Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts

BACKGROUND: Rapid, robust and reversible induction of transgene expression would significantly facilitate cancer gene therapy as well as allow the in vivo functional study of newly discovered genes in tumor formation and progression. The popularity of the ecdysone inducible gene switch system has led us to investigate whether such a system can successfully regulate gene expression in a syngeneic tumor system in vivo. RESULTS: MBT-2 and Panc02 carcinoma cells were transfected with components of a modification of the ecdysone switch system driving firefly luciferase (F-Luc). In vitro luciferase expression ± ecdysone analog GS-E indicated a robust induction with minimal baseline activity and complete decay after 24 hours without drug. In vitro selection of MBT-2 transfected cell clones which had complete absence of F-Luc expression in the absence of stimulation but which expressed this gene at high levels in response to GS-E were chosen for in vivo evaluation. Tumors from engineered MBT-2 cells were grown to 5 mm in diameter prior to GS-E administration, animals euthanized and tumors removed at 6, 12 and 24 hours after GS-E administration and assayed for F-Luc activity. GS-E resulted in a maximal induction of F-Luc activity at 6 hours in tumor tissue with almost complete reversion to control levels by 12 hours. CONCLUSIONS: This study is the first demonstration that robust and reversible transgene expression in tumors is feasible using the ecdysone system, allowing future rapid in vivo functional characterization of gene function or gene therapy applications

Regulation of smooth muscle α-actin expression and hypertrophy in cultured mesangial cells

Regulation of smooth muscle α-actin expression and hypertrophy in cultured mesangial cells.BackgroundMesangial cells during embryonic development and glomerular disease express smooth muscle α-actin (α-SMA). We were therefore surprised when cultured mesangial cells deprived of serum markedly increased expression of α-SMA. Serum-deprived mesangial cells appeared larger than serum-fed mesangial cells. We hypothesized that α-SMA expression may be more reflective of mesangial cell hypertrophy than hyperplasia.MethodsHuman mesangial cells were cultured in medium alone or with fetal bovine serum, thrombin, platelet-derived growth factor-BB (PDGF-BB) and/or transforming growth factor-β1 (TGF-β1). α-SMA expression was examined by immunofluorescence, Western blot, and Northern blot analysis. Cell size was analyzed by forward light scatter flow cytometry.Resultsα-SMA mRNA was at least tenfold more abundant after three to five days in human mesangial cells plated without serum, but β-actin mRNA was unchanged. Serum-deprived cells contained 5.3-fold more α-SMA after three days and 56-fold more after five days by Western blot. Serum deprivation also increased α-SMA in rat and mouse mesangial cells. The effects of serum deprivation on α-SMA expression were reversible. Mesangial cell mitogens, thrombin or PDGF-BB, decreased α-SMA, but TGF-β1 increased α-SMA expression and slowed mesangial cell proliferation in serum-plus medium. Flow cytometry showed that serum deprivation or TGF-β1 treatment caused mesangial cell hypertrophy. PDGF-BB, thrombin, or thrombin receptor-activating peptide blocked hypertrophy in response to serum deprivation.ConclusionsWe conclude that increased α-SMA expression in mesangial cells reflects cellular hypertrophy rather than hyperplasia

Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response

© 2019, The Author(s). Molecular mechanisms driving disease course and response to therapy in ulcerative colitis (UC) are not well understood. Here, we use RNAseq to define pre-treatment rectal gene expression, and fecal microbiota profiles, in 206 pediatric UC patients receiving standardised therapy. We validate our key findings in adult and paediatric UC cohorts of 408 participants. We observe a marked suppression of mitochondrial genes and function across cohorts in active UC, and that increasing disease severity is notable for enrichment of adenoma/adenocarcinoma and innate immune genes. A subset of severity genes improves prediction of corticosteroid-induced remission in the discovery cohort; this gene signature is also associated with response to anti-TNFα and anti-α 4 β 7 integrin in adults. The severity and therapeutic response gene signatures were in turn associated with shifts in microbes previously implicated in mucosal homeostasis. Our data provide insights into UC pathogenesis, and may prioritise future therapies for nonresponders to current approaches

Effects of Standardized Ileal Digestible Histidine:Lysine Ratio on Growth Performance of 15- to 25-lb Pigs

