The rapid emergence of drug resistance makes malaria elimination a global challenge despite the prevalence of artemisinin-based combination therapies (ACTs), thus highlighting the urgent need for the development of new antimalarials with novel modes of action. The present study aimed to develop new quinazoline hybrid antimalarials using bioactive small building blocks. The antimalarial activity results revealed that most molecular hybrids have IC50 values below 10 µM for the drug-sensitive Pf3D7 strain. The study identified molecular hybrids 19, N-(2-chloro-4-((4-(4-(((tetrahydrofuran-2-yl)methyl)amino)quinazolin-2-yl)piperazin-1-yl)sulfonyl)phenyl)acetamide and 27, 2-(4-((2-nitrophenyl)sulfonyl)piperazin-1-yl)-4-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)quinazoline as potent antimalarials with an IC50 value of 3.4 µM and 2.9 µM against Pf3D7, respectively. The cytotoxicity investigation against mammalian A549 cells and activated macrophages derived from THP1 monocytes revealed that the compounds were relatively non-cytotoxic, and their antimalarial activity was not associated with cytotoxicity. In silico studies were conducted to predict plausible drug targets of the compounds, and the results suggested that the antimalarial activity of the compounds may be due to the inhibition of zinc metalloprotease PfFLN, with concurrent inhibition of cysteine proteases PfFP2 and PfFP3. The MM-GBSA analysis revealed that the binding free energies of 19 and 27 with PfFLN were -50.3223 and -51.5066 kcal mol-1, respectively. The predicted ADME properties of the compounds fall within the Schrödinger range, which encompasses 95% of all known medications. The study thus emphasised the significance of the molecular hybridisation approach and highlighted compounds 19 and 27 as potent hit molecules that could be further optimised for the development of new antimalarials.

The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G+ granulocytes (Ly6G+Gra), IL-17, and COX2. We show that in the lungs of Mtb-infected wild-type mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G+Gra-resident 4 weeks post-infection onwards. In the genetically susceptible ifng-/- mice, excessive Ly6G+Gra infiltration correlates with severe bacteremia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G+Gra infiltration, associated pathology, and death in ifng-/- mice. Surprisingly, Ly6G+Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or ifng-/- mice. The IL-17-COX2-Ly6G+Gra interplay also operates in WT mice. Inhibiting RORγt, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G+Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCG’s protective efficacy, mainly by targeting the Ly6G+Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by RORγt inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G+Gra axis for TB prevention and therapy.

Natural killer (NK) cells contribute to early immunity against Plasmodium falciparum by recognizing and eliminating infected red blood cells (iRBCs), a process mediated in part by the integrin LFA-1. However, the cognate parasite ligand for LFA-1 has remained unknown. Here, we identify glycophorin binding protein-130 (PfGBP-130) as a surface-expressed ligand on iRBCs that binds the I-domain of LFA-1 (LFA-1 αI). Using an LFA-1 αI-Fc fusion protein, we demonstrate stage-specific binding to iRBCs, and LC-MS/MS analysis of immunoprecipitates of αI-Fc bound to iRBC revealed PfGBP-130 as a high-confidence interactor. Recombinant PfGBP-130 binds NK and THP-1 cells in an LFA-1-dependent manner. Co-culture assays show that PfGBP-130 promotes NK cell activation and degranulation and facilitates contact-dependent killing of iRBCs. Neutralizing antibodies against PfGBP-130 significantly impair these responses. Our findings establish PfGBP-130 as the LFA-1 ligand on iRBCs, providing new insight into NK cell-mediated immunity in malaria and identifying a potential target for host-directed interventions.

Macrophages are critical to maintaining intestinal homeostasis and contribute to localized inflammation once dysregulated. Here, we aimed at understanding innate macrophage dysfunction in subjects with chronic idiopathic gut inflammation Crohn’s disease (CD) and chronic infection-related gut inflammation, intestinal tuberculosis (ITB). The study population involved subjects aged 18 years or above, including males and females, who met the inclusion/exclusion criteria (CD: n = 42; ITB: n = 41). RNA-seq analysis of ex vivo Mtb-infected or uninfected monocyte-derived macrophages (MDMs) from CD, ITB and healthy controls was done. The MDMs from a cohort of patients, including CD (n = 42) and ITB (n = 41), were characterized for innate immune responses such as autophagy (CD, n = 15: ITB, n = 09), mitochondrial depolarization (CD, n = 08: ITB, n = 08) and bactericidal capacity (CD, n = 25: ITB, n = 28). Finally, the role of AXL downregulation in impaired autophagy was tested. The MDMs from CD and ITB subjects showed differential regulation of autophagy-associated genes. Functionally, CD MDMs showed significant autophagy impairment compared to ITB MDMs (average MFI-201 in CD vs. 414 in ITB). Both CD and ITB MDMs were permissive to Mtb uptake; however, CD MDMs were more permissive (44

Phenotypically drug-tolerant Mycobacterium tuberculosis ( Mtb ) subpopulations within macrophages delay bacterial clearance, contributing to prolonged therapy and treatment failure. Here, we identify phagosomal acidification as a metabolic control point linking host lipid metabolism, bacterial redox homeostasis, and antibiotic tolerance. Acidic phagosomes promote lipid droplet (LD) biogenesis in macrophages, increasing lipid availability to intracellular Mtb . Access to host lipids enables Mtb to maintain a reductive cytoplasmic redox state that supports drug tolerance. Chloroquine (CQ)-mediated phagosomal alkalinization disrupted this pH–LD axis, reducing LD accumulation, bacterial lipid access, and redox-associated drug tolerance in both H37Rv and the multidrug-resistant clinical isolate NHN1664. Transcriptomic profiling of intraphagosomal Mtb identified the Fe–S cluster transcription factor WhiB6 as a candidate regulator linking phagosomal pH, host lipid availability, and reductive stress. In C3HeB/FeJ mice infected with NHN1664, CQ monotherapy attenuated lung fibrosis and improved pulmonary function without affecting bacterial burden. In this model, where necrotic and fibrotic lesions limit anti-tuberculosis drug efficacy, moxifloxacin (MXF) alone had little effect on bacterial burden, whereas CQ–MXF combination therapy significantly improved bacterial clearance. Collectively, these findings identify phagosomal acidification as a central regulator of lipid-driven redox adaptation and establish CQ as a promising host-directed adjunct to improve tuberculosis chemotherapy.

Long-term survival in breast cancer is often limited by metastatic recurrence arising from disseminated cancer cells that persist in a dormant state. The mechanisms that enable these dormant cells to survive and subsequently reawaken remain incompletely understood. Here an unbiased genome-scale genetic screen identified Med4 as a cancer cell-intrinsic gatekeeper in metastatic reactivation. Correspondingly, MED4 haploinsufficiency was found to be prevalent in metastatic breast cancer and associated with poorer clinical outcomes. Syngeneic mouse metastasis models revealed that MED4 enforces metastatic dormancy. Mechanistically, and unexpectedly given the canonical role of the Mediator complex in transcriptional activation, MED4 suppresses enhancer priming (H3K4me1) and activation (H3K27ac). Loss of a single Med4 allele disrupts enhancer poise, leading to extracellular matrix remodelling and integrin-mediated mechanotransduction programmes that ultimately drive metastatic outgrowth. Together, these findings establish MED4 as a key regulator of breast cancer cell dormancy and nominate MED4 haploinsufficiency as a potential predictive biomarker for patients at high risk of metastatic relapse.

Mycobacterium tuberculosis (Mtb) infection of the lungs, besides producing prolonged cough with mucus, also causes progressive fatigue and cachexia with debilitating loss of muscle mass. While anti-tuberculosis (TB) drug therapy is directed toward eliminating bacilli, the treatment regimen ignores the systemic pathogenic derailments that probably dictate TB-associated mortality and morbidity. Presently, it is not understood whether Mtb spreads to metabolic organs and brings about these impairments. Here, we show that Mtb creates a replication-conducive milieu of lipid droplets in hepatocytes by upregulating transcription factor PPARγ and scavenging lipids from the host cells. In hepatocytes, Mtb shields itself against the common anti-TB drugs by inducing drug-metabolizing enzymes. Infection of the hepatocytes in the in vivo aerosol mice model can be consistently observed post-week, 4 along with enhanced expression of PPARγ and drug-metabolizing enzymes. Moreover, histopathological analysis indeed shows the presence of Mtb in hepatocytes along with granuloma-like structures in human biopsied liver sections. Hepatotropism of Mtb during the chronic infectious cycle results in immuno-metabolic dysregulation that could magnify local and systemic pathogenicity, altering clinical presentations.

