Transposon activity, local duplications and propagation of structural variants across haplotypes drive the evolution of the Drosophila S2 cell line.

BACKGROUND: Immortalized cell lines are widely used model systems whose genomes are often highly rearranged and polyploid. However, their genome structure is seldom deciphered and is thus not accounted for during analyses. We therefore used linked short- and long-read sequencing to perform haplotype-level reconstruction of the genome of a Drosophila melanogaster cell line (S2-DRSC) with a complex genome structure. RESULTS: Using a custom implementation (that is designed to use ultra-long reads in complex genomes with nested rearrangements) to call structural variants (SVs), we found that the most common SV was repetitive sequence insertion or deletion (> 80% of SVs), with Gypsy retrotransposon insertions dominating. The second most common SV was local sequence duplication. SNPs and other SVs were rarer, but several large chromosomal translocations and mitochondrial genome insertions were observed. Haplotypes were highly similar at the nucleotide level but structurally very different. Insertion SVs existed at various haplotype frequencies and were unlinked on chromosomes, demonstrating that haplotypes have different structures and suggesting the existence of a mechanism that allows SVs to propagate across haplotypes. Finally, using public short-read data, we found that transposable element insertions and local duplications are common in other D. melanogaster cell lines. CONCLUSIONS: The S2-DRSC cell line evolved through retrotransposon activity and vast local sequence duplications, that we hypothesize were the products of DNA re-replication events. Additionally, mutations can propagate across haplotypes (possibly explained by mitotic recombination), which enables fine-tuning of mutational impact and prevents accumulation of deleterious events, an inherent problem of clonal reproduction. We conclude that traditional linear homozygous genome representation conceals the complexity when dealing with rearranged and heterozygous clonal cells.

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm.

Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.

Global surgery for medical students - is it meaningful? A mixed-method study.

INTRODUCTION: There has been an increase in global health courses at medical universities in high-income countries. Their effect on students, however, is poorly understood. In 2016 an elective global surgery course was introduced for medical students at Karolinska Institutet in Sweden. The course includes a theoretical module in Sweden and a two-week clinical rotation in Uganda. The present study aimed to assess the format and determine its effect on students' knowledge of global surgery and approach towards patients of non-Swedish origin. METHOD: A mixed-methods design was used. Semi-structured case-based interviews were conducted individually with 18 students and analysed using qualitative content analysis. Examination scores and the course evaluation were analysed with Kruskal Wallis one-way analysis of variance, Pearson's Chi-square and a Wilcoxon signed-rank test as appropriate. RESULTS: The course was appreciated and students reported gained insights and interest in global surgery. Students' ability to reason about global surgery issues was improved after the course. Students considered complicating aspects in the meeting with patients of non-Swedish origin. Students with abroad clinical experience felt less compelled to act on preconceptions. DISCUSSION: The global surgery course at Karolinska Institutet is appreciated and students gained valuable knowledge. The case-based interviews acted as a catalyst for reflection and showed that students felt insecure as they lacked knowledge about globally common surgical conditions and struggled with generalized preconceptions of patients of non-Swedish origin. To further support students to integrate theoretical knowledge and professional development, we suggest the introduction of problem-based learning. CONCLUSION: The ability of the course to inspire students' commitment to global surgery is promising as this engagement is the key to reaching the goal of equitable health globally. Offering such courses is a step towards inspiring and recruiting the future clinicians and researchers needed for expanding the field of global surgery.

Randomised trial showed no difference in behavioural symptoms between surgical methods treating paediatric obstructive sleep apnoea.

AIM: Our previous randomised controlled trial of children with obstructive sleep apnoea (OSA) showed no significant differences between adenotonsillectomy (ATE) and adenotonsillotomy (ATT) in improving nocturnal respiration and quality of life after 1 year. The aim of this report was to evaluate the effects on behavioural symptoms using the Strengths and Difficulties Questionnaire (SDQ). METHODS: Children between 2 and 6 years with OSA were randomised to ATT or ATE. Parents, blinded to method, answered the SDQ while their child underwent polysomnography before and 1 year after surgery. Differences between the total SDQ scores were analysed between the treatment groups. RESULTS: The SDQ was filled out in 87% of the cases preoperatively, and in 86% postoperatively. At follow-up, the mean total SDQ score was 9.6 SD ± 5.1 in the ATE group (n = 31), and 8.2 ± 6.7 in the ATT group (n = 37), P = .09. The mean total SDQ score for all was preoperatively 10.6 ± 5.0, and postoperatively 8.8 ± 6.0, P = .0002. CONCLUSION: There were no significant differences in SDQ scores between the groups at follow-up, indicating that the more conservative ATT is a treatment option in paediatric OSA. The whole group of patients showed a significant improvement after surgery.

