Biology subjects

Hossain, M.

Publications and source records attributed to Hossain, M..

At least 19 recordsLinked to original sources

bioRxiv2026

A role for CIZ1, a Nuclear Matrix Protein Overexpressed in Cancer Cells, in Controlling the Initiation of DNA Replication

DNA replication licensing is tightly coordinated with cell-cycle progression to ensure genome stability. Here we demonstrate that CIZ1, a nuclear matrix scaffold protein in human cells, coordinates DNA replication licensing through direct interactions with the Origin Recognition Complex (ORC), CDC6, and Cyclins E/A-CDK2. The C-terminal zinc finger region of CIZ1 mediates binding to the cyclin-binding motifs of ORC1 and CDC6, and Cyclin -CDK-dependent phosphorylation regulates these interactions. Cell cycle analyses demonstrated a dynamic association of CIZ1 with ORC1 and CDC6. Functional studies showed that CIZ1 depletion impaired MCM loading, reduced DNA synthesis, delayed S-phase progression and inhibited cell proliferation. Cancer cells overexpressing CIZ1 exhibited an enhanced dependency on its expression, and CRISPR tiling screens identified the C-terminal regulatory region containing cyclin-binding motifs and CDK phosphorylation sites as essential for cell proliferation. Acute degradation of CIZ1 further revealed an unexpected role in maintaining CCCTC-binding factor (CTCF) occupancy at specific genomic loci and regulating transcriptional programs associated with cell adhesion and migration, indicating that CIZ1 links DNA replication with higher-order genome organization. Finally, depletion of CIZ1 significantly suppressed colorectal tumor growth in xenograft models. Together, these findings establish CIZ1 as a multifunctional scaffold that integrates DNA replication licensing, Cyclin -CDK signaling, and chromatin architecture.

molecular biology↗

bioRxiv2026

CRISPR-mediated Correction of Oncogenic AS-NMD in Splicing Factor Mutant Cancer

Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted a PE of a tumor suppressor (EZH2) from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, escaped AS-NMD, and restored the protein expression. This subsequently reinstated H3K27 histone methylation and rescued defective chromatin regulation associated with impaired hematopoietic stem cell differentiation. Finally, we showed the preferential advantages of CRISPR over the antisense technology we recently developed targeting the PE of EZH2. Therefore, the CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases.

cancer biology↗

bioRxiv2026

MondrianMap: Navigating Gene Set Hierarchies with Multi-Resolution Enrichment Maps

Gene Ontology encodes genes as a hierarchy, yet every enrichment visualization flattens it into a ranked list, discarding the ability to view the same process at different levels of abstraction. We present MondrianMap, a free interactive web application (https://mondrianmap.smartdrugdiscovery.org/) that organizes enrichment results into 13 semantically principled layers derived from the GOALS framework and renders them as color-encoded rectangular maps where block area reflects significance, color encodes effect direction, and spatial proximity preserves semantic relations, all within an interactive interface. Three case studies across the NIH Common Fund Data Ecosystem demonstrate that visualization facilitates recognition of patterns that are difficult to discern in flat outputs: (1) LINCS CRISPR perturbations reveal that TP53 and KRAS knockouts produce opposite color maps at a single semantic layer, the same immune recruitment processes suppressed by TP53 loss are activated by KRAS disruption; (2) GTEx aging signatures expose the inflammaging paradox as an immediate visual phenomenon, identical antimicrobial defense programs appear uniformly upregulated in aging blood yet uniformly downregulated in aging liver at matched semantic resolution; and (3) MoTrPAC exercise data capture temporal dynamics as color transitions where brown adipose tissue undergoes a threshold switch from two enriched terms to thirty-four at a single molecular layer, while cardiac tissue reverses from uniformly activated to uniformly suppressed glycolytic metabolism as adaptation progresses. MondrianMap facilitates hierarchical visual reasoning, complementing statistical enrichment reporting for biological discovery. HIGHLIGHTSO_LIMondrianMap provides multi-resolution enrichment visualization for Gene Ontology C_LIO_LILayer-specific views reveal directional oppositions difficult to discern in flat term lists C_LIO_LINavigating layers organizes one enrichment into a multi-scale biological narrative C_LIO_LIDemonstrated on cancer, aging, and exercise data across three CFDE programs C_LI IN BRIEFMondrianMap is a web application that transforms gene set enrichment results into layered visualizations encoding regulation, significance, and semantic hierarchy. Across cancer, aging, and exercise datasets, viewing enrichment at defined semantic layers exposes directional inversions, temporal switches, and multi-scale biological narratives that are difficult to extract from conventional flat enrichment outputs. THE BIGGER PICTUREWhen researchers measure gene expression changes in disease, aging, or drug response, they rely on enrichment analysis to translate thousands of molecular measurements into interpretable biological themes. The standard output is a ranked list of processes sorted by statistical significance. This format served the field well when studies examined a single condition; however, modern genomics routinely compares dozens of tissues, timepoints, and perturbations, each generating hundreds of enriched terms. The critical limitation is not statistical power; however, interpretive structure: a flat list cannot show whether two conditions activate the same biological process in opposite directions, whether a process visible at one level of abstraction disappears or transforms at another, or how a tissues functional response evolves across time. These are precisely the questions that define contemporary systems biology. Does a tumor suppressor gene silence the same immune program that an oncogene activates? Does aging drive the same defense pathway upward in the blood and downward in the liver? Does an exercise response flip from activation to suppression as the tissue adapts? Answering these questions requires a visualization framework that preserves hierarchy, encodes direction, and enables comparison at matched levels of biological resolution. MondrianMap provides this framework by organizing Gene Ontology terms into quantitatively defined semantic layers and rendering enrichment as color-encoded rectangular maps navigable from molecular mechanism to system-level theme. The result is a tool that extends enrichment analysis from a reporting step into an interactive framework for biological reasoning, hypothesis generation, and cross-dataset discovery.

Systems Biology↗

bioRxiv2026

Characterization of time-dependent and history-dependent mechanical behaviour of human masseter muscle

The human masseter muscle is one of the primary muscles responsible for mastication and mandibular movement; however, its intrinsic mechanical properties remain insufficiently characterized. In this experimental study, the nonlinear, viscoelastic, and history-dependent behaviour of the human masseter muscle was investigated using ex vivo uniaxial cyclic tensile tests. The masseter muscle samples prepared from fresh and formalin-preserved cadavers were tested under two loading protocols: a continuous stretch protocol with increasing stretch levels and a constant stretch protocol with repeated loading to a fixed maximum stretch. Tests were conducted at two strain rates, and their influences on the mechanical behaviour of the tissue were examined. The effect of formalin preservation was also investigated. The results showed that the stiffness of the tissue increases for formalin-preserved samples. Under cyclic loading, the features including energy dissipation, stress-softening, residual deformation, and cyclic conditioning progressively changed during the initial loading cycles and reached stabilization during the final cycle. These findings provide experimental evidence that the human masseter muscle exhibits nonlinear, viscoelastic, and history-dependent mechanical behaviour under cyclic tensile loading. The experimental data obtained in this study may be used for biomechanical modelling of the human masticatory system and the development of constitutive models for cranio-maxillofacial surgical simulation, prosthetic design, and facial soft-tissue biomechanics. Statement of significanceThe masseter muscle is one of the primary muscles of mastication. To address the current gap in craniofacial biomechanics that has largely focused on the mechanical characterization of the masseter muscle based on imaging techniques or monotonic loading, this study quantifies the nonlinear and viscoelastic mechanical response of masseter tissue under cyclic continuous and constant stretch loading, including strain-rate and preservation effects. The results show that the mechanical behaviour of the masseter muscle, including stiffness, hysteresis, stress-softening, and residual strain behaviour, is strongly influenced by strain-rate and formalin preservation. The experimental results provide mechanical data for constitutive modelling of the masticatory system with applications in cranio-maxillofacial surgical simulation, prosthetic design, and facial soft tissue modelling.

