Resurrection plants, which are the "gifts" of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants.

Immunosuppression in tumor microenvironments induced by regulatory T (Treg) cells is regarded a critical mechanism of tumor immune escape and poses a major impediment to cancer immunotherapy. In this study, we developed tLyp1 peptide-conjugated hybrid nanoparticles for targeting Treg cells in the tumor microenvironment. The tLyp1 peptide-modified hybrid nanoparticles presented good stability and effective targeting to Treg cells, and they enhanced the effect of imatinib in downregulating Treg cell suppression through inhibition of STAT3 and STAT5 phosphorylation. In addition, an in vivo study revealed high tumor accumulation of the hybrid nanoparticle. Specifically, prolonged survival rate, enhanced tumor inhibition, reduced intratumoral Treg cells, and elevated intratumoral CD8+ T cells against tumor were observed when combined with checkpoint-blockade by using anti-cytotoxic T-lymphocyte antigen-4 antibody. This study provided groundwork for a repertoire of nanoparticle-based drugs for targeting and modulating Treg cell function in the tumor microenvironment and for improving antitumor immunotherapy.

The use of mice that are mosaic for reporter gene expression underlies many lineage-tracing studies in stem cell biology. For example, using mosaic LacZ reporter mice, it was shown that limbal epithelial stem cells (LESCs) around the periphery of the cornea maintain radial sectors of the corneal epithelium and that radial stripe numbers declined with age. Originally, the corneal results were interpreted as progressive, age-related loss or irreversible inactivation of some LESC clones. In this study we used computer simulations to show that these results could also be explained by stochastic replacement of LESCs by neighbouring LESCs, leading to neutral drift of LESC populations. This was shown to reduce the number of coherent clones of LESCs and hence would coarsen the mosaic pattern in the corneal epithelium without reducing the absolute number of LESCs. Simulations also showed that corrected stripe numbers declined more slowly when LESCs were grouped non-randomly and that mosaicism was rarely lost unless simulated LESC numbers were unrealistically low. Possible reasons why age-related changes differ between mosaic corneal epithelia and other systems, such as adrenal cortices and intestinal crypts, are discussed.

Interest in human brown fat as a novel therapeutic target to tackle the growing obesity and diabetes epidemic has increased dramatically in recent years. While much insight into brown fat biology has been gained from murine cell lines and models, few resources are available to study human brown fat in vitro, which makes the need for new ways to derive and study human brown adipocytes imperative. Human ES cell based reporter systems present an excellent tool to identify, mark, and purify cell populations of choice. In this study, we detail the derivation and characterization of a novel human ES UCP1 reporter cell line that marks UCP1 positive adipocytes in vitro. We targeted a mCherry reporter to the UCP1 stop codon via CRISPR-Cas9 based gene targeting. The brown adipocytes derived from reporter cells express UCP1, display high mitochondrial content, multi-locular lipid morphology, and exhibit functional properties such as lipolysis. The mCherry positive cells purified after cell sorting show elevated expression of brown fat marker genes and a high similarity to isolated human brown fat via RNA-seq analysis. Finally, we demonstrate the utility of this reporter to real time monitor UCP1 expression upon stimulation. This reporter cell line thus presents new opportunities to study human brown fat biology by enabling future work to understand early human brown fat development, perform disease modeling, and facilitate drug screening.

Molecular targeted therapy for specific genes is an emerging research. Retinoic Acid Receptor (RAR-β) is a key tumor suppressor which is found to be lost drastically during much cancer progression. We hence, analyzed the expression level of RAR-β gene during B(a)P induced lung cancer development in mice and studied the lung cancer targeted action of All Trans Retinoic Acid (ATRA) in DOTAP liposomal formulation. The effect of its treatment on lung cancer was determined by histopathological analysis. RAR-β gene expression was assessed by RT-PCR and qPCR. A distinct band for RAR-β gene (density - 0.5123 for lung and 0.5160 for liver) was observed in normal mice, whereas no visible band was observed in cancer induced group, indicating loss of RAR-β gene expression. Both ATRA and lipo-ATRA treated groups showed detectable RAR-β expression with relatively lesser density than the normal group. The expression was more intense in lipo-ATRA treatment (density-0.2973) compared with free ATRA treatment (density-0.1549) in lung tissues. The qPCR results also have highlighted a highly significant (p ≤ 0.01) variation RQ values between lipo-ATRA group (15.46 ± 1.54) and free ATRA group (7.58 ± 1.30) in lung tissue sample on 30th day. The mean RQ value for normal lung on 30th day was 20.86 ± 2.58 against the cancer control. The 120th day mice also showed the similar RAR-β expression pattern with further declined expression levels as there was no treatment given after 30 days. Interestingly, the lipo-ATRA treatment could show a highly significant (p ≤ 0.001) expression (12.00 ± 2.31) when compared with free ATRA treatment (3.31 ± 0.58) which implies that the lipo-ATRA formulation could result in sustained delivery of ATRA in target site. Histopathology of lung and liver on 120th day also revealed an effective therapeutic indication in lipo-ATRA treatment compared to free ATRA treatment due to lipo-ATRA's stealth property and it efficiently inhibited the metastasis to liver. These results revealed that the lipo-ATRA treatment has efficiently delivered ATRA into target site than free ATRA and in-turn it might have induced the expression of RAR-β gene or prevented loss of RAR-β gene in cancer animals.

Based on the previously published pyrazolopyridine-based hit compound for which negative allosteric modulation of both CXCR3 and CXCR4 receptors was disclosed, we designed, synthesized and biologically evaluated a set of novel, not only negative, but also positive allosteric modulators with preserved pyrazolopyridine core. Compound 9e is a dual negative modulator, inhibiting G protein activity of both receptors. For CXCR4 receptor para-substituted aromatic group of compounds distinguishes between negative and positive modulation. Para-methoxy substitution leads to functional antagonism, while para-chloro triggers agonism. Additionally, we discovered that chemotaxis is not completely correlated with G protein pathways. This is the first work in which we have on a series of compounds successfully demonstrated that it is possible to produce selective as well as dual-acting modulators of chemokine receptors, which is very promising for future research in the field of discovery of selective or dual modulators of chemokine receptors.

With the enhancement of commercial manifestation of silver nanoparticles, concerned has risen on their accumulation in aquatic system and consequent effects on fish development and metabolism. In this study, experiments were conducted to assess the impacts of silver nanoparticles on early life cycles of fish considering Zebrafish (Danio rerio) as experimental model. Silver nanoparticles were synthesized through chemical reduction method and characterized through UV-visible spectroscopy, dynamic light scattering (DLS), and HR-TEM. Different sub lethal doses of nanosilver were applied (13.6, 21.6, 42.4, 64, and 128 μgL-1) to post-fertilization phases of Zebrafish embryos and their interaction effects were monitored up to six days period. No significant morphological variations were observed at 13.6, 21.6, 42.4 μgL-1 dose of silver nanoparticles, whereas 64 and 128 μgL-1 exposure dose exhibited bending in myotome, deformity in tail region, somites, notochord and swelling in anterior and posterior region of embryos and larva. Hatching performances analysis elicited highest hatching success in 13.6 and 21.6 μgL-1 doses of silver nanoparticles followed by positive and negative control, whereas exposure dose of 64 and 128 μgL-1 exhibited comparatively lower success. Western blot analysis were conducted on developing hatchlings with Oct4 antibody and at 13.6 and 21.6 μgL-1dose,it showed over expression elucidating stimulatory role of nanosilver in these mentioned doses. In silico analysis depicted a firm interaction of nanosilver with Oct4 revealing their key role in growth stimulation of developing embryos. The study demonstrates the function of nanosilver as a growth promoter rather only as a toxicant in fish metabolic system.

Utilization of cocoa pod husks (CPH) in animal feed is hindered by the presence of theobromine, which is variably toxic to animals. Treatment of this agro-waste to remove theobromine, while preserving its nutrient content, would allow beneficial use of the millions of metric tonnes discarded annually. The aim of this study was to assess the suitability of selected theobromine-degrading filamentous fungi for use as bio-tools in degradation of theobromine in CPH.

Estrogen-related receptors (ERRs) α, β and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRβ/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 μg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 μg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRβ/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules.

Little is known about the neural mechanism and response variability underlying neurosurgical interventions for intractable obsessive-compulsive disorder (OCD).

Although ankylosing spondylitis (AS) is a common, highly heritable arthropathy, the precise genetic mechanism underlying the disease remains elusive. Here, we investigate the disease-causing mutations in a large AS family with distinguished complexity, consisting of 23 patients covering four generations and exhibiting a mixed HLA-B27 (+) and (-) status. Linkage analysis with 32 members using three methods and whole-exome sequencing analysis with three HLA-B27 (+) patients, one HLA-B27 (-) patient, and one healthy individual did not identify a mutation common to all of the patients, strongly suggesting the existence of genetic heterogeneity in this large pedigree. However, if only B27-positive patients were analyzed, the linkage analysis located a 22-Mb region harboring the HLA gene cluster in chromosome 6 (LOD = 4.2), and the subsequent exome analysis identified two non-synonymous mutations in the TREML2 and IP6K3 genes. These genes were resequenced among 370 sporadic AS patients and 487 healthy individuals. A significantly higher mutation frequency of TREML2 was observed in AS patients (1.51% versus 0.21%). The results obtained for the AS pedigree and sporadic patients suggest that mutation of TREML2 is a major factor leading to AS for HLA-B27 (+) members in this large family and that TREML2 is also a susceptibility gene promoting the development of ankylosing spondylitis in HLA-B27 (+) individuals.

Impaired bioenergetics and oxidative damage in the mitochondria are implicated in the etiology of temporal lobe epilepsy, and hyperacetylation of mitochondrial proteins has recently emerged as a critical negative regulator of mitochondrial functions. However, the roles of mitochondrial acetylation and activity of the primary mitochondrial deacetylase, SIRT3, have not been explored in acquired epilepsy. We investigated changes in mitochondrial acetylation and SIRT3 activity in the development of chronic epilepsy in the kainic acid rat model of TLE. Hippocampal measurements were made at 48 h, 1 week and 12 weeks corresponding to the acute, latent and chronic stages of epileptogenesis. Assessment of hippocampal bioenergetics demonstrated a ≥ 27% decrease in the ATP/ADP ratio at all phases of epileptogenesis (p < 0.05), whereas cellular NAD+ levels were decreased by ≥ 41% in the acute and latent time points (p < 0.05), but not in chronically epileptic rats. In spontaneously epileptic rats, we found decreased protein expression of SIRT3 and a 60% increase in global mitochondrial acetylation, as well as enhanced acetylation of the known SIRT3 substrates MnSOD, Ndufa9 of Complex I and IDH2 (all p < 0.05), suggesting SIRT3 dysfunction in chronic epilepsy. Mass spectrometry-based acetylomics investigation of hippocampal mitochondria demonstrated a 79% increase in unique acetylated proteins from rats in the chronic phase vs. controls. Pathway analysis identified numerous mitochondrial bioenergetic pathways affected by mitochondrial acetylation. These results suggest SIRT3 dysfunction and aberrant protein acetylation may contribute to mitochondrial dysfunction in chronic epilepsy.

Trauma to the spinal cord and associated secondary inflammation can lead to permanent loss of sensory and motor function below the injury level, with the resulting environment serving as a barrier that limits regeneration. In this study, we investigate the localized expression of anti-inflammatory cytokines IL-10 and IL-4 via lentiviral transduction in multichannel bridges. Porous multichannel bridges provide physical guidance for axonal outgrowth with the cytokines hypothesized to modulate the neuroinflammatory microenvironment and enhance axonal regeneration. Gene expression analyses indicated that induced IL-10 and IL-4 expression decreased expression of pro-inflammatory genes and increased pro-regenerative genes relative to control. Moreover, these factors led to increased numbers of axons and myelination, with approximately 45% of axons myelinated and the number of oligodendrocyte myelinated axons significantly increased by 3- to 4-fold. Furthermore, the combination of a bridge with IL-10 and IL-4 expression improved locomotor function after injury to an average score of 6 relative to an average score of 3 for injury alone. Collectively, these studies highlight the potential for localized immunomodulation to decrease secondary inflammation and enhance regeneration that may have numerous applications.

REV1 is an evolutionarily conserved translesion synthesis (TLS) DNA polymerase and an assembly factor key for the recruitment of other TLS polymerases to DNA damage sites. REV1-mediated recognition of ubiquitin in the proliferative cell nuclear antigen is thought to be the trigger for TLS activation. Here we report the solution NMR structure of a 108-residue fragment of human REV1 encompassing the two putative ubiquitin-binding motifs UBM1 and UBM2 in complex with ubiquitin. While in mammals UBM1 and UBM2 are both required for optimal association of REV1 with replication factories after DNA damage, we show that only REV1 UBM2 binds ubiquitin. Structure-guided mutagenesis in Saccharomyces cerevisiae further highlights the importance of UBM2 for REV1-mediated mutagenesis and DNA damage tolerance.

Environmental enrichment (EE) is widely used to study the effects of external factors on brain development, function and health in rodent models, but very little is known of the effects of EE on the brain in a large animal model such as the pig. Twenty-four young pigs (aged 5 weeks at start of study, 1:1 male: female ratio) were housed in environmentally enriched (EE) pens and provided with additional enrichment stimulation (a bag filled with straw) once daily. Litter, weight and sex matched controls n= (24) were housed in barren (B) conditions. Behaviour was recorded on alternate days from study day 10. After 21 days, RNA-sequencing of the frontal cortex of male piglets culled one hour after the enrichment stimulation, but not those at 4 h after stimulation, showed upregulation of genes involved in neuronal activity and synaptic plasticity in the EE compared to the B condition. This result is mirrored in the behavioural response to the stimulation which showed a peak in activity around the 1 h time-point. By contrast, EE piglets displayed a signature consistent with a relative decrease in microglial activity compared to those in the B condition. These results confirm those from rodents, suggesting that EE may also confer neuronal health benefits in large mammal models, through a potential relative reduction in neuroinflammatory process and increase in neuroprotection driven by an enrichment-induced increase in behavioural activity.

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

Ordinal comparison of successively presented signal durations requires (a) the encoding of the first signal duration (standard), (b) maintenance of temporal information specific to the standard duration in memory, and (c) timing of the second signal duration (comparison) during which a comparison is made of the first and second durations. Rats were first trained to make ordinal comparisons of signal durations within three time ranges using 0.5, 1.0, and 3.0-s standard durations. Local field potentials were then recorded from the dorsal striatum and sensorimotor cortex in order to investigate the pattern of neural oscillations during each phase of the ordinal-comparison process. Increased power in delta and theta frequency ranges was observed during both the encoding and comparison stages. Active maintenance of a selected response, "shorter" or "longer" (counter-balanced across left and right levers), was represented by an increase of theta and delta oscillations in the contralateral striatum and cortex. Taken together, these data suggest that neural oscillations in the delta-theta range play an important role in the encoding, maintenance, and comparison of signal durations.

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC50 of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies.

Human induced pluripotent stem cells (iPSCs) were generated from exfoliated renal epithelial cells isolated from a urine sample of a 31-year-old healthy woman. Epithelial cells were characterized for the expression of E-cadherin and reprogrammed using non-integrating Sendai viral vectors. The urine-derived iPSC line (designated as MUSIi005-A) was karyotypically normal, expressed pluripotent markers, differentiated into cells of three embryonic germ layers, and showed no viral and transgene expressions at passage 29. Our protocol offers a non-invasive and efficient approach for iPSC generation from patients with genetic or acquired disorders.

Human induced Pluripotent Stem Cells (HiPSCs) have immense potential in research and therapeutics. Under the aegis of Department of Biotechnology funded national program entitled, "The Accelerator program for Discovery in Brain Disorders using Stem Cells (ADBS)" we have established a HiPSC biorepository (https://www.ncbs.res.in/adbs/bio-repository) with an objective to study severe mental illness. The repository comprises of HiPSC lines derived from healthy control donors and individuals with life time diagnosis of severe mental illness from dense families. In the current report we submit information regarding two population control reference lines (male = 1; female = 1) from this biorepository.