Prevalence and abundance of antibiotic-resistant genes in culturable bacteria inhabiting a non-polar passu glacier, karakorum mountains range, Pakistan.

Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; blaAmpC 6 (100%), blaVIM-1, blaSHV and blaDHA 5 (100%) each, blaOXA-1 1 (100%), blaCMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for blaKPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in blaNDM-1, blaOXA-1, blaSHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance.

Is PFAS from land applied municipal biosolids a significant source of human exposure via groundwater?

Per and polyfluoroakyl substances (PFAS) are emerging contaminants of critical concern commonly found in the bloodstream of most humans in the U.S. They are present in both Class A and B municipal biosolids. The potential for contamination of groundwater following land application of biosolids and subsequent leaching of PFAS through soil is one of several potential impacts that have generated discussions of possible bans on land application. In this commentary, we discuss the many factors that need to be considered to address the question: "Is PFAS from land applied biosolids a significant source of human exposure via groundwater?" The occurrence of PFAS in biosolids and biosolids-amended soils is discussed, as are the many factors that affect the potential for subsequent groundwater contamination. Additional critical factors are also noted.

Variant-specific SARS-CoV-2 shedding rates in wastewater.

Previous studies show that SARS-CoV-2 waste shedding rates vary by community and are influenced by multiple factors; however, differences in shedding rates across multiple variants have yet to be evaluated. The purpose of this work is to build on previous research that evaluated waste shedding rates for early SARS-CoV-2 and the Delta variant, and update population level waste shedding rates for the more-recent Omicron variant in six communities. Mean SARS-CoV-2 waste shedding rates were found to increase with the predominance of the Delta variant and subsequently decrease with Omicron infections. Interestingly, the Delta stage had the highest mean shedding rates and was associated with the most severe disease symptoms reported in other clinical studies, while Omicron, exhibiting reduced symptoms, had the lowest mean shedding rates. Additionally, shedding rates were most consistent across communities during the Omicron stage. This is the first paper to identify waste shedding rates specific to the Omicron variant and fills a knowledge gap critical to disease prevalence modeling.

Actomyosin Complex.

Formation of cross-bridges between actin and myosin occurs ubiquitously in eukaryotic cells and mediates muscle contraction, intracellular cargo transport, and cytoskeletal remodeling. Myosin motors repeatedly bind to and dissociate from actin filaments in a cycle that transduces the chemical energy from ATP hydrolysis into mechanical force generation. While the general layout of surface elements within the actin-binding interface is conserved among myosin classes, sequence divergence within these motifs alters the specific contacts involved in the actomyosin interaction as well as the kinetics of mechanochemical cycle phases. Additionally, diverse lever arm structures influence the motility and force production of myosin molecules during their actin interactions. The structural differences generated by myosin's molecular evolution have fine-tuned the kinetics of its isoforms and adapted them for their individual cellular roles. In this chapter, we will characterize the structural and biochemical basis of the actin-myosin interaction and explain its relationship with myosin's cellular roles, with emphasis on the structural variation among myosin isoforms that enables their functional specialization. We will also discuss the impact of accessory proteins, such as the troponin-tropomyosin complex and myosin-binding protein C, on the formation and regulation of actomyosin cross-bridges.

Population level SARS-CoV-2 fecal shedding rates determined via wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been utilized as an early warning tool to anticipate disease outbreaks, especially during the COVID-19 pandemic. However, COVID-19 disease models built from wastewater-collected data have been limited by the complexities involved in estimating SARS-CoV-2 fecal shedding rates. In this study, wastewater from six municipalities in Arizona and Florida with distinct demographics were monitored for SARS-CoV-2 RNA between September 2020 and December 2021. Virus concentrations with corresponding clinical case counts were utilized to estimate community-wide fecal shedding rates that encompassed all infected individuals. Analyses suggest that average SARS-CoV-2 RNA fecal shedding rates typically occurred within a consistent range (7.53-9.29 log10 gc/g-feces); and yet, were unique to each community and influenced by population demographics. Age, ethnicity, and socio-economic factors may have influenced shedding rates. Interestingly, populations with median age between 30 and 39 had the greatest fecal shedding rates. Additionally, rates remained relatively constant throughout the pandemic provided conditions related to vaccination and variants were unchanged. Rates significantly increased in some communities when the Delta variant became predominant. Findings in this study suggest that community-specific shedding rates may be appropriate in model development relating wastewater virus concentrations to clinical case counts.

Enumerating asymptomatic COVID-19 cases and estimating SARS-CoV-2 fecal shedding rates via wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) was utilized to monitor SARS-CoV-2 RNA in sewage collected from manholes specific to individual student dormitories (dorms) at the University of Arizona in the fall semester of 2020, which led to successful identification and reduction of SARS-CoV-2 transmission events. Positive wastewater samples triggered clinical testing of residents within that dorm; thus, SARS-CoV-2 infected individuals were identified regardless of symptom expression. This current study examined clinical testing data to determine the abundance of asymptomatic versus symptomatic cases in these defined communities. Nasal and nasopharyngeal swab samples processed via antigen and PCR tests indicated that 79.2% of SARS-CoV-2 infections were asymptomatic, and only 20.8% of positive cases reported COVID-19 symptoms at the time of testing. Clinical data was paired with corresponding wastewater virus concentrations, which enabled calculation of viral shedding rates in feces per infected person. Mean shedding rates averaged from positive wastewater samples across all dorms were 7.30 ± 0.67 log10 genome copies per gram of feces (gc/g-feces) based on the N1 gene. Quantification of SARS-CoV-2 fecal shedding rates from infected individuals has been the critical missing component necessary for WBE models to measure and predict SARS-CoV-2 infection prevalence in communities. The findings from this study can be utilized to create models that can be used to inform public health prevention and response actions.

Incidence of Pfas in soil following long-term application of class B biosolids.

This field study investigated the impact of long-term land application of biosolids on PFAS presence in soils that received annual repetitive land application of Class B biosolids from 1984 to 2019. Soil samples were collected from three depths of 30.5, 91 and 183 cm below land surface. Biosolid and groundwater samples used for irrigation were also collected. Concentrations measured for 18 PFAS compounds were evaluated to assess incidence rates and potential impact on groundwater. No PFAS analytes were detected at the three sampling depths for soil samples collected from undisturbed sites with no history of agriculture, irrigation, or biosolids application (background control sites). Relatively low mean concentrations of PFAS ranging from non-detect to 1.9 µg/kg were measured in soil samples collected from sites that were used for agriculture and that received irrigation with groundwater, but never received biosolids. PFAS concentrations in soils amended with biosolids were similarly low, ranging from non-detect to a mean concentration of 4.1 µg/kg. PFOS was observed at the highest concentrations, followed by PFOA for all locations. PFAS detected in the irrigation water were also present in the soil. These results indicate that biosolids and irrigation water are both important sources of PFAS present in the soils for all of the study sites. Not all PFAS detected in the biosolids were detected in the soil. Very long chain PFAS present in the biosolids were not detected or were detected at very low levels for soil, suggesting potential preferential retention within the biosolids. The precursor NMeFOSAA was present at the second highest concentrations in the biosolids but not detected in soil, indicating possible occurrence of transformation reactions. The total PFAS soil concentrations exhibited significant attenuation with depth, with a mean attenuation of 73% at the 183 cm depth. Monotonically decreasing concentrations with depth were observed for the longer-chain PFAS.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention.

Wastewater-based epidemiology has potential as an early-warning tool for determining the presence of COVID-19 in a community. The University of Arizona (UArizona) utilized WBE paired with clinical testing as a surveillance tool to monitor the UArizona community for SARS-CoV-2 in near real-time, as students re-entered campus in the fall. Positive detection of virus RNA in wastewater lead to selected clinical testing, identification, and isolation of three infected individuals (one symptomatic and two asymptomatic) that averted potential disease transmission. This case study demonstrated the value of WBE as a tool to efficiently utilize resources for COVID-19 prevention and response. Thus, WBE coupled with targeted clinical testing was further conducted on 13 dorms during the course of the Fall semester (Table 3). In total, 91 wastewater samples resulted in positive detection of SARS-CoV-2 RNA that successfully provided an early-warning for at least a single new reported case of infection (positive clinical test) among the residents living in the dorm. Overall, WBE proved to be an accurate diagnostic for new cases of COVID-19 with an 82.0% positive predictive value and an 88.9% negative predictive value. Increases in positive wastewater samples and clinical tests were noted following holiday-related activities. However, shelter-in-place policies proved to be effective in reducing the number of daily reported positive wastewater and clinical tests. This case study provides evidence for WBE paired with clinical testing and public health interventions to effectively contain potential outbreaks of COVID-19 in defined communities.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

TRIM72 promotes alveolar epithelial cell membrane repair and ameliorates lung fibrosis.

BACKGROUND: Chronic tissue injury was shown to induce progressive scarring in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), while an array of repair/regeneration and stress responses come to equilibrium to determine the outcome of injury at the organ level. In the lung, type I alveolar epithelial (ATI) cells constitute the epithelial barrier, while type II alveolar epithelial (ATII) cells play a pivotal role in regenerating the injured distal lungs. It had been demonstrated that eukaryotic cells possess repair machinery that can quickly patch the damaged plasma membrane after injury, and our previous studies discovered the membrane-mending role of Tripartite motif containing 72 (TRIM72) that expresses in a limited number of tissues including the lung. Nevertheless, the role of alveolar epithelial cell (AEC) repair in the pathogenesis of IPF has not been examined yet. METHOD: In this study, we tested the specific roles of TRIM72 in the repair of ATII cells and the development of lung fibrosis. The role of membrane repair was accessed by saponin assay on isolated primary ATII cells and rat ATII cell line. The anti-fibrotic potential of TRIM72 was tested with bleomycin-treated transgenic mice. RESULTS: We showed that TRIM72 was upregulated following various injuries and in human IPF lungs. However, TRIM72 expression in ATII cells of the IPF lungs had aberrant subcellular localization. In vitro studies showed that TRIM72 repairs membrane injury of immortalized and primary ATIIs, leading to inhibition of stress-induced p53 activation and reduction in cell apoptosis. In vivo studies demonstrated that TRIM72 protects the integrity of the alveolar epithelial layer and reduces lung fibrosis. CONCLUSION: Our results suggest that TRIM72 protects injured lungs and ameliorates fibrosis through promoting post-injury repair of AECs.

Potential indicators of virus transport and removal during soil aquifer treatment of treated wastewater effluent.

Increased water demands have led to a notable interest in the use of treated wastewater for reuse. Typically, this results from the implementation of advanced treatment of final effluent from wastewater treatment plants prior to reuse for potable or non-potable purposes. Soil aquifer treatment (SAT) is a natural treatment process in which water from sources of varying quality is infiltrated into the soil to further improve its quality. The goal of this study was to determine the log10 reduction values (LRVs) of viruses naturally present in treated effluent and evaluate two potential indicators of virus removal and transport, pepper mild mottle virus (PMMoV) and crAssphage, during SAT of treated effluent. Groundwater was sampled at three wells with different attributes within the Sweetwater Recharge Facility (SWRF) in Tucson, AZ. These sites vary greatly in operational parameters such as effluent infiltration rates and wetting/drying cycles, which may influence virus removal efficiency. Detection of adenovirus, enterovirus, PMMoV, and crAssphage were determined by qPCR/RT-qPCR and log10 reduction values (LRVs) were determined. PMMoV and crAssphage were detected in groundwater associated with a set of recharge basins that exhibited shorter wetting/drying cycles and faster infiltration rates. LRVs for crAssphage and PMMoV at this site ranged from 3.9 to 5.8, respectively. Moreover, PMMoV was detected downflow of the SAT sites, indicating the potential degradation of microbial groundwater quality in the region surrounding managed aquifer recharge facilities. Overall, PMMoV and crAssphage showed potential as conservative process indicators of virus removal during SAT, particularly for attribution of LRV credits. Moreover, the detection of these viruses indicated the potential influence of wetting/drying cycles on virus removal by SAT, a parameter that has not yet been studied with respect to biological contaminants.

Reduction of erythromycin resistance gene erm(F) and class 1 integron-integrase genes in wastewater by Bardenpho treatment.

Wastewaters routinely contain antibiotic-resistant bacteria (ARB) and genes (ARG) that are removed to a varying degree during wastewater treatment. This study investigated the removal of the erythromycin ribosome methylase class F (erm(F)) and class 1 integron-integrase (intI1) genes at each stage from two water resource recovery facilities in southern Arizona. Although genes were significantly reduced by Bardenpho treatment, erm(F) and intI1 were still observed in ≥ 9 and 7 out of 12 secondary effluent samples. Primary processes via sedimentation or dissolved air flotation, as well as chlorine disinfection, did not significantly impact erm(F) and intI1 concentrations. Therefore, Bardenpho treatment was critical to reduce erm(F) and intI1. Concentrations of erm(F) and intI1 were compared with each other and other markers for anthropogenic pollution. Results from this study support intI1 as one suitable marker to measure erythromycin resistance genes in wastewater, as intI1 was found at higher concentrations, persisted more throughout treatment, and correlated with erm(F) at nearly every treatment stage. PRACTITIONER POINTS: Bardenpho treatment was the key process responsible for the reduction of intI1 and erm(F) genes during wastewater treatment. Primary treatment and chlorine disinfection did not impact erm(F) and intI1 gene concentrations. The intI1 gene is a suitable marker for measuring erm(F) genes in wastewater.

Alveolar epithelial cell integrity: A venerable target in the lung.

Eukaryotic cells have developed a litany of conserved mechanisms to deal with membrane injuries. The first line of defense consists of homeostatic regulation of membrane tension as a preventative measure against the occurrence of injury. When these measures fail, cells can engage in elaborate signaling mechanisms aimed at quickly restoring integrity. Based on the overall direction of membrane lipid trafficking, these repair mechanisms can be divided into three broad categories: exocytosis, endocytosis, and ectocytosis. For alveolar epithelial cells (AECs), repair of endogenous cell populations is especially important for the prevention of severe lung pathologies. We provide a focus on the pulmonary setting within this chapter while incorporating relevant findings from other cell types. We emphasize the signals and molecular moieties that have demonstrated critical involvement in the repair process within AECs and other cell types that constantly encounter threats to their membrane integrity.

Countering the Modern Metabolic Disease Rampage With Ancestral Endocannabinoid System Alignment.

When primitive vertebrates evolved from ancestral members of the animal kingdom and acquired complex locomotive and neurological toolsets, a constant supply of energy became necessary for their continued survival. To help fulfill this need, the endocannabinoid (eCB) system transformed drastically with the addition of the cannabinoid-1 receptor (CB1R) to its gene repertoire. This established an eCB/CB1R signaling mechanism responsible for governing the whole organism's energy balance, with its activation triggering a shift toward energy intake and storage in the brain and the peripheral organs (i.e., liver and adipose). Although this function was of primal importance for humans during their pre-historic existence as hunter-gatherers, it became expendable following the successive lifestyle shifts of the Agricultural and Industrial Revolutions. Modernization of the world has further increased food availability and decreased energy expenditure, thus shifting the eCB/CB1R system into a state of hyperactive deregulated signaling that contributes to the 21st century metabolic disease pandemic. Studies from the literature supporting this perspective come from a variety of disciplines, including biochemistry, human medicine, evolutionary/comparative biology, anthropology, and developmental biology. Consideration of both biological and cultural evolution justifies the design of improved pharmacological treatments for obesity and Type 2 diabetes (T2D) that focus on peripheral CB1R antagonism. Blockade of peripheral CB1Rs, which universally promote energy conservation across the vertebrate lineage, represents an evolutionary medicine strategy for clinical management of present-day metabolic disorders.

Removal of fecal indicator bacteria and antibiotic resistant genes in constructed wetlands.

Wastewater discharge evidently increased bacterial diversity in the receiving waterbodies. The objective of this study was to evaluate the effectiveness of a constructed wetland in reducing fecal indicator bacteria (FIB) and antibiotic resistant genes (ARGs). We determined the prevalence and attenuation of fecal indicator bacteria including Escherichia coli and enterococci, along with ARGs, and human-associated Bacteroidales (HF183) markers by quantitative polymerase chain reaction (qPCR) method. Three types of water samples (inlet, intermediate, and outlet) from a constructed wetland were collected once a month from May to December in 2013. The overall reduction of E. coli was 50.0% based on culture method. According to the qPCR result, the overall removal rate of E. coli was only 6.7%. Enterococci were found in 62.5% of the wetland samples. HF183 genetic marker was detected in all final effluent samples with concentration ranging from 1.8 to 4.22 log10 gene copies (GC)/100 ml. Of the ARGs tested, erythromycin resistance genes (ermF) were detected in 79.2% of the wetland samples. The class 1 integrase (intI1) was detected in all water samples with concentration ranging from 0.83 to 5.54 log10 GC/100 ml. The overall removal rates of enterococci, HF183, intI1, and ermF were 84.0%, 66.6%, 67.2%, and 13.1%, respectively.

Assessing the spatial and temporal variability of bacterial communities in two Bardenpho wastewater treatment systems via Illumina MiSeq sequencing.

Next generation sequencing provides new insights into the diversity and ecophysiology of bacteria communities throughout wastewater treatment plants (WWTP), as well as the fate of pathogens in wastewater treatment system. In the present study, we investigated the bacterial communities and human-associated Bacteroidales (HF183) marker in two WWTPs in North America that utilize Bardenpho treatment processes. Although, most pathogens were eliminated during wastewater treatment, some pathogenic bacteria were still observed in final effluents. The HF183 genetic marker demonstrated significant reductions between influent and post-Bardenpho treated samples in each WWTP, which coincided with changes in bacteria relative abundances and community compositions. Consistent with previous studies, the major phyla in wastewater samples were predominantly comprised by Proteobacteria (with Gammaproteobacteria and Alphaproteobacteria among the top two classes), Actinobacteria, Bacteroidetes, and Firmicutes. Dominant genera were often members of Proteobacteria and Firmicutes, including several pathogens of public health concern, such as Pseudomonas, Serratia, Streptococcus, Mycobacterium and Arcobacter. Pearson correlations were calculated to observe the seasonal variation of relative abundances of gene sequences at different levels based on the monthly average temperature. These findings profile how changes in bacterial communities can function as a robust method for monitoring wastewater treatment quality and performance for public and environmental health purposes.

Comparative Assessment of BGM and PLC/PRF/5 Cell Lines for Enteric Virus Detection in Biosolids.

The buffalo green monkey (BGM) cell line is required for the detection of enteric viruses in biosolids through a total culturable viral assay (TCVA) by the United States Environmental Protection Agency. In the present study, BGM and PLC/PRF/5 cell lines were evaluated for TCVA and for their use in determining the incidence of adenoviruses and enteroviruses in raw sludge and Class B biosolids. Six raw sludge and 17 Class B biosolid samples were collected from 13 wastewater treatment plants from seven U.S. states. Samples were processed via organic flocculation and concentrate volumes equivalent to 4 g total solids were assayed on BGM and PLC/PRF/5 cells. Cell monolayers were observed for cytopathic effect (CPE) after two 14-days passages. Cell lysates were tested for the presence of adenoviruses and enteroviruses by PCR or RT-PCR. The PLC/PRF/5 cells detected more culturable viruses than the BGM cells by CPE (73.9% vs. 56.5%, respectively). 52% of the samples were positive for CPE using both cell lines. No viruses were detected in either cell line by PCR in flasks in which CPE was not observed. No adenoviruses were detected in 13 CPE-positive samples from BGM lysates. In contrast, of the 17 samples exhibiting CPE on PLC/PRF/5 cells, 14 were positive for adenoviruses (82.4%). In conclusion, PLC/PRF/5 cells were superior for the detection of adenoviruses in both raw sludge and Class B biosolids. Thus, the use of BGM cells alone for TCVA may underestimate the viral concentration in sludge/biosolid samples.

Near Real-Time Detection of E. coli in Reclaimed Water.

Advanced treatment of reclaimed water prior to potable reuse normally results in the inactivation of bacterial populations, however, incremental treatment failure can result in bacteria, including pathogens, remaining viable. Therefore, potential microorganisms need to be detected in real-time to preclude potential adverse human health effects. Real-time detection of microbes presents unique problems which are dependent on the water quality of the test water, including parameters such as particulate content and turbidity, and natural organic matter content. In addition, microbes are unusual in that: (i) viability and culturability are not always synonymous; (ii) viability in water can be reduced by osmotic stress; and (iii) bacteria can invoke repair mechanisms in response to UV disinfection resulting in regrowth of bacterial populations. All these issues related to bacteria affect the efficacy of real-time detection for bacteria. Here we evaluate three different sensors suitable for specific water qualities. The sensor A is an on-line, real-time sensor that allows for the continuous monitoring of particulates (including microbial contaminants) using multi-angle-light scattering (MALS) technology. The sensor B is a microbial detection system that uses optical technique, Mie light scattering, for particle sizing and fluorescence emission for viable bacteria detection. The last sensor C was based on adenosine triphosphate (ATP) production. E. coli was used a model organism and out of all tested sensors, we found the sensor C to be the most accurate. It has a great potential as a surrogate parameter for microbial loads in test waters and be useful for process control in treatment trains.

Inhaled TRIM72 Protein Protects Ventilation Injury to the Lung through Injury-guided Cell Repair.

Studies showed that TRIM72 is essential for repair of alveolar cell membrane disruptions, and exogenous recombinant human TRIM72 protein (rhT72) demonstrated tissue-mending properties in animal models of tissue injury. Here we examine the mechanisms of rhT72-mediated lung cell protection in vitro and test the efficacy of inhaled rhT72 in reducing tissue pathology in a mouse model of ventilator-induced lung injury. In vitro lung cell injury was induced by glass beads and stretching. Ventilator-induced lung injury was modeled by injurious ventilation at 30 ml/kg tidal volume. Affinity-purified rhT72 or control proteins were added into culture medium or applied through nebulization. Cellular uptake and in vivo distribution of rhT72 were detected by imaging and immunostaining. Exogenous rhT72 maintains membrane integrity of alveolar epithelial cells subjected to glass bead injury in a dose-dependent manner. Inhaled rhT72 decreases the number of fatally injured alveolar cells, and ameliorates tissue-damaging indicators and cell injury markers after injurious ventilation. Using in vitro stretching assays, we reveal that rhT72 improves both cellular resilience to membrane wounding and membrane repair after injury. Image analysis detected rhT72 uptake by rat alveolar epithelial cells, which can be inhibited by a cholesterol-disrupting agent. In addition, inhaled rhT72 distributes to the distal lungs, where it colocalizes with phosphatidylserine detection on nonpermeabilized lung slices to label wounded cells. In conclusion, our study showed that inhaled rhT72 accumulates in injured lungs and protects lung tissue from ventilator injury, the mechanisms of which include improving cell resilience to membrane wounding, localizing to injured membrane, and augmenting membrane repair.