Biology subjects

Singh, S.

Publications and source records attributed to Singh, S..

At least 307 records · Page 17Linked to original sources

bioRxiv2023

Single cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L.major infection

Hallmark for macrophages is their ability to possess plasticity which enables them to respond to changing microenvironment. Leishmania takes use of the phenotypic plasticity of macrophages to create favorable position for intracellular survival and persistent infection through regulatory cytokine like IL10. However, these effector cells can eliminate infection through modulation of critical cytokines such as IL12 and key players involved in its production. Using sophisticated tool of single cell ATAC sequencing we analyzed the regulatory axis of IL10 and IL12 in time dependent manner for L.major infection in macrophages. We recognized the cellular heterogeneity post infection with the regulators of IL10 and IL12 and observed a reciprocal relationship between them. Our prominent findings also exposed sleepy macrophages and its role in IL10 and IL12 reciprocity. To summarize, role of NFAT5 was vital in identification of sleepy macrophages which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine production and recruitment of immune cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/526191v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@f655c7org.highwire.dtl.DTLVardef@79b2adorg.highwire.dtl.DTLVardef@1c1a628org.highwire.dtl.DTLVardef@83a6a3_HPS_FORMAT_FIGEXP M_FIG Graphical abstract summary of single cell ATAC sequencing for L.major infected RAW264.7 mouse macrophage cell line. (A) Reciprocal relationship identified in cell populations which is governed through chromatin remodeling. (B) Single cell ATAC pipeline for analysis of dataset (C) Identification of sleepy macrophages which may play potential role in reciprocity. C_FIG

systems biology↗

bioRxiv2023

Recognition determinants of broad and potent HIV-1 neutralization by an affinity matured antibody from a pediatric elite-neutralizer

The structural and characteristic features of HIV-1 broadly neutralizing antibodies (bnAbs) from chronically infected pediatric donors are currently unknown. Herein, we characterized a heavy chain matured HIV-1 bnAb 44m, identified from a pediatric elite-neutralizer. Interestingly, in comparison to its wild-type AIIMS-P01 bnAb, 44m exhibited moderately higher level of somatic hypermutations of 15.2%. The 44m neutralized 79% of HIV-1 heterologous viruses (n=58) tested, with a geometric mean IC50 titer of 0.36 {micro}g/ml. The cryo-EM structure of 44m Fab in complex with fully-cleaved glycosylated native-like BG505.SOSIP.664.T332N gp140 envelope trimer at 4.4[A] resolution revealed that 44m targets the V3-glycan N332-supersite and GDIR motif to neutralize HIV-1 with improved potency and breadth, plausibly attributed by a matured heavy chain as compared to that of wild-type AIIMS-P01. This study further improves our understanding on pediatric HIV-1 bnAbs and structural basis of broad HIV-1 neutralization by 44m may be useful blueprint for vaccine design in future.

immunology↗

bioRxiv2023

An Open-Source Platform for Head-Fixed Operant and Consummatory Behavior

Head-fixed behavioral experiments in rodents permit unparalleled experimental control, precise measurement of behavior, and concurrent modulation and measurement of neural activity. Here we present OHRBETS (Open-Source Head-fixed Rodent Behavioral Experimental Training System; pronounced Orbitz), a low-cost, open-source ecosystem of hardware and software to flexibly pursue the neural basis of a variety of motivated behaviors. Head-fixed mice tested with OHRBETS displayed operant conditioning for caloric reward that replicates core behavioral phenotypes observed during freely moving conditions. OHRBETS also permits for optogenetic intracranial self-stimulation under positive or negative operant conditioning procedures and real-time place preference behavior, like that observed in freely moving assays. In a multi-spout brief-access consumption task, mice displayed licking as a function of concentration of sucrose, quinine, and sodium chloride, with licking modulated by homeostatic or circadian influences. Finally, to highlight the functionality of OHRBETS, we measured mesolimbic dopamine signals during the multi-spout brief-access task that display strong correlations with relative solution value and magnitude of consumption. All designs, programs, and instructions are provided freely online. This customizable ecosystem enables replicable operant and consummatory behaviors and can be incorporated with methods to perturb and record neural dynamics in vivo. Impact StatementA customizable open-source hardware and software ecosystem for conducting diverse head-fixed behavioral experiments in mice.

neuroscience↗

bioRxiv2023

Death by a Thousand Cuts -- Combining Kinase Inhibitors for Selective Target Inhibition and Rational Polypharmacology

Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. The high sequence and structural conservation of the catalytic kinase domain complicates the development of specific kinase inhibitors. As a consequence, most kinase inhibitors also inhibit off-target kinases which complicates the interpretation of phenotypic responses. Additionally, inhibition of off-targets may cause toxicity in patients. Therefore, highly selective kinase inhibition is a major goal in both biomedical research and clinical practice. Currently, efforts to improve selective kinase inhibition are dominated by the development of new kinase inhibitors. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target activities. We have developed a multicompound-multitarget scoring (MMS) method framework that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables rational polypharmacology by allowing optimization of inhibitor combinations against multiple selected on-targets and off-targets. Using MMS with previously published chemogenomic kinase inhibitor datasets we determine inhibitor combinations that achieve potent activity against a target kinase and that are more selective than the most selective single inhibitor against that target. We validate the calculated effect and selectivity of a combination of inhibitors using the in cellulo NanoBRET assay. The MMS framework is generalizable to other pharmacological targets where compound specificity is a challenge and diverse compound libraries are available.

pharmacology and toxicology↗

bioRxiv2023

High-dimensional phenotyping to define the genetic basis of cellular morphology

The morphology of cells is dynamic and mediated by genetic and environmental factors. Characterizing how genetic variation impacts cell morphology can provide an important link between disease association and cellular function. Here, we combined genomic and high-content imaging approaches on iPSCs from 297 unique donors to investigate the relationship between genetic variants and cellular morphology to map what we term cell morphological quantitative trait loci (cmQTLs). We identified novel associations between rare protein altering variants in WASF2, TSPAN15, and PRLR with several morphological traits related to cell shape, nucleic granularity, and mitochondrial distribution. Knockdown of these genes by CRISPRi confirmed their role in cell morphology. Analysis of common variants yielded one significant association and nominated over 300 variants with suggestive evidence (P<10-6) of association with one or more morphology traits. Our results showed that, similar to other molecular phenotypes, morphological profiling can yield insight about the function of genes and variants.

genomics↗

bioRxiv2022

Stromal resistance to castration-induced prostate regression in a mouse model of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is a non-neoplastic proliferative disease producing lower urinary tract symptoms related to the enlarged prostate. BPH is pathologically characterized by hyperplastic growth in both epithelial and stromal compartments. Androgen signaling is essential for prostate function and androgen blockade is the second-line medical therapy to relieve symptoms of BPH. Here we examined the prostates of probasin promoter-driven prolactin (Pb-PRL) transgenic mice, a robust model of BPH that spontaneously develops prostate enlargement, to investigate prostate regression in response to surgical castration. Serial ultrasound imaging demonstrated very uniform self-limited growth of Pb-PRL prostate volume that is consistent with the benign, limited cellular proliferation characteristic of BPH and that contrasts with the highly variable, exponential growth of murine prostate cancer models. Castration elicited only a partial reduction in prostate volume, relative to castration-induced regression of the normal prostate gland. The anti-androgen finasteride induced a diminished reduction of Pb-PRL prostate volume versus castration alone. The limited extent of Pb-PRL mouse prostate volume regression correlated with the initial volume of the stromal compartment, suggesting a differential sensitivity to androgen withdrawal of the epithelial and stroma compartments. Indeed, two-dimensional morphometric analyses revealed a distinctly reduced rate of regression for the stromal compartment in Pb-PRL mice. The myofibroblast component of the Pb-PRL prostate stroma appeared normal, but contained more fibroblasts and extracellular collagen deposition. Like normal prostate, the rate of regression of the Pb-PRL prostate was partially dependent on TGF{beta} and TNF signaling, but unlike the normal prostate, the extent of castration-induced regression was not affected by TGF{beta} or TNF blockade. Our studies show that androgen deprivation can effectively reduce the overall volume of hyperplastic prostate, but the stromal compartment is relatively resistant, suggesting additional therapies might be required to offer an effective treatment for the clinical manifestations of BPH.

pathology↗

bioRxiv2022

Phenotype-based single-cell transcriptomics reveal compensatory pathways involved in Golgi organization and associated transport.

The Golgi is a dynamic organelle with a unique morphology that has implications on its function. How the structural integrity of the Golgi is maintained despite its dynamic nature has been a long-standing question. Several siRNA-based screens have addressed this question and have identified a number of key players required for Golgi integrity. Interestingly, they also reported heterogeneity of phenotypic responses with regards to Golgi morphology. Although never systematically investigated, this variability has generally been attributed to poor transfection efficiency or cell cycle specific responses. Here we show that this heterogeneity is the result of differential response to the siRNA knockdown in different Golgi phenotypes, independent of transfection efficiency or cell cycle phases. To characterize the observed Golgi phenotype-specific responses at the molecular level we have developed an automated assay which enables microscopy-based phenotype classification followed by phenotype-specific single-cell transcriptome analysis. Application of this novel approach to the siRNA mediated knockdown of USO1, a key trafficking protein at the ER to Golgi boundary, surprisingly suggests a key involvement of the late endosomal/endocytic pathways in the regulation of Golgi organization. Our pipeline is the first of its kind developed to study Golgi organization, but can be applied to any biological problem that stands to gain from correlating morphology with single-cell readouts. Moreover, its automated and modular nature allows for uncomplicated scaling up, both in throughput and in complexity, helping the user achieve a systems level understanding of cellular processes.

systems biology↗

bioRxiv2022

Knockdown of mitochondrial atp1 mRNA by a custom-designed pentatricopeptide repeat protein alters F1Fo ATP synthase

We show that a custom-designed RNA-binding protein binds and specifically induces cleavage of atp1 RNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis thaliana. The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits, lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP-synthase-depleted lines showed higher respiratory rates and elevated levels of most amino acids at night, most notably serine family amino acids. The results show the value of using custom-designed PPR proteins to influence expression of specific mitochondrial transcripts to carry out reverse genetics studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis. One sentence SummaryKnockdown of mitochondrial atp1 mRNA by a custom-designed pentatricopeptide repeat protein alters F1Fo ATP synthase, plant growth and amino acid metabolism and ATP synthesis in Arabidopsis thaliana

plant biology↗

bioRxiv2022

Gradual cerebral hypoperfusion in a knock-in mouse model of Alzheimer's disease triggers cortical network dysfunctions

Alzheimers disease (AD) is characterized neuropathologically by amyloid-{beta} (A{beta}) plaques and neurofibrillary tangles. Vascular pathology caused by chronic cerebral hypoperfusion (HP) is hypothesised to exacerbate AD pathology and has emerged as an increasing cause of age-related cognitive impairment. In this study we examined the effects of gradual cerebral HP on cognitive dysfunction, A{beta} pathology, microgliosis, and cortical network dynamics in C57BL/6J mice and a single App knock-in mouse model of AD (AppNL-G-F). We performed unilateral common carotid artery gradual occlusion (UCAgO) in two-month-old mice using an ameroid constrictor. At 4 months of age, animals were tested in a behavioral battery consisting of tests of spatial learning and memory (Morris water task), recognition memory (novel object recognition task), and motor coordination (balance beam). Following behavioural testing, in vivo mesoscale wide-field voltage imaging was done to assess cortical functional connectivity and sensory-evoked cortical activity, and brains were harvested for pathology characterization using immunohistochemistry. We found that UCAgO reduced cerebral blood flow (CBF) in the occluded hemisphere (OH), however, subtle behavioural deficits were observed due to HP. A dissociative effect of HP was observed in resting-state functional connectivity analysis, where HP led to hyper-connectivity in C57 mice and hypo-connectivity in App mice. Interestingly, sensory stimulation of limbs contralateral to OH revealed hyper-cortical activations in the non-occluded hemisphere of C57 HP mice, however, hypo-cortical activations were observed in App HP mice. Furthermore, we found that the UCAgO increased cortical and hippocampal microgliosis in both hemispheres of C57 and App mice, a bilateral increase in A{beta} deposition was only observed in App mice. These results suggest that gradual cerebral HP leads to cortical network alterations in AD, which is partly mediated via activation of microglia.

neuroscience↗

bioRxiv2022

Cell types in the mouse amygdala and their transcriptional response to fear conditioning

The amygdala is one of the most widely studied regions in behavioral neuroscience. A plethora of classical, and new paradigms have dissected its precise involvement in emotional and social sensing, learning, and memory. Several important insights resulted from the use of genetic markers - yet, in the age of single cell transcriptomics, the amygdala remains molecularly underdescribed. Here, we present a molecular cell type taxonomy of the full mouse amygdala in fear learning and consolidation. We performed single-cell RNA-seq on naive and fear conditioned mice, inferred the 130 neuronal cell types distributions in silico using orthogonal spatial transcriptomic datasets, and describe the cell types transcriptional responses to learning and memory consolidation. Only a fraction of cells, within a subset of all neuronal types, were transcriptionally responsive to fear learning, memory and retrieval. These activated engram cells upregulated activity-response genes, and processes of synaptic signaling, plasticity, development and neurite outgrowth. Our transcriptome-wide data confirm known actors, and describe several new candidate genes. The atlas may help pinpoint the amygdalas circuits in performing emotional sensing and integration, and provide new insights to the global cellular processes involved.

neuroscience↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Mechanism of dual pharmacological correction and potentiation of human CFTR

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 [A] non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis. SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

biophysics↗

bioRxiv2022

Suppression of Betacellulin expression is a key mechanism for omega-3 fatty acid mediated attenuation of nonalcoholic steatohepatitis

Clinical and preclinical studies have established that supplementing diets with {omega}3 polyunsaturated fatty acids (PUFA) can reduce hepatic dysfunction in nonalcoholic steatohepatitis (NASH). Herein, we used multi-omic network analysis to unveil novel mechanistic targets of {omega}3 PUFA effects in a preclinical mouse model of western diet induced NASH. After identifying critical molecular processes responsible for the effects of {omega}3 PUFA, we next performed meta-analysis of human liver cancer transcriptomes and uncovered betacellulin as a key EGFR-binding protein that was induced in liver cancer and downregulated by {omega}3 PUFAs in animals with NASH. We then confirmed that betacellulin acts by promoting proliferation of quiescent hepatic stellate cells, stimulating transforming growth factor-{beta}2 and increasing collagen production. When used in combination with TLR2/4 agonists, betacellulin upregulated integrins in macrophages thereby potentiating inflammation and fibrosis. Taken together, our results suggest that suppression of betacellulin is one of the key mechanisms associated with anti-inflammatory and antifibrotic effects of {omega}3 PUFA during NASH. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/510635v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@b8ebe7org.highwire.dtl.DTLVardef@879e8dorg.highwire.dtl.DTLVardef@1ba56f3org.highwire.dtl.DTLVardef@a26c67_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIMulti-omic network analysis points to mitochondrial cardiolipin precursors as candidate key lipids whereby {omega}3 fatty acids restore mitochondrial functioning. C_LIO_LIMulti-omic network analysis suggests betacellulin (BTC) as one of the key mediators of NASH suppressed by {omega}3 polyunsaturated fatty acids. C_LIO_LIReduction of liver fibrosis by omega-3 fatty acids (especially by docosahexaenoic acid, DHA) is accomplished by simultaneous inhibition of betacellulin and TLR agonists. C_LIO_LIBTC promotes collagen production and induces TGFB2 in hepatic stellate cells. C_LIO_LIBTC together with TLR2/4 agonists stimulate expression of integrins in macrophages. C_LIO_LIDHA suppresses BTC-EGFR pathway in NASH animal model potentially preventing progression to hepatocellular carcinoma. C_LI

systems biology↗

bioRxiv2022

Virtual histology of Alzheimer's Disease: why are amyloid-β plaques visible with X-ray phase-contrast imaging?

Amyloid-{beta} (A{beta}) plaques from Alzheimers Disease (AD) can be visualized ex vivo in label-free brain samples using synchrotron X-ray phase-contrast tomography (XPCT). However, for XPCT to be useful as a screening method for amyloid pathology, it is essential to understand which factors drive the detection of A{beta} plaques. The current study was designed to test the hypothesis that A{beta}-related contrast in XPCT could be caused by the A{beta} fibrils and/or by metals trapped in the plaques. This study probed the fibrillar and elemental compositions of A{beta} plaques in brain samples from different types of AD patients and AD models to establish a relationship between XPCT contrast and A{beta} plaque characteristics. XPCT, micro-Fourier-Transform Infrared spectroscopy and micro-X-Ray Fluorescence spectroscopy were conducted on human samples (genetic and sporadic cases) and on four transgenic rodent strains (mouse: APPPS1, ArcA{beta}, J20; rat: TgF344). A{beta} plaques from the genetic AD patient were visible using XPCT, and had higher {beta}-sheet content and higher metal levels than the sporadic AD patient, which remained undetected by XPCT. A{beta} plaques in J20 mice and TgF344 rats appeared hyperintense on XPCT images, while they were hypointense with an hyperintense core in the case of APPPS1 and ArcA{beta} mice. In all four transgenic strains, {beta}-sheet content was similar, while metal levels were highly variable: J20 (zinc and iron) and TgF344 (copper) strains showed greater metal accumulation than APPPS1 and ArcA{beta} mice. Hence, a positive contrast formation of A{beta} plaques in XPCT images appeared driven by biometal entrapment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/509706v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@56e672org.highwire.dtl.DTLVardef@1336d85org.highwire.dtl.DTLVardef@15b6690org.highwire.dtl.DTLVardef@1aaa6cb_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAmyloid-{beta} plaques in the different forms of Alzheimers Disease have various contrasts in X-ray phase-contrast tomography C_LIO_LIIn transgenic rodents, a core-restricted, positive contrast is driven by the level of metal entrapment within plaques C_LIO_LIIn humans, greater and more diffuse metal accumulation lead to a positive contrast in a genetic case of AD C_LI

biophysics↗

bioRxiv2022

Chaperonin activity of Plasmodium prefoldin complex is essential to guard proteotoxic stress response and presents a new target for drug discovery

The intraerythrocytic growth of malaria parasite is challenged by the presence of proteotoxic stress and intrinsically unstructured proteins in the cytoplasm due to formation of toxic heme during haemoglobin digestion. To overcome the unavoidable stress and maintain the cellular protein homeostasis, parasite encodes for a number of chaperones and co-chaperones. Here, we functionally characterize the Plasmodium falciparum prefoldins (PfPFD1-6), a hexameric co-chaperone complex, for their role in protein homeostasis. We demonstrate that PfPFD1-6 localise to cytosol of the parasite and the subunits perform an orchestrated interaction (-PFD3-PFD2-PFD1-PFD5-PFD6-PFD4-) to form an active jelly-fish like complex. Biperiden, an N-propylpiperidine analogue identified by chemotype search from FDA, strongly binds and restricts the formation of prefoldin complex and inhibited its interaction with the substrates, PfMSP-1 and -tubulin-I. Biperiden treatment potently inhibited the in vitro (IC50: 1M) and in vivo growth of malaria parasite. Thus, this study provides novel virtues towards understanding the role of PfPFDs in regulating protein homeostasis and opens new avenues for drug discovery against malaria.

molecular biology↗

bioRxiv2022

Size-tunable ICG-based contrast agent platform for targeted near-infrared photoacoustic imaging

Near-infrared photoacoustic imaging (NIR-PAI) combines the advantages of optical and ultrasound imaging to provide anatomical and functional information of tissues with high resolution. Although NIR-PAI is promising, its wide application is hindered by the limited availability of NIR contrast agents. J-aggregates (JA) made of indocyanine green dye (ICG) represents an attractive class of biocompatible contrast agents for PAI. Here, we present a facile synthesis method that combines ICG and ICG-azide dyes for producing contrast agent with tunable size down to 230 nm and direct functionalization with targeting moieties. The ICG-JA platform has a detectable PA signal in vitro that is two times stronger than whole blood and high photostability. The targeting ability of ICG-JA was measured in vitro using HeLa cells. The ICG-JA platform was then injected into mice and in vivo NIR-PAI showed enhanced visualization of liver and spleen for 90 minutes post-injection with a contrast-to-noise ratio of 2.42.

bioengineering↗

bioRxiv2022

Progression of vascular function and blood pressure in a mouse model of Kawasaki disease

BackgroundKawasaki disease (KD) is a systemic vasculitis of childhood characterized by vascular damage in the acute stage, which can persist into the late stage. Aneurysms, myocardial infarction, and death are long-standing complications associated with KD. The vascular mechanisms in the cardiovascular risk of KD are not fully studied. Herein, we investigated the vascular function and blood pressure regulation in a murine model of KD. Material and methodsWe used the Candida albicans water soluble (CAWS) fraction model. Mice were injected with 4 mg CAWS for five consecutive days and separated into 3 groups. Control (Ctrl): water injected for 5 days; CAWS 7 days (C7): CAWS injected for 5 days plus 2 additional days of wait; CAWS 28 days (C28): CAWS injected for 5 days plus 23 additional days of wait. Blood pressure was analyzed via radiotelemetry. In the end, the heart and arteries were harvested for vasculitis characterization and vascular function in a wire myograph. Rat Aortic Vascular Smooth Muscle cells (RASMC) were used to dissect the molecular mechanisms in vitro. Main findingsC7 presented elevated inflammatory markers in the coronary area (CA) and abdominal aortae (AA), whereas C28 showed severe vasculitis in CA and AA. No difference was found in blood pressure and heart rate. Vascular dysfunction characterized by higher contractility to norepinephrine (NA) in C7 and C28 was abolished by blocking nitric oxide (NO) production, reactive oxygen species (ROS), and cyclooxygenase (COX)-derived products. RASMC treated with CAWS (10ug/mL) presented an increase in COX2 expression, which was prevented by pre-treating the cells with TAK-242 [Toll like receptor 4 (TLR4) antagonist, 3x10-5M]. ConclusionOur data indicate that the murine model of KD is associated with vascular dysfunction likely dependent on COX-derived products, oxidant properties, and NO bioavailability. Furthermore, VSMC may present an important role in the genesis of vascular dysfunction and vasculitis via the TLR4 pathway. Finally, the CAWS model seems not to be appropriate to study KD-associated shock. More studies are necessary to understand whether vascular dysfunction and COXs are triggers for vasculitis.

pharmacology and toxicology↗

bioRxiv2022

In vitro and in vivo activity of Gepotidacin against drug-resistant mycobacterial infections

Mycobacterial pathogens including Non-tuberculous mycobacteria (NTM) and M. tuberculosis (Mtb), are pathogens of significant worldwide interest owing to inherent drug resistance to a wide variety of FDA-approved drugs as well as causing a broad range of serious infections. Identifying new antibiotics active against mycobacterial pathogens is an urgent unmet need, especially those antibiotics that can bypass existing resistance mechanisms. In this study, we demonstrate that Gepotidacin, a first-in-class triazaacenapthylene topoisomerase inhibitor, shows potent activity against Mtb and M. fortuitum as well as against other NTMs species, including fluoroquinolone-resistant M. abscessus. Furthermore, Gepotidacin exhibits concentration-dependent bactericidal activity against various mycobacterial pathogens, synergizes with several drugs utilized for their treatment, and reduces bacterial load in macrophages in the intracellular killing assay comparable to amikacin. Additionally, M. fortuitum ATCC 6841 was unable to generate resistance to Gepotidacin in vitro. When tested in a murine neutropenic M. fortuitum infection model, Gepotidacin caused a significant reduction in bacterial load in various organs at 10 fold lower concentration than amikacin. Taken together, Gepotidacin possesses a potentially new mechanism of action that enables it to escape existing resistance mechanisms. Thus, it can be projected as a potent novel lead for the treatment of mycobacterial infections, particularly for NTM, where present therapeutic interventions are very limited.

microbiology↗