Oral cancer (OC) is a malignant tumour with high morbidity and mortality. Significant contributory factors include alcohol and tobacco abuse that dysregulate the proteome and metabolome. We assessed saliva as a noninvasive bio-sample to understand the changes in proteome and metabolome in OC, tobacco abusers (TA), and controls. OC, TA, and control samples (n = 22, 21, and 21, respectively) were subjected to LFQ-proteomics and NMR-based metabolomics analyses individually and integrated using systems biology; 292 out of 758 proteins with two or more unique peptides were significantly differently regulated. Functional annotation revealed that differentially expressed proteins are involved in important cellular metabolic processes. PLS-DA in metabolomics separated OC from the control and TA, and K-means clustering of proteomics and metabolomics profiles revealed distinguishing proteins and metabolites in OC, TA, and the control. Integrated analysis revealed convergence on molecules like transketolase (TKTT), transaldolase (TALDO), kallikrein 1 (KLK1), enolase A (ENOA), glucose-6-phosphate isomerase (G6PI), and aldolase A and C (ALDOA and ALDOC). Finally, the characteristic discriminatory features of several clusters between OC, TA, and the control remain valid only among high tobacco abusers. The results reveal metabolites that could serve as early indicators for OC, especially among chewing tobacco abusers, and therefore establish the basis for larger cohort studies to develop them as predictive OC biomarkers.

Transcriptional adaptation drives the host responses to Mycobacterium tuberculosis (Mtb) infection. However, Mtb alters host RNA splicing to quench host antibacterial responses, the mechanism for which remains unknown. Here, we report a mechanism whereby a secreted Mtb protein interferes with the biogenesis of key spliceosomal components. A high-throughput yeast-2-hybrid screen identified several Mtb-secreted proteins interacting with the host RNA splicing factors (SFs). Through custom-designed in-cell assays, we show that one of those proteins, Rv1435c/hsr1 (host splicing regulator 1), targets specific exon-skipping events. The Mtb Rv14345c/hsr1 facilitates direct interaction between Mtb phagosomes and U5 snRNA and SNRPF, key components of the snRNPs. Genetic deletion of Rv1435c/hsr1 reverses the specific exon-skipping events caused by WT Mtb infection. The Δhsr1 strain shows compromised growth during ex vivo infection in macrophages and in vivo infection in mice. Tissue sections from the WT Mtb or Δhsr1-infected mice showed significant hsr1-dependent SNRPF staining, a phenomenon also noted in the human intestinal tuberculosis (ITB) biopsies. Thus, hsr1 is a virulence factor that disrupts host snRNP biogenesis for pathogenesis. The splicing regulators from the host and pathogen are novel targets for antituberculosis therapy.

Developing an effective vaccine against malaria remains a prime goal of human health. The circumsporozoite protein (CSP) is a major surface protein of the sporozoites, and is the target of two licensed Plasmodium falciparum malaria vaccines, RTS,S/AS01, named Mosquirix and R21/Matrix-M. However, to improve the standards set by these vaccines we require a second-generation or prophylactic vaccine. Recently, monoclonal antibodies have emerged as essential biopharmaceutical prophylactic vaccines. The present study targeted recombinant Plasmodium falciparum CSP (rPfCSP) with a phage display of human single-fold scFv Tomlinson libraries I+J and picked fourteen scFvs that represented two independent clones after sequencing; CL1 and CL3. These phages were analysed for their binding to rPfCSP. The selected scFvs were cloned in a human IgG1 Fc tag vector, to generate scFv-Fc full-length antibody clones. CL1 bound rPfCSP protein with a KD of 3.8x10-6M and CL3 bound rPfCSP protein with a KD of 5.6 x 10-5 M. These antibodies detected native PfCSP on the sporozoite surface. Molecular docking simulation revealed that rPfCSP residues interacting with CL1 and CL3 were downstream of the repeat region. These antibodies inhibited the sporozoite infectivity into HepG2 cells, similar to a gold standard monoclonal antibody, 2A10. Low-dose passive transfer of the CL1 and CL3 antibodies conferred high-level protection when challenged with PfCSP-Pb transgenic parasites in the mouse infection model. The high in vitro and in vivo efficacies of the CL1 and CL3 antibodies have applications in malaria immunoprophylaxis in protecting travellers and military servicemen or as a therapeutic vaccine in malaria elimination programmes.

