bioRxiv Science⌕ Search

Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 541 records · Page 30Linked to original sources

Stochastic characterization of navigation strategies in an automated variant of the Barnes maze

Animals can use a repertoire of strategies to navigate in an environment, and it remains an intriguing question how these strategies are selected based on the nature and familiarity of environments. To investigate this question, we developed a fully automated variant of the Barnes maze, characterized by 24 vestibules distributed along the periphery of a circular arena, and monitored the trajectories of mice over 15 days as they learned to navigate towards a goal vestibule from a random start vestibule. We show that the patterns of vestibule visits can be reproduced by the combination of three stochastic processes reminiscent of random, serial and spatial strategies. The processes randomly selected vestibules based on either uniform (random) or biased (serial and spatial) probability distributions. They closely matched experimental data across a range of statistical distributions characterizing the length, distribution, step size, direction, and stereotypy of vestibule sequences, revealing a shift from random to spatial and serial strategies over time, with a strategy switch occurring approximately every six vestibule visits. Our study provides a novel apparatus and analysis toolset for tracking the repertoire of navigation strategies and demonstrates that a set of stochastic processes can largely account for exploration patterns in the Barnes maze.

animal behavior and cognition↗

Using dynamic oral dosing of rifapentine and rifabutin to simulate exposure profiles of long-acting formulations in a mouse model of tuberculosis preventive therapy

Administration of tuberculosis preventive therapy (TPT) to individuals with latent tuberculosis infection is an important facet of global tuberculosis control. The use of long-acting injectable (LAI) drug formulations may simplify and shorten regimens for this indication. Rifapentine and rifabutin have anti-tuberculosis activity and physiochemical properties suitable for LAI formulation, but there are limited data available for determining the target exposure profiles required for efficacy in TPT regimens. The objective of this study was to determine exposure-activity profiles of rifapentine and rifabutin to inform development of LAI formulations for TPT. We utilized a validated paucibacillary mouse model of TPT in combination with dynamic oral dosing of both drugs to simulate and understand exposure-activity relationships to inform posology for future LAI formulations. This work identified several LAI-like exposure profiles of rifapentine and rifabutin that, if achieved by LAI formulations, could be efficacious as TPT regimens and thus can serve as experimentally-determined targets for novel LAI formulations of these drugs. We present novel methodology to understand the exposure-response relationship and inform the value proposition for investment in development of LAI formulations that has utility beyond latent tuberculosis infection.

microbiology↗

Cell-free expressed membraneless organelles sequester RNA in synthetic cells

Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli. Synthetic minimal cells have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems.

synthetic biology↗

Revisiting the role of Acinetobacter sp. in EBPR systems

Side-stream Enhanced biological phosphorus removal (S2EBPR) has been incorporated with B-stage process to enable simultaneous phosphorus and nitrogen removal. However, the dominating phosphorus accumulating organisms (PAOs) in this novel configuration has not been evaluated. The dominance of Acinetobacter was confirmed by 16S sequencing. In addition, single cell Raman spectrum (SCRS) analysis in couple with in situ fluorescence in situ hybridization (FISH) was applied to obtain the feature spectrum and verify the phosphorus release/uptake activity of Acinetobacter spp. The phenotypic profiling further suggested the dominance of Acinetobacter-like organisms among all poly-phosphorus containing organisms and only certain phenotypic Acinetobacter (oligotype 1) contribution to P-removal in a unique HRAS-P(D)N-S2EBPR system. The findings suggest that Acinetobacter may outcompete other heterotrophic organisms in EBPR systems due to their sensitivity to operational conditions. However, stable P-removal was only observed during a specific section of the operation period, coinciding with an increase in the VFA/P ratio. Further research is needed to identify the phenotypes of Acinetobacter responsible for P-removal in EBPR systems. The study contributes to a better understanding of the microbial ecology and engineering aspects of EBPR systems and wastewater treatment in general.

bioengineering↗

Comammox and Unknown Candidate AOBs Contribute to Nitrite Accumulation in An Integrated A-B stage process that Incorporates Side-stream EBPR (S2EBPR)