Two experiments were conducted to determine the standardized ileal digestible (SID) His:Lys requirement of 15- to 25-lb nursery pigs. A total of 360 and 350 pigs (DNA 241 × 600), initially 15.6 and 14.5 lb body weight (BW), were used in Exp. 1 and 2, respectively. There were 5 pigs per pen and 12 and 10 replicates per treatment in Exp. 1 and 2, respectively. After weaning, pigs were fed a common pelleted diet for 10 d in Exp. 1 and 7 d in Exp. 2. Then, pens were assigned to treatments in a randomized complete block design with BW as the blocking factor. Dietary treatments consisted of SID His:Lys ratios of 24, 28, 32, 36, 40, and 44% in Exp. 1 and 24, 28, 30, 32, 34, 36, and 42% in Exp. 2. Experimental diets were fed in pellet form for 10 d in Exp. 1 and 14 d in Exp. 2 followed by a common mash diet for 15 d in Exp. 1 and 14 d in Exp. 2. Data were analyzed using the GLIMMIX and NLMIXED procedures of SAS. Competing statistical models were quadratic polynomial (QP), broken-line linear (BLL), and broken-line quadratic (BLQ). In Exp. 1, increasing SID His:Lys increased (quadratic, P \u3c 0.001) average daily gain (ADG), average daily feed intake (ADFI), and BW and improved (quadratic, P \u3c 0.001) feed-to-gain ratio (F/G). In Exp. 2, ADG increased (quadratic, P = 0.001) and F/G improved (quadratic, P = 0.001) and ADFI linearly increased (P = 0.001) with increasing SID His:Lys. The best-fitting model for all response variables analyzed was the BLL. In Exp. 1, requirement estimates were 29.7%, 29.1%, and 29.8% SID His:Lys for ADG, ADFI, and gain-to-feed ratio (G:F), respectively. In Exp. 2, the SID His:Lys requirements were estimated at 31.0% for ADG and 28.6% for G:F. These results suggest that the NRC may overestimate the SID His:Lys requirement for 15- to 25-lb pigs. Therefore, nursery diets can be formulated with higher inclusion of crystalline amino acids before His becomes limiting

Genetic Determinants of Variability in Glycated Hemoglobin (HbA1c) in Humans: Review of Recent Progress and Prospects for Use in Diabetes Care

Glycated hemoglobin A1c (HbA1c) indicates the percentage of total hemoglobin that is bound by glucose, produced from the nonenzymatic chemical modification by glucose of hemoglobin molecules carried in erythrocytes. HbA1c represents a surrogate marker of average blood glucose concentration over the previous 8 to 12 weeks, or the average lifespan of the erythrocyte, and thus represents a more stable indicator of glycemic status compared with fasting glucose. HbA1c levels are genetically determined, with heritability of 47% to 59%. Over the past few years, inroads into understanding genetic predisposition by glycemic and nonglycemic factors have been achieved through genome-wide analyses. Here I review current research aimed at discovering genetic determinants of HbA1c levels, discussing insights into biologic factors influencing variability in the general and diabetic population, and across different ethnicities. Furthermore, I discuss briefly the relevance of findings for diabetes monitoring and diagnosis

Extent of Height Variability Explained by Known Height-Associated Genetic Variants in an Isolated Population of the Adriatic Coast of Croatia

BACKGROUND: Human height is a classical example of a polygenic quantitative trait. Recent large-scale genome-wide association studies (GWAS) have identified more than 200 height-associated loci, though these variants explain only 2∼10% of overall variability of normal height. The objective of this study was to investigate the variance explained by these loci in a relatively isolated population of European descent with limited admixture and homogeneous genetic background from the Adriatic coast of Croatia. METHODOLOGY/PRINCIPAL FINDINGS: In a sample of 1304 individuals from the island population of Hvar, Croatia, we performed genome-wide SNP typing and assessed the variance explained by genetic scores constructed from different panels of height-associated SNPs extracted from five published studies. The combined information of the 180 SNPs reported by Lango Allen el al. explained 7.94% of phenotypic variation in our sample. Genetic scores based on 20~50 SNPs reported by the remaining individual GWA studies explained 3~5% of height variance. These percentages of variance explained were within ranges comparable to the original studies and heterogeneity tests did not detect significant differences in effect size estimates between our study and the original reports, if the estimates were obtained from populations of European descent. CONCLUSIONS/SIGNIFICANCE: We have evaluated the portability of height-associated loci and the overall fitting of estimated effect sizes reported in large cohorts to an isolated population. We found proportions of explained height variability were comparable to multiple reference GWAS in cohorts of European descent. These results indicate similar genetic architecture and comparable effect sizes of height loci among populations of European descent

