Distinct localization of chiral proofreaders resolves organellar translation conflict in plants.

Plants have two endosymbiotic organelles originated from two bacterial ancestors. The transition from an independent bacterium to a successful organelle would have required extensive rewiring of biochemical networks for its integration with archaeal host. Here, using Arabidopsis as a model system, we show that plant D-aminoacyl-tRNA deacylase 1 (DTD1), of bacterial origin, is detrimental to organellar protein synthesis owing to its changed tRNA recognition code. Plants survive this conflict by spatially restricting the conflicted DTD1 to the cytosol. In addition, plants have targeted archaeal DTD2 to both the organelles as it is compatible with their translation machinery due to its strict D-chiral specificity and lack of tRNA determinants. Intriguingly, plants have confined bacterial-derived DTD1 to work in archaeal-derived cytosolic compartment whereas archaeal DTD2 is targeted to bacterial-derived organelles. Overall, the study provides a remarkable example of the criticality of optimization of biochemical networks for survival and evolution of plant mitochondria and chloroplast.

The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination.

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1 and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.

The Fanconi Anemia (FA) pathway is the subject of intense interest owing to the role of FA as a tumor suppressor. Three FA complex proteins, FANCM, MHF1 and MHF2, were identified as factors that suppress crossover during meiosis in the model plant Arabidopsis thaliana. Here, the authors extended these findings and identified a novel anti-crossover factor and showed that it encodes the plant FANCC homolog, which was previously thought to be vertebrate-specific. They further showed that FANCC regulates meiotic crossover together with two other FA proteins, FANCE and FANCF. This suggests that the FANCC­E­F subcomplex was already regulating DNA repair in the common ancestor of all living eukaryotes.

OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA.

Among all approaches used for the semisynthesis of natural or chemically modified products, enzyme-assisted ligation is among the most promising and dynamically developing approaches. Applying an efficient C247A mutant of Oldenlandia affinis plant ligase OaAEP1 and solid-phase peptide synthesis chemistry, we present the chemoenzymatic synthesis of a complete sequence of the cysteine-rich and metal-binding cyanobacterial metallothionein Synechococcus metallothionein A (SmtA). Zn(II) and Cd(II) binding to the newly synthesized SmtA showed identical properties to the protein expressed in Escherichia coli. The presented approach is the first example of the use of OaAEP1 mutant for total protein synthesis of metallothionein, which occurs in mild conditions preventing cysteine thiol oxidation. The recognition motif of the applied enzyme could naturally occur in the protein structure or be synthetically or genetically incorporated in some loops or secondary structure elements. Therefore, we envision that this strategy can be used for efficiently obtaining SmtA and for a wide range of proteins and their derivatives.

Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3.

BACKGROUND: Mammalian metallothioneins (MTs) are small (6-7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies. RESULTS: Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recognition motif (LPETG) as removable fusion tags for high-level expression and purification of human MT3 from the bacterial system. In the first strategy, SUMOylated MT3 was expressed and purified using Ulp1-mediated cleavage. In the second strategy, SUMOylated MT3 with a sortase recognition motif at the N-terminus of MT3 was expressed and purified using sortase-mediated cleavage. In the final strategy, the fusion protein His6-SUMO-eSrtA-LPETG-MT3 was expressed and purified by one-step sortase-mediated inducible on-bead autocleavage. Using these three strategies the apo-MT3 was purified in a yield of 11.5, 11, and 10.8 mg/L, respectively, which is the highest yield achieved for MT expression and purification to date. No effect of MT3 on Ni2+-containing resin was observed. CONCLUSION: The SUMO/sortase-based strategy used as the production system for MT3 resulted in a very high expression level and protein production yield. The apo-MT3 purified by this strategy contained an additional glycine residue and had similar metal binding properties as WT-MT3. This SUMO-sortase fusion system is a simple, robust, and inexpensive one-step purification approach for various MTs as well as other toxic proteins with very high yield via immobilized metal affinity chromatography (IMAC).

Graphene oxide-Mn3O4nanocomposites for advanced electrochemical biosensor for fumonisin B1 detection.

Occurrence of mycotoxins in food samples threat to its safety issue due to the presence of high toxicity and carcinogenic behavior, thus requiring highly sensitive and selective detection. Herein, the trimanganese tetraoxide (Mn3O4) nanoparticles in combination with graphene oxide (GO) nanocomposite were used to enhance the electrochemical performance for fabrication of electrochemical biosensor for fumonisin B1 (FB1) detection. The various characterization tools were used to validate the fabrication of GOMn3O4nanocomposites. To fabricate the electrochemical biosensor on an indium tin oxide (ITO) coated glass substrate, a thin film of GOMn3O4nanocomposite was prepared using electrophoretic deposition technique, and antibodies (ab-FB1) were immobilized onto the electrode for selective FB1 detection. The differential pulse voltammetry technique was used to observe the sensing performance. The non-binding sites of the ab-FB1 on the immunoelectrode were blocked with bovine serum albumin (BSA). The biosensor electrode was fabricated as BSA/ab-FB1/GOMn3O4/ITO for the detection of FB1. The sensitivity of the biosensor was obtained as 10.08µA ml ng-1cm-2in the detection range of 1 pg ml-1to 800 ng ml-1with a limit of detection of 0.195 pg ml-1. In addition, the recovery of BSA/ab-FB1/GOMn3O4/ITO immunoelectrodes was also performed on sweet corn samples and is calculated to be 98.91%.

Effect of Imatinib treatment on renal anaemia in chronic myeloid leukemia patients.

PURPOSE: In this study, we investigate renal function and anaemia during imatinib treatment in patients with chronic myeloid leukaemia. METHODS: The patients with chronic myeloid leukaemia with chronic phase who had been treated with only imatinib for 12 months at Rajiv Gandhi Cancer Institute and Research Centre (New Delhi, India) were enrolled and prospectively analysed. The chronic renal impairment parameters, including estimated glomerular filtration rate and haemoglobin levels for anaemia from June 2020 to June 2022, were monitored in newly diagnosed in patients with chronic myeloid leukaemia-chronic phase. The data were analysed by SPSS software version 22. RESULTS: In total 55 patients with chronic myeloid leukaemia chronic phase who had been on imatinib for 12 months were monitored. The mean estimated glomerular filtration rate was significantly decreased (74 ± 14 to 59 ± 12 mL/min/1.73m2, p < 0.001) with a decrease in mean haemoglobin levels after 12 months (10.9 ± 2.01 to 9.0 ± 1.02, p < 0.004). The decreased estimated glomerular filtration rate was negatively correlated with haemoglobin levels after 1 year of imatinib administration (correlation coefficient = 0.892, R2 = 0.7976, p < 0.05). CONCLUSION: We recommended close monitoring of renal function and haemoglobin levels in patients with chronic myeloid leukaemia patients.

Rapid and label-free detection of Aflatoxin-B1viamicrofluidic electrochemical biosensor based on manganese (III) oxide (Mn3O4) synthesized by co-precipitation route at room temperature.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin, naturally occurring in food items, and it causes several types of lethal diseases. Therefore, a rapid and convenient detection method for AFB1 is the first step toward overcoming the effect of AFB1. The current work presents the development of an efficient microfluidic electrochemical-based biosensor using tri-manganese tetroxide nanoparticles (Mn3O4nps) for AFB1 detection. The Mn3O4nps were synthesized at room temperature through the co-precipitation route. Its phase purity, structural and morphological studies have been characterized through x-ray diffraction, Raman spectroscopy, energy-dispersive x-ray, Fourier transform infrared spectroscopy and transmission electron microscopy. The mask-less UV-lithography was carried out to fabricate the three-electrode chip and microfluidic channel of the microfluidic electrochemical biosensing system. The designed microfluidic immunosensor (BSA/Ab-AFB1/Mn3O4/ITO) was fabricated using the three-electrode chip, microfluidic channel in poly-dimethyl siloxane. The fabricated sensor exhibited the 3.4µA ml ng-1cm-2sensitivity and had the lowest lower detection limit of 0.295 pg ml-1with the detection range of 1 pg ml-1to 300 ng ml-1. Additionally, the spiked study was also performed with this immunoelectrode and a recovery rate was obtained of 108.2%.

Impact of imatinib treatment on renal function in chronic myeloid leukaemia patients.

BACKGROUND: Recently, multiple epidemiological studies have linked imatinib with the alteration of renal function in chronic myeloid leukaemia (CML) patients. This meta-analysis aimed to summarize the impact of imatinib use on renal function in CML patients. METHODS: A systematic search was conducted on MEDLINE and Embase to identify articles assessing the impact of imatinib exposure on renal function in CML patients. The risk of bias was assessed using the Newcastle-Ottawa scale (NOS). Two authors independently performed literature-screening, risk of bias and data extraction. The risk of renal dysfunction (chronic kidney disease or acute kidney injury) among imatinib users was computed as the primary outcome of interest. The certainty of findings was assessed using the grading of recommendations assessment, development and evaluation (GRADE) criteria. RESULTS: A total of nine articles qualified for inclusion in the systematic review, of which four articles were eligible for meta-analysis. Based on the scoring on NOS, majority of the included studies were found to be of moderate risk of bias. Majority of the studies (n = 6) reported significantly (p < .05) decrease in estimated glomerular filtration rate (eGFR) after imatinib treatment. The risk of developing renal dysfunction (chronic kidney disease or acute kidney injury) was found to be significantly higher in imatinib users as compared to other tyrosine kinase inhibitor (TKI) users with a pooled relative risk of 2.70 (95% CI: 1.49-4.91). Sensitivity analysis also revealed a consistently high risk of renal dysfunction with imatinib use. GRADE criteria revealed low certainty of evidence. CONCLUSION: This meta-analysis found an increased risk of renal dysfunction in imatinib users compared to other TKI users.

Role of the Serine/Threonine Kinase 11 (STK11) or Liver Kinase B1 (LKB1) Gene in Peutz-Jeghers Syndrome.

Peutz-Jeghers syndrome (PJS) is a well-described inherited syndrome, characterized by the development of gastrointestinal polyps and characteristic mucocutaneous freckling. PJS is an autosomal prevailing disease, due to genetic mutation on chromosome 19p, manifested by restricted mucocutaneous melanosis in association with gastrointestinal (GI) polyposis. The gene for PJS has recently been shown to be a serine/threonine kinase, known as LKB1 or STK11, which maps to chromosome subband 19p13.3. This gene has a putative coding region of 1302 bp, divided into nine exons, and acts as a tumor suppressor in the hamartomatous polyps of PJS patients and in the other neoplasms that develop in PJS patients. It is probable that these neoplasms develop from hamartomas, but it remains possible that the LKB1 or STK11 locus plays a role in a different genetic pathway of tumor growth in the cancers of PJS patients. This article focuses on the role of LKB1 or STK11 gene expression in PJS and related cancers.

No association between proton pump inhibitor use and risk of dementia: Evidence from a meta-analysis.

