Biology subjects

Ali, N.

Publications and source records attributed to Ali, N..

At least 19 recordsLinked to original sources

bioRxiv2026

The Social Cognition Paradox in Long-Duration Spaceflight: A VEN Fatigue Hypothesis for Duration-Dependent Emotion Recognition Decline

Long-duration spaceflight may alter social cognition, yet the underlying biological mechanisms remain unclear. Emotion Recognition Task (ERT) performance remains stable during typical 6-month International Space Station (ISS) missions but declines markedly during the 340-day NASA Twins Study, suggesting duration-dependent vulnerability. Here, we propose the Von Economo Neuron (VEN) Fatigue Hypothesis, which posits that microgravity increases demand on VEN-associated social cognitive networks, eliciting adaptive myelination during shorter missions before compensatory mechanisms fail with prolonged exposure. To evaluate this hypothesis, we integrated evidence from rodent, human cortical organoid, astronaut plasma proteomic, and neuroimaging datasets. ISS-flown rodent frontal cortex demonstrated increased expression of myelination-related genes, while spaceflown cortical organoids exhibited changes consistent with oligodendrocyte remodeling. Astronaut plasma transcriptomics identified reproducible alterations in VEN-associated and myelination-related proteins across independent missions, and resting-state fMRI revealed transient changes in frontal insula connectivity following long-duration spaceflight. Together, these findings provide convergent evidence supporting the VEN Fatigue Hypothesis and identify adaptive myelination and VEN-associated network remodeling as candidate mechanisms underlying duration-dependent changes in social cognition during long-duration spaceflight.

neuroscience↗

bioRxiv2026

Spaceflight-relevant microgravity triggers a sublethal Parkinson's-like dopaminergic decline in human neurons and organoids.

Spaceflight stressors may increase Parkinsons disease (PD) risk, but microgravitys specific contribution to human dopaminergic (DA) vulnerability remains undefined. Here, we exposed human iPSC-derived midbrain DA organoids and differentiated SH-SY5Y neurons to simulated microgravity for 72 hours. This exposure reduced neurite outgrowth, eroded DA identity, and activated familial-PD mitochondrial kinases without depleting extracellular dopamine. We observed severe mitochondrial dysfunction--including membrane potential loss and respiratory suppression--coupled with global translational repression. Furthermore, a sublethal, pre-degenerative state emerged, characterized by the selective release of mitochondrial cell-free DNA without apoptotic activation. Proteomic profiling revealed striking convergence with human PD transcriptomes and astronaut blood, highlighting shared suppression of mitochondrial metabolism, ribosomal translation, and altered RNA splicing. Together, these findings establish simulated microgravity as a sufficient, non-toxin trigger of early PD-like DA dysfunction, providing a robust human model for investigating prodromal neurodegeneration.

neuroscience↗

bioRxiv2026

Neonatal regulatory T cells promote alveologenesis by restraining neutrophil-mediated inflammation

Alveologenesis during early postnatal life requires tight coordination between epithelial differentiation and immune regulation, yet how immune cells contribute to this process remains unclear. Regulatory T cells (Tregs) are established mediators of immune homeostasis and tissue repair in adult lung injury, but their role in lung development is unknown. Here, we identify a transient wave of highly proliferative, activated Tregs that accumulates in the neonatal lung during an early postnatal window. Using inducible Treg ablation, we show that loss of this neonatal Treg population disrupts alveologenesis, resulting in enlarged airspaces, and persistent structural abnormalities later in life. Treg depletion also induces interferon-associated inflammatory programmes, promotes neutrophil accumulation, and is accompanied by a sustained imbalance in alveolar epithelial populations. Notably, neutrophil depletion partially rescues both epithelial composition and alveolar structure, identifying neutrophils as key downstream effectors of Treg-mediated regulation. Together, these findings show that neonatal Tregs are required for normal alveologenesis by restraining neutrophil-driven inflammation and preserving epithelial balance. Our study reveals a previously unappreciated role for immune regulation in lung organogenesis.

