The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI-H] CRC with CGI methylator phenotype, 28 MSI-H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies.

Tn antigen is a truncated O-glycan, frequently detected in colorectal cancer (CRC), but its precise role in CRC metastasis is not well addressed. Here we investigated the effects of Core 1 β3Gal-T specific molecular chaperone (Cosmc) deletion-mediated Tn antigen exposure on CRC metastasis and its underlying mechanism. We first used CRISPR/Cas9 technology to knockout Cosmc, which is required for normal O-glycosylation, and thereby obtained Tn-positive CRC cells. We then investigated the biological consequences of Tn antigen expression in CRC. The results showed that Tn-positive cells exhibited an enhanced metastatic capability both in vitro and in vivo. A further analysis indicated that Tn antigen expression induced typical activation of epithelial-mesenchymal transition (EMT). Mechanistically, we found that H-Ras, which is known to drive EMT, was markedly up-regulated in Tn-positive cells, whereas knockdown of H-Ras suppressed Tn antigen induced activation of EMT. Furthermore, we confirmed that LS174T cells (Tn-positive) transfected with wild-type Cosmc, thus expressing no Tn antigen, had down-regulation of H-Ras expression and subsequent inhibition of EMT process. In addition, analysis of 438 samples in TCGA cohort demonstrated that Cosmc expression was reversely correlated with H-Ras, underscoring the significance of Tn antigen-H-Ras signalling in CRC patients. These data demonstrated that Cosmc deletion-mediated Tn antigen exposure promotes CRC metastasis, which is possibly mediated by H-Ras-induced EMT activation.

Until recently, randomized controlled trials have not demonstrated convincing evidence that vitamin D, or vitamin D in combination with calcium supplementation could improve bone mineral density (BMD), osteoporosis and fracture. It remains unclear whether vitamin D levels are causally associated with total body BMD. Here, we performed a Mendelian randomization study to investigate the association of vitamin D levels with total body BMD using a large-scale vitamin D genome-wide association study (GWAS) dataset (including 79 366 individuals) and a large-scale total body BMD GWAS dataset (including 66,628 individuals). We selected three Mendelian randomization methods including inverse-variance weighted meta-analysis (IVW), weighted median regression and MR-Egger regression. All these three methods did not show statistically significant association of genetically increased vitamin D levels with total body BMD. Importantly, our findings are consistent with recent randomized clinical trials and Mendelian randomization study. In summary, we provide genetic evidence that increased vitamin D levels could not improve BMD in the general population. Hence, vitamin D supplementation alone may not be associated with reduced fracture incidence among community-dwelling adults without known vitamin D deficiency, osteoporosis, or prior fracture.

Microtubule (MT) regulation is involved in both neuronal function and the maintenance of neuronal structure, and MT dysregulation appears to be a general downstream indicator and effector of age-related neurodegeneration. But the role of MTs in natural aging is largely unknown. Here, we demonstrate a role of MT regulators in regulating longevity. We find that loss of EFA-6, a modulator of MT dynamics, can delay both neuronal aging and extend the lifespan of C. elegans. Through the use of genetic mutants affecting other MT-regulating genes in C. elegans, we find that loss of MT stabilizing genes (including ptrn-1 and ptl-1) shortens lifespan, while loss of MT destabilizing gene hdac-6 extends lifespan. Via the use of tissue-specific transgenes, we further show that these MT regulators can act in the nervous system to modulate lifespan. Through RNA-seq analyses, we found that genes involved in lipid metabolism were differentially expressed in MT regulator mutants, and via the use of Nile Red and Oil Red O staining, we show that the MT regulator mutants have altered fat storage. We further find that the increased fat storage and extended lifespan of the long-lived MT regulator mutants are dependent on the DAF-16/FOXO transcription factor. Our results suggest that neuronal MT status might affect organismal aging through DAF-16-regulated changes in fat metabolism, and therefore, MT-based therapies might represent a novel intervention to promote healthy aging.

To determine if nonsyndromic cleft lip with or without cleft palate (nsCL/P) genetic risk variants influence liability to nsCL/P through gene regulation pathways, such as those involving DNA methylation.

Meniscal injuries are common among both sport- and non-sport-related injuries, with over 1.7 million meniscal surgeries performed worldwide every year. As meniscal surgeries become more common, so does meniscal allograft transplantation (MAT). However, little is known about the outcomes of MAT in active patients who desire to go back to preinjury activities.

Development of vertebrate jaws involves patterning neural crest-derived mesenchyme cells into distinct subpopulations along the proximal-distal and oral-aboral axes. Although the molecular mechanisms patterning the proximal-distal axis have been well studied, little is known regarding the mechanisms patterning the oral-aboral axis. Using unbiased single-cell RNA-seq analysis followed by in situ analysis of gene expression profiles, we show that Shh and Bmp4 signaling pathways are activated in a complementary pattern along the oral-aboral axis in mouse embryonic mandibular arch. Tissue-specific inactivation of hedgehog signaling in neural crest-derived mandibular mesenchyme led to expansion of BMP signaling activity to throughout the oral-aboral axis of the distal mandibular arch and subsequently duplication of dentary bone in the oral side of the mandible at the expense of tongue formation. Further studies indicate that hedgehog signaling acts through the Foxf1/2 transcription factors to specify the oral fate and pattern the oral-aboral axis of the mandibular mesenchyme.

The mitochondrial calcium uniporter is a Ca2+ channel that regulates intracellular Ca2+ signaling, oxidative phosphorylation, and apoptosis. It contains the pore-forming MCU protein, which possesses a DIME sequence thought to form a Ca2+ selectivity filter, and also regulatory EMRE, MICU1, and MICU2 subunits. To properly carry out physiological functions, the uniporter must stay closed in resting conditions, becoming open only when stimulated by intracellular Ca2+ signals. This Ca2+-dependent activation, known to be mediated by MICU subunits, is not well understood. Here, we demonstrate that the DIME-aspartate mediates a Ca2+-modulated electrostatic interaction with MICU1, forming an MICU1 contact interface with a nearby Ser residue at the cytoplasmic entrance of the MCU pore. A mutagenesis screen of MICU1 identifies two highly-conserved Arg residues that might contact the DIME-Asp. Perturbing MCU-MICU1 interactions elicits unregulated, constitutive Ca2+ flux into mitochondria. These results indicate that MICU1 confers Ca2+-dependent gating of the uniporter by blocking/unblocking MCU.

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.

Ischemic stroke is the second most common cause of death, a major cause of acquired disability in adults. However, the pathogenesis that contributes to ischemic stroke has not been fully understood. Dual-specificity phosphatase 14 (DUSP14, also known as MKP6) is a MAP kinase phosphatase, playing important role in regulating various cellular processes, including oxidative stress and inflammation. However, its effects on cerebral ischemia/reperfusion (IR) are unclear. In the study, we found that DUSP14 expression was decreased responding to IR surgery. Over-expressing DUSP14 reduced the infarction volume of cerebral IR mice. Cognitive dysfunction was also improved in mice with DUSP14 over-expression. Promoting DUSP14 expression markedly reduced the activation of glial cells, as evidenced by the decreases in GFAP and Iba-1 expressions in mice with cerebral IR injury. In addition, inflammatory response induced by cerebral IR injury was inhibited in DUSP14 over-expressed mice, as proved by the reduced expression of tumor necrosis factor (TNF)-α and interleukin 1β (IL-1β). Furthermore, oxidative stress was markedly reduced by DUSP14 over-expression through elevating nuclear factor-erythroid 2 related factor 2 (Nrf-2) signaling pathway. Importantly, we found that DUSP14 could interact with Nrf-1, which thereby protected mice against cerebral IR injury. In vitro, we also found that repressing Nrf-2 expression abrogated DUSP14 over-expression-reduced inflammation and ROS generation. Consistent with the anti-inflammatory effect of DUSP14, reducing the production of reactive oxygen species (ROS) also down-regulated TNF-α and IL-1β expressions. Collectively, elevated DUSP14 alleviated brain damage from cerebral IR injury through Nrf-2-regulated anti-oxidant signaling pathway, and the restraining of inflammatory response. These results suggested that DUSP14 might be a potential therapeutic target to prevent ischemic stroke.

Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro, and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible.

Sphaeranthus indicus whole herb is included as a Rasayana drug in Ayurveda and is reported for the treatment of epilepsy. S. indicus flowers have anxiolytic, hypotensive, peripheral vasodilatory and cathartic activity. The objective of this study was to evaluate the anticonvulsant activity of the extract of flowers of S. indicus in various animal models of epilepsy. The anti-epileptic activity of Methanolic extract (ME) and Acetone extract (AE) of the flowers was evaluated using Maximal electro shock (MES) seizures, Pentyelenetetrazole (PTZ) induced convulsions and Picrotoxin induced convulsions. ME (50 mg/kg and 100 mg/kg) and AE (100 and 200 mg/kg respectively) protected animals against PTZ and Picrotoxin induced convulsion but did not have any effect against MES induced convulsion. In conclusion, the results of this study suggest that both the ME and AE possess promising anticonvulsant activity. It is further suggested that the flavonoids in the extract by the virtue of their effect on benzodiazepine site of GABA receptor, might be responsible for the effect, although no study is undertaken to prove this aspect. Nevertheless, the study provides pharmacological credibility to the anti-epileptic use of S. indicus suggested in Ayurveda.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex pathogenesis. Controlled release, target ability, and multi-channel synergistic treatment are key factors associated with the success of AD drugs. Herein, we report a novel mesoporous nano-selenium (MSe) release delivery system (MSe-Res/Fc-β-CD/Bor) based on the borneol (Bor) target, β-cyclodextrin nanovalves (Fc-β-CD) with loaded resveratrol (Res). Previous experiments have shown that MSe-Res/Fc-β-CD/Bor first releases Bor by interacting with blood or intracellular esterases, allowing the nanosystem to pass through the blood-brain barrier (BBB). Subsequently, the Fc-β-CD is opened by the redox (H2O2) response to the release of Res at the lesion site. We demonstrated that MSe-Res/Fc-β-CD/Bor inhibited aggregation of β-amyloid proteins (Aβ), mitigated oxidative stress, and suppressed tau hyperphosphorylation, while protecting nerve cells and successfully improving memory impairment in APP/PS1 mice. Interestingly, compared with rivastigmine (Riv) positive drugs alone, the MSe/Fc-β-CD/Bor loaded with Riv had a better pharmacokinetic index. These results indicate that MSe-Res/Fc-β-CD/Bor could be a prospective drug for treating AD.

5-Lipoxygenase (5-LOX) is a key enzyme involved in the biosynthesis of pro-inflammatory leukotrienes, leading to asthma. Developing potent 5-LOX inhibitors especially, natural product based ones, are highly attractive. Coumaperine, a natural product found in white pepper and its derivatives were herein developed as 5-LOX inhibitors. We have synthesized twenty four derivatives, characterized and evaluated their 5-LOX inhibition potential. Coumaperine derivatives substituted with multiple hydroxy and multiple methoxy groups exhibited best 5-LOX inhibition. CP-209, a catechol type dihydroxyl derivative and CP-262-F2, a vicinal trihydroxyl derivative exhibited, 82.7% and 82.5% inhibition of 5-LOX respectively at 20 µM. Their IC50 values are 2.1 ± 0.2 µM and 2.3 ± 0.2 µM respectively, and are comparable to zileuton, IC50 = 1.4 ± 0.2 µM. CP-155, a methylenedioxy derivative (a natural product) and CP-194, a 2,4,6-trimethoxy derivative showed 76.0% and 77.1% inhibition of 5-LOX respectively at 20 µM. Antioxidant study revealed that CP-209 and 262-F2 (at 20 µM) scavenged DPPH radical by 76.8% and 71.3% respectively. On the other hand, CP-155 and 194 showed very poor DPPH radical scavenging activity. Pseudo peroxidase assay confirmed that the mode of action of CP-209 and 262-F2 were by redox process, similar to zileuton, affecting the oxidation state of the metal ion in the enzyme. On the contrary, CP-155 and 194 probably act through some other mechanism which does not involve the disruption of the oxidation state of the metal in the enzyme. Molecular docking of CP-155 and 194 to the active site of 5-LOX and binding energy calculation suggested that they are non-competitive inhibitors. The In-Silico ADME/TOX analysis shows the active compounds (CP-155, 194, 209 and 262-F2) are with good drug likeliness and reduced toxicity compared to existing drug. These studies indicate that there is a great potential for coumaperine derivatives to be developed as anti-inflammatory drug.

The genus Ballota L. (Lamiaceae) comprises 33 to 35 species distributed mainly in temperate and subtropical regions of the world. Some species have been used in folk medicine as antiemetic, antispasmodic, sedative agents, vermifuge, antihemorrhoid and also in treatment of cough, etc. AIM OF THIS REVIEW: This review article aims to provide organized information on the available traditional uses, phytochemical and pharmacological studies of the genus Ballota, and to obtain new insights for further researches.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

Human-induced environmental change and habitat fragmentation pose major threats to biodiversity and require active conservation efforts to mitigate their consequences. Genetic rescue through translocation and the introduction of variation into imperiled populations has been argued as a powerful means to preserve, or even increase, the genetic diversity and evolutionary potential of endangered species [1-4]. However, factors such as outbreeding depression [5, 6] and a reduction in available genetic diversity render the success of such approaches uncertain. An improved evaluation of the consequence of genetic restoration requires knowledge of temporal changes to genetic diversity before and after the advent of management programs. To provide such information, a growing number of studies have included small numbers of genomic loci extracted from historic and even ancient specimens [7, 8]. We extend this approach to its natural conclusion, by characterizing the complete genomic sequences of modern and historic population samples of the crested ibis (Nipponia nippon), an endangered bird that is perhaps the most successful example of how conservation effort has brought a species back from the brink of extinction. Though its once tiny population has today recovered to >2,000 individuals [9], this process was accompanied by almost half of ancestral loss of genetic variation and high deleterious mutation load. We furthermore show how genetic drift coupled to inbreeding following the population bottleneck has largely purged the ancient polymorphisms from the current population. In conclusion, we demonstrate the unique promise of exploiting genomic information held within museum samples for conservation and ecological research.

How cell size is determined and maintained remains unclear, even in simple model organisms. In proliferating cells, cell size is regulated by coordinating growth and division through sizer, adder, or timer mechanisms or through some combination [1, 2]. Currently, the best-characterized example of sizer behavior is in fission yeast, Schizosaccharomyces pombe, which enters mitosis at a minimal cell size threshold. The peripheral membrane kinase Cdr2 localizes in clusters (nodes) on the medial plasma membrane and promotes mitotic entry [3]. Here, we show that the Cdr2 nodal density, which scales with cell size, is used by the cell to sense and control its size. By analyzing cells of different widths, we first show that cdr2+ cells divide at a fixed cell surface area. However, division in the cdr2Δ mutant is more closely specified by cell volume, suggesting that Cdr2 is essential for area sensing and supporting the existence of a Cdr2-independent secondary sizer mechanism more closely based on volume. To investigate how Cdr2 nodes may sense area, we derive a minimal mathematical model that incorporates the cytoplasmic kinase Ssp1 as a Cdr2 activator. The model predicts that a cdr2 mutant in an Ssp1 phosphorylation site (cdr2-T166A) [4] should form nodes whose density registers cell length. We confirm this prediction experimentally and find that thin cells now follow this new scaling by dividing at constant length instead of area. This work supports the role of Cdr2 as a sizer factor and highlights the importance of studying geometrical aspects of size control.

Hepatitis B virus (HBV) infection is a major risk factor for chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. While multiple hepatitis B drugs have been developed, build up of drug resistance during treatment or weak efficacies observed in some cases have limited their application. Therefore, there is an urgent need to develop substitutional pharmacological agents for HBV-infected individuals. Here, we identified cetylpyridinium chloride (CPC) as a novel inhibitor of HBV. Using computational docking of CPC to core protein, microscale thermophoresis analysis of CPC binding to viral nucleocapsids, and in vitro nucleocapsid formation assays, we found that CPC interacts with dimeric viral nucleocapsid protein (known as core protein or HBcAg) specifically. Compared with other HBV inhibitors, such as benzenesulfonamide (BS) and sulfanilamide (SA), CPC achieved significantly better reduction of HBV particle number in HepG2.2.15 cell line, a derivative of human HCC cells that stably expresses HBV. CPC also inhibited HBV replication in mouse hydrodynamic model system. Taken together, our results show that CPC inhibits capsid assembly and leads to reduced HBV biogenesis. Thus, CPC is an effective pharmacological agent that can reduce HBV particles.

Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells.