SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1's CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid-binding and H3K9me-binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H's target specificity.

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFβ/Activin-βA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

The casper strain of zebrafish is widely used in studies ranging from cancer to neuroscience. casper offers the advantage of relative transparency throughout adulthood, making it particularly useful for in vivo imaging by epifluorescence, confocal, and light sheet microscopy. casper was developed by selective breeding of two previously described recessive pigment mutants: 1) nacre, which harbors an inactivating mutation of the mitfa gene, rendering the fish devoid of pigmented melanocytes; and 2) roy orbison, a mutant with a so-far unidentified genetic cause that lacks reflective iridophores. To clarify the molecular nature of the roy orbison mutation, such that it can inform studies using casper, we undertook an effort to positionally clone the roy orbison mutation. We find that roy orbison is caused by an intronic defect in the gene mpv17, encoding an inner mitochondrial membrane protein that has been implicated in the human mitochondrial DNA depletion syndrome. The roy orbison mutation is phenotypically and molecularly remarkably similar to another zebrafish iridophore mutant called transparent. Using Cas9-induced crispants and germline mutants with a disrupted mpv17 open reading frame, we show in trans-heterozygote embryos that new frameshift alleles of mpv17, roy orbison, and transparent fail to complement each other. Our work provides genetic evidence that both roy orbison and transparent affect the mpv17 locus by a similar if not identical genetic lesion. Identification of mpv17 mutants will allow for further work probing the relationship between mitochondrial function and pigmentation, which has to date received little attention.

The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 2.4.1.49) produces cellodextrin oligomers from short β-1→4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.

The U.S. National Pain Strategy calls for increased population research on "high-impact chronic pain" (ie, longstanding pain that substantially limits participation in daily activities). Using data from the nationally-representative Health and Retirement Study (HRS), we investigated the prevalence of high-impact chronic pain in U.S. adults older than age 50 overall and within population subgroups. We also explored sociodemographic variation in pain-related disability within specific activity domains. Data are from a subsample of HRS respondents (n = 1,925) who were randomly selected for a supplementary pain module in 2010. Our outcome was operationalized as pain duration of ≥7 months and a disability rating of ≥7 (0-10 scale) in at least 1 domain: family/home, leisure, social activities, work, or basic activities. Overall, 8.2% (95% confidence interval = 6.7-10.1%) of adults older than age 50 met criteria for high-impact chronic pain. This proportion rose to 17.1% (95% confidence interval = 12.3-23.4%) among individuals in the lowest wealth quartile. Prevalence differences according to education, race/ethnicity, and age were not significant. Arthritis and depression were significantly associated with high-impact pain in multivariable analysis. Among adults with any chronic pain, African American and individuals in the lowest wealth quartile reported more pain-related disability across activity domains.

Accumulating evidence indicates that peroxisome functioning, catalase localization, and cellular oxidative balance are intimately interconnected. Nevertheless, it remains largely unclear why modest increases in the cellular redox state especially interfere with the subcellular localization of catalase, the most abundant peroxisomal antioxidant enzyme. This study aimed at gaining more insight into this phenomenon. Therefore, we first established a simple and powerful approach to study peroxisomal protein import and protein-protein interactions in living cells in response to changes in redox state. By employing this approach, we confirm and extend previous observations that Cys-11 of human PEX5, the shuttling import receptor for peroxisomal matrix proteins containing a C-terminal peroxisomal targeting signal (PTS1), functions as a redox switch that modulates the protein's activity in response to intracellular oxidative stress. In addition, we show that oxidative stress affects the import of catalase, a non-canonical PTS1-containing protein, more than the import of a reporter protein containing a canonical PTS1. Furthermore, we demonstrate that changes in the local redox state do not affect PEX5-substrate binding and that human PEX5 does not oligomerize in cellulo, not even when the cells are exposed to oxidative stress. Finally, we present evidence that catalase retained in the cytosol can protect against H2O2-mediated redox changes in a manner that peroxisomally targeted catalase does not. Together, these findings lend credit to the idea that inefficient catalase import, when coupled with the role of PEX5 as a redox-regulated import receptor, constitutes a cellular defense mechanism to combat oxidative insults of extra-peroxisomal origin.

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary.

Bird fancier's lung (BFL) caused by repeated inhalation of avian proteins is the most common form of hypersensitivity pneumonitis. However, the exact identification of proteins involved is unknown, and serological test use for diagnosis need to be standardized. The objectives of this study were (i) to identify antigenic proteins from pigeon droppings (ii) to provide information about their location in avian matrices and (iii) to produce them in recombinant proteins to evaluate their diagnostic performances.

Social media dedicated to health are increasingly used by patients and health professionals. They are rich textual resources with content generated through free exchange between patients. We are proposing a method to tackle the problem of retrieving clinically relevant information from such social media in order to analyze the quality of life of patients with breast cancer.

Biological compatibility, in terms of implantation of foreign mesh material in hernia surgery, still needs experimental investigation. The present study develops an experimental model using human peritoneum to study the integration between tissue and different mesh material. The ex vivo model using peritoneal tissue was studied with different mesh material, and integration was monitored over time using microscopy. The peritoneal model could be kept viable in culture for several weeks. Cell migration was seen after 7-10 days in culture and could be further monitored over several weeks. The use of a human artificial model environment enabling the investigation of tissue/mesh integration has, to our knowledge, not been described previously. This proof-of-concept model was developed for the investigation of peritoneal biology and the integration between tissue and different mesh material. It has the potential to be useful in studies on other important biological mechanisms involving the peritoneum.

Triple negative breast cancer (TNBC) is non-responsive to conventional anti-hormonal and Her2-targeted therapies, making it necessary to identify new molecular targets for therapy. Long non-coding RNA anti-differentiation ncRNA (lncRNA DANCR) was identified participating in carcinogenesis of hepatocellular carcinoma, but its expression and potential role in TNBC progression is still unclear. In the present study, our results showed that DANCR expression was increased in TNBC tissues compared with the adjacent normal tissues using quantitative real-time PCR (qRT-PCR) in 63 TNBC specimens. Patients with higher DANCR expression correlated with worse TNM stages as well as a shorter overall survival (OS) using Kaplan-Meier analysis. When the endogenous DANCR was knocked-down via specific siRNA, cell proliferation and invasion were decreased obviously in the MDA-MB-231 cells. In vivo xenograft experiments showed that knockdown of the DANCR in MDA-MB-231 cells reduced the tumor growth significantly. Furthermore, a compendium of TNBC cancer stem cell markers such as CD44, ABCG2 transporter and aldehyde dehydrogenase (ALDH1) were greatly downregulated in the MDA-MB-231 cells with DANCR knockdown. Molecular mechanistic studies revealed that knockdown of DANCR was associated with increased binding of EZH2 on the promoters of CD44 and ABCG2, and concomitant reduction of expression of these genes suggested that they may be DANCR targets in TNBC. Thus, our study demonstrated that targeting DANCR expression might be a viable therapeutic approach to treat triple negative breast cancer.

Previous studies comparing satisfaction with electronic and elastomeric infusion pumps are limited, and improvements in size and usability of electronic pumps have since occurred. The Comparing Home Infusion Devices (CHID) study plans to assess patient and nurse satisfaction with an elastomeric and electronic pump for delivering intravenous antibiotic treatment in the home. Secondary objectives are to determine pump-related complications and actual antibiotic dose administered, evaluate temperature variation and compare pump operating costs.

Our capacity for higher cognitive reasoning has a measurable limit. This limit is thought to arise from the brain's capacity to flexibly reconfigure interactions between spatially distributed networks. Recent work, however, has suggested that reconfigurations of task-related networks are modest when compared with intrinsic "resting-state" network architecture. Here we combined resting-state and task-driven functional magnetic resonance imaging to examine how flexible, task-specific reconfigurations associated with increasing reasoning demands are integrated within a stable intrinsic brain topology. Human participants (21 males and 28 females) underwent an initial resting-state scan, followed by a cognitive reasoning task involving different levels of complexity, followed by a second resting-state scan. The reasoning task required participants to deduce the identity of a missing element in a 4 × 4 matrix, and item difficulty was scaled parametrically as determined by relational complexity theory. Analyses revealed that external task engagement was characterized by a significant change in functional brain modules. Specifically, resting-state and null-task demand conditions were associated with more segregated brain-network topology, whereas increases in reasoning complexity resulted in merging of resting-state modules. Further increments in task complexity did not change the established modular architecture, but affected selective patterns of connectivity between frontoparietal, subcortical, cingulo-opercular, and default-mode networks. Larger increases in network efficiency within the newly established task modules were associated with higher reasoning accuracy. Our results shed light on the network architectures that underlie external task engagement, and highlight selective changes in brain connectivity supporting increases in task complexity.SIGNIFICANCE STATEMENT Humans have clear limits in their ability to solve complex reasoning problems. It is thought that such limitations arise from flexible, moment-to-moment reconfigurations of functional brain networks. It is less clear how such task-driven adaptive changes in connectivity relate to stable, intrinsic networks of the brain and behavioral performance. We found that increased reasoning demands rely on selective patterns of connectivity within cortical networks that emerged in addition to a more general, task-induced modular architecture. This task-driven architecture reverted to a more segregated resting-state architecture both immediately before and after the task. These findings reveal how flexibility in human brain networks is integral to achieving successful reasoning performance across different levels of cognitive demand.

The pro-apoptotic BCL-2 protein BAX commits human cells to apoptosis by permeabilizing the outer mitochondrial membrane. BAX activation has been suggested to require the separation of helix α5 from α6 - the 'latch' from the 'core' domain - among other conformational changes. Here, we show that conformational changes in this region impair BAX translocation to the mitochondria and retrotranslocation back into the cytosol, and therefore BAX inhibition, but not activation. Redirecting misregulated BAX to the mitochondria revealed an alternative mechanism of BAX inhibition. The E3 ligase parkin, which is known to trigger mitochondria-specific autophagy, ubiquitylates BAX K128 and targets the pro-apoptotic BCL-2 protein for proteasomal degradation. Retrotranslocation-deficient BAX is completely degraded in a parkin-dependent manner. Although only a minor pool of endogenous BAX escapes retrotranslocation into the cytosol, parkin-dependent targeting of misregulated BAX on the mitochondria provides substantial protection against BAX apoptotic activity.

Apoptotic cells undergo a series of morphological changes. These changes are dependent on caspase cleavage of downstream targets, but which targets are significant and how they facilitate the death process are not well understood. In Caenorhabditis elegans an increase in the refractility of the dying cell is a hallmark morphological change that is caspase dependent. We identify a presumptive transient receptor potential (TRP) cation channel, CED-11, that acts in the dying cell to promote the increase in apoptotic cell refractility. CED-11 is required for multiple other morphological changes during apoptosis, including an increase in electron density as visualized by electron microscopy and a decrease in cell volume. In ced-11 mutants, the degradation of apoptotic cells is delayed. Mutation of ced-11 does not cause an increase in cell survival but can enhance cell survival in other cell-death mutants, indicating that ced-11 facilitates the death process. In short, ced-11 acts downstream of caspase activation to promote the shrinkage, death, and degradation of apoptotic cells.

BACKGROUND To investigate associations of the CA microsatellite and rs35767, rs5742612, and rs2288377 polymorphisms and the single nucleotide polymorphism (SNP) haplotypes with and without the CA microsatellite in the IGF1 promoter with insulin sensitivity and secretion. MATERIAL AND METHODS The CA microsatellite and SNPs were genotyped in 389 type 2 diabetes mellitus (T2DM) patients. A 75 g oral glucose tolerance test (OGTT) was given to all the participants. Associations of the genotypes and haplotypes with insulin sensitivity, insulin secretion, glucose tolerance, and insulin-like growth factor 1 (IGF1) were analyzed by ANCOVA (general linear model) and multiple linear regression, after controlling for gender, age, and BMI. RESULTS The CA microsatellite, rs35767 polymorphisms, and SNP haplotypes with or without CA showed no significant association with metabolic parameters. The C allele of rs5742612 was found to be associated with decreased insulin sensitivity (HOMA-S index, β=-0.131, P=0.008; fasting insulin level, β=0.022, P=0.006) and increased insulin secretion (HOMA-B index, β=0.099, P=0.008; insulin AUC, β=0.112, P=0.012). The linear regression model also indicated that the A allele of rs2288377 was associated with decreased insulin sensitivity (HOMA-S index, β=-0.159, P=0.001; fasting insulin, β=0.143, P=0.001) and increased insulin secretion (HOMA-B index, β=0.114, P=0.017; insulin AUC, β=0.042, P=0.002). CONCLUSIONS The CA microsatellite and rs35767 have no genotype-related difference in insulin sensitivity or secretion. The rs5742612 and rs2288377 polymorphisms are significantly associated with insulin biology, with the TT genotype exhibiting higher insulin sensitivity and lower insulin secretion compared with carriers of the C allele and A allele, respectively, mostly attributed to the direct functional roles of the two loci.

Differentiation of stem cells to chondrocytes in vitro usually results in a heterogeneous phenotype. This is evident in the often detected over expression of type X collagen which, in hyaline cartilage structure is not characteristic of the mid-zone but of the deep-zone ossifying tissue. Methods to better match cartilage developed in vitro to characteristic in vivo features are therefore highly desirable in regenerative medicine. This study compares phenotype characteristics between pericytes, obtained from human adipose tissue, differentiated using diphenylalanine/serine (F2/S) peptide hydrogels with the more widely used chemical induced method for chondrogenesis. Significantly higher levels of type II collagen were noted when pericytes undergo chondrogenesis in the hydrogel in the absence of induction media. There is also a balanced expression of collagen relative to aggrecan production, a feature which was biased toward collagen production when cells were cultured with induction media. Lastly, metabolic profiles of each system show considerable overlap between both differentiation methods but subtle differences which potentially give rise to their resultant phenotype can be ascertained. The study highlights how material and chemical alterations in the cellular microenvironment have wide ranging effects on resultant tissue type.

Hypoxia-inducible factor 1 (HIF-1) has been recognized as an important mediator of the reprogramming of carbohydrate metabolic pathways from oxidative phosphorylation to accelerated glycolysis. Although this reprogramming has been associated with the antioxidant and radioresistant properties of cancer cells, gene networks triggering the HIF-1-mediated reprogramming and molecular mechanisms linking the reprogramming with radioresistance remain to be determined. Here, we show that Ubiquitin C-terminal hydrolase-L1 (UCHL1), which we previously identified as a novel HIF-1 activator, increased the radioresistance of cancer cells by producing an antioxidant, reduced glutathione (GSH), through HIF-1-mediated metabolic reprogramming. A luciferase assay to monitor HIF-1 activity demonstrated that the overexpression of UCHL1, but not its deubiquitination activity-deficient mutant (UCHL1 C90S), upregulated HIF-1 activity by stabilizing the regulatory subunit of HIF-1 (HIF-1α) in a murine breast cancer cell line, EMT6. UCHL1 overexpression induced the reprogramming of carbohydrate metabolism and increased NADPH levels in a pentose phosphate pathway (PPP)-dependent manner. The UCHL1-mediated reprogramming elevated intracellular GSH levels, and consequently induced a radioresistant phenotype in a HIF-1-dependent manner. The pharmacological inhibition of PPP canceled the UCHL1-mediated radioresistance. These results collectively suggest that cancer cells acquire antioxidant and radioresistant phenotypes through UCHL1-HIF-1-mediated metabolic reprogramming including the activation of PPP and provide a rational basis for targeting this gene network for radiosensitization.

Hypothetically, intratumoral genomic heterogeneity has the potential to foster tumor-infiltrating lymphocyte (TIL) diversity; however, no study has directly tested this hypothesis by simultaneously investigating somatic mutations, TIL diversity, and immune response activity. Thus, we performed whole-exome sequencing, immune repertoire sequencing and gene expression on ten spatially separated tumor samples obtained from two tumor masses excised from a glioblastoma multiforme (GBM) patient, and we included peripheral blood as control. We found that although the multi-region samples from one tumor shared more common mutations than those from different tumors, the TIL populations did not. TIL repertoire diversity did not significantly correlate with the number of non-synonymous mutations; however, TIL diversity was highly correlated with local immune activity, as the pathways were all immune-related pathways that highly positive correlated with local TIL diversity. Twenty-three genes with expression largely unaffected by the intratumor heterogeneity were extracted from these pathways. Fifty GBM patients were stratified into two clusters by the expression of these genes with significant difference in prognosis. This finding was validated by The Cancer Genome Atlas (TCGA) GBM dataset, which indicated that despite the heterogeneity of intra-tumor immune status, the overall level of the immune response in GBM could be connected with prognosis.

Alterations in the gut microbiome have been reported in liver cirrhosis, and probiotic interventions are considered a potential treatment strategy. This study aimed to evaluate the effects and mechanisms of Lactobacillus salivarius LI01, Pediococcus pentosaceus LI05, Lactobacillus rhamnosus GG, Clostridium butyricum MIYAIRI and Bacillus licheniformis Zhengchangsheng on CCl4-induced cirrhotic rats. Only administration of LI01 or LI05 prevented liver fibrosis and down-regulated the hepatic expression of profibrogenic genes. Serum endotoxins, bacterial translocations (BTs), and destruction of intestinal mucosal ultrastructure were reduced in rats treated with LI01 or LI05, indicating maintenance of the gut barrier as a mechanism; this was further confirmed by the reduction of not only hepatic inflammatory cytokines, such as TNF-α, IL-6, and IL-17A, but also hepatic TLR2, TLR4, TLR5 and TLR9. Metagenomic sequencing of 16S rRNA gene showed an increase in potential beneficial bacteria, such as Elusimicrobium and Prevotella, and a decrease in pathogenic bacteria, such as Escherichia. These alterations in gut microbiome were correlated with profibrogenic genes, gut barrier markers and inflammatory cytokines. In conclusion, L. salivarius LI01 and P. pentosaceus LI05 attenuated liver fibrosis by protecting the intestinal barrier and promoting microbiome health. These results suggest novel strategies for the prevention of liver cirrhosis.