bioengineering↗

bioRxiv2026

A Cross-kingdom Effector Modulates EDS1-dependent TIR-NLR-mediated Plant Immunity

In the ongoing battle between plants and pathogens, successful pathogens promote colonization by evolving new effectors that manipulate counteractive host responses, such as intracellular nucleotide-binding leucine-rich repeat (NLR)-triggered immunity. Here, we identify a secreted effector, PbSTMI, from the unique intracellular protist Plasmodiophora brassicae, that is conserved across divergent plant pathogens and broadly suppresses ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-dependent plant immunity. PbSTMI directly binds EDS1 and promotes its proteasome-mediated degradation, thereby diminishing downstream EDS1 oligomerization required for resistance. PbSTMI overexpression suppresses toll/interleukin-1 receptor (TIR) domain-containing NLR (TIR-NLR)-driven autoimmunity in Arabidopsis and further attenuates salicylic acid-mediated signaling in the autoimmune background. It blocks flg22-induced accumulation of EDS1 and regulates SA-mediated PAMP-triggered immunity (PTI) responses. Oligomerization among PbSTMI and its paralogs suggests coordinated immune suppression during different stages of P. brassicae infection and clubroot disease progression. PbSTMI suppression of EDS1-dependent TIR-NLR-mediated pathways increases susceptibility to a broad range of phytopathogens, including the biotrophs P. brassicae and Erysiphe cichoracearum and the hemibiotrophic Colletotrichum higginsianum. Together, these findings reveal a conserved pathogen strategy that disarms a central immune hub, tipping the balance of the plant-pathogen arms race decisively in favor of the pathogen.

plant biology↗

bioRxiv2026

Hematological and genetic basis of clinical heterogeneity of HemoglobinE/β-thalassemia

Patients with HbE/{beta}-thalassemia inheriting the same {beta}-globin mutations display varied clinical manifestations, the mechanism of which is only partially known. The study aimed to decipher the heterogenous basis of HbE/{beta}-thalassemia patients in more details by focusing on both hematological and genetic modifiers influencing the disease severity, which included-(i) HbF and HbE levels using Hb electrophoresis, (ii) {beta}-thalassemia mutations, (iii) anti3.7triplication using Gap-PCR, (iv) individual and cumulative effects of HbF-inducing SNPs in 4 major modifier genes, namely HBG2, BCL11A, HBSB1L_MYB intergenic-region, and HBBP1 which were genotyped using DNA sequencing and Real-time PCR-HRM methods. Accordingly, 130 diagnosed Bangladeshi patients with HbE/{beta}-thalassemia were enrolled and categorized as mild, moderate, and severe as per Mahidol scoring system. c.79G>A+IVS1_5G>C was the most predominant (73.8% of total) mutation pair across all the 3 severity groups, indicating secondary modifiers might influence the severity. Our study found both HbF and HbE protective to HbE/{beta}-thalassemia, as both were inversely related to the severity score (HbF: p<0.0001/r=-0.55; HbE: p<0.0001/r=-0.56). Four SNPs-XmnI-G{gamma}, rs2071348 (HBBP1), rs489544 and rs28384513 (HBS1L_MYB) showed significant association with the elevated HbF levels (p=0.005, 0.0001, 0.0001, 0.004 respectively). The multivariate analysis showed that the risk genotypes with single or combination of 2, 3,and 4 SNPs showed gradually increased risk [Odd Ratio (95%CI)= 2.51, 5.47, 19.5, 39.0, respectively] of less severe phenotype, suggesting that these linked SNP variants had a cumulative effect on both HbF level and clinical severity score. However, low HbE level and copresence of anti3.7triplication were found to nullify the ameliorating effect of multiple SNPs.

genetics↗

bioRxiv2026

New host and geographic range expansion of Lemuricola nycticebi in the Bengal slow loris

