The escalating crisis of multidrug-resistant bacteria demands a new generation of antibiotics. Glycocin F (GccF), a potent bacteriocin, is a promising candidate, but its function hinges on unique post-translational glycosylation. Intriguingly, a seemingly minor chemical tweak of α-methylation at Ser18 of GccF destroys its activity, reducing its potency by 1000-fold. To quantify how this subtle chemical change leads to profound functional compromise, we used an advanced molecular dynamics framework guided by Variational Autoencoder to unravel GccF's complex dynamics. Our findings reveal that native glycosylation preserves conformational plasticity to maintain functionally relevant conformations. In stark contrast, α-methylation introduces local rigidity, locking the peptide into fewer metastable basins with significantly slower transition rates. This leads to the disruptions of the α-helix structure, which perturbs the loop-helix coupling and traps the peptide into nonfunctional conformations. Together, these findings demonstrate how a subtle modification can dictate a peptide's function by profoundly altering its structural dynamics.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). Emergence of drug resistance in Mtb requires continuous enrichment of anti-tubercular medication. Inclusion of host-directed therapies holds considerable promise in this context. Sorafenib (SRB) is a multi-kinase inhibitor targeting VEGF receptor kinase, Raf, MEK, and extracellular signal-regulated kinase (ERK) signaling cascade to treat several types of cancer, including hepatocellular carcinoma. We have previously established that SRB allosterically inhibits ornithine acetyltransferase (MtArgJ), an essential enzyme in the arginine biosynthesis pathway of Mtb, thereby limiting bacterial growth in culture at a minimum inhibitory concentration of 10 µg/mL. The current work focuses on how SRB at the dose of 30 mg/kg body wt inhibits the pathogenicity and survival of bacteria in a preclinical mouse model of tuberculosis by inducing pro-apoptotic and immunomodulatory mechanisms in the host. We observed that SRB treatment promotes apoptosis in Mtb-infected and -uninfected THP-1 cells, human monocyte-derived macrophages. Concomitantly, SRB treatment reduces infection-associated necrosis in the Mtb-infected THP-1 cells. We further noted the upregulated expression of pro-apoptotic proteins during SRB treatment in preclinical mouse models. In addition, we investigated the expression of pro- and anti-inflammatory cytokines and immunomodulation in lung tissues treated with SRB. Interestingly, SRB treatment increased the number of arginase 1-positive macrophages, which are reckoned to enhance tissue healing. In conclusion, our research discloses that SRB is helpful in both lowering the tubercular burden and accelerating recovery of damaged tissue by harnessing the host immune response.IMPORTANCEHost-directed therapies hold considerable promise for treating drug-resistant Mycobacterium tuberculosis (Mtb). In this context, the induction of apoptotic and immunomodulatory responses in the host by sorafenib (SRB) is demonstrated here to compromise the survival and pathogenic potential of Mtb in a preclinical mouse model of TB and in Mtb-infected and -uninfected THP-1 cells. Concurrently, the infection-associated necrosis in the Mtb-infected THP-1 cells is also reduced. Furthermore, arginase 1-positive macrophages, which are known to enhance tissue healing, are increased in SRB-treated groups. Thus, SRB treatment not only lowers the tubercular load but also aids in healing damaged tissues by leveraging the host immunity.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by overexpression of amyloid-beta in the brain, particularly the toxic Abeta-42 form. Recent studies have identified osteocalcin, a peptide traditionally associated with bone, to modulate cognitive function in the AD brain. Osteocalcin exists in two forms: the undercarboxylated (uOC) and carboxylated (cOC) forms. This study investigates the role of uOC in modulating Abeta42 aggregation and its potential therapeutic implications for AD. Administration of uOC, but not cOC, improved both spatial learning and exploratory behavior of 5xFAD transgenic Alzheimer mice. Further investigation showed that uOC reduced the level of insoluble Abeta42 in the brain and increased the level of soluble Abeta42. There was increase in mRNA levels of CD36 in uOC treated 5xFAD transgenic brain alongside upregulation of neuroprotectants like Adipoq and Ahsg (fetuin). We explored the mechanisms underlying the influence of uOC on Aβ42 dynamics and understood that uOC interacts with Glu residues to form non-toxic early tube-like intermediates (A-O) before advancing to late mature Abeta42 fibrils. These intermediates enable Abeta42 uptake by glial cells by upregulating the cell surface expression of CD36 and reducing TNF-alpha production. Collectively, the study sheds light on the fact that uOC modulates Abeta42 dynamics and this interaction is warranted for Abeta42 uptake and clearance. The study provides a novel dimension for the treatment of amyloid disorders like AD.

Lectins are carbohydrate-binding proteins that have enormous therapeutic value because of their potent antiviral activity. However, the design of lectins for targeted intervention is marred by our poor understanding of protein-glycan recognition. Here, we focus on the mannose-specific lectin horcolin, which is nonmitogenic and shows dose-dependent inhibition of HIV infection. Saturation transfer and relaxation dispersion NMR experiments reveal that the lectin-glycan interface is conformationally frustrated, resulting in the formation of a minor state with a millisecond time-scale lifetime. There is a rearrangement of the quaternary structure of horcolin in this minor state that manifests as a noncanonical tetramer. The glycan-binding site is sequestered at the tetrameric interface, suggesting that the tetramer could serve as an autoinhibitory conformation. However, glycan recognition itself occurs via the major dimeric conformation through a "ground-state conformational selection" mechanism. We also demonstrate that the tetramer is destabilized by mannose and that conformational frustration is alleviated in the lectin-glycan complex. Our work illustrates how the architecture of biomolecular assemblies is molded in response to conflicting evolutionary signals such as folding and recognition. The work also provides insights into protein-glycan recognition that could have potential implications for deploying lectins as antiviral agents.

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis (Mtb). It is one of the major global public health problems that leads to a high morbidity and mortality rate. Drug resistance in Mycobacterium tuberculosis (Mtb) is another significant and persistent public health concern. The development of effective TB vaccines and treatments requires a better understanding of the intricate interactions between M. tuberculosis and host immunity. We previously reported that sorafenib (SRB) reduces bacterial growth by allosterically inhibiting ornithine acetyltransferase (MtArgJ), an essential enzyme in the arginine biosynthesis pathway of Mtb. Here, we report on the antimicrobial activity of sorafenib in preclinical mouse models of tuberculosis. Sorafenib is a potent drug approved by the Food and Drug Administration (FDA) for treating several types of cancer. The current study is focused on the immunomodulation that SRB induces in the host, specifically the immunological response that is triggered to combat the pathogenicity and survival of the bacteria.Here, we show that SRB significantly sterilizes the bacterial burden in chronic infection animal models of tuberculosis by reducing the number of Mtb-susceptible alveolar macrophages (AMs), and that SRB is more effective when combined with rifampicin (RIF). In the current study, we documented a new immune modulatory characteristic of sorafenib that, upon SRB treatment, markedly increased effector T cells (Teff - CD4+CD25low and CD8+CD25low) activity and decreased regulatory T cells, the immunosuppressive T cells (Treg- CD4+CD25high and CD8+CD25high) function. In conclusion, our studies revealed that SRB is beneficial for both boosting an efficient T cell response and lowering the tubercular load.

The Cus17 phloem protein, in the case of Cucumis sativus species, plays an important role in the phloem-based defense of the plant. Cus17 can bind to various carbohydrates present on insect exoskeletons or fungal cells. The recent experimental structure of chitotriose bound Cus17 elucidates the carbohydrate interacting residues of Cus17. Higher chito-oligosaccharides are also known to interact with Cus17, but the lack of experimental structure impedes our understanding of their interaction. In this study, we have employed in-silico methods to explore the binding interactions of higher chito-oligosaccharides with Cus17. Chitoheptaose forms stable interactions with canonical binding site residues Trp48 and Asp50. Smaller chito-oligosaccharides were observed to be relatively unstable at the canonical binding site of Cus17. Further, the chito-oligosaccharides were inspected for interactions with predicted ligand binding sites. We also generated different tetramers of Cus17 and docked the chito-oligosaccharides to the tetrameric Cus17. All chito-oligosaccharides were found to make persistent interactions with Cus17 tetramer. Interestingly, chitotriose shows the best binding affinity and maintains stable interactions with Cus17 tetramer upon extended simulations with the canonical site.

The escalating crisis of multi-drug resistant bacteria demand a new generation of antibiotics. Glycocin F (GccF), a potent bacteriocin, is a promising candidate, but its function hinges on unique post-translational glycosylation. Intriguingly, a seemingly minor chemical tweak of α-methylation at Ser18 of GccF destroys its activity, reducing potency by 1000-fold. To quantify how this subtle chemical change leads to profound functional compromise, we used an advanced molecular dynamics framework guided by Variational Autoencoder to unravel GccF’s complex dynamics. Our findings reveal that native glycosylation preserves conformational plasticity to maintain functionally relevant conformations. In stark contrast, α-methylation introduces local rigidity, locking the peptide into fewer metastable basins with significantly slower transition rates. This leads to the disruptions of α-helix structure which perturbs the loop-helix coupling and trapping the peptide into non-functional conformations. Together, these findings provide a critical blueprint for the rational design of next generation antibiotics, demonstrating how precise chemical modifications can dictate a peptide’s function by profoundly altering its structural dynamics. ### Competing Interest Statement The authors have declared no competing interest. Department of Atomic Energy, Government of India

Synthetic chitobiose-containing glycolipid (GL) and lipid (L) are prepared in order to secure self-assembled multivalent glycostructures, constituted with varying molar fractions of GL and L. The morphologies of glycostructures are uniform, as adjudged by dynamic light scattering (DLS) in solution and microscopies in the solid state. Presence of the ester linkage between the lipid and chitobiose moieties permit hydrolysis and disassembly of the self-assembled structures at acidic and alkaline pH. The avidity of chitobiose in the multivalent glycostructures to lysozyme follows the percentage of GL in the GL-L compositions in the order 50 % GL > 100 % GL-L > 10 % GL-L. The interaction with lysozyme occurs with fast association and slow dissociation kinetics, from which the equilibrium binding constant (Ka) is identified to be 2-4 orders of magnitude higher (Ka 105 to 107 M-1), as compared to monomeric chitobiose-lysozyme complexation in solution. When assessed for the antimicrobial lytic property of lysozyme, the multivalent chitobiose-lysozyme complex is found to delay the lytic property, when compared to the enzyme alone. The study establishes (i) the pH-sensitive multivalent chitobiose-containing glycostructures for high affinity binding to lysozyme; (ii) that the multivalent ligand presentation enables orders of magnitude higher equilibrium binding constants with enzyme lysozyme and (iii) that the lytic activity of the enzyme is delayed upon complexation with the multivalent glycostructures.

It is essential to understand the interactions and relationships between Mycobacterium tuberculosis (Mtb) and macrophages during the infection in order to design host-directed, immunomodulation-dependent therapeutics to control Mtb. We had reported previously that ornithine acetyltransferase (MtArgJ), a crucial enzyme of the arginine biosynthesis pathway of Mtb, is allosterically inhibited by pranlukast (PRK), which significantly reduces bacterial growth. The present investigation is centered on the immunomodulation in the host by PRK particularly the activation of the host’s immune response to counteract bacterial survival and pathogenicity. Here, we show that PRK decreased the bacterial burden in the lungs by upregulating the population of pro-inflammatory interstitial macrophages (IMs) and reducing the population of Mtb susceptible alveolar macrophages (AMs), dendritic cells (DCs), and monocytes (MO). Additionally, we deduce that PRK causes the host macrophages to change their metabolic pathway from fatty acid metabolism to glycolytic metabolism around the log phage of bacterial multiplication. Further, we report that PRK reduced tissue injury by downregulating the Ly6C-positive population of monocytes. Interestingly, PRK treatment improved tissue repair and inflammation resolution by increasing the populations of arginase 1 (Arg-1) and Ym1+Ym2 (chitinase 3-like 3) positive macrophages. In summary, our study found that PRK is useful not only for reducing the tubercular burden but also for promoting the healing of the diseased tissue.

FadD32, a fatty acyl-AMP ligase, plays an indispensable role in mycobacterial mycolic acid synthesis and is a validated target for tuberculosis (TB) drug development. The crystal structure of Mycobacterium tuberculosis (Mtb)FadD32 has laid the foundation of structure-based drug discovery against this crucial enzyme. Here, we screened the “isoxazole” scaffold containing molecules against MtbFadD32 and identified a compound 2,4-dibromo-6-[3-(trifluoromethyl)-1,2-oxazol-5-yl]phenol (M1) with specific inhibitory activity against Mtb. Kinetics experiments showed that M1 inhibits MtbFadD32 and MtbFadD28 activity. The transcriptomics response of Mtb disclosed M1-mediated regulation of mycobacterial decisive genes involved in cell wall synthesis, consequently creating unfavorable conditions for Mtb survival. Further, M1 curtails the Mtb survival in infected macrophages and reduces Mtb burden and tubercular granulomas in a chronic infection model of BALB/c mice. Our findings provide an effective chemical scaffold to inhibit MtbFadD32 with the potential to inhibit multiple MtbFadD family of enzymes for further development as a promising candidate for treating TB.

TRIM proteins are characterized by their conserved N-terminal RING, B-box, and coiled-coil domains. These proteins are efficient regulators of autophagy, apoptosis, and innate immune responses and confer immunity against viruses and bacteria. TRIMs function as receptors or scaffold proteins that target substrates for autophagy-mediated degradation. Most TRIMs interact with the BECN1-ULK1 complex to form TRIMosomes, thereby efficiently targeting substrates to autophagosomes. They regulate the functions of ATG proteins through physical interactions or ubiquitination. TRIMs affect the lipidation of MAP1LC3B1 to form MAP1LC3B2, which is a prerequisite for phagophore and autophagosome formation. In addition, they regulate MTOR kinase and TFEB, thereby regulating the expression of ATG genes. TRIM proteins are efficient regulators of apoptosis and are crucial for regulating cell proliferation and tumor formation. Many TRIM proteins regulate intrinsic and extrinsic apoptosis via the cell surface receptors TGFBR2, TNFRSF1A, and FAS. Mitochondria modulate the anti- and proapoptotic functions of BCL2, BAX, BAK1, and CYCS. These proteins use a multipronged approach to regulate the intrinsic and extrinsic apoptotic pathways, culminating in coordinated activation or inhibition of the initiator and executor CASPs. Furthermore, TRIMs can have a dual effect in determining cell fate and are therefore crucial for cellular homeostasis. In this review, we discuss mechanistic insights into the role of TRIM proteins in regulating autophagy and apoptosis, which can be used to better understand cellular physiology. These findings can be used to develop therapeutic interventions to prevent or treat multiple genetic and infectious diseases.

Neuropathy occurs due to damage to the peripheral/central nervous system either due to injury, disease, or drug usage. Increased endoplasmic reticulum (ER) stress is observed in neuropathy. ER stress also leads to a block in autophagy amplifying neuropathic pain. 6-Bromoindirubin-3'-oxime (6-BIO) is an inhibitor of GSK-3β which suppresses mTOR activity thereby increasing autophagy. Tunicamycin (TM)-mediated ER stress and diabetic rat models were used to elucidate the role of ER stress and autophagy in mitigation of neuropathic pain by 6-BIO. Pain was assessed by behavioral studies in ER stressed/diabetic rats having neuropathy. Western blotting, RT-PCR, and fluorescence microscopy were used to assess the level of autophagy and ER stress after TM and 6-BIO treatment in SH-SY5Y neurons. Intraplantar injection of TM in rats led to peripheral neuropathy which was reduced upon 6-BIO injection. 6-BIO also reduced pain in animals exhibiting diabetic peripheral neuropathy. Modulation in the markers of autophagy (p-mTOR, LC-3, and SQSTM1/p62) shows that 6-BIO induces autophagolysosome formation post TM treatment. Concomitantly, 6-BIO reduces ER stress and c-Fos expression-a neuronal activity and pain marker. Alleviation of pain by the inhibition of ER stress and increased formation of autolysosomes by 6-BIO can be harnessed for treating peripheral neuropathy.

Due to the uniqueness and essentiality of MEP pathway for the synthesis of crucial metabolites- isoprenoids, hopanoids, menaquinone etc. in mycobacterium, enzymes of this pathway are considered promising anti-tubercular drug targets. In the present study we seek to understand the consequences of downregulation of three of the essential genes- DXS, IspD, and IspF of MEP pathway using CRISPRi approach combined with transcriptomics in Mycobacterium smegmatis. Conditional knock down of either DXS or IspD or IspF gene showed strong bactericidal effect and a profound change in colony morphology. Impaired MEP pathway due to downregulation of these genes increased the susceptibility to frontline anti-tubercular drugs. Further, reduced EtBr accumulation in all the knock down strains in the presence and absence of efflux inhibitor indicated altered cell wall topology. Subsequently, transcriptional analysis validated by qRT-PCR of +154DXS, +128IspD, +104IspF strains showed that modifying the expression of these MEP pathway enzymes affects the regulation of mycobacterial core components. Among the DEGs, expression of small and large ribosomal binding proteins (rpsL, rpsJ, rplN, rplX, rplM, rplS, etc), essential protein translocases (secE, secY and infA, infC), transcriptional regulator (CarD and SigB) and metabolic enzymes (acpP, hydA, ald and fabD) were significantly depleted causing the bactericidal effect. However, mycobacteria survived under these damaging conditions by upregulating mostly the genes needed for the repair of DNA damage (DNA polymerase IV, dinB), synthesis of essential metabolites (serB, LeuA, atpD) and those strengthening the cell wall integrity (otsA, murA, D-alanyl-D-alanine dipeptidase etc.).

Supplementary Figure 7 from Anticancer Activity of a Combination of Cisplatin and Fisetin in Embryonal Carcinoma Cells and Xenograft Tumors

Supplementary Figures 4-5 from Anticancer Activity of a Combination of Cisplatin and Fisetin in Embryonal Carcinoma Cells and Xenograft Tumors

Phloem protein 2 (PP2) contributes crucially to phloem-based defense in plants by binding to carbohydrates displayed by pathogens. However, its three-dimensional structure and the sugar binding site remained unexplored. Here, we report the crystal structure of the dimeric PP2 Cus17 from Cucumis sativus in its apo form and complexed with nitrobenzene, N-acetyllactosamine, and chitotriose. Each protomer of Cus17 consists of two antiparallel four-stranded twisted β sheets, a β hairpin, and three short helices forming a β sandwich architectural fold. This structural fold has not been previously observed in other plant lectin families. Structure analysis of the lectin-carbohydrate complexes reveals an extended carbohydrate binding site in Cus17, composed mostly of aromatic amino acids. Our studies suggest a highly conserved tertiary structure and a versatile binding site capable of recognizing motifs common to diverse glycans on plant pathogens/pests, which makes the PP2 family suited for phloem-based plant defense.

Protein-carbohydrate interactions play a crucial role in mediating several biomolecular recognition events. We attempt to unravel its intricacies by understanding how carbohydrate-binding proteins interpret the glycan code. We aim to decipher lectin-mediated recognition in the endoplasmic reticulum (ER), which plays a crucial role in ER-mediated quality control (ER-QC). The ER-QC functions in three phases-protein folding, transport, and degradation. Altered protein QC leads to ER-related storage disorders. Cargo transport proteins-Ergic53 and Vip36-necessary for maintaining cellular homeostasis-are our primary focus. They recognize monoglucosylated/high mannose N-glycans on the folded glycoproteins. This article reports on the first dynamic investigation of the ER cargo lectins in complex with the high mannose glycans using an advanced sampling technique-replica exchange molecular dynamics to decipher the inherent conformational heterogeneity and the binding mechanism. The study involves simulations for the proteins complexed with three high mannose glycans-Man8B, Man9, and mono-glucosylated glycan. The recognition process is captured using MD simulations to achieve mechanistic insights and characterize the dynamics of glycans in their native and bound states via dihedral angle analysis. Results indicate that the flipped conformation of the glycans was crucial in differentiating their interaction with the proteins. Similar conformers of the glycans are preferred for Ergic53 and Vip36 in their glycan recognition events. Ergic53 preferred Man8B while it was Man9 for Vip36, in coherence with the previous experimental reports. These simulations provide a computational microscopic purview of the mechanism at both spatial and temporal scales. The results correlate with the published experimental data on the specificities of these lectins.

The high prevalence of oral potentially-malignant disorders exhibits diverse severity and risk of malignant transformation, which mandates a Point-of-Care diagnostic tool. Low patient compliance for biopsies underscores the need for minimally-invasive diagnosis. Oral cytology, an apt method, is not clinically applicable due to a lack of definitive diagnostic criteria and subjective interpretation. The primary objective of this study was to identify and evaluate the efficacy of biomarkers for cytology-based delineation of high-risk oral lesions. A comprehensive systematic review and meta-analysis of biomarkers recognized a panel of markers (n: 10) delineating dysplastic oral lesions. In this observational cross sectional study, immunohistochemical validation (n: 131) identified a four-marker panel, CD44, Cyclin D1, SNA-1, and MAA, with the best sensitivity (>75%; AUC>0.75) in delineating benign, hyperplasia, and mild-dysplasia (Low Risk Lesions; LRL) from moderate-severe dysplasia (High Grade Dysplasia: HGD) along with cancer. Independent validation by cytology (n: 133) showed that expression of SNA-1 and CD44 significantly delineate HGD and cancer with high sensitivity (>83%). Multiplex validation in another cohort (n: 138), integrated with a machine learning model incorporating clinical parameters, further improved the sensitivity and specificity (>88%). Additionally, image automation with SNA-1 profiled data set also provided a high sensitivity (sensitivity: 86%). In the present study, cytology with a two-marker panel, detecting aberrant glycosylation and a glycoprotein, provided efficient risk stratification of oral lesions. Our study indicated that use of a two-biomarker panel (CD44/SNA-1) integrated with clinical parameters or SNA-1 with automated image analysis (Sensitivity >85%) or multiplexed two-marker panel analysis (Sensitivity: >90%) provided efficient risk stratification of oral lesions, indicating the significance of biomarker-integrated cytopathology in the development of a Point-of-care assay.