Biology subjects

Johnson, K.

Publications and source records attributed to Johnson, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Semi-field and surveillance data define the natural diapause timeline for Culex pipiens across the United States

Culex pipiens is a globally-distributed mosquito of medical and veterinary importance, serving as a primary vector of West Nile virus (WNV). To survive winter, female Cx. pipiens mosquitoes undergo adult reproductive diapause initiated by photoperiod and temperature cues. While well-studied under laboratory conditions, the environmental signals that promote Cx. pipiens diapause induction in natural settings are less understood. Here, we evaluate Cx. pipiens in laboratory and semi-field studies to examine diapause induction, defining an approximate timeline beginning in late-August where mosquitoes become receptive to diapause. Using gravid (reproductive) mosquito surveillance data as a proxy for adult diapause incidence for locations across the United States (California, Colorado, Connecticut, Illinois, Iowa, Minnesota, Pennsylvania, and Virginia), we demonstrate consistent population declines coinciding with periods of diapause receptivity except in hybridization zones where Cx. quinquefasciatus is present, suggesting that Culex population genetics can significantly impact end-season population trends. Together, this study defines a window for diapause induction across the United States, shaped by temperature, latitude, elevation, and mosquito population genetics. Coinciding with the cessation of WNV activity, these data can have important implications for mosquito control, where targeted efforts prior to diapause induction can decrease mosquito populations and WNV overwintering to reduce mosquito-borne disease incidence the following season.

ecology↗

bioRxiv2022

In utero exposure to morphine leads to sex-specific behavioral alterations that persist into adulthood in cross-fostered mice

IntroductionThe opioid epidemic has seen an increase in drug use among women of reproductive age. It is well established that Opioid Use Disorder (OUD) can have many negative consequences for the health of mothers and their babies, both during pregnancy and after delivery, but our understanding of the impact of fetal opioid exposure on behavior during adolescence and adulthood is less understood. Preclinical studies have unveiled some of the long-term effects of in utero morphine exposure primarily using injections as the route of drug delivery. Our study utilized a model for oral, voluntary morphine self-administration to investigate neonate, adolescent, and adult offsprings behavioral phenotypes and subsequent ethanol misuse liability. MethodsWe first validated a paradigm for maternal oral intake of morphine, where female mice became morphine dependent pre-pregnancy, and continued to voluntarily consume morphine in the continuous two-bottle choice (C2BC) paradigm during pregnancy and up to offspring postnatal day 7 (PND 7). Offspring were cross-fostered to a drug-naive dam at PND 7, to model first and second trimester in utero exposure in humans and to mimic the stress associated with NOWS. Bodyweight and ultrasonic vocalizations were assessed to determine alterations in the neonates. Offspring from control and morphine-exposed dams were then tested during adolescence and adulthood in a battery of behavioral tests to assess baseline behavioral phenotypes. We also computed a global behavioral score (GBS) to integrate offsprings multiple behavioral outcomes into a composite score that could be used to identify potential vulnerable and resilient populations in offspring exposed prenatally to morphine. Offspring that were tested during adolescence were also evaluated during adulthood in the ethanol intermittent 2BC to assess ethanol misuse risk. ResultsUsing an oral maternal morphine C2BC protocol, we demonstrated that morphine dams display signs of dependence, measured by somatic signs during withdrawal, and voluntarily drink morphine throughout gestation. Neonate cross-fostered offspring display changes in spontaneous activity, body weight, and ultrasonic vocalization parameters. During adolescence, offspring display both increased baseline anxiety-like/compulsive-like behavior, while in adulthood they display increased anxiety-like behavior. No changes were found for baseline physical signs, locomotion, and depressive-like behavior during adolescence or adulthood. In addition, a greater percentage of adult male offspring exposed to maternal morphine fell into moderate and high GBS classifications, signaling a more severe behavioral phenotype, compared to male control offspring. These effects were not observed in adult female offspring exposed to morphine in utero. Additionally, male adult offspring exposed to maternal morphine reduced their 2-hour ethanol intake in the intermittent two-bottle choice (I2BC) paradigm, although no changes in 24-hour ethanol intake and preference were found. No changes were observed in female offspring of morphine-exposed dams. ConclusionOverall, maternal morphine exposure leads to sex-specific changes in neonate, adolescent, and adult behavior, including ethanol intake.

pharmacology and toxicology↗

bioRxiv2022

Development of a LAG-3 Immunohistochemistry Assay for Melanoma

AimsA robust immunohistochemistry (IHC) assay was developed to detect lymphocyte-activation gene 3 (LAG-3) expression by immune cells (ICs) in tumor tissues. LAG-3 is an immuno-oncology target with demonstrable clinical benefit, and there is a need for a standardized, well-characterized assay to measure its expression. This study aims to describe LAG-3 scoring criteria and present the specificity, sensitivity, analytical precision, and reproducibility of this assay. MethodsThe specificity of the assay was investigated by antigen competition and with LAG3 knockout cell lines. A melanin pigment removal procedure was implemented to prevent melanin interference in IHC interpretation. Formalin-fixed, paraffin-embedded (FFPE) human melanoma samples with a range of LAG-3 expression levels were used to assess the sensitivity and analytical precision of the assay with a [≥]1% cutoff to determine LAG-3-positivity. Interobserver and intraobserver reproducibility were evaluated with 60 samples in intralaboratory studies and 70 samples in interlaboratory studies. ResultsThe LAG-3 IHC method demonstrated performance suitable for analysis of LAG-3 IC expression in clinical melanoma samples. The pretreatment step effectively removed melanin pigment that could interfere with interpretation. LAG-3 antigen competition and analysis of LAG3 knockout cell lines indicated that the 17B4 antibody clone binds specifically to LAG-3. The intrarun repeatability, interday, interinstrument, interoperator, and interreagent lot reproducibility demonstrated a high scoring concordance (>95%). The interobserver and intraobserver reproducibility and overall interlaboratory and intralaboratory reproducibility also showed high scoring concordance (>90%). ConclusionsWe have demonstrated that the assay reliably assesses LAG-3 expression in FFPE human melanoma samples by IHC. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSLymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor expressed on immune cells that limits T-cell activity and is being actively explored as a target for immunotherapy. What this study addsAn immunohistochemistry assay was developed to detect the LAG-3 protein in formalin-fixed paraffin-embedded human tumor tissue specimens. This study describes scoring criteria and shows the specificity, sensitivity, analytical precision, and reproducibility of this assay as an aid to determine LAG-3 expression in melanoma patients using a [≥]1% expression on immune cells threshold. How this study might affect research, practice or policyThe study describes a key immuno-oncology checkpoint immunohistochemistry assay that is robust and suitable for clinical trials. The assay was used in RELATIVITY-047 (NCT03470922), a phase 2/3 clinical trial that compared combined nivolumab and relatlimab treatment with nivolumab monotherapy, to stratify patients based on the percentage of LAG-3-positive immune cells within the tumor region. This assay is also being used in several ongoing clinical trials evaluating clinical response to relatlimab.

pathology↗

bioRxiv2022

2-Arachodonoylglycerol-mediated endocannabinoid signaling modulates mechanical hypersensitivity associated with alcohol withdrawal in mice

Alcohol use disorder (AUDs) commonly co-occurs in patients with chronic pain, and a major barrier to achieving abstinence and preventing relapse is the emergence of hyperalgesia during alcohol withdrawal. Elucidating novel therapeutic approaches to target hyperalgesia associated with alcohol withdrawal could have important implications for the treatment of AUD. Here we examined the role of 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid (eCB) signaling in the regulation of hyperalgesia associated with alcohol withdrawal in mice and tested the hypothesis that pharmacological augmentation of 2-AG signaling could reduce hyperalgesia during withdrawal. After 72 hours of withdrawal from a continuous access two-bottle choice drinking paradigm, male and female mice exhibited increased mechanical but not thermal hypersensitivity, which normalized by 7 days. This effect was reversed by pretreatment with the monoacylglycerol lipase (MAGL) inhibitor JZL184, which elevates levels of 2-AG. The effects of JZL184 were prevented by coadministration of either a CB1 or CB2 antagonist. Inhibition of the 2-AG synthetic enzyme diacylglycerol lipase (DAGL) with DO34 exacerbated mechanical hypersensitivity during alcohol withdrawal, causing an earlier onset and persistent hypersensitivity even one week into withdrawal. Our findings demonstrate the critical role of 2-AG signaling in the bidirectional regulation of mechanical sensitivity during alcohol withdrawal, with enhancement of 2-AG levels reducing sensitivity, and inhibition of 2-AG synthesis exacerbating sensitivity. These data suggest 2-AG augmentation could represent a novel approach to the treatment of alcohol withdrawal-associated hyperalgesia and AUD in patients with comorbid pain disorders.

neuroscience↗

bioRxiv2021

Biological regulatory networks are less nonlinear than expected by chance

The extent to which the components of a biological system are (non)linearly regulated determines how amenable they are to therapy and control. To better understand this property termed regulatory nonlinearity, we analyzed a suite of 137 published Boolean network models, containing a variety of complex nonlinear regulatory interactions, using a probabilistic generalization of Boolean logic that George Boole himself had proposed. Leveraging the continuous-nature of this formulation, we used Taylor decomposition to approximate the models with various levels of regulatory nonlinearity. A comparison of the resulting series of approximations of the biological models with appropriate random ensembles revealed that biological regulation tends to be less nonlinear than expected, meaning that higher-order interactions among the regulatory inputs tend to be less pronounced. A further categorical analysis of the biological models revealed that the regulatory nonlinearity of cancer and disease networks could not only be sometimes higher than expected but also relatively more variable. We show that this variation is caused by differences in the apportioning of information among the various orders of regulatory nonlinearity. Our results suggest that there may have been a weak but discernible selection pressure for biological systems to evolve linear regulation on average, but for certain systems such as cancer, on the other hand, to also evolve more nonlinear rules.

systems biology↗

bioRxiv2021

Nipah virus Bangladesh infection elicits organ-specific innate and inflammatory responses in the marmoset model

The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal non-human primate (NHP) at high-biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all four infected marmosets. Three developed pulmonary edema and hemorrhage as well as multi-focal hemorrhagic lymphadenopathy, while one recapitulated neurologic clinical symptoms and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA-seq in six different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological symptoms. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.

microbiology↗

bioRxiv2021

CHRONIC INFLAMMATION IN ULCERATIVE COLITIS CAUSES LONG TERM CHANGES IN GOBLETCELL FUNCTION

ObjectiveOne of the features of ulcerative colitis (UC) is a defect in the protective mucus layer. This has been attributed to a reduced number of goblet cells (GC). However, it is not known whether abnormal GC mucus secretion also contributes to the reduced mucus layer. Our aims were to test the hypothesis that GC secretion was abnormal in UC with the changes persistent in colonoids even in the absence of immune cells. DesignColonoids were established from intestinal stem cells of healthy subjects (HS) and from patients with UC (inactive and active sites). Colonoids were maintained as undifferentiated (UD) or induced to differentiate (DF) and studied as 3D or monolayers on Transwell filters. Total RNA was extracted for quantitative real-time PCR analysis. Carbachol and PGE2 mediated stimulation followed by examination of mucus layer by MUC2 IF/confocal microscopy and TEM were performed. ResultsColonoids derived from patients with UC can be propagated over many passages; however, they exhibit a reduced rate of growth and TEER compared with colonoids from HS. Differentiated UC colonoid monolayers form a thin and non-continuous mucus layer. UC colonoids have increased expression of secretory lineage markers: ATOH1 and SPDEF, including MUC2 positive GCs and ChgA positive enteroendocrine cells but failed to secrete mucin when exposed to the cholinergic agonist carbachol and PGE2, which caused increased secretion in HS. Exposure to TNF- (5days), reduced the number of GC with a greater percentage decrease in UC colonoids compared to HS. ConclusionsAbnormal mucus layer in UC is due to long term changes in epithelial cells that lead to abnormal mucus secretion as well as effects of the inflammatory environment to reduce the number of GC. This continued defect in GC mucus secretion may be involved in UC recurrence.

physiology↗

bioRxiv2021

Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections

SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3 of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. One Sentence SummaryThe recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.

genomics↗

bioRxiv2021

Argonaute Binding within Human Nuclear RNA and its Impact on Alternative Splicing

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNAseq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically-significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and were recapitulated by synthetic duplex RNAs and miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be a natural regulatory mechanism, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

molecular biology↗

bioRxiv2020

Reduced activity of Adenylyl Cyclase 1 Attenuates Morphine Induced Hyperalgesia and Inflammatory Pain in Mice

Opioid tolerance and opioid-induced hyperalgesia during repeated opioid administration and chronic pain are associated with upregulation of adenylyl cyclase activity. The objective of this study was to test the hypothesis that a reduction in adenylyl cyclase 1 (AC1) activity or expression would attenuate morphine tolerance and hypersensitivity, and inflammatory pain using murine models. To investigate opioid tolerance and opioid-induced hyperalgesia, mice were subjected to twice daily treatments of saline or morphine using either a static (15 mg/kg, 5 days) or an escalating tolerance paradigm (10-40 mg/kg, 4 days). Systemic treatment with an AC1 inhibitor, ST03437 (5 mg/kg, ip), reduced morphine tolerance and morphine hyperalgesia in mice. Lumbar intrathecal administration of a vector incorporating adeno-associated virus and short-hairpin RNA against Adcy1 reduced morphine induced hypersensitivity compared to control vector treated mice. In contrast, morphine antinociception, along with baseline thermal paw withdrawal latencies, motor performance, exploration in an open field test, and burrowing behaviors were not affected by intrathecal Adcy1 knockdown. Knockdown of Adcy1 by intrathecal injection also attenuated inflammatory mechanical hyperalgesia after intraplantar administration of Complete Freunds Adjuvant (CFA) after one week post injection. This Adcy1 knockdown strategy also increased burrowing and nesting activity after CFA injection when compared to controls. Together, these data indicate targeting AC1 to mitigate opioid-induced adverse effects, or as a method to treat chronic pain, are appropriate as a clinical approach and further development into generating pharmaceuticals targeting these genes/proteins may prove beneficial in the future.

neuroscience↗

bioRxiv2020

Loss of SUR1 subtype KATP channels alters antinociception and locomotor activity after opioid administration

BackgroundOpioid signaling can occur through several downstream mediators and influence analgesia as well as reward mechanisms in the nervous system. KATP channels are downstream targets of the opioid receptor and contribute to morphine-induced antinociception. AimsThe aim of the present work was to assess the role of SUR1-subtype KATP channels in antinocicpetion and hyperlocomotion of synthetic and semi-synthetic opioids. MethodsAdult male and female mice wild-type (WT) and SUR1 deficient (KO) mice were assessed for mechanical and thermal antinociception after administration of either buprenorphine, fentanyl, or DAMGO. Potassium flux was assessed in the dorsal root ganglia and superficial dorsal horn cells in WT and KO mice. Hyperlocomotion was also assessed in WT and KO animals after buprenorphine, fentanyl, or DAMGO administration. ResultsSUR1 KO mice had attenuated mechanical antinociception after systemic administration of buprenorphine, fentanyl, and DAMGO. Potassium flux was also attenuated in the dorsal root ganglia and spinal cord cells after acute administration of buprenorphine and fentanyl. Hyperlocomotion after administration of morphine and buprenorphine was potentiated in SUR1 KO mice, but was not seen after administration of fentanyl or DAMGO. ConclusionsThese results suggest SUR1-subtype KATP channels mediate the antinociceptive response of several classes of opioids (alkaloid and synthetic/semi-synthetic), but may not contribute to the "drug-seeking" behaviors of all classes of opioids.

neuroscience↗

bioRxiv2020

The human ApoE4 variant reduces functional recovery and neuronal sprouting after incomplete spinal cord injury in male mice

Spinal cord injury (SCI) is a devastating form of neurotrauma. Patients who carry one or two ApoE4 alleles show worse functional outcomes and longer hospital stays after SCI but the cellular and molecular underpinnings for this genetic link remain poorly understood. Thus, there is a great need to generate animal models to accurately replicate the genetic determinants of outcomes after SCI to spur development of treatments that improve physical function. Here, we examined outcomes after a moderate contusion SCI of transgenic mice expressing human ApoE3 or ApoE4. ApoE4 mice have worse locomotor function and coordination after SCI. Histological examination revealed greater glial staining in ApoE4 mice after SCI associated with reduced levels of neuronal sprouting markers. Bulk RNA sequencing revealed that subcellular processes (SCPs), such as extracellular matrix organization and inflammatory responses, were highly-ranked among upregulated genes at 7 days after SCI in ApoE4 variants. Conversely, SCPs related to neuronal action potential and neuron projection development were increased in ApoE3 mice at 21 days. In summary, our results reveal a clinically relevant SCI mouse model that recapitulates the influence of ApoE genotypes on post-SCI function in individuals who carry these alleles and suggest that the mechanisms underlying worse recovery for ApoE4 animals involve glial activation and loss of sprouting and synaptic activity.

neuroscience↗

bioRxiv2020

Loop Extrusion Mediates Physiological Locus Contraction for V(D)J Recombination

Immunoglobulin heavy chain locus (Igh) VH, D, and JH gene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a JH-recombination signal sequence (RSS) within a chromatin-based recombination center (RC) and then, in an orientation-dependent process, scans upstream D-containing chromatin presented by cohesin-mediated loop extrusion for convergent D-RSSs to initiate DJH-RC formation1,2. In primary pro-B cells, 100s of upstream VH-associated RSSs, embedded in convergent orientation to the DJH-RC-RSS, gain proximity to the DJH-RC for VH-to-DJH joining via a mechanistically-undefined VH-locus contraction process3-7. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent VH-RSSs and cryptic RSSs, even though locus contraction per se is maintained. Moreover, this inversion activated rearrangement of both cryptic VH-locus RSSs normally in the opposite orientation and, unexpectedly, of normally-oriented cryptic RSSs within multiple, sequential upstream convergent-CBE domains. Primary pro-B cells had significantly reduced transcription of Wapl8, a cohesin-unloading factor, versus levels in v-Abl pro-B lines that lack marked locus contraction or distal VH rearrangements2,9-11. Correspondingly, Wapl depletion in v-Abl lines activated VH-locus contraction and orientation-specific RAG-scanning across the VH-locus. Our findings indicate that locus contraction and physiological VH-to-DJH joining both are regulated via circumvention of CBE scanning impediments.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "sub-genomic RNAs". sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5 UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5 end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

bioinformatics↗

bioRxiv2020

Multi-Locus Molecular Phylogeny of Solenopsis (Hymenoptera: Formicidae)

The myrmicine ant genus Solenopsis is species-rich, globally distributed, and is often a common and ecologically important faunal element of the leaf litter. The genus is also well-known for containing several widely distributed tramp species and some of the worst invasive species in the World (the Red Imported Fire Ant, S. invicta, and the Tropical Fire Ant, S. geminata). Although not hyper-diverse and despite its ecological and economic importance, Solenopsis has long frustrated systematists due its lack of reliable diagnostic characters and no phylogenetic hypothesis exists to date. We present a preliminary multi-locus molecular phylogenetic analysis of Solenopsis to address this knowledge gap. Our analyses recover mostly well-supported phylogenetic hypothesis, which suggests Solenopsis arose in the Neotropics and spread to all other continents (except Antarctica). Importantly, it demonstrates problems with current systematic understanding of the genus, but provides an evolutionary framework upon which to build future research.

evolutionary biology↗

bioRxiv2020

Obesity phenotypes are preserved in intestinal stem cell enteroids from morbidly obese patients

Obesity and obesity-related comorbidities are significant health care challenges. Bariatric surgery (BS) is the most effective therapy for treating obesity and type 2 diabetes. A barrier in the development of therapeutic alternatives is incomplete mechanistic understanding of the benefits of BS and the lack of human intestinal models that recapitulate the pathophysiology of obesity. Using adult intestinal stem cell-derived enteroid cultures established from healthy lean subjects and morbidly obese patients, including post-BS cases, four phenotypes correlating patient BMI and intestinal glucose absorption were identified suggesting that enteroids retain patient phenotype heterogeneity associated with healthy and diseased state. In a sub-population of obese patients, increased dietary glucose absorption and gluconeogenesis was due to significantly higher expression of intestinal carbohydrate transporters (SGLT1, GLUT2 and GLUT5) and gluconeogenic enzymes (PEPCK1 and G6Pase) compared to enteroids from lean subjects that demonstrated low glucose absorption and lacked gluconeogenesis. Enteroids established from successful BS cases exhibited low glucose absorption similar to that observed in lean subjects. These data show that human enteroids preserve the patient phenotype in long-term cultures and represent a reliable preclinical model to study the heterogeneity of the obesity mechanisms, which is necessary to determine the efficacy of therapeutic interventions.

physiology↗

bioRxiv2020

In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

Skin color patterns are ubiquitous in nature, evolve rapidly, and impact social behavior1, predator avoidance2, and protection from ultraviolet irradiation3. A leading model system for vertebrate skin patterning is the zebrafish4-7; its alternating blue stripes and yellow interstripes depend on guanine crystal-containing cells called iridophores that reflect light. It was suggested that the zebrafishs alternating color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes7-9. When we tracked iridophores, however, we found they did not migrate between stripes and interstripes but instead differentiated and proliferated in place based on their micro-environment. RNA seq analysis further revealed stripe and interstripe iridophores had different transcriptomic states, while cryogenic scanning electron microscopy and micro-X-ray diffraction showed they had different guanine crystal organizations and responsiveness to norepinephrine, all indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present a new model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, subcellular organelles arise in stripe and interstripe zones by in situ differentiation. In this model, pattern phenotype depends not only on interactions among pigment cells that affect their arrangements, but also on factors that specify subcellular organization and physiological responsiveness of specialized organelles.

developmental biology↗

bioRxiv2020

Argonaute Binding within 3'-Untranslated Regions Does Not Predict Gene Repression

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: 1) Knockout of argonaute (AGO) variants; 2) RNA sequencing analysis of gene expression changes; and 3) Crosslinking Immunoprecipitation Sequencing (CLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2, and AGO3 are necessary to achieve full impact on gene expression. CLIP-seq reveals several hundred significant AGO2 associations within the 3-untranslated regions of cytoplasmic transcripts. The standard mechanism of miRNA action would suggest that these associations repress gene expression. Contrary to this expectation, clusters are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased gene expression in wild-type versus knock out cells, including the strongest cluster within the MYC 3-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

molecular biology↗