Convergent evolution in the supercoiling of prokaryotic flagellar filaments.

The supercoiling of bacterial and archaeal flagellar filaments is required for motility. Archaeal flagellar filaments have no homology to their bacterial counterparts and are instead homologs of bacterial type IV pili. How these prokaryotic flagellar filaments, each composed of thousands of copies of identical subunits, can form stable supercoils under torsional stress is a fascinating puzzle for which structural insights have been elusive. Advances in cryoelectron microscopy (cryo-EM) make it now possible to directly visualize the basis for supercoiling, and here, we show the atomic structures of supercoiled bacterial and archaeal flagellar filaments. For the bacterial flagellar filament, we identify 11 distinct protofilament conformations with three broad classes of inter-protomer interface. For the archaeal flagellar filament, 10 protofilaments form a supercoil geometry supported by 10 distinct conformations, with one inter-protomer discontinuity creating a seam inside of the curve. Our results suggest that convergent evolution has yielded stable superhelical geometries that enable microbial locomotion.

Cyclic AMP binding to a universal stress protein in Mycobacterium tuberculosis is essential for viability.

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.

Trehalose-Grafted Glycopolymer: Synthesis via the Staudinger Reaction and Capture of Mycobacteria.

A new trehalose-grafted poly(2-hydroxyethyl methacrylate) (HEMA) glycopolymer was synthesized via the perfluorophenyl azide (PFPA)-mediated Staudinger reaction between poly(HEMA-co-HEMA-PFPA) and a diphenylphosphine-derivatized trehalose. The reaction occurred rapidly at room temperature without the use of any catalyst, giving the trehalose glycopolymers over 68% yield after 1 h. The grafting density of trehalose can be controlled by the copolymer composition in poly(HEMA-co-HEMA-PFPA), resulting in 6.1% (TP1) or 37% (TP2) at 10:1 and 1:1 HEMA/HEMA-PFPA feed ratio, respectively. The trehalose glycopolymer was covalently attached on glass slides or silicon wafers using a thin film of poly(HEMA-co-HEMA-PFPA) as the adhesion layer, achieved through the C-H insertion reaction of the photogenerated singlet perfluorophenyl nitrene. To demonstrate the ability of the trehalose glycopolymer to capture mycobacteria, arrays of the trehalose glycopolymer were fabricated and treated with Mycobacterium smegmatis. Results from the optical, fluorescence, and scanning electron microscopy showed that mycobacteria were indeed captured on the trehalose glycopolymer. The amount of mycobacteria captured increased with the percent trehalose in the trehalose glycopolymer and also with the concentration of the trehalose glycopolymer. In addition, the captured bacteria could be visualized by the naked eye under the illumination of a hand-held UV lamp.

Mycobacterial STAND adenylyl cyclases: The HTH domain binds DNA to form biocrystallized nucleoids.

Mycobacteria harbor a unique class of adenylyl cyclases with a complex domain organization consisting of an N-terminal putative adenylyl cyclase domain fused to a nucleotide-binding adaptor shared by apoptotic protease-activating factor-1, plant resistance proteins, and CED-4 (NB-ARC) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal helix-turn-helix (HTH) domain. The products of the rv0891c-rv0890c genes represent a split gene pair, where Rv0891c has sequence similarity to adenylyl cyclases, and Rv0890c harbors the NB-ARC-TPR-HTH domains. Rv0891c had very low adenylyl cyclase activity so it could represent a pseudoenzyme. By analyzing the genomic locus, we could express and purify Rv0890c and find that the NB-ARC domain binds ATP and ADP, but does not hydrolyze these nucleotides. Using systematic evolution of ligands by exponential enrichment (SELEX), we identified DNA sequences that bound to the HTH domain of Rv0890c. Uniquely, the HTH domain could also bind RNA. Atomic force microscopy revealed that binding of Rv0890c to DNA was sequence independent, and binding of adenine nucleotides to the protein induced the formation of higher order structures that may represent biocrystalline nucleoids. This represents the first characterization of this group of proteins and their unusual biochemical properties warrant further studies into their physiological roles in future.

A universal stress protein in Mycobacterium smegmatis sequesters the cAMP-regulated lysine acyltransferase and is essential for biofilm formation.

Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP4207) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp4207-deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a Δusp4207Δkatms strain. We identified several proteins whose acetylation was lost in the Δkatms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP4207, suggesting that USP4207 insulates KATms from its other substrates in the cell. We propose that USP4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses.

Confinement driven anomalous freezing in nano porous spray dried microspheres.