A novel integrated pilot-scale A-stage high rate activated sludge, B-stage short-cut biological nitrogen removal and side-stream enhanced biological phosphorus removal (A/B-shortcut N- S2EBPR) process for treating municipal wastewater was demonstrated with the aim to achieve simultaneous and carbon- and energy-efficient N and P removal. In this studied period, an average of 7.62 {+/-} 2.17 mg-N/L nitrite accumulation was achieved through atypical partial nitrification without canonical known NOB out-selection. Network analysis confirms the central hub of microbial community as Nitrospira, which was one to two orders of magnitude higher than canonical aerobic oxidizing bacteria (AOB) in a B-stage nitrification tank. The contribution of comammox Nitrospira as AOB was evidenced by the increased amoB/nxr ratio and higher ammonia oxidation activity. Furthermore, oligotyping analysis of Nitrospira revealed two dominant sub-clusters (microdiveristy) within the Nitrospira. The relative abundance of oligotype II, which is phylogenetically close to Nitrospira_midas_s_31566, exhibited a positive correlation with nitrite accumulation in the same operational period, suggesting its role as comammox Nitrospira. Additionally, the phylogenetic investigation suggested that heterotrophic organisms from the family Comamonadacea and the order Rhodocyclaceae embedding ammonia monooxygenase and hydroxylamine oxidase may function as heterotrophic nitrifiers. This is the first study that elucidated the impact of integrating the S2EBPR on nitrifying populations with implications on short-cut N removal. The unique conditions in the side-stream reactor, such as low ORP, favorable VFA concentrations and composition, seemed to exert different selective forces on nitrifying populations from those in conventional biological nutrient removal processes. The results provide new insights for integrating EBPR with short-cut N removal process for mainstream wastewater treatment.

bioengineering↗

Click chemistry selectively activates an auristatin protodrug with either intratumoral or systemic tumor-targeting agents

The Click Activated Protodrugs Against Cancer (CAPAC) platform enables activation of powerful cancer drugs at tumor sites, maximizing efficacy and minimizing systemic toxicity. CAPAC utilizes a potent click chemistry reaction between tetrazine and trans-cyclooctene, called tetrazine ligation. The reaction between the activator, linked to a tumor targeting agent, and the protodrug leads to targeted activation of the drug. In this study, activation is accomplished either by intratumoral injection of a tetrazine-modified hyaluronic acid biopolymer (SQL70) or by systemic infusion of a tetrazine-modified HER2-targeting antigen-binding fragment (SQT01). The drug used is monomethyl auristatin E (MMAE), a cytotoxic agent hindered in its clinical use by severe toxicity. MMAE modification with a trans-cyclooctene moiety to form the protodrug SQP22 reduced its cytotoxicity in vitro and in vivo. Treatment of SQP22 paired with SQL70 biopolymer demonstrated anti-tumor effects in Karpas 299 and RENCA murine tumor models, establishing the requirement of click chemistry for protodrug activation. Furthermore, SQP22 paired with SQT01 induced anti-tumor effects in the HER2-positive NCI-N87 murine tumor model, showing that activation could be accomplished by systemic dosing of a tumor-targeting antibody conjugate. Observed toxicities were limited to modest, transient myelosuppression and moderate body weight loss in these tumor models. This study further delineates the capabilities of the CAPAC platform by demonstrating anti-tumor activity of SQP22 with two differentiated targeting approaches and underscores the power of click chemistry to precisely control the activation of cancer drugs at tumor sites. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/534625v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1563a70org.highwire.dtl.DTLVardef@16ac78org.highwire.dtl.DTLVardef@95214eorg.highwire.dtl.DTLVardef@bdbb78_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisClick chemistry efficiently activates protodrugs at tumors. A novel protodrug delivering monomethyl auristatin E, SQP22, and preclinical experiments with a biopolymer and antibody fragment as targeting agents are reported.

pharmacology and toxicology↗

Molecular Evidence of Internal Carbon-Driven Partial Denitrification Annamox (PdNA) in a mainstream Pilot A-B System Coupled with Side-stream EBPR treating municipal wastewater

Achieving mainstream short-cut nitrogen removal via nitrite has been a carbon and energy-efficient goal which wastewater engineers are dedicated to explore. This study applied a novel pilot-scale A-B-S2EBPR system process integrated with sidestream enhanced biological phosphorus removal) to achieve the nitrite accumulation and downstream anammox for treating municipal wastewater. Nitrite accumulated to 5.5 {+/-} 0.3 mg N/L in the intermittently aerated tanks of B-stage with the nitrite accumulation ratio (NAR) of 79.1 {+/-} 6.5%. The final effluent concentration and removal efficiency of total inorganic nitrogen (TIN) were 4.6 {+/-} 1.8 mg N/L and 84.9 {+/-} 5.6%, respectively. Batch nitrification/denitrification activity tests and functional gene abundance of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) suggested that the nitrite accumulation was mostly caused by partial denitrification without NOB- selection. The unique features of S2EBPR (longer anaerobic HRT/SRT, lower ORPs, high and more complex VFAs etc.) seemed to impact the nitrogen microbial communities: the conventional AOB kept at a very low level of 0.13 {+/-} 0.13% during the operation period, and the dominant candidate internal carbon-accumulating heterotrophic genera of Acinetobacter (17.8 {+/-} 15.5)% and Comamonadaceae (6.7 {+/-} 3.4%) were highly enriched. Furthermore, the single-cell Raman spectroscopy-based intracellular polymer analysis revealed the dominate microorganisms that could utilize polyhydroxyalkanoates (PHA) as the potential internal carbon source to drive partial denitrification. This study provides insights and a new direction for implementing the mainstream PdNA short-cut nitrogen removal via incorporating S2EBPR into sustainable A-B process.

bioengineering↗

Mind of a dauer: Comparative connectomics reveals developmental plasticity

A fundamental question in neurodevelopmental biology is how flexibly the nervous system changes during development. To address this, we reconstructed the complete connectome of dauer, an alternative developmental stage of nematodes with distinct behavioral characteristics, by volumetric reconstruction and automated synapse detection using deep learning. With the basic architecture of the nervous system preserved, structural changes in neurons, large or small, were closely associated with connectivity changes, which in turn evoked dauer-specific behaviors such as nictation. Graph theoretical analyses revealed significant dauer-specific rewiring of sensory neuron connectivity and increased clustering within motor neurons in the dauer connectome. We suggest that the nervous system in the nematode, probably animals in general, has evolved to respond to harsh environments by reversibly developing a quantitatively and qualitatively differentiated connectome.

neuroscience↗

Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

molecular biology↗

Loss-of-SIRT7 sensitizes hepatocellular carcinoma to sorafenib through the regulation of ERK Phosphorylation

The FDA-approved oral multi-kinase inhibitor, sorafenib (BAY 43-9006, Nexavar), is the first approved systemic therapy for patients with unresectable hepatocellular carcinoma (HCC). Although it has been shown to significantly improve the overall survival of patients with HCC, drug resistance limits the response rate to this therapeutic. Here, we report that acquired sorafenib resistance is associated with overexpression of the deacetylase, SIRT7, and a high level of ERK phosphorylation. Further, we identify that the hyperactivation of ERK is controlled by SIRT7-mediated deacetylation of DDX3X. The inhibition of SIRT7 combined with sorafenib resulted in a marked reduction of cell viability in vitro and of tumor growth in vivo. It seems plausible that SIRT7 is responsible for the acquired sorafenib resistance and its inhibition is most likely beneficial together in conjunction with sorafenib by suppressing ERK signaling. HighlightsO_LISorafenib resistance in HCC is associated with SIRT7 and ERK hyperactivation. C_LIO_LISuppression of SIRT7 combined with sorafenib restores sensitivity to sorafenib. C_LIO_LISIRT7 controls sorafenib resistance through ERK activation by mediating DDX3X deacetylation. C_LI

cancer biology↗

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling

Initiation of B-cell receptor (BCR)1 signaling, and subsequent antigen-encounter in germinal centers2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL)4 and lymphoma5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells6-9, the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKC{delta} and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKC{delta}10-12, SHIP113,14 and SHP114, 15,16, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKC{delta}-mediated phosphorylation of CD25 on its cytoplasmic tail (S268). Genetic rescue experiments identified CD25-S268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

immunology↗

Targeted engagement of β-catenin-Ikaros complexes in refractory B-cell malignancies

In most cell types, nuclear {beta}-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of {beta}-catenin but critically depended on GSK3{beta} for effective {beta}-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that {beta}-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, {beta}-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive {beta}-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3{beta} small molecule inhibitors to subvert {beta}-catenin degradation. Clinically approved GSK3{beta}-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of {beta}-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3{beta}-inhibitors for targeted engagement of lymphoid-specific {beta}-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTSO_LIUnlike other cell lineages, B-cells express nuclear {beta}-catenin protein at low baseline levels and depend on GSK3{beta} for its degradation. C_LIO_LIIn B-cells, {beta}-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. C_LIO_LICRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed {beta}-catenin-dependent Myc repression and induction of cell death. C_LIO_LIThe discovery of GSK3{beta}-dependent degradation of {beta}-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3{beta}-inhibitors for the treatment of refractory B-cell malignancies. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/532152v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1b70e4dorg.highwire.dtl.DTLVardef@10bfcc8org.highwire.dtl.DTLVardef@b10eorg.highwire.dtl.DTLVardef@1683a3e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAbundant nuclear {beta}-catenin C_LIO_LI{beta}-catenin pairs with TCF7 factors for transcriptional activation of MYC C_LIO_LIB-cells rely on efficient degradation of {beta}-catenin by GSK3{beta} C_LIO_LIB-cell-specific expression of Ikaros factors C_LI Unique vulnerability in B-cell tumors: O_LIGSK3{beta}-inhibitors induce nuclear accumulation of {beta}-catenin. C_LIO_LI{beta}-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC C_LI

cancer biology↗

An ancestral vaccine induces anti-Omicron antibodies by hypermutation

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyzed blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyzed their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) was achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counter protective mechanism against virus variant immune escape.

bioengineering↗

A Living Organoid Biobank of Crohn's Disease Patients Reveals Molecular Subtypes for Personalized Therapeutics

ABSTRACT (Structured)Crohns disease (CD) is a complex, clinically heterogeneous disease of multifactorial origin; there is no perfect pre-clinical model, little insight into the basis for such heterogeneity, and still no cure. To address these unmet needs, we sought to explore the translational potential of adult stem cell-derived organoids that not only retain their tissue identity, but also their genetic and epigenetic disease-driving traits. We prospectively created a biobank of CD patient-derived organoid cultures (PDOs) using biopsied tissues from colons of 34 consecutive subjects representing all clinical subtypes (Montreal Classification B1-B3 and perianal disease). PDOs were generated also from healthy subjects. Comparative gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and revealed that despite the clinical heterogeneity there are two major molecular subtypes: immune-deficient infectious-CD [IDICD] and stress and senescence-induced fibrostenotic-CD [S2FCD]. The transcriptome, genome and phenome show a surprising degree of internal consistency within each molecular subtype. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. These insights enabled drug screens that reversed subtype-specific phenotypes, e.g., impaired microbial clearance in IDICD was reversed using agonists for nuclear receptors, and senescence in S2FCD was rectified using senotherapeutics, but not vice versa. Phenotyped-genotyped CD-PDOs may fill the gap between basic biology and patient trials by enabling pre-clinical Phase 0 human trials for personalized therapeutics. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/532245v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1edcea0org.highwire.dtl.DTLVardef@198a3c8org.highwire.dtl.DTLVardef@28d394org.highwire.dtl.DTLVardef@5a9dbb_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefThis work creates a prospectively biobanked phenotyped-genotyped Crohns disease patient-derived organoids (CD-PDOs) as platforms for molecular subtyping of disease and for ushering personalized therapeutics. HIGHLIGHTSO_LIProspectively biobanked CD-organoids recapitulate the disease epithelium in patients C_LIO_LIThe phenome-transcriptome-genome of CD-organoids converge on two molecular subtypes C_LIO_LIOne subtype shows impaired microbial clearance, another increased cellular senescence C_LIO_LIPhenotyped-genotyped PDOs are then used for integrative and personalized therapeutics C_LI

immunology↗

Proformer: a hybrid macaron transformer model predicts expression values from promoter sequences

The breakthrough high-throughput measurement of the cis-regulatory activity of millions of randomly generated promoters provides an unprecedented opportunity to systematically decode the cis-regulatory logic that determines the expression values. We developed an end-to-end transformer encoder architecture named Proformer to predict the expression values from DNA sequences. Proformer used a Macaron-like Transformer encoder architecture, where two half-step feed forward (FFN) layers were placed at the beginning and the end of each encoder block, and a separable 1D convolution layer was inserted after the first FFN layer and in front of the multi-head attention layer. The sliding k-mers from one-hot encoded sequences were mapped onto a continuous embedding, combined with the learned positional embedding and strand embedding (forward strand vs. reverse complemented strand) as the sequence input. Moreover, Proformer introduced multiple expression heads with mask filling to prevent the transformer models from collapsing when training on relatively small amount of data. We empirically determined that this design had significantly better performance than the conventional design such as using the global pooling layer as the output layer for the regression task. These analyses support the notion that Proformer provides a novel method of learning and enhances our understanding of how cis-regulatory sequences determine the expression values.

bioinformatics↗

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimers disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A{beta} deposition. Genotype, age, and A{beta} deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse-EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD. Graphical AbstractHippocampal EVISF response to amyloid beta (A{beta}) is sexually dimorphic and related to the microglial EVISF proteome. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/532133v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1a275d6org.highwire.dtl.DTLVardef@e63da5org.highwire.dtl.DTLVardef@1d93cecorg.highwire.dtl.DTLVardef@12ed0db_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling

Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit1, the anti-inflammatory immune system2-4, wound healing5-7, normal social behavior in mice8-10, and prevents bone reabsorption11-17. Each of these functions is impacted by oxytocin18-22, and oxytocin signaling is required for L. reuteri-mediated wound healing5 and social behavior9; however, the initiating events in the gut that lead to oxytocin stimulation and related beneficial functions remain unknown. Here we found evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We subsequently found that L. reuteri facilitates oxytocin secretion directly from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells23, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.

microbiology↗

Condition for successful artificial selection on microbial collectives and optimal experimental setup

Collectives, such as microbial communities, can perform functions beyond the capability of individual members. Enhancing these collective functions through artificial selection, however, presents significant challenges. Here, we explore the rafting-a-waterfall phenomenon, a metaphor illustrating how the success in achieving a target population composition in microbial collectives depends on both the target characteristics and initial conditions. Specifically, collectives comprising fast-growing (F) and slow-growing (S) individuals were grown for a period of "maturation" time, and the collective with S-frequency closest to the target value is chosen to "reproduce" (inoculate) offspring collectives. Such collective selection is dictated by two opposing forces: during collective maturation, intra-collective selection acts like a waterfall, relentlessly driving the S-frequency to lower values, while during collective reproduction, inter-collective selection resembles a rafter striving to reach the target frequency. Due to this model structure, maintaining a target frequency requires the continued action of inter-collective selection. Using simulations and analytical calculations, we show that intermediate target S frequencies are the most challenging, akin to a target within the vertical drop of a waterfall, rather than above or below it. This arises because intra-collective selection is the strongest at intermediate S-frequencies, which can overpower inter-collective selection. While achieving low target S frequencies is consistently feasible, attaining high target S-frequencies requires an initially high S-frequency -- much like a raft that can descend but not ascend a waterfall. The range of attainable target frequencies depends on the initial population size of the collectives: as the population size in Newborn collectives increases, the region of achievable target frequency is reduced until no frequency is achievable. In contrast, the number of collectives under selection plays a less critical role. In scenarios involving more than two populations, the evolutionary trajectory must navigate entirely away from the metaphorical waterfall drop. Our findings illustrate that the strength of intra-collective evolution is frequency-dependent, with implications in experimental planning.

evolutionary biology↗