Acetaminophen influences musculoskeletal signaling but not adaptations to endurance exercise training.

Acetaminophen (ACE) is a widely used analgesic and antipyretic drug with various applications, from pain relief to fever reduction. Recent studies have reported equivocal effects of habitual ACE intake on exercise performance, muscle growth, and risks to bone health. Thus, this study aimed to assess the impact of a 6-week, low-dose ACE regimen on muscle and bone adaptations in exercising and non-exercising rats. Nine-week-old Wistar rats (n = 40) were randomized to an exercise or control (no exercise) condition with ACE or without (placebo). For the exercise condition, rats ran 5 days per week for 6 weeks at a 5% incline for 2 min at 15 cm/s, 2 min at 20 cm/s, and 26 min at 25 cm/s. A human equivalent dose of ACE was administered (379 mg/kg body weight) in drinking water and adjusted each week based on body weight. Food, water intake, and body weight were measured daily. At the beginning of week 6, animals in the exercise group completed a maximal treadmill test. At the end of week 6, rats were euthanized, and muscle cross-sectional area (CSA), fiber type, and signaling pathways were measured. Additionally, three-point bending and microcomputer tomography were measured in the femur. Follow-up experiments in human primary muscle cells were used to explore supra-physiological effects of ACE. Data were analyzed using a two-way ANOVA for treatment (ACE or placebo) and condition (exercise or non-exercise) for all animal outcomes. Data for cell culture experiments were analyzed via ANOVA. If omnibus significance was found in either ANOVA, a post hoc analysis was completed, and a Tukey's adjustment was used. ACE did not alter body weight, water intake, food intake, or treadmill performance (p > .05). There was a treatment-by-condition effect for Young's Modulus where placebo exercise was significantly lower than placebo control (p < .05). There was no treatment by condition effects for microCT measures, muscle CSA, fiber type, or mRNA expression. Phosphorylated-AMPK was significantly increased with exercise (p < .05) and this was attenuated with ACE treatment. Furthermore, phospho-4EBP1 was depressed in the exercise group compared to the control (p < .05) and increased in the ACE control and ACE exercise group compared to placebo exercise (p < .05). A low dose of ACE did not influence chronic musculoskeletal adaptations in exercising rodents but acutely attenuated AMPK phosphorylation and 4EBP1 dephosphorylation post-exercise.

Replacement of dietary carbohydrate with protein increases fat mass and reduces hepatic triglyceride synthesis and content in female obese Zucker rats.

Previous studies have demonstrated both energy restriction (ER) and higher protein (HP), lower carbohydrate (LC) diets downregulate hepatic de novo lipogenesis. Little is known about the independent and combined impact of ER and HP/LC diets on tissue-specific lipid kinetics in leptin receptor-deficient, obese rodents. This study investigated the effects of ER and dietary macronutrient content on body composition; hepatic, subcutaneous adipose tissue (SAT), and visceral AT (VAT) lipid metabolic flux (2 H2 O-labeling); and blood and liver measures of cardiometabolic health in six-week-old female obese Zucker rats (Leprfa+/fa+ ). Animals were randomized to a 10-week feeding intervention: ad libitum (AL)-HC/LP (76% carbohydrate/15% protein), AL-HP/LC (35% protein/56% carbohydrate), ER-HC/LP, or ER-HP/LC. ER groups consumed 60% of the feed consumed by AL. AL gained more fat mass than ER (P-energy = 0.012) and HP/LC gained more fat mass than HC/LP (P-diet = 0.025). Hepatic triglyceride (TG) concentrations (P-interaction = 0.0091) and absolute hepatic TG synthesis (P-interaction = 0.012) were lower in ER-HP/LC versus ER-HC/LP. ER had increased hepatic, SAT, and VAT de novo cholesterol fractional synthesis, absolute hepatic cholesterol synthesis, and serum cholesterol (P-energy≤0.0035). A HP/LC diet, independent of energy intake, led to greater gains in fat mass. A HP/LC diet, in the context of ER, led to reductions in absolute hepatic TG synthesis and TG content. However, ER worsened cholesterol metabolism. Increased adipose tissue TG retention with the HP/LC diet may reflect improved lipid storage capacity and be beneficial in this genetic model of obesity.

Contact time has limited impact on the efficacy of disinfectant towelettes when tested under conditions reflective of realistic use.

BACKGROUND: Disinfectant towelettes are increasingly being used as a means to prevent transmission of clinically important pathogens which could lead to healthcare-associated infections (HAIs). However, the efficacy of disinfectant towelette products when tested under realistic use conditions is understudied. A test model was designed to replicate realistic wiping conditions. The objective of this study was to determine the impact of varied contact time on disinfectant towelette efficacy under these conditions. METHODS: Five product types were tested against Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15,442) at five contact times (30 s, one min, two min, three min, and 10 min) on hard, non-porous laminate templates to determine the impact of contact time on disinfectant towelette efficacy when tested under realistic use. RESULTS: Product type had a significant impact on the efficacy of disinfectant towelettes when tested under conditions reflective of realistic use. The effect of contact time was limited and no differences in efficacy were seen at a contact time of one min compared with the other contact times tested. Only one disinfectant towelette product achieved a mean 5-log reduction under the tested conditions. CONCLUSION: Efficacy of disinfectant towelettes was primarily impacted by product type when applied in a model designed to replicate realistic use in which only a limited effect of contact time was observed. There is a need for further investigation into which factors have the greatest impact on disinfectant towelette efficacy when applied in clinical settings.

Contact time and disinfectant formulation significantly impact the efficacies of disinfectant towelettes against Candida auris on hard, non-porous surfaces.

There has been an increase in Candida auris healthcare-associated infections, which result from cross-contamination from surfaces and equipment. In this study, we tested the efficacies of EPA-registered disinfectant towelettes products that are increasingly used for infection control against C. auris at a range of contact times following modifications to standard EPA protocol MB-33-00. Hydrogen peroxide (HP)-based disinfectant towelettes were more efficacious against C. auris than the quaternary ammonium chloride (QAC)-alcohol-based disinfectant towelettes irrespective of tested contact times. Thirty s contact time was significantly less effective in reducing C. auris compared to 1-, 2-, 3-, and 10-min contact times. However, there were no statistically significant differences in the level of disinfection among 1-min and longer contact times regardless of product chemistry. None of the products achieved a standard six-log10 reduction at any tested contact times. Overall, the HP-based disinfectant towelette was significantly more fungicidal than the QAC-alcohol-based disinfectant towelette. For all product types, 30 s contact time did not achieve the same level of disinfection as 1-min or longer contact times. Overall, disinfectant towelette efficacy is dependent upon product formulation and contact time.

Divergent effects of sex and calcium/vitamin D supplementation on serum magnesium and markers of bone structure and function during initial military training.

Maintaining Mg status may be important for military recruits, a population that experiences high rates of stress fracture during initial military training (IMT). The objectives of this secondary analysis were to (1) compare dietary Mg intake and serum Mg in female and male recruits pre- and post-IMT, (2) determine whether serum Mg was related to parameters of bone health pre-IMT, and (3) whether Ca and vitamin D supplementation (Ca/vitamin D) during IMT modified serum Mg. Females (n 62) and males (n 51) consumed 2000 mg of Ca and 25 µg of vitamin D/d or placebo during IMT (12 weeks). Dietary Mg intakes were estimated using FFQ, serum Mg was assessed and peripheral quantitative computed tomography was performed on the tibia. Dietary Mg intakes for females and males pre-IMT were below the estimated average requirement and did not change with training. Serum Mg increased during IMT in females (0·06 ± 0·08 mmol/l) compared with males (-0·02 ± 0·10 mmol/l; P < 0·001) and in those consuming Ca/vitamin D (0·05 ± 0·09 mmol/l) compared with placebo (0·001 ± 0·11 mmol/l; P = 0·015). In females, serum Mg was associated with total bone mineral content (BMC, ß = 0·367, P = 0·004) and robustness (ß = 0·393, P = 0·006) at the distal 4 % site, stress-strain index of the polaris axis (ß = 0·334, P = 0·009) and robustness (ß = 0·420, P = 0·004) at the 14 % diaphyseal site, and BMC (ß = 0·309, P = 0·009) and stress-strain index of the polaris axis (ß = 0·314, P = 0·006) at the 66 % diaphyseal site pre-IMT. No significant relationships between serum Mg and bone measures were observed in males. Findings suggest that serum Mg may be modulated by Ca/vitamin D intake and may impact tibial bone health during training in female military recruits.

Slc39a4 in the small intestine predicts zinc absorption and utilization: a comprehensive analysis of zinc transporter expression in response to diets of varied zinc content in young mice.

Zinc homeostasis is primarily maintained by zinc transporters that regulate zinc uptake and efflux in the small intestine; however, the relative contribution of the many zinc transporters identified (Slc39a1-14, Slc30a1-10) to dietary zinc absorption and utilization remains unknown. The objective of this study was to determine the expression of Slc39a1-14 and Slc30a1-10 in the small intestine and their relative contribution to dietary zinc absorption in mice. Five-week-old male C57BL/6J mice were fed modified AIN-93G diets containing <1, 30, or 100ppm zinc (n=15 mice/diet). Following 1 week of feeding, mice were given an oral gavage containing 67Zn and liver and plasma isotope appearance was determined 6-h later by ICP-MS. Expression of Slc39a1-14 and Slc30a1-10 was determined in mucosa from duodenum, jejunum, and ileum. Plasma and liver total zinc concentrations were not different after one week of feeding (P>.05). Liver and plasma appearance of 67Zn was greater in mice fed <1ppm compared to the 30ppm (P<.0001) and 100ppm (P<.0001) zinc diets. With the exception of Slc39a2, Slc39a12, Slc30a3, and Slc30a8, the remaining zinc transporters were expressed across all diets and intestinal segments. Expression of Slc39a4, Slc39a11, and Slc30a6 changed with diet (Pdiet<.05 for all); expression of Slc39a5, Slc39a7, Slc39a11, Slc39a14, Slc30a1, Slc30a2, Slc30a4, Slc30a5, Slc30a7, and Slc30a10 changed by intestinal segment (Psegment<.05 for all). Slc39a4 was the only transporter positively associated with liver (r2=0.316, P<.001) and plasma (r2=0.189, P<.01) 67Zn appearance. Although most zinc transporters are expressed in the small intestine, intestinal Slc39a4 predicts fractional zinc absorption and utilization in young mice.

Comparative Genomic and Transcriptomic Analysis of Naegleria fowleri Clinical and Environmental Isolates.

Out of over 40 species of Naegleria, which are free-living thermophilic amebae found in freshwater and soil worldwide, only Naegleria fowleri infects humans, causing primary amebic meningoencephalitis (PAM), a typically fatal brain disease. To understand the population structure of Naegleria species and the genetic relationships between N. fowleri isolates and to detect pathogenic factors, we characterized 52 novel clinical and environmental N. fowleri genomes and a single Naegleria lovaniensis strain, along with transcriptomic data for a subset of 37 N. fowleri isolates. Whole-genome analysis of 56 isolates from three Naegleria species (N. fowleri, N. lovaniensis, and Naegleria gruberi) identified several genes unique to N. fowleri that have previously been linked to the pathogenicity of N. fowleri, while other unique genes could be associated with novel pathogenicity factors in this highly fatal pathogen. Population structure analysis estimated the presence of 10 populations within the three Naegleria species, of which 7 populations were within N. fowleri. The whole-nuclear-genome (WNG) phylogenetic analysis showed an overall geographical clustering of N. fowleri isolates, with few exceptions, and provided higher resolution in identifying potential clusters of isolates beyond that of the traditional locus typing. There were only 34 genes that showed significant differences in gene expression between the clinical and environmental isolates. Genomic data generated in this study can be used for developing rapid molecular assays and to conduct future population-based global genomic analysis and will also be a valuable addition to genomic reference databases, where shotgun metagenomics data from routine water samples could be searched for the presence of N. fowleri strains. IMPORTANCE N. fowleri, the only known Naegleria species to infect humans, causes fatal brain disease. PAM cases from 1965 to 2016 showed <20 cases per year globally. Out of approximately 150 cases in North America since 1962, only four PAM survivors are known, yielding a >97% case fatality rate, which is critically high. Although the pathogenesis of N. fowleri has been studied for the last 50 years, pathogenetic factors that lead to human infection and breaching the blood-brain barrier remain unknown. In addition, little is known regarding the genomic diversity both within N. fowleri isolates and among Naegleria species. In this study, we generated novel genome sequences and performed comparative genomic and transcriptomic analysis of a set of 52 N. fowleri draft genome sequences from clinical and environmental isolates derived from all over the world in the last 53 years, which will help shape future genome-wide studies and develop sensitive assays for routine surveillance.

Draft Chromosome Sequences of a Clinical Isolate of the Free-Living Ameba Naegleria fowleri.

We present the chromosome sequences of a Naegleria fowleri isolate from a human primary amebic meningoencephalitis (PAM) case. The genome sequences were assembled from Illumina HiSeq and PacBio sequencing data and verified with the optical mapping data. This led to the identification of 37 contigs representing 37 chromosomes in N. fowleri.

SneakerNet: A modular quality assurance and quality check workflow for primary genomic and metagenomic read data.

Laboratories that run Whole Genome Sequencing (WGS) produce a tremendous amount of data, up to 10 gigabytes for some common instruments. There is a need to standardize the quality assurance and quality control process (QA/QC). Therefore we have created SneakerNet to automate the QA/QC for WGS.

Sensitivity and reliability of zinc transporter and metallothionein gene expression in peripheral blood mononuclear cells as indicators of zinc status: responses to ex vivo zinc exposure and habitual zinc intake in humans.

Zn is an essential nutrient for humans; however, a sensitive biomarker to assess Zn status has not been identified. The objective of this study was to determine the reliability and sensitivity of Zn transporter and metallothionein (MT) genes in peripheral blood mononuclear cells (PBMCs) to Zn exposure ex vivo and to habitual Zn intake in human subjects. In study 1, human PBMCs were cultured for 24 h with 0-50 µm ZnSO4 with or without 5 µm N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and mRNA expression of SLC30A1-10, SLC39A1-14, MT1 subtypes (A, B, E, F, G, H, L, M and X), MT2A, MT3 and MT4 mRNA was determined. In study 2, fifty-four healthy male and female volunteers (31·9 (sd 13·8) years, BMI 25·7 (sd 2·9) kg/m2) completed a FFQ, blood was collected, PBMCs were isolated and mRNA expression of selected Zn transporters and MT isoforms was determined. Study 1: MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A and SLC30A1 increased with increasing concentrations of Zn and declined with the addition of TPEN. Study 2: Average daily Zn intake was 16·0 (sd 5·3) mg/d (range: 9-31 mg/d), and plasma Zn concentrations were 15·5 (SD 2·8) µmol/l (range 11-23 µmol/l). PBMC MT2A was positively correlated with dietary Zn intake (r 0·306, P = 0·03) and total Zn intake (r 0·382, P < 0·01), whereas plasma Zn was not (P > 0·05 for both). Findings suggest that MT2A mRNA in PBMCs reflects dietary Zn intake in healthy adults and may be a component in determining Zn status.

Spatial signatures identify immune escape via PD-1 as a defining feature of T-cell/histiocyte-rich large B-cell lymphoma.

T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types.

High-Altitude Acclimatization Suppresses Hepcidin Expression During Severe Energy Deficit.

Hennigar, Stephen R., Claire E. Berryman, Alyssa M. Kelley, Bradley J. Anderson, Andrew J. Young, James P. McClung, and Stefan M. Pasiakos. High-altitude acclimatization suppresses hepcidin expression during severe energy deficit. High Alt Med Biol. 21:232-236, 2020. Background: The erythropoietic cells in the bone marrow require iron to synthesize heme for incorporation into hemoglobin. Exposure to hypoxic conditions, such as extended sojourns to high altitude (HA), results in increased erythropoiesis and an increased physiological requirement for iron. In addition to increasing iron requirements, hypoxic conditions suppress appetite and often lead to decreased energy intake. The objective of this study was to determine the combined effects of severe energy deficit and hypoxia on hepcidin and measures of iron status in lowlanders sojourning to HA. Methods: Iron status indicators and hepcidin were determined in 17 healthy male volunteers (mean ± standard deviation, age 23 ± 6 years, body mass index 27 ± 4 kg/m2) fed a controlled diet (12 ± 1.2 mg iron/day) during a 20-day sojourn to 4300 m above sea level. Results: Chronic exposure to HA during severe energy deficit increased hematocrit by 12% (p < 0.01) and decreased serum hepcidin by 37% (p < 0.01) compared with baseline. Ferritin declined by 18% (p = 0.02) and transferrin saturation and soluble transferrin receptor increased by 55% and 83%, respectively (p < 0.01 for both) compared with baseline. Conclusions: HA acclimatization suppresses hepcidin expression to increase iron availability during severe energy deficit. Registered at ClinicalTrials.gov as NCT02731066.

Comparative genomic analysis of the IId subtype family of Cryptosporidium parvum.

Host adaptation is known to occur in Cryptosporidium parvum, with IIa and IId subtype families preferentially infecting calves and lambs, respectively. To improve our understanding of the genetic basis of host adaptation in Cryptosporidium parvum, we sequenced the genomes of two IId specimens and one IIa specimen from China and Egypt using the Illumina technique and compared them with the published IIa IOWA genome. Sequence data were obtained for >99.3% of the expected genome. Comparative genomic analysis identified differences in numbers of three subtelomeric gene families between sequenced genomes and the reference genome, including those encoding SKSR secretory proteins, the MEDLE family of secretory proteins, and insulinase-like proteases. These gene gains and losses compared with the reference genome were confirmed by PCR analysis. Altogether, 5,191-5,766 single nucleotide variants were seen between genomes sequenced in this study and the reference genome, with most SNVs occurring in subtelomeric regions of chromosomes 1, 4, and 6. The most highly polymorphic genes between IIa and IId encode mainly invasion-associated and immunodominant mucin proteins, and other families of secretory proteins. Further studies are needed to verify the biological significance of these genomic differences.

Metallothionein and Zinc Transporter Expression in Circulating Human Blood Cells as Biomarkers of Zinc Status: a Systematic Review.

Zinc is an essential nutrient for humans; however, a sensitive biomarker to assess zinc status has not been identified. The objective of this systematic review was to compile and assess studies that determined zinc transporter and/or metallothionein expression in various blood cell types and to determine their reliability and sensitivity to changes in dietary zinc. Sixteen studies were identified that determined the expression of zrt-, irt-like protein (ZIP) 1 [solute carrier family (SLC) 39A1], ZIP3 (SLC39A3), ZIP5 (SLC39A5), ZIP6 (SLC39A6), ZIP7 (SLC39A7), ZIP8 (SLC39A8), ZIP10 (SLC39A10), ZIP14 (SLC39A14), zinc transporter (ZnT)1 (SLC30A1), ZnT2 (SLC30A2), ZnT4 (SLC30A4), ZnT5 (SLC30A5), ZnT6 (SLC30A6), ZnT7 (SLC30A7), ZnT9 (SLC30A9), and/or metallothionein in various blood cells isolated from healthy adult men and women in response to zinc supplementation or depletion. Cell types included leukocytes, peripheral blood mononuclear cells, T lymphocytes, monocytes, and erythrocytes. ZIP1, ZnT1, and metallothionein were the most commonly measured proteins. Changes in ZIP1 and ZnT1 in response to zinc supplementation or depletion were not consistent across studies. Leukocyte metallothionein decreased with zinc depletion (-39% change from baseline, <5 mg Zn/d, n = 2 studies) and increased with zinc supplementation in a dose-dependent manner (35%, 15-22 mg Zn/d, n = 7 studies; 267%, 50 mg Zn/d, n = 2 studies) and at the earliest time points measured; however, no change or delayed response was observed in metallothionein in erythrocytes. A greater percentage of studies demonstrated that metallothionein in leukocyte subtypes was a more reliable (100%, n = 12; 69%, n = 16) and responsive (92%, n = 12; 82%, n = 11) indicator of zinc exposure than was plasma zinc, respectively. In conclusion, current evidence indicates that metallothionein in leukocyte subtypes may be a component in determining zinc status.

Genetic Basis for PD-L1 Expression in Squamous Cell Carcinomas of the Cervix and Vulva.

IMPORTANCE: Patients with squamous cell carcinoma (SCC) of the cervix or vulva have limited therapeutic options, and the potential for immunotherapy for this population has not been evaluated. Recent trials suggest that tumors with a genetic basis for PD-1 (programmed cell death protein 1) ligand expression are highly sensitive to therapeutic antibodies targeting PD-1. OBJECTIVE: To determine the genetic status of CD274 (encoding PD-L1 [programmed cell death 1 ligand 1]) and PDCD1LG2 (encoding PD-L2 [programmed cell death 1 ligand 2]) in SCCs of the cervix and vulva and to correlate the findings with PD-L1 protein expression. DESIGN, SETTING, AND PARTICIPANTS: We performed fluorescence in situ hybridization (FISH) using probes targeting CD274, PDCD1LG2, and the centromeric portion of chromosome 9, and immunohistochemistry (IHC) using an antibody recognizing PD-L1 on formalin-fixed, paraffin-embedded (FFPE) biopsy specimens from 48 cervical SCCs and 23 vulvar SCCs. MAIN OUTCOMES AND MEASURES: Tumors were categorized according to the genetic abnormality in CD274 and PDCD1LG2 (coamplification > cogain > polysomy > disomy) as detected by FISH, and evaluated on a semiquantitative scale (modified H score, the product of the percentage of tumor cells with positive staining and the maximum intensity of positive staining) for PD-L1 protein expression as detected by IHC. RESULTS: Overall, 71 samples of FFPE tissue from cases of cervical SCCs (n = 48) and vulvar SCCs (n = 23) were retrieved from the archives of Brigham and Women's Hospital and included in this study. We observed cogain or coamplification of CD274 and PDCD1LG2 in 32 of 48 cervical SCCs (67%) and 10 of 23 vulvar SCCs (43%). Median PD-L1 protein expression was highest among tumors with CD274 and PDCD1LG2 coamplification and lowest among tumors with disomy. CONCLUSIONS AND RELEVANCE: Recurrent copy number gain of the genes encoding the PD-1 ligands provides a genetic basis for PD-L1 expression in a subset of cervical and vulvar SCCs and identifies a class of patients that are rational candidates for therapies targeting PD-1.