Biology subjects

Azhar, M.

Publications and source records attributed to Azhar, M..

12 recordsLinked to original sources

bioRxiv2026

Organotypic artery-graft culture enables label-free multiphoton tracking of remodeling that links to long-term graft microarchitecture

The long-term performance of tissue-engineered scaffolds, particularly small-diameter vascular grafts, is shaped by remodeling events at the tissue-graft interface, yet these processes remain difficult to resolve longitudinally and at microstructural resolution in conventional implantation models. Here we develop an organotypic artery-graft model that preserves cylindrical vessel geometry and enables non-destructive label-free multiphoton monitoring of interface remodeling. Using second harmonic generation and two-photon excited fluorescence, we capture evolving fibrillar collagen architecture and cellularization over time, demonstrate compatibility with multiple biomaterial classes, and show integration with rat and mouse explants, live-cell dyes, and fluorescent reporter tissues. The platform resolved distinct remodeling responses to transforming growth factor-{beta} isoforms (TGF-{beta}1, -{beta}2, and -{beta}3), with differential shifts in collagen-fiber distributions, accompanied by changes in matrix-remodeling and contractile gene expression. Across two graft designs, culture-derived remodeling phenotypes, collagen fiber distributions, and initial trajectories agreed with those observed in long-term 6-month interpositional explants. Together, these results establish an accessible intermediate platform for interrogating artery-graft remodeling, tracking these trajectories, and prioritizing graft designs through interface-resolved outcomes before and alongside animal implantation studies.

bioengineering↗

bioRxiv2025

Smooth Muscle Cell-Specific TGFβ2 Protects Against Thoracic Aortic Aneurysm and Dissection in Mice

ObjectiveThoracic aortic aneurysm and dissection (TAAD) are major complications of Loeys- Dietz syndrome caused by heterozygous TGFB2 mutations. While Tgfb2 knockout mice die at birth and adult heterozygotes develop late, non-dissecting or non-rupturing aneurysms, the role of vascular smooth muscle cell (SMC)-derived TGF{beta}2 in postnatal aortic homeostasis and disease remains undefined. Approach and ResultsWe generated tamoxifen-inducible, SMC-specific Tgfb2 conditional knockout mice (Tgfb2cKO) by crossing Tgfb2flox alleles with Myh11CreERT2 and ROSAmT/mG lineage reporter mice. Tgfb2 deletion was induced at 4 weeks of age. Tgfb2cKO mice developed rapidly progressive aneurysms involving both ascending and descending aortas, with intramural dissection and/or rupture at the proximal descending aorta. Lineage tracing confirmed loss of Tgfb2-deficient SMCs during disease progression. Histological and morphometric analyses revealed elastic fiber fragmentation, SMC loss and de-differentiation, medial thickening, adventitial fibrosis, and accumulation of collagen and proteoglycans. Molecular profiling demonstrated reduced expression of SMC contractile genes (Acta2, Myh11), increased fibrillar collagen (Col1a1) expression, early suppression of SMAD2/3 phosphorylation and increased non-canonical TGF{beta} signaling via p38 and pERK1/2 MAPK pathways. ConclusionsThese findings demonstrate that TGF{beta}2 derived from vascular SMCs is essential for postnatal aortic wall homeostasis by preserving SMC differentiation, maintaining extracellular matrix integrity, and supporting and preserving a proper balance of both canonical and non-canonical TGF{beta} signaling. Loss of SMC-specific Tgfb2 precipitates medial degeneration, aneurysm formation, dissection, and rupture, providing direct mechanistic insight into TGFB2-associated aortopathy and establishing a robust novel genetic mouse model for evaluating targeted therapies in TAAD. HighlightsO_LIPostnatal, SMC-specific Tgfb2 deletion in mice caused rapidly progressive thoracic aortic aneurysms, dissections, and fatal rupture. C_LIO_LILoss of Tgfb2 disrupts SMC contractile phenotype and ECM homeostasis, leading to medial degeneration, elastin fragmentation, and abnormal collagen/proteoglycan accumulation. C_LIO_LICanonical TGF{beta}-SMAD signaling is suppressed, while MAPK pathways are activated, indicating ligand-specific signaling imbalance. C_LIO_LIFindings highlight TGF{beta}2 as a central regulator of postnatal aortic homeostasis and suggest that targeted ligand-specific therapeutic strategies may better preserve aortic wall stability. C_LI SignificanceThis study identifies smooth muscle cell-derived TGF{beta}2 as a critical, nonredundant regulator of postnatal aortic wall integrity, linking its loss to thoracic aortic aneurysm, dissection, and rupture, and highlighting TGF{beta}2 ligand-specific signaling as a targeted therapeutic target. Graphical AbstractSmooth muscle cell-derived TGF{beta}2 maintains postnatal aortic wall homeostasis by preserving contractile gene expression, elastin architecture, and balanced ECM remodeling. Conditional deletion of Tgfb2 in SMCs shifts signaling from canonical SMAD2/3 to MAPK pathways, leading to medial degeneration, progressive aneurysm, dissection, and rupture--highlighting TGF{beta}2 as a nonredundant, ligand-specific regulator and potential therapeutic target in thoracic aortopathy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/679917v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ef41d3org.highwire.dtl.DTLVardef@ca8feborg.highwire.dtl.DTLVardef@aebce4org.highwire.dtl.DTLVardef@1f3599b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

Transforming growth factor {beta} (TGF{beta}) is a secreted growth factor that is sequestered to the extracellular matrix (ECM) as a latent complex. In adult disease TGF{beta} release in the heart transforms fibroblasts into a differentiated state that synthesizes more ECM. However, it is not known how TGF{beta} functions in the early developing heart to impact resident fibroblasts. Here, we observe that deletion of the Tgfb1, Tgfb2, and Tgfb3 genes (TGF{beta} ligands) from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte-deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness. Gene expression profiling of hearts from myocyte-specific Tgfb1/2/3 deleted mice reveal defects in both cardiomyocyte and fibroblast maturation with ectopic expression of multiple skeletal muscle-specific genes beginning at embryonic day 17.5 and progressing with age. However, cardiomyocyte-specific deletion of TGF{beta} receptors I/II encoding genes (Tgfbr1/2) or Smad2/3 encoding genes (Smad2/3) do not recapitulate this phenotype suggesting that TGF{beta} directly programs early heart fibroblast development that in turn specifies cardiomyocyte maturation. Importantly, Col1a2-/-;Col6a2-/- mice with defective cardiac ECM stiffness, mice lacking cardiomyocyte Itgb1 with reduced ECM load sensing, and Tcf21-/- embryos at E17.5 lacking cardiac fibroblasts each fail to generate the same pathologic ECM program with ectopic cardiomyocyte differentiation observed with Tgfb1/2/3 myocyte-specific deletion. These and additional results indicate that TGF{beta} generated by cardiomyocytes in the embryonic heart mediates fibroblast differentiation that co-evolves the ECM environment that in turn programs cardiomyocyte maturation to establish their identity.

developmental biology↗

bioRxiv2024

HEMATO-CHEMICAL VARIATIONS- A DIAGNOSTIC TOOL FOR BABESIA, THEILERIA AND ANAPLASMA INFECTIONS IN CAPTIVE HOG DEER (AXIS PORCINUS)

Hog deer are an essential component of Pakistans biodiversity. Their conservation helps maintain integrity of local ecosystems, contributing to overall stability and balance of regions wildlife populations. In Pakistan, they are classified as an endangered species according to IUCN. Theileria, babesia and anaplasma are among most prevalent blood parasites in ruminants which require diagnosis at early stages of the infection so that treatment protocols can be followed and more severe pathological alterations can be prevented. The changes in hemato-chemical parameters caused by Theileria, babesia and anaplasma were recorded individually and then compared with healthy group of hog deer respectively. Alterations in hemato-chemical parameters produced by these parasite-infected hog deer were assessed as potential diagnostic tools for distinguishing between individual blood parasites. Total leukocyte count, monocytes and eosinophils were observed to be higher than normal in babesia infected hog deer while level was lower than normal hog deer in anaplasma and theileria infected hog deer. The main objective of this study is to promote early diagnosis of parasitic infections in hog deer to facilitate prompt treatment, pathological prevention and allocate resources for further research on hemato-chemical parameters as diagnostic tools, enhancing understanding and management of blood parasite infections in hog deer.

pathology↗

bioRxiv2021

Momelotinib is a highly potent inhibitor of FLT3-mutant AML

Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogenic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to a more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues, D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL) and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation. Key pointsO_LIMomelotinib shows high efficacy against FLT3 mutated AML cells including quizartinib-resistant activation loop variants. C_LIO_LIMomelotinib effectively suppresses intrinsic resistance conferred by FLT3 ligand and hematopoietic cytokines (GM-CSF and IL3). C_LI

cancer biology↗

bioRxiv2021

Effects of Emodin, a Plant-Derived Anthraquinone, on TGFβ1-Induced Cardiac Fibroblast Activation and Function

