Biology subjects

Tam, J.

Publications and source records attributed to Tam, J..

30 records · Page 2Linked to original sources

bioRxiv2022

Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC

The ability to differentiate stem cells into human cell types is essential to define basic mechanisms and therapeutics, especially for cell types not routinely accessible by biopsies. But while engineered expression of transcription factors (TFs) identified through TF screens has been found to rapidly and efficiently produce some cell types, generation of other cell types that require complex combinations of TFs has been elusive. Here we develop an iterative, pooled single-cell TF screening method that improves the identification of effective TF combinations using the generation of human microglia-like cells as a testbed: Two iterations identified a combination of SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8 as sufficient to differentiate human iPSC into microglia-like cells in 4 days. Characterization of TF-induced microglia demonstrated molecular and functional similarity to primary microglia. We explore the use of single-cell atlas reference datasets to confirm identified TFs and how combining single-cell TF perturbation and gene expression data can enable the construction of causal gene regulatory networks. We describe what will be needed to fashion these methods into a generalized integrated pipeline, further ideas for enhancement, and possible applications.

bioengineering↗

bioRxiv2022

Tauroursodeoxycholic acid inhibits Clostridioides difficile toxin induced apoptosis

C. difficile infection (CDI) is a highly inflammatory disease mediated by the production of two large toxins that weaken the intestinal epithelium and cause extensive colonic tissue damage. Antibiotic alternative therapies for CDI are urgently needed as current antibiotic regimens prolong the perturbation of the microbiota and lead to high disease recurrence rates. Inflammation is more closely correlated with CDI severity than bacterial burden, thus therapies that target the host response represent a promising yet unexplored strategy for treating CDI. Intestinal bile acids are key regulators of gut physiology that exert cytoprotective roles in cellular stress, inflammation and barrier integrity, yet the dynamics between bile acids and host cellular processes during CDI have not been investigated. Here we show that several bile acids are protective against apoptosis caused by C. difficile toxins in Caco-2 cells and that protection is dependent on conjugation. Out of 20 tested bile acids, taurine conjugated ursodeoxycholic acid (TUDCA) was the most potent inhibitor yet unconjugated UDCA did not alter toxin-induced apoptosis. TUDCA treatment decreased expression of genes in lysosome associated and cytokine signaling pathways. TUDCA did not affect C. difficile growth or toxin activity in vitro whereas UDCA significantly reduced toxin activity in a Vero cell assay and decreased tcdA gene expression. These results demonstrate that bile acid conjugation can have profound effects on C. difficile as well as the host and that conjugated and unconjugated bile acids may exert different therapeutic mechanisms against CDI.

microbiology↗

bioRxiv2022

Dual inhibition of vacuolar ATPase and TMPRSS2 is required for complete blockade of SARS-CoV-2 entry into cells

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using non-specific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

cell biology↗

bioRxiv2022

What the flip? What the P-N flip can tell us about proactive suppression

It has been debated whether salient distractors in visual search can be proactively suppressed to completely prevent attentional capture, as the occurrence of proactive suppression implies that the initial shift of attention is not entirely driven by physical salience. While the presence of a Pd component in the EEG (associated with suppression) without a preceding N2pc component (associated with selection) has been used as evidence for proactive suppression, the link between these ERPs and the underlying mechanisms is not always clear. This is exemplified in two recent papers that observed the same waveform pattern, where an early Pd-like component flipped to a N2pc-like component, but provided very different interpretations (Drisdelle & Eimer, Psychophysiology, 58(9), 1-12, 2021; Kerzel & Burra, Journal of Cognitive Neuroscience, 32(6), 1170-1183, 2020). Using RAGNAROC (Wyble et al., Psychological Review, 127(6), 1163-1198, 2020), a computational model of reflexive attention, we successfully simulated this ERP pattern with minimal changes to its existing architecture, providing a parsimonious and mechanistic explanation for this flip in the EEG that is unique from both of the previous interpretations. Our account supports the occurrence of proactive suppression and demonstrates the benefits of incorporating computational modelling into theory building.

neuroscience↗

bioRxiv2021

Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in PSC.

ObjectiveConflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. Goal: Define complex interactions between resident microbes and their association in PSC patients by studying antibiotic-treated specific pathogen-free (SPF) and germ-free (GF) multi-drug-resistant 2 deficient (mdr2-/-) mice. DesignWe measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, fecal 16s rRNA gene profiling, and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. ResultsGF mdr2-/- mice had exaggerated hepatic inflammation and fibrosis with 100% mortality by 8 weeks; early SPF autologous stool transplantation rescued liver-related mortality. Broad-spectrum antibiotics and vancomycin alone accelerated disease in weanling SPF mdr2-/- mice, indicating that vancomycin-sensitive resident microbiota protect against hepatobiliary disease. Vancomycin treatment selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased cytolysin-expressing E. faecalis and E. coli liver translocation; colonization of gnotobiotic mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated liver inflammation and mortality. Lachnospiraceae colonization of antibiotic pre-treated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, while Lachnospiraceae-produced SCFA decreased fibrosis. Fecal E. faecalis/ Enterobacteriaceae was positively and Lachnospiraceae was negatively associated with PSC patients clinical severity Mayo risk scores. ConclusionsWe identified specific functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical outcomes. These insights may guide personalized targeted therapeutic interventions in PSC patients.

microbiology↗

bioRxiv2021

Transcriptomic analysis of pathways associated with alpha(v) integrin-related non-canonical autophagy in human B cells

Autophagy proteins have been linked with development of immune-mediated diseases including lupus, but the mechanisms for this are unclear. We have previously shown that non-canonical autophagy induced by v-integrins regulates B cell activation by viral and self-antigens in mice. Here we investigated the involvement of this pathway in B cells from human tissue. Our data revealed that autophagy is specifically induced in germinal-center and memory B cell sub-populations from human tonsil and spleen. Transcriptomic analysis showed that induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of non-canonical autophagy in B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells we found that v-integrin-related non-canonical autophagy limits activation of specific pathways while promoting others. These data provide new mechanistic links for autophagy and immune dysregulation in diseases such as lupus.

immunology↗

bioRxiv2021

A new drug candidate for glycogen storage disorders enhances glycogen catabolism: Lessons from Adult Polyglucosan Body Disease models

This work employs Adult Polyglucosan Body Disease (APBD) models to explore the efficacy and mechanism of action of 144DG11, a new polyglucosan-reducing lead compound discovered by a high-throughput screen (HTS). APBD is an adult onset glycogen storage disorder (GSD) manifesting as a debilitating progressive axonopathic leukodystrophy. APBD is caused by glycogen branching enzyme (GBE) deficiency leading to poorly branched and insoluble glycogen inclusions, which precipitate as neuropathogenic polyglucosans (PG). 144DG11 led to prolonged survival and improved motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. Histopathologically, 144DG11 reduced PG and glycogen levels in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic PG. These results were also reflected at the cellular level by increased glycolytic, mitochondrial and total ATP production. Mechanistically, we show that the molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. Enhanced mitochondrial activity and lysosomal modifications were also the most pronounced effects of 144DG11 in APBD patient fibroblasts as discovered by image-based multiparametric phenotyping analysis and corroborated by proteomics. In summary, this work presents a broad mechanistic and target-based characterization of 144DG11 in in vivo and cell models of the prototypical GSD APBD. This investigation warrants development of 144DG11 into a safe and efficacious GSD therapy. One Sentence SummaryA new compound, demonstrated to ameliorate APBD in vivo and ex vivo by autophagic catabolism of glycogen, may potentially become a universal drug for glycogen storage disorders.

neuroscience↗

bioRxiv2020

Peripheral sgp130-mediated trans-signaling blockade induces obesity and insulin resistance in mice via PPARα suppression

IL-6 signaling via its receptor (IL-6R) and co-receptor (gp130) performs multiple roles in regulating metabolic homeostasis. However, gp130 is also expressed systemically in a soluble form (sgp130), which limits soluble IL-6 receptor (sIL-6R)-mediated signaling - also called trans-signaling. Here we find that transgenic peripheral sgp130-mediated trans-signaling blockade induces mature-onset obesity, while differentially affecting age-dependent behavioral determinants of energy expenditure. In youth, trans-signaling blockade increases feeding associated with reduced leptin sensitivity but increases energy expenditure to maintain metabolic balance. In aging, reduced physical activity predisposes mice to adiposity, adipose tissue macrophage recruitment, hepatosteatosis, hyperglycemia, and insulin resistance. Mechanistically, trans-signaling blockade reduces hepatic Stat3 phosphorylation and suppresses PPAR, associated with miR-21 upregulation, while pharmacological activation of PPAR prevents obesity and hepatosteatosis, and rescues insulin sensitivity. Together these experiments reveal a role for peripheral IL-6 trans-signaling in metabolic homeostasis and provide clinical significance to elevated sgp130 levels found in some obese and diabetic patients.

physiology↗

bioRxiv2020

TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multi-modular scaffold, GIV (a.k.a Girdin) titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (LPS, a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress pro-inflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene depletion studies confirmed that the presence of GIV ameliorates DSS-induced colitis and sepsis-induced death. The anti-inflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL)-motif which binds the cytoplasmic TIR-modules of TLR4 in a manner that precludes receptor dimerization; the latter is a pre-requisite for pro-inflammatory signaling. Binding of GIVs TILL motif to other TIR modules inhibits pro-inflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses. SignificanceTo ensure immunity, and yet limit pathology, inflammatory responses must be confined within the proverbial Goldilocks zone. TLR4 is the prototypical sensor that orchestrates inflammatory responses through a series of well characterized downstream cascades. How TLR4 signals are confined remain incompletely understood. Using trans-scale approaches ranging from disease modeling in live animals, through cell-based interventional studies, to structure-guided biochemical studies that offer an atomic-level resolution, this study unravels the existence of a brake within the TLR4 signaling cascade, i.e., GIV; the latter is a prototypical member of an emerging class of scaffold proteins. By showing that GIV uses conserved mechanisms to impact multi-TLR signaling, this work unravels a multi-scale point of convergence of immune signaling of broader impact beyond TLR4.

immunology↗

bioRxiv2020

Electrostatic Fields Have Strong Repellency Effects Against Culex pipiens

Currently, the most effective mosquito repellents employ the use of the chemical N, N-Diethyl-meta-toluamide (DEET), often considered the gold standard of repellents. However, as DEET continues to be widely used, it is beginning to lose efficacy as mosquitoes gradually gain resistance to it. Additionally, DEET only protects the skin that it is applied on, and therefore, it is difficult to achieve 100% repellency for the entire body. As urbanization and climate change continue to increase the threat of widespread mosquito-borne illnesses, new forms of repellents must be developed to combat the possibility of an epidemic. Previous studies indicate that a multitude of insects, including spiders, flies, and mosquitoes, avoid electrostatic fields at sufficiently high voltages. In this study, I evaluate if electrostatic fields can be used as a potential personal repellent against mosquitoes and an alternative to chemical sprays. I found that by charging the surface of a persons body with a high voltage (9800V) but minuscule current, I created a safe and effective method that repels mosquitoes from landing on the skin. This method has similar efficacy to DEET, with near 100% repellency after 20 minutes of charging. It also has the advantage of not needing to be reapplied and provides full-body protection, allowing greater protection to the user than DEET. These results introduce a promising method of repelling mosquitoes, that used together with present-day chemical repellents, can help provide greater protection to malaria-endemic countries.

zoology↗

bioRxiv2020

CB1R Regulates Soluble Leptin Receptor Levels via CHOP, Contributing to Hepatic Leptin Resistance

The soluble isoform of leptin receptor (sOb-R), secreted by the liver, regulates leptin bioavailability and bioactivity. Its reduced levels in diet-induced obesity (DIO) contributes to hyperleptinemia and leptin resistance, effects that are known to be regulated by the endocannabinoid (eCB)/CB1R system. Here we show that pharmacological activation/blockade as well as genetic overexpression/deletion of hepatic CB1R modulates sOb-R levels and consequently hepatic leptin resistance. Interestingly, peripheral CB1R blockade failed to reverse DIO-induced reduction of sOb-R levels, fat mass, dyslipidemia, and hepatic steatosis in mice lacking C/EBP homologous protein (CHOP), whereas direct activation of CB1R in hepatocytes reduced sOb-R levels in a CHOP-dependent manner. Moreover, CHOP stimulation increased sOb-R expression and release via a direct regulation of its promoter, while CHOP deletion reduced leptin sensitivity. Our findings highlight a novel molecular aspect by which the hepatic eCB/CB1R system involves in the development of hepatic leptin resistance by regulating sOb-R levels via CHOP. SummaryHere we describe a novel molecular aspect by which the hepatic endocannabinoid/CB1R system contributes to hepatic leptin resistance by regulating soluble leptin receptor levels via CHOP.

molecular biology↗

bioRxiv2020

Regulated repression, and not activation, governs the cell fate promoter controlling yeast meiosis

Intrinsic signals and cues from the external environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1. How nutrient signals control IME1 expression remains poorly understood. Here we show that IME1 transcription is regulated by multiple sequence-specific transcription factors that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight transcription factors bind the IME1 promoter when nutrients are ample. Remarkably, association of these transcription factors is highly regulated by different nutrient cues. Mutant cells lacking three transcription factors (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple transcription factors mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.

molecular biology↗