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Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or controlmice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare. [ABSTRACT FROM AUTHOR]

Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is characterized by high molecular genetic heterogeneity, metastatic activity and unfavorable prognosis. MicroRNAs (miRNA) are 22-nucleotide noncoding RNAs that are aberrantly expressed in cancer cells and have gained serious consideration as non-invasive cancer biomarkers. We investigated possible differential miRNA signatures that may differentiate high-grade ccRCC from primary disease stages. High-throughput miRNAs expression profiling, using TaqMan OpenArray Human MicroRNA panel, was performed in a group of 21 ccRCC patients. The obtained data was validated in 47 ccRCC patients. We identified nine dysregulated miRNAs (miRNA-210, -642, -18a, -483-5p, -455-3p, -487b, -582-3p, -199b and -200c) in tumor ccRCC tissue compared to normal renal parenchyma. Our results show that the combination of miRNA-210, miRNA-483-5p, miRNA-455 and miRNA-200c is able to distinguish low and high TNM ccRCC stages. Additionally, miRNA-18a, -210, -483-5p and -642 showed statistically significant differences between the low stage tumor ccRCC tissue and normal renal tissue. Contrariwise, the high stages of the tumor process were accompanied by alteration in the expression levels of miRNA-200c, -455-3p and -582-3p. Although the biological roles of these miRNAs in ccRCC are not totally clear, our findings need additional investigations into their involvement in the pathogenesis of ccRCC. Prospective studies with large study cohorts of ccRCC patients are important to further establish the clinical validity of our miRNA markers to predict ccRCC. [ABSTRACT FROM AUTHOR]

Simple Summary: A novel coronavirus that causes a worldwide pandemic poses a significant threat to patients. The virus can induce not only a cytokine storm and thrombosis but also various extra-respiratory diseases such as liver dysfunction, strong headaches, loss of smell and even psychiatric disorders. However, information on whether SARS-CoV-2 can infect liver or brain tissues is still contradictory. Here, we show that the coronavirus efficiently infects liver cancer cells but does not replicate in non-tumor hepatocyte-like cells. SARS-CoV-2 was also found to infect some glioblastoma cells, which is the most common type of brain tumor. In conclusion, we show that SARS-CoV-2 can infect tumor tissues. Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection. [ABSTRACT FROM AUTHOR]

The phenotypic characteristics of animals are result of the complex interaction of a number of genetic and non-genetic factors, which usually act simultaneously and it is difficult to determine the degree of influence of each of them. Early identification of the genetic traits of individuals enables more efficient management of selection. Fertility determins the cost-effectiveness of sheep farming, regardless of productive direction. The individual fertility of the animals is determined by the number of lambs born per sheep. This trait is characterized by a low inheritance rate, hence conventional breeding methods shows slow progress. The introduction of new molecular-based technologies to improve productivity contributes to a greater and faster effect on the realization of animal genetic resources. The assessment of the breeding value of animals at the earliest possible age makes it possible to maximize the effect of the selection and to increase the use of animals with the highest genetic potential. The molecular markers and the application of RFLP-PCR method allow the determination of allelic variants of genes related to the quantity and quality of animal productivity. By identifying the different polymorphic variations and their phenotypic manifestation, a database can be accumulated to manage the genetic progression of economically important traits. Many mutations have been found in sheep that affect fertility and ovulation rates to varying degrees. Large number of mutations which affects ovulation have been found in genes expressing different proteins, such as the BMPR1B or FecB (Booroola gene or the bone morphogenetic protein receptor type 1B), GDF9 or FecG (the growth differentiation factor 9b) and BMP15 or FecX (a bone morphogenetic protein 15). [ABSTRACT FROM AUTHOR]

Background: DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods: In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results: We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions: The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology. [ABSTRACT FROM AUTHOR]

The effect of noopept (N-phenylacetyl-L-prolyl-glycine ethyl ester) on the DNA-binding activity of HIF-1 in SH-SH5Y cells and the mechanisms of stabilization of this transcription factor were studied in vitro. Noopept was shown to increase both the basal DNA-binding activity of HIF-1 and the activity induced by various hypoxia mimetics. The mechanism of stabilization of the oxygen-sensitive HIF1α subunit by noopept involves the inhibition of HIF-1 prolyl hydroxylase, which is indirectly indicated by the data obtained using the ODD-Luc reporter, and the positive effect on the level of the HIF1α protein. It was revealed that the effect of noopept is accompanied by changes in gene expression, which belong to different metabolic pathways and are controlled by the transcription factor HIF-1. [ABSTRACT FROM AUTHOR]

Study Question: Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients?Summary Answer: No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts.What Is Known Already: Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity.Study Design, Size, Duration: For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts.Participants/materials, Setting, Methods: COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3-5 days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24 h containing C-type natriuretic peptide), followed by an IVM step (30 h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets.Main Results and the Role Of Chance: CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups.Limitations, Reasons For Caution: The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known.Wider Implications Of the Findings: A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS.Study Funding/competing Interest(s): IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.'s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is a malignant kidney tumour with a poor prognosis and difficult to treat. Despite significant advances in the treatment of ccRCC, immune checkpoint inhibitors (ICI) still have limited therapeutic efficacy. A growing number of investigations has demonstrated that exosomal miRNAs are key modulators of tumour signaling and determinants of the tumour microenvironment. Disruption of miRNA regulation may affect ccRCC immunogenicity and response to ICI therapy, making them attractive for use as prognostic molecular genetic biomarkers. We evaluated exosomal miRNAs (miRNA-424, -146a, -503, -144) expression levels before and after ICI therapy in plasma samples obtained from 42 ccRCC patients. Expression analysis was performed using real-time PCR method. The results showed that the expression levels of miRNA-424 and miRNA-146a were upregulated after ICI therapy treatment (miRNA-424 = Mean ± SEM 1.202 ± 0.15 and miRNA-146a = 12.22 ± 1.45) compared expression levels before therapy (miRNA-424 = Mean ± SEM 0.63 ± 0.17; p-value = 0.03 and miRNA-146a = 7.03 ± 0.90; p-value = 0.006). miRNA-424 and miRNA-146a can be used to create a panel of molecular markers for evaluating the effectiveness of immune checkpoint inhibitors therapy. Even though the results are preliminary and requires further studying on a larger cohorts, it further increases the interest in using miRNAs, as additional ICI therapeutic markers capable of modulating immune tolerance. [ABSTRACT FROM AUTHOR]

