Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event

BACKGROUND: Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage λ red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step. RESULTS: We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion. CONCLUSION: This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacteria, making moving transgenes from plasmids to a chromosomal location a simple matter. The non-requirement for selection is particularly well suited for use in development of unmarked strains for environmental release, such as live-vector vaccine strains, and also for promoter-fusion studies, and experiments in which every bacterial cell must express a transgene construct

Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7

The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein

The Impact of HAART on the Respiratory Complications of HIV Infection: Longitudinal Trends in the MACS and WIHS Cohorts

Objective: To review the incidence of respiratory conditions and their effect on mortality in HIV-infected and uninfected individuals prior to and during the era of highly active antiretroviral therapy (HAART). Design: Two large observational cohorts of HIV-infected and HIV-uninfected men (Multicenter AIDS Cohort Study [MACS]) and women (Women's Interagency HIV Study [WIHS]), followed since 1984 and 1994, respectively. Methods: Adjusted odds or hazards ratios for incident respiratory infections or non-infectious respiratory diagnoses, respectively, in HIV-infected compared to HIV-uninfected individuals in both the pre-HAART (MACS only) and HAART eras; and adjusted Cox proportional hazard ratios for mortality in HIV-infected persons with lung disease during the HAART era. Results: Compared to HIV-uninfected participants, HIV-infected individuals had more incident respiratory infections both pre-HAART (MACS, odds ratio [adjusted-OR], 2.4; 95% confidence interval [CI], 2.2-2.7; p<0.001) and after HAART availability (MACS, adjusted-OR, 1.5; 95%CI 1.3-1.7; p<0.001; WIHS adjusted-OR, 2.2; 95%CI 1.8-2.7; p<0.001). Chronic obstructive pulmonary disease was more common in MACS HIV-infected vs. HIV-uninfected participants pre-HAART (hazard ratio [adjusted-HR] 2.9; 95%CI, 1.02-8.4; p = 0.046). After HAART availability, non-infectious lung diseases were not significantly more common in HIV-infected participants in either MACS or WIHS participants. HIV-infected participants in the HAART era with respiratory infections had an increased risk of death compared to those without infections (MACS adjusted-HR, 1.5; 95%CI, 1.3-1.7; p<0.001; WIHS adjusted-HR, 1.9; 95%CI, 1.5-2.4; p<0.001). Conclusion: HIV infection remained a significant risk for infectious respiratory diseases after the introduction of HAART, and infectious respiratory diseases were associated with an increased risk of mortality. © 2013 Gingo et al

American Association of Endocrine Surgeons Guidelines for Adrenalectomy: Executive Summary.

IMPORTANCE: Adrenalectomy is the definitive treatment for multiple adrenal abnormalities. Advances in technology and genomics and an improved understanding of adrenal pathophysiology have altered operative techniques and indications. OBJECTIVE: To develop evidence-based recommendations to enhance the appropriate, safe, and effective approaches to adrenalectomy. EVIDENCE REVIEW: A multidisciplinary panel identified and investigated 7 categories of relevant clinical concern to practicing surgeons. Questions were structured in the framework Population, Intervention/Exposure, Comparison, and Outcome, and a guided review of medical literature from PubMed and/or Embase from 1980 to 2021 was performed. Recommendations were developed using Grading of Recommendations, Assessment, Development and Evaluation methodology and were discussed until consensus, and patient advocacy representation was included. FINDINGS: Patients with an adrenal incidentaloma 1 cm or larger should undergo biochemical testing and further imaging characterization. Adrenal protocol computed tomography (CT) should be used to stratify malignancy risk and concern for pheochromocytoma. Routine scheduled follow-up of a nonfunctional adrenal nodule with benign imaging characteristics and unenhanced CT with Hounsfield units less than 10 is not suggested. When unilateral disease is present, laparoscopic adrenalectomy is recommended for patients with primary aldosteronism or autonomous cortisol secretion. Patients with clinical and radiographic findings consistent with adrenocortical carcinoma should be treated at high-volume multidisciplinary centers to optimize outcomes, including, when possible, a complete R0 resection without tumor disruption, which may require en bloc radical resection. Selective or nonselective α blockade can be used to safely prepare patients for surgical resection of paraganglioma/pheochromocytoma. Empirical perioperative glucocorticoid replacement therapy is indicated for patients with overt Cushing syndrome, but for patients with mild autonomous cortisol secretion, postoperative day 1 morning cortisol or cosyntropin stimulation testing can be used to determine the need for glucocorticoid replacement therapy. When patient and tumor variables are appropriate, we recommend minimally invasive adrenalectomy over open adrenalectomy because of improved perioperative morbidity. Minimally invasive adrenalectomy can be achieved either via a retroperitoneal or transperitoneal approach depending on surgeon expertise, as well as tumor and patient characteristics. CONCLUSIONS AND RELEVANCE: Twenty-six clinically relevant and evidence-based recommendations are provided to assist surgeons with perioperative adrenal care

Placing the library at the heart of plagiarism prevention: The University of Bradford experience.

yesPlagiarism is a vexed issue for Higher Education, affecting student transition, retention and attainment. This paper reports on two initiatives from the University of Bradford library aimed at reducing student plagiarism. The first initiative is an intensive course for students who have contravened plagiarism regulations. The second course introduces new students to the concepts surrounding plagiarism with the aim to prevent plagiarism breaches. Since the Plagiarism Avoidance for New Students course was introduced there has been a significant drop in students referred to the disciplinary programme. This paper discusses the background to both courses and the challenges of implementation

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

The genetics of blood pressure regulation and its target organs from association studies in 342,415 individuals

To dissect the genetic architecture of blood pressure and assess effects on target-organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry and genotypes from an additional 140,886 individuals were used for validation. We identified 66 blood pressure loci, of which 17 were novel and 15 harbored multiple distinct association signals. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target-organ damage in multiple tissues, with minor effects in the kidney. Our findings expand current knowledge of blood pressure pathways and highlight tissues beyond the classic renal system in blood pressure regulation

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure