Evaluation of synbiotic combinations of commercial probiotic strains with different prebiotics in in vitro and ex vivo human gut microcosm model.

Exploring probiotics for their crosstalk with the host microbiome through the fermentation of non-digestible dietary fibers (prebiotics) for their potential metabolic end-products, particularly short-chain fatty acids (SCFAs), is important for understanding the endogenous host-gut microbe interaction. This study was aimed at a systematic comparison of commercially available probiotics to understand their synergistic role with specific prebiotics in SCFAs production both in vitro and in the ex vivo gut microcosm model. Probiotic strains isolated from pharmacy products including Lactobacillus sporogenes (strain not labeled), Lactobacillus rhamnosus GG (ATCC53103), Streptococcus faecalis (T-110 JPC), Bacillus mesentericus (TO-AJPC), Bacillus clausii (SIN) and Saccharomyces boulardii (CNCM I-745) were assessed for their probiotic traits including survival, antibiotic susceptibility, and antibacterial activity against pathogenic strains. Our results showed that the microorganisms under study had strain-specific abilities to persist in human gastrointestinal conditions and varied anti-infective efficacy and antibiotic susceptibility. The probiotic strains displayed variation in the utilization of six different prebiotic substrates for their growth under aerobic and anaerobic conditions. Their prebiotic scores (PS) revealed which were the most suitable prebiotic carbohydrates for the growth of each strain and suggested xylooligosaccharide (XOS) was the poorest utilized among all. HPLC analysis revealed a versatile pattern of SCFAs produced as end-products of prebiotic fermentation by the strains which was influenced by growth conditions. Selected synbiotic (prebiotic and probiotic) combinations showing high PS and high total SCFAs production were tested in an ex vivo human gut microcosm model. Interestingly, significantly higher butyrate and propionate production was found only when synbiotics were applied as against when individual probiotic or prebiotics were applied alone. qRT-PCR analysis with specific primers showed that there was a significant increase in the abundance of lactobacilli and bifidobacteria with synbiotic blends compared to pre-, or probiotics alone. In conclusion, this work presents findings to suggest prebiotic combinations with different well-established probiotic strains that may be useful for developing effective synbiotic blends.

Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis, biological and molecular characterization.

The increasing demands of keratinases for biodegradation of recalcitrant keratinaceous waste like chicken feathers has lead to research on newer potential bacterial keratinases to produce high-value products with biological activities. The present study reports a novel keratinolytic bacterium Bacillus velezensis strain ZBE1 isolated from deep forest soil of Western Ghats of Karnataka, which possessed efficient feather keratin degradation capability and induced keratinase production. Production kinetics depicts maximum keratinase production (11.65 U/mL) on 4th day with protein concentration of 0.61 mg/mL. Effect of various physico-chemical factors such as, inoculum size, metal ions, carbon and nitrogen sources, pH and temperature influencing keratinase production were optimized and 3.74 folds enhancement was evidenced through response surface methodology. Silver (AgNP) and zinc oxide (ZnONP) nanoparticles with keratin hydrolysate produced from chicken feathers by the action of keratinase were synthesized and verified with UV-Visible spectroscopy that revealed biological activities like, antibacterial action against Bacillus cereus and Escherichia coli. AgNP and ZnONP also showed potential antioxidant activities through radical scavenging activities by ABTS and DPPH. AgNP and ZnONP revealed cytotoxic effect against MCF-7 breast cancer cell lines with IC50 of 5.47 µg/ml and 62.26 µg/ml respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the thermostability, structure and surface attributes. The study suggests the prospective applications of keratinase to trigger the production of bioactive value-added products and significant application in nanotechnology in biomedicine.

In-vitro and computational analysis of Urolithin-A for anti-inflammatory activity on Cyclooxygenase 2 (COX-2).

Cyclooxygenase 2 (COX-2) participates in the inflammation process by converting arachidonic acid into prostaglandin G2 which increases inflammation, pain and fever. COX-2 has an active site and a heme pocket and blocking these sites stops the inflammation. Urolithin A is metabolite of ellagitannin produced from humans and animals gut microbes. In the current study, Urolithin A showed good pharmacokinetic properties. Molecular docking of the complex of Urolithin A and COX-2 revealed the ligand affinity of -7.97 kcal/mol with the ligand binding sites at TYR355, PHE518, ILE517 and GLN192 with the 4-H bonds at a distance of 2.8 Å, 2.3 Å, 2.5 Å and 1.9 Å. The RMSD plot for Urolithin A and COX-2 complex was observed to be constant throughout the duration of dynamics. A total of 3 pair of hydrogen bonds was largely observed on average of 3 simulation positions for dynamics duration of 500 ns. The MMPBSA analysis showed that active site amino acids had a binding energy of -22.0368 kJ/mol indicating that throughout the simulation the protein of target was bounded by Urolithin A. In-silico results were validated by biological assays. Urolithin A strongly revealed to exhibit anti-inflammatory effect on COX-2 with an IC50 value of 44.04 µg/mL. The anti-inflammatory capability was also depicted through reduction of protein denaturation that showed 37.6 ± 0.1 % and 43.2 ± 0.07 % reduction of protein denaturation for BSA and egg albumin respectively at 500 µg/mL. The present study, suggests Urolithin A to be an effective anti-inflammatory compound for therapeutic use.

Daily consumption of galactooligosaccharide gummies ameliorates constipation symptoms, gut dysbiosis, degree of depression and quality of life among sedentary university teaching staff: A double-blind randomized placebo control clinical trial.

BACKGROUND: Functional constipation affects approximately 10% of the Indian population and may reduce the quality of life (QOL) and increase gut dysbiosis. PURPOSE OF STUDY: The study aimed at assessing the impact of galactooligosaccharide (GOS) gummy supplementation on gut health, depression status and QOL of constipated subjects. METHODS: A double-blind placebo control clinical trial (CTRI/2021/10/037474) was conducted on sedentary constipated adults (n = 35), who were split into an experimental group (n = 17) and a control group (n = 18), supplemented with 10 g GOS and sugar gummies, respectively, for 30 days. Relative abundance of fecal gut microbes, including Bifidobacterium, Lactobacillus, Clostridium and Bacteroides and phyla Bacteroidetes and Firmicutes using real-time polymerase chain reaction and short-chain fatty acids, was analyzed pre and post supplementation. Constipation profile was studied using Rome IV criteria and the Bristol stool chart. Depression status was studied using the Becks Depression Inventory. The QOL was assessed using patient assessment of constipation. RESULTS: GOS gummy supplementation increased Bifidobacterium and Lactobacillus by 1230% and 322%, respectively, (p < 0.001; p < 0.01) with reduced Clostridium by 63%, phylum Firmicutes by 73% and Bacteroidetes by 85% (p < 0.01). The GOS-supplemented group demonstrated a higher F/B ratio (4.2) indicating improved gut health (p < 0.01) with reduced gut dysbiosis and constipation severity. GOS gummies enhanced acetic acid and butyric acid levels compared to the control group (p < 0.01; p < 0.001). Post supplementation, there was 40% reduction in depression (p < 0.01) and 22% improvement in QOL (p < 0.05). CONCLUSIONS: This research validates the predicted beneficial benefits of short-term GOS consumption on constipation profile, gut microflora, depression status and quality of life of constipated subjects.

Synchronization of the circadian clock to the environment tracked in real time.

The circadian system of the cyanobacterium Synechococcus elongatus PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.

Molecular docking and simulation studies against nucleoside diphosphate kinase (NDK) of Pseudomonas aeruginosa with secondary metabolite identified by genome mining from paenibacillusehimensis.

Pseudomonas aeruginosa is one of the leading opportunistic pathogens that causes nosocomial pneumonia and mostly in people with cystic fibrosis. In the present study, an in-silicoapproach was adopted to identify the novel drug target against Pseudomonas aeruginosa by employing subtractive genomics and molecular docking studies. Each step in the subtractive genomics scrutinized the bacterial proteome and determined a potential drug target against Pseudomonas aeruginosa. 71 essential proteins were obtained from the subcellular localization method that resides in the extracellular region. Metabolic pathways were studied to elucidate the unique pathways where the involvement of proteins present in the pathogen was predicted and a total of 6 unique pathways were determined. By, Genome mining of the source organism Paenibacillusehimensis, 9 ligands were obtained. The molecular docking analysis between the binding site of target protein NDK and ligands was carried out by employing the AutoDock Vina tool. Based on the highest binding affinity, Paenibactin, AnabaenopeptinNZ857 and Nostamide A complex with NDK protein with a lower binding energy of -7.5 kcal/mol, -7.4and -7.2 kcal/molrespectively were considered for the simulation studies. Molecular dynamics simulation studies showed the ligand in complex with protein was highly stable and rigid for a duration of 150 ns. For Paenibactin, AnabaenopeptinNZ857 and Nostamide Acomplex with protein, RMSD plot showed a deviation of ∼0.2-0.3 nm till ∼30ns/50 ns-110ns and further stabilized. The radius of the gyration plot clearly showed that the values stayed at ∼1.45 nm- 1.55 nm showing compactness and stability. The SASA stayed at the range ∼80nm2 and at least one total number of hydrogen bonds was shown throughout the 150 ns simulation for all three possible ligand-protein complexes. In the RMSF plot, the maximum fluctuation was ranged from ∼0.4-0.42 nm at the range between ∼57ns-60ns.The Paenibactin, AnabaenopeptinNZ857 and Nostamide A complex with NDK protein showed a stable, rigid and compact interaction throughout the simulation of duration 150 ns.Communicated by Ramaswamy H. Sarma.

Health and Lifestyle Issues among Youth: A Record Analysis of Contributing Factors among Beneficiaries Attending Youth Mental Health Promotion Clinics (Yuva Spandana Kendras) in Karnataka, India.

Background: Youth are consideren to be most vulnerable to health and lifestyle issues (HLS) in India. The current study aims to investigate the factors that contribute to health and lifestyle issues among youth attending mental health promotion clinics (YMHP) in Karnataka. Method: Three-year first-visit data from beneficiaries (aged 15-35 years) attending YMHP clinics in Karnataka between 2017 and 2020 were analyzed. Multivariable logistic regression analysis included beneficiaries reporting any HLS issue as the outcome and a host of 57 hypothesized variables as exposures. Results: Overall, 2,615 (25%) beneficiaries reported HLS issues. Years of schooling (AOR 5-7 years = 0.89; 95% confidence interval [CI] =0.60-1.31), (AOR 8-10 years = 0.65; 95% CI = 0.46-0.91), (AOR >10 years = 0.67; 95% CI = 0.49-0.93)], unemployed youth (AOR = 0.52; 95% CI = 0.45-0.61) business and salaried workers (AOR = 1.69; 95% CI = 1.33-2.13), and other occupations (AOR = 2.11; 95% CI = 1.73-2.56), junk food consumption (AOR = 0.76;95% CI = 0.68-0.84), having issues related to relationships with parents (AOR = 3.01; 95% CI = 2.47-3.68) and intergenerational issues (AOR = 1.71; 95% CI = 1.19-2.45), self-development issues (AOR low-self-awareness = 1.57; 95% CI = 1.33-1.85), (AOR low-self-esteem = 1.29; 95% CI = 1.062-1.57), (AOR emotional issues = 1.57; 95% CI = 1.31-1.89), education and academics (AOR = 1.23; 95% CI = 1.09-1.39), safety issues (AOR = 4.11; 95% CI = 3.07-5.50), gender sex and sexuality issues (AOR = 2.44; 95% CI = 1.43-4.15), suicidal ideation (AOR = 1.91; 95% CI = 1.44-2.54), substance use (AOR tobacco chewing = 1.45; 95% CI = 1.09-1.93), (AOR tobacco-smoking = 1.66; 95% CI = 1.18-2.32), (AOR smoking = 4.94; 95% CI = 3.52-6.93) and experiencing emotions (AOR feel anxious = 1.63; 95% CI = 1.41-1.88), (AOR forgetfulness = 1.50; 95% CI = 1.41-1.98), (AOR difficulty in concentration = 1.37; 95% CI = 1.035-1.81), (AOR anger = 1.61; 95% CI = 1.25-2.07), (AOR feel worthless = 2.21; 95% CI = 1.71-2.86) were associated with HLS issues among beneficiaries. Conclusion: This analysis addresses an important but neglected component of HLS issues among youth highlighting the importance of early intervention among youth to prevent the development of diseases later in life. The study has important implications for youth health promotion in India and countries such as India. Health and Lifestyle Issues Among Youth: A record analysis of contributing factors among beneficiaries attending Youth Mental Health promotion clinics (Yuva Spandana Kendras) in Karnataka, India.

Assays for L-type voltage gated calcium channels.

Voltage gated calcium channels (VGCCs) are pursued as drug targets for neurodegenerative and cardiovascular diseases. High throughput drug screening targeting VGCCs depends on patch-clamp electrophysiology or fluorophore-based calcium imaging that requires powerful equipment and specialized expertise thus leading to cost escalation. Moreover, VGCC needs to be transfected into cell lines such as HEK-293. We report the presence of L-type VGCC (L-VGCC) subunit proteins, Cav1.2, α2δ and ß in HEK-293 cells and the application of simple methods for its assay. Endogenous expression of the channel in HEK-293 cells overcomes the need for transfection. L-VGCC in HEK-293 cells was activated either by the agonist, BayK8644 or by KCl-mediated depolarization. Activity was detected using the calcium sensing probe, GCaMP6m by live imaging. L-VGCC activity induced enhancement in GCaMP6m fluorescence returned to baseline corresponding to channel-closure. Activity was also shown using a methodology involving end-point detection of the calcium dependent interaction of α-CaMKII with NMDA receptor subunit GluN2B sequence. This methodology further simplifies the assay as it eliminates the need for real time imaging. Activation was blocked by the specific L-type VGCC antagonist, nifedipine. Finding the protein and activity of L-VGCC in HEK-293 cells offers commercially viable assays for drug screening.

Role of Ca2+/Calmodulin-Dependent Protein Kinase Type II in Mediating Function and Dysfunction at Glutamatergic Synapses.

Glutamatergic synapses harbor abundant amounts of the multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMKII). Both in the postsynaptic density as well as in the cytosolic compartment of postsynaptic terminals, CaMKII plays major roles. In addition to its Ca2+-stimulated kinase activity, it can also bind to a variety of membrane proteins at the synapse and thus exert spatially restricted activity. The abundance of CaMKII in glutamatergic synapse is akin to scaffolding proteins although its prominent function still appears to be that of a kinase. The multimeric structure of CaMKII also confers several functional capabilities on the enzyme. The versatility of the enzyme has prompted hypotheses proposing several roles for the enzyme such as Ca2+ signal transduction, memory molecule function and scaffolding. The article will review the multiple roles played by CaMKII in glutamatergic synapses and how they are affected in disease conditions.

Coupling of distant ATPase domains in the circadian clock protein KaiC.

The AAA+ family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.

Micro-Raman spectroscopy of the light-harvesting pigments in Chlamydomonas reinhardtii under salinity stress.

