Body Mass Index and Gut Microbiome: A Cluster-Randomized, Controlled, Pilot Feasibility Study.

Background: Prebiotic fiber has been examined as a way to foster gut bacteria less associated with obesity. Tests of prebiotic fiber in reducing obesity have been conducted mainly in animals, adults, and Caucasians when the highest obesity rates are in African American and Latinx youth. Response to prebiotic fiber is determined by the pre-existing intestinal microbiota. The type of microbiota varies based on diet and physical activity (PA), so it is important to examine acceptability and response to prebiotic fiber in those most at risk for obesity. Methods: This cluster-randomized, controlled feasibility trial included an online program designed to improve diet and PA along with administration of a prebiotic fiber for 12 weeks in 123 students of 4th and 5th grade where 98% were eligible for free or reduced-fee lunch. Of these 56% were male; 71% Latinx; 15% African American; and 14% other. Results: Decrease in body fat (BF) was associated with higher pretest BF. Lower body mass index (BMI) was associated with decrease in fecal Tenericutes and increase in Actinobacteria. Conclusions: Prebiotic fiber can be helpful in supporting healthy weight, so inclusion in culturally congruent foods usually eaten by children from groups at high risk for obesity should be considered following additional studies. Determining those most responsive to prebiotic fibers can also permit individual recommendations for greater inclusion in usual diet choices. Clinical Trial Registration Number NCT05671731.

Longitudinal changes in the gut microbiome of infants on total parenteral nutrition.

BACKGROUND: Animal studies suggest that total parenteral nutrition (TPN) may alter bacterial colonization of the intestinal tract and contribute to complications. Progressive changes in gut microbiome of infants receiving TPN are not well understood. METHODS: Infants with and without TPN/soy lipid were enrolled in a prospective, longitudinal study. Weekly fecal samples were obtained for the first 4 weeks of life. High throughput pyrosequencing of 16S rDNA was used for compositional analysis of the gut microbiome. RESULTS: 47 infants were eligible for analyses, 25 infants received TPN, and 22 infants did not (control). Although similar between TPN and control groups in the first week, fecal bacterial alpha diversity was significantly lower in the TPN group compared to controls at week 4 (Shannon index 1.0 vs 1.5, P-value = 0.03). The TPN group had significantly lower Bacteroidetes and higher Verrucomicrobia abundance compared to controls (P-values < 0.05), and these differences became more pronounced over time. At the genus level, TPN was associated with lower abundance of Bacteroides and Bifidobacterium in all weeks. CONCLUSIONS: TPN is associated with significant loss of biodiversity and alterations in the pattern of gut microbial colonization of infants over time. TPN-associated dysbiosis may predispose infants to adverse NICU outcomes.

Influence of the time of day on axial length and choroidal thickness changes to hyperopic and myopic defocus in human eyes.