BACKGROUND AND AIM: A growing body of literature suggests the association between dementia risk and proton pump inhibitor (PPI) use. Therefore, we aimed to investigate the association between PPI use and dementia risk. METHODS: An extensive literature search was performed in PubMed, Embase, and Cochrane till March 31, 2019. All the studies (cohort and case-control) assessing the association between PPI use and dementia risk were eligible for inclusion. Articles were selected based on the screening of title and abstract, data were extracted, and risk of bias was assessed using Newcastle-Ottawa scale. The primary outcome was pooled risk of dementia among PPI user as compared with non-PPI user. Secondary outcomes include dementia risk based on subgroups. Statistical analysis was performed using review manager software. RESULTS: Twelve studies (eight cohort and four case-control) were found to be eligible for inclusion. Majority of the studies were of high quality. Dementia was diagnosed based on International Classification of Diseases 9/10 codes in majority of the included studies. PPI use was not associated with the dementia risk, with a pooled relative risk (RR) of 1.05 (95% confidence interval [CI]: 0.96-1.15), P = 0.31. Subgroup analysis based on study design (cohort: P = 0.14; case-control: P = 0.14), sex (RR 1.25 [95% CI: 0.97-1.60], P = 0.08), histamine 2 receptor antagonist blockers (P = 0.93), and Alzheimer's disease (RR 1.00 [95% CI: 0.91-1.09], P = 0.93) revealed no significant association between PPI use and dementia risk. CONCLUSION: We found no significant association between PPI use and the risk of dementia or Alzheimer's disease.

Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference.

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Solvation and hydrogen bonding aided efficient non-radiative deactivation of polar excited state of 5-aminoquinoline.

The fluorescence of 5-aminoquinoline (5AQ) is quenched strongly in alcoholic solvents, in stark contrast to the enhancement in fluorescence observed earlier for its positional isomer, 3-aminoquinoline (3AQ). This phenomenon has been explained by the involvement of a highly dipolar excited state, which is manifested in the solvatochromism of 5AQ. A marked wavelength dependence of fluorescence decays of 5AQ in alcoholic solvents is ascribed to the ultrafast solvation of the highly dipolar excited state in these solvents. The resultant stabilization of this state leads to a decrease in the gap between its energy and lower lying triplet as well as ground singlet states, resulting in an efficient non-radiative relaxation and consequently, fluorescence quenching. Solvation dynamics reported for 5AQ is somewhat slower than earlier reports with coumarin probes, due to the involvement of solute-solvent hydrogen bonds, especially in the excited state of the solute. At lower temperatures, solvation is slowed down. An extreme case of this phenomenon is the absence of solvent relaxation at liquid nitrogen temperature. The fluorescence lifetime increases from tens of picoseconds at room temperature to tens of nanoseconds at cryogenic temperature, lending credence to the proposed model.

Value of CD16/CD66b/CD45 in comparison to CD55/CD59/CD45 in diagnosis of paroxysmal nocturnal haemoglobinuria: An Indian experience.

BACKGROUND & OBJECTIVES: Diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), a rare haematopoietic stem cell disorder, is challenging in patients with bone marrow failure (BMF) syndrome like aplastic anaemia (AA). This study was conducted with the aim to test the efficacy of the newly recommended markers viz. anti-CD16 and CD66b antibody over the existing anti-CD55 and CD59 antibody for PNH diagnosis in India. METHODS: This study was conducted on 193 suspected cases of PNH by flow cytometry using lyse wash technique to stain the granulocytes with CD16/CD66b and CD55/CD59. RESULTS: Of the 193 suspected cases, 62 patients showed the presence of PNH clone. Forty six patients were detected by CD55/CD59/CD45, whereas 61 were detected by CD16/CD66b/CD45. CD16/CD66b detected 16 (25.8%) additional patients over CD55/CD59 (P<0.05) and was more sensitive in detecting the PNH clone with higher negative predictive value. Most of the patients (11/16) who were picked up by CD16/CD66b were of AA who had small clone sizes. Further, the PNH clones were more discreetly identified in CD16/CD66b plots than by CD55/CD59. Clone size assessed by CD16/CD66b which reflects the clinical severity of classical PNH (thrombosis/haemolysis), was more representative of the underlying clinical condition than CD55/59. INTERPRETATION & CONCLUSIONS: In our experience of 62 patients of PNH, CD16/CD66b proved to be more efficacious in detecting PNH. The new panel was especially useful in monitoring PNH associated with BMF which had small clone sizes.

Radial Artery Occlusion - Incidence, Predictors and Long-term outcome after TRAnsradial Catheterization: clinico-Doppler ultrasound-based study (RAIL-TRAC study).

Background and aim The purpose of this study was to assess incidence, predictors and outcome of radial artery occlusion (RAO) after transradial catheterization (TRC) based on clinical and Doppler ultrasound study. Methods A total of 1,945 consecutive patients undergoing transradial catheterization for diagnostic evaluation or intervention were included. Radial artery examination was based on palpation and colour Doppler study on the day before, 1 day (D1), 1 month (D30) and 6 months (D180) following the procedure. RAO was defined as absence of pulse on palpation and forward flow on Doppler study. Predictors of RAO were found by logistic regression analysis. Results Baseline demographic and procedural data were recorded. The mean radial arterial diameter was 2.56 ± 0.29 mm. On D1, radial artery Doppler examination revealed RAO in 339 patients (17.4%) but pulse was still palpable in 115 (34%) of them. At D30, these were 221 (11.4%) and 114 (52%), respectively, as no new RAO were noted. Interestingly, 118 (34.8%) patients had spontaneous recanalization of their radial artery as shown by catch-up in patency rate. At D180, these were 99 (5.1%) and 68 (69%), respectively, meaning further new catch-up implying further recanalization. Patients with persistent RAO remained asymptomatic. On multivariate analysis, female sex, diabetes, lower BMI, radial artery diameter ≤2.2 mm and radial artery-to-sheath ratio (AS ratio) < 1 were predictors of RAO. Conclusion TRC for coronary angiography, ad hoc and staged angioplasty can be performed with similar efficacy and safety though RAO occurs more frequently in patients with prior radial artery cannulation and with larger sheath size. Persistent RAO remains asymptomatic.

Encoding temporal information in deep convolution neural network.

A recent development in deep learning techniques has attracted attention to the decoding and classification of electroencephalogram (EEG) signals. Despite several efforts to utilize different features in EEG signals, a significant research challenge is using time-dependent features in combination with local and global features. Several attempts have been made to remodel the deep learning convolution neural networks (CNNs) to capture time-dependency information. These features are usually either handcrafted features, such as power ratios, or splitting data into smaller-sized windows related to specific properties, such as a peak at 300 ms. However, these approaches partially solve the problem but simultaneously hinder CNNs' capability to learn from unknown information that might be present in the data. Other approaches, like recurrent neural networks, are very suitable for learning time-dependent information from EEG signals in the presence of unrelated sequential data. To solve this, we have proposed an encoding kernel (EnK), a novel time-encoding approach, which uniquely introduces time decomposition information during the vertical convolution operation in CNNs. The encoded information lets CNNs learn time-dependent features in addition to local and global features. We performed extensive experiments on several EEG data sets-physical human-robot collaborations, P300 visual-evoked potentials, motor imagery, movement-related cortical potentials, and the Dataset for Emotion Analysis Using Physiological Signals. The EnK outperforms the state of the art with an up to 6.5% reduction in mean squared error (MSE) and a 9.5% improvement in F1-scores compared to the average for all data sets together compared to base models. These results support our approach and show a high potential to improve the performance of physiological and non-physiological data. Moreover, the EnK can be applied to virtually any deep learning architecture with minimal effort.

Nano-modified screen-printed electrode-based electrochemical immunosensors for oral cancer biomarker detection in undiluted human serum and saliva samples.

