Modifying the maternal microbiota alters the gut-brain metabolome and prevents emotional dysfunction in the adult offspring of obese dams.

Maternal obesity disturbs brain-gut-microbiota interactions and induces negative affect in the offspring, but its impact on gut and brain metabolism in the offspring (F1) are unknown. Here, we tested whether perinatal intake of a multispecies probiotic could mitigate the abnormal emotional behavior in the juvenile and adult offspring of obese dams. Untargeted NMR-based metabolomic profiling and gene-expression analysis throughout the gut-brain axis were then used to investigate the biology underpinning behavioral changes in the dams and their offspring. Prolonged high-fat diet feeding reduced maternal gut short-chain fatty acid abundance, increased markers of peripheral inflammation, and decreased the abundance of neuroactive metabolites in maternal milk during nursing. Both juvenile (postnatal day [PND] 21) and adult (PND112) offspring of obese dams exhibited increased anxiety-like behavior, which were prevented by perinatal probiotic exposure. Maternal probiotic treatment increased gut butyrate and brain lactate in the juvenile and adult offspring and increased the expression of prefrontal cortex PFKFB3, a marker of glycolytic metabolism in astrocytes. PFKFB3 expression correlated with the increase in gut butyrate in the juvenile and adult offspring. Maternal obesity reduced synaptophysin expression in the adult offspring, while perinatal probiotic exposure increased expression of brain-derived neurotrophic factor. Finally, we showed that the resilience of juvenile and adult offspring to anxiety-like behavior was most prominently associated with increased brain lactate abundance, independent of maternal group. Taken together, we show that maternal probiotic supplementation exerts a long-lasting effect on offspring neuroplasticity and the offspring gut-liver-brain metabolome, increasing resilience to emotional dysfunction induced by maternal obesity.

Platelet mass cytometry reveals dysregulation of prothrombotic pathways in essential thrombocythemia.

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.

Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.

Multispecies probiotic administration reduces emotional salience and improves mood in subjects with moderate depression: a randomised, double-blind, placebo-controlled study.

BACKGROUND: The potential antidepressant properties of probiotics have been suggested, but their influence on the emotional processes that may underlie this effect is unclear. METHODS: Depressed volunteers (n = 71) were recruited into a randomised double-blind, placebo-controlled study to explore the effects of a daily, 4-week intake of a multispecies probiotic or placebo on emotional processing and cognition. Mood, anxiety, positive and negative affect, sleep, salivary cortisol and serum C-reactive peptide (CRP) were assessed before and after supplementation. RESULTS: Compared with placebo, probiotic intake increased accuracy at identifying faces expressing all emotions (+12%, p < 0.05, total n = 51) and vigilance to neutral faces (mean difference between groups = 12.28 ms ± 6.1, p < 0.05, total n = 51). Probiotic supplementation also reduced reward learning (-9%, p < 0.05, total n = 51), and interference word recall on the auditory verbal learning task (-18%, p < 0.05, total n = 50), but did not affect other aspects of cognitive performance. Although actigraphy revealed a significant group × night-time activity interaction, follow up analysis was not significant (p = 0.094). Supplementation did not alter salivary cortisol or circulating CRP concentrations. Probiotic intake significantly reduced (-50% from baseline, p < 0.05, n = 35) depression scores on the Patient Health Questionnaire-9, but these did not correlate with the changes in emotional processing. CONCLUSIONS: The impartiality to positive and negative emotional stimuli or reward after probiotic supplementation have not been observed with conventional antidepressant therapies. Further studies are required to elucidate the significance of these changes with regard to the mood-improving action of the current probiotic.

Gut dysbiosis in severe mental illness and chronic fatigue: a novel trans-diagnostic construct? A systematic review and meta-analysis.

Reduced gut-microbial diversity ("gut dysbiosis") has been associated with an anhedonic/amotivational syndrome ("sickness behavior") that manifests across severe mental disorders and represent the key clinical feature of chronic fatigue. In this systematic review and meta-analysis, we investigated differences in proxy biomarkers of gut dysbiosis in patients with severe mental illness and chronic fatigue vs. controls and the association of these biomarkers with sickness behavior across diagnostic categories. Following PRISMA guidelines, we searched from inception to April 2020 for all the studies investigating proxy biomarkers of gut dysbiosis in patients with severe mental illness and chronic fatigue. Data were independently extracted by multiple observers, and a random-mixed model was used for the analysis. Heterogeneity was assessed with the I2 index. Thirty-three studies were included in the systematic review; nineteen in the meta-analysis (N = 2758 patients and N = 1847 healthy controls). When compared to controls, patients showed increased levels of zonulin (four studies reporting data on bipolar disorder and depression, SMD = 0.97; 95% Cl = 0.10-1.85; P = 0.03, I2 = 86.61%), lipopolysaccharide (two studies reporting data on chronic fatigue and depression, SMD = 0.77; 95% Cl = 0.42-1.12; P < 0.01; I2 = 0%), antibodies against endotoxin (seven studies reporting data on bipolar disorder, depression, schizophrenia, and chronic fatigue, SMD = 0.99; 95% CI = 0.27-1.70; P < 0.01, I2 = 97.14%), sCD14 (six studies reporting data on bipolar disorder, depression, schizophrenia, and chronic fatigue, SMD = 0.54; 95% Cl 0.16-0.81; P < 0.01, I2 = 90.68%), LBP (LBP, two studies reporting data on chronic fatigue and depression, SMD = 0.87; 95% Cl = 0.25-1.48; P < 0.01; I2 = 56.80%), alpha-1-antitripsin (six studies reporting data on bipolar disorder, depression, and schizophrenia, SMD = 1.23; 95% Cl = 0.57-1.88; P < 0.01, I2: 89.25%). Elevated levels of gut dysbiosis markers positively correlated with severity of sickness behavior in patients with severe mental illness and chronic fatigue. Our findings suggest that gut dysbiosis may underlie symptoms of sickness behavior across traditional diagnostic boundaries. Future investigations should validate these findings comparing the performances of the trans-diagnostic vs. categorical approach. This will facilitate treatment breakthrough in an area of unmet clinical need.

Endocannabinoid system mediates the association between gut-microbial diversity and anhedonia/amotivation in a general population cohort.

Anhedonia and amotivation are debilitating symptoms and represent unmet therapeutic needs in a range of clinical conditions. The gut-microbiome-endocannabinoid axis might represent a potential modifiable target for interventions. Based on results obtained from animal models, we tested the hypothesis that the endocannabinoid system mediates the association between gut-microbiome diversity and anhedonia/amotivation in a general population cohort. We used longitudinal data collected from 786 volunteer twins recruited as part the TwinsUK register. Our hypothesis was tested with a multilevel mediation model using family structure as random intercept. The model was set using alpha diversity (within-individual gut-microbial diversity) as predictor, serum and faecal levels of the endocannabinoid palmitoylethanolamide (PEA) as mediator, and anhedonia/amotivation as outcome. PEA is considered the endogenous equivalent of cannabidiol, with increased serum levels believed to have anti-depressive effects, while increased stool PEA levels, reflecting increased excretion, are believed to have opposite, detrimental, effects on mental health. We therefore expected that either reduced serum PEA or increased stool PEA would mediate the association between microbial diversity and anhedonia amotivation. Analyses were adjusted for obesity, diet, antidepressant use, sociodemographic and technical covariates. Data were imputed using multiple imputation by chained equations. Mean age was 65.2 ± 7.6; 93% of the sample were females. We found a direct, significant, association between alpha diversity and anhedonia/amotivation (ß = -0.37; 95%CI: -0.71 to -0.03; P = 0.03). Faecal, but not serum, levels of the endocannabinoid palmitoylethanolamide (PEA) mediated this association: the indirect effect was significant (ß = -0.13; 95%CI: -0.24 to -0.01; P = 0.03), as was the total effect (ß = -0.38; 95%CI: -0.72 to -0.04; P = 0.03), whereas the direct effect of alpha diversity on anhedonia/amotivation was attenuated fully (ß = -0.25; 95%CI: -0.60 to 0.09; P = 0.16). Our results suggest that gut-microbial diversity might contribute to anhedonia/amotivation via the endocannabinoid system. These findings shed light on the biological underpinnings of anhedonia/amotivation and suggest the gut microbiota-endocannabinoid axis as a promising therapeutic target in an area of unmet clinical need.

Ligand-activated RXFP1 gene therapy ameliorates pressure overload-induced cardiac dysfunction.

Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an in vivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. In vitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from ß-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.

Mom's diet matters: Maternal prebiotic intake in mice reduces anxiety and alters brain gene expression and the fecal microbiome in offspring.

Compelling evidence links enteric microbes to brain function and behavior. Galacto-oligosaccharide prebiotics have been shown to modulate the composition of gut flora and induce metabolic, neurochemical, and behavioral changes in adult rodents. Despite the brain being most susceptible to environmental factors, such as nutrients and toxins, during the earliest stages of development, it is unknown whether maternal prebiotic supplementation during gestation and lactation influences the offspring gut microbiome, brain, or behavior. The aim of this study was to test whether maternal galacto-oligosaccharide intake during pregnancy and lactation alters the brain and behavior in naïve and endotoxin-challenged offspring. CD1 female mice received either normal drinking water or water supplemented with Bimuno® galacto-oligosaccharides (B-GOS) during gestation and suckling. Offspring behavior was tested at weaning age or adulthood, and a cross-foster design was employed in a separate cohort to differentiate between effects of prenatal and postnatal maternal B-GOS intake. Lipopolysaccharide was also administered to pups at postnatal day 9 to determine whether maternal B-GOS influences the neurobiological and behavioral effects of a neonatal pro-inflammatory challenge in adulthood. Fecal microbiome composition and metabolites were analyzed to explore potential relationships between the maternal microbiome, the offspring gut microbiome, and the offspring brain and behavior. Maternal B-GOS supplementation increased exploratory behavior and reduced expression of hippocampal glutamate receptor genes in young, weaning-age offspring. In addition, postnatal, but not prenatal, B-GOS supplementation increased fecal butyrate and propionate levels. Finally, in adult offspring, perinatal B-GOS intake increased cortical glutamate receptor subunits in females, increased social preference, and reduced anxiety. We provide novel and comprehensive evidence for the influence of maternal prebiotic intake on offspring behavior, brain gene expression, and gut microbiome composition in mice.

Percutaneous mitral valve repair in recurrent severe mitral valve regurgitation after mitral annuloplasty : MitraClip-in-the-ring as a complementary strategy.

BACKGROUND: Patients with reduced left ventricular (LV) function undergoing coronary artery bypass graft surgery or/and aortic valve replacement occasionally show severe mitral valve (MV) regurgitation and thus also undergo surgical mitral annuloplasty. Over time, further deterioration of LV function and additional ischemic events cause recurrence of severe MV regurgitation due to the Carpentier IIIb morphology of the MV that is not adequately addressed by the previously implanted annuloplasty ring. METHODS: Seven patients (Society of Thoracic Surgeons score: 7.5 ± 1.5%) with Carpentier type-IIIb recurrent severe MV regurgitation, having undergone prior cardiothoracic surgery (median: 40 months) including mitral annuloplasty, were treated with the MitraClip device. RESULTS: MitraClip implantation resulted in significantly reduced MV regurgitation and improved New York Heart Association functional state, translating into an increased exercise capability and improved cardiac biomarkers. The morphology of the MV was adequately addressed without causing relevant MV stenosis, while the MV annulus area remained unaltered. The procedure was safe with a 30-day mortality rate of 0%. CONCLUSION: MitraClip-in-the-ring is feasible and in principle safe for treating Carpentier type IIIb severe MV regurgitation after surgical MV repair using mitral annuloplasty. MitraClip-in-the-ring resulted in immediate amelioration of clinical symptoms and increased physical exercise capacity.

Opposing effects of antibiotics and germ-free status on neuropeptide systems involved in social behaviour and pain regulation.

BACKGROUND: Recent research has revealed that the community of microorganisms inhabiting the gut affects brain development, function and behaviour. In particular, disruption of the gut microbiome during critical developmental windows can have lasting effects on host physiology. Both antibiotic exposure and germ-free conditions impact the central nervous system and can alter multiple aspects of behaviour. Social impairments are typically displayed by antibiotic-treated and germ-free animals, yet there is a lack of understanding of the underlying neurobiological changes. Since the µ-opioid, oxytocin and vasopressin systems are key modulators of mammalian social behaviour, here we investigate the effect of experimentally manipulating the gut microbiome on the expression of these pathways. RESULTS: We show that social neuropeptide signalling is disrupted in germ-free and antibiotic-treated mice, which may contribute to the behavioural deficits observed in these animal models. The most notable finding is the reduction in neuroreceptor gene expression in the frontal cortex of mice administered an antibiotic cocktail post-weaning. Additionally, the changes observed in germ-free mice were generally in the opposite direction to the antibiotic-treated mice. CONCLUSIONS: Antibiotic treatment when young can impact brain signalling pathways underpinning social behaviour and pain regulation. Since antibiotic administration is common in childhood and adolescence, our findings highlight the potential adverse effects that antibiotic exposure during these key neurodevelopmental periods may have on the human brain, including the possible increased risk of neuropsychiatric conditions later in life. In addition, since antibiotics are often considered a more amenable alternative to germ-free conditions, our contrasting results for these two treatments suggest that they should be viewed as distinct models.

Neonatal prebiotic (BGOS) supplementation increases the levels of synaptophysin, GluN2A-subunits and BDNF proteins in the adult rat hippocampus.

Compelling data suggest that perturbations in microbial colonization of the gut in early-life, influences neurodevelopment and adult brain function. If this is the case, then ensuring the growth of beneficial bacteria at an early age will lead to optimal brain development and maturation. We have tested whether feeding neonatal rats daily (from post-natal days 3-21) with a galacto-oligosaccharide prebiotic (Bimuno®, BGOS) or a control solution, alters the levels of hippocampal N-Methyl-D-Aspartate receptor (NMDAR) subunits (GluN1, GluN2A, GluN2B), synaptic proteins (synaptophysin, MAP2, and GAP43) and brain-derived-neurotrophic factor (BDNF), at post-natal days 22 and 56. The administration of BGOS significantly elevated GluN2A subunits, synaptophysin and BDNF in the hippocampus of 22 day old rats. The effect was also observed on day 56 (26 days after the feeding ceased). The levels of all other proteins (GluN1, GluN2B, MAP2, GAP43) remained unaltered. Increased GluN2A, synaptophysin, BDNF, but not MAP2, may suggest that neonatal BGOS feeding alters neurotransmission rather than synaptic architecture. Although the functional consequences of our findings require further investigation, the current study confirms that the manipulation of gut bacteria in early-life, has central effects that persist until at least young adulthood.

Prebiotic administration normalizes lipopolysaccharide (LPS)-induced anxiety and cortical 5-HT2A receptor and IL1-ß levels in male mice.

The manipulation of the enteric microbiota with specific prebiotics and probiotics, has been shown to reduce the host's inflammatory response, alter brain chemistry, and modulate anxiety behaviour in both rodents and humans. However, the neuro-immune and behavioural effects of prebiotics on sickness behaviour have not been explored. Here, adult male CD1 mice were fed with a specific mix of non-digestible galacto-oligosaccharides (Bimuno®, BGOS) for 3 weeks, before receiving a single injection of lipopolysaccharide (LPS), which induces sickness behaviour and anxiety. Locomotor and marble burying activities were assessed 4h after LPS injection, and after 24h, anxiety in the light-dark box was assessed. Cytokine expression, and key components of the serotonergic (5-Hydroxytryptamine, 5-HT) and glutamatergic system were evaluated in the frontal cortex to determine the impact of BGOS administration at a molecular level. BGOS-fed mice were less anxious in the light-dark box compared to controls 24h after the LPS injection. Elevated cortical IL-1ß concentrations in control mice 28 h after LPS were not observed in BGOS-fed animals. This significant BGOS×LPS interaction was also observed for 5HT2A receptors, but not for 5HT1A receptors, 5HT, 5HIAA, NMDA receptor subunits, or other cytokines. The intake of BGOS did not influence LPS-mediated reductions in marble burying behaviour, and its effect on locomotor activity was equivocal. Together, our data show that the prebiotic BGOS has an anxiolytic effect, which may be related to the modulation of cortical IL-1ß and 5-HT2A receptor expression. Our data suggest a potential role for prebiotics in the treatment of neuropsychiatric disorders where anxiety and neuroinflammation are prominent clinical features.

Increased burst-firing of ventral tegmental area dopaminergic neurons in D-amino acid oxidase knockout mice in vivo.

d-Amino acid oxidase (DAO) degrades the N-methyl-d-aspartate (NMDA) receptor co-agonist d-serine, and is implicated in schizophrenia as a risk gene and therapeutic target. In schizophrenia, the critical neurochemical abnormality affects dopamine, but to date there is little evidence that DAO impacts on the dopamine system. To address this issue, we measured the electrophysiological properties of dopaminergic (DA) and non-DA neurons in the ventral tegmental area (VTA) of anaesthetised DAO knockout (DAO(-/-) ) and DAO heterozygote (DAO(+/-) ) mice as compared with their wild-type (DAO(+/+) ) littermates. Genotype was confirmed at the protein level by western blotting and immunohistochemistry. One hundred and thirty-nine VTA neurons were recorded in total, and juxtacellular labelling of a subset revealed that neurons immunopositive for tyrosine hydroxylase had DA-like electrophysiological properties that were distinct from those of neurons that were tyrosine hydroxylase-immunonegative. In DAO(-/-) mice, approximately twice as many DA-like neurons fired in a bursting pattern than in DAO(+/-) or DAO(+/+) mice, but other electrophysiological properties did not differ between genotypes. In contrast, non-DA-like neurons had a lower firing rate in DAO(-/-) mice than in DAO(+/-) or DAO(+/+) mice. These data provide the first direct evidence that DAO modulates VTA DA neuron activity, which is of interest for understanding both the glutamatergic regulation of dopamine function and the therapeutic potential of DAO inhibitors. The increased DA neuron burst-firing probably reflects increased availability of d-serine at VTA NMDA receptors, but the site, mechanism and mediation of the effect requires further investigation, and may include both direct and indirect processes.

AAV6.ßARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model.

AIMS: G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the ßARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term ßARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). METHODS AND RESULTS: A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.ßARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term ßARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.ßARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.ßARKct gene therapy. CONCLUSION: These data--showing sustained amelioration of cardiac function in a post-MI pig HF model--demonstrate the therapeutic potential of ßARKct gene therapy for HF.

Importance of good hosting: reviewing the bi-directionality of the microbiome-gut-brain-axis.

Gut microorganisms have been shown to significantly impact on central function and studies that have associated brain disorders with specific bacterial genera have advocated an anomalous gut microbiome as the pathophysiological basis of several psychiatric and neurological conditions. Thus, our knowledge of brain-to-gut-to microbiome communication in this bidirectional axis seems to have been overlooked. This review examines the known mechanisms of the microbiome-to-gut-to-brain axis, highlighting how brain-to-gut-to-microbiome signaling may be key to understanding the cause of disrupted gut microbial communities. We show that brain disorders can alter the function of the brain-to-gut-to-microbiome axis, which will in turn contribute to disease progression, while the microbiome-to gut-to brain direction presents as a more versatile therapeutic axis, since current psychotropic/neurosurgical interventions may have unwanted side effects that further cause disruption to the gut microbiome. A consideration of the brain-to-gut-to-microbiome axis is imperative to better understand how the microbiome-gut-brain axis overall is involved in brain illnesses, and how it may be utilized as a preventive and therapeutic tool.

Psycho-Pharmacomicrobiomics: A Systematic Review and Meta-Analysis.

BACKGROUND: Understanding the interactions between the gut microbiome and psychotropic medications (psycho-pharmacomicrobiomics) could improve treatment stratification strategies in psychiatry. In this systematic review and meta-analysis, we first explored whether psychotropics modify the gut microbiome; second, we investigated whether the gut microbiome affects the efficacy and tolerability of psychotropics. METHODS: Following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, we searched (November 2022) for longitudinal and cross-sectional studies that investigated the effect of psychotropics on the gut microbiome. The primary outcome was the difference in diversity metrics (alpha and beta) before and after treatment with psychotropics (longitudinal studies) and in medicated compared with unmedicated individuals (cross-sectional studies). Secondary outcomes included the association between gut microbiome and efficacy and tolerability outcomes. Random effect meta-analyses were conducted on alpha diversity metrics, while beta diversity metrics were pooled using distance data extracted from graphs. Summary statistics included standardized mean difference and Higgins I2 for alpha diversity metrics and F and R values for beta diversity metrics. RESULTS: Nineteen studies were included in our synthesis; 12 investigated antipsychotics and 7 investigated antidepressants. Results showed significant changes in alpha (4 studies; standard mean difference: 0.12; 95% CI: 0.01-0.23; p = .04; I2: 14%) and beta (F = 15.59; R2 = 0.05; p < .001) diversity metrics following treatment with antipsychotics and antidepressants, respectively. Altered gut microbiome composition at baseline was associated with tolerability and efficacy outcomes across studies, including response to antidepressants (2 studies; alpha diversity; standard mean difference: 2.45; 95% CI: 0.50-4.40; p < .001, I2: 0%). CONCLUSIONS: Treatment with psychotropic medications is associated with altered gut microbiome composition, and the gut microbiome may in turn influence the efficacy and tolerability of these medications.

Reticulated Platelets Predict Cardiovascular Death and Adverse Events in Coronary Artery Disease: A Systematic Review and Meta-analysis.

BACKGROUND: The pro-thrombotic immature or reticulated platelets (RPs) are known to be elevated in high-risk patients and in different pathological settings. It has been shown that RPs correlate with an insufficient antiplatelet response to antiplatelet agents. RPs are emerging novel predictors of adverse cardiovascular events in cardiovascular disease. This study, using the totality of existing evidence, evaluated the prognostic role of RPs in patients with coronary artery disease. METHODS: We performed a systematic review and meta-analysis including trials of acute and chronic coronary syndrome reporting clinical outcomes according to RPs levels in the peripheral blood. We compared patients with elevated RPs (RPshigh) to patients without elevated RPs (RPslow). Odds ratios (ORs) and 95% CIs were used as metric of choice for treatment effects with random-effects models. The primary endpoint was major adverse cardiovascular and cerebrovascular events (MACCE). Secondary endpoints were cardiovascular death, myocardial infarction, ischemic stroke, urgent coronary revascularization and bleedings. RESULTS: A total of 7 studies, including 2213 patients, were included. The risk for MACCE was significantly higher in RPshigh compared to RPslow patients (OR 2.67 [1.87; 3.81], I2 = 43.8%). RPshigh were associated with cardiovascular death (OR 2.09 [1.36; 3.22], I2 = 40.4%). No associations for RPshigh were detected with the other singular components of MACCE: myocardial infarction (OR 1.73 [0.89; 3.38] I2 = 60.5%) and stroke (OR 1.72 [0.59; 4.96] I2 = 21%). The risk of bleeding did not differ between groups(OR 0.58 [0.15; 2.22] I2 = 86.1%). CONCLUSION: Elevated RPs are significantly associated with increased risk of cardiovascular events and cardiovascular death.

The importance of using food and nutrient intake data to identify appropriate vehicles and estimate potential benefits of food fortification in Uganda.

BACKGROUND: Concern over micronutrient inadequacies in Uganda has prompted the introduction of mass fortification. OBJECTIVE: To use food intake to determine nutrient inadequacies in children aged 24 to 59 months and nonpregnant women of reproductive age, and to model the adequacy of mass fortification. METHODS: Data were collected by the 24-hour recall method in three regions. Usual nutrient intakes were calculated by adjusting actual intake distribution for the intraindividual variance. The impact of fortification on intake adequacy was simulated. RESULTS: The nutrients with the highest prevalence of inadequate intake across regions were vitamin A (30% to 99%), vitamin B12 (32% to 100%), iron (55% to 89%), zinc (18% to 82%), and calcium (84% to 100%). According to simulations, fortification of vegetable oil and sugar with vitamin A would reduce the prevalence of vitamin A inadequacy in the Western and Northern regions; in Kampala it would eliminate vitamin A inadequacy but would cause 2% to 48% of children to exceed the Tolerable Upper Intake Level (UL). The proposed fortification of wheat flour would reduce the prevalence of inadequate intakes of thiamine, riboflavin, folate, and niacin in Kampala, but would have little impact in the other two regions due to low flour consumption. CONCLUSIONS: Micronutrient fortification of vegetable oil and sugar in all regions and of wheat flour in Kampala would reduce the prevalence of micronutrient inadequacies. However, the wheat flour formulation should be modified to better meet requirements, and the vitamin A content in sugar should be reduced to minimize the risk of high intakes. Maize flour may be suitable for targeted fortification, but prior consolidation of the industry would be required for maize flour to become a good vehicle for mass fortification.

Live or heat-killed probiotic administration reduces anxiety and central cytokine expression in BALB/c mice, but differentially alters brain neurotransmitter gene expression.

While the potential for probiotic supplements to act as adjunctive treatments for mood disorders has been widely demonstrated, the precise mode of action remains unclear. To investigate the psychotropic effects of a multi-species probiotic on emotional behaviour in male BALB/c mice, we explored the potential mechanisms of action relating to the temporal changes in the mRNA expression of brain cytokines, BDNF, central 5HT receptor and serotonin transporter (SERT) and GABA receptor in the context of probiotic induced behavioural changes. The effects of a heat-killed probiotic, independent of microbial metabolic processes were also evaluated on the same outcomes to understand whether the host response to the bacteria is more or less important than the contribution of the metabolic activity of the bacteria themselves. Results showed that probiotic supplementation reduced anxiety-like behaviours, increased time spent in the light area of the light-dark box, and decreased the expression of pro-inflammatory cytokines in the brain. Furthermore, probiotic administration elevated hippocampal BDNF and decreased GABAB1ß expression. Interestingly, the heat-killed probiotic and its membrane fraction had similar effects on emotional behaviours and gene expression in the brain. The ingestion of live and heat-killed probiotic preparations also reduced TLR2 expression in the gut. Thus, the present study reveals that the anxiolytic action of a multispecies probiotic in BALB/c mice is independent of bacterial viability. This suggests that it is the host response to probiotics, rather than microbial metabolism that facilitates the molecular changes in the brain and downstream behaviours. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

D-amino acid oxidase knockdown in the mouse cerebellum reduces NR2A mRNA.

Virus mediated RNA-interference (RNAi) is a powerful approach to study genes in vivo. Here we report a method using lentivirus-delivered RNAi to knockdown the glial enzyme, D-amino acid oxidase (DAO), in the mouse cerebellum. After initial characterisation in vitro, we achieved a 40-50% reduction of DAO mRNA in the cerebellum 7 and 28 days after a single injection of lentivirus encoding a DAO-specific, short-hairpin RNA. Injections also decreased DAO immunoreactivity (-33%). The major substrate for DAO is D-serine, an N-methyl-D-aspartate receptor (NMDAR) co-agonist. Thus, we also measured whether DAO knockdown impacted on d-serine, or expression of NMDAR subunits, and found that DAO RNAi led to increased cerebellar D-serine levels (+77%), and decreased NMDAR subunit NR2A mRNA (-22%), but did not affect NR1 or NR2C mRNAs. These data demonstrate the utility of lentiviruses to deliver RNAi to glial cells within the cerebellum, and confirm the role of DAO in D-serine metabolism. They also provide a tool to investigate DAO, an enzyme currently of considerable interest in the pathophysiology and therapy of schizophrenia.

Cardiac Gene Delivery in Large Animal Models: Selective Retrograde Venous Injection.

Delivery of viral vectors to the heart represents a challenging endeavor. Besides vector design, the route of substrate administration is significantly influencing gene delivery success. The selective retrograde venous injection (SRVI) represents one of the most efficient percutaneous delivery strategies for transduction of the anterior left ventricular myocardium. In this chapter, we discuss the advantages and limitations of this vector delivery approach and provide a protocol for selective retrograde venous injection in a preclinical large animal model. As limited transgene expression frequently hampers generation of reliable proof-of-principle data and thus translation, this technique provides a valuable tool to ensure high myocardial transduction in preclinical research.