Research in animal models have shown that exposing the eye to positive or negative spectacle lenses can lead to predictable changes in eye growth. Recent research indicates that brief periods (1-2 h) of monocular defocus results in small, but significant changes in axial length and choroidal thickness of human subjects. However, the effects of the time of day on these ocular changes with defocus are not known. In this study, we examined the effects of monocular myopic and hyperopic defocus on axial length and choroidal thickness when applied in the morning (change between 10 a.m. and 12 p.m.) vs the evening (change between 5 and 7 p.m.) in young adult human participants (mean age, 23.44 ±â€¯4.52 years). A series of axial length (using an IOL Master) and choroidal thickness (using an optical coherence tomographer) measurements were obtained over three consecutive days in both eyes. Day 1 (no defocus) examined the baseline ocular measurements in the morning (10 a.m. and 12 p.m.) and in the evening (5 and 7 p.m.), day 2 investigated the effects of hyperopic and myopic defocus on ocular parameters in the morning (subjects wore a spectacle lens with +3 or -3 DS over the right eye and a plano lens over the left eye between 10 a.m. and 12 p.m.), and day 3 examined the effects of defocus in the evening (+3 or -3 DS spectacle lens over the right eye between 5 and 7 p.m.). Exposure to myopic defocus caused a significant reduction in axial length and thickening of the subfoveal choroid at both times; but, compared to baseline data from day 1, the relative change in axial length (-0.021 ± 0.009 vs +0.004 ± 0.003 mm, p = 0.009) and choroidal thickness (+0.027 ± 0.006 vs +0.007 ± 0.006 mm, p = 0.011) with defocus were significantly greater for evening exposure to defocus than for the morning session. On the contrary, introduction of hyperopic defocus resulted in a significant increase in axial length when given in the morning (+0.026 ± 0.006 mm), but not in the evening (+0.001 ± 0.003 mm) (p = 0.047). Furthermore, hyperopic defocus resulted in a significant thinning of the choroid (p = 0.005), but there was no significant influence of the time of day on choroidal changes associated with hyperopic defocus (p = 0.672). Exposure to hyperopic and myopic defocus at different times of the day was also associated with changes in the parafoveal regions of the choroid (measured across 1.5 mm nasal and temporal choroidal regions on either side of the fovea). Our results show that ocular response to optical defocus varies significantly depending on the time of day in human subjects. These findings represent a potential interaction between the signal associated with the eye's natural diurnal rhythm and the visual signal associated with the optical defocus, making the eye perhaps more responsive to hyperopic defocus (or 'go' signal) in the morning, and to myopic defocus (or 'stop' signal) in the latter half of the day.

Ceftriaxone Administration Disrupts Intestinal Homeostasis, Mediating Noninflammatory Proliferation and Dissemination of Commensal Enterococci.

Enterococci are Gram-positive commensals of the mammalian intestinal tract and harbor intrinsic resistance to broad-spectrum cephalosporins. Disruption of colonization resistance in humans by antibiotics allows enterococci to proliferate in the gut and cause disseminated infections. In this study, we used Enterococcus faecalis (EF)-colonized mice to study the dynamics of enterococci, commensal microbiota, and the host in response to systemic ceftriaxone administration. We found that the mouse model recapitulates intestinal proliferation and dissemination of enterococci seen in humans. Employing a ceftriaxone-sensitive strain of enterococci (E. faecalis JL308), we showed that increased intestinal abundance is critical for the systemic dissemination of enterococci. Investigation of the impact of ceftriaxone on the mucosal barrier defenses and integrity suggested that translocation of enterococci across the intestinal mucosa was not associated with intestinal pathology or increased permeability. Ceftriaxone-induced alteration of intestinal microbial composition was associated with transient increase in the abundance of multiple bacterial operational taxonomic units (OTUs) in addition to enterococci, for example, lactobacilli, which also disseminated to the extraintestinal organs. Collectively, these results emphasize that ceftriaxone-induced disruption of colonization resistance and alteration of mucosal homeostasis facilitate increased intestinal abundance of a limited number of commensals along with enterococci, allowing their translocation and systemic dissemination in a healthy host.

Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production.

Alterations in gastrointestinal microbiota indirectly modulate the risk of atopic disease, but effects on respiratory viral infections are less clear. Using the murine paramyxoviral virus type 1, Sendai virus (SeV), we examined the effect of altering gastrointestinal microbiota on the pulmonary antiviral immune response. C57BL6 mice were treated with streptomycin before or during infection with SeV and resulting immune response studied. Ingestion of the non-absorbable antibiotic streptomycin led to a marked reduction in intestinal microbial diversity without a significant effect on lung microbiota. Reduction in diversity in the gastrointestinal tract was followed by greatly increased mortality to respiratory viral infection (p < 0.0001). This increase in mortality was associated with a dysregulated immune response characterized by decreased lung (p = 0.01) and intestinal (p = 0.03) regulatory T cells (Tregs), and increased lung IFNγ (p = 0.049), IL-6 (p = 0.015), and CCL2 (p = 0.037). Adoptive transfer of Treg cells or neutralization of IFNγ prevented increased mortality. Furthermore, Lin-CD4+ cells appeared to be a potential source of the increased IFNγ. Together, these results demonstrate gastrointestinal microbiota modulate immune responses at distant mucosal sites and have the ability to significantly impact mortality in response to a respiratory viral infection.

Modulation of the diet and gastrointestinal microbiota normalizes systemic inflammation and ß-cell chemokine expression associated with autoimmune diabetes susceptibility.

Environmental changes associated with modern lifestyles may underlie the rising incidence of Type 1 diabetes (T1D). Our previous studies of T1D families and the BioBreeding (BB) rat model have identified a peripheral inflammatory state that is associated with diabetes susceptibility, consistent with pattern recognition receptor ligation, but is independent of disease progression. Here, compared to control strains, islets of spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ weanlings provided a standard cereal diet expressed a robust proinflammatory transcriptional program consistent with microbial antigen exposure that included numerous cytokines/chemokines. The dependence of this phenotype on diet and gastrointestinal microbiota was investigated by transitioning DR+/+ weanlings to a gluten-free hydrolyzed casein diet (HCD) or treating them with antibiotics to alter/reduce pattern recognition receptor ligand exposure. Bacterial 16S rRNA gene sequencing revealed that these treatments altered the ileal and cecal microbiota, increasing the Firmicutes:Bacteriodetes ratio and the relative abundances of lactobacilli and butyrate producing taxa. While these conditions did not normalize the inherent hyper-responsiveness of DR+/+ rat leukocytes to ex vivo TLR stimulation, they normalized plasma cytokine levels, plasma TLR4 activity levels, the proinflammatory islet transcriptome, and ß-cell chemokine expression. In lymphopenic DRlyp/lyp rats, HCD reduced T1D incidence, and the introduction of gluten to this diet induced islet chemokine expression and abrogated protection from diabetes. Overall, these studies link BB rat islet-level immunocyte recruiting potential, as measured by ß-cell chemokine expression, to a genetically controlled immune hyper-responsiveness and innate inflammatory state that can be modulated by diet and the intestinal microbiota.

Intestinal alkaline phosphatase deficiency leads to dysbiosis and bacterial translocation in the newborn intestine.

BACKGROUND: Intestinal alkaline phosphatase (IAP) has been shown to help maintain intestinal homeostasis. Decreased expression of IAP has been linked with pediatric intestinal diseases associated with bacterial overgrowth and subsequent inflammation. We hypothesize that the absence of IAP leads to dysbiosis, with increased inflammation and permeability of the newborn intestine. METHODS: Sprague-Dawley heterozygote IAP cross-matches were bred. Pups were dam fed ad lib and euthanized at weaning. The microbiotas of terminal ileum (TI) and colon was determined by quantitative real-time polymerase chain reaction (qRT-PCR) of subphylum-specific bacterial 16S ribosomal RNA. RT-PCR was performed on TI for inflammatory cytokines. Intestinal permeability was quantified by fluorescein isothiocyanate-dextran permeability and bacterial translocation by qRT-PCR for bacterial 16S ribosomal RNA in mesenteric lymph nodes. Statistical analysis was done by chi-square analysis. RESULTS: All three genotypes had similar concentrations of bacteria in the TI and colon. However, IAP knockout (IAP-KO) had significantly decreased diversity of bacterial species in their colonic stool compared with heterozygous and wild-type (WT). IAP-KO pups had a nonstatistically significant 3.9-fold increased inducible nitric oxide synthase messenger RNA expression compared with WT (IAP-KO, 3.92 ± 1.36; WT, 1.0 ± 0.27; P = 0.03). IAP-KO also had significantly increased bacterial translocation to mesenteric lymph nodes occurred in IAP-KO (IAP-KO, 7625 RFU/g ± 3469; WT, 4957 RFU/g ± 1552; P = 0.04). Furthermore, IAP-KO had increased permeability (IAP-KO, 0.297 mg/mL ± 0.2; WT, 0.189 mg/mL ± 0.15 P = 0.07), but was not statistically significant. CONCLUSIONS: Deficiency of IAP in the newborn intestine is associated with dysbiosis and increased inflammation, permeability, and bacterial translocation.

Black Raspberries and Their Anthocyanin and Fiber Fractions Alter the Composition and Diversity of Gut Microbiota in F-344 Rats.

Natural compounds can alter the diversity and composition of the gut microbiome, potentially benefiting our health. We previously demonstrated chemopreventive effects of black raspberries (BRBs) in colorectal cancer, which is associated with gut dysbiosis. To investigate the effects of whole BRBs and their fractions on gut microbiota, we fed F-344 rats a control diet, 5% BRBs, the BRB anthocyanin fraction, or the BRB residue fraction for 6 weeks. Feces were collected at baseline and at weeks 3 and 6, and bacterial sequence counts were analyzed. We observed distinct patterns of microbiota from different diet groups. Beta diversity analysis suggested that all diet groups exerted time-dependent changes in the bacterial diversity. Hierarchical clustering analysis revealed that post-diet fecal microbiota was segregated from baseline fecal microbiota within each diet. It is interesting to note that fractions of BRBs induced different changes in gut bacteria compared to whole BRBs. The abundance of specific microbial species known to have anti-inflammatory effects, such as Akkermansia and Desulfovibrio, was increased by whole BRBs and their residue. Further, butyrate-producing bacteria, e.g., Anaerostipes, were increased by whole BRBs. Our results suggest that whole BRBs and their fractions alter the gut microbiota in ways that could significantly influence human health.

Paneth cell defects in Crohn's disease patients promote dysbiosis.

BACKGROUND: Paneth cell dysfunction has been implicated in a subset of Crohn's disease (CD) patients. We previously stratified clinical outcomes of CD patients by using Paneth cell phenotypes, which we defined by the intracellular distribution of antimicrobial proteins. Animal studies suggest that Paneth cells shape the intestinal microbiome. However, it is unclear whether Paneth cell phenotypes alter the microbiome complexity in CD subjects. Therefore, we analyzed the correlation of Paneth cell phenotypes with mucosal microbiome composition and ileal RNA expression in pediatric CD and noninflammatory bowel disease (non-IBD) patients. METHODS: Pediatric CD (n = 44) and non-IBD (n = 62) patients aged 4 to 18 were recruited prior to routine endoscopic biopsy. Ileal mucosal samples were analyzed for Paneth cell phenotypes, mucosal microbiome composition, and RNA transcriptome. RESULTS: The prevalence of abnormal Paneth cells was higher in pediatric versus adult CD cohorts. For pediatric CD patients, those with abnormal Paneth cells showed significant changes in their ileal mucosal microbiome, highlighted by reduced protective microbes and enriched proinflammatory microbes. Ileal transcriptome profiles showed reduced transcripts for genes that control oxidative phosphorylation in CD patients with abnormal Paneth cells. These transcriptional changes in turn were correlated with specific microbiome alterations. In non-IBD patients, a subset contained abnormal Paneth cells. However, this subset was not associated with alterations in the microbiome or host transcriptome. CONCLUSION: Paneth cell abnormalities in human subjects are associated with mucosal dysbiosis in the context of CD, and these changes are associated with alterations in oxidative phosphorylation, potentially in a feedback loop. FUNDING: The research was funded by Helmsley Charitable Trust (to T.S. Stappenbeck, R.J. Xavier, and D.P.B. McGovern), Crohn's and Colitis Foundation of America (to N.H. Salzman, T.S. Stappenbeck, R.J. Xavier, and C. Huttenhower), and Doris Duke Charitable Foundation grant 2014103 (to T.C. Liu).

Intestinal Microbial Metabolites Are Linked to Severity of Myocardial Infarction in Rats.

Intestinal microbiota determine severity of myocardial infarction in rats. We determined whether low molecular weight metabolites derived from intestinal microbiota and transported to the systemic circulation are linked to severity of myocardial infarction. Plasma from rats treated for seven days with the non-absorbed antibiotic vancomycin or a mixture of streptomycin, neomycin, polymyxin B and bacitracin was analyzed using mass spectrometry-based metabolite profiling platforms. Antibiotic-induced changes in the abundance of individual groups of intestinal microbiota dramatically altered the host's metabolism. Hierarchical clustering of dissimilarities separated the levels of 284 identified metabolites from treated vs. untreated rats; 193 were altered by the antibiotic treatments with a tendency towards decreased metabolite levels. Catabolism of the aromatic amino acids phenylalanine, tryptophan and tyrosine was the most affected pathway comprising 33 affected metabolites. Both antibiotic treatments decreased the severity of an induced myocardial infarction in vivo by 27% and 29%, respectively. We then determined whether microbial metabolites of the amino acids phenylalanine, tryptophan and tyrosine were linked to decreased severity of myocardial infarction. Vancomycin-treated rats were administered amino acid metabolites prior to ischemia/reperfusion studies. Oral or intravenous pretreatment of rats with these amino acid metabolites abolished the decrease in infarct size conferred by vancomycin. Inhibition of JAK-2 (AG-490, 10 µM), Src kinase (PP1, 20 µM), Akt/PI3 kinase (Wortmannin, 100 nM), p44/42 MAPK (PD98059, 10 µM), p38 MAPK (SB203580, 10 µM), or KATP channels (glibenclamide, 3 µM) abolished cardioprotection by vancomycin, indicating microbial metabolites are interacting with cell surface receptors to transduce their signals through Src kinase, cell survival pathways and KATP channels. These inhibitors have no effect on myocardial infarct size in untreated rats. This study links gut microbiota metabolites to severity of myocardial infarction and may provide future opportunities for novel diagnostic tests and interventions for the prevention of cardiovascular disease.

Radiation-induced changes in intestinal and tissue-nonspecific alkaline phosphatase: implications for recovery after radiation therapy.

BACKGROUND: Exogenous replacement of depleted enterocyte intestinal alkaline phosphatase (IAP) decreases intestinal injury in models of colitis. We determined whether radiation-induced intestinal injury could be mitigated by oral IAP supplementation and the impact on tissue-nonspecific AP. METHODS: WAG/RjjCmcr rats (n = 5 per group) received lower hemibody irradiation (13 Gy) followed by daily gavage with phosphate-buffered saline or IAP (40 U/kg/d) for 4 days. Real-time polymerase chain reaction, AP activity, and microbiota analysis were performed on intestine. Lipopolysaccharide and cytokine analysis was performed on serum. Data were expressed as a mean ± SEM with P greater than .05 considered significant. RESULTS: Intestine of irradiated animals demonstrates lower hemibody irradiation and is associated with upregulation of tissue-nonspecific AP, downregulation of IAP, decreased AP activity, and altered composition of the intestinal microbiome. CONCLUSIONS: Supplemental IAP after radiation may be beneficial in mitigating intestinal radiation syndrome as evidenced by improved histologic injury, decreased acute intestinal inflammation, and normalization of intestinal microbiome.

Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract.

Enterococcus faecalis is both a common commensal of the human gastrointestinal tract and a leading cause of hospital-acquired infections. Systemic infections with multidrug-resistant enterococci occur subsequent to gastrointestinal colonization. Preventing colonization by multidrug-resistant E. faecalis could therefore be a valuable approach towards limiting infection. However, little is known about the mechanisms E. faecalis uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive conjugative plasmids encoding bacteriocins are common among enterococcal strains and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of colonization of the mouse gut with E. faecalis, without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 21 (ref. 4) in enterococcal colonization. Here we show that E. faecalis harbouring pPD1 replaces indigenous enterococci and outcompetes E. faecalis lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other E. faecalis strains by conjugation, enhancing their survival. Colonization with an E. faecalis strain carrying a conjugation-defective pPD1 mutant subsequently resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore, bacteriocin expression by commensal bacteria can influence niche competition in the gastrointestinal tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multidrug-resistant bacteria, without profound disruption of the indigenous microbiota.

Cardiac injury after 10 gy total body irradiation: indirect role of effects on abdominal organs.

The objective of this study was to determine whether radiation-induced injury to the heart after 10 Gy total body irradiation (TBI) is direct or indirect. Young male WAG/RijCmcr rats received a 10 Gy single dose using TBI, upper hemi-body (UHB) irradiation, lower hemi-body (LHB) irradiation, TBI with the kidneys shielded or LHB irradiation with the intestines shielded. Age-matched, sham-irradiated rats served as controls. The lipid profile, kidney injury, heart and liver morphology and cardiac function were determined up to 120 days after irradiation. LHB, but not UHB irradiation, increased the risk factors for cardiac disease as well as the occurrence of cardiac and kidney injury in a way that was quantitatively and qualitatively similar to that observed after TBI. Shielding of the kidneys prevented the increases in risk factors for cardiac disease. Shielding of the intestines did not prevent the increases in risk factors for cardiac disease. There was no histological evidence of liver injury 120 days after irradiation. Injury to the heart from irradiation appears to be indirect, supporting the notion that injury to abdominal organs, principally the kidneys, is responsible for the increased risk factors for and the occurrence of cardiac disease after TBI and LHB irradiation.

A method for quantifying mechanical properties of tissue following viral infection.

Viral infection and replication involves the reorganization of the actin network within the host cell. Actin plays a central role in the mechanical properties of cells. We have demonstrated a method to quantify changes in mechanical properties of fabricated model three-dimensional (3D) connective tissue following viral infection. Using this method, we have characterized the impact of infection by the human herpesvirus, cytomegalovirus (HCMV). HCMV is a member of the herpesvirus family and infects a variety of cell types including fibroblasts. In the body, fibroblasts are necessary for maintaining connective tissue and function by creating mechanical force. Using this 3D connective tissue model, we observed that infection disrupted the cell's ability to generate force and reduced the cumulative contractile force of the tissue. The addition of HCMV viral particles in the absence of both viral gene expression and DNA replication was sufficient to disrupt tissue function. We observed that alterations of the mechanical properties are, in part, due to a disruption of the underlying complex actin microfilament network established by the embedded fibroblasts. Finally, we were able to prevent HCMV-mediated disruption of tissue function by the addition of human immune globulin against HCMV. This study demonstrates a method to quantify the impact of viral infection on mechanical properties which are not evident using conventional cell culture systems.

Intestinal microbiota as novel biomarkers of prior radiation exposure.

There is an urgent need for rapid, accurate, and sensitive diagnostic platforms to confirm exposure to radiation and estimate the dose absorbed by individuals subjected to acts of radiological terrorism, nuclear power plant accidents, or nuclear warfare. Clinical symptoms and physical dosimeters, even when available, do not provide adequate diagnostic information to triage and treat life-threatening radiation injuries. We hypothesized that intestinal microbiota act as novel biomarkers of prior radiation exposure. Adult male Wistar rats (n = 5/group) received single or multiple fraction total-body irradiation of 10.0 Gy and 18.0 Gy, respectively. Fresh fecal pellets were obtained from each rat prior to (day 0) and at days 4, 11, and 21 post-irradiation. Fecal microbiota composition was determined using microarray and quantitative PCR (polymerase chain reaction) analyses. The radiation exposure biomarkers consisted of increased 16S rRNA levels of 12 members of the Bacteroidales, Lactobacillaceae, and Streptococcaceae after radiation exposure, unchanged levels of 98 Clostridiaceae and Peptostreptococcaceae, and decreased levels of 47 separate Clostridiaceae members; these biomarkers are present in human and rat feces. As a result of the ubiquity of these biomarkers, this biomarker technique is non-invasive; microbiota provide a sustained level of reporting signals that are increased several-fold following exposure to radiation, and intestinal microbiota that are unaffected by radiation serve as internal controls. We conclude that intestinal microbiota serve as novel biomarkers of prior radiation exposure, and may be able to complement conventional chromosome aberrational analysis to significantly enhance biological dose assessments.

Intestinal microbiota determine severity of myocardial infarction in rats.

Signals from the intestinal microbiota are important for normal host physiology; alteration of the microbiota (dysbiosis) is associated with multiple disease states. We determined the effect of antibiotic-induced intestinal dysbiosis on circulating cytokine levels and severity of ischemia/reperfusion injury in the heart. Treatment of Dahl S rats with a minimally absorbed antibiotic vancomycin, in the drinking water, decreased circulating leptin levels by 38%, resulted in smaller myocardial infarcts (27% reduction), and improved recovery of postischemic mechanical function (35%) as compared with untreated controls. Vancomycin altered the abundance of intestinal bacteria and fungi, measured by 16S and 18S ribosomal DNA quantity. Pretreatment with leptin (0.12 µg/kg i.v.) 24 h before ischemia/reperfusion abolished cardioprotection produced by vancomycin treatment. Dahl S rats fed the commercially available probiotic product Goodbelly, which contains the leptin-suppressing bacteria Lactobacillus plantarum 299v, also resulted in decreased circulating leptin levels by 41%, smaller myocardial infarcts (29% reduction), and greater recovery of postischemic mechanical function (23%). Pretreatment with leptin (0.12 µg/kg i.v.) abolished cardioprotection produced by Goodbelly. This proof-of-concept study is the first to identify a mechanistic link between changes in intestinal microbiota and myocardial infarction and demonstrates that a probiotic supplement can reduce myocardial infarct size.

Hydrogel tissue construct-based high-content compound screening.

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)-based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds--rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)--for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

High-throughput measurements of hydrogel tissue construct mechanics.

Engineered tissues represent a natural environment for studying cell physiology, mechanics, and function. Cellular interactions with the extracellular matrix proteins are important determinants of cell physiology and tissue mechanics. Dysregulation of these parameters can result in diseases such as cardiac fibrosis and atherosclerosis. In this report we present a novel system to produce hydrogel tissue constructs (HTCs) and to characterize their mechanical properties. HTCs are grown in custom chambers and a robotic system is used to indent them and measure the resulting forces. Force measurements are then used to estimate HTC pretension (cellular contractility). Pretension was reduced in a dose-dependent manner by cytochalasin D (CD) treatment; the highest concentration (2microM) resulted in 10-fold decrease. On the other hand, treatment with fetal bovine serum (20%) resulted in approximately threefold increase in pretension. Excellent repeatability and precision were observed in measurements from replicate HTCs. The coefficient of statistical variance of quantified pretension ranged from 7% to 15% (n=4). Due to the small size (4x4x0.8mm) of the HTCs, this system of profiling HTC mechanics can readily be used in high-throughput applications. In particular, it can be used for screening chemical libraries in search of drugs that can alter tissue mechanics.

Regulation of ENaC expression at the cell surface by Rab11.

The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption. Coexpression of Rab11a and Rab3a small G proteins with ENaC results in a significant increase in channel activity. In contrast, coexpression of Rab5, Rab27a, and Arf-1 had no effect or slightly decreased ENaC activity. Inhibition of MEK with PD98059, Rho-kinase with Y27632 or PI3-kinase with LY294002 had no effect on ENaC activity in Rab11a-transfected CHO cells. Fluorescence imaging methods demonstrate that Rab11a colocalized with ENaC. Rab11a increases ENaC activity in an additive manner with dominant-negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular pool in a Rab11-dependent manner.

Image-guided modeling of virus growth and spread.

Although many tools of cellular and molecular biology have been used to characterize single intracellular cycles of virus growth, few culture methods exist to study the dynamics of spatially spreading viruses over multiple generations. We have previously developed a method that addresses this need by tracking the spread of focal infections using immunocytochemical labeling and digital imaging. Here, we build reaction-diffusion models to account for spatio-temporal patterns formed by the spreading viral infection front as well as data from a single cycle of virus growth (one-step growth). Systems with and without the interferon-mediated antiviral response of the host cells are considered. Dynamic images of the spreading infections guide iterative model refinement steps that lead to reproduction of all of the salient features contained in the images, not just the velocity of the infection front. The optimal fits provide estimates for key parameters such as virus-host binding and the production rate of interferon. For the examined data, highly-lumped infection models that ignore the one-step growth dynamics provide a comparable fit to models that more accurately account for these dynamics, highlighting the fact that increased model complexity does not necessarily translate to improved fit. This work demonstrates how model building can facilitate the interpretation of experiments by highlighting contributions from both biological and methodological factors.