Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2'-phosphotransferase (Tpt1) 'poison' that traps the enzyme on its abortive RNA-2'-PO4-(ADP-2″OMe-ribose) reaction intermediate.

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.

Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end.

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO4 and 5'-PO4 RNA ends to form a 2'-PO4,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO4 RNA terminus to form an RNA-adenylate intermediate (A5'pp5'RNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO4 Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA2'p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.

Tracking heterogenous protein aggregation at nanoscale through fluorescence correlation spectroscopy.

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.

Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

Inositol pyrophosphate 1,5-IP8 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-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 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-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 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 IP8 toxicosis, whereby excessive 1,5-IP8 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-IP8 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-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 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-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.

A Misdiagnosed Case of Malignant Melanoma in an Infected Nail: A Case Report.

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.

Challenges in Diagnosis and Management of Unusual Cases of Eosinophilic Enteritis in Rural Health Settings: A Comprehensive Review.

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.

Exploring the determinants of internet addiction among Peri-urban adolescents (aged 13-18) in Delhi-NCR, India: an ordered logit model analysis.

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.

De novo designed aliphatic and aromatic peptides assemble into amyloid-like cytotoxic supramolecular nanofibrils.

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.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 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'-PO4 and 5'-PO4 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'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 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'-PO4 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.

Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-ß42 aggregation and fibril formation.

The chaperone Hsp104, a member of the Hsp100/Clp family of translocases, prevents fibril formation of a variety of amyloidogenic peptides in a paradoxically substoichiometric manner. To understand the mechanism whereby Hsp104 inhibits fibril formation, we probed the interaction of Hsp104 with the Alzheimer's amyloid-ß42 (Aß42) peptide using a variety of biophysical techniques. Hsp104 is highly effective at suppressing the formation of Thioflavin T (ThT) reactive mature fibrils that are readily observed by atomic force (AFM) and electron (EM) microscopies. Quantitative kinetic analysis and global fitting was performed on serially recorded 1H-15N correlation spectra to monitor the disappearance of Aß42 monomers during the course of aggregation over a wide range of Hsp104 concentrations. Under the conditions employed (50 µM Aß42 at 20 °C), Aß42 aggregation occurs by a branching mechanism: an irreversible on-pathway leading to mature fibrils that entails primary and secondary nucleation and saturating elongation; and a reversible off-pathway to form nonfibrillar oligomers, unreactive to ThT and too large to be observed directly by NMR, but too small to be visualized by AFM or EM. Hsp104 binds reversibly with nanomolar affinity to sparsely populated Aß42 nuclei present in nanomolar concentrations, generated by primary and secondary nucleation, thereby completely inhibiting on-pathway fibril formation at substoichiometric ratios of Hsp104 to Aß42 monomers. Tight binding to sparsely populated nuclei likely constitutes a general mechanism for substoichiometric inhibition of fibrillization by a variety of chaperones. Hsp104 also impacts off-pathway oligomerization but to a much smaller degree initially reducing and then increasing the rate of off-pathway oligomerization.

Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis.

Translesion synthesis by translesion polymerases is a conserved mechanism of DNA damage tolerance. In bacteria, DinB enzymes are the widely distributed promutagenic translesion polymerases. The role of DinBs in mycobacterial mutagenesis was unclear until recent studies revealed a role for mycobacterial DinB1 in substitution and frameshift mutagenesis, overlapping with that of translesion polymerase DnaE2. Mycobacterium smegmatis encodes two additional DinBs (DinB2 and DinB3) and Mycobacterium tuberculosis encodes DinB2, but the roles of these polymerases in mycobacterial damage tolerance and mutagenesis is unknown. The biochemical properties of DinB2, including facile utilization of ribonucleotides and 8-oxo-guanine, suggest that DinB2 could be a promutagenic polymerase. Here, we examine the effects of DinB2 and DinB3 overexpression in mycobacterial cells. We demonstrate that DinB2 can drive diverse substitution mutations conferring antibiotic resistance. DinB2 induces frameshift mutations in homopolymeric sequences, both in vitro and in vivo. DinB2 switches from less to more mutagenic in the presence of manganese in vitro. This study indicates that DinB2 may contribute to mycobacterial mutagenesis and antibiotic resistance acquisition in combination with DinB1 and DnaE2.

A Critical Review on the Recovery of Base and Critical Elements from Electronic Waste-Contaminated Streams Using Microbial Biotechnology.

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.

Influence of Chronic Electroconvulsive Seizures on Plasticity-Associated Gene Expression and Perineuronal Nets Within the Hippocampi of Young Adult and Middle-Aged Sprague-Dawley Rats.

BACKGROUND: Electroconvulsive seizure therapy is often used in both treatment-resistant and geriatric depression. However, preclinical studies identifying targets of chronic electroconvulsive seizure (ECS) are predominantly focused on animal models in young adulthood. Given that putative transcriptional, neurogenic, and neuroplastic mechanisms implicated in the behavioral effects of chronic ECS themselves exhibit age-dependent modulation, it remains unknown whether the molecular and cellular targets of chronic ECS vary with age. METHODS: We subjected young adult (2-3 months) and middle-aged (12-13 months), male Sprague Dawley rats to sham or chronic ECS and assessed for despair-like behavior, hippocampal gene expression, hippocampal neurogenesis, and neuroplastic changes in the extracellular matrix, reelin, and perineuronal net numbers. RESULTS: Chronic ECS reduced despair-like behavior at both ages, accompanied by overlapping and unique changes in activity-dependent and trophic factor gene expression. Although chronic ECS had a similar impact on quiescent neural progenitor numbers at both ages, the eventual increase in hippocampal progenitor proliferation was substantially higher in young adulthood. We noted a decline in reelin⁺ cell numbers following chronic ECS only in young adulthood. In contrast, an age-invariant, robust dissolution of perineuronal net numbers that encapsulate parvalbumin⁺ neurons in the hippocampus were observed following chronic ECS. CONCLUSION: Our findings indicate that age is a key variable in determining the nature of chronic ECS-evoked molecular and cellular changes in the hippocampus. This raises the intriguing possibility that chronic ECS may recruit distinct, as well as overlapping, mechanisms to drive antidepressant-like behavioral changes in an age-dependent manner.

Amyloid deposition in granuloma of tuberculosis patients: A single-center pilot study.

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.

Distinctive roles of translesion polymerases DinB1 and DnaE2 in diversification of the mycobacterial genome through substitution and frameshift mutagenesis.

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.

STROVE: spatial data infrastructure enabled cloud-fog-edge computing framework for combating COVID-19 pandemic.

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.

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator.

Understanding the structural and mechanistic details of protein-DNA interactions that lead to cellular defence against toxic metal ions in pathogenic bacteria can lead to new ways of combating their virulence. Herein, we examine the Copper Efflux Regulator (CueR) protein, a transcription factor which interacts with DNA to generate proteins that ameliorate excess free Cu(i). We exploit site directed Cu(ii) labeling to measure the conformational changes in DNA as a function of protein and Cu(i) concentration. Unexpectedly, the EPR data indicate that the protein can bend the DNA at high protein concentrations even in the Cu(i)-free state. On the other hand, the bent state of the DNA is accessed at a low protein concentration in the presence of Cu(i). Such bending enables the coordination of the DNA with RNA polymerase. Taken together, the results lead to a structural understanding of how transcription is activated in response to Cu(i) stress and how Cu(i)-free CueR can replace Cu(i)-bound CueR in the protein-DNA complex to terminate transcription. This work also highlights the utility of EPR to measure structural data under conditions that are difficult to access in order to shed light on protein function.

A compact photonic resonator absorption microscope for point of care digital resolution nucleic acid molecular diagnostics.

Rapid, sensitive, and selective detection of nucleic acid biomarkers for health diagnostic applications becomes feasible for point of care scenarios when the detection instrument is inexpensive, simple, and robust. Here, we report the design, implementation, and characterization of a point of care instrument for photonic resonator absorption microscopy (PRAM) that takes advantage of resonant optical coupling between plasmonic gold nanoparticle tags and a photonic crystal (PC) surface. Matching the PC resonant wavelength to the gold nanoparticle's surface plasmon wavelength generates localized and efficient quenching of the PC resonant reflection intensity, resulting in the ability to clearly detect and count individual gold nanoparticles when they are captured on the PC surface. Surface-captured nanoparticles are observed by illuminating the PC at normal incidence with polarized light from a low-intensity red LED, and recording of PC reflected intensity on an inexpensive CMOS image sensor. A contrast limited adaptive histogram equalization (CLAHE) image processing algorithm was applied to derive counts of captured nanoparticles. The instrument is utilized in the context of an activate capture + digital counting (AC + DC) assay for a specific miRNA sequence, using nucleic acid toehold probes applied to gold nano-urchin (AuNU) nanoparticles to achieve 160 aM detection limits in a 30 min. assay.

Erratum: A compact photonic resonator absorption microscope for point of care digital resolution nucleic acid molecular diagnostics: publisher's note.

[This corrects the article on p. 4637 in vol. 12.].

Structure and mechanism of Mycobacterium smegmatis polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.