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Obesity is a risk factor for cardio-metabolic and neurological disease. The contribution of gut microbiota to derangements of the gut-brain axis in the context of obesity has been acknowledged, particularly through physiology modulation by short-chain fatty acids (SCFAs). Thus, probiotic interventions and administration of SCFAs have been employed with the purpose of alleviating symptoms in both metabolic and neurological disease. We investigated the effects of four butyrate-producing bacteria from the Lachnospiraceae family on the development of metabolic syndrome and behavioural alterations in a mouse model of diet-induced obesity. Male mice were fed either a high-fat diet (HFD) or an ingredient-matched control diet for 2 months, and bacteria cultures or culture medium were given by gavage to HFD-fed mice every second day. Mice were assessed through a battery of metabolic and behaviour tests, and fluxes through the gut barrier and blood-brain barrier were determined using Dextran-based tracers. One of the administered bacteria from the Coprococcus genus, which produces butyrate and formate, afforded some degree of protection against the development of obesity and its complications. Results from this study, however, are insufficient to support brain health benefits of the bacteria tested. None of the bacteria modulated permeability through the gut or blood-brain barriers. Our results suggest health benefits of a bacteria from Lachnospiraceae family, and encourage further exploration of its use as probiotic.

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (P r ). Surprisingly, VGCC levels are highly predictive of heterogeneous P r among individual synapses of either low- or high-P r inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-P r synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ-3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-P r inputs, yet positively correlate with P r among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses., Competing Interests: The authors declare no competing interests.

The preeminence of Drosophila genetics has led to key discoveries in biology across a variety of fields and disciplines. The advent of CRISPR gene editing has expanded the toolkit of genetic reagents that can be applied to manipulate and observe genes, RNAs, and proteins in an in vivo context. This review describes CRISPR and its use as a transformative gene editing tool in Drosophila We focus on the canonical pathway in which the Cas9 nuclease is directed to specific sequences by guide RNA (gRNA), where cleavage leads to DNA repair by one of two main cellular pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway can be appropriated to disrupt targeted sequences, enabling a variety of loss-of-function studies. Induction of the HDR pathway allows precise editing, including defined deletions, the introduction of specific sequence changes, and the incorporation of fluorescent and epitope tags. These approaches have increased the power of Drosophila genetics and been successfully used to conduct in vivo structure-function studies, study disease-associated variants, and follow protein dynamics., (© 2024 Cold Spring Harbor Laboratory Press.)

CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing., (© 2024 Cold Spring Harbor Laboratory Press.)

At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca 2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Mycotoxins are important food contaminants that commonly co-occur with modified mycotoxins such as mycotoxin-glucosides in contaminated cereal grains. These masked mycotoxins are less toxic, but their breakdown and release of unconjugated mycotoxins has been shown by mixed gut microbiota of humans and animals. The role of different bacteria in hydrolysing mycotoxin-glucosides is unknown, and this study therefore investigated fourteen strains of human gut bacteria for their ability to break down masked mycotoxins. Individual bacterial strains were incubated anaerobically with masked mycotoxins (deoxynivalenol-3-β-glucoside, DON-Glc; nivalenol-3-β-glucoside, NIV-Glc; HT-2-β-glucoside, HT-2-Glc; diacetoxyscirpenol-α-glucoside, DAS-Glc), or unconjugated mycotoxins (DON, NIV, HT-2, T-2, and DAS) for up to 48 h. Bacterial growth, hydrolysis of mycotoxin-glucosides and further metabolism of mycotoxins were assessed. We found no impact of any mycotoxin on bacterial growth. We have demonstrated that Butyrivibrio fibrisolvens , Roseburia intestinalis and Eubacterium rectale hydrolyse DON-Glc, HT-2 Glc, and NIV-Glc efficiently and have confirmed this activity in Bifidobacterium adolescentis and Lactiplantibacillus plantarum (DON-Glc only). Prevotella copri and B. fibrisolvens efficiently de-acetylated T-2 and DAS, but none of the bacteria were capable of de-epoxydation or hydrolysis of α-glucosides. In summary we have identified key bacteria involved in hydrolysing mycotoxin-glucosides and de-acetylating type A trichothecenes in the human gut.

Background: The aim of this retrospective study was to compare the targeting of "pure" osteomyelitis (i.e., without surrounding soft tissue infection) by directly 99mTc-labelled complete immunoglobulin G (IgG) monoclonal antibody (MAb) ([99mTc]besilesomab) and by directly 99mTc-labelled fragment antigen-binding (FAb) MAb ([99mTc]sulesomab) in relation to their kinetic fate. A total of 73 patients with "pure" osteomyelitis were examined with [99mTc]besilesomab, (Scintimun®, IBA/CIS bio international, Saclay, France; N.=38) and [99mTc]sulesomab (LeukoScan®, Immunomedics Inc., Morris Plains, NJ, USA; N.=35)., Methods: Kinetic data were deduced from whole-body and single-photon emission computed tomographic scans, performed 10 minutes to 24 hour p.i. (region-of-interest technique [ROI])., Results: In targeting "pure" osteomyelitis, sensitivities at 1-4 hours were found to be higher for [99mTc]sulesomab (44% and 80% for [99mTc]besilesomab and [99mTc]sulesomab, respectively) but at significantly lower target/background (T/B) ratios than with [99mTc]besilesomab (1.8±0.3 versus 1.4±0.5 for [99mTc]besilesomab and [99mTc]sulesomab respectively; P<0.01). With [99mTc]besilesomab, there was a continuous osteomyelitis uptake over 24 hours, whereas with [99mTc]sulesomab, the maximal uptake occurred mostly within 1-4 hours, with subsequent clearance being slower for antigen-bound activity than for nonspecific background. Hence, diagnosis was possible mostly after 4h with [99mTc]sulesomab but often not before 24 hours with [99mTc]besilesomab, the later increasing significantly (P<0.01) in sensitivity (87% and 84% for [99mTc]besilesomab and [99mTc]sulesomab, respectively)., Conclusions: These results show that the higher sensitivity of [99mTc]sulesomab in osteomyelitis targeting at earlier p.i. times does not rely on an increased antibody uptake but on a more rapid clearance of nonspecific background activity due to faster metabolism and excretion. Intact [99mTc]besilesomab show a slow, continuous uptake, leading to higher T/B at later p.i. times, often beyond the imaging possibilities of 99mTc.