Heel ulcers do heal in patients with diabetes.

A heel ulcer is considered to be a serious complication in patients with diabetes, and there is limited information regarding outcome. In most of the literature, a poor prognosis is described. The aim of this study was to investigate a large cohort of ulcers located in the heel in patients with diabetes. Seven hundred and sixty-eight patients [median age 73 (17-98)], presenting with a heel ulcer at a multidisciplinary diabetes foot clinic, fulfilled the inclusion criteria and were followed-up until final outcome. Fifty-eight per cent of the patients healed primarily; 7% healed after major debridement; 9% healed after amputation and 25% died unhealed. Median healing time was 17 weeks. Ulcer progression was seen in 19% of patients. Thirty-one percent of patients had severe peripheral vascular disease. A creatinine level below 91 µmol/l was related to a higher probability for healing without major debridement or amputation, whereas vascular surgery, nephropathy and oedema were related to a lower probability for healing without major debridement or amputation. Two thirds of heel ulcers do heal in patients with diabetes despite patients being elderly and with extensive comorbidity. The extent of peripheral vascular disease, nephropathy, oedema and decreased renal function are important factors influencing outcome.

Early laryngeal outcome of prolonged intubation using an anatomical tube: a double blind, randomised study.

The objective of this study was to study the short-term impact on larynx by a newly designed anatomical tube. A prospective randomised trial of a newly designed anatomical tube versus a standard endotracheal tube in patients operated under general anaesthesia for at least 12 h. Seventy adults were included and randomised to either type of tube. The patients were evaluated by means of fibre-optic laryngoscopy and VAS-rating of symptoms on two occasions in the first post-operative week. The evaluating investigators and the patients were blinded to the type of tube used. 27 cases and 23 controls had complete data for evaluation. Age, gender and intubation times were comparable. Symptoms such as hoarseness, coughing, and pain were rated above 30 % of maximum during at least one of the first post-operative days by 21 and 19 patients, respectively. At the first examination (within 24 h), 38 % of patients in the anatomical tube group stated no hoarseness; compared to 13 % of the controls (p = 0.057). Fibre-optic laryngoscopy showed some kind of pathology in all the patients examined within 24 h of extubation. After 3-6 days, seven patients with the anatomical tube and four patients in the control group showed complete resolution of the lesions, and the changes were limited to redness in the vocal process area in another seven and four, respectively. The differences between the groups did not attain statistical significance. The study shows considerable short-term laryngeal morbidity after prolonged intubation, and the anatomical tube only showed an advantage concerning hoarseness. Further improvement of the endotracheal tubes and intubation routines are still needed.

High probability of healing without amputation of plantar forefoot ulcers in patients with diabetes.

Diabetic foot ulcer is an important entity which in many cases is the first serious complication in diabetes. Although a plantar forefoot location is common, there are few studies on larger cohorts and in such studies there is often a combination of various types of ulcer and ulcer locations. The purpose of this study is to discern the outcome of plantar forefoot ulcers and their specific characteristics in a large cohort. All patients (n = 770), presenting with a plantar forefoot ulcer at a multidisciplinary diabetes foot clinic from January 1, 1983 to December 31, 2012 were considered for the study. Seven hundred one patients (median age 67 [22-95]) fulfilled the inclusion criteria and were followed according to a preset protocol until final outcome (healing or death). Severe peripheral vascular disease was present in 26% of the patients and 14% had evidence of deep infection upon arrival at the foot clinic. Fifty-five percent (385/701) of the patients healed without foot surgery, 25% (173/701) healed after major debridement, 9% (60/701) healed after minor or major amputation and 12% (83/701) died unhealed. Median healing time was 17 weeks. An ulcer classified as Wagner grade 1 or 2 at inclusion and independent living were factors associated with a higher healing rate. Seventy-nine percent of 701 patients with diabetes and a plantar forefoot ulcer treated at a multidisciplinary diabetes foot clinic healed without amputation. For one third some form of foot surgery was needed to achieve healing.

Screening for depressed mood in an adolescent psychiatric context by brief self-assessment scales--testing psychometric validity of WHO-5 and BDI-6 indices by latent trait analyses.

BACKGROUND: Major depressive disorder is prevalent in the adolescent psychiatric clinical setting and often comorbid with other primary psychiatric diagnoses such as ADHD or social anxiety disorder. Systematic manual-based diagnostic procedures are recommended to identify such comorbidity but they are time-consuming and often not fully implemented in clinical practice. Screening for depressive symptoms in the child psychiatric context using brief, user-friendly and easily managed self-assessment scales may be of clinical value and utility. The aim of the study is to test the psychometric validity of two such scales, which may be used in a two-step screening procedure, the WHO-Five Well-being Index (WHO-5) and the six-item version of Beck's Depression Inventory (BDI-6). METHOD: 66 adolescent psychiatric patients with a clinical diagnosis of major depressive disorder (MDD), 60 girls and 6 boys, aged 14-18 years, mean age 16.8 years, completed the WHO-5 scale as well as the BDI-6. Statistical validity was tested by Mokken and Rasch analyses. RESULTS: The correlation between WHO-5 and BDI-6 was -0.49 (p=0.0001). Mokken analyses showed a coefficient of homogeneity for the WHO-5 of 0.52 and for the BDI-6 of 0.46. Rasch analysis also accepted unidimensionality when testing males versus females (p > 0.05). CONCLUSIONS: The WHO-5 is psychometrically valid in an adolescent psychiatric context including both genders to assess the wellness dimension and applicable as a first step in screening for MDD. The BDI-6 may be recommended as a second step in the screening procedure, since it is statistically valid and has the ability to unidimensionally capture the severity of depressed mood.

Buffering and proteolysis are induced by segmental monosomy in Drosophila melanogaster.

Variation in the number of individual chromosomes (chromosomal aneuploidy) or chromosome segments (segmental aneuploidy) is associated with developmental abnormalities and reduced fitness in all species examined; it is the leading cause of miscarriages and mental retardation and a hallmark of cancer. However, despite their documented importance in disease, the effects of aneuploidies on the transcriptome remain largely unknown. We have examined the expression effects of seven heterozygous chromosomal deficiencies, both singly and in all pairwise combinations, in Drosophila melanogaster. The results show that genes in one copy are buffered, i.e. expressed more strongly than the expected 50% of wild-type level, the buffering is general and not influenced by other monosomic regions. Furthermore, long genes are significantly more highly buffered than short genes and gene length appears to be the primary determinant of the buffering degree. For short genes the degree of buffering depends on expression level and expression pattern. Furthermore, the results show that in deficiency heterozygotes the expression of genes involved in proteolysis is enhanced and negatively correlates with the degree of buffering. Thus, enhanced proteolysis appears to be a general response to aneuploidy.

Psychiatric problems associated with subthreshold ADHD and disruptive behaviour diagnoses in teenagers.

AIM: To study the coexistence of subthreshold diagnoses of both attention deficit hyperactivity disorder (ADHD) and disruptive behaviour disorders (DBD) with other symptoms of child and adolescent psychiatric disorders as well as risk behaviours associated with smoking, alcohol and drug use. METHODS: A population-based sample of twins including 177 girls and 135 boys was interviewed using the Swedish version of Kiddie-SADS Present and Lifetime Version (K-SADS-PL). Subthreshold diagnoses were compiled based on the ADHD and DBD criteria, where each criterion was assessed as 'possible' or 'certain' according to K-SADS-PL. The odds ratios (OR) between the subthreshold diagnoses and each of the screening questions in K-SADS-PL were calculated. RESULTS: Subthreshold diagnoses of ADHD and DBD coexisted with the screening questions concerning depression, mania, panic attack, phobias, anorexia nervosa, motor tics and posttraumatic stress disorder (PTSD) in girls. In boys, these subthreshold diagnoses coexisted with symptoms of depression and PTSD. For both boys and girls, smoking and high alcohol consumption contributed to a high OR with regard to ADHD and DBD. CONCLUSION: Subthreshold diagnoses of ADHD and DBD were risk factors for several other psychiatric symptoms as well as smoking and high alcohol consumption. Thus, a broad clinical assessment is needed for adolescents with such preliminary diagnoses.

Factors related to outcome of neuroischemic/ischemic foot ulcer in diabetic patients.

OBJECTIVES: Peripheral vascular disease (PVD) is an important limiting factor for healing in neuroischemic or ischemic diabetic foot ulcer. The purpose of this study was to identify factors related to healing in patients with diabetes with foot ulcers and severe PVD. METHODS: Patients with diabetes with a foot ulcer, consecutively presenting at a multidisciplinary foot center with a systolic toe pressure <45 mm Hg or an ankle pressure <80 mm Hg were prospectively included, followed according to a preset program, and with the exception of specified exclusions, subjected to angiography offered vascular intervention when applicable. All patients had continuous follow-up until healing or death irrespective of the type of vascular intervention. RESULTS: One thousand one hundred fifty-one patients were included. Eighty-two percent had a toe pressure <45 mm Hg and 49% had an ankle pressure <80 mm Hg. Eight hundred one patients (70%) underwent an angiography. Out of these, 63% had vascular intervention, either percutaneous transluminal angioplasty (PTA; 39%) or reconstructive surgery (24%). Nine percent of the patients had one or more complications after angiography. PTA was multisegmental in 46% and to the crural arteries in 46%. Reconstructive surgery was distal in 51%. Age (P < .001), renal function impairment (P = .005), congestive heart failure (P = .01), number and type of ulcer (P < .001), and severity of PVD (P = .003) affected the outcome of ulcers. PTA and reconstructive vascular surgery increased the probability of healing without amputation (odds ratio [OR], 1.77 and 2.05, respectively). CONCLUSION: Probability of ulcer healing is strongly related to comorbidity, extent of tissue involvement, and severity of PVD in patients with diabetes with severe PVD.