In recent years, nanotechnology has significantly impacted various technological domains, especially drug delivery systems. As modern drug delivery techniques evolve, Solid Lipid Nanoparticles (SLNs) have emerged as a promising platform in the fields of biotechnology, hiomedical engineering, and nanomedicine for their potential in healthcare and diagnostics. SLNs address critical challenges in drug formulation, particularly the poor solubility and bioavailability of many newly developed drugs.Lipid-based nanoparticles have shown great promise in overcoming these limitations. Compared to other colloidal carriers, lipid nanoparticles are biocompatible. Biodegradable, and mainly composed of components generally recognized as safe (GRAS). Since their emergence in the early 1990s, SLNs have demonstrated their capability to surpass the shortcomings of traditional drug delivery systems. Many effective pharmaceutical formulations fail in clinical practice due to low absorption, high metabolism, wide systemic distribution, and poor bioavailability especially in the case of BCS Class II and IV drugs, peptides, and proteins. Instead of developing new drugs from scratch, repurposing existing drugsusing carriers like SLNs proves to be more cost-effective and efficient. SLNs consist of solid lipids such as triglycerides, waxes, and glyceride blends that remain solid at room and body temperature. They also contain surfactants and co-surfactants (0.5%-5%) for stabilization. The unique nanoscale size of SLNs (typically 50-200 nm) allows for reduced toxicity, sustained release, and protection against enzymatic degradation, Furthermore, their surfaces can be functionalized with ligands or polymers for targeted delivery, and both hydrophilic and lipophilic drugs can be efficiently incorporated into their matrices.Due to these attributes, SLNs offer a powerful and flexible nanocarrier system with applications in various therapeutic areas, especially for drugs with poor water solubility

The search for new anti-tubercular agents is vital for the fight against Mycobacterium tuberculosis, particularly given the rise of drug-resistant strains. DRILS-1398, a pyrazolo[4,3-d]pyrimidine derivative, was discovered as a potent inhibitor of M.tb chorismate mutase (M.tb-CM) with an IC50 = 3.0 ± 0.2 μM (n = 3) and IC90 = 10 μM. The compound demonstrated efficacy against multi-drug resistant M.tb strains (MIC = 4 μg/mL, ∼10.0 μM) and effective inhibition of intracellular M.tb in THP-1 macrophages. With favorable pharmacokinetics, moderate stability in vitro, and a promising safety profile, DRILS-1398 showed no toxicity at doses up to 500 mg/kg b.w./day when dosed orally daily once for 7 consecutive days in mice. Both DRILS-1398 and its formulation DRILS-1398(F) were successful in clearing M.tb infection from the lungs and spleen in murine models. These findings suggest DRILS-1398 as a promising lead candidate for developing a first-in-class anti-tubercular drug.

