Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling.

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.

Deciphering the mannose transfer mechanism of mycobacterial PimE by molecular dynamics simulations.

Phosphatidyl-myo-inositol mannosides (PIMs), Lipomannan (LM), and Lipoarabinomannan (LAM) are essential components of the cell envelopes of mycobacteria. At the beginning of the biosynthesis of these compounds, phosphatidylinositol (PI) is mannosylated and acylated by various enzymes to produce Ac1/2PIM4, which is used to synthesize either Ac1/2PIM6 or LM/LAM. The protein PimE, a membrane-bound glycosyltransferase (GT-C), catalyzes the addition of a mannose group to Ac1PIM4 to produce Ac1PIM5, using polyprenolphosphate mannose (PPM) as the mannose donor. PimE-deleted Mycobacterium smegmatis (Msmeg) showed structural deformity and increased antibiotic and copper sensitivity. Despite knowing that the mutation D58A caused inactivity in Msmeg, how PimE catalyzes the transfer of mannose from PPM to Ac1/2PIM4 remains unknown. In this study, analyzing the AlphaFold structure of PimE revealed the presence of a tunnel through the D58 residue with two differently charged gates. Molecular docking suggested PPM binds to the hydrophobic tunnel gate, whereas Ac1PIM4 binds to the positively charged tunnel gate. Molecular dynamics (MD) simulations further demonstrated the critical roles of the residues N55, F87, L89, Y163, Q165, K197, L198, R251, F277, W324, H326, and I375 in binding PPM and Ac1PIM4. The mutation D58A caused a faster release of PPM from the catalytic tunnel, explaining the loss of PimE activity. Along with a hypothetical mechanism of mannose transfer by PimE, we also observe the presence of tunnels through a negatively charged aspartate or glutamate with two differently-charged gates among most GT-C enzymes. Common hydrophobic gates of GT-C enzymes probably harbor sugar donors, whereas, differently-charged tunnel gates accommodate various sugar-acceptors.

Crystal structure of a thiolase from Archaeal Pyrococcus furiosus and its in silico functional annotation.

In most of the eukaryotes and archaea, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) essential building blocks of all isoprenoids synthesized in the mevalonate pathway. Here, the first enzyme of this pathway, acetoacetyl CoA thiolase (PFC_04095) from an archaea Pyrococcus furiosus is structurally characterized. The crystal structure of PFC_04095 is determined at 2.7 Å resolution, and the crystal structure reveals the absence of catalytic acid/base cysteine in its active site, which is uncommon in thiolases. In place of cysteine, His285 of HDAF motif performs both protonation and abstraction of proton during the reaction. The crystal structure shows that the distance between Cys83 and His335 is 5.4 Å. So, His335 could not abstract a proton from nucleophilic cysteine (Cys83), resulting in the loss of enzymatic activity of PFC_04095. MD simulations of the docked PFC_04095-acetyl CoA complex show substrate binding instability to the active site pocket. Here, we have reported that the stable binding of acetyl CoA to the PFC_04095 pocket requires the involvement of three protein complexes, i.e., thiolase (PFC_04095), DUF35 (PFC_04100), and HMGCS (PFC_04090).

Predictors for Lymph Node Metastasis and Survival of Patients with T1b Esophageal Adenocarcinoma Treated with Surgery and Endoscopic Therapy: An Analysis of Surveillance, Epidemiology, and End Results Database.

BACKGROUND AND AIMS: Limited data exists regarding the long-term outcomes of endoscopic therapy (ET) with or without chemoradiation therapy (CRT) for T1b esophageal adenocarcinoma (EAC). Our aim was to identify the risk factors for lymph node metastasis (LNM) in T1b EAC and assess how the chosen treatment modality affects overall survival (OS) and cancer-specific survival (CSS). METHODS: We analyzed histologically confirmed T1b EAC patients diagnosed between 2004 and 2018 using Surveillance Epidemiology and End Results database. Focusing on T1bN0M0 staging, we divided the patients into two groups: ET (n=174) and surgery (n=769), and calculated OS and CSS rates. RESULTS: Out of 1418 patients with T1b EAC, 228 cases (16.1%) exhibited LNM at diagnosis. Notable risk factors for LNM included poorly differentiated tumor and lesion size ≥20 mm. For T1bN0M0 cases, ET was commonly performed from 2009 to 2018 (OR 4.3), especially for patients aged ≥ 65 years (OR 3.1) with tumor size <20mm (OR 2.3). During 50 months median follow-up, age ≥ 65 years (HR 1.9), ET (HR 1.5), and CRT (HR 1.4) were associated with poorer OS. Factors linked to decreased CSS were age ≥ 65 years (sub hazard ratio (SHR) 1.6), poorly differentiated tumors (SHR 1.5), and CRT (SHR 1.5). CONCLUSION: In T1b EAC, tumor size ≥20mm and poor differentiation are notable risk factors for LNM. ET showed comparable CSS outcomes to surgery for carefully selected T1bN0M0 lesions. CRT did not provide additional survival benefit for these lesions; however, large scale studies are required to validate this finding.

Effect of non-linear strain stiffening in eDAH and unjamming.

In cell clusters, the prominent factors at play encompass contractility-based enhanced tissue surface tension and cell unjamming transition. The former effect pertains to the boundary effect, while the latter constitutes a bulk effect. Both effects share outcomes of inducing significant elongation in cells. This elongation is so substantial that it surpasses the limits of linear elasticity, thereby giving rise to additional effects. To investigate these effects, we employ atomic force microscopy (AFM) to analyze how the mechanical properties of individual cells change under such considerable elongation. Our selection of cell lines includes MCF-10A, chosen for its pronounced demonstration of the extended differential adhesion hypothesis (eDAH), and MDA-MB-436, selected due to its manifestation of cell unjamming behavior. In the AFM analyses, we observe a common trend in both cases: as elongation increases, both cell lines exhibit strain stiffening. Notably, this effect is more prominent in MCF-10A compared to MDA-MB-436. Subsequently, we employ AFM on a dynamic range of 1-200 Hz to probe the mechanical characteristics of cell spheroids, focusing on both surface and bulk mechanics. Our findings align with the results from single cell investigations. Specifically, MCF-10A cells, characterized by strong contractile tissue tension, exhibit the greatest stiffness on their surface. Conversely, MDA-MB-436 cells, which experience significant elongation, showcase their highest stiffness within the bulk region. Consequently, the concept of single cell strain stiffening emerges as a crucial element in understanding the mechanics of multicellular spheroids (MCSs), even in the case of MDA-MB-436 cells, which are comparatively softer in nature.

Tuneable quantised conductance memory states in TiO2based resistive switching devices in crossbar geometry for high density memory applications.

The tunability and controllability of conductance quantization mediated multilevel resistive switching (RS) memory devices, fabricated in crossbar geometry can be a promising alternative for boosting storage density. Here, we report fabrication of Cu/TiO2/Pt based RS devices in 8 × 8 crossbar geometry, which showed reliable bipolar RS operations. The crossbar devices showed excellent spatial and temporal variability, time retention and low switching voltage (<1 V) and current (∼100µA). Furthermore, during the reset switching, highly repeatable and reliable integral and half-integral quantized conductance (QC) was observed. The observed QC phenomenon was attributed to the two dimensional confinement of electrons as lateral width of the conducting filament (CF) matches the fermi wavelength. The magnitude and number of the QC steps were found to increase from ∼2.5 to 12.5 and from 5 to 18, respectively by increasing the compliance current (IC) from 50 to 800µA which also increased the diameter of the CF from ∼1.2 to 3.3 nm. The enhancement in both number and magnitude of QC states was explained using electrochemical dissolution mechanism of CF of varying diameter. A thicker CF, formed at higherIC, undergoes a gradual rupture during reset process yielding a greater number of QC steps compared to a thinner CF. The realisation of QC states in the crossbar Cu/TiO2/Pt device as well asICmediated tunability of their magnitude and number may find applications in high-density resistive memory storage devices and neuromorphic computing.

Peptide-drug conjugate designated for targeted delivery to HER2-expressing cancer cells.

Targeted therapy of the highest globally incident breast cancer shall resolve the issue of off-target toxicity concurring with augmented killing of specific diseased cells. Thus, the goal of this study was to prepare a peptide-drug conjugate targeting elevated expression of HER2 receptors in breast cancer. Towards this, the rL-A9 peptide was conjugated with the chemotherapeutic drug doxorubicin (DOX) through a N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. The synthesized peptide-drug conjugate, rL-A9-DOX, was characterized by mass spectrometry. Molecular docking studies, based on binding energy data, suggested a stronger interaction of rL-A9-DOX with the HER2 receptor in comparison to the unconjugated peptide, rL-A9. The cytotoxic effect of the rL-A9-DOX conjugate was observed to be higher in HER2-positive SKOV3 cells compared to HER2-negative MDA-MB-231 cells, indicating selective cell killing. Cellular internalization of the rL-A9-DOX conjugate was evident from the flow cytometry analysis, where a noticeable shift in mean fluorescent intensity (MFI) was observed for the conjugate compared to the control group. This data was further validated by confocal microscopy, where the fluorescent signal ascertained nuclear accumulation of rL-A9-DOX. The present studies highlight the promising potential of rL-A9-DOX for targeted delivery of the drug into a defined group of cancer cells.

Crystal structure of a mycobacterial secretory protein Rv0398c and in silico prediction of its export pathway.

Secretory proteins are used by pathogenic bacteria to manipulate the host systems and compete with other microorganisms, thereby enabling their survival in their host. Similar to other bacteria, secretory proteins of Mycobacterium tuberculosis also play a pivotal role in evading immune response within hosts, thereby leading to acute and latent tuberculosis infection. Prokaryotes have several classes of bacterial secretory systems out of which the Sec and Tat pathways are the most conserved in Mtb to transport proteins across the cytoplasmic membrane. Here, we report the crystal structure of a secretory protein, Rv0398c determined to 1.9 Å resolution. The protein comprises a core of antiparallel ß sheets surrounded by α helices adopting a unique ß sandwich fold. Structural comparison with other secretory proteins in Mtb and other pathogenic bacteria reveals that Rv0398c may be secreted via the Sec pathway. Our structural and in silico analyses thus provide mechanistic insights into the pathway adopted by Mtb to transport out secretory protein, Rv0398c which will facilitate the invasion to the host immune system.

Staphylococcal superantigen-like protein 10 enhances the amyloidogenic biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is a highly infectious pathogen that represents a significant burden on the current healthcare system. Bacterial attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in chronic diseases such as endocarditis, osteomyelitis and wound infections. These biofilms decrease bacterial susceptibility to antibiotics and immune defences, making the infections challenging to treatment. S. aureus produces numerous exotoxins that contribute to the pathogenesis of the bacteria. In this study, we have identified a novel function of staphylococcal superantigen-like protein 10 (SSL10) in enhancing the formation of staphylococcal biofilms. Biofilm biomass is significantly increased when SSL10 is added exogenously to bacterial cultures, whereas SSL2 and SSL12 are found to be less active. Exogenously added SSL10 mask the surface charge of the bacterial cells and lowers their zeta potential, leading to the aggregation of the cells. Moreover, the biofilm formation by SSL10 is governed by amyloid aggregation, as evident from spectroscopic and microscopic studies. These findings thereby give the first overview of the SSL-mediated amyloid-based biofilm formation and further drive the future research in identifying potential molecules for developing new antibacterial therapies against Staphylococcus aureus.

Synthesis and 177Lu Labeling of the First Retro Analog of the HER2-Targeting A9 Peptide: A Superior Variant.

The retro analog of the HER2-targeting A9 peptide was synthesized by coupling amino acids in a reverse fashion and switching the N-terminal in the original sequence of the L-A9 peptide (QDVNTAVAW) to the C-terminal in rL-A9 (WAVATNVDQ). Modification in the backbone resulted in higher conformational stability of the retro peptide as evident from CD spectra. Molecular docking analysis revealed a higher HER2 binding affinity of [177Lu]Lu-DOTA-rL-A9 than the original radiopeptide [177Lu]Lu-DOTA-L-A9. Enormously enhanced metabolic stability of the retro analog led to significant elevation in tumor uptake and retention. SPECT imaging studies corroborated biodistribution results demonstrating a remarkably higher tumor signal for [177Lu]Lu-DOTA-rL-A9. The presently studied retro probe has promising efficiency for clinical screening.

Tunable, reversible resistive switching behavior of PVA-zirconia nanocomposite films and validation of the trap-assisted switching mechanism by the selective application of external bias voltages.

Flexible, free-standing polyvinyl alcohol (PVA)-zirconia (mean particle size ∼24 nm) nanocomposite films have been synthesized and their performance as a potential next-generation resistive switching device material has been assessed in this report. The nanocomposite films switch from a high resistive state (HRS) to a low resistive state (LRS) at the SET potential and from LRS to HRS at the RESET potential within the voltage window of 5 V. The origination of trap-assisted SET/RESET potentials has been experimentally validated by analyzing the experimental data and invoking various theoretical models. The impregnation of zirconium dioxide (ZrO2) nanoparticles considerably enhances the interfacial charges facilitated by the formation of dangling bonds. The current (I)-voltage (V) characteristics elucidate how the alteration of free volumetric space in the nanocomposites can modify the SET-RESET potential. This leads to tunable SET/RESET potential, good resistance ratio (∼80), and extensive cycling ability of these PVA-ZrO2 organic flexible nanocomposite films. Herein, we have also investigated the effect of applying external bias voltage (equal to the RESET potential) for possible energy bandgap modification and polymer chain orientation. The impedance spectra differ considerably when the sample is subjected to SET, RESET, and zero voltage bias. The observations have been correlated with the UV-vis absorption spectra and electrical studies. The adopted analysis method and obtained results can open up new avenues for designing and analyzing resistive switching-based random-access memory devices.

Targeting HER2-receptors with 177Lu-labeled triazole stapled cyclic peptidomimetic.

In this study on-resin Cu(I)-catalyzed click reaction was performed to synthesize triazole-stapled cyclic peptidomimetic, DOTA-c[TZ]A9 targeting HER2 receptor expression in breast cancers. Spectroscopic (circular dichroism) and docking analysis provided evidence of enhanced helicity and secondary structure stabilization along with improved HER2 affinity in comparison to the corresponding linear peptide, DOTA-[Pra1, Aza7]A9. 177Lu-labeled cyclic peptide, 177Lu-DOTA-c[TZ]A9 displayed higher in vitro serum stability and in vivo metabolic stability and better HER2 binding affinity {Kd of 16.93 ± 3.02 nM} than the linear counterpart, [177Lu]DOTA-[Pra1, Aza7]A9 {Kd of 26.28 ± 2.87 nM}. Biodistribution profile in SKBR3 tumor bearing SCID mice demonstrated elevated radioactivity levels and prolonged retention of cyclic peptide in the tumor compared to the linear peptide. Thus, solid phase click cyclization technique can be extended towards preparation of triazole-stapled peptides targeting different receptors with improved stability and efficacy.

Residues at the interface between zinc binding and winged helix domains of human RECQ1 play a significant role in DNA strand annealing activity.

RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a ß-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the ß-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2-Zn-WH domains harbouring mutations on the ß-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Role of regional diffusion tensor imaging (DTI)-derived tensor metrics in the evaluation of intracranial gliomas and its histopathological correlation.

Background: The imaging of brain tumours has significantly improved with the use of advanced magnetic resonance (MR) techniques like diffusion tensor imaging (DTI). This study was conducted to analyse the utility of DTI-derived tensor metrics in the evaluation of intracranial gliomas with histopathological correlation and further adoption of these image-data analyses in clinical setting. Methods: A total of 50 patients with suspected diagnosis of intracranial gliomas underwent DTI along with conventional MR examination. The study correlated various DTI parameters in the enhancing part of the tumour and the peritumoral region with the histopathological grades of the intracranial gliomas. Results: The study revealed higher values of Cl (linear anisotropy), Cp (planar anisotropy), AD (axial diffusivity), FA (fractional anisotropy) and RA (relative anisotropy) and lower values of Cs (spherical anisotropy), MD (mean diffusivity) and RD (radial diffusivity) in the enhancing part of the tumour in case of high-grade gliomas. However, in the peritumoral region, the values of Cl, Cp, AD, FA and RA were less whereas values of Cs, MD and RD were more in high-grade gliomas than in the low-grade gliomas. The various cutoff values of these DTI-derived tensor metrics were found to be statistically significant. Conclusion: DTI-derived tensor metrics can be a valuable tool in differentiation between high-grade and low-grade gliomas which might be accepted in clinical practice in near future.

In-silico identification of critical residues in the mannose-transfer mechanism of phosphatidyl-myo-inositol mannosyltransferase B'.

The complex cellular envelope is one of the major reasons behind the survival in hostile conditions and the emergence of the drug-resisting properties of mycobacteria. Phosphatidyl-myo-inositol hexamannoside (PIM6), Lipomannan (LM), and Lipoarabinomannan (LAM) are important structural constituents of the cell envelope and have roles in modulating host immune functions. Phosphatidyl-myo-inositol (PI) is first mannosylated at the 2-position of the inositol group by phosphatidyl-myo-inositol mannosyltransferase A (PimA) to produce phosphatidyl-myo-inositol monomannoside (PIM1). This PIM1 is then further mannosylated at the 6-position of the inositol group by phosphatidyl-myo-inositol mannosyltransferase B' (PimB') utilizing GDP-mannose as the mannose-donor to synthesize phosphatidyl-myo-inositol dimannoside (PIM2) and GDP. Further mannosylation and acylation on PIM2 produce Ac1/2PIM4, which can then be converted to either Ac1/2PIM6 or LM/LAM. Detailed functional mechanism of how PimB' transfers the mannose sugar to PIM1 is not understood. Using molecular docking, the interactions of PimB' with the substrate PIM1 and the product PIM2 are analyzed here. Molecular dynamics (MD) simulations of PimB' with the substrates and the products were performed for 300ns to find out critical residues involved in the mannose-transfer reaction. Docking and MD analyses indicated the residues R206 and R210 bind both PIM1 and PIM2 and are critical in the mannose-transfer reaction. The residues 120HEVGWSMLPGS130 and 281RTRGGGL288 were involved in the transfer of PIM1 from the active site. The residues 18IGG20, K211, E290, G291, 294IV295, and E298 were also important in the mannosylation reaction. The crucial residues obtained from this study may help design novel drugs against mycobacterial PimB'.

A subtractive proteomics and immunoinformatics approach towards designing a potential multi-epitope vaccine against pathogenic Listeriamonocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, which is dangerous for pregnant women, the elderly or individuals with a weakened immune system. Individuals with leukaemia, cancer, HIV/AIDS, kidney transplant and steroid therapy suffer from immunological damage are menaced. World Health Organization (WHO) reports that human listeriosis has a high mortality rate of 20-30% every year. To date, no vaccine is available to treat listeriosis. Thereby, it is high time to design novel vaccines against L. monocytogenes. Here, we present computational approaches to design an antigenic, stable and safe vaccine against the L. monocytogenes that could help to control the infections associated with the pathogen. Three vital pathogenic proteins of L. monocytogenes, such as Listeriolysin O (LLO), Phosphatidylinositol-specific phospholipase C (PI-PLC), and Actin polymerization protein (ActA), were selected using a subtractive proteomics approach to design the multi-epitope vaccine (MEV). A total of 5 Cytotoxic T-lymphocyte (CTL) and 9 Helper T-lymphocyte (HTL) epitopes were predicted from these selected proteins. To design the multi-epitope vaccine (MEV) from the selected proteins, CTL epitopes were joined with the AAY linker, and HTL epitopes were joined with the GPGPG linker. Additionally, a human ß-defensin-3 (hBD-3) adjuvant was added to the N-terminal side of the final MEV construct to increase the immune response to the vaccine. The final MEV was predicted to be antigenic, non-allergen and non-toxic in nature. Physicochemical property analysis suggested that the MEV construct is stable and could be easily purified through the E. coli expression system. This in-silico study showed that MEV has a robust binding interaction with Toll-like receptor 2 (TLR2), a key player in the innate immune system. Current subtractive proteomics and immunoinformatics study provides a background for designing a suitable, safe and effective vaccine against pathogenic L. monocytogenes.

Fluorescently labelled thioacetazone for detecting the interaction with Mycobacterium dehydratases HadAB and HadBC.

Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes.

Stochastic gene transcription with non-competitive transcription regulatory architecture.

The transcription factors, such as activators and repressors, can interact with the promoter of gene either in a competitive or non-competitive way. In this paper, we construct a stochastic model with non-competitive transcriptional regulatory architecture and develop an analytical theory that re-establishes the experimental results with an improved data fitting. The analytical expressions in the theory allow us to study the nature of the system corresponding to any of its parameters and hence, enable us to find out the factors that govern the regulation of gene expression for that architecture. We notice that, along with transcriptional reinitiation and repressors, there are other parameters that can control the noisiness of this network. We also observe that, the Fano factor (at mRNA level) varies from sub-Poissonian regime to super-Poissonian regime. In addition to the aforementioned properties, we observe some anomalous characteristics of the Fano factor (at mRNA level) and that of the variance of protein at lower activator concentrations in the presence of repressor molecules. This model is useful to understand the architecture of interactions which may buffer the stochasticity inherent to gene transcription.

A Study on Clinical Profile of Hemifacial Spasm in India and the Therapeutic Response to Botulinum Toxin Type A Injection as well as Pre and Postinjection Quality of Life.

BACKGROUND: Hemifacial spasm (HFS) is a distressing, involuntary, irregular tonic-clonic contraction of the facial muscles innervated by the seventh cranial nerve. It affects the quality of life. Botulinum toxin is a preferred symptomatic treatment option for the condition. However, there is a lack of study in the Indian scenario. Therefore, we observed the demographic profile, clinical spectrum, therapeutic response, and adverse effects of botulinum toxin and assessed the quality of life in the pre and postinjection phases in our subjects with HFS. MATERIALS AND METHODS: The study design is a prospective open-label observational study. Consecutive cases of HFS were selected from the general neurology outpatient department (OPD) and movement disorder clinic of a medical college hospital in Eastern India. Clinical and relevant neuroimaging studies excluded mimickers and secondary causes of HFS. Institutional Ethics Committee's permission was obtained. Informed consent was taken from patients before botulinum toxin injection. The pre and postinjection assessment tools were spasm rate for a specific period of time, quantification of facial asymmetry, widening palpebral fissure by visual analog scale, Jankovic disability rating scale, HFS-7 scale, and videography. RESULTS: A total of 250 cases of HFS (F:M = 138:112) were studied. The mean age of presentation was 47 years. The mean dose of botulinum toxin injection was 24.2 units per patient. The mean duration of improvement was 4 months. The spasm frequency was decreased by 90%, and the facial asymmetry was improved by 86%. The improvement in quality of life was 86%. Local adverse effects are seen in 10.4% of cases, and all were reversible. CONCLUSION: This is one of the largest studies on the effects of botulinum toxin in subjects with HFS in the Indian population. Periodic injection of botulinum toxin is a safe and effective therapy for subjects with HFS. There is a significant improvement in the quality of life following botulinum toxin therapy in subjects with HFS. Adverse effects were local, mild, well-tolerated, and reversible.