Microalgae are a rich source of carotenoids with enhanced yields during biotic or abiotic stresses, which often impose survival challenges on the cells. Using a non-invasive pigment profiling approach with micro-Raman spectroscopy, we have analyzed the effect of salinity stress on carotenoids in autotrophic Chlamydomonas reinhardtii. Raman spectral analysis of ν(C = C) mode indicates an increase in the carotenoids with lower conjugation length (lutein and zeaxanthin) compared to ß-carotene, as the function of culture age and salinity stress, but especially when salinity stress was imposed in two-stage mode (stress imposed on 2nd day, D2_100, and 4th day, D4_100, during exponential phase). Population-scale heterogeneities in carotenoid Raman mode peak center, quantified with heterogeneity index (HI), were highest during the stationary phase of the cultures and under salinity stress. Although the Raman signal was obtained from a randomly selected small focal volume in the cell, a decrease in chlorophyll Raman mode intensities with age and salinity stress was well corroborated by single-cell population fraction measurements by microscopy. Raman intensity fluctuations (If) were high for both chlorophyll and carotenoid modes under salinity stress, which can arise due to variations in chlorophyll/carotenoid content and composition, or conformational changes in the pigments in C. reinhardtii cells. Interestingly, in all growth conditions, chlorophyll a Raman mode intensity was found to show a high correlation to that of ß-carotene, pointing out a high degree of cooperativity in the light-harvesting complex pigments even during salinity stress. Thus, we demonstrate the usefulness of non-invasive pigment profiling with micro-Raman spectroscopy for developing an optimization for salinity stress conditions for high biomass yield and proper harvest time to obtain carotenoids with desired chemical composition.

Label free detection of SARS CoV-2 Receptor Binding Domain (RBD) protein by fabrication of gold nanorods deposited on electrochemical immunosensor (GDEI).

Coronavirus Disease 2019 (COVID-19) pandemic has shown the need for early diagnosis to manage infectious disease outbreaks. Here, we report a label free electrochemical Fluorine-Doped Tin Oxide (FTO) Immunosensor coupled with gold nanorods (GNRs) as an electron carrier for ultrasensitive detection of the Receptor Binding Domain (RBD) of SARS CoV-2 Spike protein. The RBD gene was cloned, and expressed in-house with confirmed molecular weight of ∼31 kDa via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). RBD antibodies (Ab) were generated to be used as a bioreceptor for sensor fabrication, and characterized using SDS-PAGE, Western Blot, and Enzyme-Linked Immunosorbent Assay (ELISA). GNRs were fabricated on the electrode surface, followed by immobilization of RBD Ab. The conjugation steps were confirmed by UV-Vis Spectroscopy, Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Cyclic Voltammetry (CV), and Differential Pulse Voltammetry (DPV). The fabricated electrode was further optimized for maximum efficiency and output. The detection limit of the developed electrode was determined as 0.73 fM for RBD antigen (Ag). Furthermore, the patient nasopharyngeal samples were collected in Viral Transport Media (VTM), and tested on the sensor surface that resulted in detection of SARS CoV-2 within 30 s, which was further validated via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, the immunosensor showed good repeatability, storage stability, and minimal cross reactivity against Middle East Respiratory Syndrome (MERS) spike protein. Along with ease of fabrication, the electrodes show future miniaturization potential for extensive and rapid screening of populations for COVID-19.

Linkage of Microbial Parameters with Sediment Physicochemical Properties in Subsurface Fluvial Sediment Deposits of the Mahi River Basin, Western India.

The linkage between sediment physicochemical and microbial parameters within river terrace sedimentary ecosystems of semiarid regions is still in infancy. Here, we investigated microbial enzyme activities involved in C, N, P, and S cycling, and quantitative PCR (qPCR) based gene abundance of two laterally deposited sediment cores (28 and 25 m deep) comprising the Late Quaternary sediments of the Mahi River (Gujarat, India). Gene abundance indicates the presence of a sustainable bacterial population throughout both cores. The stratified subsurface sediments had notable microbial enzyme activities indicating an important role of both cores in biogeochemical cycling. Correlation between microbial and geological parameters revealed that various trace elements, rare earth elements, K2O, P2O5, EC, TDS, and salinity link significantly with microbial parameters. However, the direction and magnitude of the correlation differ in both cores under study. These results emphasize that sediment physicochemical properties influence microbial parameters differently in the laterally deposited subsurface sediments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00998-4.

A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy.

As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as a model system for detailed mechanistic studies of day-night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the "bulk" and "edge" KaiBC sites in stoichiometric and substoichiometric KaiBiC6 complexes (i = 1-5). We provide kinetic evidence to support the intermediacy of the "edge" KaiBC sites as bridges and nucleation sites between free KaiB and the "bulk" KaiBC sites. Furthermore, we show that the relative abundance of "edge" and "bulk" sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.

Alterations of Primary Metabolites in Root Exudates of Intercropped Cajanus cajan-Zea mays Modulate the Adaptation and Proteome of Ensifer (Sinorhizobium) fredii NGR234.

Legume-cereal intercropping systems, in the context of diversity, ecological function, and better yield have been widely studied. Such systems enhance nutrient phytoavailability by balancing root-rhizosphere interactions. Root exudates (RE) play an important role in the rhizospheric interactions of plant-plant and/or plant-microbiome interaction. However, the influence of the primary metabolites of RE on plant-rhizobia interactions in a legume-cereal intercrop system is not known. To understand the plant communication with rhizobia, Cajanus cajan-Zea mays intercropped plants and the broad host range legume nodulating Ensifer fredii NGR234 as the model plants and rhizobium used respectively. A metabolomics-based approach revealed a clear separation between intercropped and monocropped RE of the two plants. Intercropped C. cajan showed an increase in the myo-inositol, and proline, while intercropped Z. mays showed enhanced galactose, D-glucopyranoside, and arginine in the RE. Physiological assays of NGR234 with the RE of intercropped C. cajan exhibited a significant enhancement in biofilm formation, while intercropped Z. mays RE accelerated the bacterial growth in the late log phase. Further, using label-free proteomics, we identified a total of 2570 proteins of NGR234 covering 50% annotated protein sequences upon exposure to Z. mays RE. Furthermore, intercropped Z. mays RE upregulated bacterioferritin comigratory protein (BCP), putative nitroreductase, IlvD, LeuC, D (branched-chain amino acid proteins), and chaperonin proteins GroEL2. Identification offered new insights into the metabolome of the legume-cereal intercrop and proteome of NGR234-Z. mays interactions that underline the new molecular candidates likely to be involved in the fitness of rhizobium in the intercropping system.

Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.

Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.

Evaluation of bacterial strains isolated from Late Quaternary alluvial sediments spanning ~ 28 m in depth for heavy metal tolerance and Cr(VI) removal ability.

Microbial heavy metal tolerance in subsurface samples is indicative of long-term ecotoxicological impact of metals and could also reflect metal contamination of groundwater. However, the heavy metal tolerance characteristics of microbes isolated from subsurface river sediment profiles are still obscure. In the present study, determination of heavy metal tolerance of bacterial strains isolated from two Late Quaternary sediment profiles (~ 28 m and ~25 m deep) located at the Mahi river basin, Western India, was carried out. Identification of bacterial isolates by the 16S rRNA gene sequencing revealed that bacterial isolates affiliated with phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes were dominant in both sediment profiles. Heavy metal tolerance of bacterial strains as determined by plate diffusion assay revealed order of metal tolerance as follows: Hg(II)

Monitoring Protein-Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy.

The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.

Glu60 of α-Calcium/calmodulin dependent protein kinase II mediates crosstalk between the regulatory T-site and protein substrate binding region of the active site.

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.

Recovery of proteins from coconut milk whey employing ultrafiltration and spray drying.

The coconut whey (CW) (underutilized by-product of virgin coconut oil production, with 2-3% protein) was subjected to ultrafiltration for concentration of protein and sugar removal prior to spray drying. The process parameters of ultrafiltration were standardised with respect to transmembrane flux, protein retention efficiency and removal of sugar by using 300 kDa membrane cut off, feed pH 4 and 2 bar transmembrane pressure at temperature 25 ± 2 °C. The protein content was found to increase in the retentate after ultrafiltration (termed as concentrated coconut whey, CCW) (from 21 to 46%, w/w) and sugar content to reduce (from 59 to 34%, w/w). The color lightness (L*) values of the CW and CCW powders obtained by spray drying were found to be 83.81 ± 0.33 and 80.70 ± 0.47, respectively. Both the samples of coconut protein powders were found to be microbiologically safe having water activity index for CCWP of 0.27 and CWP of 0.26. Carr index values for CCW (32.95) and CW (33.14) powders indicate both of them to have fair flowability. The powders have high potential as new source of protein or as a functional ingredient in the food processing industries.