Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase

Genomic integration by the Streptomyces bacteriophage ϕC31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the ϕC31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34⁺ haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of ϕC31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in ϕC31 integrase transfected A549 lung than Jurkat T cells. When the ϕC31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the ϕC31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of ϕC31 integrase activity.     

Malakoplakia of bone. A case report

BACKGROUND: Malakoplakia is an uncommon but distinctive granulomatous disease, characterized by an accumulation of histiocytes or Von Hansemann cells containing intracytoplasmic, laminated Michaelis-Gutmann bodies. CASE: A 3-year-old male presented with a tender swelling in the left gluteal region that had been present for one month. Smears made from a fine needle aspirate showed large histiocytic cells containing intracytoplasmic, basophilic, laminated, targetoid Michaelis-Gutmann bodies resembling Von Hansemann cells in malakoplakia. Histopathology confirmed the diagnosis of malakoplakia of bone. CONCLUSION: This case, histologically proven to be malakoplakia, demonstrated regression of the lesion following therapy. The characteristic cytologic features and presence of Von Hansemann cells may in themselves be diagnostic and obviate the need for biopsy.     

The validity of self-reported weight change among adolescents and young adults

Objective: To assess the association of weight change based on serial self‐reported vs. measured weights. Research Methods and Procedures: Two thousand two hundred eighty‐four male and 2476 female participants in the National Longitudinal Study of Adolescent Health who provided information on weight at Waves II and III and were at least 16 years of age were studied. Linear regression was used to assess predictors of the discrepancy between weight change based on self‐reported vs. measured weights. Logistic regression was used to identify predictors of self‐report correctly classifying participants in terms of weight change category. Results: Self‐reported weight was slightly lower than measured weight at Waves II and III, but weight change based on self‐reported weights underestimated true weight change by only 2.1 (female participants) to 2.8 (male participants) pounds. Overweight and obese female participants were consistent in their under‐reporting of their weight more than their leaner peers; thus, the discrepancy between weight change from Wave II to Wave III based on serial self‐reports vs. measured weights was significantly smaller among the obese female vs. healthy‐weight female participants (0.3‐ vs. 2.3‐pound underestimation, p < 0.05). Among the male participants, the same pattern was evident. African‐American ethnicity, Hispanic ethnicity, the level of physical activity, the hours per week watching television, and weight change efforts were not related to the discrepancy between weight change based on self‐reported vs. measured weights. Discussion: The discrepancy between weight change based on self‐report vs. measured weights was minor and not related to race, weight change efforts, activity, or inactivity, thus suggesting that much of the error is random.     

Race and gender differences in the association of dieting and gains in BMI among young adults

Objective: To assess the relationship between dieting and subsequent weight change and whether the association varies by gender or race/ethnicity. Research Methods and Procedures: Male (n = 4100) and female (n = 4302) participants in the National Longitudinal Study of Adolescent Health who provided information on weight and height at baseline and two follow‐up assessments and were not missing information on weight control strategies or race were studied. Generalized estimating equations were used to assess whether dieting to lose or maintain weight at Wave I or II predicted BMI (kg/m²) change between adolescence and young adulthood (Wave II to III). Analyses were stratified by gender and took sampling weights and clustering into account. Results: At Wave I, the mean age of the participants was 14.9 years. Approximately 29.3% of female participants and 9.8% of male participants reported dieting in Wave I or II. Fewer African Americans than whites (6.2% vs. 10.0% and 25.5% vs. 31.2%, p = 0.007 and p = 0.02, among males and females, respectively) reported dieting. Between Waves II and III, participants gained on average 3.3 kg/m². Independent of BMI gain during adolescence (Waves I to II), female participants who dieted to lose or maintain weight during adolescence made larger gains in BMI during the 5 years between Waves II and III (mean additional gain, 0.39 kg/m²; 95% confidence interval, 0.08 to 0.71) than their nondieting peers. The association was not significant among the male participants. The association was largest among African‐American female participants. Discussion: The results suggest that not only is dieting to lose weight ineffective, it is actually associated with greater weight gain, particularly among female adolescents. Female African‐American dieters made the largest BMI gains.     

Marker Assisted Transfer of Two Powdery Mildew Resistance Genes PmTb7A.1 and PmTb7A.2 from Triticum boeoticum (Boiss.) to Triticum aestivum (L.)

Powdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the important wheat diseases, worldwide. Two PM resistance genes, designated as PmTb7A.1 and PmTb7A.2, were identified in T. boeoticum acc. pau5088 and mapped on chromosome 7AL approximately 48cM apart. Two resistance gene analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were identified to be linked to the PmTb7A.1 and PmTb7A.2, at a distance of 0.6cM and 6.0cM, respectively. In the present study, following marker assisted selection (MAS), the two genes were transferred to T. aestivum using T. durum as bridging species. As many as 12,317 florets of F₁ of the cross T. durum /T. boeoticum were pollinated with T. aestivum lines PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively, of three-way F₁. The resulting F₁s of the cross T. durum/T. boeoticum//T. aestivum were screened with marker flanking both the PM resistance genes PmTb7A.1 and PmTb7A.2 (foreground selection) and the selected plants were backcrossed to generate BC₁F₁. Marker assisted selection was carried both in BC₁F₁ and the BC₂F₁ generations. Introgression of alien chromatin in BC₂F₁ plants varied from 15.4 - 62.9 percent. Out of more than 110 BC₂F₁ plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants with minimum of the alien introgression. Cytological analysis showed that most plants have chromosome number ranging from 40-42. The BC₂F₂ plants homozygous for the two genes have been identified. These will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. The PM resistance gene PmTb7A.1 maps in a region very close to Sr22, a stem rust resistance gene effective against the race Ug99. Analysis of selected plants with markers linked to Sr22 showed introgression of Sr22 from T. boeoticum in several BC₂F₁ plants. Thus, in addition to PM resistance, these progeny might also carry resistance to stem rust race Ug99.     

Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux

Oilseeds provide a unique platform for the production of high-value fatty acids that can replace non-sustainable petroleum and oceanic sources of specialty chemicals and aquaculture feed. However, recent efforts to engineer the seeds of crop and model plant species to produce new types of fatty acids, including hydroxy and conjugated fatty acids for industrial uses and long-chain omega-3 polyunsaturated fatty acids for farmed fish feed, have met with only modest success. The collective results from these studies point to metabolic 'bottlenecks' in the engineered plant seeds that substantially limit the efficient or selective flux of unusual fatty acids between different substrate pools and ultimately into storage triacylglycerol. Evidence is emerging that diacylglycerol acyltransferase 2, which catalyzes the final step in triacylglycerol assembly, is an important contributor to the synthesis of unusual fatty acid-containing oils, and is likely to be a key target for future oilseed metabolic engineering efforts.     

Cloning,expression analysis and In silico characterization of HSP101: a potential player conferring heat stress in Aegilops speltoides (Tausch) Gren

Heat shock protein (HSP101) function as molecular chaperones and confer thermotolerance to plants. In the present investigation, identification, comprehensive expression analysis, phylogeny and protein modelling of HSP101 gene has been done in Aegilops speltoides accession Pau3583. In the present study, we cloned and in silico characterized a HSP101C gene designated as AsHSP101C-Pau3583. AsHSP101C-Pau3583 is 4180 bp long with seven exons and six introns and encoded a polypeptide of 910 amino acids predicted by FGENESH. We have identified 58 SNPs between the AsHSP101C-Pau3583 and reference gene sequence extracted from Ae. speltoides TGAC assembly. Real-time RT-PCR analysis of expression levels of HSP101 gene in two wheat genotypes under heat stress revealed that gene namely HSP101C was up-regulated in Aegilops speltoides acc. Pau3583 by > fourfold in comparison to Triticum aestivum cv. PBW343 under heat stress signifies that it plays a role in conferring heat tolerance. Sequence comparison and phylogenetic analysis of AsHSP101C-Pau3583 with seven wheat homologs Triticum aestivum, Aegilops speltoides (TGAC), Triticum durum cv Cappelli, Triticum durum cv Strongfield, Triticum monococcum, Aegilops tauschii and Triticum urartu showed significant similarities with highly conserved coding regions and functional domains (AAA, AAA + 2, ClpB domains), suggesting the conserved function of HSP101C in different species. The illustration of the protein models of HSP101C in homologs provided information for the ATP-binding motifs within the nucleotide binding domains (NBD), specific for the chaperone activity. These findings are important and identified SNPs could be used for designing markers for ensuring the transfer of AsHSP101C-Pau3583 gene into hexaploid wheat and its role in heat tolerance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01005-2.