ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.

Reversible changes in structure and function of photosynthetic apparatus of pea (Pisum sativum) leaves under drought stress.

The effects of drought on photosynthesis have been extensively studied, whereas those on thylakoid organization are limited. We observed a significant decline in gas exchange parameters of pea (Pisum sativum) leaves under progressive drought stress. Chl a fluorescence kinetics revealed the reduction of photochemical efficiency of photosystem (PS)II and PSI. The non-photochemical quenching (NPQ) and the levels of PSII subunit PSBS increased. Furthermore, the light-harvesting complexes (LHCs) and some of the PSI and PSII core proteins were disassembled in drought conditions, whereas these complexes were reassociated during recovery. By contrast, the abundance of supercomplexes of PSII-LHCII and PSII dimer were reduced, whereas LHCII monomers increased following the change in the macro-organization of thylakoids. The stacks of thylakoids were loosely arranged in drought-affected plants, which could be attributed to changes in the supercomplexes of thylakoids. Severe drought stress caused a reduction of both LHCI and LHCII and a few reaction center proteins of PSI and PSII, indicating significant disorganization of the photosynthetic machinery. After 7 days of rewatering, plants recovered well, with restored chloroplast thylakoid structure and photosynthetic efficiency. The correlation of structural changes with leaf reactive oxygen species levels indicated that these changes were associated with the production of reactive oxygen species.

Protective Role of Nanoceria-Infused Nanofibrous Scaffold toward Bone Tissue Regeneration with Senescent Cells.

The presence of oxidative stress in bone defects leads to delayed regeneration, especially in the aged population and patients receiving cancer treatment. This delay is attributed to the increased levels of reactive oxygen species (ROS) in these populations due to the accumulation of senescent cells. Tissue-engineered scaffolds are emerging as an alternative method to treat bone defects. In this study, we engineered tissue scaffolds tailored to modulate the adverse effects of oxidative stress and promote bone regeneration. We used polycaprolactone to fabricate nanofibrous mats by using electrospinning. We exploited the ROS-scavenging properties of cerium oxide nanoparticles to alleviate the high oxidative stress microenvironment caused by the presence of senescent cells. We characterized the nanofibers for their physical and mechanical properties and utilized an ionization-radiation-based model to induce senescence in bone cells. We demonstrate that the presence of ceria can modulate ROS levels, thereby reducing the level of senescence and promoting osteogenesis. Overall, this study demonstrates that ceria-infused nanofibrous scaffolds can be used for augmenting the osteogenic activity of senescent progenitor cells, which has important implications for engineering bone tissue scaffolds for patients with low regeneration capabilities.

The senescent mesothelial matrix accentuates colonization by ovarian cancer cells.

Ovarian cancer is amongst the most morbid of gynecological malignancies due to its diagnosis at an advanced stage, a transcoelomic mode of metastasis, and rapid transition to chemotherapeutic resistance. Like all other malignancies, the progression of ovarian cancer may be interpreted as an emergent outcome of the conflict between metastasizing cancer cells and the natural defense mounted by microenvironmental barriers to such migration. Here, we asked whether senescence in coelom-lining mesothelia, brought about by drug exposure, affects their interaction with disseminated ovarian cancer cells. We observed that cancer cells adhered faster on senescent human and murine mesothelial monolayers than on non-senescent controls. Time-lapse epifluorescence microscopy showed that mesothelial cells were cleared by a host of cancer cells that surrounded the former, even under sub-confluent conditions. A multiscale computational model predicted that such colocalized mesothelial clearance under sub-confluence requires greater adhesion between cancer cells and senescent mesothelia. Consistent with the prediction, we observed that senescent mesothelia expressed an extracellular matrix with higher levels of fibronectin, laminins and hyaluronan than non-senescent controls. On senescent matrix, cancer cells adhered more efficiently, spread better, and moved faster and persistently, aiding the spread of cancer. Inhibition assays using RGD cyclopeptides suggested the adhesion was predominantly contributed by fibronectin and laminin. These findings led us to propose that the senescence-associated matrisomal phenotype of peritoneal barriers enhances the colonization of invading ovarian cancer cells contributing to the metastatic burden associated with the disease.

Acetylcholine Structure-Based Small Activatable Fluorogenic Probe for Specific Detection of Acetylcholinesterase.

Early detection of Alzheimer's disease (AD) is important for taking proper measures against AD pathogenesis. Acetylcholinesterase (AChE) is widely reported to be associated with the pathogenicity of AD. Here, employing the "acetylcholine-mimic" approach, we designed and synthesized a new class of naphthalimide (Naph)-based fluorogenic probes for specific detection of AChE and avoiding interference of butyrylcholinesterase (BuChE), the pseudocholinesterase. We investigated the action of the probes on Electrophorus electricus AChE, and the native human brain AChE that we expressed in Escherichia coli and purified in the active form for the first time. The probe Naph-3 exhibited a substantial fluorescence enhancement with AChE and majorly avoided BuChE. Naph-3 successfully crossed the cell membrane of the Neuro-2a cells and fluoresced upon reaction with endogenous AChE. We further established that the probe could be effectively used for screening AChE inhibitors. Our study provides a new avenue for the specific detection of AChE, which can be extended to the diagnosis of AChE-related complications.

Asymmetric total synthesis of diosniponols A and B.

A concise asymmetric total synthesis of diosniponols A and B has been achieved based on an enantioselective Jacobsen kinetic resolution of racemic epoxide and the important 2,3-dihydro-4H-pyran-4-one moiety being installed by the metal-free δ-hydroxyalkynone rearrangement catalyzed by p-TsOH. A diastereoselective catalytic hydrogenation set the required all-syn stereochemistry leading to diosniponol A, which then, under the Mitsunobu inversion conditions, provided diosniponol B. The structure and absolute stereochemistry of the natural products were further confirmed.

Paradoxical behaviour of rainfall and temperature over ecologically sensitive areas along the Western Ghats.

Initial reports signify some specific isolated locations in different latitudes, revealing a paradoxical increase in both heavy and very heavy rainfall events and also an increment in total, i.e., in both rainfall and temperature, over ecologically sensitive areas along the Western Ghats (WG). This paper presents a coherent study of the full-scale of daily rainfall and temperature over 27 well-spaced stations in the study area to determine its extent and investigate whether or not this contradictory behaviour is real. Also, an attempt has been made to assess the differential behaviour of rainfall, temperature, and heavy rainfall events in association with land use and land cover change (LULC). The analysis revealed that rainfall and temperature over the study area are increasing, whereas heavy rainfall events have increased during 1981-2020 with strong peaks after 2000 around 18-19°N (Mumbai metropolitan region), 14-16°N (mining and quarrying regions in Goa), and 9-12°N (a narrow strip of land spanning across the coastal towns of Karnataka and Kerala) latitudes. The majority of the rainfall excess years coincided with El Nino years, indicating that El Nino does not affect rainfall negatively. However, rainfall over the WG is influenced by local relief and cascading topography. The spatial pattern of average annual rainfall shows a decreasing trend from south to north because the elevation and span of rainfall occurrence are higher in the southern part of WG. The findings of the current research will help in building a strategy to address trends and patterns of climatic variables in association with LULC.

Suppression of photorespiratory metabolism by low O2 and presence of aminooxyacetic acid induces oxidative stress in Arabidopsis thaliana leaves.

Photorespiration, an essential component of plant metabolism, was upregulated under abiotic stress conditions, such as high light or drought. One of the signals for such upregulation was the rise in reactive oxygen species (ROS). Photorespiration was expected to mitigate oxidative stress by reducing ROS levels. However, it was unclear if ROS levels would increase when photorespiration was lowered. Our goal was to examine the redox status in leaves when photorespiratory metabolism was restricted under low O2 (medium flushed with N2 gas) or by adding aminooxyacetic acid (AOA), a photorespiratory inhibitor. We examined the impact of low O2 and AOA in leaves of Arabidopsis thaliana under dark, moderate, or high light. Downregulation of typical photorespiratory enzymes, including catalase (CAT), glycolate oxidase (GO), and phosphoglycolate phosphatase (PGLP) under low O2 or with AOA confirmed the lowering of photorespiratory metabolism. A marked increase in ROS levels (superoxide and H2O2) indicated the induction of oxidative stress. Thus, our results demonstrated for the first time that restricted photorespiratory conditions increased the extent of oxidative stress. We propose that photorespiration is essential to sustain normal ROS levels and optimize metabolism in cellular compartments of Arabidopsis leaves. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01388-4.

Culture-Independent Raman Spectroscopic Identification of Bacterial Pathogens from Clinical Samples Using Deep Transfer Learning.

The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

Exopolyphosphatases PPX1 and PPX2 from Mycobacterium tuberculosis regulate dormancy response and pathogenesis.

Stress adaptation and virulence of various bacterial pathogens require stringent response pathways involving guanosine pentaphosphate and inorganic polyphosphate (PolyP). In M. tuberculosis, intracellular PolyP levels are maintained by the activities of polyphosphate kinase (PPK-1, PPK-2) and exopolyphosphatases (PPX-1, PPX-2). We demonstrate that these exopolyphosphatases cumulatively contribute to biofilm formation and survival of M. tuberculosis in nutrient limiting, low oxygen growth conditions and in macrophages. Characterization of single (Δppx2) and double knock out strain (dkppx) of M. tuberculosis demonstrated that these exopolyphosphatases are essential for establishing infection in guinea pigs and mice. Transcriptional profiling revealed that relative to the parental strain the expression of genes belonging to DosR regulon were significantly reduced in mid-log phase cultures of dkppx strain. We also show that PolyP inhibited the autophosphorylation activities associated with DosT and DosS sensor kinases. Host RNA-seq analysis revealed that transcripts involved in various antimicrobial pathways such as apoptosis, autophagy, macrophage activation, calcium signalling, innate and T-cell response were differentially expressed in lung tissues of dkppx strain infected mice. Taken together, we demonstrate that enzymes involved in PolyP homeostasis play a critical role in physiology and virulence of M. tuberculosis. These enzymes are attractive targets for developing novel interventions that might be active against drug-sensitive and drug-resistant M. tuberculosis.

Rapid Discrimination of Bacterial Drug Resistivity by Array-Based Cross-Validation Using 2D MoS2.

The precise discrimination of microbes based on family, class and drug resistivity is essential for the early diagnosis of infectious diseases. Information about the type and strength of drug resistivity can help the analyst to prescribe a suitable antibiotic at the proper dosage to completely eradicate microbes without giving them a chance to gain further resistance. Herein, we propose a sensor array based on the use of cationic two-dimensional MoS2 units and green fluorescence protein as building blocks. Cationic surfaces of receptors with various functionality were suitable for tunable interaction with anionic surfaces of microbes. The array successfully discriminates six different bacterial strains. The versatile ability of the receptors to bind with the wild-type as well as the corresponding ampicillin-resistant strain contributed significantly to rapid detection with high sensitivity. The optimized array was able to classify five different types and three different extents of drug-resistant variants of Escherichia coli by using bacteria cells and lysates. Finally, we have introduced the cross identification method using both bacteria cells and lysates and we found a great enhancement of detection in sensitivity and accuracy. This is the first report of this approach, which can be extended to many other methods for better accuracy in array-based detection.

Cyclic di-GMP sensing histidine kinase PdtaS controls mycobacterial adaptation to carbon sources.

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.

Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness.

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free way to characterize cell-substrate interactions and heterogeneities in cell populations. We quantified the critical adhesion strengths of MCF-7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower critical adhesion strengths (τ50) than their non-invasive counterparts, HPL1D and MCF-7. Detachment dynamics inversely correlated with cell invasion potentials. A theoretical model, based on τ50 values and the distribution of cell areas on substrates, provided good fits to results from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions; non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. Fluorescence intensity of actin was significantly lower in invasive cells as compared to non invasive cells. These correlations in adhesion strengths and traction stresses with cell invasiveness may be useful in cancer diagnostics and other pathologies featuring mis-regulation in adhesion.

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

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

A matrix targeted fluorescent probe to monitor mitochondrial dynamics.

Mitochondria are an indispensable organelle for energy production and regulation of cellular metabolism. The structural and functional alterations to mitochondria instigate pathological conditions of cancer, and aging-associated and neurodegenerative disorders. The normal functioning of mitochondria is maintained by quality control mechanisms involving dynamic fission, fusion, biogenesis and mitophagy. Under conditions of mitophagy and neurodegenerative diseases, mitochondria are exposed to different acidic environments and high levels of reactive oxygen species (ROS). Therefore stable molecular tools and methods are required to monitor the pathways linked to mitochondrial dysfunction and disease conditions. Herein, we report a far-red fluorescent probe (Mito-TG) with excellent biocompatibility, biostability, photostability, chemical stability and turn on emission for selective targeting of the mitochondrial matrix in different live cells. The probe was successfully employed for monitoring dynamic processes of mitophagy and amyloid beta (Aß) induced mitochondrial structural changes.

Inorganic polyphosphate accumulation suppresses the dormancy response and virulence in Mycobacterium tuberculosis.

Stringent response pathways involving inorganic polyphosphate (PolyP) play an essential role in bacterial stress adaptation and virulence. The intracellular levels of PolyP are modulated by the activities of polyphosphate kinase-1 (PPK1), polyphosphate kinase-2 (PPK2), and exopolyphosphatases (PPXs). The genome of Mycobacterium tuberculosis encodes two functional PPXs, and simultaneous deletion of ppx1 and ppx2 results in a defect in biofilm formation. We demonstrate here that these PPXs cumulatively contribute to the ability of M. tuberculosis to survive in nutrient-limiting, low-oxygen growth conditions and also in macrophages. Characterization of single (Δppx2) and double knockout (dkppx) strains of M. tuberculosis indicated that PPX-mediated PolyP degradation is essential for establishing bacterial infection in guinea pigs. RNA-Seq-based transcriptional profiling revealed that relative to the parental strain, the expression levels of DosR regulon-regulated dormancy genes were significantly reduced in the dkppx mutant strain. In concordance, we also provide evidence that PolyP inhibits the autophosphorylation activities associated with DosT and DosS sensor kinases. The results in this study uncover that enzymes involved in PolyP homeostasis play a critical role in M. tuberculosis physiology and virulence and are attractive targets for developing more effective therapeutic interventions.

Rapid detection of bacterial infection and viability assessment with high specificity and sensitivity using Raman microspectroscopy.

Infectious diseases caused by bacteria still pose major diagnostic challenges in spite of the availability of various molecular approaches. Irrespective of the type of infection, rapid identification of the causative pathogen with a high degree of sensitivity and specificity is essential for initiating appropriate treatment. While existing methods like PCR possess high sensitivity, they are incapable of identifying the viability status of the pathogen and those which can, like culturing, are inherently slow. To overcome these limitations, we developed a diagnostic platform based on Raman microspectroscopy, capable of detecting biochemical signatures from a single bacterium for identification as well as viability assessment. The study also establishes a decontamination protocol for handling live pathogenic bacteria which does not affect identification and viability testing, showing applicability in the analysis of sputum samples containing pathogenic mycobacterial strains. The minimal sample processing along with multivariate analysis of spectroscopic signatures provides an interface for automatic classification, allowing the prediction of unknown samples by mapping signatures onto available datasets. Also, the novelty of the current work is the demonstration of simultaneous identification and viability assessment at a single bacterial level for pathogenic bacteria. Graphical abstract.

Nanomotors Sense Local Physicochemical Heterogeneities in Tumor Microenvironments*.

The invasion of cancer is brought about by continuous interaction of malignant cells with their surrounding tissue microenvironment. Investigating the remodeling of local extracellular matrix (ECM) by invading cells can thus provide fundamental insights into the dynamics of cancer progression. In this paper, we use an active untethered nanomechanical tool, realized as magnetically driven nanomotors, to locally probe a 3D tissue culture environment. We observed that nanomotors preferentially adhere to the cancer-proximal ECM and magnitude of the adhesive force increased with cell lines of higher metastatic ability. We experimentally confirmed that sialic acid linkage specific to cancer-secreted ECM makes it differently charged, which causes this adhesion. In an assay consisting of both cancerous and non-cancerous epithelia, that mimics the in vivo histopathological milieu of a malignant breast tumor, we find that nanomotors preferentially decorate the region around the cancer cells.

NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G.

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.

Photorespiration is complemented by cyclic electron flow and the alternative oxidase pathway to optimize photosynthesis and protect against abiotic stress.

Optimization of photosynthetic performance and protection against abiotic stress are essential to sustain plant growth. Photorespiratory metabolism can help plants to adapt to abiotic stress. The beneficial role of photorespiration under abiotic stress is further strengthened by cyclic electron flow (CEF) and alternative oxidase (AOX) pathways. We have attempted to critically assess the literature on the responses of these three phenomena-photorespiration, CEF and AOX, to different stress situations. We emphasize that photorespiration is the key player to protect photosynthesis and upregulates CEF as well as AOX. Then these three processes work in coordination to protect the plants against photoinhibition and maintain an optimal redox state in the cell, while providing ATP for metabolism and protein repair. H2O2 generated during photorespiratory metabolism seems to be an important signal to upregulate CEF or AOX. Further experiments are necessary to identify the signals originating from CEF or AOX to modulate photorespiration. The mutants deficient in CEF or AOX or both could be useful in this regard. The mutual interactions between CEF and AOX, so as to keep their complementarity, are also to be examined further.