The HITRAN2020 Molecular Spectroscopic Database

The HITRAN database is a compilation of molecular spectroscopic parameters. It was established in the early 1970s and is used by various computer codes to predict and simulate the transmission and emission of light in gaseous media (with an emphasis on terrestrial and planetary atmospheres). The HITRAN compilation is composed of five major components: the line-by-line spectroscopic parameters required for high-resolution radiative-transfer codes, experimental infrared absorption cross-sections (for molecules where it is not yet feasible for representation in a line-by-line form), collision-induced absorption data, aerosol indices of refraction, and general tables (including partition sums) that apply globally to the data. This paper describes the contents of the 2020 quadrennial edition of HITRAN. The HITRAN2020 edition takes advantage of recent experimental and theoretical data that were meticulously validated, in particular, against laboratory and atmospheric spectra. The new edition replaces the previous HITRAN edition of 2016 (including its updates during the intervening years). All five components of HITRAN have undergone major updates. In particular, the extent of the updates in the HITRAN2020 edition range from updating a few lines of specific molecules to complete replacements of the lists, and also the introduction of additional isotopologues and new (to HITRAN) molecules: SO, CH3F, GeH4, CS2, CH3I and NF3. Many new vibrational bands were added, extending the spectral coverage and completeness of the line lists. Also, the accuracy of the parameters for major atmospheric absorbers has been increased substantially, often featuring sub-percent uncertainties. Broadening parameters associated with the ambient pressure of water vapor were introduced to HITRAN for the first time and are now available for several molecules. The HITRAN2020 edition continues to take advantage of the relational structure and efficient interface available at www.hitran.org and the HITRAN Application Programming Interface (HAPI). The functionality of both tools has been extended for the new edition

The HITRAN2020 molecular spectroscopic database

The HITRAN database is a compilation of molecular spectroscopic parameters. It was established in the early 1970s and is used by various computer codes to predict and simulate the transmission and emission of light in gaseous media (with an emphasis on terrestrial and planetary atmospheres). The HITRAN compilation is composed of five major components: the line-by-line spectroscopic parameters required for high-resolution radiative-transfer codes, experimental infrared absorption cross-sections (for molecules where it is not yet feasible for representation in a line-by-line form), collision-induced absorption data, aerosol indices of refraction, and general tables (including partition sums) that apply globally to the data. This paper describes the contents of the 2020 quadrennial edition of HITRAN. The HITRAN2020 edition takes advantage of recent experimental and theoretical data that were meticulously validated, in particular, against laboratory and atmospheric spectra. The new edition replaces the previous HITRAN edition of 2016 (including its updates during the intervening years). All five components of HITRAN have undergone major updates. In particular, the extent of the updates in the HITRAN2020 edition range from updating a few lines of specific molecules to complete replacements of the lists, and also the introduction of additional isotopologues and new (to HITRAN) molecules: SO, CH3F, GeH4, CS2, CH3I and NF3. Many new vibrational bands were added, extending the spectral coverage and completeness of the line lists. Also, the accuracy of the parameters for major atmospheric absorbers has been increased substantially, often featuring sub-percent uncertainties. Broadening parameters associated with the ambient pressure of water vapor were introduced to HITRAN for the first time and are now available for several molecules. The HITRAN2020 edition continues to take advantage of the relational structure and efficient interface available at www.hitran.org and the HITRAN Application Programming Interface (HAPI). The functionality of both tools has been extended for the new edition

Association of the Type 2 Diabetes Mellitus Susceptibility Gene, TCF7L2, with Schizophrenia in an Arab-Israeli Family Sample

Many reports in different populations have demonstrated linkage of the 10q24–q26 region to schizophrenia, thus encouraging further analysis of this locus for detection of specific schizophrenia genes. Our group previously reported linkage of the 10q24–q26 region to schizophrenia in a unique, homogeneous sample of Arab-Israeli families with multiple schizophrenia-affected individuals, under a dominant model of inheritance. To further explore this candidate region and identify specific susceptibility variants within it, we performed re-analysis of the 10q24-26 genotype data, taken from our previous genome-wide association study (GWAS) (Alkelai et al, 2011). We analyzed 2089 SNPs in an extended sample of 57 Arab Israeli families (189 genotyped individuals), under the dominant model of inheritance, which best fits this locus according to previously performed MOD score analysis. We found significant association with schizophrenia of the TCF7L2 gene intronic SNP, rs12573128, (p = 7.01×10−6) and of the nearby intergenic SNP, rs1033772, (p = 6.59×10−6) which is positioned between TCF7L2 and HABP2. TCF7L2 is one of the best confirmed susceptibility genes for type 2 diabetes (T2D) among different ethnic groups, has a role in pancreatic beta cell function and may contribute to the comorbidity of schizophrenia and T2D. These preliminary results independently support previous findings regarding a possible role of TCF7L2 in susceptibility to schizophrenia, and strengthen the importance of integrating linkage analysis models of inheritance while performing association analyses in regions of interest. Further validation studies in additional populations are required

Haematopoietic stem cells in perisinusoidal niches are protected from ageing.

With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing