Microbiological safety of popular recreation swimming sites in Central California.

The objective of the study was to assess the microbiological safety of popular recreational swimming sites in Central California. Water samples were collected from eleven monitoring sites across the lower reaches of two watersheds for two consecutive swimming seasons (2012-2013), and levels of indicator and pathogenic microorganisms were determined. Data on ambient weather and water chemistry were collected for analyzing their associations with microorganisms in water. All water samples were positive for indicator E. coli with mean concentrations per site ranging from 3.07 to 216.11 MPN/100 ml in 2012 and 13.4 to 226.97 MPN/100 ml in 2013. Mean E. coli concentrations in 27% and 36% samplings sites exceeded the EPA 2012 Recreational Water Quality Criteria recommended mean concentration of ≤ 126 CFU/100 ml of E. coli, in 2012 and 2013, respectively. Cryptosporidium spp. oocysts were detected in all water samples from all sampling sites, with an overall prevalence of 50% and mean concentrations of 0.08 oocysts/l in 2012 and 0.19 oocysts/l in 2013. Giardia spp. cysts were detected at eight sites, with an overall prevalence of 28.8% and mean concentration of 0.2 cysts/l in both years. The majority of the detected Cryptosporidium spp. oocysts and Giardia spp. cysts appeared damaged under microscopy. E. coli O157:H7 was detected in 9% of water samples, with positive samples limited to three sites. Salmonella spp. were detected in all but one site across the two years, with mean concentrations of 0.94 MPN/l in 2012 and 1.85 MPN/l in 2013. Cryptosporidium spp. oocyst concentrations were negatively associated with 30-day mean wind speed and cumulative precipitation and dissolved oxygen in water. Giardia spp. cyst concentrations were positively associated with turbidity and pH of water and negatively associated with E. coli concentrations and 24-h mean air temperature. Salmonella spp. concentrations were positively associated with 30-day mean air temperature. The occurrence of E. coli O157:H7 was positively associated with previous 30-day cumulative precipitation.

Mumps Outbreak in a Marshallese Community - Denver Metropolitan Area, Colorado, 2016-2017.

In January 2017, the Colorado Department of Public Health and Environment (CDPHE) identified four epidemiologically linked cases of mumps among persons from a Marshallese community who were members of the same church in the Denver metropolitan area. During 2016-2017, sizable outbreaks of mumps reported in Arkansas, Hawaii, and Washington also affected the Marshallese population (1). CDPHE, the Tri-County Health Department (TCHD), and Denver Public Health collaborated to conduct an outbreak investigation during January-March 2017 using active and passive surveillance that identified 17 confirmed and 30 probable cases. Public health actions included conducting measles-mumps-rubella (MMR) vaccination clinics at local Marshallese churches; these resulted in the vaccination of 126 persons with ≥1 doses of MMR vaccine. Implementation of active surveillance and support from local Marshallese church leaders in promoting vaccination programs likely contributed to interruption of the outbreak.

Spatiotemporal Variability in Microbial Quality of Western US Agricultural Water Supplies: A Multistate Study.

In 2011, the US Congress passed the Food Safety Modernization Act, which tasks the US Food and Drug Administration to establish microbiological standards for agricultural water. However, little data are available for the microbiological quality of surface water irrigation supplies. During the 2015 irrigation season, we conducted a baseline study on the microbial water quality of large irrigation districts in California ( = 2) and Washington ( 4). Monthly samples ( 517) were analyzed for bacterial indicators (fecal coliforms, enterococci, and ) and pathogens ( spp., O157, and non-O157 Shiga toxin-producing [STEC]). Although there was a high degree of variability (µ ± SD = 59.13 ± 106.0), only 11% of samples (56/517) exceeded 126 colony-forming units (CFU) 100 mL, and only six samples exceeded 410 CFU 100 mL. Two volumes of water were collected for pathogen analysis (1 L and 10 L); prevalence of in 10-L samples (68149) was nearly double of that found in 1-L samples (132/517). We found STEC during ∼9% of sampling events (58/517); serotypes O26 and O45 were the most common at 31 and 26%, respectively. Pathogens were not associated with exceedance of the regulatory threshold, yet the odds of detecting increased approximately threefold (odds ration [O.R.] = 3.14, 0.0001) for every log increase in turbidity. Microbiological outcomes were highly district-specific, suggesting drivers of water quality vary across spatiotemporal scales. The true risk of contamination of produce from irrigation water supplies remains unknown, along with the optimal monitoring strategy to improve food safety.

Prevalence and Genomic Characterization of Escherichia coli O157:H7 in Cow-Calf Herds throughout California.

Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

Public Health Economic Burden Associated with Two Single Measles Case Investigations - Colorado, 2016-2017.

During July 2016-January 2017, two unrelated measles cases were identified in the Denver, Colorado area after patients traveled to countries with endemic measles transmission. Each case resulted in multiple exposures at health care facilities and public venues, and activated an immediate and complex response by local and state public health agencies, with activities led by the Tri-County Health Department (TCHD), which serves Adams, Arapahoe, and Douglas counties. To track the economic burden associated with investigating and responding to single measles cases, personnel hours and supply costs incurred during each investigation were tracked prospectively. No secondary cases of measles were identified in either investigation. Postexposure prophylaxis (PEP) was administered to 31 contacts involving the first case; no contacts of the second case were eligible for PEP because of a delay in diagnosing measles disease. Public health costs of disease investigation in the first and second case were estimated at $49,769 and $18,423, respectively. Single measles cases prompted coordinated public health action and were costly and resource-intensive for local public health agencies.

Multistate Evaluation of Microbial Water and Sediment Quality from Agricultural Recovery Basins.

Agricultural recovery basins are an important conservation practice designed to provide temporary storage of sediment and water on farms before low-volume discharge. However, food safety concerns have been raised regarding redistribution of captured sediment and water to fields used for human food production. The purpose of this study was to examine the potential microbiological risk that recovery basins may contribute to nearby produce fields and to evaluate characteristics that may influence or mitigate those risks. Water and sediment samples were collected from participating farms in three states and evaluated for bacterial indicators and pathogens over several months. Overall, 45% ( = 48) of water samples and less than 15% ( = 13) of sediment samples were positive for spp. In water samples, the occurrence of was positively associated with the use of surface water as a source of irrigation compared with groundwater as well as log-scale increases in concentration. In sediment samples, was associated with basin location (region) and basin fill levels. Sediment exposed to drying during dewatering had lower concentrations of indicator and a lower proportion of positives than submerged sediment from the same pond. Surrounding landscape characteristics, including vegetative coverage, proximity to livestock operations, and evidence of wildlife, were not correlated with pathogen occurrence in either sediment or water samples, suggesting that although habitat surrounding ponds may be an attractant to wildlife, those features may not contribute to increased pathogen occurrence in agricultural recovery basins.

Fecal Indicator and Pathogenic Bacteria and Their Antibiotic Resistance in Alluvial Groundwater of an Irrigated Agricultural Region with Dairies.

Surveys of microbiological groundwater quality were conducted in a region with intensive animal agriculture in California, USA. The survey included monitoring and domestic wells in eight concentrated animal feeding operations (CAFOs) and 200 small (domestic and community supply district) supply wells across the region. was not detected in groundwater, whereas O157:H7 and were each detected in 2 of 190 CAFO monitoring well samples. Nonpathogenic generic and spp. were detected in 24.2% (46/190) and 97.4% (185/190) groundwater samples from CAFO monitoring wells and in 4.2% (1/24) and 87.5% (21/24) of CAFO domestic wells, respectively. Concentrations of both generic and spp. were significantly associated with well depth, season, and the type of adjacent land use in the CAFO. No pathogenic bacteria were detected in groundwater from 200 small supply wells in the extended survey. However, 4.5 to 10.3% groundwater samples were positive for generic and . Concentrations of generic were not significantly associated with any factors, but concentrations of were significantly associated with proximity to CAFOs, seasons, and concentrations of potassium in water. Among a subset of and isolates from both surveys, the majority of (63.6%) and (86.1%) isolates exhibited resistance to multiple (≥3) antibiotics. Findings confirm significant microbial and antibiotic resistance loading to CAFO groundwater. Results also demonstrate significant attenuative capacity of the unconfined alluvial aquifer system with respect to microbial transport.

Genomic Surveillance of SARS-CoV-2 Sequence Variants at Universities in Southwest Idaho.

Although the impact of the SARS-CoV-2 pandemic on major metropolitan areas is broadly reported and readily available, regions with lower populations and more remote areas in the United States are understudied. The objective of this study is to determine the progression of SARS-CoV-2 sequence variants in a frontier and remote intermountain west state among university-associated communities. This study was conducted at two intermountain west universities from 2020 to 2022. Positive SARS-CoV-2 samples were confirmed by quantitative real-time reverse transcription-polymerase chain reaction and variants were identified by the next-generation sequencing of viral genomes. Positive results were obtained for 5355 samples, representing a positivity rate of 3.5% overall. The median age was 22 years. Viral genomic sequence data were analyzed for 1717 samples and phylogeny was presented. Associations between viral variants, age, sex, and reported symptoms among 1522 samples indicated a significant association between age and the Delta variant (B 1.167.2), consistent with the findings for other regions. An outbreak event of AY122 was detected August-October 2021. A 2-month delay was observed with respect to the timing of the first documented viral infection within this region compared to major metropolitan regions of the US.

IFN-ß mediates suppression of IL-12p40 in human dendritic cells following infection with virulent Francisella tularensis.

Active suppression of inflammation is a strategy used by many viral and bacterial pathogens, including virulent strains of the bacterium Francisella tularensis, to enable colonization and infection in susceptible hosts. In this study, we demonstrated that virulent F. tularensis strain SchuS4 selectively inhibits production of IL-12p40 in primary human cells via induction of IFN-ß. In contrast to the attenuated live vaccine strain, infection of human dendritic cells with virulent SchuS4 failed to induce production of many cytokines associated with inflammation (e.g., TNF-α and IL-12p40). Furthermore, SchuS4 actively suppressed secretion of these cytokines. Assessment of changes in the expression of host genes associated with suppression of inflammatory responses revealed that SchuS4, but not live vaccine strain, induced IFN-ß following infection of human dendritic cells. Phagocytosis of SchuS4 and endosomal acidification were required for induction of IFN-ß. Further, using a defined mutant of SchuS4, we demonstrated that the presence of bacteria in the cytosol was required, but not sufficient, for induction of IFN-ß. Surprisingly, unlike previous reports, induction of IFN-ß by F. tularensis was not required for activation of the inflammasome, was not associated with exacerbation of inflammatory responses, and did not control SchuS4 replication when added exogenously. Rather, IFN-ß selectively suppressed the ability of SchuS4-infected dendritic cells to produce IL-12p40. Together, these data demonstrated a novel mechanism by which virulent bacteria, in contrast to attenuated strains, modulate human cells to cause disease.

Zinc-Acetate-Amine Complexes as Precursors to ZnO and the Effect of the Amine on Nanoparticle Morphology, Size, and Photocatalytic Activity.

Zinc oxide is an environmentally friendly and readily synthesized semiconductor with many industrial applications. ZnO powders were prepared by alkali precipitation using different [Zn(acetate)2(amine)x] compounds to alter the particle size and aspect ratio. Slow precipitations from 95 °C solutions produced micron-scale particles with morphologies of hexagonal plates, rods, and needles, depending on the precursor used. Powders prepared at 65 °C with rapid precipitation yielded particles with minimal morphology differences, but particle size was dependent on the precursor used. The smallest particles were produced using precursors that yielded crystals with low aspect ratios during high-temperature synthesis. Particles produced during rapid synthesis had sizes ranging from 21-45 nm. The materials were characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, BET, and diffuse reflectance. The materials prepared using precursors with less-volatile amines were found to retain more organic material than ZnO produced using precursors with more volatile amines. The amount of organic material associated with the nanoparticles influenced the photocatalytic activity of the ZnO, with powders containing less organic material producing faster rate constants for the decolorizing of malachite green solutions under ultraviolet illumination, independent of particle size. [Zn(acetate)2(hydrazine)2] produced ZnO with the fastest rate constant and was recycled five times for dye degradation studies that revealed minimal to no reduction in catalytic efficiency.

Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline.

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis.

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1's functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.

Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.

MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9-DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

The presence of CD14 overcomes evasion of innate immune responses by virulent Francisella tularensis in human dendritic cells in vitro and pulmonary cells in vivo.

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.

Estradiol and progesterone affect enzymes but not glucose consumption in a mink uterine cell line (GMMe).

Cells lining the uterus are responsible for storage and secretion of carbohydrates to support early embryonic development. Histotrophic secretions contain glycogen and glycolytic products such as lactate and pyruvate. Insufficient carbohydrate storage as glycogen has been correlated with infertility in women. While it is clear that changes in estrogen (17-ß-estradiol (E2)) and progesterone (P4) in vivo affect the distribution of glucose in the uterine cells and secretions, the biochemical mechanism(s) by which they affect this crucial allocation is not well understood. Furthermore, in cultured uterine cells, neither E2 nor P4 affect glycogen storage without insulin present. We hypothesized that P4 and E2 alone affect the activity of glycolytic enzymes, glucose and glycolytic flux to increase glycogen storage (E2) and catabolism (P4) and increase pyruvate and lactate levels in culture. We measured the rate of glucose uptake and glycolysis in a mink immortalized epithelial cell line (GMMe) after 24-h exposure to 10 µM P4 and 10 nM E2 (pharmacologic levels) at 5 mM glucose and determined the kinetic parameters (Vmax, Km) of all enzymes. While the activities of many glycolytic enzymes in GMMe cells were shown to be decreased by E2 treatment, in contrast, glucose uptake, glycolytic flux and metabolites levels were not affected by the treatments. The cellular rationale for P4- and E2-induced decreases in the activity of enzymes may be to prime the system for other regulators such as insulin. In vivo, E2 and P4 may be necessary but not sufficient signals for uterine cycle carbohydrate allocation.

An Insight into Surface Topographical Parameters and Bacterial Adhesion: A Case Study of Listeria monocytogenes Scott A Attachment on 304 Stainless Steel.

ABSTRACT: Bacterial attachment on surfaces is an important biological and industrial concern. Many parameters affect cell attachment behavior, including surface roughness and other topographical features. An understanding of these relationships is critical in the light of recent outbreaks caused by foodborne bacteria. Postharvest packing lines have been identified as a potential source of cross-contamination with pathogens, which can cause subsequent foodborne illness. The objective of this article is to evaluate the influence of surface topographical features on bacterial attachment at various processing temperatures to determine the extent of bacterial colonization. Type 304 stainless steel surfaces and pathogenic Listeria monocytogenes Scott A were used for a detailed investigation. Two commonly used surface types, extruded and ground, were evaluated to determine differences in bacterial attachment on the same type of material. Fifteen surface topography parameters at three processing temperatures were studied to evaluate possible correlations with microbial attachment on these surfaces. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and confocal microscopy were used for both qualitative and quantitative analyses of surfaces. An analysis of variance and multivariate regression analysis were used to predict the attachment behavior of L. monocytogenes Scott A on stainless steel surfaces. Surface isotropy, average surface roughness, surface spacing, and processing temperatures were strongly correlated with bacterial attachment on 304 stainless steel material.

Bacillus subtilis: a potential growth promoter in weaned pigs in comparison to carbadox.

The study was conducted to investigate the efficacy of a probiotic Bacillus subtilis strain on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli and to compare the efficacy of B. subtilis with that of carbadox. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of four dietary treatments: negative control (NC, control diet without E. coli challenge), positive control (PC, control diet with E. coli challenge), and supplementation of 50 mg/kg of carbadox (antibiotic growth promotor [AGP]) or 2.56 × 109 CFU/kg of B. subtilis probiotics (PRO). The experiment lasted for 28 d with 7 d before and 21 d after the first E. coli inoculation. Fecal and blood samples were collected on days 0, 3, 7, 14, and 21 post inoculation (PI) to analyze ß-hemolytic coliforms and complete blood cell count, respectively. Diarrhea score was recorded daily for each pig to calculate the frequency of diarrhea. All pigs were euthanized at day 21 PI to collect jejunal and ileal mucosa for gene expression analysis. Pigs in AGP had greater (P < 0.05) BW on days 7, 14, and 21 PI than pigs in PC and PRO groups. Supplementation of PRO enhanced pigs' BW on day 21 PI compared with the PC. Escherichia coli F18 challenge reduced (P < 0.05) average daily gain (ADG) and feed efficiency from day 0 to 21 PI, while supplementation of carbadox or PRO enhanced ADG and feed efficiency in E. coli F18-challenged pigs from day 0 to 21 PI. Pigs in AGP and PRO groups had reduced (P < 0.05) frequency of diarrhea throughout the experiment and fecal ß-hemolytic coliforms on day 7 PI than pigs in the PC. Pigs in PRO had greater (P < 0.05) gene expression of CLDN1 in jejunal mucosa than pigs in the PC. Supplementation of carbadox or PRO reduced (P < 0.05) the gene expression of IL6 and PTGS2 in ileal mucosa of E. coli-infected pigs compared with pigs in the PC. Pigs in the PRO group had lower (P < 0.05) white blood cell number and neutrophil count, and serum haptoglobin concentration on day 7 PI, and less (P < 0.05) monocyte count on day 14 PI, compared with PC. In conclusion, supplementation of probiotic B. subtilis could enhance disease resistance and promote the growth performance of weaned pigs under disease challenge conditions. The potential mechanisms include but not limited to enhanced gut barrier integrity and local and systemic immune responses of weaned pigs.

Direct and indirect impairment of human dendritic cell function by virulent Francisella tularensis Schu S4.

The gram-negative, facultative intracellular bacterium Francisella tularensis causes acute, lethal pneumonic disease following infection with only 10 CFU. The mechanisms used by the bacterium to accomplish this in humans are unknown. Here, we demonstrate that virulent, type A F. tularensis strain Schu S4 efficiently infects and replicates in human myeloid dendritic cells (DCs). Despite exponential replication over time, Schu S4 failed to stimulate transforming growth factor beta, interleukin-10 (IL-10), IL-6, IL-1beta, IL-12, tumor necrosis factor alpha, alpha interferon (IFN-alpha), and IFN-beta throughout the course of infection. Schu S4 also suppressed the ability of directly infected DCs to respond to different Toll-like receptor agonists. Furthermore, we also observed functional inhibition of uninfected bystander cells. This inhibition was mediated, in part, by a heat-stable bacterial component. Lipopolysaccharide (LPS) from Schu S4 was present in Schu S4-conditioned medium. However, Schu S4 LPS was weakly inflammatory and failed to induce suppression of DCs at concentrations below 10 microg/ml, and depletion of Schu S4 LPS did not significantly alleviate the inhibitory effect of Schu S4-conditioned medium in uninfected human DCs. Together, these data show that type A F. tularensis interferes with the ability of a central cell type of the immune system, DCs, to alert the host of infection both intra- and extracellularly. This suggests that immune dysregulation by F. tularensis operates on a broader and more comprehensive scale than previously appreciated.

Contribution of NADH increases to ethanol's inhibition of retinol oxidation by human ADH isoforms.

BACKGROUND: A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans-retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free reduced nicotinamide adenine dinucleotide (NADH) in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms. METHODS: To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of 4 reactions: (1) ADH oxidation of ethanol and NAD(+), (2) ADH oxidation of retinol and NAD(+), (3) oxidation of ethanol by a generalized Ethanol(oxidase) that uses NAD(+), (4) NADH(oxidase) which carries out NADH turnover. RESULTS: Using the metabolic modeling package ScrumPy, we have shown that the ethanol-induced increase in NADH contributes from 0% to 90% of the inhibition by ethanol, depending on (ethanol) and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanol(oxidase) and the NADH(oxidase) contribute as well. CONCLUSIONS: Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases.