p53-mediated cellular senescence has been intensively investigated, because it is important for tumor suppressive function. In addition, p16/INK4A is well known to be critical for cellular senescence. However, detailed molecular mechanism or relevance between p53 and p16-mediated senescence has not been demonstrated yet. Here we show that p53 induces p16 through Lamin A/C stabilization via direct interaction. Stabilized Lamin A/C promotes degradation of BMI-1 and MEL-18 (Polycomb repressor complex 1, PRC1), which sequesters p16 promotor. Increased p53 can reduce BMI-1/MEL-18 and induce p16 expression via Lamin A/C. Elimination of Lamin A/C can abolish p53-induced p16 expression and BMI-1/MEL-18 reduction. As Lamin A/C expression is increased during cell differentiation, this mechanism seems to be very useful for selective induction of senescence in non-stem cells. Our results suggest that Lamin A/C-p53 network is important for p16/INK4A-mediated cellular senescence.

Axis formation is a fundamental issue in developmental biology. Axis formation and patterning in plant leaves is crucial for morphology and crop productivity. Here, we reveal the basis of proximal-distal patterning in rice leaves, which consist of a proximal sheath, a distal blade, and boundary organs formed between these two regions. Analysis of the three rice homologs of the Arabidopsis BLADE-ON-PETIOLE1 (BOP1) gene indicates that OsBOPs activate proximal sheath differentiation and suppress distal blade differentiation. Temporal expression changes of OsBOPs are responsible for the developmental changes in the sheath:blade ratio. We further identify that the change in the sheath:blade ratio during the juvenile phase is controlled by the miR156/SPL pathway, which modifies the level and pattern of expression of OsBOPs. OsBOPs are also essential for differentiation of the boundary organs. We propose that OsBOPs, the main regulators of proximal-distal patterning, control temporal changes in the sheath:blade ratio of rice leaves.

A definitive diagnosis of tuberculosis serositis (TS) is still challenging. Our preliminary practice found that Serous Fluid Drainage Flocky Precipitate (SFDFP) was a useful testing sample to diagnose TS. We designed this study to assess the diagnostic performance of SFDPF for TS compared with conventional bacteriology methods on serous fluid (SF). A cohort study was conducted from July 2014 to April 2016. Patients with suspected TS were consecutively screened. SF and SFDFP were collected and tested by Ziehl-Neelsen stain, MTB culture, and Xpert/RIF assay. We compared the diagnostic performance of SF and SFDFP in several test settings. Through this study, 85 patients were enrolled, of whom 70 (82.4%) were confirmed TS or highly probable TS, 13 (15.3%) were none-TS and 2 (2.4%) indeterminate results were ruled out. The overall sensitivity using both SFDFP and SF was significantly higher than each (60% vs. 48% and 41%, p < 0.05). SFDFP and SF samples had similar diagnostic performance (p < 0.05). No false positive was detected in this study. We concluded that SFDFP is a reliable testing sample for diagnosing tuberculous serositis. SFDFP may significantly improve the diagnostic yield as a supplement to conventional tests.

Thrombopoietin (TPO) enhances platelet activation through activation of the tyrosine kinase; JAK2 and the lipid kinase phosphatidylinositide 3-kinase (PI3K). The aim of our study was to identify the PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. We found that p110β plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin αIIbβ3 activation and α-granule secretion, (ii) synergistic enhancement of PAR-mediated activation of the small GTPase RAP1, a regulator of integrin activation and (iii) phosphorylation of the PI3K effector Akt. More importantly, the synergistic effect of TPO on phosphorylation of extracellular-regulated kinase (ERK1/2) and thromboxane (TxA2) synthesis was dependent on both p110β and p110γ. p110β inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110β and p110γ completely prevented the synergistic effect of TPO on ERK1/2 phosphorylation and TxA2 synthesis. The latter was ablated by inhibition of MEK, but not p38, confirming a role for ERK1/2 in regulating TPO-mediated increases in TxA2 synthesis. In conclusion, the synergistic effect of TPO on RAP1 and integrin activation is largely mediated by p110β, whereas p110β and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation.

We have previously demonstrated the pro-tumoral role of neutrophils using a kras-induced zebrafish hepatocarcinogenesis model. To further illustrate the molecular basis of the pro-tumoral role, Tumor-associated neutrophils (TANs) were isolated by fluorescence-activated cell sorting (FACS) and transcriptomic analyses were carried out by RNA-Seq. Differentially expressed gene profiles of TANs from larvae, male and female livers indicate great variations during liver tumorigenesis, but the common responsive canonical pathways included an immune pathway (Acute Phase Response Signaling), a liver metabolism-related pathway (LXR/RXR Activation) and Thrombin Signaling. Consistent with the pro-tumoral role of TANs, gene module analysis identified a consistent down-regulation of Cytotoxicity module, which may allow continued proliferation of malignant cells. Gene Set Enrichment Analysis indicated up-regulation of several genes promoting angiogenesis. Consistent with this, we found decreased density of blood vessels accompanied with decreased oncogenic liver sizes in neutrophil-depleted larvae. Collectively, our study has indicated some molecular mechanisms of the pro-tumoral roles of TANs in hepatocarcinogenesis, including weakened immune clearance against tumor cells and enhanced function in angiogenesis.

Endothelial dysfunction is the common molecular basis of multiple human diseases, such as atherosclerosis, diabetes, hypertension, and acute lung injury. Therefore, primary isolation of high-purity endothelial cells (ECs) is crucial to study the mechanisms of endothelial function and disease pathogenesis. Mouse lung ECs (MLECs) are widely used in vascular biology and lung cell biology studies such as pulmonary inflammation, angiogenesis, vessel permeability, leukocyte/EC interaction, nitric oxide production, and mechanotransduction. Thus, in this paper, we describe a simple, and reproducible protocol for the isolation and culture of MLECs from adult mice using collagenase I-based enzymatic digestion, followed by sequential sorting with PECAM1 (also known as CD31)- and ICAM2 (also known as CD102)-coated microbeads. The morphology of isolated MLECs were observed with phase contrast microscope. MLECs were authenticated by CD31 immunoblotting, and immunofluorescent staining of established EC markers VE-cadherin and von Willebrand factor (vWF). Cultured MLECs also showed functional characteristics of ECs, evidenced by DiI-oxLDL uptake assay and THP-1 monocyte adhesion assay. Finally, we used MLECs from endothelium-specific enhancer of zeste homolog 2 (EZH2) knockout mice to show the general applicability of our protocol. To conclude, we describe here a simple and reproducible protocol to isolate highly pure and functional ECs from adult mouse lungs. Isolation of ECs from genetically engineered mice is important for downstream phenotypic, genetic, or proteomic studies.

Organotypic brain culture is an experimental tool widely used in neuroscience studies. One major drawback of this technique is reduced neuronal survival across time, which is likely exacerbated by the loss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into the commonly-used, standard semi-permeable membrane culture methodology which has significantly enhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states: the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removes waste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Using immunohistochemistry and electrophysiology, our system was found superior to standard techniques in preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provide evidence for the first time that the LDT can be preserved in organotypic slice culture, and further, our technical improvements of adding a flow system, which likely enhanced perfusion to the slice, were associated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypic experiments focused on chronic stimulations and multielectrode recordings in the LDT, as well as enhance neuronal survival in slice cultures originating from other brain regions.

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.

In soybeans, eighteen members constitute the serine hydroxymethyltransferase (GmSHMT) gene family, of which the cytosolic-targeted GmSHMT08c member has been reported to mediate resistance to soybean cyst nematode (SCN). This work presents a comprehensive study of the SHMT gene family members, including synteny, phylogeny, subcellular localizations, haplotypes, protein homology modeling, mutational, and expression analyses. Phylogenetic analysis showed that SHMT genes are divided into four classes reflecting their subcellular distribution (cytosol, nucleus, mitochondrion, and chloroplast). Subcellular localization of selected GmSHMT members supports their in-silico predictions and phylogenetic distribution. Expression and functional analyses showed that GmSHMT genes display many overlapping, but some divergent responses during SCN infection. Furthermore, mutational analysis reveals that all isolated EMS mutants that lose their resistance to SCN carry missense and nonsense mutations at the GmSHMT08c, but none of the Gmshmt08c mutants carried mutations in the other GmSHMT genes. Haplotype clustering analysis using the whole genome resequencing data from a collection of 106 diverse soybean germplams (15X) was performed to identify allelic variants and haplotypes within the GmSHMT gene family. Interestingly, only the cytosolic-localized GmSHMT08c presented SNP clusters that were associated with SCN resistance, supporting our mutational analysis. Although eight GmSHMT members respond to the nematode infestation, functional and mutational analysis has shown the absence of functional redundancy in resistance to SCN. Structural analysis and protein homology modeling showed the presence of spontaneous mutations at important residues within the GmSHMT proteins, suggesting the presence of altered enzyme activities based on substrate affinities. Due to the accumulation of mutations during the evolution of the soybean genome, the other GmSHMT members have undergone neofunctionalization and subfunctionalization events.

Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility in both cellular and model bilayers has been extensively investigated in recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat of fluorescence techniques is the need to use dye-labeled variants of the lipid of interest, thus potentially perturbing the structural and dynamic properties of the native species. Generally, the effect of the dye/tracer molecule is implicitly assumed to be negligible. Nevertheless, in view of the widespread use of optically modified lipids for studying lipid bilayer dynamics, it is highly desirable to well assess this point. Here, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations have been combined together to uncover subtle structural and dynamic effects in DOPC planar membranes enriched with a standard Rhodamine-labeled lipid. Our findings support a non-neutral role of the dye-labeled lipids in diffusion experiments, quantitatively estimating a decrease in lipid mobility of up to 20% with respect to the unlabeled species. Moreover, results highlight the existing interplay between dye concentration, lipid lateral diffusion and membrane permeability, thus suggesting possible implications for future optical microscopy studies of biophysical processes occurring at the membrane level.

The aim of this study was to measure and compare the biomechanical properties of two different graft link preparation techniques for anterior cruciate ligament reconstruction. We hypothesised that there would be differences in elongation, load at failure and failure mode due to the different graft link preparation techniques. Thirty fresh-frozen anatomical specimen knees were used. Both tendons (semitendinosus and gracilis) were harvested and randomly assigned to two groups. Graft links prepared with a continuous loop technique were allocated to group 1, whereas those prepared with a buried-knot technique were allocated to group 2. The mechanical properties of both techniques were measured. A mean load to failure of 731 N and an overall graft elongation of 6 mm was found in the continuous loop group. In the buried-knot group, a higher load to failure (848 N) and a lower mean overall elongation (5 mm) was found. The buried-knot technique showed better results with significantly higher load to failure and significantly less elongation compared to the continuous loop technique. It is essential in clinical practice to choose the most accurate technique for graft link preparation to ensure graft stability, especially in the early phase of recovery.

There is an urgent need to develop new drugs against tuberculosis. In particular, it is critical to target drug tolerant Mycobacterium tuberculosis (M. tuberculosis), responsible, in part, for the lengthy antibiotic regimen required for treatment. We previously postulated that the presence of in vivo biofilm-like communities of M. tuberculosis could contribute to this drug tolerance. Consistent with this hypothesis, certain 2-aminoimidazole (2-AIs) molecules with anti-biofilm activity were shown to revert mycobacterial drug tolerance in an in vitro M. tuberculosis biofilm model. While exploring their mechanism of action, it was serendipitously observed that these 2-AI molecules also potentiated β-lactam antibiotics by affecting mycobacterial protein secretion and lipid export. As these two bacterial processes are energy-dependent, herein it was evaluated if 2-AI compounds affect mycobacterial bioenergetics. At low concentrations, 2B8, the lead 2-AI compound, collapsed both components of the proton motive force, similar to other cationic amphiphiles. Interestingly, however, the minimum inhibitory concentration of 2B8 against M. tuberculosis correlated with a higher drug concentration determined to interfere with the mycobacterial electron transport chain. Collectively, this study elucidates the mechanism of action of 2-AIs against M. tuberculosis, providing a tool to better understand mycobacterial bioenergetics and develop compounds with improved anti-mycobacterial activity.

CD97/ADGRE5 is an adhesion G protein-coupled receptor (aGPCR) involved in tumor cell adhesion, migration, angiogenesis, and apoptosis. CD97 has been shown previously to stimulate angiogenesis by interacting with integrins on endothelial cells via an Arginine-Glycine-Aspartic acid (RGD) motif. In this report, the role of the RGD motif in tumor cell adhesion and apoptosis was investigated using a previously-established HT1080 cell-based system. We found that the RGD motif is critical in CD97-promoted cell adhesion, in part due to the up-regulation of αvβ5 and α2β1 integrins, and that CD97 mediates its anti-apoptotic effect in extrinsic apoptosis via RGD-dependent cell adhesion. In contrast, CD97-modulated anti-apoptotic effect in intrinsic apoptosis is mediated by RGD-independent, N-cadherin-induced homotypic cell aggregation. Hence, CD97 promotes tumorigenesis via RGD-dependent and -independent mechanisms.

The objective of this study was to evaluate the most effective method of DNA extraction of oral mouthwash samples for use in microbiome studies that utilize next generation sequencing (NGS). Eight enzymatic and mechanical DNA extraction methods were tested. Extracted DNA was amplified using barcoded primers targeting the V6 variable region of the bacterial 16S rRNA gene and the ITS1 region of the fungal ribosomal gene cluster and sequenced using the Illumina NGS platform. Sequenced reads were analyzed using QIIME and R. The eight methods yielded significantly different quantities of DNA (p < 0.001), with the phenol-chloroform extraction method producing the highest total yield. There were no significant differences in observed bacterial or fungal Shannon diversity (p = 0.64, p = 0.93 respectively) by extraction method. Bray-Curtis beta-diversity did not demonstrate statistically significant differences between the eight extraction methods based on bacterial (R2 = 0.086, p = 1.00) and fungal (R2 = 0.039, p = 1.00) assays. No differences were seen between methods with or without bead-beating. These data indicate that choice of DNA extraction method affect total DNA recovery without significantly affecting the observed microbiome.

The significant role of microRNAs (miRNAs) in various biological processes and diseases has been widely studied and reported in recent years. Several computational methods associated with mature miRNA identification suffer various limitations involving canonical biological features extraction, class imbalance, and classifier performance. The proposed classifier, miRFinder, is an accurate alternative for the identification of mature miRNAs. The structured-sequence features were proposed to precisely extract miRNA biological features, and three algorithms were selected to obtain the canonical features based on the classifier performance. Moreover, the center of mass near distance training based on K-means was provided to improve the class imbalance problem. In particular, the AdaBoost-SVM algorithm was used to construct the classifier. The classifier training process focuses on incorrectly classified samples, and the integrated results use the common decision strategies of the weak classifier with different weights. In addition, the all mature miRNA sites were predicted by different classifiers based on the features of different sites. Compared with other methods, the performance of the classifiers has a high degree of efficacy for the identification of mature miRNAs. MiRFinder is freely available at https://github.com/wangying0128/miRFinder .

Western hemisphere goats have European, African and Central Asian origins, and some local or rare breeds are reported to be adapted to their environments and economically important. By-in-large these genetic resources have not been quantified. Using 50 K SNP genotypes of 244 animals from 12 goat populations in United States, Costa Rica, Brazil and Argentina, we evaluated the genetic diversity, population structure and selective sweeps documenting goat migration to the "New World". Our findings suggest the concept of breed, particularly among "locally adapted" breeds, is not a meaningful way to characterize goat populations. The USA Spanish goats were found to be an important genetic reservoir, sharing genomic composition with the wild ancestor and with specialized breeds (e.g. Angora, Lamancha and Saanen). Results suggest goats in the Americas have substantial genetic diversity to use in selection and promote environmental adaptation or product driven specialization. These findings highlight the importance of maintaining goat conservation programs and suggest an awaiting reservoir of genetic diversity for breeding and research while simultaneously discarding concerns about breed designations.

The exploration of Earth's terrestrial subsurface biosphere has led to the discovery of several new archaeal lineages of evolutionary significance. Similarly, the deep subseafloor crustal biosphere also harbors many unique, uncultured archaeal taxa, including those belonging to Candidatus Hydrothermarchaeota, formerly known as Marine Benthic Group-E. Recently, Hydrothermarchaeota was identified as an abundant lineage of Juan de Fuca Ridge flank crustal fluids, suggesting its adaptation to this extreme environment. Through the investigation of single-cell and metagenome-assembled genomes, we provide insight into the lineage's evolutionary history and metabolic potential. Phylogenomic analysis reveals the Hydrothermarchaeota to be an early-branching archaeal phylum, branching between the superphylum DPANN, Euryarchaeota, and Asgard lineages. Hydrothermarchaeota genomes suggest a potential for dissimilative and assimilative carbon monoxide oxidation (carboxydotrophy), as well as sulfate and nitrate reduction. There is also a prevalence of chemotaxis and motility genes, indicating adaptive strategies for this nutrient-limited fluid-rock environment. These findings provide the first genomic interpretations of the Hydrothermarchaeota phylum and highlight the anoxic, hot, deep marine crustal biosphere as an important habitat for understanding the evolution of early life.

The Deepwater Horizon oil spill (April 20, 2010) caused concern regarding Gulf seafood safety. Communities were skeptical of governmental risk assessments because they did not take into account the higher consumption of seafood along coastal areas. The objective of this study was to perform a probabilistic risk assessment based on the consumption rates of high-end consumers of Gulf seafood. We utilized seafood consumption data from five communities across the northeastern Gulf of Mexico. This study collected finfish, shrimp, blue crab, and oysters from these communities and analyzed their tissues for polynuclear aromatic hydrocarbons (PAHs). A probabilistic risk assessment was performed using population-specific seafood consumption rates and body weights for commercial fishers, recreational fishers, and a Filipino-American community. For non-cancer effects, 95th percentile hazard quotients for these targeted populations ranged between 1.84E-04 to 5.39E-03 for individual seafood types. The 95th percentile hazard indices for total seafood consumption ranged from 3.45E-03 to 8.41E-03. Based on total seafood consumption, highest hazard indices were modeled for the Filipino-American community followed by commercial and recreational fishers. Despite higher consumption rates, hazard indices for the high-end consumers targeted in this study were two to three orders of magnitude below the regulatory limit of 1.

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

Glutinoglossum is one of the earth tongue genera with viscid or glutinous ascocarps. Based on morphology and ITS1-5.8S-ITS2, nrLSU and tef1 sequence data, seven new species are described: G. circinatum, G. lumbricale, G. orientale, G. peregrinans, G. proliferatum, G. pseudoglutinosum, and G. triseptatum. The lectotypes for Geoglossum glutinosum var. lubricum and for Geoglossum glabrum var. majus as well as the epitype for Glutinoglossum glutinosum are designated. The comprehensive morphological study of G. heptaseptatum resulted in the discovery of ascospores germinating by conidia inside the asci, which is first noted for Glutinoglossum species. The status of Cibalocoryne is discussed.