PurposeThe Bengal slow loris (Nycticebus bengalensis) is a globally Endangered nocturnal primate with limited health data across its South and Southeast Asian range. MethodsDuring necropsies of two deceased individuals from northeastern Bangladesh, numerous live adult pinworms were recovered. ResultsMorphological examination combined with mitochondrial cox1 gene sequencing identified the parasites as Lemuricola (Protenterobius) nycticebi. Both hosts exhibited severe gastrointestinal and peritoneal lesions consistent with high-intensity helminth infection. This represents the first confirmed record of L. nycticebi in the Bengal slow loris and extends the parasites known geographic range by approximately 3,400 km. ConclusionOur findings highlight an overlooked parasitic threat to slow lorises. Given that many Bengal slow lorises are rescued or confiscated from the illegal wildlife trade and forest-edge areas in Bangladesh every year and subsequently released into the wild, the results underscore the need for systematic health assessments, including parasitic screening, before repatriation into the wild.

zoology↗

bioRxiv2025

Direct and indirect regulation of fetal globin transcript by RNA-binding protein IGF2BP1

Despite extensive investigation, the molecular control of developmental hemoglobin expression remains incompletely elucidated. Hemoglobin switching is controlled by transcription factors, miRNAs, and RNA-binding proteins (RBPs) that enforce gene regulatory changes through development. Here we examine the role of the heterochronically silenced N-6 methyladenosine (m6A) RNA-binding protein IGF2BP1 that was previously described to regulate HBG1/2 indirectly by suppressing BCL11A expression through an unknown mechanism. We find that IGF2BP1 binds and activates HIC2, itself a BCL11A repressor. Furthermore, we identify that IGF2BP1 plays a BCL11A-independent role by direct binding to HBG1/2 to promote its translation. Stop codon-proximal m6A-modified coding sequences within HBG2 transcripts are necessary and sufficient for direct positive regulation mediated by IGF2BP1. This work deepens the mechanistic understanding of hemoglobin switching and suggests a physical relationship between heterochronic RBPs and globin transcripts.

genetics↗

bioRxiv2025

L2 loop engineering enhances the enzymatic activity and synergism property of the AA16 family Lytic polysaccharide monooxygenase enzyme from Aspergillus fumigatus.

Lytic polysaccharide monooxygenase (LPMO) is an enzyme that has enormous potential for industrial applications. It has a synergistic effect with the cellulase enzyme complex. The LPMO enzymes typically adopt a compact {beta}-sandwich fold that consists of a 7 to 9 {beta}-strand with a flat active site containing copper in its active site. There are a few loops in LPMO, among them the L2 loop is reported to take a key role in shaping the active site and substrate binding. Now, in this work, we want to investigate the role of the L2 loop in enzymatic activity and synergistic effect. In achieving our goal, we have replaced the L2 loop of our concerning AfLPMO16 with other L2 loops from different LPMOs: HiLPMO9B (PDB: 5NNS), McLPMO9 (PDB: 7NTL) and CsLPMO9 (PDB: 7EXK). Interestingly, L2 loop replacement from CsLPMO9 (PDB: 7EXK) showed enhanced activity and synergism compared to others. The secondary structural analysis by circular dichroism also suggested that it changed the structure significantly. Moreover, this is the first report of complete L2 loop engineering in LPMO.

biochemistry↗

bioRxiv2025

AAV gene therapy for GBA-PD and Gaucher Disease

Mutations in GBA1, the gene encoding glucocerebrosidase (GCase), are the most common risk factor for Parkinsons Disease (PD). GBA-PD patients are a genetic subpopulation of PD carrying heterozygous mutations in GBA1. Additionally, bi-allelic mutations in GBA1 cause Gaucher Disease (GD), a lysosomal storage disorder. Loss of GCase activity, a lysosomal enzyme leads to the accumulation of lipid substrates, disrupting lipid homeostasis and promoting cellular toxicity. Here, we report an AAV-mediated GBA1 replacement strategy to treat GD and GBA-PD by a one-time infusion via intravenous (GD Type 1) or intra-CSF (GBA-PD) route of administration. We engineered human GCase to be readily secretable to facilitate broad cross-correction. We developed CBE (conduritol {beta}-epoxide) induced lipid accumulation models to assess efficacy in mice and non-human primates (NHPs) to assess efficacy of our engineered constructs. Based on data across species, across different routes of administration, we nominated AAV.GMU01 SS3-GBA1 as our lead candidate. SS3-GBA1 is robustly secreted, cross-corrected across tissues and promotes lipid clearance. By comparing human GCase levels in AAV-treated NHP brains to healthy human donor brains, we demonstrate that AAV.GMU01 SS3-GBA1 replenishes the GCase deficit seen in GBA-PD patients, thus, restoring GCase to near-physiological levels Importantly, AAV.GMU01 SS3-GBA1 is well-tolerated with no adverse findings. Collectively, we establish a therapeutic strategy for the treatment of Gaucher Disease and GBA-PD with a single gene therapy product. One Sentence SummaryA novel gene therapy strategy for GBA1-PD and Gaucher disease with an engineered payload that robustly cross-corrects enhancing therapeutic footprint

neuroscience↗

bioRxiv2025

AAV-mediated ARSA replacement for the treatment of Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder caused by mutations in the arylsulfatase A (ARSA) gene, resulting in lower sulfatase activity and the toxic accumulation of sulfatides in the central and peripheral nervous system. Children account for 70% of cases and become progressively disabled with death occurring within 10 years of disease onset. Gene therapy approaches to restore ARSA expression via adeno-associated viral vectors (AAV) have been promising but hampered by limited brain biodistribution. We report the development of a novel capsid AAV.GMU01, demonstrating superior biodistribution and transgene expression in the central nervous system of non-human primates (NHPs). Next, we show that AAV.GMU01-ARSA treated MLD mice exhibit persistent, normal levels of sulfatase activity and a concomitant reduction in toxic sulfatides. Treated mice also show a reduction in MLD-associated pathology and auditory dysfunction. Lastly, we demonstrate that treatment with AAV.GMU01-ARSA in NHPs is well-tolerated and results in potentially therapeutic ARSA expression in the brain. In summary, we propose AAV.GMU01-ARSA mediated gene replacement as a clinically viable approach to achieve broad and therapeutic levels of ARSA.

neuroscience↗

bioRxiv2025

Interactive effects of xylem and phloem damage under drought on plant water and carbon dynamics: Mimicking the impact of plant canker disease

O_LIIt is often stated that trees experiencing climate change may be more vulnerable to damaging effects of pest and disease, but experimental tests are still rare. In this study, we examined the separate and combined impact of experimentally applied xylem and phloem damage in an Australian Eucalypt species (Corymbia calophylla), which is increasingly suffering from phloem and xylem damage by a canker disease caused by Quambalaria coyrecup. C_LIO_LICut treatments were applied to saplings under controlled conditions by removing xylem only ([~]56%), phloem only ([~]70%) or both in the main stem under well-watered and drought treatments. C_LIO_LIAs expected, xylem damage reduced whole-plant conductance, stomatal conductance and photosynthesis, and hence growth. Phloem damage limited phloem transport, increased leaf non-structural carbohydrate concentrations (NSC), and reduced root NSC, photosynthesis and growth. Contrary to expectations, effects were larger in well-watered than droughted plants. The combined effects of damage to xylem and phloem were generally less than additive. Plants were remarkably resilient to significant loss of xylem and phloem, although xylem damage had a greater impact on most of the physiological parameters than phloem damage. C_LIO_LIThese results reveal potential consequences of xylem-phloem dysfunction due to biotic attack, leading to whole tree mortality, in association with drought stress. C_LI

plant biology↗

bioRxiv2024

Identification of potential inhibitors against Inosine 5'-Monophosphate Dehydrogenase of Cryptosporidium parvum through an integrated in silico approach

The protozoan parasite Cryptosporidium, found in several vertebrates, including humans, is the source of the global infection known as cryptosporidiosis, which manifests as acute gastroenteritis, abdominal pain, and diarrhea. Although infections in certain individuals have been linked to other species, Cryptosporidium parvum is the main cause of illnesses in humans. Lactate Dehydrogenase, Inosine 5'-Monophosphate Dehydrogenase (IMPDH), and several other targets have been identified by the genome sequencing of C. parvum. Bioactive phytochemicals derived from nature have enormous potential as anti-cryptosporidiosis agents. The study aimed to identify new anti-cryptosporidial agents that work against the IMPDH of the parasite by using integrated in silico approaches. In this study, a total of 24 bioactive phytochemicals were screened virtually through molecular docking and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analyses. Four lead compounds were identified, including Brevelin A (-8.9 kcal/mol), Vernodalin (-8.7 kcal/mol), Luteolin (-8.6 kcal/mol), and Pectolinarigenin (-8.1 kcal/mol), against the IMPDH protein (PDB ID: 4IXH) from the parasite. All the lead compounds had excellent pharmacokinetic and pharmacodynamic characteristics. The toxicity analysis showed satisfactory results with no major side effects. All of the selected compounds showed no violation of Lipinskis rules of five, indicating the possibility of oral bioavailability as potential drug candidates. In the majority of cases, target class prediction-identified enzymes, as well as investigational and experimental drugs, have been found to have structural similarities to the lead compounds. With significant biochemical interactions, all of the targeted phytochemical compounds have demonstrated excellent pharmacokinetics and better bioavailabilities. The findings strongly recommend in vitro experimental studies to aid in the development of novel therapeutics against Cryptosporidium parvum.

bioinformatics↗

bioRxiv2024

DNA damage causes ATM-dependent heterochromatin loss leading to nuclear softening, blebbing, and rupture

The nucleus must maintain stiffness to protect the shape and integrity of the nucleus to ensure proper function. Defects in nuclear stiffness caused from chromatin and lamin perturbations produce abnormal nuclear shapes common in aging, heart disease, and cancer. Loss of nuclear shape via protrusions called blebs leads to nuclear rupture that is well-established to cause nuclear dysfunction, including DNA damage. However, it remains unknown how increased DNA damage affects nuclear stiffness, shape, and ruptures, which could create a negative feedback loop. To determine if increased DNA damage alters nuclear physical properties, we treated MEF cells with DNA damage drugs cisplatin and bleomycin. DNA damage drugs caused increased nuclear blebbing and rupture in interphase nuclei within a few hours and independent of mitosis. Micromanipulation force measurements reveal that DNA damage decreased chromatin-based nuclear mechanics but did not change lamin-based strain stiffening at long extensions relative to wild type. Immunofluorescence measurements of DNA damage treatments reveal the mechanism is an ATM-dependent decrease in heterochromatin leading to nuclear weaken, blebbing, and rupture which can be rescued upon ATM inhibition treatment. Thus, DNA damage drugs cause ATM-dependent heterochromatin loss resulting in nuclear softening, blebbing, and rupture.

cell biology↗

bioRxiv2024

Spatial analysis reveals combinative role for natural killer and CD8 T cells in antitumor immunity despite profound MHC class I loss in non-small cell lung cancer

BackgroundMHC class I (MHC-I) loss is frequent in non-small cell lung cancer (NSCLC) rendering tumor cells resistant to T cell lysis. NK cells kill MHC-I-deficient tumor cells, and although previous work indicated their presence at NSCLC margins, they were functionally impaired. Within, we evaluated whether NK cell and CD8 T cell infiltration and activation vary with MHC-I expression. MethodsWe used single-stain immunohistochemistry (IHC) and Kaplan-Meier analysis to test the effect of NK cell and CD8 T cell infiltration on overall and disease-free survival. To delineate immune covariates of MHC-I-disparate lung cancers, we used multiplexed immunofluorescence (mIF) imaging followed by multivariate statistical modeling. To identify differences in infiltration and intercellular communication between IFN{gamma}-activated and non-activated lymphocytes, we developed a computational pipeline to enumerate single cell neighborhoods from mIF images followed by multivariate discriminant analysis ResultsSpatial quantitation of tumor cell MHC-I expression revealed intra- and inter-tumoral heterogeneity, which was associated with the local lymphocyte landscape. IHC analysis revealed that high CD56+ cell numbers in patient tumors were positively associated with disease-free survival (DFS) (HR=0.58, p=0.064) and over-all survival (OS) (HR=0.496, p=0.041). The OS association strengthened with high counts of both CD56+ and CD8+ cells (HR=0.199, p<1x10-3). mIF imaging and multivariate discriminant analysis revealed enrichment of both CD3+CD8+ T cells and CD3-CD56+ NK cells in MHC-I-bearing tumors (p<0.05). To infer associations of functional cell states and local cell-cell communication, we analyzed spatial single cell neighborhood profiles to delineate the cellular environments of IFN{gamma}+/- NK cells and T cells. We discovered that both IFN{gamma}+ NK and CD8 T cells were more frequently associated with other IFN{gamma}+ lymphocytes in comparison to IFN{gamma}- NK cells and CD8 T cells (p<1x10-30). Moreover, IFN{gamma}+ lymphocytes were most often found clustered near MHC-I+ tumor cells. ConclusionsTumor-infiltrating NK cells and CD8 T cells jointly affected control of NSCLC tumor progression. Co-association of NK and CD8 T cells was most evident in MHC-I-bearing tumors, especially in the presence of IFN{gamma}. Frequent co-localization of IFN{gamma}+ NK cells with other IFN{gamma}+ lymphocytes in near-neighbor analysis suggests NSCLC lymphocyte activation is coordinately regulated. What is already known on this topicMHC-I loss occurs frequently in NSCLC and corresponds with waning immunity in the tumor microenvironment (TME). NK cells recognize "missing-self" targets and could be leveraged to target NSCLC tumors with MHC-I loss. While NK cell presence at tumor margins has been documented in NSCLC, they were shown to lose function in this environment. What this study addsWe developed spatial analysis pipelines leveraging the local heterogeneity of the TME at single cell resolution to test whether NK cells and T cells together contribute antitumoral immunity in NSCLC. We discovered that a high density of tumor-infiltrating NK cells corresponded with DFS, and this association was increased in patients with high coincident CD8 T cells, especially those in central tumor. Intriguingly, both cell types were found clustered together in MHC-I-bearing tumors, especially when both expressed IFN{gamma}, suggesting coordinated lymphocyte activities may enhance immune control of NSCLC. How this study might affect research, practice, or policyThis study provides a rationale for developing novel immunotherapies that simultaneously increase NK and T cell anti-tumoral immunity. Associations linking NK cells with patient survival and increased immune effector activity in NSCLC, even in MHC-I-deficient tumors, further highlights the need to devise and deploy NK cell activating strategies which may be highly efficacious in CD8 T cell refractory NSCLC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/581048v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1e383c0org.highwire.dtl.DTLVardef@1dee268org.highwire.dtl.DTLVardef@1e21bd4org.highwire.dtl.DTLVardef@1910a9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Designer antisense circRNAGFP reduces GFP protein abundance in transgenic Arabidopsis protoplasts in a sequence-specific manner, independent of RNAi pathways

Circular RNAs (circRNAs) are single-stranded RNA molecules characterised by their covalently closed structure and are emerging as key regulators of cellular processes in mammals, including gene expression, protein function and immune responses. Recent evidence suggests that circRNAs also play significant roles in plants, influencing development, nutrition, biotic stress resistance, and abiotic stress tolerance. However, the potential of circRNAs to modulate target protein abundance in plants remains largely unexplored. In this study, we investigated the potential of designer circRNAs to modulate target protein abundance in plants using Arabidopsis as a model system. We demonstrate that treatment with a 50 nt circRNAGFP, containing a 30 nt GFP antisense sequence stretch, results in reduced GFP reporter target protein abundance in a dose- and sequence-dependent manner. Notably, a single-stranded open isoform of circRNAGFP had little effect on protein abundance, indicating the importance of the closed circular structure. Additionally, circRNAGFP also reduced GFP abundance in Arabidopsis mutants defective in RNA interference (RNAi), suggesting that circRNA activity is independent of the RNAi pathway. We also show that circRNA, unlike dsRNA, does not induce pattern-triggered immunity (PTI) in plants. Findings of this proof-of-principle study together are crucial first steps in understanding the potential of circRNAs as versatile tools for modulating gene expression and offer exciting prospects for their application in agronomy, particularly for enhancing crop traits through metabolic pathway manipulation. HighlightsWe demonstrate the potential of non-immunogenic circRNA as a tool for targeted gene regulation in plants, where circRNA acts in an isoform- and sequence-specific manner, paving the way for future agronomic applications.

plant biology↗

bioRxiv2023

Intercalation of the disulfide bond between the A2 and A4 loop of Cellobiohydrolase (Cel7A) of Aspergillus fumigatus enhances catalytic activity and thermostability

Disulfide bond is important for maintaining the structural conformation and stability of the protein. Introducing new disulfide bond is a promising strategy for rational protein design. In this report, disulfide bond engineering has been applied to improve the stability of an industrially important enzyme, Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of A fumigatus origin. Disulfide by Design 2.0 (DbD2), an online tool, was used for the detection of the mutation sites and created four mutations (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) both inside and outside of the peripheral loops but, not in the catalytic region. The disulfide bond formed between the A2 and A4 loop of DSB3 showed higher thermostability (70% activity at 700C), higher substrate affinity (Km= 0.081mM) and higher catalytic activity (Kcat =9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild type AfCel7A (50% activity at 700C; Km= 0.128mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min -1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3:13.6 [A]; Wt: 5.3 [A]) at the product exit site; giving flexibility in the entrance region and mobility of the substrate. It may facilitate substrate entry into the catalytic tunnel and releases the product faster than the wild type. Whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop. ImportanceCellulases are important for their role in the production of bioethanol, the cleanest renewable replacement of fossil fuels. Engineering of the cellulases is a chalange to increase their catalytic activity and thermostability for production of cheap ethanol. In this report we have introduced disulfide bond and successfully increased the both thermostabilty and catalytic activity of AfCel7A.

biochemistry↗

bioRxiv2023

Tandem mobilization of anti-phage defenses alongside SCCmec cassettes

Bacterial viruses (phages) and the immune systems targeted against them significantly impact bacterial survival, evolution, and the emergence of pathogenic strains. While recent research has made spectacular strides towards discovering and validating new defenses in a few model organisms1-3, the inventory of immune systems in clinically-relevant bacteria remains under-explored, and little is known about the mechanisms by which these systems horizontally spread. Such pathways not only impact the evolutionary trajectory of bacterial pathogens, but also threaten to undermine the effectiveness of phage-based therapeutics. Here, we investigate the battery of defenses in staphylococci, opportunistic pathogens that constitute leading causes of antibiotic-resistant infections. We show that these organisms harbor a variety of anti-phage defenses encoded within/near the infamous SCC (staphylococcal cassette chromosome) mec cassettes, mobile genomic islands that confer methicillin resistance. Importantly, we demonstrate that SCCmec-encoded recombinases mobilize not only SCCmec, but also tandem cassettes enriched with diverse defenses. Further, we show that phage infection potentiates cassette mobilization. Taken together, our findings reveal that beyond spreading antibiotic resistance, SCCmec cassettes play a central role in disseminating anti-phage defenses. This work underscores the urgent need for developing adjunctive treatments that target this pathway to save the burgeoning phage therapeutics from suffering the same fate as conventional antibiotics.

microbiology↗