bioRxiv : the preprint server for biology最新文献

Nanobodies have recently emerged as alternatives to classical antibodies in therapeutic and diagnostic contexts from parasites to bacteria to viruses, promising improved stability and simpler manufacturing. To improve nanobody discovery efficiency, we developed an integrated experimental and computational pipeline for detailed characterization of the target binding properties of complete alpaca immune repertoires using our custom Nanobody Meta-clustering Analysis Platform (NanoMAP). We tested our pipeline on three distinct pools of targets, immunizing two alpacas with each pool and generating cDNA and phage display libraries from their immune repertoires. We then panned the phage libraries on each target. To produce more detailed binding information, we performed panning variations using subunits, natural variants, intact pathogens, and binding site competitors. Deep sequencing reads from nanobody libraries before and after each panning were pooled and analyzed with NanoMAP to identify nanobody clonal families and assess their levels of enrichment from the library in each panning, reflecting their affinities. NanoMAP outperformed standard clustering methods, producing clonal families that are coherent in sequence and function and detecting rare but high affinity families. By aggregating sequencing data within clonal families, NanoMAP produced reliable and rich data on nanobody repertoire binding phenotypes for each antigen, enhancing nanobody discovery capabilities.

The gut ecosystem is shaped by multiple factors with the immune system being one of the major determinants in shaping its composition in health and disease. On the other hand, the immune system regulates its responses through the action of the sympathetic nervous system (SNS) in part through beta-adrenergic receptors 1/2 (ADRB1/2). In the past years, a clear link has been established between the immune system, SNS, and the modification of nutrient absorption by the gut microbiota in the development of diet-induced obesity. We have previously shown in male mice transplanted with bone marrow cells ADRB1/2 knock-out mice (KD) showed mild immunosuppression and microbiota changes. Post-recovery, mice were challenged with high-fat diet (HFD) for two weeks ad libitum . Our findings show that KD mice are protected against diet-induced adiposity and weight gain. Additionally, these mice showed an increase in residual calorific values and a decreased expression of the fatty acid transporter FAT/CD36. Suggesting a decreased absorption of lipids in the KD mice. Gut microbiota analysis showed that KD microbiota composition on a HFD remained stable with a significant enrichment in the Bacteroidetes phylum , which is depleted in obesity. This was associated with a switch from triglycerides to diglyceride fecal profile. Moreover, microbiome culture showed a decrease in triglycerides after an incubation with 0.1% of HFD lipid extract. Suggesting a potential role of the Bacteroidetes phylum in the metabolism of these lipids. Our findings demonstrate not only that the gut microbiota can modify nutrient absorption and susceptibility to diet-induced obesity but also that the immune system contributes to selective depletion of microbial members that would otherwise thrive on dietary lipids. Revealing a novel mechanism by which host immunity sculpts the gut ecosystem in ways that influence metabolic outcomes.

mTORC1 integrates growth factor and nutrient signals to regulate cellular metabolism, yet there are no metabolites known to directly regulate mTORC1 activity in cells. Cryo-EM studies revealed that inositol hexakisphosphate (IP ₆ ) associates with the FAT domain of mTOR, suggesting that inositol phosphates may directly modulate mTOR activity. We previously showed that higher-order inositol phosphates enhance mTORC1 kinase activity and stability in vitro. Here, we investigated whether inositol phosphate metabolism regulates mTORC1 signaling in pancreatic β-cells. Suppression or acute inhibition of inositol phosphate multikinase (IPMK), as well as knockdown of inositol trisphosphate kinase 1 (ITPK1), selectively reduced cellular IP ₅ levels without altering IP ₆ and resulted in impaired basal and insulin-stimulated mTORC1 signaling, particularly under physiological glucose and low growth factor conditions. Combined inhibition of IPMK and ITPK1 nearly abolished IP ₅ and reduced IP ₆ , demonstrating that these enzymes compensate to supply IP ₅ for IP ₆ synthesis. Importantly, depletion of IP ₅ did not impair PI3K/Akt activation but accelerated termination of the mTORC1 signal, indicating a role for IP ₅ in stabilizing the active mTORC1 complex. Reduction of inositol phosphate levels did not prevent insulin- or glucose-induced mTORC1 activation, revealing that IP ₅ primarily regulates signal persistence rather than initiation. Together, these findings identify IP ₅ as a metabolic regulator that prolong mTORC1 activity in β-cells, providing a mechanism by which cellular metabolic state modulates sustained mTORC1 signaling.

Significance statement: mTORC1 is a central metabolic regulator whose chronic activation contributes to metabolic disease, yet mechanisms that sustain mTORC1 activity after its activation are poorly understood. We show that enzymes controlling inositol phosphate metabolism regulate the stability of mTORC1 signaling in pancreatic β-cells by maintaining cellular levels of inositol pentakisphosphate (IP ₅ ). Reducing IP ₅ impairs basal and sustained mTORC1 signaling without affecting upstream growth factor or energy-sensing pathways, revealing a mechanism that controls signal duration rather than activation. These findings identify IP ₅ as a metabolic regulator of mTORC1 and suggest that targeting inositol phosphate metabolism may provide a strategy to modulate mTORC1 activity in metabolic disease.

The enteric nervous system (ENS) orchestrates critical gastrointestinal functions including peristalsis, fluid exchange, and blood flow regulation, and develops from vagal neural crest (vNC) progenitors that colonize the gut. While the gut epithelium and mesenchyme exhibit pronounced anterior-posterior (A-P) transcriptional patterning and lineage diversification after mid-gestation, whether the ENS itself undergoes comparable regional embryonic transcriptional diversification has remained unclear. Here, we use multiplexed single-cell RNA sequencing and functional perturbations to dissect how the ENS is patterned between E13.5 and E18.5 within the context of a regionally specialized gut. We find that, while the epithelium and mesenchyme display strong and enduring AP-graded gene expression programs, the ENS lacks intrinsic regionalization and instead follows a predominantly temporal maturation trajectory characterized by neuronal and glial differentiation states. Integrative ligand-receptor analyses reveal that mesenchymal populations express A-P patterned microenvironmental cues that correlate with subtle, region-linked transcriptional tuning in ENS cells, despite the absence of intrinsic A-P identities. Among these signals, PTN/MDK-PTPRZ1 signaling emerges as a major spatial and temporal input to the ENS, with gradients that track both small intestinal region and developmental time. To test the relevance of PTPRZ1 signaling for human ENS development, we perturbed pluripotent stem cell-derived ENS cultures and found that modulating PTPRZ1 signaling impacts proliferative, neurogenic, and neurotransmitter-specification programs, confirming that niche-derived cues fine-tune ENS development. Together, our findings support a model in which the small intestine establishes A-P regionalization through epithelial and mesenchymal patterning, whereas the ENS maintains a relatively uniform core neuroglial program that is secondarily refined by localized microenvironmental signals. This framework highlights how extrinsic, region-specific cues, rather than intrinsic regional transcriptional codes, shape ENS maturation within the small intestine.

Anti-amyloid antibody treatment for Alzheimer's disease is linked to Amyloid-Related Imaging Abnormalities (ARIA), including vasogenic edema (ARIA-E) and microhemorrhages (ARIA-H), especially in ApoE ε4/4 carriers. To investigate mechanisms underlying ARIA, we examined the binding and temporal vascular effects of immunization with 3D6, the precursor to the anti-amyloid antibody bapineuzumab, in two aged Alzheimer's disease amyloid mouse models. Acutely, 3D6 bound to cerebral amyloid angiopathy (CAA), resulting in C1q binding and classical complement activation. Weekly short-term immunization over 7 weeks resulted in elevated CAA- and plaque-associated complement deposition, red blood cell extravasation and microhemorrhages, and was accompanied by significant transcriptomic changes in genes related to complement, inflammation, vascular dysfunction, and endothelial lipid responses. Longer-term dosing over 13-15 weeks further increased complement deposition and was associated with blood-brain barrier disruption, MMP-9 upregulation, and microhemorrhages, accompanied by reduced amyloid burden and modest CAA clearance. C3 levels correlated with microhemorrhage severity. Perivascular macrophages co-localized with complement-decorated CAA in 3D6-treated mice. These findings implicate complement activation as an early key driver of ARIA and suggest that therapeutic targeting of complement may reduce ARIA risk.

Abstract figure:

Intron length is a fascinating example of form without function. The vast majority of intronic space within genomes remains without a provided utility. It often fascinates us to find introns performing any function at all, establishing an attention bias against the vast lacking of utility of the remaining intergenic space. In an attempt to better understand the greater breadth of intronic length, I investigate here what I term The Kinetic Intron Hypothesis. This hypothesis investigates hypothetical dynamics of intron RNA synthesis and degradation. It explores how NTPs stored within intron RNA might function in mitosis and NTP resource management. Preliminary testing of the hypothesis leads to trends that warrant further exploration and validation by the scientific community.

Significance: Currently no widely acknowledged model exists to characterize the length of introns within genes, yet intron length is massively abundant in eukaryotic genomes. Here I present an attempt to model the length of introns. In doing so, I explore novel hypothesized intron dynamics, presenting preliminary data for previously uncharacterized intron characteristics. The new data and model have the protentional to unveil new avenues of utility for introns at the intracellular level.

On evolutionary timescales, brain circuits adapt to support survival in each species' ecological niche. While some anatomical aspects of neural circuitry are conserved across species with distant evolutionary origins, each species also exhibits specific circuit adaptations that enable its behavioral repertoire. It remains unclear whether homologous brain regions leverage analogous neural computations as different species perform common behaviors such as reaching and manipulating objects. Here, we directly assessed conservation of neural computations using intracortical recordings from mouse, monkey, and human motor cortex-a homologous region across many mammals-during motor behaviors crucial for survival. We hypothesized that, despite their phylogenetic distance, rodents and primates produce movements through conserved neural computations implemented by motor cortical population dynamics. Remarkably, we found that movement-related neural dynamics were highly conserved across species, while variations in behavioral output were uniquely captured in neural trajectory geometries. Strikingly, neural dynamics during movement across species were more conserved than those across brain regions in the same human and between motor preparation and execution in the same monkeys. Lastly, through manipulation of neural network models trained to perform reaching movements, we reinforce that conservation of neural dynamics across species likely stems from shared circuit constraints. We thus assert that evolution maintains neural computations across phylogeny even as behavioral repertoires expand.

Obesity is a major risk factor for severe influenza A virus (IAV) infection, however, the innate immune mechanisms underlying this increased vulnerability remain unclear. Here, we identify significant defects in natural killer (NK) cell antiviral responses in mice with diet-induced obesity. In lean mice, NK cells are critical for protection as NK cell depletion during IAV infection led to increased lung viral load, morbidity, and mortality. In contrast, in obese mice NK cell depletion had minimal impact on viral replication or survival. Notably, IAV infection in obese mice recapitulated the phenotype observed in NK cell-depleted lean mice, indicating that obesity is associated with preexisting NK cell dysfunction. Following IAV infection, obese NK cells in the lung were functionally impaired with diminished activation (CD69 ⁺ ), cytokine production (IFN-γ), and cytolytic activity (Granzyme B) accompanied by defects in the mTOR signaling pathway and reduced glycolytic and oxidative metabolism. Bulk and spatial lipidomics revealed obesity and infection-driven remodeling of the lung lipidome. We observed increased triglyceride accumulation, abundance of long-chain free fatty acids, and a shift toward monounsaturated phospholipid species, reshaping the lung microenvironment that coincides with NK cell metabolic dysfunction. Consistent with this lipid-rich environment, obese NK cells sustained high expression of the lipid transporter CD36 post-IAV infection and accumulation of intracellular lipids (LipidTOX ⁺ ), consistent with mechanisms known to suppress NK cell function. Notably, short-term weight loss (4 weeks) was sufficient to restore NK cell metabolism, antiviral function, and survival following IAV infection. These findings uncover a lipid-associated mechanism regulating NK cell function and show it plays a critical role in defense against infection and that it is dysfunctional in obesity. We suggest that targeting immunometabolism could lead to new antiviral therapies and potentially improve vaccine efficacy, especially in high-risk populations such as obesity.

Graphical abstract:

Obesity is the most common cause of hypertension. We have previously shown that high levels of circulating leptin in diet-induced obese (DIO) mice induced hypertension by increasing expression of Transient Receptor Potential Melastatin-subfamily member 7 (TRPM7) in the carotid bodies (CB). In addition, we demonstrated in rat PC12 cells that leptin increases Trpm7 gene expression by inducing CpG site-specific demethylation within the 5' regulatory region containing a signal transducer and activator of transcription 3 (STAT3) binding site. This leptin-induced Trpm7 upregulation was prevented by inhibition of JAK-STAT3 signaling. Based on these findings, we hypothesized that reversing region-specific methylation at the Trpm7 promoter in the CB could attenuate obesity-associated hypertension. Compared with lean controls, DIO mice exhibited increased Trpm7 expression and the STAT3- binding site-specific promoter demethylation in the CB. Administration of methylated DNA oligonucleotides targeting the STAT3 binding site attenuated CpG site-specific DNA demethylation and reduced Trpm7 transcription in the CB of DIO mice. This intervention resulted in decreased carotid sinus nerve activity and reduced arterial blood pressure, especially during the light phase. Our results suggest that targeted modulation of CpG site-specific DNA methylation at the Trpm7 promoter using DNA oligonucleotide may represent a novel therapeutic strategy for obesity-induced hypertension.

Cell fate transitions require coordinated remodeling of intracellular organelles, but how organelle morphology and interactions rewire during neurogenesis remains unclear. Here we combine multispectral imaging with quantitative organelle signature analysis to simultaneously map eight organelles as human induced pluripotent stem cells differentiate into forebrain-like neurons. We find compartment and time-specific rescaling of organelles and a progressive increase in higher-order membrane contacts, with mitochondria emerging as an early interaction hub. Later, endoplasmic reticulum (ER)-organelle contacts dominate with ER-peroxisome contacts promoting ether lipid biosynthesis, membrane homeostasis and synapse formation. Disrupting this contact impairs plasmalogen production, synaptic organization, and neuronal activity, identifying the ER-peroxisome axis as a key regulator of neuronal maturation.