Ca²⁺ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca²⁺ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca²⁺ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms.

The interferon (IFN)-inducible protein TRIM22 (Staf50) is a member of the tripartite motif protein family and has been suggested a role in the regulation of viral replication as well as of protein ubiquitylation. In addition, we have previously shown that TRIM22 is a direct target gene for the tumour suppressor p53. Consistently, over-expression of TRIM22 inhibits the clonogenic growth of monoblastic U937 cells, suggesting anti-proliferative or cell death-inducing effects.

Since several neuroprotectives failed to reproduce promising preclinical results under clinical conditions, efforts emerged to implement clinically relevant endpoints in animal stroke studies. Thereby, insufficient attention was given on autonomic reactions due to experimental stroke, although clinical trials reported on high functional and prognostic impact. This study focused on autonomic consequences and body weight changes in a translational relevant stroke model and investigated interrelations to different outcome measurements.

Annelids and arthropods each possess a segmented body. Whether this similarity represents an evolutionary convergence or inheritance from a common segmented ancestor is the subject of ongoing investigation.

The gene expression profiles of cancer cells are closely related to their aggressiveness and metastatic potential. Antibody-based immunohistochemistry (IHC) of tissue specimens is a common method of identifying expressed proteins in cancer cells and increasingly inform treatment decisions. Molecular imaging is a potential method of performing similar IHC studies in vivo without the requirement for biopsy or tumor excision. To date, antibody-based imaging has been limited by high background levels related to slow clearance, making such imaging practical. However, optically activatable imaging agents, which are only fluorescent when bound to their cognate receptor, open the possibility of doing in vivo multi-color IHC.

Iron oxide magnetic nanoparticles (MNP's) have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe(3)O(4) and surface coated with poly(ethylenimine) (PEI) and poly(ethylene glycol) (PEG) to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM), was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS) production and lipid peroxidation (LPO), LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines.

Although human epidermal growth factor receptor 2 (HER2) positive or estrogen receptor (ER) positive breast cancers are treated with clinically validated anti-HER2 or anti-estrogen therapies, intrinsic and acquired resistance to these therapies appears in a substantial proportion of breast cancer patients and new therapies are needed. Identification of additional molecular factors, especially those characterized by aggressive behavior and poor prognosis, could prioritize interventional opportunities to improve the diagnosis and treatment of breast cancer.

Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy.

Mucopolysaccharidosis VII (MPS VII) is due to deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral disks, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that were not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some disks. Histochemical staining demonstrated that disks did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth.

Cancer cells are frequently addicted to deregulated oncogenic protein translation. The small molecule 4EG-I selectively inhibits the cap-dependent translation of mRNAs. As multiple myeloma is an incurable disease that requires new therapeutic approaches, we investigated whether targeting the translation initiation pathway could be a target for myeloma therapy.

Environmental estrogens alter hormone signaling in the body that can induce reproductive abnormalities in both humans and wildlife. Available testing systems for estrogens are focused on specific systems such as reproduction. Crucially, however, the potential for significant health impacts of environmental estrogen exposures on a variety of body systems may have been overlooked.

To sense numerous odorants and chemicals, animals have evolved a large number of olfactory receptor genes (Olfrs) in their genome. In particular, the house mouse has ~1,100 genes in the Olfr gene family. This makes the mouse a good model organism to study Olfr genes and olfaction-related genes. To date, whether male and female mice possess the same ability in detecting environmental odorants is still unknown. Using the next generation sequencing technology (paired-end mRNA-seq), we detected 1,088 expressed Olfr genes in both male and female olfactory epithelium. We found that not only Olfr genes but also odorant-binding protein (Obp) genes have evolved rapidly in the mouse lineage. Interestingly, Olfr genes tend to express at a higher level in males than in females, whereas the Obp genes clustered on the X chromosome show the opposite trend. These observations may imply a more efficient odorant-transporting system in females, whereas a more active Olfr gene expressing system in males. In addition, we detected the expression of two genes encoding major urinary proteins, which have been proposed to bind and transport pheromones or act as pheromones in mouse urine. This observation suggests a role of main olfactory system (MOS) in pheromone detection, contrary to the view that only accessory olfactory system (AOS) is involved in pheromone detection. This study suggests the sexual differences in detecting environmental odorants in MOS and demonstrates that mRNA-seq provides a powerful tool for detecting genes with low expression levels and with high sequence similarities.

11β-Hydroxysteroid dehydrogenase type 1 (11βHSD1) is a therapeutic target in metabolic syndrome because it catalyses reductase regeneration of cortisol from cortisone in adipose and liver. 11βHSD1 can also catalyze the reverse dehydrogenase reaction in vitro (e.g., if cofactor is limited). We used stable isotope tracers to test the hypothesis that both 11βHSD1-reductase and -dehydrogenase activities occur in human metabolic tissues in vivo. 1,2-[(2)H](2)-Cortisone (d2-cortisone) was validated as a tracer for 11β-dehydrogenase activity and its inhibition by licorice. d2-Cortisone and 9,11,12,12-[(2)H](4)-cortisol (d4-cortisol) (to measure 11β-reductase activity) were coinfused and venous samples obtained from skeletal muscle, subcutaneous adipose (n = 6), and liver (n = 4). Steroids were measured by liquid chromatography-tandem mass spectrometry and arteriovenous differences adjusted for blood flow. Data are means ± SEM. 11β-Reductase and -dehydrogenase activities were detected in muscle (cortisol release 19.7 ± 4.1 pmol/100 mL/min, d3-cortisol 5.9 ± 1.8 pmol/100 mL/min, and cortisone 15.2 ± 5.8 pmol/100 mL/min) and splanchnic (cortisol 64.0 ± 11.4 nmol/min, d3-cortisol 12.9 ± 2.1 nmol/min, and cortisone 19.5 ± 2.8 nmol/min) circulations. In adipose, dehydrogenase was more readily detected than reductase (cortisone release 38.7 ± 5.8 pmol/100 g/min). Active recycling between cortisol and cortisone in metabolic tissues in vivo may facilitate dynamic control of intracellular cortisol but makes consequences of dysregulation of 11βHSD1 transcription in obesity and diabetes unpredictable. Disappointing efficacy of 11βHSD1 inhibitors in phase II studies could be explained by lack of selectivity for 11β-reductase.

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.

Inappropriate contact lens (CL) use and care often lead to corneal neovascularization (corneal NV). We used mouse eyes which wore CL as the animal model to assess the reason for corneal NV with CL wear. The similar and overlapping activity of vascular endothelial growth factor (VEGF) and the potent angiogenic hypoxia-inducible factor 1α (HIF-1α) called for a study of the temporal relationship in the expression of these two autocoids. We determined the time dependent expression of HIF-1α and correlated it to that of VEGF expression in the mouse model of closed eye with CL wear.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

Biofilms are dense microbial communities. Although widely distributed and medically important, how biofilm cells interact with one another is poorly understood. Recently, we described a novel process whereby myxobacterial biofilm cells exchange their outer membrane (OM) lipoproteins. For the first time we report here the identification of two host proteins, TraAB, required for transfer. These proteins are predicted to localize in the cell envelope; and TraA encodes a distant PA14 lectin-like domain, a cysteine-rich tandem repeat region, and a putative C-terminal protein sorting tag named MYXO-CTERM, while TraB encodes an OmpA-like domain. Importantly, TraAB are required in donors and recipients, suggesting bidirectional transfer. By use of a lipophilic fluorescent dye, we also discovered that OM lipids are exchanged. Similar to lipoproteins, dye transfer requires TraAB function, gliding motility and a structured biofilm. Importantly, OM exchange was found to regulate swarming and development behaviors, suggesting a new role in cell-cell communication. A working model proposes TraA is a cell surface receptor that mediates cell-cell adhesion for OM fusion, in which lipoproteins/lipids are transferred by lateral diffusion. We further hypothesize that cell contact-dependent exchange helps myxobacteria to coordinate their social behaviors.

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process.

Although autism has a clear genetic component, the high genetic heterogeneity of the disorder has been a challenge for the identification of causative genes. We used homozygosity analysis to identify probands from nonconsanguineous families that showed evidence of distant shared ancestry, suggesting potentially recessive mutations. Whole-exome sequencing of 16 probands revealed validated homozygous, potentially pathogenic recessive mutations that segregated perfectly with disease in 4/16 families. The candidate genes (UBE3B, CLTCL1, NCKAP5L, ZNF18) encode proteins involved in proteolysis, GTPase-mediated signaling, cytoskeletal organization, and other pathways. Furthermore, neuronal depolarization regulated the transcription of these genes, suggesting potential activity-dependent roles in neurons. We present a multidimensional strategy for filtering whole-exome sequence data to find candidate recessive mutations in autism, which may have broader applicability to other complex, heterogeneous disorders.

Using ~60,000 SNPs selected for minimal linkage disequilibrium, we perform population structure analysis of 1,374 unrelated Hispanic individuals from the Multi-Ethnic Study of Atherosclerosis (MESA), with self-identification corresponding to Central America (n = 93), Cuba (n = 50), the Dominican Republic (n = 203), Mexico (n = 708), Puerto Rico (n = 192), and South America (n = 111). By projection of principal components (PCs) of ancestry to samples from the HapMap phase III and the Human Genome Diversity Panel (HGDP), we show the first two PCs quantify the Caucasian, African, and Native American origins, while the third and fourth PCs bring out an axis that aligns with known South-to-North geographic location of HGDP Native American samples and further separates MESA Mexican versus Central/South American samples along the same axis. Using k-means clustering computed from the first four PCs, we define four subgroups of the MESA Hispanic cohort that show close agreement with self-identification, labeling the clusters as primarily Dominican/Cuban, Mexican, Central/South American, and Puerto Rican. To demonstrate our recommendations for genetic analysis in the MESA Hispanic cohort, we present pooled and stratified association analysis of triglycerides for selected SNPs in the LPL and TRIB1 gene regions, previously reported in GWAS of triglycerides in Caucasians but as yet unconfirmed in Hispanic populations. We report statistically significant evidence for genetic association in both genes, and we further demonstrate the importance of considering population substructure and genetic heterogeneity in genetic association studies performed in the United States Hispanic population.