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Manganese (Mn) ranks twelfth among the most exuberant metal present in the earth's crust and finds its imperative application in the manufacturing steel, chemical, tannery, glass, and battery industries. Solubilisation of Mn can be performed by several bacterial strains which are useful in developing environmental friendly solutions for mining activities. The present investigation aims to isolate and characterize Mn solubilising bacteria from low grade ores from Sanindipur Manganese mine of Sundargh district in Odisha state of India. Four morphologically distinct bacterial strains showing visible growth on Mn supplemented plates were isolated. Mn solubilising ability of the bacterial strains was assessed by visualizing the lightening of the medium appearing around the growing colonies. Three isolates were gram negative and rod shaped while the remaining one was gram positive, coccobacilli. Molecular identification of the isolates was carried out by 16S rRNA sequencing and the bacterial isolates were taxonomically classified as Bacillus anthrasis MSB 2, Acinetobacter sp. MSB 5, Lysinibacillus sp. MSB 11, and Bacillus sp. MMR-1 using BLAST algorithm. The sequences were deposited in NCBI GenBank with the accession number KP635223, KP635224, KP635225 and JQ936966, respectively. Manganese solubilisation efficiency of 40, 96, 97.5 and 48.5% were achieved by MMR-1, MSB 2, MSB 5 and MSB 11 respectively. The efficiency of Mn solubilisation is suggested with the help of a pH variation study. The results are discussed in relation to the possible mechanisms involved in Manganese solubilisation efficiency of bacterial isolates. [ABSTRACT FROM AUTHOR]

The formation of granuloma is one of the characteristic features of tuberculosis. Besides, elevated serum amyloid A (SAA) protein level is the indicator for chronic inflammation associated with tuberculosis. The linkage between tuberculosis and SAA-driven secondary amyloidosis is well documented. However, SAA-derived amyloid onset and deposition start sites are not well understood in tuberculosis. We hypothesized that granuloma could be a potential site for amyloid deposition because of the presence of SAA protein and proteases, cleaving SAA into aggregation-prone fragments. 150 tuberculosis patients were identified and biopsies were collected from the affected organs. Patients showing eosinophilic hyaline-rich deposits within granuloma and its periphery were further screened for the presence of amyloid deposits. Upon Congo red staining, these hyaline deposits exhibited characteristic apple-green birefringence under polarized light, confirming their amyloid nature in 20 patients. Further upon Immuno-histochemical staining with anti-SAA antibody, the amyloid enriched areas showed positive immunoreactivity. In this pilot study, we have shown granuloma as a potential site for serum amyloid A derived amyloid deposition in tuberculosis patients. This study would expand the clinical and fundamental research for understanding the mechanism of amyloid formation in granuloma underlying tuberculosis and other chronic inflammatory conditions. • Granuloma is the potential site for serum amyloid A derived amyloid deposition in tuberculosis patients. • Granuloma and SAA protein might have synergistic or independent role in driving amyloid formation in tuberculosis patients. • Congo red staining of tuberculous granuloma might help to diagnose amyloidosis onset even in early stages of tuberculosis. [ABSTRACT FROM AUTHOR]

CuII‐‐NTA spin labeling of the transcription regulator CueR, an E. coli copper‐sensing protein, enabled the high‐resolution detection of its various conformational states, as described by S. Ruthstein and co‐workers in their Communication on page 3053 ff. Two of the conformations, activators 1 and 2, represent CueR as it binds DNA and CuI ions. The computed structures suggest that one is more compressed on the DNA than the other and the transition between those states depends on CuI concentration in the bacterial cell. [ABSTRACT FROM AUTHOR]

Macromolecular crowding experiments bridge the gap between in-vivo and in-vitro studies by mimicking some of the cellular complexities like high viscosity and limited space, while still manageable for experiments and analysis. Macromolecular crowding impacts all biological processes and is a focus of contemporary research. Recent reviews have highlighted the effect of crowding on various protein properties. One of the essential characteristics of protein is its dynamic nature; however, how protein dynamics get modulated in the crowded milieu has been largely ignored. This article discusses how protein translational, rotational, conformational, and solvation dynamics change under crowded conditions, summarizing key observations in the literature. We emphasize our research on microsecond conformational and water dynamics in crowded milieus and their impact on enzymatic activity and stability. Lastly, we provided our outlook on how this field might move forward in the future., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO4 and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for anti-fungal drug discovery. Mucorales species, deemed high priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO4 and 5'-PO4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg2+ that illuminates the basis for guanosine nucleotide specificity., (Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2'-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Colorectal cancer (CRC) continues to be a global health concern, necessitating further research into its complex biology and innovative treatment approaches. The etiology, pathogenesis, diagnosis, and treatment of colorectal cancer are summarized in this thorough review along with recent developments. The multifactorial nature of colorectal cancer is examined, including genetic predispositions, environmental factors, and lifestyle decisions. The focus is on deciphering the complex interactions between signaling pathways such as Wnt/β-catenin, MAPK, TGF-β as well as PI3K/AKT that participate in the onset, growth, and metastasis of CRC. There is a discussion of various diagnostic modalities that span from traditional colonoscopy to sophisticated molecular techniques like liquid biopsy and radiomics, emphasizing their functions in early identification, prognostication, and treatment stratification. The potential of artificial intelligence as well as machine learning algorithms in improving accuracy as well as efficiency in colorectal cancer diagnosis and management is also explored. Regarding therapy, the review provides a thorough overview of well-known treatments like radiation, chemotherapy, and surgery as well as delves into the newly-emerging areas of targeted therapies as well as immunotherapies. Immune checkpoint inhibitors as well as other molecularly targeted treatments, such as anti-epidermal growth factor receptor (anti-EGFR) as well as anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibodies, show promise in improving the prognosis of colorectal cancer patients, in particular, those suffering from metastatic disease. This review focuses on giving readers a thorough understanding of colorectal cancer by considering its complexities, the present status of treatment, and potential future paths for therapeutic interventions. Through unraveling the intricate web of this disease, we can develop a more tailored and effective approach to treating CRC., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inositol pyrophosphate 1,5-IP 8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1 , pho84 , and tgp1 , via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP 8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP 7 to 1,5-IP 8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP 7 , 1-IP 7 , and 1,5-IP 8 . Aps1 displays a ~twofold preference for hydrolysis of 1-IP 7 versus 5-IP 7 and aps1 ∆ cells have twofold higher levels of 1-IP 7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1 ∆ siw14 ∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP 8 toxicosis, whereby excessive 1,5-IP 8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1 ∆ siw14 ∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1 ∆ siw14 ∆ on YES by deleting tgp1 . However, the severe growth defect of an aps1 ∆ asp1-H397A strain could not be alleviated by deleting tgp1 , suggesting that 1,5-IP 8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity., Importance: Repression of the fission yeast PHO genes tgp1 , pho1 , and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP 8 , a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP 8 is formed by phosphorylation of 5-IP 7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo . Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP 7 , 1-IP 7 , and 1,5-IP 8 in vitro , with a ~twofold preference for 1-IP 7 over 5-IP 7 . aps1 ∆ cells have twofold higher levels of 1-IP 7 than wild-type cells. An aps1 ∆ siw14 ∆ double mutation is lethal because excessive 1,5-IP 8 triggers derepression of tgp1 , leading to toxic uptake of glycerophosphocholine., Competing Interests: The authors declare no conflict of interest.

Various biophysical techniques have been extensively employed to study protein aggregation due to its significance. Traditionally, these methods detect aggregation at micrometer length scales and micromolar concentrations. However, unlike in vitro, protein aggregation typically occurs at nanomolar concentrations in vivo. Here, using fluorescence correlation spectroscopy (FCS), we captured bromelain aggregation at concentrations as low as ~20 nM, surpassing the detection limit of traditional methods like thioflavin T fluorescence, scattering, and fluorescence microscopy by more than one order of magnitude. Moreover, using thioflavin T fluorescence-based FCS, we have detected larger aggregates at higher bromelain concentrations, which is undetectable in FCS otherwise. Importantly, our study reveals inherent heterogeneity in bromelain aggregation, inaccessible to ensemble-averaged techniques. The presented report may provide a platform for the characterization of premature aggregates at very low protein concentrations, which are thought to be functionally significant species in protein aggregation-induced diseases., (© 2024 American Society for Photobiology.)

Subungual melanoma is associated with the highest mortality among all skin cancers and is strongly linked to acquired mutations caused by exposure to ultraviolet radiation in sunlight. The commonest sites of occurrence are the great toe and thumb. Diagnosis of melanoma often becomes a challenge as it is difficult to differentiate it from other pigmented disorders. A histopathological evaluation of the lesion with adequate nail matrix biopsy can address the diagnostic dilemma. Additionally, an early diagnosis of melanoma is critical as once detected early, it is often treatable. We present a case of a 72-year-old diabetic male patient with a pigmented lesion over the right great toe. In view of the patient's age and history of diabetes, the initial presentation was mistaken as onychomycosis which created a diagnostic dilemma. Hence, we present this case to shed light upon the fact that these lesions can mimic several other benign conditions like fungal melanonychia, lentigo, and subungual hemorrhage. To avoid misdiagnosis and subsequent delay in management, early clinical, dermoscopic, and very pertinently, histopathological and radiological co-relations are extremely important., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Chatterjee et al.)

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO 4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO 4 and 5'-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO 4 terminus in the end-joining reaction, whereby the 2'-PO 4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO 4 installed by RNA ligase is removed by a dedicated NAD + -dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD + -dependent RNA 2'-phosphotransferase activity in vitro., (© 2024 Ghosh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Objectives: In recent years, there has been phenomenal growth in internet usage worldwide, with a substantial proportion of children and adolescents actively engaging with online platforms. While the internet presents numerous opportunities for children and adolescents, the lack of digital literacy and adequate online safety measures exposes them to various cybercrimes, including cyberbullying, cyberstalking, identity theft, and sexual predation. Moreover, there is growing concern regarding internet addiction among this population., Methods: To investigate the determinants of internet addiction among adolescents, we conducted a cross-sectional study in peri-urban Delhi-NCR, India. We used a self-administered questionnaire to gather information on internet usage, and 630 adolescents aged 13-18 participated in the study, also completing an Internet Addiction Test., Results: The findings indicate that 415 adolescents (65.9 %) exhibited no signs of internet addiction, suggesting a healthy relationship with the internet. However, 215 adolescents (33.1 %) displayed symptoms of internet addiction. Among those exhibiting internet addictions, 159 (74.0 %) were classified as mild internet addicts, indicating moderate levels of internet usage. Furthermore, 56 (26.0 %) adolescents were classified as moderate internet addicts, reflecting a higher level of internet addiction., Conclusions: Our study highlights the significant influence of various factors, including family dynamics, environmental factors, and personal experiences, on internet addiction among adolescents. Based on these findings, we propose implementing measures at different levels to foster responsible internet use among adolescents, thereby substantially reducing internet addiction., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

This comprehensive review delves into the challenges associated with diagnosing and managing unusual cases of eosinophilic enteritis in rural health settings. Eosinophilic enteritis, characterized by an abnormal accumulation of eosinophils in the gastrointestinal (GI) tract, poses distinct difficulties in diagnosis due to its varied presentations. In rural contexts, limited access to specialized diagnostic tools, a shortage of healthcare professionals, and geographical constraints compound these challenges. This abstract encapsulates the critical issues explored in the review, emphasizing the importance of addressing atypical cases and rural healthcare's unique hurdles. The conclusion is a rallying call for collaborative action, advocating for improved education, telemedicine solutions, and enhanced access to specialized care. The implications extend beyond eosinophilic enteritis, with the potential to instigate systemic improvements in rural healthcare globally. This review is a crucial contribution to understanding eosinophilic enteritis in rural settings and advocates for transformative measures to improve diagnosis, management, and overall healthcare outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Ghosh et al.)

Peptides are very interesting biomolecules that upon self-association form a variety of thermodynamically stable supramolecular structures of nanometric dimension e.g. nanotubes, nanorods, nanovesicles, nanofibrils, nanowires and many others. Herein, we report six peptide molecules having a general chemical structure, H-Gaba-X-X-OH (Gaba: γ-aminobutyric acid, X: amino acid). Out of these six peptides, three are aromatic and the others are aliphatic. Atomic force microscopic (AFM) studies reveal that except peptide 6 (H-Gaba-Trp-Trp-OH), all the reported peptides adopt nanofibrillar morphology upon aggregation in aqueous medium. These supramolecular assemblies can recognize amyloid-specific molecular probe congo red (CR) and thioflavine t (ThT) and exhibit all the characteristic properties of amyloids. The MTT cell viability assay reveals that the toxicity of both aliphatic and aromatic peptides increases with increasing concentration of the peptides to both cancer (HeLa) and non-cancer (HEK 293) cells. Of note, the aromatic peptides show a slightly higher cytotoxic effect compared to the aliphatic peptides. Overall, the studies highlight the self-assembling nature of the de novo designed aliphatic and aromatic peptides and pave the way towards elucidating the intricacies of pathogenic amyloid assemblies., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

Pollution by end-of-life electronics is a rapid ever-increasing threat and is a universal concern with production of million metric tons of these wastes per annum. Electronic wastes (E-waste) are rejected electric or electronic equipment which have no other applications. The aggrandized unproper land filling of E-waste may generate hazardous effects on living organisms and ecosystem. At present, millions of tons of E-waste await the advancement of more efficient and worthwhile recycling techniques. Recovery of base and critical elements from electronic scraps will not only reduce the mining of these elements from natural resources but also reduces the contamination caused by the hazardous chemicals (mostly organic micropollutants) released from these wastes when unproperly disposed of. Bioleaching is reported to be the most eco-friendly process for metal recycling from spent electronic goods. A detailed investigation of microbial biodiversity and a molecular understanding of the metabolic pathways of bioleaching microorganisms will play a vital function in extraction of valuable minerals from the end-of-life scraps. Bioleaching technique as an economic and green technology costs around 7 USD per kg for effective reusing of E-waste as compared to other physical and chemical techniques. This review provides a summary of worldwide scenario of electronic pollutants; generation, composition and hazardous components of electronic waste; recycling of valuable elements through bioleaching; mechanism of bioleaching; microorganisms involved in base and critical element recovery from E-waste; commercial bioleaching operations; and upcoming aspects of this eco-friendly technique., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Antibiotic resistance of Mycobacterium tuberculosis is exclusively a consequence of chromosomal mutations. Translesion synthesis (TLS) is a widely conserved mechanism of DNA damage tolerance and mutagenesis, executed by translesion polymerases such as DinBs. In mycobacteria, DnaE2 is the only known agent of TLS and the role of DinB polymerases is unknown. Here we demonstrate that, when overexpressed, DinB1 promotes missense mutations conferring resistance to rifampicin, with a mutational signature distinct from that of DnaE2, and abets insertion and deletion frameshift mutagenesis in homo-oligonucleotide runs. DinB1 is the primary mediator of spontaneous -1 frameshift mutations in homo-oligonucleotide runs whereas DnaE2 and DinBs are redundant in DNA damage-induced -1 frameshift mutagenesis. These results highlight DinB1 and DnaE2 as drivers of mycobacterial genome diversification with relevance to antimicrobial resistance and host adaptation., (© 2022. The Author(s).)

The outbreak of 2019 novel coronavirus (COVID-19) has triggered unprecedented challenges and put the whole world in a parlous condition. The impacts of COVID-19 is a matter of grave concern in terms of fatality rate, socio-economical condition, health infrastructure. It is obvious that only pharmaceutical solutions (vaccine) cannot eradicate this pandemic completely, and effective strategies regarding lockdown measures, restricted mobility, emergency services to users-in brief data-driven decision system is of utmost importance. This necessitates an efficient data analytics framework, data infrastructure to store, manage pandemic related information, and distributed computing platform to support such data-driven operations. In the past few decades, Internet of Things-based devices and applications have emerged significantly in various sectors including healthcare and time-critical applications. To be specific, health-sensors help to accumulate health-related parameters at different time-instances of a day, the movement sensors keep track of mobility traces of the user, and helps to assist them in varied conditions. The smartphones are equipped with several such sensors and the ability of low-cost connected sensors to cover large areas makes it the most useful component to combat pandemics such as COVID-19. However, analysing and managing the huge amount of data generated by these sensors is a big challenge. In this paper we have proposed a unified framework which has three major components: (i) Spatial Data Infrastructure to manage, store, analyse and share spatio-temporal information with stakeholders efficiently, (ii) Cloud-Fog-Edge-based hierarchical architecture to support preliminary diagnosis, monitoring patients' mobility, health parameters and activities while they are in quarantine or home-based treatment, and (iii) Assisting users in varied emergency situation leveraging efficient data-driven techniques at low-latency and energy consumption. The mobility data analytics along with SDI is required to interpret the movement dynamics of the region and correlate with COVID-19 hotspots. Further, Cloud-Fog-Edge-based system architecture is required to provision healthcare services efficiently and in timely manner. The proposed framework yields encouraging results in taking decisions based on the COVID-19 context and assisting users effectively by enhancing accuracy of detecting suspected infected people by ∼ 24% and reducing delay by ∼ 55% compared to cloud-only system., (© The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature 2022.)

There has been alarming depletion of manganese (Mn) reserves owing to the ongoing extensive mining operations for catering the massive industrial demand of this element. Moreover, the mining operations have been leading to the generation of Mn-rich waste, thereby contaminating both terrestrial and aquatic bodies. The current scenario necessitates the development of alternative processes for bioremediation as well as economic recovery of Mn from mining wastes. The present investigation aims to report the bioleaching of Mn by Lysinibacillus sp. from mining waste residues in the context of mine waste remediation. Results confirmed that the native isolate had a high Mn biosolubilization potential with a solubilizing efficiency of 84% at the end of a 21-day study under optimized conditions of pulp density 2% (< 150-μm particle size), pH 6.5, and temperature 30 °C. Fourier transform infrared spectroscopy (FTIR) studies followed by liquid chromatography mass spectrometry (LC-MS) analysis were used to ascertain the change in microbial protein conformation, configuration, and protein identification. The results revealed the expression of heat shock proteins (HSP) from the family HSP which is predominantly expressed in bacteria during stress conditions. This study represents the application of native bacterial strain in Mn biosolubilization. We foresee the utility of proteomics-based studies to provide a methodological framework to the underlying mechanism of metal solubilization, thereby facilitating the two-tier benefit of recovery of Mn from alternative sources as well as bioremediation of waste having high manganese content., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

Biofilm associated infections are the major contributor of mortality, morbidity and financial burden in patients with a bacterial infection. About 65% of all bacterial infections are associated with the information of bacterial biofilms. Bacterial biofilms not only reduce the efficacy of antibacterial treatment but also increases the threat of developing antibacterial resistance. Recently, our group has discovered the antibacterial activity of Fmoc-phenylalanine (Fmoc-F) and other Fmoc-amino acids (Fmoc-AA). Fmoc-F and other Fmoc-AA showed antibacterial activity due to their surfactant properties. Surfactants are known to eradicate biofilm and enhance antimicrobial activity in biofilm. Thus, in the present study, we evaluated the anti-biofilm activity of Fmoc-F against clinically relevant bacteria. We found that Fmoc-F not only inhibits the biofilm formation in Staphylococcus aureus and Pseudomonas aeruginosa, but also eradicates the already formed biofilms over the surface. Further, Fmoc-F coated glass surface resists S. aureus and P. aeruginosa biofilm formation and attachment, when biofilm is grown over the surface. The mechanistic investigation suggests that Fmoc-F reduces the extracellular matrix (ECM) components such as proteins, carbohydrates and eDNA in the biofilm and affect its stability via direct interactions with ECM components and/ or indirectly through reducing bacterial cell population. Finally, we showed that Fmoc-F treatment in combination with vancomycin and ampicillin synergistically inhibit biofilm formation. Overall, the study demonstrates the potential application of Fmoc-F and other Fmoc-AA molecules individually as well as in combination as anti-biofilm coating material for treating biofilm associated infections.

Rationale: Senile systemic amyloidosis, a disease of elderly is caused by amyloid deposition of wild-type transthyretin. The symptoms often overlap with other heart diseases. Hence it is either misdiagnosed or considered as a normal aging process in majority of cases., Patient Concerns: We present a young patient of wild-type transthyretin amyloidosis, contradicting its only senile presence. The 34-year-old man presented with dyspnoea on exertion. He was suffering from hypertension for consecutive 3 years., Diagnosis: Echocardiography demonstrated left ventricular hypertrophy with reduced global longitudinal strain and apical sparing. Congo red staining and immuno-histochemical staining of the abdominal fat biopsy confirmed transthyretin amyloid deposition. Genetic analysis revealed absence of any mutant variant/s of transthyretin gene, confirming wild-type transthyretin amyloidosis., Intervention: A combination of amlodipine 5 mg, telmisartan 40 mg, and chlorthalidone 12.5 mg once daily was given to control the blood pressure of the patient., Outcome: Blood pressure was controlled but he continued to have exertional dyspnoea. The patient expired in December 2019., Lessons: A systematic diagnosis for wild type transthyretin amyloid cardiomyopathy (ATTR-CM) shall be considered in young cardiac patients suffering from cardiac distress with unknown etiology., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

In this Account, we showcase site-directed Cu 2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu 2+ complex. Pulsed EPR measurements on such Cu 2+ -labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin label-the approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at β-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu 2+ -Cu 2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu 2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu 2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein-DNA interactions.

Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3' and 5' single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3'-OH and 3'-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Growing evidence suggests that prebiotics may induce weight loss and alleviate non-alcoholic fatty liver disease (NAFLD) via modulation of the gut microbiota. However, key members of the gut microbiota that may mediate the beneficial effects of prebiotics remain elusive. Here, we find that restricted prebiotic feeding during active phase (HF-ARP) induced weight-independent alleviation of liver steatosis and reduced serum cholesterol in high-fat diet (HF) fed mice more significantly than unrestricted feeding (HF-UP). HF-ARP mice also showed concomitantly altered gut microbiota structure that was different from HF-UP group along with significantly increased production of total short-chain fatty-acids (SCFAs). Amplicon sequence variants (ASVs) were clustered into co-abundant groups (CAGs) as potential functional groups that may respond distinctively to prebiotic consumption and prebiotic feeding regime. Prebiotic feeding induces significant alterations in CAG abundances by day 7. Eight of 32 CAGs were promoted by prebiotics, including CAG17 with the most abundant ASV from Parabacteroides, CAG22 with Bacteroides thetaiotamicron and CAG32 with Fecalibaculum and Akkermansia . Among the prebiotic-promoted CAGs, CAG20 with ASVs from Lachnospiraceae and CAG21 with ASVs from Bifidobacterium and Lachnospiraceae were significantly enhanced in HF-ARP compared to HF-UP. Moreover, most of the prebiotic-promoted CAGs were also significantly associated with improvements in hepatic steatosis, reduction in serum cholesterol and increased cecal propionate production. Together, these results suggest that the impact of prebiotics on weight-independent alleviation of liver steatosis and cholesterol-lowering effect can be optimized by restricting prebiotic intake to active phase and is associated with a distinct change of gut microbiota with increased SCFA production., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu2+-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu2+-DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu2+-DPA on four different DNA duplexes. The distance between the Cu2+ centers, extracted from the 2 μs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu2+-DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu2+-DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu2+-DPA in a DNA depends on the position of the label in the duplex, and a 2 μs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electron resonance (DEER) results. The lack of orientational selectivity effects of the Cu2+-DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu2+-DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein-DNA interactions.

Electron paramagnetic resonance (EPR) in combination with the recently developed double-histidine (dHis)-based Cu 2+ spin labeling has provided valuable insights into protein structure and conformational dynamics. To relate sparse distance constraints measured by EPR to protein fluctuations in solution, modeling techniques are needed. In this work, we have developed force field parameters for Cu 2+ -nitrilotriacetic and Cu 2+ -iminodiacetic acid spin labels. We employed molecular dynamics (MD) simulations to capture the atomic-level details of dHis-labeled protein fluctuations. The interspin distances extracted from 200 ns MD trajectories show good agreement with the experimental results. The MD simulations also illustrate the dramatic rigidity of the Cu 2+ labels compared to the standard nitroxide spin label. Further, the relative orientations between spin-labeled sites were measured to provide insight into the use of double electron-electron resonance (DEER) methods for such labels. The relative mean angles, as well as the standard deviations of the relative angles, agree well in general with the spectral simulations published previously. The fluctuations of relative orientations help rationalize why orientation selectivity effects are minimal at X-band frequencies, but observable at the Q-band for such labels. In summary, the results show that by combining the experimental results with MD simulations precise information about protein conformations as well as flexibility can be obtained.

In this work, we explore the potential of a rigid Cu 2+ spin-labeling technique, the double histidine (dHis) motif, along with Q-band electron paramagnetic resonance to report on the relative orientations of the spin labels. We show that the precision of the dHis motif, coupled with the sensitivity and resolution of Q-band frequencies, may allow for the straightforward determination of the relative orientation of the dHis-Cu 2+ labels using double electron-electron resonance (DEER). We performed Q-band DEER measurements at different magnetic fields on a protein containing two dHis Cu 2+ sites. These measurements exhibited orientational selectivity such that each discrete magnetic field yielded a unique DEER signal. We determined the relative orientation of the two metal centers by simulating the orientationally selective DEER data. These relative orientations were validated by visual analysis of the protein crystal structure modified with dHis sites. The simple visual analysis was shown to agree well with the angular values determined via simulation of the experimental data. The combination of the dHis-Cu 2+ motif along with the advantages of the Q-band can aid in the accurate measurement of protein structural and conformational dynamics.

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu 2+ . This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu 2+ -based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu 2+ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu 2+ -IDA complexation. Herein, we introduce an alternative method of dHis-Cu 2+ loading using the nitrilotriacetic acid (NTA)-Cu 2+ complex. We find that the Cu 2+ -NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu 2+ -NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu 2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand β-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu 2+ -NTA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlabelled: Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc(2)155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc(2)155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2., Importance: Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)