Metastasis in cancer is influenced by epigenetic factors. Using an in vivo screen, we demonstrate that several subunits of the polybromo-associated BAF (PBAF) chromatin remodeling complex, particularly Brd7, are required for maintaining breast cancer metastatic dormancy in the lungs of female mice. Brd7 loss induces metastatic reawakening, along with modifications in epigenomic landscapes and upregulated oncogenic signaling. Breast cancer cells harboring Brd7 inactivation also reprogram the surrounding immune microenvironment by downregulating MHC-1 expression and promoting a pro-metastatic cytokine profile. Flow cytometric and single-cell analyses reveal increased levels of pro-tumorigenic inflammatory and transitional neutrophils, CD8+ exhausted T cells, and CD4+ stress response T cells in lungs from female mice harboring Brd7-deficient metastases. Finally, attenuating this immunosuppressive milieu by neutrophil depletion, neutrophil extracellular trap (NET) inhibition, or immune checkpoint therapy abrogates metastatic outgrowth. These findings implicate Brd7 and PBAF in triggering metastatic outgrowth in cancer, pointing to targetable underlying mechanisms involving specific immune cell compartments. Metastasis-initiating cells can reawaken from a dormant state that initially allowed them to survive, triggering metastatic outgrowth. Here, authors show that loss of Brd7 promotes an immunosuppressive tumor microenvironment that drives breast cancer metastatic reawakening from dormancy in the lung.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is responsible for extreme mortality and morbidity across the globe. The bacteria have evolved multiple strategies for their successful prevalence. The emergence of multidrug-resistant TB (MDR-TB) has established the importance of eliciting host-pathogen interactions at cellular and molecular levels. Various pattern recognition receptors play determinant roles when encountering M. tb infection. Here, we investigated the regulation of key defense responses from Dectin-1 and Mincle during mycobacterial infection in THP-1-derived macrophages, the long-term hosts for mycobacteria. Our data indicate that infection of THP-1 macrophages with either M. bovis Bacillus Calmette-Guérin (BCG) or M. tb H37Rv increases the surface expression of Dectin-1 and Mincle. This increase translated directly to increased intracellular bacterial survival within macrophages. Likewise, M. bovis BCG infection of human peripheral blood mononuclear cell-derived macrophages also led to an increased expression of Dectin-1 and Mincle. Stimulation of Dectin-1 or Mincle along with BCG infection induces suppressor responses such as an attenuated oxidative burst and mitochondrial membrane potential intactness. In addition, decreased apoptosis and autophagy induction was also observed following stimulation of Dectin-1 and Mincle. Conversely, RNA interference-mediated knockdown of Dectin-1 or Mincle reversed the previous responses, resulting in higher oxidative burst, mitochondrial membrane potential disruption, increased mitochondrial reactive oxygen species production, and increased apoptosis. This results in a significant decrease in intracellular mycobacterial survival. These results point toward a well-orchestrated strategy of fine-tuning the host's defense machinery of Dectin-1 and Mincle adopted by mycobacteria to suppress protective responses mounted against it and prepare the macrophages for prolonged persistent infection.

DNA repair pathways play an essential role in maintaining the genomic integrity of bacteria, and a perturbation in their biological activity helps bacteria survive under duress. In drug-resistant clinical strains, we identified a Q135K mutation in the uvrA gene, a DNA repair pathway gene. To delineate the role of uvrA and the Q135K mutation, we generated the gene replacement mutant of UvrA (RvΔuvrA) in Mycobacterium tuberculosis H37Rv (Mtb-Rv). While the lack of UvrA function in RvΔuvrA could be restored upon complementation with uvrA, the uvrA-Q135K mutant identified in clinical drug-resistant strains failed to do so. This was reflected in higher mutation rates in RvΔuvrA and RvΔuvrA::uvrAQ135A, compared with wild-type Rv or RvΔuvrA::uvrA complemented strains in the presence and absence of oxidative stress. Killing kinetics experiments with anti-TB drugs showed increased survival of RvΔuvrA and RvΔuvrA::uvrAQ135K, strains compared with Rv or RvΔuvrA::uvrA. Importantly, RvΔuvrA and RvΔuvrA::uvrAQ135K showed enhanced survival in peritoneal macrophages and murine infection model of infection. Together, data suggests that acquiring Q135K mutation benefits the pathogen, which helps enhance the host's survival adaptability. ### Competing Interest Statement The authors have declared no competing interest.

Abstract The limited availability of molecularly targeted low‐molecular‐weight imaging agents for monitoring multiple myeloma (MM)‐targeted therapies has been a significant challenge in the field. In response, a first‐in‐class peptide‐based radiotracer, [68Ga]Ga‐AJ206, is developed that can be seamlessly integrated into the standard clinical workflow and is specifically designed to noninvasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). A bicyclic peptide, AJ206, is synthesized and exhibits high affinity to CD38 (KD: 19.1 ± 0.99 × 10−9 m) by surface plasmon resonance. Further, [68Ga]Ga‐AJ206‐PET shows high contrast within 60 min and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga‐AJ206 detects CD38 expression in cell line‐derived xenografts, patient‐derived xenografts (PDXs), and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga‐AJ206‐PET successfully quantifies CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following all‐trans retinoic acid (ATRA) therapy. In conclusion, [68Ga]Ga‐AJ206 exhibits the salient features required for clinical translation, providing CD38‐specific high‐contrast images in multiple models of MM. [68Ga]Ga‐AJ206‐PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement.