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The degree of lymph-node metastasis in prostate cancer is crucial for both staging the disease and planning treatment. Here, Burton and colleagues describe a one-step, non-invasive imaging technology using prostate-specific adenoviral vectors that express imaging reporter genes. This set-up specifically and accurately detects lymph-node metastases in a model of human prostate cancer and eliminates the need for invasive lymphadenectomy required by the current lymphoscintigraphy method. The accurate assessment of nodal involvement in prostate cancer is crucial to planning treatment, yet there is a shortage of noninvasive imaging techniques capable of visualizing nodal lesions directly. This study demonstrates the feasibility of using recombinant human adenoviral vectors to detect nodal metastases in a human prostate cancer model. This was achieved by the prostate-restricted expression of optical and positron emission tomography (PET) imaging reporter genes by the viral vector coupled with the innate lymphotropic properties of adenovirus. We show that peritumoral administration of these vectors results in the direct detection of reporter gene expression in metastatic lesions within sentinel lymph nodes. Notably, this approach parallels the current lymphoscintigraphy method but enables the direct PET visualization of sentinel lymph node metastases, eliminating the need for invasive lymphadenectomy. These findings may lead to more effective diagnostic and therapeutic strategies for individuals with advanced-stage prostate cancer.

Introduction: Metastatic disease is a major cause of prostate cancer mortality. Lymph node involvement is an important early prognostic indicator of patient survival. The extent of tumor- associated lymphangiogenesis can dictate lymphatic metastasis in many tumors. Limited experience on this issue in prostate cancer leads us to study tumor models, which recapitulate many of characteristics of the human disease including androgen regulation and PSA expression. Results: By applying in vivo bioluminescence imaging, we observed that prostate cancer xenografts display distinctive metastatic potentials. Whereas LAPC-4 and CWR22RV-1 tumors metastasized to lymph nodes and lungs, LAPC-9 tumors did not. Quantitative real-time PCR revealed that the expression of pro-lymphangiogenic factor, VEGF-C, was elevated in LAPC-4 and CWR22RV-1 compared to LAPC-9 tumors. Immunohistochemistry showed that LAPC-4 tumors contained patent intratumoral lymphatics, often filled with tumor cells. To examine the correlation of tumor lymphangiogenesis and metastasis, we over-expressed VEGF-C in the non-metastatic LAPC-9 model, and we down- regulated lymphangiogenesis in CWR22RV-1 by expressing soluble VEGF-R3 to sequester VEGF-C. Recombinant lentiviral vectors were applied to express luciferase and vascular growth factor genes in the tumors. The effects of VEGF-C, VEGF-C C156S (a point mutant of VEGF-C that induces lymphangiogenesis but not angiogenesis), or VEGF-A were compared to control vector. While VEGF-A increased the number of blood vessels and the rate of growth, we observed limited enhancement of lymph node (signals 10-fold greater photons/s/cm 2 /sr over control) and lung metastasis. On the other hand, VEGF-CC156S and VEGF-C over-expression resulted in enhanced intratumoral lymphangiogenesis and significant dissemination to ipsilateral lymph nodes (200-to 1000-fold over control) and lungs (25-fold enhancement). The VEGF-CC156S tumors exhibited no increase in blood vessels. The expression of soluble VEGF-R3 in CWR22RV-1 resulted in a significant reduction in intratumoral vasculatures as well as metastasis to both regional lymph node and lung. The extent of metastasis in all tumor models assessed by optical imaging correlated well with immunocytochemical analyses of metastasis by cytokeratin marker and GFP. Conclusion: Bioluminescence imaging is a valuable tool to track metastasis in both the VEGF-C and VEGF-A expressing tumors. Our results indicate that tumor lymphangiogenesis is a key factor contributing to not only local regional lymph nodes but also to lungs in prostate cancer models. Inhibiting tumor lymphangiogenesis may be a promising therapeutic strategy to suppress the deadly consequence of systemic metastasis.

A poem is presented.

Nonalcoholic fatty liver disease (NAFLD) is an obesity-related disorder that is rapidly increasing in incidence and is considered the hepatic manifestation of the metabolic syndrome. The gut microbiome plays a role in metabolism and maintaining gut barrier integrity. Studies have found differences in the microbiota between NAFLD and healthy patients and increased intestinal permeability in patients with NAFLD. Fecal microbiota transplantation (FMT) can be used to alter the gut microbiome. It was hypothesized that an FMT from a thin and healthy donor given to patients with NAFLD would improve insulin resistance (IR), hepatic proton density fat fraction (PDFF), and intestinal permeability. Twenty-one patients with NAFLD were recruited and randomized in a ratio of 3:1 to either an allogenic (n = 15) or an autologous (n = 6) FMT delivered by using an endoscope to the distal duodenum. IR was calculated by HOMA-IR, hepatic PDFF was measured by MRI, and intestinal permeability was tested using the lactulose:mannitol urine test. Additional markers of metabolic syndrome and the gut microbiota were examined. Patient visits occurred at baseline, 2, 6 weeks, and 6 months post-FMT. There were no significant changes in HOMA-IR or hepatic PDFF in patients who received the allogenic or autologous FMT. Allogenic FMT patients with elevated small intestinal permeability (>0.025 lactulose:mannitol, n = 7) at baseline had a significant reduction 6 weeks after allogenic FMT. FMT did not improve IR as measured by HOMA-IR or hepatic PDFF but did have the potential to reduce small intestinal permeability in patients with NAFLD.

Exposure to environmental toxins is a 21st century global health problem that is often the result of dietary intake. Although efforts are made to reduce dietary toxin levels, they are often unsuccessful, warranting research into novel methods to reduce host exposure. Food-grade microbes that can be delivered to the gastrointestinal tract and that are capable of sequestering toxins present a safe and cost-effective intervention. We sought to investigate the potential for probiotic-supplemented yogurt to lower heavy metal levels in at-risk populations of pregnant women and in children in Mwanza, Tanzania, and to examine the microbiome in relation to toxin levels. Two populations suspected to have high toxic metal exposures were studied. A group of 44 school-aged children was followed over 25 days, and 60 pregnant women were followed over their last two trimesters until birth. A yogurt containing 10 10  CFU Lactobacillus rhamnosus GR-1 per 250 g was administered, while control groups received either whole milk or no intervention. Changes in blood metal levels were assessed, and the gut microbiomes of the children were profiled by analyzing 16S rRNA sequencing via the Ion Torrent platform. The children and pregnant women in the study were found to have elevated blood levels of lead and mercury compared to age- and sex-matched Canadians. Consumption of probiotic yogurt had a protective effect against further increases in mercury (3.2 nmol/liter; P = 0.035) and arsenic (2.3 nmol/liter; P = 0.011) blood levels in the pregnant women, but this trend was not statistically significant in the children. Elevated blood lead was associated with increases in Succinivibrionaceae and Gammaproteobacteria relative abundance levels in stool. IMPORTANCE Probiotic food produced locally represents a nutritious and affordable means for people in some developing countries to counter exposures to toxic metals. Further research and field trials are warranted to explore this approach in countries where communities are located near mining sites and agricultural areas, two types of areas where toxins are likely to be elevated. Probiotic food produced locally represents a nutritious and affordable means for people in some developing countries to counter exposures to toxic metals. Further research and field trials are warranted to explore this approach in countries where communities are located near mining sites and agricultural areas, two types of areas where toxins are likely to be elevated.

A lactobacilli dominated microbiota in most pre and post-menopausal women is an indicator of vaginal health. The objective of this double blinded, placebo-controlled crossover study was to evaluate in 14 post-menopausal women with an intermediate Nugent score, the effect of 3 days of vaginal administration of probiotic L. rhamnosus GR-1 and L. reuteri RC-14 (2.5×109 CFU each) on the microbiota and host response. The probiotic treatment did not result in an improved Nugent score when compared to when placebo. Analysis using 16S rRNA sequencing and metabolomics profiling revealed that the relative abundance of Lactobacillus was increased following probiotic administration as compared to placebo, which was weakly associated with an increase in lactate levels. A decrease in Atopobium was also observed. Analysis of host responses by microarray showed the probiotics had an immune-modulatory response including effects on pattern recognition receptors such as TLR2 while also affecting epithelial barrier function. This is the first study to use an interactomic approach for the study of vaginal probiotic administration in post-menopausal women. It shows that in some cases multifaceted approaches are required to detect the subtle molecular changes induced by the host to instillation of probiotic strains. ClinicalTrials.gov NCT02139839.

Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18's persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.

Background Inquiry of microbiota involvement in kidney stone disease (KSD) has largely focussed on potential oxalate handling abilities by gut bacteria and the increased association with antibiotic exposure. By systematically comparing the gut, urinary, and oral microbiota of 83 stone formers (SF) and 30 healthy controls (HC), we provide a unified assessment of the bacterial contribution to KSD. Results Amplicon and shotgun metagenomic sequencing approaches were consistent in identifying multi-site microbiota disturbances in SF relative to HC. Biomarker taxa, reduced taxonomic and functional diversity, functional replacement of core bioenergetic pathways with virulence-associated gene markers, and community network collapse defined SF, but differences between cohorts did not extend to oxalate metabolism. Conclusions We conclude that multi-site microbiota alteration is a hallmark of SF, and KSD treatment should consider microbial functional restoration and the avoidance of aberrant modulators such as poor diet and antibiotics where applicable to prevent stone recurrence. 7-LZNFESG25-7M4uP2eh2i Video Abstract

People evaluate a stranger’s trustworthiness from their facial features in a fraction of a second, despite common advice “not to judge a book by its cover.” Evaluations of trustworthiness have critical and widespread social impact, predicting financial lending, mate selection, and even criminal justice outcomes. Consequently, understanding how people perceive trustworthiness from faces has been a major focus of scientific inquiry, and detailed models explain how consensus impressions of trustworthiness are driven by facial attributes. However, facial impression models do not consider variation between observers. Here, we develop a sensitive test of trustworthiness evaluation and use it to document substantial, stable individual differences in trustworthiness impressions. Via a twin study, we show that these individual differences are largely shaped by variation in personal experience, rather than genes or shared environments. Finally, using multivariate twin modeling, we show that variation in trustworthiness evaluation is specific, dissociating from other key facial evaluations of dominance and attractiveness. Our finding that variation in facial trustworthiness evaluation is driven mostly by personal experience represents a rare example of a core social perceptual capacity being predominantly shaped by a person’s unique environment. Notably, it stands in sharp contrast to variation in facial recognition ability, which is driven mostly by genes. Our study provides insights into the development of the social brain, offers a different perspective on disagreement in trust in wider society, and motivates new research into the origins and potential malleability of face evaluation, a critical aspect of human social cognition.

Membrane fusion triggered by Ca 2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes 1 – 4 . For neurotransmitter release, the Ca 2+ sensitivity is introduced by interactions between the Ca 2+ sensor synaptotagmin and the SNARE complex 5 , and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion 6 . Disruption of Ca 2+ -triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis 7 – 11 . Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca 2+ -triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca 2+ -binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca 2+ -triggered fusion at physiological Ca 2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca 2+ -triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways. Peptides that disrupt Ca 2+ -triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.