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability to. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT. [ABSTRACT FROM AUTHOR]

Release of neurotransmitter is executed by complex multiprotein machinery, which is assembled around the presynaptic cytomatrix at the active zone. One well-established function of this proteinaceous scaffold is the spatial organization of synaptic vesicle cluster, the protein complexes that execute membrane fusion and compensatory endocytosis, and the transmembrane molecules important for alignment of pre- and postsynaptic structures. The presynaptic cytomatrix proteins function also in processes other than the formation of a static frame for assembly of the release apparatus and synaptic vesicle cycling. They actively contribute to the regulation of multiple steps in this process and are themselves an important subject of regulation during neuronal plasticity. We are only beginning to understand the mechanisms and signalling pathways controlling these regulations. They are mainly dependent on posttranslational modifications, including phosphorylation and small-molecules conjugation, such as ubiquitination. Ubiquitination of presynaptic proteins might lead to their degradation by proteasomes, but evidence is growing that this modification also affects their function independently of their degradation. Signalling from presynapse to nucleus, which works on a much slower time scale and more globally, emerged as an important mechanism for persistent usage-dependent and homeostatic neuronal plasticity. Recently, two new functions for the largest presynaptic scaffolding proteins bassoon and piccolo emerged. They were implied (1) in the regulation of specific protein ubiquitination and proteasome-mediated proteolysis that potentially contributes to short-term plasticity at the presynapse and (2) in the coupling of activity-induced molecular rearrangements at the presynapse with reprogramming of expression of neuronal activity-regulated genes. [ABSTRACT FROM AUTHOR]

Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in the PTDSS1 gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism. [ABSTRACT FROM AUTHOR]

Rhizobia are able to establish a beneficial interaction with legumes by forming a new organ, called the symbiotic root nodule, which is a unique ecological niche for rhizobial nitrogen fixation. Rhizobial infection has many similarities with pathogenic infection and induction of defence responses accompanies both interactions, but defence responses are induced to a lesser extent during rhizobial infection. However, strong defence responses may result from incompatible interactions between legumes and rhizobia due to a mutation in either macro- or microsymbiont. The aim of this research was to analyse different plant defence reactions in response to Rhizobium infection for several pea ( Pisum sativum) mutants that result in ineffective symbiosis . Pea mutants were examined by histochemical and immunocytochemical analyses, light, fluorescence and transmission electron microscopy and quantitative real-time PCR gene expression analysis. It was observed that mutations in pea symbiotic genes sym33 ( PsIPD3/PsCYCLOPS encoding a transcriptional factor) and sym40 ( PsEFD encoding a putative negative regulator of the cytokinin response) led to suberin depositions in ineffective nodules, and in the sym42 there were callose depositions in infection thread (IT) and host cell walls. The increase in deposition of unesterified pectin in IT walls was observed for mutants in the sym33 and sym42; for mutant in the sym42, unesterified pectin was also found around degrading bacteroids. In mutants in the genes sym33 and sym40, an increase in the expression level of a gene encoding peroxidase was observed . In the genes sym40 and sym42, an increase in the expression levels of genes encoding a marker of hypersensitive reaction and PR10 protein was demonstrated. Thus, a range of plant defence responses like suberisation, callose and unesterified pectin deposition as well as activation of defence genes can be triggered by different pea single mutations that cause perception of an otherwise beneficial strain of Rhizobium as a pathogen. [ABSTRACT FROM AUTHOR]

Purpose: This study was conducted to investigate the effect of fish oil (FO) and krill oil (KO) supplementation on glucose tolerance in obese New Zealand white rabbits. Methods: The experiments were carried out with 24 male rabbits randomly divided into four groups: KO-castrated, treated with KO; FO-castrated, treated with FO; C-castrated, non-treated; NC-non-castrated, non-treated. At the end of treatment period (2 months), an intravenous glucose tolerance test (IVGTT) was performed in all rabbits. Results: Fasting blood glucose concentrations in FO and KO animals were significantly lower than in group C. The blood glucose concentrations in FO- and KO-treated animals returned to initial values after 30 and 60 min of IVGTT, respectively. In liver, carnitine palmitoyltransferase 2 ( Cpt2) and 3-hydroxy-3-methyl-glutaryl-CoA synthase 2 ( Hmgcs2) genes were significantly increased in FO-fed rabbits compared with the C group. Acetyl-CoA carboxylase alpha ( Acaca) expression was significantly reduced in both KO- and FO-fed rabbits. In skeletal muscle, Hmgcs2 and Cd36 were significantly higher in KO-fed rabbits compared with the C group. Acaca expression was significantly lower in KO- and FO-fed rabbits compared with the C group. Conclusion: The present results indicate that FO and KO supplementation decreases fasting blood glucose and improves glucose tolerance in obese New Zealand white rabbits. This could be ascribed to the ameliorated insulin sensitivity and insulin secretion and modified gene expressions of some key enzymes involved in β-oxidation and lipogenesis in liver and skeletal muscle. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive histological type of cancer in this location. Distant metastases are present in approximately 30% of patients at the time of first examination. Therefore, the ability to predict the occurrence of metastases in patients at early stages of the disease is an urgent task aimed at personalized treatment. Samples of tumor and paired histologically normal kidney tissue from patients with metastatic and non-metastatic ccRCC were studied. Gene expression was analyzed using real-time PCR. The level of gene methylation was evaluated using bisulfite conversion followed by quantitative methylation-specific PCR. Two groups of genes were analyzed in this study. The first group includes genes whose expression is significantly reduced during metastasis: CA9, NDUFA4L2, EGLN3, and BHLHE41 (p < 0.001, ROC analysis). The second group includes microRNA genes: MIR125B-1, MIR137, MIR375, MIR193A, and MIR34B/C, whose increased methylation levels are associated with the development of distant metastases (p = 0.002 to <0.001, ROC analysis). Based on the data obtained, a combined panel of genes was formed to identify patients whose tumors have a high metastatic potential. The panel can estimate the probability of metastasis with an accuracy of up to 92%. [ABSTRACT FROM AUTHOR]

Recent publications on gall formation induced on the leaves of dicotyledonous flowering plants by eriophyoid mites (Eriophyoidea) and representatives of four insect orders (Diptera, Hemiptera, Hymenoptera, Lepidoptera) are analyzed. Cellular and molecular level data on the stimuli that induce and sustain the development of both mite and insect galls, the expression of host plant genes during gallogenesis, and the effects of these galling arthropods on photosynthesis are considered. A hypothesis is proposed for the relationship between the size of galls and the volume of secretions injected by a parasite. Multistep, varying patterns of plant gene expression and accompanying histo-morphological changes in the transformed gall tissues are apparent. The main obstacle to better elucidating the nature of the induction of gallogenesis is the impossibility of collecting a sufficient amount of saliva for analysis, which is especially important in the case of microscopic eriophyoids. The use of modern omics technologies at the organismal level has revealed a spectrum of genetic mechanisms of gall formation at the molecular level but has not yet answered the questions regarding the nature of gall-inducing agents and the features of events occurring in plant cells at the very beginning of gall growth. [ABSTRACT FROM AUTHOR]

Persistent experience-driven adaptation of brain function is associated with alterations in gene expression patterns, resulting in structural and functional neuronal remodeling. How synaptic activity-in particular presynaptic performance-is coupled to gene expression in nucleus remains incompletely understood. Here, we report on a role of Ct BP1, a transcriptional co-repressor enriched in presynapses and nuclei, in the activity-driven reconfiguration of gene expression in neurons. We demonstrate that presynaptic and nuclear pools of Ct BP1 are interconnected and that both synaptic retention and shuttling of Ct BP1 between cytoplasm and nucleus are co-regulated by neuronal activity. Finally, we show that Ct BP1 is targeted and/or anchored to presynapses by direct interaction with the active zone scaffolding proteins Bassoon and Piccolo. This association is regulated by neuronal activity via modulation of cellular NAD/ NADH levels and restrains the size of the Ct BP1 pool available for nuclear import, thus contributing to the control of activity-dependent gene expression. Our combined results reveal a mechanism for coupling activity-induced molecular rearrangements in the presynapse with reconfiguration of neuronal gene expression. [ABSTRACT FROM AUTHOR]

Purpose: Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme with extracellular catalytic domain that is overexpressed in a variety of cancers including breast cancer and plays a crucial role in maintaining favourable intracellular pH and reducing extracellular pH. The purpose of the current study was to elucidate the prognostic significance of CAIX in the intrinsic subtypes of breast cancer and to characterise CAIX as a drug target in breast cancer. Methods: The prognostic significance of CAIX mRNA expression was interrogated in a cohort of 3,455 breast tumours by using an online tool, Kaplan-Meier plotter. The functional effects of stable CAIX depletion by shRNA in three breast cancer cell lines-MDA-MB-231, MCF7 and SKBR-3, representing basal-like, luminal A and HER2+ subtypes, respectively-were studied by proliferation, invasion, clonal spheroid formation and chemosensitivity assays under normoxia and hypoxia. Finally, the effect of pharmacological CA inhibition alone or in the combination with doxorubicin on self-renewal was assessed by spheroid-forming assay. Results: High CAIX mRNA expression was significantly associated with poor survival in patients with basal-like, luminal B and triple-negative breast cancer, but not luminal A and HER+ subtypes. Silencing of CAIX expression had no significant effect on the cell proliferation or viability upon treatment with doxorubicin in any of the cell lines studied, while it inhibited spheroid formation in hypoxic conditions. Furthermore, pharmacological inhibition of CAs using acetazolamide had a synergistic effect with doxorubicin on decreasing the spheroid-forming efficiency in MDA-MB-231 cells. Conclusions: Inhibition of CAIX reduces the self-renewal capacity of breast cancer cells, and the combination of doxorubicin and CAIX inhibition is an attractive therapeutic strategy in basal-like and triple-negative breast cancer, which warrants further investigations. [ABSTRACT FROM AUTHOR]

The initial phases of molecular and cellular maladaptive bone responses in early chronic kidney disease (CKD) remain mostly unknown. We induced mild CKD in spontaneously hypertensive rats (SHR) by either causing arterial hypertension lasting six months (sham-operated rats, SO6) or in its' combination with 3/4 nephrectomy lasting two and six months (Nx2 and Nx6, respectively). Sham-operated SHRs (SO2) and Wistar Kyoto rats (WKY2) with a two-month follow-up served as controls. Animals were fed standard chow containing 0.6% phosphate. Upon follow-up completion in each animal, we measured creatinine clearance, urine albumin-to-creatinine ratio, renal interstitial fibrosis, inorganic phosphate (Pi) exchange, intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho, Dickkopf-1, sclerostin, and assessed bone response by static histomorphometry and gene expression profiles. The mild CKD groups had no increase in renal Pi excretion, FGF23, or PTH levels. Serum Pi, Dickkopf-1, and sclerostin were higher in Nx6. A decrease in trabecular bone area and osteocyte number was obvious in SO6. Nx2 and Nx6 had additionally lower osteoblast numbers. The decline in eroded perimeter, a resorption index, was only apparent in Nx6. Significant downregulation of genes related to Pi transport, MAPK, WNT, and BMP signaling accompanied histological alterations in Nx2 and Nx6. We found an association between mild CKD and histological and molecular features suggesting lower bone turnover, which occurred at normal levels of systemic Pi-regulating factors. [ABSTRACT FROM AUTHOR]

The photoreceptors of red light (phytochromes) and blue light (cryptochromes) impact plant growth and metabolism. However, their action has been barely studied, especially in coniferous plants. Therefore, the influence of blue (maximum 450 nm), red (maximum 660 nm), white light (maxima 450 nm + 575 nm), far-red light (maximum 730 nm), white fluorescent light and dark on seed germination, growth, chlorophyll and carotenoid contents, as well as the transcript levels of genes involved in reception, photosynthesis, light and hormonal signaling of Scots pine plantlets, was investigated. The highest values of dry weight, root length and photosynthetic pigment contents were characteristic of 9-day-old plantlets grown under red light, whereas in the dark plantlet length, seed vigor, seed germination, dry weight and pigment contents were decreased. Under blue and white lights, the main studied morphological parameters were decreased or close to red light. The cotyledons were undeveloped under dark conditions, likely due to the reduced content of photosynthetic pigments, which agrees with the low transcript levels of genes encoding protochlorophyllide oxidoreductase (PORA) and phytoene synthase (PSY). The transcript levels of a number of genes involved in phytohormone biosynthesis and signaling, such as GA3ox, RRa, KAO and JazA, were enhanced under red light, unlike under dark conditions. We suggest that the observed phenomena of red light are the most important for the germination of the plantlets and may be based on earlier and enhanced expression of auxin, cytokinin, gibberellin and jasmonate signaling genes activated by corresponding photoreceptors. The obtained results may help to improve reforestation technology; however, this problem needs further study. [ABSTRACT FROM AUTHOR]

Abstract: Cold acclimation is important for crop survival in environments undergoing seasonal low temperatures. It involves the induction of defensive mechanisms including the accumulation of different cryoprotective molecules among which are dehydrins (DHN). Recently several sequences coding for dehydrins were identified in white clover (Trifolium repens). This work aimed to select the most responsive to cold stress DHN analogues in search for cold stress diagnostic markers. The assessment of dehydrin transcript accumulation via RT-PCR and immunodetection performed with three antibodies against the conserved K-, Y-, and S-segment allowed to outline different dehydrin types presented in the tested samples. Both analyses confirmed that YnKn dehydrins were underrepresented in the controls but exposure to low temperature specifically induced their accumulation. Strong immunosignals corresponding to 37–40 kDa with antibodies against Y- and K-segment were revealed in cold-stressed leaves. Another ‘cold-specific’ band at position 52–55 kDa was documented on membranes probed with antibodies against K-segment. Real time RT-qPCR confirmed that low temperatures induced the accumulation of SKn and YnSKn transcripts in leaves and reduced their expression in roots. Results suggest that a YnKn dehydrin transcript with GenBank ID: KC247805 and the immunosignal at 37–40 kDa, obtained with antibodies against Y- and K-segment are reliable markers for cold stress in white clover. The assessment of SKn (GenBank ID: EU846208) and YnSKn (GenBank ID: KC247804) transcript levels in leaves could serve as additional diagnostic tools. [Copyright &y& Elsevier]

A precise balance between protein degradation and synthesis is essential to preserve skeletal muscle mass. Here, we found that TP53INP2, a homolog of the Drosophila melanogaster DOR protein that regulates autophagy in cellular models, has a direct impact on skeletal muscle mass in vivo. Using different transgenic mouse models, we demonstrated that muscle-specific overexpression of Tp53inp2 reduced muscle mass, while deletion of Tp53inp2 resulted in muscle hypertrophy. TP53INP2 activated basal autophagy in skeletal muscle and sustained p62-independent autophagic degradation of ubiquitinated proteins. Animals with muscle-specific overexpression of Tp53inp2 exhibited enhanced muscle wasting in streptozotocin-induced diabetes that was dependent on autophagy; however, TP53INP2 ablation mitigated experimental diabetes-associated muscle loss. The overexpression or absence of TP53INP2 did not affect muscle wasting in response to denervation, a condition in which autophagy is blocked, further indicating that TP53INP2 alters muscle mass by activating autophagy. Moreover, TP53INP2 expression was markedly repressed in muscle from patients with type 2 diabetes and in murine models of diabetes. Our results indicate that TP53INP2 negatively regulates skeletal muscle mass through activation of autophagy. Furthermore, we propose that TP53INP2 repression is part of an adaptive mechanism aimed at preserving muscle mass under conditions in which insulin action is deficient. [ABSTRACT FROM AUTHOR]

We studied the involvement of angiotensin (Ang) II AT receptors in the pathophysiology of kainate (KA)-induced neurotoxicity, focusing on the regulation of the oxidative stress state and expression of HSP 72 in the frontal cortex and hippocampus in two strains, spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. The KA injection was executed after the rats were infused subcutaneously via osmotic mini-pumps with losartan (10 mg/kg day) for 14 days. Losartan delayed the onset of KA-induced seizures in SHRs but not in Wistar rats without affecting the seizure intensity score. This selective AT receptor antagonist decreased the lipid peroxidation only in naive SHRs. However, it attenuated the KA-induced increase in lipid peroxidation in both SHRs and Wistar rats. The adaptive enhancement of cytosolic superoxide dismutase (SOD) activity in KA-treated SHRs was recovered to control level after sub-chronic losartan infusion while no change in mitochondrial SOD activity was detected in the two strains. Both losartan and KA produced a higher expression of HSP 72 in the hippocampus of the two strains compared to naive rats infused with vehicle. Taken together, our findings demonstrate that the efficacy of a sub-chronic systemic losartan infusion in preventing the KA-induced seizure activity and neurotoxicity is more pronounced in SHRs, considered as a model of essential hypertension, than in normotenisve Wistar rats. The results suggest that the blockade of AT1 receptors, commonly used as a strategy for prevention of high blood pressure, may be useful as an adjunctive treatment in status epilepticus to reduce oxidative stress and neurotoxicity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Distinct mouse OFF bipolar cell types express different ionotropic glutamate receptor types. [•] Some OFF bipolar cell types express either AMPA or kainate receptors. [•] Some OFF bipolar cell types express both AMPA and kainate receptors. [•] AMPA and kainate receptors are expressed at separate synaptic sites. [ABSTRACT FROM AUTHOR]

Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration. One potential approach for restoring retinal light sensitivity after photoreceptor degeneration is to express optogenetic tools in retinal ganglion cells (RGCs). For this approach, restoration of ON and OFF center-surround receptive fields in RGCs, a key feature of visual information processing, may be important. A possible solution is to differentially express depolarizing and hyperpolarizing optogenetic tools, such as channelrhodopsin-2 and halorhodopsin, to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs. Recombinant adeno-associated virus (rAAV) vectors have proven to be a powerful vehicle for in vitro and in vivo gene delivery, including in the retina. Therefore, the search for protein targeting motifs that can achieve rAAV-mediated subcellular targeted expression would be particularly valuable for developing therapeutic applications. In this study, we identified two protein motifs that are suitable for rAAV-mediated subcellular targeting for generating center-surround receptive fields while reducing the axonal expression in RGCs. Resulting morphological dendritic field and physiological response field by center-targeting were significantly smaller than those produced by surround-targeting. rAAV motif-mediated protein targeting could also be a valuable tool for studying physiological function and clinical applications in other areas of the central nervous system. [ABSTRACT FROM AUTHOR]

Objective Cisplatin is a widely used gastric cancer (GC) chemotherapy; however, genetic factors regulating GC responses to cisplatin remain obscure. Identifying genes regulating cisplatin resistance could aid clinicians in tailoring treatments, by distinguishing cisplatin sensitive patients from those who might benefit from alternative platinum therapies, and highlight novel targeted strategies for overcoming cisplatin resistance. Here integrated epigenomics is applied to identify genes associated with GC cisplatin resistance Design 20 GC cell lines were subjected to gene expression profiling, DNA methylation profiling and drug response assays. The molecular data were integrated to identify genes highly expressed and unmethylated specifically in cisplatin-resistant lines. Candidate genes were functionally tested by several in vitro and in vivo assays. Clinical impact of candidate genes was also assessed in a cohort of 197 GC patients. Results Epigenomic analysis identified bone morphogenetic protein 4 (BMP4) as an epigenetically regulated gene highly expressed in cisplatin-resistant lines. Functional assays confirmed that BMP4 is necessary and sufficient for the expression of several prooncogenic traits, likely mediated through stimulation of the epithelial-mesenchymal transition. In primary tumours, BMP4 promoter methylation levels were inversely correlated with BMP4 expression, and patients with high BMP4-expressing tumours exhibited significantly worse prognosis. Therapeutically, targeted genetic inhibition of BMP4 caused significant sensitisation of GC cells to cisplatin. Notably, BMP4- expressing GCs also did not exhibit cross resistance to oxaliplatin. Conclusions BMP4 epigenetic and expression status may represent promising biomarkers for GC cisplatin resistance. Targeting BMP4 may sensitise GC cells to cisplatin. Oxaliplatin, a clinically acceptable cisplatin alternative, may represent a potential therapeutic option for BMP4-positive GCs. [ABSTRACT FROM AUTHOR]

Abstract: Retinal bipolar cells and ganglion cells are known to possess voltage-gated T-type Ca2+ channels. Previous electrophysiological recording studies suggested that there is differential expression of different T-type Ca2+ channel α1 subunits among bipolar cells. The detailed expression patterns of the individual T-type Ca2+ channel subunits in the retina, however, remain unknown. In this study, we examined the expression of the CaV3.2 Ca2+ channel α1 subunit in the mouse retina using immunohistochemical analysis and patch-clamp recordings together with a CaV3.2 knock out (KO) mouse line. The specificity of a CaV3.2 Ca2+ channel antibody was first confirmed in recombinant T-type Ca2+ channels expressed in human embryonic kidney (HEK) cells and in CaV3.2 KO mice. Our immunohistochemical analysis indicates that the CaV3.2 antibody labels a subgroup of type-3 cone bipolar cells (CBCs), the PKAβII-immunopositive type-3 CBCs. The labeling was observed throughout the cell including dendrites and axon terminals. Our patch-clamp recording results further demonstrate that CaV3.2 Ca2+ channels contribute to the T-type Ca2+ current in a subpopulation of type-3 CBCs. The findings of this study provide new insights into understanding the functional roles of T-type Ca2+ channels in retinal processing. [Copyright &y& Elsevier]

Memory formation is a complex process involving changes in the synaptic activity and gene expression encoding the insulin-like growth factors. We analyzed changes in the expression of genes encoding the insulin/insulin-like growth factors' proteins at the early period of learning in the CA1 region and dentate gyrus of the dorsal and ventral hippocampus in mice 1 hour after presentation of a new context (contextual fear conditioning) with and without negative reinforcement. It was found that in addition to changes in the expression of immediate early genes c-Fos (in all studied hippocampal fields) and Arc (in dorsal and ventral CA1, as well as in dorsal dentate gyrus), exposure to a new context significantly altered expression of the insulin receptor substrate 2 gene (Irs2) in dorsal CA1 and ventral dentate gyrus irrespectively of the negative reinforcement, which suggests participation of the insulin/IGF system in the early stages of neural activation during learning. [ABSTRACT FROM AUTHOR]

Genomic imprinting is an important and enigmatic form of gene regulation in mammals in which one copy of a gene is silenced in a manner determined by its parental history. Imprinted genes range from those with constitutive monoallelic silencing to those, typically more remote from imprinting control regions, that display developmentally regulated, tissue-specific or partial monoallelic expression. This diversity may make these genes, and the processes they control, more or less sensitive to factors that modify or disrupt epigenetic marks. Imprinted genes have important functions in development and physiology, including major endocrine/neuroendocrine axes. Owing to is central role in coordinating growth, metabolism and reproduction, as well as evidence from genetic and knockout studies, the hypothalamus may be a focus for imprinted gene action. Are there unifying principles that explain why a gene should be imprinted? Conflict between parental genomes over limiting maternal resources, but also co-adaptation between mothers and offspring, have been invoked to explain the evolution of imprinting. Recent reports suggest there may be many more genes imprinted in the hypothalamus than hitherto expected, and it will be important for these new candidates to be validated and to determine whether they conform to current notions of how imprinting is regulated. In fully evaluating the role of imprinted genes in the hypothalamus, much work needs to be done to identify the specific neuronal populations in which particular genes are expressed, establish whether there are pathways in common and whether imprinted genes are involved in long-term programming of hypothalamic functions. [ABSTRACT FROM AUTHOR]

mino acid sequence of the drosophila retrovirus MDG4 ( gypsy) structural protein Gag does not contain a canonical motif known for the majority of vertebrate retroviruses. Moreover, protein translation can theoretically begin with two separated initiation codons located within its unique open reading frame. We designed constructs for expression of two theoretically possible variants of Gag polypeptide and investigated the ability of each product to form virus-like particles in the bacterial cell, i.e., in the absence of eukaryotic cell factors. The results obtained showed that both variants of the gypsy protein Gag form globular particles in the bacterial cell. [ABSTRACT FROM AUTHOR]

Abstract: Acquiring the behavioral significance of sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 min (24 h separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in situ hybridization (FISH) to assess the layer-specific subcellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3–6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1–2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical subcellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. [Copyright &y& Elsevier]

Abstract: The membrane-bound guanylyl cyclases A and B (GC-A/B), which are receptors for natriuretic peptides, are expressed in cancer cells including melanomas and may represent new anticancer targets. Here, we report down-regulation of GC-A/B expression in human metastatic melanoma cells at simulated weightlessness in comparison to 1g conditions, suggesting attenuation of metastatic potential in weightlessness. [Copyright &y& Elsevier]

In the present work, we established a rapid, cost-effective and high-throughput method for genotyping using a multiplexed microsphere-based suspension array platform - Luminex × MAP™, which enabled us to analyse two SNPs in the promoter of IL-6 gene, determining haplotypes associated with different levels of expression. Using this system, IL-6 diversity in four different ethnic groups - East Asians, Caucasians, Hispanic and African Americans - was assessed. Results showed a significant variability in terms of allele, genotype and haplotype distribution. Considering the important immunoregulatory role of this cytokine and as a clinically relevant marker, this genotyping approach will provide a powerful tool for disease association or transplant outcome studies. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian retina consists of five major classes of neuronal cells, as well as glial cells, and it contains more than 50 cell types. The ability to manipulate gene expression in specific cell type(s) in the retina is important for understanding the molecular mechanisms of retinal function and diseases. The Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in vivo in a cell- and tissue-specific fashion. The key to this tool is the availability of Cre mouse lines with cell- or tissue-type specific expression of Cre recombinase. To date, a large number of Cre-transgenic mouse lines have been generated to target Cre recombinase expression to specific neuronal and glial cell populations in the central nervous system; however, information about the expression patterns of Cre recombinase lines in the retina is largely lacking. In this study, we examined and characterized the expression patterns of Cre recombinase in the retinas of 15 Cre-transgenic mouse lines. Significant Cre-induced recombination or expression of Cre recombinase was observed in the majority of these lines. In particular, we found several Cre lines in which the Cre-induced recombination was found to target exclusively or predominantly a single type or class of retinal cells, including bistratified retinal ganglion cells, starburst amacrine cells, rod bipolar cells, and Müller glial cells. In other lines, the Cre-induced recombination was found in several retinal cell types. These Cre lines provide a valuable resource for retinal research. [Copyright &y& Elsevier]

Background: Thrombopoietin (Thpo) and its receptor (Mpl), which regulate megakaryopoiesis, are expressed in the central nervous system (CNS), where Thpo is thought to exert pro-apoptotic effects on newly generated neurons. Mpl expression has been analysed in brain tissue on transcript level and in cultured primary rat neurons and astrocytes on protein level. Herein, we analysed Mpl expression in the developing and adult murine CNS by immunohistochemistry and investigated the brain of mice with homozygous Mpl deficiency (Mpl-/-) by MRI. Results: Mpl was not detectable at developmental stages E12 to E15 in any resident cells of the CNS. From E18 onwards, robust Mpl expression was found in various brain areas, including cerebral cortex, olfactory bulb, thalamus, hypothalamus, medulla, pons, and the grey matter of spinal cord. However, major developmental changes became obvious: In the subventricular zone of the cerebral cortex Mpl expression occurred only during late gestation, while in the hippocampus Mpl expression was detectable for first time at stage P4. In the white matter of the cerebellum Mpl expression was restricted to the perinatal period. In the adult cerebellum, Mpl expression switched to Purkinje cell. The majority of other Mpl-positive cells were NeuN-positive neurons. None of the cells could be double-labelled with astrocyte marker GFAP. Mpl-/- mice showed no gross abnormalities of the brain. Conclusions: Our data locate Mpl expression to neurons at different subdivisions of the spinal cord, rhombencephalon, midbrain and prosencephalon. Besides neuronal cells Mpl protein is also expressed in Purkinje cells of the adult cerebellum. [ABSTRACT FROM AUTHOR]

Background: Reference genes are widely used to normalise transcript abundance data determined by quantitative RTPCR and microarrays. However, the approaches taken to define reference genes can be variable. Although Oryza sativa (rice) is a widely used model plant and important crop specie, there has been no comprehensive analysis carried out to define superior reference genes. Results: Analysis of 136 Affymetrix transcriptome datasets comprising of 373 genome microarrays from studies in rice that encompass tissue, developmental, abiotic, biotic and hormonal transcriptome datasets identified 151 genes whose expression was considered relatively stable under all conditions. A sub-set of 12 of these genes were validated by quantitative RT-PCR and were seen to be stable under a number of conditions. All except one gene that has been previously proposed as a stably expressed gene for rice, were observed to change significantly under some treatment. Conclusion: A new set of reference genes that are stable across tissue, development, stress and hormonal treatments have been identified in rice. This provides a superior set of reference genes for future studies in rice. It confirms the approach of mining large scale datasets as a robust method to define reference genes, but cautions against using gene orthology or counterparts of reference genes in other plant species as a means of defining reference genes. [ABSTRACT FROM AUTHOR]

Abstract: Retinal bipolar cells relay visual information from photoreceptors to third-order retinal neurons. Bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The identification of molecular markers that can label specific retinal bipolar cells could facilitate the investigation of bipolar cell functions in the retina. Transgenic mice with specific cell type(s) labeled with green fluorescent protein (GFP) have become a powerful tool for morphological and functional studies of neurons in the CNS, including the retina. In this study, we report a 5-hydroxytryptamine receptor 2a (5-HTR2a) transgenic mouse line in which expression of GFP was observed in two populations of bipolar cells in the retina. Based on the terminal stratification and immunostaining, all the strongly GFP-labeled bipolar cells were found to be type 4 cone bipolar cells. A small population of weakly labeled bipolar cells was also observed, which may represent type 8 or 9 cone bipolar cells. GFP expression in retinal cone bipolar cells was seen as early as postnatal day 5. In addition, despite severe retinal degeneration due to the presence of the rd1 mutation in this transgenic line, the density of GFP-labeled cone bipolar cells remained stable up to at least 6 months of age. This transgenic mouse line will be a useful tool for the study of type 4 cone bipolar cells in the retina under both normal and disease conditions. [Copyright &y& Elsevier]

Many solid cancers are known to exhibit a high degree of heterogeneity in their deregulation of different oncogenic pathways. We sought to identify major oncogenic pathways in gastric cancer (GC) with significant relationships to patient survival. Using gene expression signatures, we devised an in silico strategy to map patterns of oncogenic pathway activation in 301 primary gastric cancers, the second highest cause of global cancer mortality. We identified three oncogenic pathways (proliferation/stem cell, NF-κB, and Wnt/β-catenin) deregulated in the majority (>70%) of gastric cancers. We functionally validated these pathway predictions in a panel of gastric cancer cell lines. Patient stratification by oncogenic pathway combinations showed reproducible and significant survival differences in multiple cohorts, suggesting that pathway interactions may play an important role in influencing disease behavior. Individual GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Predicting pathway activity by expression signatures thus permits the study of multiple cancer-related pathways interacting simultaneously in primary cancers, at a scale not currently achievable by other platforms. [ABSTRACT FROM AUTHOR]

Abstract: Gender differences in lung disease and cancer are well-established. We reported estrogenic transcriptional responses in lung adenocarcinoma cells from females but not males despite similar estrogen receptor (ER) expression. Here we tested the hypothesis that normal human bronchial epithelial cells (HBECs) show gender-independent estrogenic responses. We report that a small sample of HBECs express ∼twice as much ERβ as ERα. ERα and ERβ were located in the cytoplasm, nucleus, and mitochondria. In contrast to lung adenocarcinoma cells, estradiol (E2) induced estrogen response element (ERE)-mediated luciferase reporter activity in transiently transfected HBECs regardless of donor gender. Overexpression of ERα-VP16 increased ERE-mediated transcriptional activity in all HBECs. E2 increased and 4-hydroxytamoxifen and ICI 182,780 inhibited HBEC proliferation and cyclin D1 expression in a cell line-specific manner. In conclusion, the response of HBECs to ER ligands is gender-independent suggesting that estrogenic sensitivity may be acquired during lung carcinogenesis. [Copyright &y& Elsevier]

Abstract: Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A–C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A–C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B. [Copyright &y& Elsevier]

The system of transformation of heterologous genes under the method of agrobacterial transfer into Acremonium chrysogenum ATCC 11550 wild-type strains, natural producents of beta-lactam antibiotic cephalosporin C, and strains highly producing cephalosporin C no. 26/8 revealed by the multistage selection on its basis were developed. Vectors for agrobacterial transformation of A. chrysogenum containing expression cassettes of genes encoding resistance to geneticin (G418) and bleomicin (ZeocinTM) antibiotics under control of Ashbya gossypii and Saccharomyces cerevisiae TEF1 promoters were constructed. A comparable assessment of agrotransformation methods while co-cultivating fungi and agrobacterial cells on filters and in deep culture was conducted. Transformants, selected by resistance to geneticin and bleomicin, were characterized by PCR and Southern blot analyses. [ABSTRACT FROM AUTHOR]

In this study, we aimed to explore the expression of genes associated with neuroinflammation in patients with benign and highly active multiple sclerosis (MS) and healthy controls, to define gene signatures associated with MS as well as disease activity and progression. We identified differences in the expression of 89 genes in benign and highly active MS patients and in healthy controls (q < 0.05). Twenty-eight genes related to myeloid cells function, the innate immune response, apoptosis, and autophagy were differentially expressed in patients with benign and highly active MS. Time to second relapse and expanded disability status scale (EDSS) scores were correlated with the expression of genes associated with myeloid cells function, innate immunity, and apoptosis. Our results could indicate the importance of innate immunity-associated pathways in maintaining high disease activity in MS and their crucial role in disease progression. [Display omitted] • The neuroinflammatory gene expression differed between MS patients and healthy controls. • The most distinct expression profile was observed in highly active multiple sclerosis. • Differentially expressed genes were mostly linked to apoptosis, autophagy, myeloid cells function, and innate immunity. • Myeloid cells function, innate immunity, and apoptosis-related genes expression was correlated with EDSS scores. [ABSTRACT FROM AUTHOR]

Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40–50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly β- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1. [ABSTRACT FROM AUTHOR]

Abstract: The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (AMV). v-Myb is thought to exert its biological effects by deregulating the expression of specific target genes. Here we have used DNaseI hypersensitive site mapping and reporter gene assays to study the activation of three Myb target genes – mim-1, the lysozyme gene and the C/EBPβ gene – all of which are activated by Myb in myelomonocytic cells but not in other hematopoietic lineages. We have found that these genes are activated by Myb via more than one cis-regulatory region. Our data suggest that all three genes are activated by Myb by dual mechanisms involving the promoters as well as enhancers. Using a cell line that expresses an estrogen-inducible v-Myb/estrogen receptor fusion protein we have also determined the effect of Myb on the expression of the C/EBPα gene. Our results show that C/EBPα expression is down-regulated by v-Myb. Thus, v-Myb affects the expression of two C/EBP family members in opposite directions. [Copyright &y& Elsevier]

Chronic allograft nephropathy (CAN) is one of the main causes of graft loss in renal transplantation. Polymorphisms with functional significance in the promoter and coding regions of cytokine genes have been suggested as a possible factor for graft rejection. The aim of this study was to investigate the impact of cytokine gene polymorphism of pro and anti-inflammatory cytokines on development of CAN in a group of renal transplant patients and donors. Eight single nucleotide polymorphisms (SNPs) including TNFA (-308), TGFB1 (cdns10, 25), IL-10 (-1082, -819, -592), IL-6 (- 174) and IFNG (+874) were analyzed in 56 patients with stable graft function (SGF), 10 with CAN and 28 kidney donors by PCR-SSP method. CAN was significantly associated with the recipient TGFB1 cod10 T/T and combination of cods 10, 25 T/T G/G genotypes (high producer), (p <0.05). Influence of patient's TNFA genotype correlated with high level of gene expression on the development of CAN was further demonstrated when the patients were stratified according to the HLA mismatches (HLA-DRB MMs). Additionally donor TNFA-308 G/A (high) and IL-6- 174 CC (low) genotypes were increased in cases with CAN. No statistically significant differences in distribution of IL-10, IL-6 and IFNG genotypes between recipients with SGF and CAN were found. In conclusion our data suggest that the high producer genotype of profibrogenetic TGF-β1, pro-inflammatory TNF-α and genetically determined low production of immunoregulatory IL-6 cytokine might be risk factors for CAN development. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate and analyze some peculiarities in the age specificity of protein expression in the fat body of A. mellifera during the ontogenesis of the individuals. By using 7.5% ployacrylamide gel electrophoresis, 189 individual samples of fat body extracts were investigated in the course of their development in the following stages and ages: larvae-third, fourth and fifth larval age (L3, L4, L5); prepupae (PP) and pupae-white-eyed and dark-eyed pupae (WP, DP). A total of 25 protein fractions were expressed in the fat body of male and female individuals during their development. Some sex-differences in studied fat body proteins were noticed. The age and stage specific dynamics in the expression of established proteins of the fat body spectrum were analyzed and commented. [ABSTRACT FROM AUTHOR]

The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over ~37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1α expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdhQ111/Q111 cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1α-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdh111/Q111 cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-jB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD. [ABSTRACT FROM AUTHOR]

Trans-activation of HIV-1 transcription is triggered by the interaction of the protein Tat and host cellular factors with a 59-residue stem-loop RNA known as the trans-activation responsive element (TAR). Here we compare the trans-activation steric block inhibitory activity of 16-mer oligonucleotides targeted to TAR containing tricyclo-DNAs, and their mixmers with LNA or OMe residues, with LNA/OMe oligonucleotide. Despite generally weaker TAR RNA binding affinity, all tricyclo-DNA oligonucleotides showed similarly good activity levels to OMe/LNA oligonucleotide in a HeLa Tat-dependent trans-activation cell reporter assay with cationic lipid delivery, but mixmers of tricyclo-DNA were inactive. Tricyclo-DNA 16-mer showed sequence-specific inhibition of β-galactosidase expression in an anti-HIV HeLa cell reporter assay. [ABSTRACT FROM AUTHOR]

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells. [ABSTRACT FROM AUTHOR]

Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stern cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells. [ABSTRACT FROM AUTHOR]