Cardiac fibrosis accompanies a number of pathological conditions and results in altered myocardial structure, biomechanical properties and function. The signaling networks leading to fibrosis are complex, contributing to the general lack of progress in identifying effective therapeutic approaches to prevent or reverse this condition. Several studies have shown protective effects of emodin, a plant-derived anthraquinone, in animal models of fibrosis. A number of questions remain regarding the mechanisms whereby emodin impacts fibrosis. TGF-{beta}1 is a potent stimulus of fibrosis and fibroblast activation. In the present study, experiments were performed to evaluate the effects of emodin on activation and function of cardiac fibroblasts following treatment with TGF-{beta}1. We demonstrate that emodin attenuates TGF-{beta}1-induced fibroblast activation and collagen accumulation in vitro. Emodin also inhibits activation of several canonical (SMAD2/3) and non-canonical (Erk1/2) TGF-{beta} signaling pathways, while activating the p38 pathway. These results suggest that emodin may provide an effective therapeutic agent for fibrosis that functions via specific TGF-{beta} signaling pathways.

cell biology↗

bioRxiv2021

Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localised in the Polymerase (3Dpol) Encoding Region.

AbstractRNA structure plays a crucial role in the replication of positive sense RNA viruses and can form functional elements within the untranslated regions (UTRs) and the protein coding sequences (or open reading frames (ORFs)). While RNA structures in the UTRs of several picornaviruses have been functionally characterised, the roles of putative RNA structures predicted for the ORF remain largely undefined. Here we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome and predicted the existence of 53 evolutionarily conserved RNA structures within the ORF. Forty-five (45) of these structures were located in the regions encoding the non-structural proteins (nsps). To investigate if the structures in the regions encoding the nsps are required for FMDV replication we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimise RNA secondary structures while preserving protein coding, native dinucleotide frequencies and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV sub-genomic replicon. We found that three of the RNA structures, all at the 3 termini of the FMDV ORF, were critical for replicon replication. Contrastingly, disruption of the other 42 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. ImportanceSome RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 45 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the non-structural proteins, only three are essential for replication of an FMDV sub-genomic replicon. Replicons replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify if the remaining 42 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

evolutionary biology↗

bioRxiv2020

NG2+/Nestin+ mesenchymal stem cells dictate DTC dormancy in the bone marrow through TGFβ2

In the bone marrow (BM) microenvironment, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell (HSC) quiescence1,2. Importantly, the BM can also harbour disseminated tumour cells (DTCs) from multiple cancers, which, like HSCs, can remain dormant3. The BM signals are so growth-restrictive that dormant BM DTCs can persist for years to decades only to awaken and fuel lethal metastasis3-10. The mechanisms and niche components regulating DTC dormancy remain largely unknown. Here, we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can instruct breast cancer (BC) DTCs to enter dormancy. NG2+/Nestin+ MSCs produce TGF{beta}2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27-CDK inhibitor induction. Importantly, genetic depletion of the NG2+/Nestin+ MSCs or conditional knock-out of TGF{beta}2 in the NG2+/Nestin+ MSCs led to awakening and bone metastatic expansion of otherwise dormant p27+/Ki67- DTCs. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy. Given that aged NG2+/Nestin+ MSCs can lose homeostatic control of HSC dormancy, our results suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche may result in a break from dormancy and BC bone relapse.

cancer biology↗

bioRxiv2020

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

immunology↗

bioRxiv2020

Rapid, field-deployable nucleobase detection and identification using FnCas9

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids has provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19.

molecular biology↗

bioRxiv2019

TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9

Locus-specific interrogation of the genome using programmable CRISPR-based technologies is tremendously useful in dissecting the molecular basis of target gene function and modulating its downstream output. Although these tools are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using this technique is not well developed due to inadequate labeling technologies. Here, we report the development of a modular technology, sgRNA-Click (sgR-CLK), which harnesses the power of bioorthogonal click chemistry for remodeling CRISPR to display synthetic molecules on target genes. A terminal uridylyl transferase (TUTase) was repurposed to construct an sgRNA containing multiple minimally invasive bioorthogonal clickable handles, which served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Our results demonstrate that sgR-CLK could provide a simplified solution for site-directed display of small molecules to study as well as modulate the function of gene targets.

biochemistry↗

bioRxiv2019

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.\n\nSIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 from Francisella novicida (FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.

molecular biology↗