Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.     

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.     

Manipulating the Level of Sensorimotor Stimulation during LI-rTMS Can Improve Visual Circuit Reorganisation in Adult Ephrin-A2A5-/- Mice

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject’s brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5^-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5^-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.     

Pial Vessel-Associated Microglia/Macrophages Increase in Female Dahl-SS/Jr Rats Independent of Pregnancy History

As the resident immune cells of the central nervous system, microglia have a wide range of functions such as surveillance, phagocytosis, and signaling through production of chemokines and cytokines. Recent studies have identified and characterized macrophages residing at the meninges, a series of layers surrounding the brain and spinal cord. While perivascular microglia within the brain parenchyma increase following chronic hypertension, there are no reports of changes at the meninges, and specifically, associated with the pial vasculature. Thus, we used female Sprague Dawley and Dahl salt-sensitive (SS/Jr) rat brains, stained for ionized calcium-binding adapter molecule (Iba1), and characterized microglia/macrophages associated with pial vessels in the posterior brain. Results indicate that Iba1⁺ pial vessel-associated microglia (PVAM) completely surrounded the vessels in brains from the Dahl-SS/Jr rats. PVAM density was significantly higher and distance between PVAMs lower in Dahl-SS/Jr compared to the Sprague Dawley rat brains. Pregnancy history did not affect these findings. While the functional role of these cells are not known, we contextualize our novel findings with that of other studies assessing or characterizing myeloid cells at the borders of the CNS (meninges and choroid plexus) and perivascular macrophages and propose their possible origin in the Dahl-SS/Jr model of chronic hypertension.     

Lake Champlain offshore benthic invertebrate community before and after zebra mussel invasion

Benthic invertebrates are important bio-indicators of water quality and play a significant role in aquatic systems. Lake Champlain has limited benthic invertebrate data which hinders development of food web models, assessment of invasive species impacts, and evaluation of management actions. In June 2016, we assessed benthic invertebrates along three transects in the main basin of Lake Champlain ranging from 5 to 100?m, and then compared results to densities from a limited survey in 1991 prior to the zebra mussel (Dreissena polymorpha) invasion. In 2016, total biomass and density were 1–2 orders of magnitude greater at 5?m than at 20–100?m. Zebra mussels, chironomids, oligochaetes, and gastropods were dominant at 5?m, and oligochaetes and sphaeriids were dominant at 20–100?m. Total density at the 5-m site was 94% lower in 2016 compared to 1991, but similar at the 100-m site. Diporeia, while abundant in many freshwater bodies, is historically rare in Lake Champlain and was not detected in our sampling. Because Lake Champlain benthic invertebrate densities are low and display dissimilar distributions to the Great Lakes, we hypothesize the offshore fish community is likely much more reliant on pelagic rather than benthic production. Although the current composition and biomass suggest the benthic community in Lake Champlain may not be greatly impacted by an invasion of quagga mussel (D. rostriformis bugensis), the potential for quaggas to re-route energy from pelagic to benthic habitats, as it has in the Great Lakes, could limit the Lake Champlain offshore fish community.     

β-Selective C−H Arylation of Electron-Deficient Thiophenes,Pyrroles, and Furans

A general β-C−H arylation of electron-deficient thiophenes, pyrroles, and furans has been developed using ligand-modulated palladium catalyst. The use of a modified norbornene is crucial for reversing the conventional α-selectivity of these substrates. This method features good yields, high β-selectivity, and good tolerance of functional groups.     

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

Infections from antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial-resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β-lactam antibiotics against Gram-positive bacteria. BPEI binds cell-wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-β-lactam co-drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.     

5-Benzylidene-4-Oxazolidinones Are Synergistic with Antibiotics for the Treatment of Staphylococcus aureus Biofilms

The failure of frontline antibiotics in the clinic is one of the most serious threats to human health and requires a multitude of novel therapeutics and innovative approaches to treatment so as to curtail the growing crisis. In addition to traditional resistance mechanisms resulting in the lack of efficacy of many antibiotics, most chronic and recurring infections are further made tolerant to antibiotic action by the presence of biofilms. Herein, we report an expanded set of 5-benzylidene-4-oxazolidinones that are able to inhibit the formation of Staphylococcus aureus biofilms, disperse preformed biofilms, and, in combination with common antibiotics, are able to significantly reduce the bacterial load in a robust collagen-matrix model of biofilm infection.