This proposed work reports the development of in-house made conductive ink-based screen-printed electrodes (SPEs) for label-free detection of oral cancer biomarkers. Carbon ink synthesis includes graphite powder, gum arabic, and water. The selectivity test of the fabricated SPE involves immobilizing antibodies specific to biomarkers and challenges with redox-active interference, other serum molecules, and non-target biomarkers. Three different biomarkers, cytokeratin-19 fragment (CYFRA 21-1), interleukin 8 (IL-8), and tumor protein p53 (TP-53), act as target entities for the detection of oral cancer in patients' samples (serum, N = 28, and saliva, N = 16) at an early stage. The standard technique enzyme-linked immunosorbent assay (ELISA) was employed to estimate the concentration of the biomarkers in serum and saliva samples. SPEs contain amine (-NH2) functional groups involved in covalent bonding with the carboxyl (-COOH) groups of antibody molecules. These immunosensors exhibited remarkably lower detection limits of 829.5 pg mL-1, 0.543 pg mL-1, and 1.165 pg mL-1, and excellent sensitivity of 0.935 µA mL pg-1 cm-1, 0.039 µA mL pg-1 cm-1, and 0.008 µA mL pg-1 cm-1 for CYFRA 21-1, IL-8, and TP-53 biomarkers, respectively. This sensing platform does not require any functionalization for biomolecule immobilization. Thus, it is a cost-effective, disposable, flexible, miniaturized, and sensitive strip to detect oral cancer biomarkers.

A translation proofreader of archaeal origin imparts multi-aldehyde stress tolerance to land plants.

Aldehydes, being an integral part of carbon metabolism, energy generation, and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes, i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

The connection of α- and ß-domains in mammalian metallothionein-2 differentiates Zn(II) binding affinities, affects folding, and determines zinc buffering properties.

Mammalian metallothioneins (MTs) are small Cys-rich proteins involved in Zn(II) and Cu(I) homeostasis. They bind seven Zn(II) ions in two distinct ß- and α-domains, forming Zn3Cys9 and Zn4Cys11 clusters, respectively. After six decades of research, their role in cellular buffering of Zn(II) ions has begun to be understood recently. This is because of different affinities of bound ions and the proteins' coexistence in variously Zn(II)-loaded Zn4-7MT species in the cell. To date, it has remained unclear how these mechanisms of action occur and how the affinities are differentiated despite the Zn(S-Cys)4 coordination environment being the same. Here, we dissect the molecular basis of these phenomena by using several MT2 mutants, hybrid protein, and isolated domains. Through a combination of spectroscopic and stability studies, thiol(ate) reactivity, and steered molecular dynamics, we demonstrate that both protein folding and thermodynamics of Zn(II) ion (un)binding significantly differ between isolated domains and the whole protein. Close proximity reduces the degrees of freedom of separated domains, making them less dynamic. It is caused by the formation of intra- and interdomain electrostatic interactions. The energetic consequence of domains connection has a critical impact on the role of MTs in the cellular environment, where they function not only as a zinc sponge but also as a zinc buffering system keeping free Zn(II) in the right concentrations. Any change of that subtle system affects the folding mechanism, zinc site stabilities, and cellular zinc buffer components.

Prevalence of Anemia among Chronic Myeloid Leukemia Patients Treated with Imatinib: A Evidence-based Meta-analysis.

BACKGROUND: Imatinib is one of the tyrosine kinase inhibitors used for the treatment of chronic myeloid leukemia (CML) patients. The exact association of imatinib with anemia in CML patients is still unclear. AIM: The current study aimed to find the prevalence of anemia in chronic myeloid leukemia patients treated with imatinib. METHODS: The relevant articles were searched in PubMed, Google scholar, and Clinical trials registries till 31st July, 2021. The quality of the articles was assessed using the Newcastle-Ottawa Scale. The prevalence rate with 95% CI was calculated using StatsDirect Statistical analysis software V.3. RESULTS: A total of 18 studies containing 3537 patients were found relevant for the analysis. The pooled prevalence of anemia in CML was found to be 34% (95% CI: 23%-46%). However, the heterogeneity among studies was found to be high. CONCLUSION: The monitoring of hemoglobin levels and identifying the cause of anemia are major concerns for the CML patients treated with Imatinib.