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Nuclear factor-kappa B (NF-kB) plays a crucial role in numerous cellular processes, such as inflammation, immunological responses to infection, cell division, apoptosis, and the development of embryos and neurons. Cytokines, plays an important role in positive feedback loop and leads to inflammatory cell death through the release of pathogenic cytokine known to be cytokine storm which causes diseases like Acute Respiratory Disorder (ARD), multi-organ disorder, Hyperinflammation syndrome and may cause death. This cytochrome storm was identified in the people severely affected by covid-19. NF-kB presents a promising therapeutic opportunity to mitigate covid-19-induced cytokine storm and reduce the risk of severe morbidity and mortality resulting from the diseases. This paper therefore explores the modulation of the NF-kB pathway by inhibiting the binding of the transcription factor as a potential strategy to mitigate the morbidity and mortality caused by cytokine storms. To identify small molecule inhibitors of NF-kB signaling, we screened approximately 101 molecules in two identified pockets of NF-kB (p50/p65)-DNA complex. Each molecule was virtually screened in two pockets (A1 and A2). The focus library was developed starting from chemical structures obtained from the literature (Angelicin and Psolaren) which shows the inhibition of NF-kB signaling, as well as using artificial intelligence (WADDAICA) and rationally designed molecules. Among the 3 highest-scored ligands (NFAI64, NF30 and NF49) selected from the docking studies and further molecular dynamic investigations. The identified compound NF30 showed significantly higher binding affinity (ΔG binding ) in A2 pocket (60.92 ± 1.83 kJ/mol) as compared to the rest of the molecules, making it a promising molecule for the inhibition of NF-kB. The discovered novel compounds by computational studies could be of relevance to identify more potent inhibitors of NF-kB dependent biological functions beneficial to control the cytokine storm occurring in the patients affected with Covid-19.

Proteins possessing double active sites have the potential to revolutionise enzyme design strategies. This study extensively explored an enzyme that contains both a natural active site (NAS) and an engineered active site (EAS), focusing on understanding its structural and functional properties. Metadynamics simulations were employed to investigate how substrates interacted with their respective active sites. The results revealed that both the NAS and EAS exhibited similar minimum energy states, indicating comparable binding affinities. However, it became apparent that the EAS had a weaker binding site for the substrate due to its smaller pocket and constrained conformation. Interestingly, the EAS also displayed dynamic behaviour, with the substrate observed to move outside the pocket, suggesting the possibility of substrate translocation. To gain further insights, steered molecular dynamics (SMD) simulations were conducted to study the conformational changes of the substrate and its interactions with catalytic residues. Notably, the substrate adopted distinct conformations, including near-attack conformations, in both the EAS and NAS. Nevertheless, the NAS demonstrated superior binding minima for the substrate compared to the EAS, reinforcing the observation that the engineered active site was less favourable for substrate binding due to its limitations. The QM/MM (Quantum mechanics and molecular mechanics) analyses highlight the energy disparity between NAS and EAS. Specifically, EAS exhibited elevated energy levels due to its engineered active site being located on the surface. This positioning exposes the substrate to solvents and water molecules, adding to the energy challenge. Consequently, the engineered enzyme did not provide a significant advantage in substrate binding over the single active site protein. Further, the investigation of internal channels and tunnels within the protein shed light on the pathways facilitating transport between the two active sites. By unravelling the complex dynamics and functional characteristics of this double-active site protein, this study offers valuable insights into novel strategies of enzyme engineering. These findings establish a solid foundation for future research endeavours aimed at harnessing the potential of double-active site proteins in diverse biotechnological applications.

Penicillin G Acylase (PGA) has emerged as a critical biocatalyst in pharmaceutical sciences, exceeding its traditional role in penicillin synthesis. Despite its industrial significance, challenges, including substrate specificity, stability under industrial conditions, and efficiency in immobilization, persist. Engineering enhanced enzyme variants and developing advanced immobilization techniques along with process optimization shall be possible solutions to further improve reaction efficiency and scalability. Green chemistry integration can make PGA-based processes more sustainable. Moreover, the use of computational tools, including AI-driven optimization, can guide enzyme design and reaction condition refinement. A review synthesizing these advancements not only consolidates existing knowledge but also identifies opportunities for further innovation, ensuring the enzyme's continued industrial and scientific relevance. The review discusses the structure and functionality of PGA, highlighting its diverse applications beyond penicillin production. Beyond antibiotic synthesis, PGA's usefulness extends to ester synthesis, resolving racemic mixtures and peptide bond formation, underlining its importance in various bioconversions and synthetic reactions. This adaptability is crucial for green chemistry, promoting sustainable practices in industrial processes. The kinetic parameters of PGA are discussed, providing insights into its operational efficiency. Despite its significant potential, PGA faces limitations in commercial applications, primarily due to stability issues under industrial conditions. Efforts to enhance PGA's stability, including engineering approaches, are explored to improve its industrial applicability. The review concludes by emphasizing PGA's role as a catalyst with vast implications in science and medicine, particularly in an era of rising antibiotic resistance. It underscores the enzyme's interconnected roles in production and therapeutics, its broad spectrum of applications, and the shift from traditional penicillin synthesis to broad-spectrum bioconversions. The scope of PGA engineering is also highlighted, indicating future directions for research and application in the pharmaceutical industry.

The biosynthesis of plumbagin is known to occur via the acetate polymalonate pathway; however there are several intermediary steps that remain unidentified that leads to its synthesis. The study identifies enzyme naphthoate synthase to catalyze the cyclization of O-malonyl benzoyl CoA to form an intermediate that is acted upon by thioesterase before the reaction proceeds to form plumbagin. Two possible structures were predicted for this intermediate using quantum mechanics studies. A total of 60 ns molecular dynamics simulations revealed the most probable intermediate structure of the predicted two.

The fluorinase enzyme, the only known biocatalyst forming stable carbon–fluorine bonds, operates with extremely low efficiency, catalyzing one reaction every 2–12 minutes. This severely limits its utility for sustainable biofluorination, and its sluggish activity remains poorly understood. We suppressed its aggregation through directed mutagenesis and elucidated the kinetic mechanism using a novel mathematical framework that fits complex kinetic and oligomerization data. This analysis revealed that >80% of enzyme molecules are inactive under standard conditions due to two dead-end pathways. The designed W50F+A279R mutant preferentially formed hexamers and displayed enhanced catalytic efficiency in this oligomeric state. When coupled with mechanism-based optimization of the reaction medium, including enzymatic removal of the inhibitory product, the catalytic turnover rate reached 12.5 ± 2.1 min⁻¹, representing ∼60-fold increase compared with previously reported turnover rates of the wild-type enzyme. Our work provides a mechanistic blueprint for fluorinase enhancement and a generalizable mathematical framework for analyzing kinetics of multimeric enzymes.

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery. Competing Interest Statement The authors have declared no competing interest.

Multiple myeloma (MM) is a rare hematological malignancy caused by monoclonal proliferation of plasma cells in the marrow of various bones. It is more common in men in the sixth and seventh decade of life. Patients usually present with bone pain, fatigue, recurrent infections, renal failure and nervous system dysfunction. Rarely, oral lesions may be the initial sign of multiple myeloma presenting with pain, jaw swelling, tooth mobility, multiple punched out radiolucencies and parasthesia. A case of multiple myeloma occurring in a 71 year old male patient who presented with a solitary lesion in the mandible is presented here. This paper highlights the importance of knowing oral manifestations of multiple myeloma and interdisciplinary approach required for early diagnosis.

In this paper, we propose a technique for rebalancing link weights in decentralized credit networks. Credit networks are peer-to-peer trust-based networks that enable fast and inexpensive cross-currency transactions compared to traditional bank wire transfers, which has led to their increasing popularity and use. Although researchers have studied security of transactions and privacy of users of such networks, and have invested significant efforts into designing efficient routing algorithms for credit networks, comparatively little work has been done in the area of replenishing credit links of users in the network. Replenishing links at regular intervals in a credit network is important to keep users solvent, the network viable with enough liquidity, and to prevent transaction failures. This is achieved by a process called rebalancing that enables a poorly funded user to create incoming as well as outgoing credit links. We propose a system where a user with zero or no link weights can create incoming links with existing, trusted users in the network, in a procedure we call balance transfer, followed by creating outgoing links to existing or new users that would like to join the network, a process we call bailout. Both these processes together constitute our proposed rebalancing mechanism. Our techniques would also serve to make the network more competitive by offering users lower rates of interest, and enable users to earn routing fees-based revenue by participating in high throughput transaction paths.

The present study analyzed 53 consecutive patients with oral squamous cell carcinoma (OSCC) over a period of 6 years (from 2006 to 2012) to determine if epidemiological differences correspond to different stages of the disease and affected the survival rate. In this cohort study, medical records were reviewed retrospectively. The epidemiological data included age, gender, residence, tobacco habit, duration of tobacco use, blood group, and hemoglobin. The clinicopathological features noted were tumor site, tumor size, nodal status, histopathological grade, metastasis, clinical grade, and treatment. We analyzed the relationship of epidemiological characteristics with treatment by the Chi-square test and survival analysis using the Kaplan-Meier curve. The statistical test significance level was set at P < 0.05. Of the 53 patients of OSCC confirmed through histopathological diagnosis, the ratio of female to male was 1.65:1 and mean age was 51.83 ± 12.57 years. Majority of the cases, i.e., 43 (81.13%) were from rural area. 51 (96.22%) patients were tobacco habituates, of which 41 (80.40%) were smokeless tobacco chewers. Buccal mucosa was the most common tumor site, i.e., 23 (43.40%). The clinical presentation of ulcer/ ulceroproliferative cases, i.e., 43 (81.13%) was most frequent. Moreover, statistical significance was observed with univariate association analysis between clinical presentation and treatment; in patients who underwent surgical treatment (P = 0.04). Of 35 (66.04%) patients who underwent surgical treatment, only 7 (20%) patients had recurrence. Survival rate was 90.57% at 3-year follow-up. The clinical presentation and treatment outcome of OSCC patients with the habit of smokeless tobacco is presented. The improved survival rate in our patients could be due to surgery being the main treatment modality and buccal mucosa being the most frequent site.