Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.

Leukotriene B4 enhances innate immune defense against the puerperal sepsis agent Streptococcus pyogenes.

Puerperal sepsis is a leading cause of maternal mortality worldwide. Streptococcus pyogenes [group A Streptococcus; (GAS)] is a major etiologic agent of severe postpartum sepsis, yet little is known regarding the pathogenesis of these infections. Tissue macrophages provide innate defense against GAS, and their actions are highly regulated. The intracellular second messenger cAMP can negatively regulate macrophage actions against GAS. Because leukotriene (LT) B(4) has been shown to suppress intracellular cAMP in macrophages, we hypothesized that it could enhance innate defenses against GAS. We assessed the capacity of LTB(4) to modulate antistreptococcal actions of human macrophages, including placental and decidual macrophages and used a novel intrauterine infection model of GAS in mice lacking the 5-lipoxygenase enzyme to determine the role of endogenous LTs in host defense against this pathogen. Animals lacking 5-lipoxygenase were significantly more vulnerable to intrauterine GAS infection than were wild-type mice and showed enhanced dissemination of bacteria out of the uterus and a more robust inflammatory response than did wild-type mice. In addition, LTB(4) reduced intracellular cAMP levels via the BLT1 receptor and was a potent stimulant of macrophage phagocytosis and NADPH oxidase-dependent intracellular killing of GAS. Importantly, interference was observed between the macrophage immunomodulatory actions of LTB(4) and the cAMP-inducing lipid PGE(2), suggesting that interplay between pro- and anti-inflammatory compounds may be important in vivo. This work underscores the potential for pharmacological targeting of lipid mediator signaling cascades in the treatment of invasive GAS infections.

Total parenteral nutrition-associated lamina propria inflammation in mice is mediated by a MyD88-dependent mechanism.

Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88(-/-) mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88(-/-) mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80(+)CD11b(+)CD11c(dull/-) macrophage-derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88(-/-) TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.

Intrauterine group A streptococcal infections are exacerbated by prostaglandin E2.

Streptococcus pyogenes (Group A Streptococcus; GAS) is a major cause of severe postpartum sepsis, a re-emerging cause of maternal morbidity and mortality worldwide. Immunological alterations occur during pregnancy to promote maternofetal tolerance, which may increase the risk for puerperal infection. PGE2 is an immunomodulatory lipid that regulates maternofetal tolerance, parturition, and innate immunity. The extent to which PGE2 regulates host immune responses to GAS infections in the context of endometritis is unknown. To address this, both an in vivo mouse intrauterine (i.u.) GAS infection model and an in vitro human macrophage-GAS interaction model were used. In C57BL/6 mice, i.u. GAS inoculation resulted in local and systemic inflammatory responses and triggered extensive changes in the expression of eicosanoid pathway genes. The i.u. administration of PGE2 increased the mortality of infected mice, suppressed local IL-6 and IL-17A levels, enhanced neutrophilic inflammation, reduced uterine macrophage populations, and increased bacterial dissemination. A role for endogenous PGE2 in the modulation of antistreptococcal host defense was suggested, because mice lacking the genes encoding the microsomal PGE2 synthase-1 or the EP2 receptor were protected from death, as were mice treated with the EP4 receptor antagonist, GW627368X. PGE2 also regulated GAS-macrophage interactions. In GAS-infected human THP-1 (macrophage-like) cells, PGE2 inhibited the production of MCP-1 and TNF-α while augmenting IL-10 expression. PGE2 also impaired the phagocytic ability of human placental macrophages, THP-1 cells, and mouse peritoneal macrophages in vitro. Exploring the targeted disruption of PGE2 synthesis and signaling to optimize existing antimicrobial therapies against GAS may be warranted.

Effects of protein concentration and beta-adrenergic agonists on ruminal bacterial communities in finishing beef heifers.