Buffering of segmental and chromosomal aneuploidies in Drosophila melanogaster.

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).

Carbon monoxide concentration in donated blood: relation to cigarette smoking and other sources.

BACKGROUND: Carbon monoxide (CO) is normally present in the human body due to endogenous production of CO. CO can also be inhaled by exposure to external sources such as cigarette smoke, car exhaust, and fire. The purpose of this study was to investigate CO concentrations in blood from 410 blood donors at the blood center in Umeå, Sweden. To further evaluate the effects of cigarette smoking on CO concentrations, the elimination time for CO was examined in six volunteer smokers after a smoked cigarette. STUDY DESIGN AND METHODS: Blood samples from whole blood donors were obtained during the blood center's routine operation. In connection with blood donations, demographic and behavioral data were collected from the donors. The CO concentration was determined using gas chromatography. RESULTS: The majority of blood donors had approximately the same CO concentration (mean, 84.5 micromol/L). In 6 percent of the samples, the concentrations were higher than 130 micromol per L. The highest CO concentration was 561 micromol per L. The main source for these high CO concentrations appeared to be cigarette smoking. In the volunteer smokers, the elimination time after a smoked cigarette varied significantly, with elimination half-lives from 4.7 to 8.4 hours. CONCLUSION: These results show that blood bank red blood cell bags may have CO concentrations above the physiologic level. The time interval between cigarette smoking and blood donation seems to be a particularly important factor for elevated CO concentrations.

Organization and regulation of sex-specific thioredoxin encoding genes in the genus Drosophila.

Thioredoxins are small thiol proteins that have a conserved active site sequence, WCGPC, and reduce disulfide bonds in various proteins using the two active site cysteines, a reaction that oxidizes thioredoxin and renders it inactive. Thioredoxin reductase returns thioredoxin to its reduced, active form in a reaction that converts NADPH to NADP(+). The biological functions of thioredoxins vary widely; they have roles in oxidative stress protection, act as electron donors for ribonucleotide reductase, and form structural components of enzymes. To date, three thioredoxin genes have been characterized in Drosophila melanogaster: the generally expressed Thioredoxin-2 (Trx-2) and the two sex-specific genes ThioredoxinT (TrxT) and deadhead (dhd). The male-specific TrxT and the female-specific dhd are located as a gene pair, transcribed in opposite directions, with only 470 bp between their transcription start points. We show in this study that all three D. melanogaster thioredoxins are conserved in 11 other Drosophilid species, which are believed to have diverged up to 40 Ma ago and that Trx-2 is conserved all the way to Tribolium castaneum. We have found that the intriguing gene organization and regulation of TrxT and dhd is remarkably well conserved and identified potential conserved regulatory sequences. In addition, we show that the 50-70 C terminal amino acids of TrxT constitute a hyper-variable domain, which could play a role in sexual conflict and male-female co-evolution.

A new strategy for the improvement of photophysical properties in ruthenium(II) polypyridyl complexes. Synthesis and photophysical and electrochemical characterization of six mononuclear ruthenium(II) bisterpyridine-type complexes.

The synthesis and characterization of six ruthenium(II) bistridentate polypyridyl complexes is described. These were designed on the basis of a new approach to increase the excited-state lifetime of ruthenium(II) bisterpyridine-type complexes. By the use of a bipyridylpyridyl methane ligand in place of terpyridine, the coordination environment of the metal ion becomes nearly octahedral and the rate of deactivation via ligand-field (i.e., metal-centered) states was reduced as shown by temperature-dependent emission lifetime studies. Still, the possibility to make quasi-linear donor-ruthenium-acceptor triads is maintained in the complexes. The most promising complex shows an excited-state lifetime of tau = 15 ns in alcohol solutions at room temperature, which should be compared to a lifetime of tau = 0.25 ns for [Ru(tpy)2]2+. The X-ray structure of the new complex indeed shows a more octahedral geometry than that of [Ru(tpy)2]2+. Most importantly, the high excited-state energy was retained, and thus, so was the potential high reactivity of the excited complex, which has not been the case with previously published strategies based on bistridentate complexes.

Rapid and sensitive method for the analysis of carbon monoxide in blood using gas chromatography with flame ionisation detection.

In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.