immunology↗

bioRxiv2026

Chronological Aging Stiffens Cells, Alters Mitochondria, and Impairs Mitochondrial Mechanical Responsiveness in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes, play a critical role for maintaining bone homeostasis. Although, aging impairs MSC function and contributes to several complications, the effects of aging on subcellular structure and related gene expression need further investigation. Here we established an in vitro system to study MSCs isolated from bones of 5, 12, and 24 months old mice which showed significantly decreased bone volume and exercise performance with age. RNA sequencing revealed downregulation of genes related to cell-matrix interactions, cell metabolism, and division. Functionally MSCs extracted from 24mo showed significantly increased adipogenesis and reduced osteogenesis compared to 5mo, which was accompanied by reduced levels of cell proliferative marker Ki67 and increased expression of senescence protein p16. Data-driven segmentation of F-actin architecture revealed no cell-wide or nuclei-associated alteration, while 24mo MSCs showed decreased nuclear volume and increased spreading and higher nuclear stiffness compared to 5mo. Mitochondria from 12mo and 24mo showed increased length and volume of fibers when compared to 5mo, which was accompanied by gene expression changes associated with mitochondrial inflammation and oxidative phosphorylation. To test the functional consequences, MSCs were subjected to mechanical stress for 72 hours using 90Hz, 07g low-intensity vibration (LIV). While 5mo MSCs showed a robust fusing of individual mitochondrial fibers, LIV response of 12mo and 24mo MSCs were progressively less, indicating an already stressed mitochondria compromised to mechanical challenge. In summary, this study has established an in vitro assay system, revealing age-associated impairments in differentiation, mitochondrial function, and mechanotransduction capacity.

bioengineering↗

bioRxiv2026

p70 ribosomal protein S6 Kinase (p70S6K) as a potential peripheral biomarker for mental symptoms in 16p11.2 deletion and duplication syndromes

Neurodevelopmental disorders (NDDs) encompass heterogeneous cognitive, motor, and psychiatric manifestations that typically require extensive behavioural assessments for characterization. Individuals carrying 16p11.2 copy number variations (CNVs), including both deletions and duplications, represent a relatively common NDD subgroup marked by wide variability in psychiatric symptoms, as well as metabolic and peripheral abnormalities, complicating prediction of disease trajectory and treatment response. The identification of biomarkers in easily accessible tissues, such as peripheral blood mononuclear cells (PBMCs), could provide valuable tools for diagnosis and prognosis, yet remains an unmet clinical need. Converging evidence implicates dysregulation of ubiquitous signalling pathways, including ERK and mTOR cascades, in altered protein synthesis across both idiopathic and genetic NDDs. Here, using a hypothesis-driven screening approach, we examined candidate peripheral biomarkers in individuals with 16p11.2 deletions and duplications. We identified a significant reduction of ribosomal protein S6 kinase (p70S6K) levels in PBMCs across both genotypes. Notably, the magnitude of p70S6K reduction correlated with the severity of autistic symptoms independently of CNV genotype. In parallel, MAPK3/ERK1 protein levels mirrored gene dosage, showing increased expression in duplication carriers and reduced levels in deletion carriers, in accordance with the genotype. Collectively, these findings indicate that peripheral molecular alterations may support clinical stratification and suggest that p70S6K represents a promising biomarker for symptom severity and potentially treatment responsiveness in NDD patients carrying 16p11.2 CNVs.

neuroscience↗

bioRxiv2025

Single-cell Analysis of Attenuation-Driven Transcription Reveals New Principles of Bacterial Gene Regulation

Transcription attenuation fine-tunes biosynthetic gene expression in bacteria via premature termination upon metabolic signals. In transcription initiation-controlled bacterial systems, promoter architecture and transcription factor binding sets the size of transcriptional bursts at {sigma}70 promoters, while distal enhancer elements and associated transcriptional activators modulate burst frequency at {sigma}54 promoters. Using the tryptophan biosynthesis operon as a model, we show that transcription attenuation, acting post-initiation and alongside transcriptional repression, simultaneously modulates both burst size and frequency from a {sigma}70 promoter. This challenges the view that frequency modulation requires distal enhancer input and reveals that post-initiation mechanisms can shape divergent transcriptional bursting. We also uncover that bacteria use cross-feeding as a previously unrecognised strategy for controlling cell-to-cell variation in gene expression, with implications for metabolic coordination among cells. These findings redefine transcription dynamics within cell populations and suggest new principles by which bacteria regulate gene expression to adapt to environmental change.

microbiology↗

bioRxiv2025

Development of a Clinically Relevant Rabbit Model of Acute Laryngeal Injury

ObjectivesAcute Laryngeal Injury (ALgI) is created as a result of endotracheal tube pressure ulcer formation leading to fibrosis and inflammation. This condition often leads to airway obstruction and voice and swallowing dysfunction. This study demonstrates a reliable animal model of ALgI to reproduce the acute wound process seen clinically, to explore the pathophysiology of this disease process, and serve as a reproducible injury suitable for the evaluation of therapeutic interventions. MethodsAn ALgI model was developed in New Zealand White rabbits using precise mucosal stripping of the posterior larynx, followed by intubation with an oversized 4.0 endotracheal tube for one hour to mimic intubation-associated trauma and pressure ischemia. Laryngoscopy and laryngeal harvest were performed two weeks post-injury for histologic and immunofluorescent evaluation. ResultsInjured rabbits demonstrated an eight-fold increase in posterior glottic thickness (1.57mm vs. 0.19mm in controls; p=0.0004) and eleven-fold increase in collagen content (1.93mm2 vs. 0.17mm2; p=0.005). Collagen subtype analysis revealed a shift toward active collagen within the injured larynx compared to the uninjured, with increased type III collagen (69.0%% vs. 26.1%; p<0.0001) and reduced type I collagen (27.2% vs. 73.9%; p<0.0001) in the posterior glottis, consistent with the proliferative phase of wound healing. Collagen fiber alignment analysis demonstrated increased coherency in injured tissues (0.36 vs. 0.21; p=0.023), indicating early organized collagen formation consistent with scar formation within the posterior glottis. ConclusionsThe model offers a robust platform for studying the acute pathogenesis of laryngeal injury and for testing the treatment options in the management of ALgI. Level of EvidenceLevel III

bioengineering↗

bioRxiv2025

Evaluation of Leishmania Homologue of Activated C Kinase (LACK) of Leishmania donovani in comparison to glycoprotein 63 as vaccine candidate against visceral leishmaniasis

Leishmaniasis, caused by Leishmania protozoa transmitted via sand fly bites, affects over 12 million people annually, manifesting as self-limiting cutaneous lesions or fatal visceral leishmaniasis (VL). The diseases immune response involves a Th1/Th2 paradigm, with Th1 promoting resistance and Th2 linked to susceptibility. Despite no available human vaccine, the Leishmania homologue of activated C kinase (LACK) protein has shown promise as a candidate due to its conservation across species. In this study, we purified native 34-kDa LACK protein from Leishmania donovani promastigotes and compared its efficacy with gp63 in cationic DSPC liposomes. While gp63 exhibited protective efficacy, LACK failed to protect BALB/c mice. Recombinant LACK, which was cloned, expressed, and purified, also did not confer protection. Immunological assays revealed a Th2-biased immune response characterized by a high IgG1/IgG2a ratio, elevated Th2 cytokines, and an unaltered delayed-type hypersensitivity (DTH) response, highlighting its limited potential. ImportanceThis study underscores the limitations of LACK as a standalone vaccine candidate against VL. Both native and recombinant LACK forms, despite liposomal encapsulation, elicited a Th2- dominated immune response, inadequate for protection. These findings suggest that LACKs intrinsic immunogenic properties may not support the robust Th1 response crucial for combating Leishmania donovani infections. Future research should prioritize alternative antigens or combination formulations to promote Th1-driven immunity. This work contributes to the growing body of knowledge guiding vaccine development for VL, emphasizing the need for immunogens that effectively target protective immune pathways.

immunology↗

bioRxiv2025

A Dual-Action Liposome-Peptide Formulation Synergistically Counteracts A Gain-of-Function p53 Mutant

ObjectiveInactivation of p53 tumor suppressor functions, often through missense mutations, is essential for carcinogenesis. A sub-class of such p53 missense mutations gains new functions, including drug resistance and enhanced proliferation, in addition to its loss of function. Among the most frequent gain-of-function p53 mutants, R273H occurs in tumors of many tissue origins and imparts aggressive character and resistance to drugs to the tumor. Tumors bearing p53R273H are generally resistant to all available therapies, and need for novel interventions are urgently needed. Interaction of p53R273H with Positive Coactivator 4 (PC4), an abundant chromatin-associated protein, is essential for acquiring the gain-of-function properties. Previously, we developed a chemically modified peptide, NLS-p53(380-386), targeting PC4 that abrogated the interaction of p53R273H with PC4 and reversed many of its gain-of-function properties. We earlier demonstrated that cationic phosphatidylcholine-stearylamine (PC-SA) liposomes possess inherent anti-tumor properties. To improve efficacy, pharmacokinetics, and delivery, we entrapped the PC4-targeted peptide into PC-SA liposome. MethodsWe synthesized the NLS-p53(380-386) peptide and entrapped in PC-SA liposome. We used MTT assay, confocal microscopy, flow cytometry, qRT-PCR, and western blotting to investigate the biological effects of the p53-entrapped PC-SA. ResultsPre-treatment with the PC-SA liposome entrapped peptide enhanced the chemosensitivity of widely used anticancer drug doxorubicin in cell lines bearing p53R273H mutation. The doxorubicin-induced cell-killing effect was much more enhanced when pre- treated with the liposome-entrapped peptide than when pre-treated with either the free peptide or the liposome alone. ConclusionThe liposome-encapsulated peptide is a promising formulation for developing therapies targeting tumors bearing the p53R273H.

cancer biology↗

bioRxiv2025

Structure and Activity of the Essential UCH Family Deubiquitinase DUB16 from Leishmania donovani

In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the ubiquitin C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of ubiquitin consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric ubiquitin from small molecule ubiquitin adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type deubiquitinase fold, and a relatively short and disordered crossover loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits deubiquitinase activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal ubiquitin and the C-terminus of the distal ubiquitin. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 M, comparable to the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. Together these results suggest on-target activity and that DUB16 may be a druggable target to develop new anti-leishmania compounds.

biochemistry↗

bioRxiv2024

Computational studies reveal structural characterization and novel families of Puccinia striiformis f. sp. tritici effectors

Understanding the biological functions of Puccinia striiformis f. sp. tritici (Pst) effectors is fundamental for uncovering the mechanisms of pathogenicity and variability, thereby paving the way for developing durable and effective control strategies for stripe rust. However, due to the lack of an efficient genetic transformation system in Pst, progress in effector function studies has been slow. Here, we modeled the structures of 15,201 effectors from twelve Pst races or isolates, a Puccinia striiformis isolate, and one Puccinia striiformis f. sp. hordei isolate using AlphaFold2. Of these, 8,102 folds were successfully predicted, and we performed sequence- and structure-based annotations of these effectors. These effectors were classified into 410 structure clusters and 1,005 sequence clusters. Sequence lengths varied widely, with a concentration between 101-250 amino acids, and motif analysis revealed the presence of known effector motifs such as [Y/F/W]xC and RxLR. Subcellular localization predictions indicated a predominant cytoplasmic localization, with notable chloroplast and nuclear presence. Clear annotations based on sequence and structure included superoxide dismutase and trehalose-6-phosphate phosphatase. A common feature observed was the formation of similar structures from different sequences. In our study, one of the comparative structural analyses revealed a new structure family with a core structure of four helices, including Pst27791, PstGSRE4, and PstSIE1, which target key wheat immune pathway proteins, impacting the host immune function. Further comparative structural analysis showed similarities between Pst effectors and effectors from other pathogens such as AvrSr35, AvrSr50, Zt-KP4-1, and MoHrip2, highlighting convergent evolutionary strategies. This comprehensive analysis provides novel insights into Pst effectors structural and functional characterization, advancing our understanding of Pst pathogenicity and evolution. Author SummaryStripe rust, caused by the fungus Puccinia striiformis f. sp. tritici (Pst), is a major threat to wheat crops worldwide. The fungus uses special proteins, called effectors, to bypass the plants immune defenses and establish infection. To better understand how these effectors work, we used a computational tool, AlphaFold2, to predict the structures of over 15,000 Pst effector proteins. Interestingly, some of the effectors resemble proteins found in other plant pathogens, suggesting that different fungi may evolve similar ways to infect plants. Our research offers new insights into the infection strategies of Pst and could lead to new methods for protecting wheat from stripe rust.

microbiology↗

bioRxiv2024

Jagged-1+ Tregs Mediate Lymphatic Remodeling in Tumour-Draining Lymph Nodes

The tumour-draining lymph nodes (tdLNs) are crucial sites for early immune surveillance of tumours. During tumour development, tdLNs undergo significant stromal remodelling that impacts lymph node (LN) architecture. The reconstruction of tdLNs is dependent on the crosstalk between regulatory T cells (Tregs) and resident stromal cells. The Notch ligand Jagged1 (Jag1), highly expressed in skin Tregs, is critical for facilitating stem cell-mediated tissue regeneration. However, the role of Jag1pos Tregs in mediating the activity of tdLN-resident immune cells and stromal cells remains unexplored. Here, we used the B16-F10 tumour model to assess the role of Jag1pos Tregs during tdLN development. During tumor progression, late-stage tdLN Tregs expressed higher levels of Jag1 than early-stage tdLNs. Conditional deletion of Jag1 in Tregs markedly restrained tdLN expansion, without influencing effector T cell abundance or activation profile. Transcriptomic analysis of tdLNs revealed downregulation of lymphatic endothelial cell (LEC)-related markers in mice with Treg-specific Jag1 ablation. Disruption of lymphatic networks in the tdLN was further confirmed by flow cytometric profiling and histological analysis. In vivo permeability assessment by FITC-dextran demonstrated perturbed lymphatic drainage, likely due to suppressed LN lymphangiogenesis. Finally, we identify the presence of Jag1pos Tregs in tdLNs of human melanoma patients. Overall, our results highlight that crosstalk between Jag1pos Tregs and LECs is required for lymphatic sinus expansion, which is critical for the formation of the tdLN niche during melanoma progression.

immunology↗

bioRxiv2024

Jagged-1+ Skin Tregs Modulate the Innate Immune Response to Wound Healing

Skin-resident regulatory T cells (Tregs) play an irreplaceable role in orchestrating cutaneous immune homeostasis and repair, including the promotion of hair regeneration via the Notch signaling ligand Jagged-1 (Jag1). While skin Tregs are indispensable for facilitating tissue repair post-wounding, it remains unknown if Jag1-expressing skin Tregs impact wound healing. Using a tamoxifen inducible Foxp3creERT2Jag1fl/fl model, we show that loss of functional Jag1 in Tregs significantly delays the rate of full-thickness wound closure. Unlike in hair regeneration, skin Tregs do not utilize Jag1 to impact epithelial stem cells during wound healing. Instead, mice with Treg-specific Jag1 ablation exhibit a significant reduction in Ly6G+ neutrophil accumulation at the wound site. However, during both homeostasis and wound healing, the loss of Jag1 in Tregs does not impact the overall abundance or activation profile of immune cell targets in the skin, such as CD4+ and CD8+ T cells, or pro-inflammatory macrophages. This collectively suggests that skin Tregs may utilize Jag1-Notch signalling to co-ordinate innate cell recruitment under conditions of injury but not homeostasis. Overall, our study demonstrates the importance of Jag1 expression in Tregs to facilitate adequate wound repair in the skin.

immunology↗

bioRxiv2024

Mutant HTT protein decreases with CAG repeat expansion: implications for therapeutics and bioassays.

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in the HTT protein. The mutant CAG repeat is unstable and expands in specific brain cells and peripheral tissues throughout life. Genes involved in the DNA mismatch repair pathways, known to act on expansion, have been identified as genetics modifiers, therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of disease progression. In the context of an expanded CAG repeat, the HTT pre-mRNA can be alternatively processed to generate the HTT1a transcript, that encodes the aggregation prone and highly pathogenic HTT1a protein. This may be a mechanism through which somatic CAG repeat expansion exerts its pathogenic effects, as the longer the CAG repeat, the more HTT1a and HTT1a is produced. The allelic series of knock-in mouse models: HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175 with polyQ expansions of 20, 50, 80, 111 140 and [~]190 can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. By western blot of cortical lysates, we found that mutant HTT levels decreased with increasing CAG repeat length; mutant HTT was only 23% and 10% of wild-type levels in CAG140 and zQ175 cortices, respectively. To identify the optimal bioassays for detecting the full-length HTT and HTT1a isoforms, we interrogated the pairwise combinations of seven well-characterized antibodies on both the HTRF and MSD platforms. In total we tested 32 HTRF and 32 MSD assays to detect full-length mutant HTT, HTT1a, total mutant HTT (full-length HTT and HTT1a) and total full-length HTT (mutant and wild type). None of these assays recapitulated the full-length mutant HTT levels as measured by western blot. We recommend using isoform- and species-specific assays that detect either full-length mutant HTT, HTT1a or wild-type HTT as opposed to those that detect more than one isoform simultaneously. Our finding that as the CAG repeat expands, full-length mutant HTT levels decrease, whilst HTT1a and HTT1a levels increase has implications for therapeutic strategies. If mutant HTT levels in cells containing (CAG)200 are only 10% of wild-type, HTT-lowering strategies targeting full-length HTT at sequences 3 to intron 1 HTT will predominantly lower wild-type HTT, as mutant HTT levels in these cells are already depleted. These data support a therapeutic strategy that lowers HTT1a and depletes levels of the HTT1a protein.

neuroscience↗

bioRxiv2023

Proteomics of Patient-derived Breast Tumours Identifies a Pro-migratory Osteomodulin-Cyclin Dependent Kinase 1 Axis which Drives Bone Metastasis

Metastasis to different organs remains the main cause of mortality in breast cancer. Molecular predictors of metastasis are limited as well as therapeutic options. Here, we conducted quantitative proteomics and phosphoproteomics analysis of patient-derived tumours, identifying osteomodulin (OMD) as a dysregulated protein and associated with bone metastases. Cancer-associated fibroblasts secrete OMD which increases breast cancer migration in vitro and promotes the formation of bone metastases in vivo. Downstream of OMD, phosphoproteomics identified the activation of cyclin-dependent kinase 1 (CDK1). The OMD-CDK1 signalling axis drives a pro-migratory and pro-survival phenotype in vitro and bone metastasis in vivo. Our findings highlight the importance of OMD and CDK1 in breast cancer bone metastasis and proposes an alternative therapeutic avenue for the treatment and the prevention of organ-specific metastases.

cancer biology↗

bioRxiv2023

Early life regulatory T cells modulate PPARγ-dependent skin pigmentation

The maintenance of adult tissue homeostasis is dependent on the functional cross-talk between stem cells (SCs) and tissue-resident immune cells. This reciprocal relationship is also essential for tissue organogenesis during early life. The skin harbors a relatively large population of Regulatory T cells (Tregs) that accumulate within the first two weeks after birth. A functional role for early skin seeding Tregs (ETregs) during the first week of life is currently unexplored. Here, we show that skin Tregs are detected early as postnatal day 3 (P3) where they localize to hair follicle (HF) structures and enter a dynamic flux of activation marker expression. Punctual ETreg depletion from P6-P8, but not later, results in defective HF melanocyte SC (MeSC) mediated skin pigmentation in juvenile life. Transcriptomic analysis of the whole skin on P9 exhibited immediate and pronounced changes in MeSC markers and perturbation of Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) target genes in the HF. Accordingly, punctual ETreg depletion combined with short-term PPAR{gamma} agonization restored skin pigmentation. Single cell profiling of P9 skin revealed that PPAR{gamma} signalling activity is preferentially diminished in the HF epithelium upon loss of ETregs. Finally, we explored changes in the single cell transcriptome of the human tissue disorder, vitiligo, characterized by a lack of melanin and consequent skin depigmentation. These analyses showed that the HF cells from lesional vitiligo skin exhibited a significant downregulation in PPAR{gamma} pathway activation, relative to heathy controls. Overall, ETregs in neonatal skin are critical for sustaining HF PPAR{gamma} signaling, which is vital for facilitating MeSC mediated skin pigmentation during postnatal development. One Sentence SummaryPPAR{gamma} pathway functions downstream of neonatal Tregs to regulate melanocyte stem cell function.

immunology↗

bioRxiv2023

Recyclable CRISPR/Cas9 mediated gene disruption and deletions in Histoplasma

Targeted gene disruption is challenging in the dimorphic fungal pathogen Histoplasma due to the low frequency of homologous recombination. Transformed DNA is either integrated ectopically into the genome or maintained extra chromosomally by de novo addition of telomeric sequences. Based on a system developed in Blastomyces, we adapted a CRISPR/Cas9 system to facilitate targeted gene disruption in Histoplasma with high efficiency. We express a codon-optimized version of Cas9 as well as guide RNAs from a single ectopic vector carrying a selectable marker. Once the desired mutation is verified, one can screen for isolates that have lost the Cas9 vector by simply removing the selective pressure. Multiple mutations can then be generated in the same strain by retransforming the Cas9 vector carrying different guides. We used this system to disrupt a number of target genes including RYP2 and SRE1 where loss-of-function mutations could be monitored visually by colony morphology or color, respectively. Interestingly, expression of two guide RNAs targeting the 5 and 3 ends of a gene allowed isolation of deletion mutants where the sequence between the guide RNAs was removed from the genome. Whole-genome sequencing showed that the frequency of off-target mutations associated with the Cas9 nuclease was negligible. Finally, we increased the frequency of gene disruption by using an endogenous Histoplasma regulatory sequence to drive guide RNA expression. These tools transform our ability to generate targeted mutations in Histoplasma. ImportanceHistoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.

microbiology↗

bioRxiv2023

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homolog ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

developmental biology↗