One-step evaporative jamming of colloidal silica particles in contact-free spray droplets resulted in well-defined powder micro-granules with interstitial nanopores. This paper reports the anomalous freezing behaviour of confined water in the microspheres synthesized using spray drying. It has been revealed that the freezing point of water in these microspheres gets significantly lowered (∼-45 °C) owing to the confinement effect. Thermoporometry results are corroborated with the structural details obtained using complementary techniques of gas adsorption measurements and small-angle x-ray scattering.

Do children use language structure to discover the recursive rules of counting?

We test the hypothesis that children acquire knowledge of the successor function - a foundational principle stating that every natural number n has a successor n + 1 - by learning the productive linguistic rules that govern verbal counting. Previous studies report that speakers of languages with less complex count list morphology have greater counting and mathematical knowledge at earlier ages in comparison to speakers of more complex languages (e.g., Miller & Stigler, 1987). Here, we tested whether differences in count list transparency affected children's acquisition of the successor function in three languages with relatively transparent count lists (Cantonese, Slovenian, and English) and two languages with relatively opaque count lists (Hindi and Gujarati). We measured 3.5- to 6.5-year-old children's mastery of their count list's recursive structure with two tasks assessing productive counting, which we then related to a measure of successor function knowledge. While the more opaque languages were associated with lower counting proficiency and successor function task performance in comparison to the more transparent languages, a unique within-language analytic approach revealed a robust relationship between measures of productive counting and successor knowledge in almost every language. We conclude that learning productive rules of counting is a critical step in acquiring knowledge of recursive successor function across languages, and that the timeline for this learning varies as a function of count list transparency.

Mycobacterial phenolic glycolipid synthesis is regulated by cAMP-dependent lysine acylation of FadD22.

The mycobacterial cell envelope is unique in its chemical composition, and has an important role to play in pathogenesis. Phthiocerol dimycocerosates (PDIMs) and glycosylated phenolphthiocerol dimycocerosates, also known as phenolic glycolipids (PGLs), contribute significantly to the virulence of Mycobacterium tuberculosis. FadD22 is essential for PGL biosynthesis. We have recently shown in vitro that FadD22 is a substrate for lysine acylation by a unique cAMP-dependent, protein lysine acyltransferase found only in mycobacteria. The lysine residue that is acylated is at the active site of FadD22. Therefore, acylation is likely to inhibit FadD22 activity and reduce PGL biosynthesis. Here, we show accumulation of PGLs in a strain of M. bovis BCG deleted for the gene encoding the cAMP-dependent acyltransferase, katbcg, with no change seen in PDIM synthesis. Complementation using KATbcg mutants that are deficient in cAMP-binding or acyltransferase activity shows that PGL accumulation is regulated by cAMP-dependent protein acylation in vivo. Expression of FadD22 and KATbcg mutants in Mycobacterium smegmatis confirmed that FadD22 is a substrate for lysine acylation by KATbcg. We have therefore described a mechanism by which cAMP can regulate mycobacterial virulence as a result of the ability of this second messenger to modulate critical cell wall components that affect the host immune response.

Temperature Mediated Morphological Transition during Drying of Spray Colloidal Droplets.

Understanding how a tiny dilute evaporative colloidal spray droplet gets transformed into a microgranule with a characteristic morphology is crucial from scientific as well as technological points of view. In the present work, it is demonstrated that the morphology and the size distribution of the microcapsules can be tuned simply by adjusting the drying temperature. Shape and size of the capsules are quantified at four different drying temperatures. It is shown that the morphology transits gradually from sphere to toroid with increasing temperature keeping the average volume-fraction of the correlated nanoparticles nearly unaffected for the synthesized granules. A plausible mechanism for the chronological pathway of such morphological transformation is illustrated. Computer simulation corroborates the experimentally observed morphological transition. The variation in hollowness and buckling tendency of the capsules are elucidated by scattering and imaging techniques.

Low-Loss Dielectric Ink for Printed Radio Frequency and Microwave Devices.

Direct write printing is restricted by the lack of dielectric materials that can be printed with high resolution and offer dissipation factors at radio frequency (RF) within the range of commercial RF laminates. Herein, we outline the development of dielectric materials with dielectric loss below 0.006 in X and Ku frequency bands (8.2-18 GHz), the range required for radio frequency and microwave applications. The described materials were designed for printability and processability, specifically a prolonged viscosity below 1000 cps and a robust cure procedure, which requires minimal heat treatment. In the first stage of this work, nonpolar ring-opening metathesis polymerization (ROMP) is demonstrated at room temperature in an open-air environment with a low-viscosity monomer, 5-vinyl-2-norbornene, using the second-generation Grubbs catalyst (G-II). Differential scanning calorimetry (DSC) was used to study how the catalyst activity is increased with heating at various stages in the reaction, which is then used as a strategy to cure the material after printing. The resulting cured poly(5-vinyl-2-norbornene) material is then characterized for dielectric and mechanical performance before and after a secondary heat treatment, which mimics processing procedures to incorporate subsequent printed conductor layers for multilayer applications. After the secondary heat treatment, the material exhibits a 55.0% reduction in the coefficient of thermal expansion (CTE), an increase in glass-transition temperature (Tg) from 32.4 to 46.1 °C, and an increased 25 °C storage modulus from 428 to 1031 MPa while demonstrating a minimal change in dielectric loss. Lastly, samples of the developed dielectric material are printed with silver overtop to demonstrate how the material can be effectively incorporated into fully printed, multilayer RF applications.

Polymorphic haplotypes of CRELD1 differentially predispose Down syndrome and euploids individuals to atrioventricular septal defect.

To explore the role of CRELD1 variants on congenital heart defects, we sequenced the entire reading frame of CRELD1 in the samples from Kolkata and adjoining areas. Nearly, 400 participants were included in the genetic association study and they were stratified as Down syndrome (DS) with atrioventricular septal defect (AVSD), DS without AVSD, euploid with AVSD, and euploid without AVSD. A significant association was found between AVSD and three polymorphisms, namely rs9878047 (c.1049-129T > C), rs3774207 (c.1119C > T), and rs73118372 (c.1136T > C) among the Down syndrome and euploid individuals. The polymorphism rs73118372, involves a transition (c.1136T > C) that leads to change in amino acid methionine to threonine which alters protein secondary structure as confirmed by the bioinformatics software SOPMA. In addition, two haplotypes, C-T-C and C-T-T, in the order of loci rs9878047-rs3774207-rs73118372 were associated with incidence of AVSD among euploid and Down syndrome, with a slightly higher odds ratio in the later group. We hypothesize that these haplotypes increase the risk of AVSD, and the susceptibility is exacerbated in DS, possibly due to the trisomy 21 genetic background. Moreover, we report for the first time on an interaction between the mutant alleles of rs3774207 and rs73118372 which could disrupt the delicate balance between different CRELD1 isoforms.

Effectiveness of BBV152/Covaxin and AZD1222/Covishield vaccines against severe COVID-19 and B.1.617.2/Delta variant in India, 2021: a multi-centric hospital-based case-control study.

OBJECTIVES: India introduced BBV152/Covaxin and AZD1222/Covishield vaccines in January 2021. We estimated the effectiveness of these vaccines against severe COVID-19 among individuals aged ≥45 years. METHODS: We did a multi-centric, hospital-based, case-control study between May and July 2021. Cases were severe COVID-19 patients, and controls were COVID-19 negative individuals from 11 hospitals. Vaccine effectiveness (VE) was estimated for complete (2 doses ≥ 14 days) and partial (1 dose ≥ 21 days) vaccination; interval between two vaccine doses and vaccination against the Delta variant. We used the random effects logistic regression model to calculate the adjusted odds ratios (aOR) with a 95% confidence interval (CI) after adjusting for relevant known confounders. RESULTS: We enrolled 1143 cases and 2541 control patients. The VE of complete vaccination was 85% (95% CI: 79-89%) with AZD1222/Covishield and 71% (95% CI: 57-81%) with BBV152/Covaxin. The VE was highest for 6-8 weeks between two doses of AZD1222/Covishield (94%, 95% CI: 86-97%) and BBV152/Covaxin (93%, 95% CI: 34-99%). The VE estimates were similar against the Delta strain and sub-lineages. CONCLUSION: BBV152/Covaxin and AZD1222/Covishield were effective against severe COVID-19 among the Indian population during the period of dominance of the highly transmissible Delta variant in the second wave of the pandemic. An escalation of two-dose coverage with COVID-19 vaccines is critical to reduce severe COVID-19 and further mitigate the pandemic in the country.

Variety-specific arsenic accumulation in 44 different rice cultivars (O. sativa L.) and human health risks due to co-exposure of arsenic-contaminated rice and drinking water.

Arsenic (carcinogenic) is a global health concern due to its presence in groundwater and subsequent accumulation in cultivated-rice via irrigation. The present work focused on the evaluation of arsenic concentration in groundwater, different cultivated-rice varieties (studied together for the first-time) and related health-risks. Arsenic in groundwater (0.26-0.73 mg/L) exceeded the World Health Organization limit for drinking water (0.01 mg/L). Arsenic concentration in rice-grains was found in the range: < 0.0003-2.6 mg/kg dry-weights, where 42 rice varieties (out of total 44) exceeded the Codex Alimentarius Commission limit of polished-rice (0.2 mg/kg). The variety-specific differential-response of arsenic-accumulation was observed (first-time report), where high yielding rice varieties (HYV) were more prone to accumulate arsenic in comparison to local varieties (LV), however, 'Radhunipagol' (an aromatic LV) exhibited as a moderate arsenic-accumulator (BCF = 2.8). The cumulative estimated-daily-intakes (EDICumulative) of arsenic in central-tendency-exposure were observed to be 0.029, 0.031 and 0.04 mg/kg-day among children, teenagers and adults, respectively. The EDICumulative for possible reasonable-maximum-exposure among the above mentioned subpopulation was 0.038, 0.04 and 0.05 mg/kg-day, respectively. The evaluated Cumulative Hazard Index and Individual Excess Lifetime Cancer Risk values suggested that the studied population is under extremely severe cancerous and noncancerous risks to arsenic co-exposures via drinking water and rice.

A Portable Micro-Gas Chromatography with Integrated Photonic Crystal Slab Sensors on Chip.

The miniaturization of gas chromatography (GC) systems has made it possible to utilize the analytical technique in various on-site applications to rapidly analyze complex gas samples. Various types of miniaturized sensors have been developed for micro-gas chromatography (µGC). However, the integration of an appropriate detector in µGC systems still faces a significant challenge. We present a solution to the problem through integration of µGC with photonic crystal slab (PCS) sensors using transfer printing technology. This integration offers an opportunity to utilize the advantages of optical sensors, such as high sensitivity and rapid response time, and at the same time, compensate for the lack of detection specificity from which label-free optical sensors suffer. We transfer printed a 2D defect free PCS on a borofloat glass, bonded it to a silicon microfluidic gas cell or directly to a microfabricated GC column, and then coated it with a gas responsive polymer. Realtime spectral shift in Fano resonance of the PCS sensor was used to quantitatively detect analytes over a mass range of three orders. The integrated µGC-PCS system was used to demonstrate separation and detection of a complex mixture of 10 chemicals. Fast separation and detection (4 min) and a low detection limit (ng) was demonstrated.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica.

BACKGROUND: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. OBJECTIVE: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. METHODS: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. RESULTS: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. CONCLUSION: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two - state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

Anisotropic interaction driven surface modulation on spray-dried microgranules.

Rapid evaporation of solvent from spray colloidal droplets induces directed self-assembly among the nanoparticles, eventually interlocking them into correlated granular structures. In this work, it is demonstrated that anisotropy in colloidal interparticle interaction plays a key role in governing the surface topology of spray-dried granules. Colloidal dispersion comprised of spherical nanosilica (NS) and cylindrical carbon nanotubes (CNT) was chosen as a model system in this regard. For identical polarities of the colloidal components, granules with prominent wrinkle-like modulations are obtained, which is in drastic contrast with the case of opposite polarities. The extent of surface modulation depends on the relative concentration of CNT with respective to NS. A plausible mechanism for the formation of surface modulation is elucidated on the basis of the evolving anisotropic interparticle interactions during assembly. Electron microscopy, small-angle scattering, Raman spectroscopic techniques have been used for quantitative characterization of these micro-granules.

Optofluidic vapor sensing with free-space coupled 2D photonic crystal slabs.

We report here a compact vapor sensor based on polymer coated two-dimensional (2D) defect-free photonic crystal slabs (PCS). The sensing mechanism is based on the resonance spectral shift associated with the Fano resonance mode in the PCS due to the vapor molecule adsorption and desorption induced changes in both polymer thickness and polymer refractive index (RI). Sensitivity due to RI and thickness change were theoretically investigated respectively. With three different thicknesses of OV-101 polymer coating, sensitivity and response time were experimentally evaluated for hexane and ethanol vapors. The polymer demonstrated roughly four times higher sensitivity towards the hexane vapor than ethanol vapor. The PCS sensor with thicker polymer coating showed higher sensitivity to both hexane and ethanol vapors but exhibiting longer response time.

Porous nano-structured micro-granules from silica-milk bi-colloidal suspension: Synthesis and characterization.

Synthesis and characterization of nano-structured porous granules, with fairly defined morphology and porosity, is crucial because such granules are widely utilized for various technological applications. However, an easy, one-step, economic synthesis protocol for large scale production is extremely desirable. In the present work, we have reported the synthesis and characterization of the nano-structured micro-granules using aerosol drying of bi-colloidal suspension of nano-silica and milk. Removal of soft organic component from the granules results in formation of meso and macro pores with moderate specific surface area. Granule morphology and porosity depends strongly on the concentration ratio of the individual components in the drying aerosol as well as the interaction between them.

Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci.

BACKGROUND: The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression. MATERIALS AND METHODS: A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes. RESULTS: Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only. CONCLUSION: Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their capacity to form biofilms.