Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules.

Platelet α-granules regulate hemostasis and myriad other physiological processes, but their biogenesis is unclear. Mutations in only 3 proteins are known to cause α-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized α-granule proteins through megakaryocyte (MK) endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Syntaxin 12 (Stx12), a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12-deficient MKs display reduced α-granule numbers and overall levels of α-granule proteins, thus revealing Stx12 as a new component of the α-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B, suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in MKs contains COMMD3, one of 10 COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced α-granule numbers and overall levels of α-granule proteins, establishing the COMMD3/CCC complex as a new factor in α-granule biogenesis. Furthermore, P-selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in α-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of α-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in MKs to make α-granules.

Cargo-mediated recruitment of the endocytic adaptor protein Sla1 in S. cerevisiae.

Endocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests that adaptor protein recruitment to the plasma membrane may depend on binding to endocytic cargo. To test this idea, we analyzed the yeast adaptor protein Sla1, which binds membrane proteins harboring the endocytic signal NPFxD via the Sla1 SHD1 domain. Consistently, SHD1 domain point mutations that disrupted NPFxD binding caused a proportional reduction in Sla1-GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 domain point mutation and deletion of the C-terminal LxxQxTG repeat (SR) region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1-GFP recruitment to the plasma membrane. These data suggest that multiple interactions are needed for recruitment of Sla1 to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain, which binds ubiquitin, and the SHD1 domain displayed broad surface localization, suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitylated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region might regulate the Sla1-cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.

Gut microbiota regulates cardiac ischemic tolerance and aortic stiffness in obesity.

The gut microbiota has emerged as an important regulator of host physiology, with recent data suggesting a role in modulating cardiovascular health. The present study determined if gut microbial signatures could transfer cardiovascular risk phenotypes between lean and obese mice using cecal microbiota transplantation (CMT). Pooled cecal contents collected from obese leptin-deficient (Ob) mice or C57Bl/6j control (Con) mice were transplanted by oral gavage into cohorts of recipient Ob and Con mice maintained on identical low-fat diets for 8 wk (n = 9-11/group). Cardiovascular pathology was assessed as the degree of arterial stiffness (aortic pulse wave velocity) and myocardial infarct size following a 45/120 min ex vivo global cardiac ischemia-reperfusion protocol. Gut microbiota was characterized by 16S rDNA sequencing, along with measures of intestinal barrier function and cecal short-chain fatty acid (SCFA) composition. Following CMT, the gut microbiota of recipient mice was altered to resemble that of the donors. Ob CMT to Con mice increased arterial stiffness, left ventricular (LV) mass, and myocardial infarct size, which were associated with greater gut permeability and reduced cecal SCFA concentrations. Conversely, Con CMT to Ob mice increased cecal SCFA, reduced LV mass, and attenuated myocardial infarct size, with no effects on gut permeability or arterial stiffness. Collectively, these data demonstrate that obesity-related changes in the gut microbiota, independent of dietary manipulation, regulate hallmark measures of cardiovascular pathology in mice and highlight the potential of microbiota-targeted therapeutics for reducing cardiovascular pathology and risk in obesity.NEW & NOTEWORTHY These data are the first to demonstrate that cecal microbiota transplantation (CMT) can alter cardiovascular pathology in lean and obese mice independent from alterations in dietary intake. Myocardial infarct size was reduced in obese mice receiving lean CMT and worsened in lean mice receiving obese CMT. Lean mice receiving obese CMT also displayed increased aortic stiffness. These changes were accompanied by alterations in short-chain fatty acids and gut permeability.

PHAGE Study: Effects of Supplemental Bacteriophage Intake on Inflammation and Gut Microbiota in Healthy Adults.

The gut microbiota is increasingly recognized as an important modulator of human health. As such, there is a growing need to identify effective means of selectively modifying gut microbial communities. Bacteriophages, which were briefly utilized as clinical antimicrobials in the early 20th century, present an opportunity to selectively reduce populations of undesirable microorganisms. However, whether intentional consumption of specific bacteriophages affects overall gut ecology is not yet known. Using a commercial cocktail of Escherichia coli-targeting bacteriophages, we examined their effects on gut microbiota and markers of intestinal and systemic inflammation in a healthy human population. In a double-blinded, placebo-controlled crossover trial, normal to overweight adults consumed bacteriophages for 28 days. Stool and blood samples were collected and used to examine inflammatory markers, lipid metabolism, and gut microbiota. Reductions in fecal E. coli loads were observed with phage consumption. However, there were no significant changes to alpha and beta diversity parameters, suggesting that consumed phages did not globally disrupt the microbiota. However, specific populations were altered in response to treatment, including increases in members of the butyrate-producing genera Eubacterium and a decreased proportion of taxa most closely related to Clostridium perfringens. Short-chain fatty acid production, inflammatory markers, and lipid metabolism were largely unaltered, but there was a small but significant decrease in circulating interleukin-4 (Il-4). Together, these data demonstrate the potential of bacteriophages to selectively reduce target organisms without global disruption of the gut community.

Distinct Physiological Roles of the Three Ferredoxins Encoded in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins-an abundant class of Fe-S-containing small proteins-are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology.IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.

A linear pathway for mevalonate production supports growth of Thermococcus kodakarensis.

The sole unifying feature of Archaea is the use of isoprenoid-based glycerol lipid ethers to compose cellular membranes. The branched hydrocarbon tails of archaeal lipids are synthesized via the polymerization of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but many questions still surround the pathway(s) that result in production of IPP and DMAPP in archaeal species. Isotopic-labeling strategies argue for multiple biological routes for production of mevalonate, but biochemical and bioinformatic studies support only a linear pathway for mevalonate production. Here, we use a combination of genetic and biochemical assays to detail the production of mevalonate in the model archaeon Thermococcus kodakarensis. We demonstrate that a single, linear pathway to mevalonate biosynthesis is essential and that alternative routes of mevalonate production, if present, are not biologically sufficient to support growth in the absence of the classical mevalonate pathway resulting in IPP production from acetyl-CoA. Archaeal species provide an ideal platform for production of high-value isoprenoids in large quantities, and the results obtained provide avenues to further increase the production of mevalonate to drive isoprenoid production in archaeal hosts.

Bacteriophage for Gastrointestinal Health (PHAGE) Study: Evaluating the Safety and Tolerability of Supplemental Bacteriophage Consumption.

OBJECTIVE: The gut microbiota has been recognized as a critical regulator of human health, and novel interventions to selectively modulate the microbiota are actively being sought. Bacteriophages (bacterial viruses) have the potential to selectively eliminate specific detrimental microbes while enhancing beneficial microbe populations. The Bacteriophage for Gastrointestinal Health (PHAGE) study aimed to determine the safety and tolerability of supplemental bacteriophage consumption in a population of healthy adults with mild to moderate gastrointestinal distress. METHODS: The PHAGE study was a randomized, double-blind, placebo-controlled crossover intervention. Healthy adults with self-reported gastrointestinal distress were recruited and asked to consume one 15-mg capsule containing 4 strains of bacteriophages (LH01-Myoviridae, LL5-Siphoviridae, T4D-Myoviridae, and LL12-Myoviridae) and a placebo, each for 28 days. Participants were randomly assigned to the starting treatment, which was followed by a 2-week washout period before they began the second arm of the intervention. Primary outcome measures included a comprehensive metabolic panel and gastrointestinal health questionnaire. In addition, samples were collected for future analysis of several secondary outcome measures, including global microbiota profiles, plasma lipids, and markers of local and systemic inflammation. RESULTS: Forty-three individuals met all study criteria and consented to participate. Of these participants, 36 completed at least one arm of the trial and 32 completed the study. There were no effects of treatment sequence on comprehensive metabolic panel outcomes, but there were 1- and 2-way carryover effects on gastrointestinal questionnaire data. Levels of aspartate aminotransferase significantly decreased while participants were taking the treatment but not placebo; however, all mean values remained within clinically acceptable ranges. Participants also reported significant improvements in several symptoms of gastrointestinal distress while taking both the treatment and the placebo. CONCLUSIONS: Consumption of therapeutic doses of a mixture of 4 bacteriophages was both safe and tolerable in a target human population.

Impact of Edible Cricket Consumption on Gut Microbiota in Healthy Adults, a Double-blind, Randomized Crossover Trial.

Edible insects are often considered a nutritious, protein-rich, environmentally sustainable alternative to traditional livestock with growing popularity among North American consumers. While the nutrient composition of several insects is characterized, all potential health impacts have not been evaluated. In addition to high protein levels, crickets contain chitin and other fibers that may influence gut health. In this study, we evaluated the effects of consuming 25 grams/day whole cricket powder on gut microbiota composition, while assessing safety and tolerability. Twenty healthy adults participated in this six-week, double-blind, crossover dietary intervention. Participants were randomized into two study arms and consumed either cricket-containing or control breakfast foods for 14 days, followed by a washout period and assignment to the opposite treatment. Blood and stool samples were collected at baseline and after each treatment period to assess liver function and microbiota changes. Results demonstrate cricket consumption is tolerable and non-toxic at the studied dose. Cricket powder supported growth of the probiotic bacterium, Bifidobacterium animalis, which increased 5.7-fold. Cricket consumption was also associated with reduced plasma TNF-α. These data suggest that eating crickets may improve gut health and reduce systemic inflammation; however, more research is needed to understand these effects and underlying mechanisms.