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Singh, A.

Publications and source records attributed to Singh, A..

At least 343 records · Page 19Linked to original sources

A Tumour-Specific Molecular Network Promotes Tumour Growth in Drosophila by Enforcing a JNK-YKI Feedforward Loop

Cancer cells expand rapidly in response to altered intercellular and signalling interactions to achieve hallmarks of cancer. Impaired cell polarity combined with activated oncogenes is known to promote several hallmarks of cancer e.g., activating invasion by increased activity of Jun N-terminal kinase (JNK), and sustained proliferative signalling by increased activity of Hippo effector Yorkie (Yki). Thus, JNK, Yki, and their downstream transcription factors have emerged as synergistic drivers of tumour growth through pro-tumour signalling and intercellular interactions like cell-competition. However, little is known about the signals that converge onto JNK and Yki in tumour cells that enable the tumour cells to achieve hallmarks of cancer. Here, using mosaic models of cooperative oncogenesis (RasV12, scrib-) in Drosophila, we show that RasV12, scrib- tumour cells grow by activation of a previously unidentified network comprising Wingless (Wg), Dronc, JNK and Yki. We show that RasV12, scrib- cells show increased Wg, Dronc, JNK, and Yki signalling, and all of these signals are required for the growth of RasV12, scrib- tumours. We report that Wg and Dronc converge onto a JNK-Yki self-reinforcing positive feedback signal-amplification loop that promotes tumour growth. We found that Wg-Dronc-Yki-JNK molecular network is specifically activated in polarity-impaired tumour cells and not in normal cells where apical basal polarity is intact. Our findings suggest that identification of molecular networks may provide significant insights about the key biologically meaningful changes in signalling pathways, and paradoxical signals that promote Tumourigenesis.

cancer biology↗

Telomeres control regulation of the human Telomerase (hTERT) gene through non-telomeric TRF2 and independent of Telomere looping

The function of the human telomerase reverse transcriptase (hTERT) in the synthesis and maintenance of chromosome ends, or telomeres, is widely understood. Whether and how telomeres, on the other hand, influence hTERT regulation is relatively less studied. We found hTERT was transcriptionally altered depending on telomere length (TL). This resulted from TL-dependent binding of TRF2 between telomeres and the hTERT promoter. hTERT promoter-bound TRF2 was non-telomeric and did not involve the looping of telomeres to the hTERT promoter. Cell lines from different tissue types (fibrosarcoma (HT1080), colon cancer (HCT116), and breast cancer (MDA-MB-231), engineered for either telomere elongation/shortening gave increase/decrease in hTERT, respectively. Mechanistically, we show hTERT promoter-bound non-telomeric TRF2 recruits the canonical PRC2-complex inducing repressor histone H3K27-trimethylation in a TL-dependent fashion. This was further supported by TL-dependent promoter activity from an exogenously inserted hTERT reporter. Increase in TL over days followed by gradual decline, resulted in activation followed by repression of hTERT in a concerted manner, further implicating TL as a key factor for hTERT regulation. Notably on reprogramming primary fibroblasts to induced pluripotent stem cells (iPSCs), TRF2 loss from the hTERT promoter was evident along with telomere elongation and hTERT upregulation. Conversely, on telomere shortening in iPSCs, hTERT promoter-bound TRF2 was restored with marked reduction in hTERT further supporting the causal role of TL in hTERT transcription. Mechanisms of tight control of hTERT by TL shown here are likely to have major implications in telomere-related physiologies, particularly, cancer, ageing and pluripotency. TeaserTelomere length controls hTERT expression by modulating TRF2 distribution and PRC2-mediated repression, highlighting a self-regulatory mechanism in cancer.

cancer biology↗

How did UGA codon translation as tryptophan evolve in certain ciliates? A critique of Kachale et al. 2023 Nature

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition1. All non-standard ciliate genetic codes involve stop codon reassignments1,2,3. Two of these codes are ambiguous1-3, with "stop" codons either translated or terminating translation depending on their context2,3. Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia4 and an alveolate species from the genus Amoebophrya5. Two ambiguous genetic codes in ciliates share translation of UGA "stop" codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons1,2. Furthermore, though a UGA-cognate tRNACysUCA was reported in Euplotes6, a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNATrpUCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNATrpCCAs, rather than the typical 5 base pair stem tRNAs7. New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination7. Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing "stop" codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

evolutionary biology↗

Separate orexigenic hippocampal ensembles shape dietary choice by enhancing contextual memory and motivation

The hippocampus (HPC), traditionally known for its role in learning and memory, has emerged as a controller of food intake. While prior studies primarily associated the HPC with food intake inhibition, recent research suggests a critical role in appetitive processes. We hypothesized that orexigenic HPC neurons differentially respond to fats and/or sugars, potent natural reinforcers that contribute to obesity development. Results uncover previously-unrecognized, spatially-distinct neuronal ensembles within the dorsal HPC (dHPC) that are responsive to separate nutrient signals originating from the gut. Using activity-dependent genetic capture of nutrient-responsive HPC neurons, we demonstrate a causal role of both populations in promoting nutrient-specific preference through different mechanisms. Sugar-responsive neurons encode an appetitive spatial memory engram for meal location, whereas fat-responsive neurons selectively enhance the preference and motivation for fat intake. Collectively, these findings uncover a neural basis for the exquisite specificity in processing macronutrient signals from a meal that shape dietary choices.

animal behavior and cognition↗

Characterizing the role of autaptic feedback in enhancing precision of neuronal firing times

In a chemical synapse, information flow occurs via the release of neurotransmitters from a presynaptic neuron that triggers an Action potential (AP) in the postsynaptic neuron. At its core, this occurs via the postsynaptic membrane potential integrating neurotransmitter-induced synaptic currents, and AP generation occurs when potential reaches a critical threshold. This manuscript investigates feedback implementation via an autapse, where the axon from the postsynaptic neuron forms an inhibitory synapse onto itself. Using a stochastic model of neuronal synaptic transmission, we formulate AP generation as a first-passage time problem and derive expressions for both the mean and noise of AP-firing times. Our analytical results supported by stochastic simulations identify parameter regimes where autaptic feedback transmission enhances the precision of AP firing times consistent with experimental data. These noise attenuating regimes are intuitively based on two orthogonal mechanisms - either expanding the time window to integrate noisy upstream signals; or by linearizing the mean voltage increase over time. Interestingly, we find regimes for noise amplification that specifically occur when the inhibitory synapse has a low probability of release for synaptic vesicles. In summary, this work explores feedback modulation of the stochastic dynamics of autaptic neurotransmission and reveals its function of creating more regular AP firing patterns.

neuroscience↗

Salmonella Typhimurium employ spermidine to exert protection against ROS-mediated cytotoxicity and rewires host polyamine metabolism to ameliorate its survival in macrophages

Salmonella infection involves a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by the macrophages, inside which, the bacteria face multiple stresses and, consequently, employ appropriate countermeasures. We show that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which is known to prevent the oxidation of proteins in E.coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro, and lesser organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. Interestingly, Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which also solves the mystery of the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D,L--difluoromethylornithine (DFMO), reduces Salmonella colonization and tissue damage in the mouse model of infection, while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a strategy of the bacteria in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

microbiology↗

Microevolution of clade II isolates of Candida auris highlights multifaceted intra-clade heterogeneity in acquiring resistance towards amphotericin B

Candida auris exhibits high-level resistance to amphotericin B (AmB). Mechanisms such as ergosterol biosynthesis malfunction, oxidative damage mismanagement, and increased drug efflux contribute to AmB resistance in C. auris. In this study, we experimentally evolved two East Asian drug-susceptible clade II isolates of C. auris (P2428 and CBS10913T) isolated from different geographical locations to develop resistance against AmB. We analysed alterations in karyotype, genome sequence, and gene expression profiles to uncover the mechanisms driving AmB resistance. The independently evolved clade II adaptors displayed up to 4-16-fold higher MIC50, as compared to the parental cells. AOX2 (alternative oxidase) and the cell wall integrity pathway have been identified as critical in the development of AmB resistance. However, we noted certain intra-clade heterogeneity in the associated mechanisms. While in P2428 adaptors (P-lines), the ergosterol and sphingolipid pathways appear to play a crucial role, this was not the case for CBS10913T adaptors (A-lines), which acquired resistance independent of lipid-associated changes. The transcriptomic, WGS, and phenotypic analyses also confirm that the evolved AmB-resistant isolates follow distinct trajectories for adaptation, Furthermore, unlike the fluconazole-resistant isolates, as reported previously, changes in ploidy do not seem to contribute to the differential mechanisms of AmB resistance. Overall, this study not only provides insights into the mechanisms and pathways involved in AmB resistance but also highlights intra-clade-heterogeneity that exists within clade II of C. auris. ImportanceCandida auris demonstrates significant resistance to amphotericin B (AmB) that stems from factors like alteration of ergosterol biosynthesis, perturbation of the oxidative damage response, etc. A comprehensive understanding of underlying mechanisms can be studied in a holistic manner by subjecting resistant as well as susceptible clinical isolates to a comparative genome-level analysis. An alternate and more dynamic approach is to expose susceptible isolates to a certain concentration of drug which is not lethal but can trigger the resistance mechanisms. In the present study, we evolved C. auris towards AmB and observed novel and differential mechanisms of resistance development, in two different isolates despite belonging to the same clade. This study provides insights into the intra-clade heterogeneous behavior of C. auris towards AmB.

microbiology↗

Klebsiella Pneumoniae turns more virulent under flow stresses in capillary like microchannels

Fluidic habitats are very common to bacterial life, however, very little is known about the effect of the flow stresses on the virulence of the bacteria. In the present work, we conduct microfluidic experiments to understand the consequence of stresses generated by flowing fluid on the bacterial morphology and virulence. We consider Klebsiella pneumoniae (KP), an ESKAPE pathogen as the model bacteria that are responsible for blood stream infections like bacteremia apart from pneumonia, urinary tract infections and more. We generate four different stress conditions by changing the flow rate and channel geometry subsequently altering the shear rate and stressing time ({tau}). We observe significant changes in the structural aspects of the stressed bacteria. With an increase in stressing parameters, the viability of the bacterial sample deteriorated. Most importantly, these stressed samples proliferate much more than unstressed samples inside the RAW264.7 murine macrophages. The results shed light on the complex relationship between flow stresses and bacterial virulence. Furthermore, we challenge the bacterial samples with ciprofloxacin to see how they behave under different stress conditions. The present study can be extended to model deadly diseases like bacteremia using organ-on-a-chip technology and help understand bacterial pathogenicity under realistic environments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/558194v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1111ea1org.highwire.dtl.DTLVardef@f20494org.highwire.dtl.DTLVardef@10ac86dorg.highwire.dtl.DTLVardef@ec6eb1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure:C_FLOATNO A schematic representation of the present work. Figure created with BioRender (www.biorender.com) C_FIG

biophysics↗

Mechanisms of Cell Size Regulation in Slow-Growing Escherichia coli Cells: Discriminating Models Beyond the Adder

Under ideal conditions, Escherichia coli cells divide after adding a fixed cell size, a strategy known as the adder. This concept applies to various microbes and is often explained as the division that occurs after a certain number of stages, associated with the accumulation of precursor proteins at a rate proportional to cell size. However, under poor media conditions, E. coli cells exhibit a different size regulation. They are smaller and follow a sizer-like division strategy where the added size is inversely proportional to the size at birth. We explore three potential causes for this deviation: precursor protein degradation, nonlinear accumulation rate, and a threshold size termed the commitment size. These models fit mean trends but predict different distributions given the birth size. To validate these models, we used the Akaike information criterion and compared them to open datasets of slow-growing E. coli cells in different media. the degradation model could explain the division strategy for media where cells are larger, while the commitment size model could account for smaller cells. The power-law model, finally, better fits the data at intermediate regimes.

systems biology↗

SIRT1 and SIRT3 impact host mitochondrial function and host- Salmonella pH balance during infection

Mitochondria are an important organelle regulating energy homeostasis. Mitochondrial health and dynamics are crucial determinants of the outcome of several bacterial infections. SIRT3, a major mitochondrial sirtuin, along with SIRT1 regulates key mitochondrial functions. This led to considerable interest in understanding the role of SIRT1 and SIRT3 in governing mitochondrial functions during Salmonella infection. Here, we show that loss of SIRT1 and SIRT3 function either by shRNA-mediated knockdown or inhibitor treatment led to increased mitochondrial dysfunction with alteration in mitochondrial bioenergetics alongside increased mitochondrial superoxide generation in the Salmonella-infected macrophages. Consistent with dysfunctional mitochondria, mitophagy was induced along with altered mitochondrial fusion-fission dynamics in S. Typhimurium-infected macrophages. Additionally, the mitochondrial bioenergetic alteration promotes acidification of the infected macrophage cytosolic pH. This host cytosolic pH imbalance skewed the intra-phagosomal and intra- bacterial pH in the absence of SIRT1 and SIRT3, resulting in decreased SPI-2 gene expression. Our results suggest a novel role of SIRT1 and SIRT3 in maintaining the intracellular Salmonella niche by modulating the mitochondrial bioenergetics and dynamics in the infected macrophages. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/557159v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@486cbaorg.highwire.dtl.DTLVardef@da2fb0org.highwire.dtl.DTLVardef@70cd46org.highwire.dtl.DTLVardef@1b4d0bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Targeting YAP mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology

Pathologic alterations in the biomechanical properties of the Schlemms canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared to that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell YAP mechanosignaling in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP activity in primary human SC cells, and whether disruption of YAP mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Lastly, we found that perfusion of the clinically-used, small molecule YAP inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP mechanosignaling in SC cell dysfunction and suggest that YAP inhibition has therapeutic value for treating ocular hypertension in glaucoma.

cell biology↗

Urolithin A induces cardioprotection and enhanced mitochondrial quality during natural aging and heart failure

Cardiovascular diseases remain the primary cause of global mortality, necessitating effective strategies to alleviate their burden. Mitochondrial dysfunction is a driving force behind aging and chronic conditions, including heart disease. Here, we investigate the potential of Urolithin A (UA), a gut microbiome-derived postbiotic that enhances mitophagy, to ameliorate both age-related decline in cardiac function and cardiac failure. We highlight the significance of targeting mitochondria, by comparing gene expression changes in aging human hearts and cardiomyopathies. UA oral administration successfully counteracts mitochondrial and cardiac dysfunctions in preclinical models of aging and heart failure. UA improves both systolic and diastolic heart functions, distinguishing it from other mitochondrial interventions. In cardiomyocytes, UA recovers mitochondrial ultrastructural defects and decline in mitochondrial biomarkers occurring with aging and disease. These findings extend UAs benefits to heart health, making UA a promising nutritional intervention to evaluate in the clinic to promote healthy cardiovascular function as we age.

cell biology↗

Evaluating single-cell variability in proteasomal decay

Gene expression is a stochastic process that leads to variability in mRNA and protein abundances even within an isogenic population of cells grown in the same environment. This variation, often called gene-expression noise, has typically been attributed to transcriptional and translational processes while ignoring the contributions of protein decay variability across cells. Here we estimate the single-cell protein decay rates of two degron GFPs in Saccharomyces cerevisiae using time-lapse microscopy. We find substantial cell-to-cell variability in the decay rates of the degron GFPs. We evaluate cellular features that explain the variability in the proteasomal decay and find that the amount of 20s catalytic beta subunit of the proteasome marginally explains the observed variability in the degron GFP half-lives. We propose alternate hypotheses that might explain the observed variability in the decay of the two degron GFPs. Overall, our study highlights the importance of studying the kinetics of the decay process at single-cell resolution and that decay rates vary at the single-cell level, and that the decay process is stochastic. A complex model of decay dynamics must be included when modeling stochastic gene expression to estimate gene expression noise.

systems biology↗

Phenotypic variation in maize can be largely explained by genetic variation at transcription factor binding sites

Comprehensive maps of functional variation at transcription factor (TF) binding sites (cis-elements) are crucial for elucidating how genotype shapes phenotype. Here we report the construction of a pan-cistrome of the maize leaf under well-watered and drought conditions. We quantified haplotype-specific TF footprints across a pan-genome of 25 maize hybrids and mapped over two-hundred thousand genetic variants (termed binding-QTL) linked to cis-element occupancy. Three lines of evidence support the functional significance of binding-QTL: i) they coincide with numerous known causative loci that regulate traits, including VGT1, Trehalase1, and the MITE transposon near ZmNAC111 under drought; ii) their footprint bias is mirrored between inbred parents and by ChIP-seq; iii) partitioning genetic variation across genomic regions demonstrates that binding-QTL capture the majority of heritable trait variation across [~]70% of 143 phenotypes. Our study provides a promising approach to make previously hidden cis-variation more accessible for genetic studies and multi-target engineering of complex traits.

genomics↗

ISWI1 complex proteins facilitate developmental genome editing in Paramecium

Chromatin remodeling is required for essential cellular processes, including DNA replication, DNA repair, and transcription regulation. The ciliate germline and soma are partitioned into two distinct nuclei within the same cell. During a massive editing process that forms a somatic genome, ciliates eliminate thousands of DNA sequences from a germline genome copy in the form of internal eliminated sequences (IESs). Recently we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia. Here we describe two paralogous proteins, ICOP1 and ICOP2, essential for DNA elimination. ICOP1 and ICOP2 are highly divergent from known proteins; the only domain detected showed distant homology to the WSD motif. We show that both ICOP1 and ICOP2 interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative IES excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOP1 and ICOP2 are needed for chromatin remodeling and accurate DNA elimination in Paramecium.

molecular biology↗

A genome-wide atlas of human cell morphology

A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting1,2) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale.

genomics↗

Ultrasound Imaging Enables Longitudinal Tracking of Vascular Changes that Correlate with Immune Cell Infiltration After Radiotherapy

BackgroundWhile immunotherapy shows great promise in patients with triple negative breast cancer, many will not respond to treatment. Radiotherapy has the potential to prime the tumor-immune microenvironment for immunotherapy. However, predicting response is difficult due to tumor heterogeneity across patients, which necessitates personalized medicine strategies that incorporate tumor tracking into the therapeutic approach. Here, we investigated the use of ultrasound (US) imaging of the tumor vasculature to monitor the tumor response to treatment. MethodsWe utilized ultrafast power doppler US to track the vascular response to radiotherapy over time. We used 4T1 (metastatic) and 67NR (non-metastatic) breast cancer models to determine if US measurements corroborate conventional immunostaining analysis of the tumor vasculature. To evaluate the effects of radiation, tumor volume and vascular index were calculated using US, and the correlation between vascular changes and immune cell infiltration was determined. ResultsUS tumor measurements and the quantified vascular response to radiation were confirmed with caliper measurements and immunostaining, respectively, demonstrating a proof-of-principle method for non-invasive vascular monitoring. Additionally, we found significant infiltration of CD8+ T cells into irradiated tumors 10 days after radiation, which followed a sustained decline in vascular index and an increase in splenic CD8+ T cells that was first observed 1 day post-radiation. ConclusionsOur findings reveal that ultrafast power doppler US can evaluate changes in tumor vasculature that are indicative of shifts in the tumor-immune microenvironment. This work may lead to improved patient outcomes through observing and predicting response to therapy.

cancer biology↗

A Titratable Necrotizing Enterocolitis Mouse Model

BackgroundNecrotizing enterocolitis (NEC) is an inflammatory gastrointestinal process that afflicts approximately 10% of preterm infants born in the United States each year, with a mortality rate of 30%. NEC severity is graded using Bells classification system, from stage I mild NEC to stage III severe NEC. Over half of NEC survivors present with neurodevelopmental impairment during adolescence, a long-term complication that is poorly understood but can occur even after mild NEC. Although multiple animal models exist, none allow the experimenter to control nor represent the gradient of symptom severities seen in NEC patients. We bridge this knowledge gap by developing a graded murine model of NEC and studying its relationship with neuroinflammation across a range of NEC severities. MethodsPostnatal day 3 (P3) C57BL/6 mice were fed a formula containing different concentrations (0% control, 0.25%, 1%, 2%, and 3%) of dextran sodium sulfate (DSS). P3 mice were fed every 3 hours for 72-hours. We collected data on weight gain and behavior (activity, response, body color) during feeding. At the end of the experiment, we collected tissues (intestine, liver, plasma, brain) for immunohistochemistry, immunofluorescence, and cytokine and chemokine analysis. ResultsThroughout NEC induction, mice fed higher concentrations of DSS died sooner, lost weight faster, and became sick or lethargic earlier. Intestinal characteristics (dilation, color, friability) were worse in mice fed with higher DSS concentrations. Histology revealed small intestinal disarray among mice fed all DSS concentrations, while higher DSS concentrations resulted in reduced small intestinal cellular proliferation and increased hepatic and systemic inflammation. In the brain, IL-2, G-CSF, and CXCL1 concentrations increased with higher DSS concentrations. Although the number of neurons and microglia in the CA1 hippocampal region did not differ, microglial branching was significantly reduced in DSS-fed mice. ConclusionWe characterize a novel graded model of NEC that recapitulates the full range of NEC severities. We show that mild NEC is sufficient to initiate neuroinflammation and microglia activation. This model will facilitate studies on the neurodevelopmental effects of NEC.

physiology↗