To improve animal performance and modify growth by increasing lean tissue accretion, beef cattle production has relied on use of growth promoting technologies such as beta-adrenergic agonists. These synthetic catecholamines, combined with the variable inclusion of rumen degradable (RDP) and undegradable protein (RUP), improve feed efficiency and rate of gain in finishing beef cattle. However, research regarding the impact of beta-adrenergic agonists, protein level, and source on the ruminal microbiome is limited. The objective of this study was to determine the effect of different protein concentrations and beta-adrenergic agonist (ractopamine hydrochloride; RAC) on ruminal bacterial communities in finishing beef heifers. Heifers (n = 140) were ranked according to body weight and assigned to pens in a generalized complete block design with a 3 × 2 factorial arrangement of treatments of 6 different treatment combinations, containing 3 protein treatments (Control: 13.9% CP, 8.9% RDP, and 5.0% RUP; High RDP: 20.9% CP, 14.4% RDP, 6.5% RUP; or High RUP: 20.9% CP, 9.7% RDP, 11.2% RUP) and 2 RAC treatments (0 and 400 mg/day). Rumen samples were collected via orogastric tubing 7 days before harvest. DNA from rumen samples were sequenced to identify bacteria based on the V1-V3 hypervariable regions of the 16S rRNA gene. Reads from treatments were analyzed using the packages 'phyloseq' and 'dada2' within the R environment. Beta diversity was analyzed based on Bray-Curtis distances and was significantly different among protein and RAC treatments (P < 0.05). Alpha diversity metrics, such as Chao1 and Shannon diversity indices, were not significantly different (P > 0.05). Bacterial differences among treatments after analyses using PROC MIXED in SAS 9 were identified for the main effects of protein concentration (P < 0.05), rather than their interaction. These results suggest possible effects on microbial communities with different concentrations of protein but limited impact with RAC. However, both may potentially act synergistically to improve performance in finishing beef cattle.

Promoting public engagement in interdisciplinary biological systems education by leveraging American sports-inspired bracket contests on social media and web.

The complexity of modern biology poses challenges in fostering interdisciplinary understanding, particularly between practicing scientists and the public. Furthermore, scientists often lack formal training in science communication, despite various motivations to engage the public. The science literacy of the public in the biological sciences can also vary across socio-economic and cultural backgrounds. Leveraging popular culture and informal learning practices to promote active learning offers promising avenues to enhance public understanding of biological systems. Organized sports hold collective recognition across various communities and cultures, serving as a means to bring people together. Notably, the NCAA March Madness event holds widespread national and international popularity, presenting an opportunity to laterally apply this concept to promote science communication within STEM and biology education. An educational social media and web-based contest tool was developed integrating NCAA-inspired brackets with animal biological systems concepts. The tool featured tournament-style matchups based on animal biological systems, interesting animal facts, and a voting system, all housed within a user-friendly interface. To encourage regular user access to the tool, graphic designs were developed for all social media posts to aid in visual recruitment to the voting website. Based on online metrics, the use of social media garnered repeat users across both the public and educators. The latter noted the tool's simplicity and informative content. Application of this social media and web-based bracket contest tool, which leverages informal settings for active learning for use in biology education, can foster science communication to engage audiences, improve comprehension, and promote interdisciplinary biology education.

Beta-Adrenergic Agonists, Dietary Protein, and Rumen Bacterial Community Interactions in Beef Cattle: A Review.

Improving beef production efficiency, sustainability, and food security is crucial for meeting the growing global demand for beef while minimizing environmental impact, conserving resources, ensuring economic viability, and promoting animal welfare. Beta-adrenergic agonists and dietary protein have been critical factors in beef cattle production. Beta-agonists enhance growth, improve feed efficiency, and influence carcass composition, while dietary protein provides the necessary nutrients for muscle development and overall health. A balanced approach to their use and incorporation into cattle diets can lead to more efficient and sustainable beef production. However, microbiome technologies play an increasingly important role in beef cattle production, particularly by optimizing rumen fermentation, enhancing nutrient utilization, supporting gut health, and enhancing feed efficiency. Therefore, optimizing rumen fermentation, diet, and growth-promoting technologies has the potential to increase energy capture and improve performance. This review addresses the interactions among beta-adrenergic agonists, protein level and source, and the ruminal microbiome. By adopting innovative technologies, sustainable practices, and responsible management strategies, the beef industry can contribute to a more secure and sustainable food future. Continued research and development in this field can lead to innovative solutions that benefit both producers and the environment.

Interplay between the gastric bacterial microbiota and Candida albicans during postantibiotic recolonization and gastritis.

The indigenous bacterial microbiome of the stomach, including lactobacilli, is vital in promoting colonization resistance against Candida albicans. However, there are gaps in our understanding about C. albicans gastric colonization versus disease, especially during the postantibiotic recovery phase. This study compared the gastric responses to C. albicans strains CHN1 and SC5314 in microbiome-disturbed and germfree mice to elucidate the contribution of the indigenous microbiota in C. albicans colonization versus disease and yeast-bacterium antagonism during the post-cefoperazone recolonization period. C. albicans can prevent the regrowth of Lactobacillus spp. in the stomach after cefoperazone and promote increased colonization by Enterococcus spp. Using a culture-independent analysis, the effects of oral cefoperazone on the gastric bacterial microbiota were observed to last at least 3 weeks after the cessation of the antibiotic. Disturbance of the gastric bacterial community by cefoperazone alone was not sufficient to cause gastritis, C. albicans colonization was also needed. Gastritis was not evident until after day 7 in cefoperazone-treated infected mice. In contrast, in germfree mice which lack a gastric microbiota, C. albicans induced gastric inflammation within 1 week of inoculation. Therefore, the gastric bacterial community in cefoperazone-treated mice during the first week of postantibiotic recolonization was sufficient to prevent the development of gastritis, despite being ineffective at conferring colonization resistance against C. albicans. Altogether, these data implicate a dichotomy between C. albicans colonization and gastric disease that is bacterial microbiome dependent.

Candida albicans and bacterial microbiota interactions in the cecum during recolonization following broad-spectrum antibiotic therapy.

Candida albicans is a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota, C. albicans can disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role of C. albicans in shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventing C. albicans colonization. Histological analysis demonstrated that colonization with C. albicans during antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence of C. albicans during antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishes Bacteroidetes populations long term in the ceca of mice, but the presence of C. albicans during cefoperazone recovery promoted Bacteroidetes population recovery. However, the presence of C. albicans resulted in a long-term reduction in Lactobacillus spp. and promoted Enterococcus faecalis populations. Previous studies have focused on the ability of bacteria to alter C. albicans; this study addresses the ability of C. albicans to alter the bacterial microbiota during nonpathogenic colonization.

Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme (DUB), is a potential drug target in various cancers, and liver and lung fibrosis. However, bona fide functions and substrates of UCHL1 remain poorly understood. Herein, we report the characterization of UCHL1 covalent inhibitor MT16-001 based on a thiazole cyanopyrrolidine scaffold. In combination with chemical proteomics, a closely related activity-based probe (MT16-205) was used to generate a comprehensive quantitative profile for on- and off-targets at endogenous cellular abundance. Both compounds are selective for UCHL1 over other DUBs in intact cells but also engage a range of other targets with good selectivity over the wider proteome, including aldehyde dehydrogenases, redox-sensitive Parkinson's disease related protein PARK7, and glutamine amidotransferase. Taken together, these results underline the importance of robust profiling of activity-based probes as chemical tools and highlight the cyanopyrrolidine warhead as a versatile platform for liganding diverse classes of protein with reactive cysteine residues which can be used for further inhibitor screening, and as a starting point for inhibitor development.

Control of mucosal polymicrobial populations by innate immunity.

The gastrointestinal tract carries out the complex process of localizing the polymicrobial populations of the indigenous microbiota to the lumenal side of the GI mucosa while absorbing nutrients from the lumen and preventing damage to the mucosa. This process is accomplished through a combination of physical, innate and adaptive host defences and a 'strategic alliance' with members of the microbiota. To cope with the constant exposure to a diverse microbial community, the GI tract, through the actions of a number of specialized cells in the epithelium and lamina propria, has layers of humoral, physical and cellular defences that limit attachment, invasion and dissemination of the indigenous microbiota. However, the role of the microbiota in this dynamic balance is vital and serves as another level of 'innate' defence. We are just beginning to understand how bacterial metabolites aid in the control of potential pathogens within the microbiota and limit inflammatory responses to the microbiota, concepts that will impact our understanding of the biological effects of antibiotics, diet and probiotics on mucosal inflammatory responses.

Cow-calf performance, forage utilization, and economics of warm-season annual baleage in beef cattle winter feeding systems.

A 52-d winter feeding trial was conducted to determine animal performance, utilization, and economics of pearl millet (PM) baleage, sorghum × sudangrass (SS) baleage, and "Tifton 85" bermudagrass (B) hay for lactating beef cow-calf pairs. Cone (C) and open-shaped (O) rings were evaluated for potential to minimize forage wastage. The experiment was a completely randomized design with a 3 × 2 factorial arrangement of treatments for each forage type × hay ring (3 cow-calf pairs per treatment; 2 replications per treatment). Animal response measures included cow body weight (BW) change and body condition score (BCS) over the 52-d trial, initial and final calf BW, and cow milk production at the midpoint and end of the study. Forage nutritive value parameters evaluated for each forage type included ash, crude protein (CP), in vitro true digestibility (IVTD), neutral detergent fiber (NDF), acid detergent fiber, and acid detergent lignin (ADL). Forage wastage was estimated for each forage × ring treatment as the percentage of the bale weight remaining in feeding rings at the time of bale replacement. An economic evaluation of the relative costs associated with production and utilization of each forage type was calculated. There were no differences (P ≥ 0.10) in cow BW change or BCS change among forage types, between ring shapes, or an interaction observed for these response variables. Proportion of waste from PM and SS baleage was greater (P < 0.10) than for B hay, although there was no forage type × hay ring interaction or differences between O and C hay ring treatments for forage waste (P ≥ 0.10, respectively). Cow milk production and calf BW gain did not differ among forage type (P ≥ 0.10, respectively); however, beef calves in pens containing the O ring feeder weighed 6 kg more (P ≤ 0.05) than calves whose dams were fed using C rings. The economic analysis implies that it is more costly to feed warm-season annual forage baleage to cow-calf pairs than dry hay, largely due to greater costs of production, lack of difference in animal performance responses, and less utilization of baleage compared with feeding bermudagrass hay in this trial.

Overview of gut immunology.

The gastrointestinal tract (GI tract) plays dual roles in human physiology: digestion and uptake of nutrients and the more daunting task of maintaining immune homeostasis (protecting the body from potentially harmful microbes, while inducing tolerogenic responses to innocuous food, commensals and self-antigens). The unique architecture of the GI tract facilitates both of these functions; multiple levels of infolding results in an immense overall surface area that allows maximal nutrient absorption while housing the largest number of immune cells in the body. This review will focus on how mucosal immune responses generated in the GI tract are organized and controlled. The gastro-intestinal associated lymphoid tissue (GALT), which is composed of discrete inductive and effectors sites, is able to discriminate between harmful and harmless antigens while maintaining homeostasis. Inductive sites are organized into specialized aggregations oflymphoid follicles called Peyer's patches (PP), while effector sites are more diffusely dispersed. The separation of these sites serves to limit and control immune responses. In addition to its distinct architecture, the GI tract has specialized immune cells that aid in promoting a tolerogenic response to orally introduced antigens, (e.g. subsets ofdendritic cells (DCs) and regulatory T-cells (T(R. Secretory IgA (sIgA),which is produced in appreciable quantities at mucosal surfaces, also promotes an anti-inflammatory environment by neutralizing immune stimulatory antigens. The mechanisms of induction tolerance are currently poorly understood; however, this tolerant environment limits potentially damaging inflammatory responses to inappropriate stimuli.

Modulation of post-antibiotic bacterial community reassembly and host response by Candida albicans.

The introduction of Candida albicans into cefoperazone-treated mice results in changes in bacterial community reassembly. Our objective was to use high-throughput sequencing to characterize at much greater depth the specific changes in the bacterial microbiome. The colonization of C. albicans significantly altered bacterial community reassembly that was evident at multiple taxonomic levels of resolution. There were marked changes in the levels of Bacteriodetes and Lactobacillaceae. Lachnospiraceae and Ruminococcaceae, the two most abundant bacterial families, did not change in relative proportions after antibiotics, but there were marked genera-level shifts within these two bacterial families. The microbiome shifts occurred in the absence of overt intestinal inflammation. Overall, these experiments demonstrate that the introduction of a single new microbe in numerically inferior numbers into the bacterial microbiome during a broad community disturbance has the potential to significantly alter the subsequent reassembly of the bacterial community as it recovers from that disturbance.

Postpartum group a Streptococcus sepsis and maternal immunology.

Group A Streptococcus (GAS) is an historically important agent of puerperal infections and sepsis. The inception of hand-washing and improved hospital hygiene drastically reduced the incidence of puerperal sepsis, but recently the incidence and severity of postpartum GAS infections has been rising for uncertain reasons. Several epidemiological, host, and microbial factors contribute to the risk for GAS infection and mortality in postpartum women. These include the mode of delivery (vaginal versus cesarean section), the location where labor and delivery occurred, exposure to GAS carriers, the altered immune status associated with pregnancy, the genetic background of the host, the virulence of the infecting GAS strain, and highly specialized immune responses associated with female reproductive tract tissues and organs. This review will discuss the complicated factors that contribute to the increased susceptibility to GAS after delivery and potential reasons for the recent increase observed in morbidity and mortality.

From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta.

UNLABELLED: A dynamic homeostasis is maintained between the host and native bacteria of the gastrointestinal tract in animals, but migration of bacteria from the gut to other organs can lead to disease or death. Enterococcus faecalis is a commensal of the gastrointestinal tract; however, Enterococcus spp. are increasingly frequent causes of nosocomial infections with a high mortality rate. We investigated the commensal-to-pathogen switch undergone by E. faecalis OG1RF in the lepidopteran model host Manduca sexta associated with its location in the host. E. faecalis persists in the harsh midgut environment of M. sexta larvae without causing apparent illness, but injection of E. faecalis directly into the larval hemocoel is followed by rapid death. Additionally, oral ingestion of E. faecalis in the presence of Bacillus thuringiensis insecticidal toxin, a pore-forming toxin that targets the midgut epithelium, induces an elevated mortality rate. We show that the loss of gut integrity due to B. thuringiensis toxin correlates with the translocation of E. faecalis from the gastrointestinal tract into the hemolymph. Upon gaining access to the hemolymph, E. faecalis induces an innate immune response, illustrated by hemocyte aggregation, in larvae prior to death. The degree of hemocyte aggregation is dependent upon the route of E. faecalis entry. Our data demonstrate the efficacy of the M. sexta larval model system in investigating E. faecalis-induced sepsis and clarifies controversies in the field regarding the events leading to larval death following B. thuringiensis toxin exposure. IMPORTANCE: This study advances our knowledge of Enterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate that E. faecalis is a commensal in the gut of Manduca sexta and a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the "commensal-to-pathogen" switch. While controversy remains regarding Bacillus thuringiensis toxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential for B. thuringiensis toxin killing of Lymantria dispar (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196-15199, 2006; B. Raymond, et al., Environ. Microbiol. 11:2556-2563, 2009; P. R. Johnston, and N. Crickmore, Appl. Environ. Microbiol. 75:5094-5099, 2009). We and others have demonstrated that the role of the midgut microbiota in B. thuringiensis toxin killing is dependent upon the lepidopteran species and formulation of B. thuringiensis toxin (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196-15199, 2006; N. A. Broderick, et al., BMC Biol. 7:11, 2009). This work reconciles much of the apparently contradictory previous data and reveals that the M. sexta-E. faecalis system provides a model for mammalian sepsis.