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Background: The link between inflammation and carcinogenesis is indisputable. In trying to understand key factors at play, cancer research has developed an interest in the toll-like receptors (TLRs), which have shown signs of having prognostic value in various adenocarcinomas. We began investigating the expression of toll-like receptors 1, 3, 5, 7, and 9 to evaluate their prognostic value of patients with pancreatic ductal adenocarcinoma (PDAC). Methods: We collected tumor biopsies from 154 stage I-III PDAC patients surgically treated at Helsinki University Hospital between 2002 and 2011, excluding patients undergoing neoadjuvant therapy. We used tissue microarray slides and immunohistochemistry to assess expression of TLRs 1, 3, 5, 7, and 9 in PDAC tissue. Immunopositivity scores and clinicopathological characteristics were subjected to Fisher’s exact test or the linear-by-linear association test. For the survival analysis, we applied the Kaplan-Meier method and log-rank test, and the Cox regression proportional hazard model served for univariate and multivariate analyses. Results: Strong TLR1 expression was observable in 60 (39%), strong TLR3 in 48 (31%), strong TLR5 in 58 (38%), strong TLR7 in 14 (9%), and strong TLR9 in 22 (14%) patients. The multivariate analysis showed strong TLR1 expression to associate with better survival than moderate, low, or negative expression (HR = 0.68; 95% CI 0.47–0.99; p = 0.044). Additionally, those few patients with tumors negative for TLR1, TLR3, TLR7, or TLR9 fared poorly (HR = 2.41; 95% CI 1.31–4.43; p = 0.005; n = 13). Conclusion: Strong TLR1 expression suggested better prognosis in PDAC patients, whereas negative expression of TLR1, TLR3, TLR7, or TLR9 was a sign of poor prognosis. [ABSTRACT FROM AUTHOR]

The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly is critically important, as misregulation or delay of cilia loss leads to cell cycle defects. The physical means by which cilia are lost are poorly understood but are thought to involve resorption of ciliary components into the cell body. To investigate cilium loss in mammalian cells, we used live-cell imaging to comprehensively characterize individual events. The predominant mode of cilium loss was rapid deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of Gradual resorption was followed by rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently of calcium. Together, these results suggest that mammalian ciliary loss involves a tunable decision between deciliation and resorption. [ABSTRACT FROM AUTHOR]

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection. [ABSTRACT FROM AUTHOR]

Objectives: The current study aims to identify markers that would reflect the number of Leydig cells present in the testis, to help determine whether labour-intensive methods such as stereology are necessary. We used our well-characterised Sertoli cell ablation model in which we have empirically established the size of the Leydig cell population, to try to identify transcriptional biomarkers indicative of population size. Results: Following characterisation of the Leydig cell population after Sertoli cell ablation in neonatal life or adulthood, we identified Hsd3b1 transcript levels as a potential indicator of Leydig cell number with utility for informing decision-making on whether to engage in time-consuming stereological cell counting analysis. [ABSTRACT FROM AUTHOR]

The light-promoted recovery of epidermal barrier of skin was evaluated by the associated recovery of transepidermal potential (TEP), the potential difference between the surface and dermis of skin, by using porcine skin samples. An accelerated recovery of TEP was observed by irradiation of red light with the irradiance of 40 mW/cm2 and a duration of > 10 min. The influence of the light stimulation to the surroundings (~ 20 mm) was also observed. The irradiations of blue and purple lights were ineffective in accelerating the barrier recovery. These characteristics of the light stimulation would be useful for the design of effective and safe phototherapy devices for skin. The present study proves that the TEP can serve as a spatiotemporal indicator of the epidermal barrier function. [ABSTRACT FROM AUTHOR]

Accumulation of amyloid ß (Aß) peptides, the major component of amyloid fibrils in senile plaques, is one of the main causes of Alzheimer’s disease. Docosahexaenoic acid (DHA) is a fatty acid abundant in the brain, and is reported to have protective effects against Alzheimer’s disease, although the mechanistic effects of DHA against Alzheimer’s pathophysiology remain unclear. Because dietary supplementation of DHA in Aß precursor protein transgenic mice ameliorates Aß pathology and behavioral deficits, we hypothesize that DHA may affect the fibrillization and deposition of Aß. Here we studied the effect of different types of fatty acids on Aß fibril formation by in vitro Aß fibrillization assay. Formation of amyloid fibrils consists of two steps, i.e., the initial nucleation phase and the following elongation phase. We found that unsaturated fatty acids, especially DHA, accelerated the formation of Aß fibrils with a unique short and curved morphology in its nucleation phase, which did not elongate further into the long and straight, mature Aß fibrils. Addition of DHA afterwards did not modify the morphology of the mature Aß(1–40) fibrils. The short and curved Aß fibrils formed in the presence of DHA did not facilitate the elongation phase of Aß fibril formation, suggesting that DHA promotes the formation of “off-pathway” conformers of Aß. Our study unravels a possible mechanism of how DHA acts protectively against the pathophysiology of Alzheimer’s disease. [ABSTRACT FROM AUTHOR]

The subset of pro-inflammatory B cells, called late memory, tissue-like or double negative (DN), accumulates in the blood of elderly individuals. Here we show that DN B cells do not proliferate and do not make antibodies to influenza antigens, but they secrete antibodies with autoimmune reactivity, in agreement with their membrane phenotype (CD95+CD21-CD11c+) and their spontaneous expression of the transcription factor T-bet. These cells also increase in the blood of individuals with obesity and autoimmune diseases, but causative mechanisms and signaling pathways involved are known only in part. In the present paper we compare frequencies and metabolic requirements of these cells in the blood of healthy individuals of different ages and in the blood and the subcutaneous adipose tissue (SAT) of individuals with obesity. Results show that DN B cells from young individuals have minimal metabolic requirements, DN B cells from elderly and obese individuals utilize higher amounts of glucose to perform autoimmune antibody production and enroll in aerobic glycolysis to support their function. DN B cells from the SAT have the highest metabolic requirements as they activate oxidative phosphorylation, aerobic glycolysis and fatty acid oxidation. DN B cells from the SAT also show the highest levels of ROS and the highest levels of phosphorylated AMPK (5’-AMP activated kinase) and Sestrin 1, both able to mitigate stress and cell death. This metabolic advantage drives DN B cell survival and function (secretion of autoimmune antibodies). [ABSTRACT FROM AUTHOR]

Despite its poor bioavailability, curcumin is a promising natural polyphenol targeting NF-κβ. NF-κβ is a target for new therapeutics because it plays a pivotal role in the pathophysiology of Alzheimer disease (AD). In contrast, ambrsoin, a sesquiterpene lactone which is a potent NF-κβ inhibitor, is scarcely studied in AD models. The current work aims to assess the efficacy of ambrosin as a possible remedy for AD. In silico studies showed that bioavailability and BBB permeability could be favorable for ambrosin over curcumin. Memory impairment was induced in mice by single intraperitoneal injection of LPS (0.4 mg/kg). Treated groups received curcumin (100 mg/kg) or ambrosin at doses (5 or 10 mg/kg) for 7 days. Mice in treated groups showed a significant improvement in memory functions during Morris water maze and object recognition tests. Curcumin and ambrosin (10 mg/kg) inhibited the upsurge of NF-κβp65 transcript and protein levels. Consequently, downstream pro-inflammatory and nitrosative mediators were inhibited, namely, TNF-α, IL-1β, COX-2 and iNOS. BACE1 was inhibited, thereby reducing amyloid plaques (Aβ) deposition and eventually reducing inflammation and apoptosis of neurons as revealed by immunohistopathological examination. In conclusion, ambrosin can be repurposed as AD remedy after further pharmacokinetic/pharamacodynamic assessments. It could serve as an additional lead drug for AD therapeutics. [ABSTRACT FROM AUTHOR]

The overall survival for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) remains low, with little progress made over decades. Cisplatin, most frequently used for HNSCC treatment, activates mitochondria-dependent apoptosis through the BCL-2 family proteins. We have previously demonstrated that the pro-apoptotic BH3-only protein, NOXA plays a critical role in this process. NOXA binds and inactivates anti-apoptotic MCL-1, while the BCL-2 inhibitor ABT-263 is capable of inactivating anti-apoptotic BCL-2 and BCL-XL. We hypothesized that combination of NOXA and ABT-263 treatment increases cell death by simultaneously inhibiting anti-apoptotic BCL-2 family proteins in HNSCC cells. Here, we demonstrated that combination of ectopic NOXA expression and ABT-263 enhanced apoptosis in p53-inactive, p53 wild-type, and human papillomavirus (HPV)-positive HNSCC cell lines. Furthermore, a retinoid derivative and an endoplasmic reticulum stress inducer, fenretinide, induced NOXA, and combination of fenretinide and ABT-263 strongly induced apoptosis in HNSCC cells regardless of the HPV or p53 statuses. We also found that MCL-1 and BCL-XL are the primary targets of apoptosis induced by the combinations. These results will develop novel and alternative therapeutic strategies to directly modify the cell death machinery in HNSCC. [ABSTRACT FROM AUTHOR]

Background: The identification of patients with high-risk prostate cancer (PC) is a major challenge for clinicians, and the improvement of current prognostic parameters is an unmet clinical need. We and others have identified an association between the nuclear localization of NF-κB p65 and biochemical recurrence (BCR) in PC in small and/or single-centre cohorts of patients.Methods and Findings: In this study, we accessed 2 different multi-centre tissue microarrays (TMAs) representing cohorts of patients (Test-TMA and Validation-TMA series) of the Canadian Prostate Cancer Biomarker Network (CPCBN) to validate the association between p65 nuclear frequency and PC outcomes. Immunohistochemical staining of p65 was performed on the Test-TMA and Validation-TMA series, which include PC tissues from patients treated by first-line radical prostatectomy (n = 250 and n = 1,262, respectively). Two independent observers evaluated the p65 nuclear frequency in digital images of cancer tissue and benign adjacent gland tissue. Kaplan-Meier curves coupled with a log-rank test and univariate and multivariate Cox regression models were used for statistical analyses of continuous values and dichotomized data (cutoff of 3%). Multivariate analysis of the Validation-TMA cohort showed that p65 nuclear frequency in cancer cells was an independent predictor of BCR using continuous (hazard ratio [HR] 1.02 [95% CI 1.00-1.03], p = 0.004) and dichotomized data (HR 1.33 [95% CI 1.09-1.62], p = 0.005). Using a cutoff of 3%, we found that this biomarker was also associated with the development of bone metastases (HR 1.82 [95% CI 1.05-3.16], p = 0.033) and PC-specific mortality (HR 2.63 [95% CI 1.30-5.31], p = 0.004), independent of clinical parameters. BCR-free survival, bone-metastasis-free survival, and PC-specific survival were shorter for patients with higher p65 nuclear frequency (p < 0.005). As the small cores on TMAs are a limitation of the study, a backward validation of whole PC tissue section will be necessary for the implementation of p65 nuclear frequency as a PC biomarker in the clinical workflow.Conclusions: We report the first study using the pan-Canadian multi-centre cohorts of CPCBN and validate the association between increased frequency of nuclear p65 frequency and a risk of disease progression. [ABSTRACT FROM AUTHOR]

In humans and mice, the detailed phenotypic and functional characterization of peripheral blood monocytes allows for identification of three monocyte subsets. There are also evidences of monocyte phenotypic heterogeneity in other species, including cattle, sheep, pig and horse. However, little is known about such variability in dogs. The aim of the study was to determine whether and how peripheral blood monocytes of healthy dogs differ in the presence of MHCII and CD4 and in the basal production of reactive oxygen species (ROS). Three distinct subsets of CD11b+CD14+ monocytes were found in peripheral blood samples of healthy dogs, based on the variations in the density of MHCII and CD4 surface molecules: MHCII+CD4– (Mo1), MHCII+CD4+ (Mo2) and MHCII–CD4+ (Mo3). The Mo2 and Mo3 were significantly lower in percentage than Mo1 but their basal ROS production was higher. Within the Mo2 and Mo3 subsets, the percentage of cells producing ROS was significantly higher comparing to cells lacking this activity. Canine peripheral blood monocytes vary in the expression of MHCII and CD4 and in the activity suggesting that cells within the three identified subsets carry out different functions. The higher production of ROS in non-activated cells within small subsets of Mo2 and Mo3 monocytes might indicate their immunomodulatory potential. [ABSTRACT FROM AUTHOR]

Mung bean (Vigna radiata), an important legume crop, has the property of desiccation tolerance (DT), which is lost in the final stage of germination (preimbibition, 18 h-24 h). We compared parameters related to the programmed cell death (PCD) of mung bean seeds before and after dehydration at different imbibition stages through various detection methods. The results of Evans blue and TTC staining methods showed that the dehydration process could lead to cell death. The results of optical and subcellular morphology showed that PCD occurred after dehydration. The destruction of DNA integrity and the activity changes in caspase and total nuclease in mung bean seeds after dehydration treatment indicated that the loss of desiccation tolerance was related to PCD. Dehydration resulted in the destruction of the mitochondrial structure, reversal of the membrane potential, and the entrance of cytochrome C into the cytoplasm. These processes all indicate that the mitochondrial apoptosis pathway was the main form of dehydration-induced PCD. The results of cytoplasmic Ca2+ concentration showed that Ca2+ signaling also played a role in inducing PCD, with the upstream signal being dehydration-induced changes in water potential and the downstream signal being the ROS and mitochondrial PT channel, according to the order in which these signals happened. The mitochondrial apoptosis pathway can be considered the main mechanism of dehydration-induced PCD based on our analysis of the sequence of major events in PCD. The main processes include dehydration induction, changes in Ca2+ and mitochondrial respiratory electron transport, the reversal of mitochondrial membrane potential induced by ROS and Ca2+, and the transmission and execution of PCD downstream signals induced by cytochrome C release. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a global health problem with an estimated report of 2 million new cases every year and more than 1 billion people at risk of contracting this disease in endemic areas. The innate immune system plays a central role in controlling L. major infection by initiating a signaling cascade that results in production of pro-inflammatory cytokines and recruitment of both innate and adaptive immune cells. Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils to the site of infection. Herein, we report that L. major specifically targets murine CXCL1 for degradation. The degradation of CXCL1 is not dependent on host factors as L. major can directly degrade recombinant CXCL1 in a cell-free system. Using mass spectrometry, we discovered that the L. major protease cleaves at the C-terminal end of murine CXCL1. Finally, our data suggest that L. major metalloproteases are involved in the direct cleavage and degradation of CXCL1, and a synthetic peptide spanning the CXCL1 cleavage site can be used to inhibit L. major metalloprotease activity. In conclusion, our study has identified an immune evasion strategy employed by L. major to evade innate immune responses in mice, likely reservoirs in the endemic areas, and further highlights that targeting these L. major metalloproteases may be important in controlling infection within the reservoir population and transmittance of the disease. [ABSTRACT FROM AUTHOR]

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT /GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells. [ABSTRACT FROM AUTHOR]

In unilateral ureteral obstruction (UUO), both oxidative stress and mitochondrial dysfunction are related to cell death. The aim of this study has been to characterize profiles of enzyme antioxidant activities and mitochondrial functioning of the contralateral (CL) compared to UUO and Sham (false-operated) kidneys of Balb/c mice. Kidneys were resected 14 days after obstruction for immunohistochemical and cortical mitochondrial functioning assays. Antioxidant enzymes activities were investigated in mitochondria and cytosol. Oxygen consumption (QO2) and formation of O2 reactive species (ROS) were assessed with pyruvate plus malate or succinate as the respiratory substrates. QO2 decreased in CL and UUO in all states using substrates for complex II, whereas it was affected only in UUO when substrates for complex I were used. Progressive decrease in mitochondrial ROS formation–in the forward and reverse pathway at complex I–correlates well with the inhibition of QO2 and, therefore, with decreased electron transfer at the level of complexes upstream of cytochrome c oxidase. CL and UUO transmembrane potential responses to ADP were impaired with succinate. Intense Ca2+-induced swelling was elicited in CL and UUO mitochondria. Important and selective differences exist in CL antioxidant enzymes with respect to either Sham or UUO kidneys: CL kidneys had increased mitochondrial glutathione peroxidase and cytosolic catalase activities, indicative of compensatory responses in the face of an early altered ROS homeostasis (as detected by 4-hydroxynonenal), and of a significant tendency to apoptosis. In CL and UUO, upregulation of nuclear (erythroid-derived 2)-like 2 transcription factor (Nrf2), as well as of cytoplasmic and nuclear Kelch-like ECH-associated protein 1 (Keap1) in opposition to decreased heme oxygenase-1 (HO-1), suggest impairment of the Nrf2/Keap1/HO-1 system. It is concluded that chronic obstruction impairs mitochondrial function in CL and UUO, preferentially affecting complex II. [ABSTRACT FROM AUTHOR]

The immunomodulatory potential of multipotent mesenchymal stromal cells (MSC) provides a basis for current and future regenerative therapies. In this study, we established an approach that allows to address the effects of pro-inflammatory stimulation and co-culture with MSC on different specific leukocyte subpopulations. Equine peripheral blood leukocyte recovery was optimized to preserve all leukocyte subpopulations and leukocyte activation regimes were evaluated. Allogeneic labeled equine adipose-derived MSC were then subjected to direct co-culture with either non-stimulated, concanavalin A (ConA)-activated or phosphate 12-myristate 13-acetate and ionomycin (PMA/I)-activated leukocytes. Subsequently, production of the cytokines interferon-γ (IFN- γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) and presence of FoxP3 were determined in specific cell populations using multicolor flow cytometry. Prostaglandin E2 (PGE2) was measured in the supernatants. ConA-stimulation induced mild activation of leukocytes, whereas PMA/I-stimulation led to strong activation. In T cells, PMA/I promoted production of all cytokines, with no distinct suppressive effects of MSC. However, increased numbers of CD25/FoxP3-positive cells indicated that MSC supported regulatory T cell differentiation in PMA/I-activated leukocyte cultures. MSC also reduced numbers of cytokine-producing B cells and granulocytes, mostly irrespective of preceding leukocyte activation, and reversed the stimulatory effect of ConA on IFN-γ production in monocytes. Illustrating the possible suppressive mechanisms, higher numbers of MSC produced IL-10 when co-cultured with non-stimulated or ConA-activated leukocytes. This was not observed in co-culture with PMA/I-activated leukocytes. However, PGE2 concentration in the supernatant was highest in the co-culture with PMA/I-activated leukocytes, suggesting that PGE2 could still mediate modulatory effects in strongly inflammatory environment. These context- and cell type-specific modulatory effects observed give insight into the interactions between MSC and different types of immune cells and highlight the roles of IL-10 and PGE2 in MSC-mediated immunomodulation. The approach presented could provide a basis for further functional MSC characterization and the development of potency assays. [ABSTRACT FROM AUTHOR]

Fes1 is a conserved armadillo repeat-containing Hsp70 nucleotide exchange factor important for growth at high temperature, proteasomal protein degradation and prion propagation. Depleting or mutating Fes1 induces a stress response and causes defects in these processes that are ascribed solely to disruption of Fes1 regulation of Hsp70. Here, we find Fes1 was essential for degradation of gluconeogenic enzymes by the vacuole import and degradation (Vid) pathway and for cell wall integrity (CWI), which is crucial for growth at high temperature. Unexpectedly, Fes1 mutants defective in physical or functional interaction with Hsp70 retained activities that support Vid and CWI. Fes1 and the Fes1 mutants bound to the Vid substrate Fbp1 in vitro and captured Slt2, a signaling kinase that regulates CWI, from cell lysates. Our data show that the armadillo domain of Fes1 binds proteins other than Hsp70, that Fes1 has important Hsp70-independent roles in the cell, and that major growth defects caused by depleting Fes1 are due to loss of these functions rather than to loss of Hsp70 regulation. We uncovered diverse functions of Fes1 beyond its defined role in regulating Hsp70, which points to possible multi-functionality among its conserved counterparts in other organisms or organelles. [ABSTRACT FROM AUTHOR]

Activation of SIRT1, an NAD+-dependent protein deacetylase, ameliorates muscular pathophysiology of δ-sarcoglycan-deficient TO-2 hamsters and dystrophin-deficient mdx mice. We found that SIRT1 was highly expressed beneath the cellular membranes of muscle cells. To elucidate functional roles of SIRT1 on muscles, skeletal muscle-specific SIRT1 knockout mice (SIRT1-MKO) were generated. SIRT1-MKO mice showed muscular pathology similar to mild muscular dystrophies with increased numbers of centrally nucleated small myofibers and decreased numbers of middle-sized (2000–3001 μm2) myofibers compared to those of wild-type (WT) mice. Accordingly, SIRT1-MKO mice showed significantly decreased exercise capacity in treadmill and inverted hanging tests with higher levels of serum creatine kinase activities compared with those in WT mice. Evans blue dye uptake after exercise was greater in the muscles of SIRT1-MKO than those of WT mice, suggesting membrane fragility in SIRT1-MKO mice. Because SIRT1 was dominantly localized beneath the membranes of muscular cells, SIRT1 may have a new role in the membranes. We found that levels of fluorescent FM1-43 dye intake after laser-induced membrane disruption in C2C12 cells were significantly increased by SIRT1 inhibitors or Sirt1-siRNA compared with those of control cells. Inhibition of SIRT1 or SIRT1-knockdown severely disturbed the dynamic aggregation of membrane vesicles under the injured site but did not affect expression levels of membrane repair proteins. These data suggested that SIRT1 had a critical role in the resealing of membrane-ruptured muscle cells, which could affect phenotypes of SIRT1-MKO mice. To our knowledge, this report is the first to demonstrate that SIRT1 affected plasma-membrane repair mechanisms. [ABSTRACT FROM AUTHOR]

Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ≥ 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention. [ABSTRACT FROM AUTHOR]

Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D. dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D. dianthicola, which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase, of D. dianthicola. Assay specificity was assessed using strains present in inclusivity (16 D. dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D. dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection. [ABSTRACT FROM AUTHOR]

Semiconductor nanowires are increasingly used in optoelectronic devices. However, their effects on human health have not been assessed fully. Here, we investigate the effects of gallium phosphide nanowires on human lung adenocarcinoma cells. Four different geometries of nanowires were suspended in the cell culture for 48 hours. We show that cells internalize the nanowires and that the nanowires have no effect on cell proliferation rate, motility, viability and intracellular ROS levels. By blocking specific internalization pathways, we demonstrate that the nanowire uptake is the result of a combination of processes, requiring dynamin and actin polymerization, which suggests an internalization through macropinocytosis and phagocytosis. [ABSTRACT FROM AUTHOR]

Transparency of the cornea is essential for vision and is maintained by the corneal endothelium. Consequently, corneal endothelial decompensation arising from irreversible damage to the corneal endothelium causes severe vision impairment. Until recently, transplantation of donor corneas was the only therapeutic choice for treatment of endothelial decompensation. In 2013, we initiated clinical research into cell-based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs), in combination with Rho kinase inhibitor, into the anterior chamber. The aim of the present study was to establish a protocol for cryopreservation of HCECs to allow large-scale commercial manufacturing of these cells. This study focused on the effects of various cryopreservation reagents on HCEC viability. Screening of several commercially available cryopreservation reagents identified Bambanker hRM as an effective agent that maintained a cell viability of 89.4% after 14 days of cryopreservation, equivalent to the cell viability of 89.2% for non-cryopreserved control cells. The use of Bambanker hRM and HCECs at a similar grade to that used clinically for cell based therapy (passage 3–5 and a cell density higher than 2000 cells/mm2) gave a similar cell density for cryopreserved HCECs to that of non-preserved control HCECs after 28 days of cultivation (2099 cells/mm2 and 2111 cells/mm2, respectively). HCECs preserved using Bambanker hRM grew in a similar fashion to non-preserved control HCECs and formed a monolayer sheet-like structure. Cryopreservation of HCECs has multiple advantages including the ability to accumulate stocks of master cells, to transport HCEC stocks, and to manufacture HCECs on demand for use in cell-based treatment of endothelial decompensation. [ABSTRACT FROM AUTHOR]

The repair of white matter damage is of paramount importance for functional recovery after brain injuries. Here, we report that interleukin-4 (IL-4) promotes oligodendrocyte regeneration and remyelination. IL-4 receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. IL-4 deficiency in knockout mice resulted in greater deterioration of white matter over 14 d after stroke. Consistent with these findings, intranasal delivery of IL-4 nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. The selective effect of IL-4 on remyelination was verified in an ex vivo organotypic model of demyelination. By leveraging primary oligodendrocyte progenitor cells (OPCs), microglia-depleted mice, and conditional OPC-specific peroxisome proliferator-activated receptor gamma (PPARγ) knockout mice, we discovered a direct salutary effect of IL-4 on oligodendrocyte differentiation that was mediated by the PPARγ axis. Our findings reveal a new regenerative role of IL-4 in the central nervous system (CNS), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. Therefore, intranasal IL-4 delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries. [ABSTRACT FROM AUTHOR]

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a translation factor selectively expressed by heart, skeletal muscle, nervous system and some specialized cells. Its ectopic expression relates with tumorigenesis in several types of human cancer. No data are available about the role of eEF1A2 in Triple Negative Breast Cancers (TNBC). This study investigated the relation between eEF1A2 protein levels and the prognosis of TNBC. A total of 84 TNBC diagnosed in the period 2002–2011 were included in the study. eEF1A2 protein level was measured in formalin-fixed paraffin-embedded tissues by immunohistochemistry in a semi-quantitative manner (sum of the percentage of positive cells x staining intensity) on a scale from 0 to 300. Cox regression assessed the association between eEF1A2 levels and disease-free survival (DFS) and breast cancer-specific survival (BCSS). Elevated values of eEF1A2 were associated with older age at diagnosis (p = 0.003), and androgen receptors positivity (p = 0.002). At univariate Cox analysis, eEF1A2 levels were not significantly associated with DFS and BCSS (p = 0.11 and p = 0.08, respectively) whereas adjusting for stage of disease, elevated levels of eEF1A2 protein resulted associated with poor prognosis (HR = 1.05, 95% CI: 1.01–1.11, p = 0.04 and HR = 1.07, 95% CI: 1.01–1.14, p = 0.03 for DFS and BCSS, respectively). This trend was confirmed analyzing negative versus positive samples by using categorized scores. Our data showed a negative prognostic role of eEF1A2 protein in TNBC, sustaining further investigations to confirm this result by wider and independent cohorts of patients. [ABSTRACT FROM AUTHOR]

Autophagy plays a vital role in tumor therapy and survival of dormant tumor cells. Here we describe a novel function of a protein known as Transmembrane 219 (TM219) as an autophagy activator. TM219 is a small membrane protein expressed in all known human tissues except the thymus. We used biochemical approaches to identify calmodulin and calmodulin dependent protein kinase II as a part of TM219 protein complex. Then, we employed in vitro reconstitution system and fluorescence anisotropy to study the requirements of TM219 to bind calmodulin in vitro. We also used this system to study the effects of a synthetic peptide derived from the sequence of the short cytoplasmic tail of TM219 (SCTT) on calmodulin-TM219 receptor interactions. We conjugated SCTT peptide with a pH Low Insertion peptide (pHLIP) for optimal cellular delivery. We finally tested the effects of SCTT-pHLIP on triple negative human breast cancer cells in three dimension culture. Our data defined a novel function of TM219 protein and an efficient approach to inhibit it. [ABSTRACT FROM AUTHOR]

In this study, we report on the synthesis of silver nanoparticles (AgNPs) from the leaf extracts of Cynara scolymus (Artichoke) using microwave irradiation and the evaluation of its anti-cancer potential with photodynamic therapy (PDT). Silver nanoparticles formation was characterized by scanning electron microscopy with energy dispersive x-ray spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Silver nanoparticles formation was also investigated the surface charge, particle size and distribution using zetasizer analysis. The cytotoxic effect of AgNPs and/or PDT was studied by MTT assay and migration by the scratch assay. The apoptotic inducing ability of the AgNPs and/or PDT was investigated by intracellular ROS analysis, antioxidant enzyme levels (SOD, CAT, GPx and GSH), Hoechst staining and Bax/Bcl-2 analysis using western blotting. The mean particle size of produced AgNPs was found 98.47±2.04 nm with low polydispersity (0.301±0.033). Zeta potential values of AgNPs show -32.3± 0.8 mV. These results clearly indicate the successful formation of AgNPs for cellular uptake. Mitochondrial damage and intracellular ROS production were observed upon treatment with AgNPs (10μg/mL) and PDT (0.5 mJ/cm2) showed significant reducing cell migration, expression of Bax and suppression of Bcl-2. Significantly, biosynthesized AgNPs showed a broad-spectrum anti-cancer activity with PDT therapy and therefore represent promoting ROS generation by modulating mitochondrial apoptosis induction in MCF7 breast cancer cells. [ABSTRACT FROM AUTHOR]

In an ever-aging society the demand for bone-defect filling grafts continues to gain in importance. While autologous grafting still prevails as the gold standard, allografts and xenografts present viable alternatives with promising results. Physiochemical properties of a graft strongly depend on the processing method such as the decellularization protocol. In addition, the physiochemical characteristics are critical factors for a successful integration of the graft after the implantation and might influence mesenchymal stem cell function in therapeutic approaches combining grafts and autologous mesenchymal stem cells (MSCs). Several decellularization methods have been proposed, however it still remains unclear which method results in favorable physiochemical properties or might be preferred in stem cell applications. In the first part of this study we compared two decellularization approaches resulting in chemically processed allografts (CPAs) or sonication-based processed allografts (SPAs). Each decellularization approach was compared for its decellularization efficacy and its influence on the grafts’ surface texture and composition. In the second part of this study biocompatibility of grafts was assessed by testing the effect of extraction medium on MSC viability and comparing them to commercially available allografts and xenografts. Additionally, grafts’ performance in terms of MSC functionality was assessed by reseeding with MSCs pre-differentiated in osteogenic medium and determining cell adhesion, proliferation, as well as alkaline phosphatase (ALP) activity and the degree of mineralization. In summary, results indicate a more effective decellularization for the SPA approach in comparison to the CPA approach. Even though SPA extracts induced a decrease in MSC viability, MSC performance after reseeding was comparable to commercially available grafts based on DNA quantification, alkaline phosphatase activity and quantification of mineralization. Commercial Tutoplast allografts showed overall the best effects on MSC functionality as indicated by extraction biocompatibility testing as well as by comparing proliferation and osteogenic differentiation. [ABSTRACT FROM AUTHOR]

We previously reported the presence of memory CD4+ T cells that express low levels of SAMHD1 (SAMHD1low) in peripheral blood and lymph nodes from both HIV-1 infected and uninfected individuals. These cells are enriched in Th17 and Tfh subsets, two populations known to be preferentially targeted by HIV-1. Here we investigated whether SAMHD1low CD4+ T-cells harbour replication-competent virus and compartimentalized HIV-1 genomes. We sorted memory CD4+CD45RO+SAMHD1low, CD4+CD45RO+SAMHD1+ and naive CD4+CD45RO-SAMHD1+ cells from HIV-1-infected patients on anti-retroviral therapy (c-ART) and performed HIV-1 DNA quantification, ultra-deep-sequencing of partial env (C2/V3) sequences and phenotypic characterization of the cells. We show that SAMHD1low cells include novel Th17 CCR6+ subsets that lack CXCR3 and CCR4 (CCR6+DN). There is a decrease of the % of Th17 in SAMHD1low compartment in infected compared to uninfected individuals (41% vs 55%, p<0.05), whereas the % of CCR6+DN increases (7.95% vs 3.8%, p<0.05). Moreover, in HIV-1 infected patients, memory SAMHD1low cells harbour high levels of HIV-1 DNA compared to memory SAMHD1+ cells (4.5 vs 3.8 log/106cells, respectively, p<0.001), while naïve SAMHD1+ showed significantly lower levels (3.1 log/106cells, p<0.0001). Importantly, we show that SAMHD1low cells contain p24-producing cells. Moreover, phylogenetic analyses revealed well-segregated HIV-1 DNA populations with compartmentalization between SAMHD1low and SAMHD1+ memory cells, and limited viral exchange. As expected, the % of Ki67+ cells was significantly higher in SAMHD1low compared to SAMHD1+ cells. There was positive association between levels of HIV-1 DNA and Ki67+ in memory SAMHD1low cells, but not in memory and naïve SAMHD1+ CD4+ T-cells. Altogether, these data suggest that proliferative memory SAMHD1low cells contribute to viral persistence. [ABSTRACT FROM AUTHOR]

During viral infection, tight regulation of CD8+ T-cell functions determines the outcome of the disease. Recently, others and we determined that the natural killer (NK) cells kill hyperproliferative CD8+ T cells in the context of viral infection, but molecules that are involved in shaping the regulatory capability of NK cells remain virtually unknown. Here we used mice lacking the Fc-receptor common gamma chain (FcRγ, FcεRIγ, Fcer1g–/–mice) to determine the role of Fc-receptor and NK-receptor signaling in the process of CD8+ T-cell regulation. We found that the lack of FcRγ on NK cells limits their ability to restrain virus-specific CD8+ T cells and that the lack of FcRγ in Fcer1g–/–mice leads to enhanced CD8+ T-cell responses and rapid control of the chronic docile strain of the lymphocytic choriomeningitis virus (LCMV). Mechanistically, FcRγ stabilized the expression of NKp46 but not that of other killer cell–activating receptors on NK cells. Although FcRγ did not influence the development or activation of NK cell during LCMV infection, it specifically limited their ability to modulate CD8+ T-cell functions. In conclusion, we determined that FcRγ plays an important role in regulating CD8+ T-cell functions during chronic LCMV infection. [ABSTRACT FROM AUTHOR]

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages. [ABSTRACT FROM AUTHOR]

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness. [ABSTRACT FROM AUTHOR]

The interaction between atherosclerosis and commensal microbes through leaky gut syndrome (LGS), which is characterized by impaired intestinal permeability and the introduction of undesired pathogens into the body, has not been fully elucidated. Our aim was to investigate the potential role of a ClC-2 chloride channel activator, lubiprostone, which is reported to have beneficial effects on LGS, in the development of atherosclerosis in apolipoprotein E–deficient (ApoE-/-) mice. After a 15-week feeding period of a Western diet (WD), ApoE-/- mice were treated with a Western-type diet (WD) alone or WD with oral supplementation of lubiprostone for 10 weeks. This feeding protocol was followed by experimental evaluation of LGS and atherosclerotic lesions in the aorta. In mice with lubiprostone, in vivo translocation of orally administered 4-kDa FITC-dextran was significantly improved, and RNA expression of the epithelial tight junction proteins, Zo-1 and occludin, was significantly up-regulated in the ileum, compared to the WD alone group, suggesting a possible reversal of WD-induced intestinal barrier dysfunction. As a result, WD-induced exacerbation of atherosclerotic lesion formation was reduced by 69% in longitudinally opened aortas and 26% in aortic root regions. In addition, there was a significant decrease in circulating immunoglobulin level, followed by an attenuation of inflammatory responses in the perivascular adipose tissue, as evidenced by reduced expression of pro-inflammatory cytokines and chemokines. Lubiprostone attenuates atherosclerosis by ameliorating LGS-induced inflammation through the restoration of the intestinal barrier. These findings raise the possibility of targeting LGS for the treatment of atherosclerosis. [ABSTRACT FROM AUTHOR]

Aggrecan is an integral component of the extracellular matrix in cartilaginous tissues, including the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation. The mechanisms accounting for this phenotype remain unknown. We used ATDC5 cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knockdown cell lines (AggKD) were generated using lentiviral shRNA transduction particles. Cells were stimulated with insulin/transferrin/selenium for up to 21 days to induce chondrogenesis. Control ATDC5 cells showed induction of Col2a1 starting at day 8 and induction of Col10a1 starting at day 12. AggKD cells had significantly reduced expression of Col2a1 and Col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. The induction of Col2a1 and Col10a1 was not rescued by culturing of AggKD cells in wells pre-conditioned with ATDC5 extracellular matrix or in co-culture with wild-type ATDC5 cells. We interpret our studies as indicating that aggrecan has an integral role in chondrogenesis that may be mediated through intracellular mechanisms. [ABSTRACT FROM AUTHOR]

Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E. coli, S. cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S. meliloti that enable DNA transformation with up to 1.4 x 105 and 2.1 x 103 CFU μg-1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum. We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. The development of S. meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) corrupts the outcome of about 50% of all critically ill patients. We investigated the possible contribution of the pathology acidemia on the development of AKI. Pigs were exposed to acidemia, acidemia plus hypoxemia or a normal acid-base balance in an experimental setup, which included mechanical ventilation and renal replacement therapy to facilitate biotrauma caused by extracorporeal therapies. Interestingly, extensive histomorphological changes like a tubular loss of cell barriers occurred in the kidneys after just 5 hours exposure to acidemia. The additional exposure to hypoxemia aggravated these findings. These ‘early’ microscopic pathologies opposed intra vitam data of kidney function. They did not mirror cellular or systemic patterns of proinflammatory molecules (like TNF-α or IL 18) nor were they detectable by new, sensitive markers of AKI like Neutrophil gelatinase-associated lipocalin. Instead, the data suggest that the increased renal proton excretion during acidemia could be an ‘early’ first hit in the multifactorial pathogenesis of AKI. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase-8 (CA8) is an intracellular protein that functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) critical to intracellular Ca++ release, synaptic functions and neuronal excitability. We showed previously that murine nociception and analgesic responses are regulated by the expression of this gene in dorsal root ganglion (DRG) associated with a cis-eQTL. In this report, we identify an exon-level cis-eQTL (rs6471859) that regulates human DRG CA8 alternative splicing, producing a truncated 1,697bp transcript (e.g., CA8-204). Our functional genomic studies show the “G” allele at rs6471859 produces a cryptic 3’UTR splice site regulating expression of CA8-204. We developed constructs to study the expression and function of the naturally occurring CA8-204G transcript (G allele at rs6471859), CA8-204C (C allele at rs6471859 reversion mutation) and CA8-201 (full length transcript). CA8-204G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204C expression was restricted to neuronal derived cells (NBL) in vitro. CA8-204G produced a stable truncated transcript in HEK293 cells that was barely detectable in NBL cells. We also show CA8-204 produces a stable peptide that inhibits pITPR1 and Ca++ release in HEK293 cells. These results imply homozygous G/G individuals at rs6471859, which are common in the general population, produce exclusively CA8-204G that is barely detectable in neuronal cells. CA8 null mutations that greatly impact neuronal functions are associated with severe forms of spinal cerebellar ataxia, and our data suggest G/G homozygotes should display a similar phenotype. To address this question, we show in vivo using AAV8-FLAG-CA8-204G and AAV8-V5-CA8-201 gene transfer delivered via intra-neural sciatic nerve injection (SN), that these viral constructs are able to transduce DRG cells and produce similar analgesic and anti-hyperalgesic responses to inflammatory pain. Immunohistochemistry (IHC) examinations of DRG tissues further show CA8-204G peptide is expressed in advillin expressing neuronal cells, but to a lesser extent compared to glial cells. These findings explain why G/G homozygotes that exclusively produce this truncated functional peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses. [ABSTRACT FROM AUTHOR]

The beneficial effect of mesenchymal stem cells (MSCs) on wound healing is mostly attributed to a trophic effect that promotes angiogenesis. Whether MSCs can contribute to the formation of new blood vessels by direct differentiation is still controversial. Pelvic floor dysfunction (PFD) is a group of disorders that negatively affect the quality of women’s lives. Traditional vaginal surgical repair provides disappointing anatomical outcome. Stem cell transplantation may be used to supplement surgery and improve its outcome. Here we aimed to examine the engraftment, survival, differentiation and angiogenic effect of transplanted MSCs in a vaginal injury rat model. MSCs were obtained from the bone marrow of Sprague Drawley (SD) rats, expanded and characterized in vitro. The MSCs expressed CD90 and CD29, did not express CD45, CD34, CD11b and CD31 and could differentiate into osteogenic, chondrogenic and adipogenic lineages. Cells were labeled with either PKH-26 or GFP and transplanted systemically or locally to female SD rats, just after a standardized vaginal incision was made. Engraftment after local transplantation was less efficient at all-time points compared to systemic administration. In the systemically transplanted animal group, MSCs migrated to the injury site and were present in the healed vagina for at least 30 days. Both systemic and local MSCs transplantation promoted host angiogenesis. Systemically transplanted MSCs created new vascular-like structures by direct differentiation into endothelium. These findings pave the way to further studies of the potential role of MSCs transplantation in improving surgical outcome in women with PFD. [ABSTRACT FROM AUTHOR]

Expanding the southern range of herbaceous peony (Paeonia lactiflora Pall.) is a meaningful and worthwhile horticultural endeavor in the Northern Hemisphere. However, high temperatures in winter seriously hinder the bud dormancy release and flowering of peony in the more southern areas of subtropical and tropical regions. Resource introduction and hybridization can contribute to creating new cultivars with high adaptability in a warmer winter climate. In this study, three representative cultivars of P. lactiflora were screened for flowering capabilities and their annual growth cycles were observed to provide information needed for hybridization. Among these three cultivars, ‘Hang Baishao’ is the best adapted cultivar for southern growing regions and is unique in its ability to thrive in southern areas of N 30°00’. Pollen viability of ‘Hang Baishao’ was 55.60% based on five measuring methods, which makes it an excellent male parent in hybridization. Hybrid plants among these three cultivars grew well, but all of their flower buds aborted. Additionally, the ability of three growth regulators that advance the flowering of ‘Hang Baishao’ to promote an indoor cultivation strategy for improving peony application as a potted or cut-flower plant was tested. 5-azacytidine could impact the growth of ‘Hang Baishao’ and induce dwarfism and small flowers but not advance the flowering time. Gibberellin A3 promoted the sprouting and growth significantly, but all plants eventually withered. Chilling at 0–4°C for four weeks and irrigation with 300 mg/L humic acid was the optimal combination used to hasten flowering and ensure flowering quality simultaneously. These results can lay the foundation for future studies on the chilling requirement trait, bud dormancy release and key functional gene exploration of herbaceous peony. Additionally, this study can also provide guidance for expanding the range of economically important plants with the winter dormancy trait to the low-latitude regions. [ABSTRACT FROM AUTHOR]

A 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 has a multitude of biological functions and plays an important role in the regulation of inflammatory responses and tumor biology. Our work has focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and we have recently developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which has promising safety profile and clinical activity in patients with malignant pleural mesothelioma. The development of an anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in formalin-fixed paraffin-embedded (FFPE) tissues in the clinical setting is therefore of the utmost importance. To develop novel anti-CD26 mAbs capable of binding to denatured CD26, we immunized mice with urea-treated CD26 protein. Hybridoma supernatants were screened for specific reactivity with human CD26 by immunostaining through the use of a set of FFPE human CD26-positive or negative tumor cell lines. This screening method enables us to develop novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in FFPE non-tumor and tumor tissue sections with reliable clarity and intensity. Specifically, these mAbs display strong binding affinity to denatured human CD26 rather than undenatured human CD26, and are capable of detecting denatured human CD26 in decalcified specimens. These novel anti-CD26 mAbs are potentially useful for the analysis of CD26 expression in cancer patients with bony metastasis, and may help decide the appropriateness of YS110 therapy for future cancer patients. [ABSTRACT FROM AUTHOR]

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs. [ABSTRACT FROM AUTHOR]

Recently identified broadly neutralizing antibodies (bnAbs) show great potential for clinical interventions against HIV-1 infection. However, resistant strains may impose substantialchallenges. Here, we report on the identification and characterization of a panel of HIV-1 strains with broad and potent resistance against a large number of bnAbs, particularly those targeting the CD4-binding site (CD4bs). Site-directed mutagenesis revealedthat several key epitope mutations facilitate resistance and arelocated in the inner domain, loop D, and β23/loop V5/β24 of HIV-1 gp120. The resistance is largely correlated with binding affinity of antibodies to the envelope trimers expressed on the cell surface. Our results therefore demonstrate the existence of broadly resistant HIV-1 strains against CD4bs neutralizing antibodies. Treatment strategies based on the CD4bs bnAbs must overcome such resistance to achieve optimal clinical outcomes. [ABSTRACT FROM AUTHOR]

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization. This was verified by disruption of vacuolar structure and membrane integrity of yeast cells with auroramycin treatment. Addition of salt but not sorbitol to the medium rescued the growth of auroramycin-treated yeast cells suggesting that auroramycin causes ionic stress. Furthermore, auroramycin caused hyperpolarization of the yeast plasma membrane and displayed a synergistic interaction with cationic hygromycin. Our data strongly suggest that auroramycin inhibits yeast cells by causing leakage of cations from the cytoplasm. Thus, auroramycin’s mode-of-action is distinct from known antifungal polyenes, reinforcing the importance of natural products in the discovery of new anti-infectives. [ABSTRACT FROM AUTHOR]

The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103+ dendritic cells (DCs). We show here that CD103+ DCs are strictly required for gastric Th1 responses to H. pylori and for H. pylori infection control. A similar dependence of type 1 immunity on CD103+ DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3+ effector and regulatory T-cells to target tissues and in their local expansion. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin, and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis. [ABSTRACT FROM AUTHOR]

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata. [ABSTRACT FROM AUTHOR]

Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119–127, enables us to reliably detect abnormal isoform prion protein (PrPSc) in cells or frozen tissue sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrPSc-specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrPC) are close enough to allow bivalent binding, mAb 132 binds to PrPC. These results indicate that weak monovalent binding to monomeric PrPC diminishes PrPC signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrPSc in a bivalent manner. [ABSTRACT FROM AUTHOR]

COPD patients are prone to acute infectious exacerbations that impair their quality of life and hamper prognosis. The purpose of the present study was to investigate the in situ IFN-Ǝ response in the lungs of stable COPD and non-COPD patients. Lung samples from 70 subjects (9 control never smokers, 19 control smokers without COPD, 21 patients with moderate COPD and 21 patients with very severe COPD) were studied for the expression of IFN-Ǝ, its main transcription factor, IRF-7, and two products of its autocrine function, namely RIG-I and MDA-5, by immunohistochemical techniques and quantitative real-time PCR. IFN-β, IRF-7, RIG-I and MDA-5 were widely detected in most lung cell types. In epithelial tissues and alveolar macrophages, IFN-Ǝ and IRF-7 labeling scores were decreased up to 65% and 74%, respectively, for COPD patients, paralleling an analogous reduction (43% and 65%, respectively) in the amount of their lung mRNA. Moreover, this decreased production of IFN-Ǝ in COPD patients correlated with a similar decrease in the expression of RIG-I and MDA-5, two essential members of the innate immune system. Our study indicates that most lung cells from stable COPD patients show a constitutive decreased expression of IFN-β, IRF-7, RIG-I and MDA-5, suggesting that this deficiency is the main cause of their acute viral exacerbations. [ABSTRACT FROM AUTHOR]

Background: We aimed to determine demographic and clinicopathological predictors for residual disease in women with cervical intraepithelial neoplasia (CIN 2/3) with endocervical cone margin involvement. Methods and findings: A cross-sectional study was conducted. The eligible patients were women who underwent hysterectomy as a treatment option after having a positive endocervical margin for CIN 2/3 in cervix conization specimens from 2000 to 2015. The patients were divided into two groups based on the persistence of CIN 2/3 and absence of CIN 2/3 in hysterectomy specimens. Demographic, clinical and histology information were collected in both groups. A total of 80 patients were eligible for the study; 37 (46.3%) had no persistence of CIN 2/3 and 43 (53.7%) had persistence of CIN 2/3 in the hysterectomy specimens. Demographic, clinical, and cone specimen characteristics, and a visible squamocolumnar junction and type of conization were analyzed as possible risk factors for the presence of residual lesions at hysterectomy, and none of these variables were associated with residual disease. Menopausal status was strongly associated with a high risk of persistent residual disease 81.2% (OR 4.9, CI 1.27–18.9), P = 0.014. In the multivariate analysis, only a menopausal status (P = 0.04) was associated with a high risk of persistent lesions. Conclusion: This analysis found that menopausal status exhibited an important association with persistent residual disease. Menopausal women with endocervical margin involvement exhibit a greater than 80% risk of persistent lesions. [ABSTRACT FROM AUTHOR]

Fast calcium transients (<10 ms) remain difficult to analyse in cellular microdomains, yet they can modulate key cellular events such as trafficking, local ATP production by endoplasmic reticulum-mitochondria complex (ER-mitochondria complex), or spontaneous activity in astrocytes. In dendritic spines receiving synaptic inputs, we show here that in the presence of a spine apparatus (SA), which is an extension of the smooth ER, a calcium-induced calcium release (CICR) is triggered at the base of the spine by the fastest calcium ions arriving at a Ryanodyne receptor (RyR). The mechanism relies on the asymmetric distributions of RyRs and sarco/ER calcium-ATPase (SERCA) pumps that we predict using a computational model and further confirm experimentally in culture and slice hippocampal neurons. The present mechanism for which the statistics of the fastest particles arriving at a small target, followed by an amplification, is likely to be generic in molecular transduction across cellular microcompartments, such as thin neuronal processes, astrocytes, endfeets, or protrusions. [ABSTRACT FROM AUTHOR]

Hyperthermia (HT) has shown feasibility and potency as an anticancer therapy. Administration of HT in the chemotherapy has previously enhanced the cytotoxicity of drugs against pancreatic cancer. However, the drugs used when conducting these studies are substantially conventional chemotherapeutic agents that may cause unwanted side effects. Additionally, the thermal dosage in the treatment of cancer cells could also probably harm the healthy cells. The purpose of this work was to investigate the potential of the two natural polyphenolic compounds, epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), as heat synergizers in the thermal treatment of the PANC-1 cells. Furthermore, we have introduced a unique strategy entitled the thermal cycling-hyperthermia (TC-HT) that is capable of providing a maximum synergy and minimal side effect with the anticancer compounds. Our results demonstrate that the combination of the TC-HT and the CGA or EGCG markedly exerts the anticancer effect against the PANC-1 cells, while none of the single treatment induced such changes. The synergistic activity was attributed to the cell cycle arrest at the G2/M phase and the induction of the ROS-dependent mitochondria-mediated apoptosis. These findings not only represent the first in vitro thermal synergistic study of natural compounds in the treatment of pancreatic cancer, but also highlight the potential of the TC-HT as an alternative strategy in thermal treatment. [ABSTRACT FROM AUTHOR]