl phenyl sulfoxide (PMSO), carbamazepine, and caffeine as probe TrOCs, we observed that the apparent second-order rate constants (kapp) between TrOCs and Fe(VI) determined with the initial kinetics data were strongly dependent on the initial molar ratios of TrOCs to Fe(VI). Furthermore, the kapp value increases gradually as the reaction proceeds. Several lines of evidence suggested that these phenomena were ascribed to the accumulation of Fe(IV) and Fe(V) arising from Fe(VI) decay. Kinetic models were built and employed to simulate the kinetics of Fe(VI) self-decay and the kinetics of PMSO degradation by Fe(VI). The modeling results revealed that PMSO was mainly degraded by Fe(IV) and Fe(V) rather than by Fe(VI) per se and Fe(V) played a dominant role, which was also supported by the density functional theory calculation results. Given that Fe(IV) and Fe(V) have much greater oxidizing reactivity than Fe(VI), this work urges the development of Fe(V)/Fe(IV)-based oxidation technology for efficient degradation of TrOCs.

Urban closed lakes: Nutrient sources, assimilative capacity and pollutant reduction under different precipitation frequencies.

Many natural and man-made urban lakes have been developed under urbanization. A unique feature of these lakes is the lack of an outlet; thus, they are defined as urban closed lakes (UCLs). UCLs are facing unexpected eutrophication under climate change and human activities. Our study assessed the trophic state, assimilative capacity (AC) and pollutant reduction of UCLs under different precipitation frequencies in Wuhan, China based on Carlson's Trophic State Index, assimilative capacity modelling, field investigations and observed data. The UCLs in Wuhan are nearly eutrophic in summer. Three primary nutrient sources are atmospheric deposition, pollutants carried in rainfall and nutrients released by sediments. TN and TP in the UCL water column are primarily contributed by surface runoff. The ACs of TN and TP in 2015 for Lingjiao Lake, Yue Lake, and Houxianghe Lake were 3472.07 kg, 13,800.99 kg, and 2805.58 kg, respectively, and 641.66 kg, 8386.79 kg, and 800.14 kg, respectively. The ACs of TN and TP were much higher at a 25% precipitation frequency (wet year) compared with a 50% frequency, and the lowest AC was observed at a 75% precipitation frequency (dry year). A comparison of the pollution load and AC showed that TN and TP reduction was highest in the dry and wet years, respectively. We found that specific meteorological conditions in the early stage led to the algal bloom. These results can facilitate governmental decision making in the future.

Plasmid-mediated tigecycline-resistant gene tet(X4) in Escherichia coli from food-producing animals, China, 2008-2018.

The recent emergence of plasmid-mediated tigecycline resistance genes, tet(X3) and tet(X4), in animals and humans in China would pose a foreseeable threat to public health. To illustrate this paradigm shift in tigecycline resistance, here, covering the period 2008-2018, we retrospectively analysed a national strain collection of Escherichia coli (n = 2254), obtained from chickens and pigs, in six representative provinces of China. The gene tet(X4) was identified in five pig isolates collected in 2016 and 2018 from the provinces of Sichuan (3/15, 2018), Henan (1/25, 2018) and Guangdong (1/28, 2016), but not in the isolates prior to 2016. None of the isolates was detected harbouring tet(X3). All tet(X4)-positive E. coli exhibited high levels of tigecycline resistance (MICs, 16-64 mg/L), and two were confirmed as colistin resistant, harbouring chromosome-borne mcr-1 gene. The gene tet(X4) was detected on a plasmid in all five isolates, whereas a co-location of tet(X4) on the chromosome of one isolate was observed. Diverse host strains and novel plasmids related to the tet(X4) gene were observed. Our timely findings of the recent emergence of tet(X4) gene in food animal support the rapid surveillance and eradication of this gene before it is established.

Cytosolic delivery of inhibitory antibodies with cationic lipids.

Antibodies can be developed to directly inhibit almost any protein, but their inability to enter the cytosol limits inhibitory antibodies to membrane-associated or extracellular targets. Developing a cytosolic antibody delivery system would offer unique opportunities to directly inhibit and study intracellular protein function. Here we demonstrate that IgG antibodies that are conjugated with anionic polypeptides (ApPs) can be complexed with cationic lipids originally designed for nucleic acid delivery through electrostatic interactions, enabling close to 90% cytosolic delivery efficiency with only 500 nM IgG. The ApP is fused to a small photoreactive antibody-binding domain (pAbBD) that can be site-specifically photocrosslinked to nearly all off-the-shelf IgGs, enabling easy exchange of cargo IgGs. We show that cytosolically delivered IgGs can inhibit the drug efflux pump multidrug resistance-associated protein 1 (MRP1) and the transcription factor NFκB. This work establishes an approach for using existing antibody collections to modulate intracellular protein function.

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases.

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating investigators to analyze and interpret genes, samples and clinical data across different types of cancer. Herein, we describe a practical operation procedure, taking ID1 (Inhibitor of DNA binding proteins 1) as an example, to characterize the expression patterns of biomarker and survival predictors of breast cancer based on pooled clinical datasets derived from online accessible databases, including ONCOMINE, bcGenExMiner v4.0 (Breast cancer gene-expression miner v4.0), GOBO (Gene expression-based Outcome for Breast cancer Online), HPA (The human protein atlas), and Kaplan-Meier plotter. The analysis began with querying the expression pattern of the gene of interest (e.g., ID1) in cancerous samples vs. normal samples. Then, the correlation analysis between ID1 and clinicopathological characteristics in breast cancer was performed. Next, the expression profiles of ID1 was stratified according to different subgroups. Finally, the association between ID1 expression and survival outcome was analyzed. The operation procedure simplifies the concept to integrate multidimensional data types at the gene level from different databases and test hypotheses regarding recurrence and genomic context of gene alteration events in breast cancer. This method can improve the credibility and representativeness of the conclusions, thereby, present informative perspective on a gene of interest.

Site-Specific C-Terminal Labeling of Recombinant Proteins with Proximity-Based Sortase-Mediated Ligation (PBSL).

S. aureus sortase A (SrtA), a calcium-dependent transpeptidase, is frequently employed to site-specifically label the C-terminus of recombinant proteins bearing an LPXTG SrtA recognition motif. Unfortunately, SrtA suffers from low turnover rates, resulting in poor ligation efficiencies even with optimized reaction conditions. In this chapter, we describe proximity-based sortase-mediated ligation (PBSL), which uses the SpyTag-SpyCatcher peptide-protein pair to link SrtA to target proteins and dramatically improves reaction rate and ligation efficiency.

Overcoming the Limitations of Sortase with Proximity-Based Sortase-Mediated Ligation (PBSL).

S. aureus sortase A (SrtA), a calcium-dependent bacterial transpeptidase, is commonly used to site-specifically label proteins containing a LPXTG SrtA recognition motif with a wide array of chemical moieties. A major limitation of sortase-mediated labeling, however, is SrtA's poor binding affinity to its recognition motif, resulting in long reaction times and poor ligation efficiencies. Here we describe proximity-based sortase-mediated ligation (PBSL), which utilizes the SpyTag-SpyCatcher peptide-protein pair to tether target proteins with a SrtA recognition motif to SrtA, dramatically increasing their local concentrations and overcoming this limitation.

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing.

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.