Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.

Incident autoimmune diseases in association with SARS-CoV-2 infection: a matched cohort study.

OBJECTIVES: To investigate whether the risk of developing an incident autoimmune disease is increased in patients with prior COVID-19 disease compared to those without COVID-19, a large cohort study was conducted. METHOD: A cohort was selected from German routine health care data. Based on documented diagnoses, we identified individuals with polymerase chain reaction (PCR)-confirmed COVID-19 through December 31, 2020. Patients were matched 1:3 to control patients without COVID-19. Both groups were followed up until June 30, 2021. We used the four quarters preceding the index date until the end of follow-up to analyze the onset of autoimmune diseases during the post-acute period. Incidence rates (IR) per 1000 person-years were calculated for each outcome and patient group. Poisson models were deployed to estimate the incidence rate ratios (IRRs) of developing an autoimmune disease conditional on a preceding diagnosis of COVID-19. RESULTS: In total, 641,704 patients with COVID-19 were included. Comparing the incidence rates in the COVID-19 (IR=15.05, 95% CI: 14.69-15.42) and matched control groups (IR=10.55, 95% CI: 10.25-10.86), we found a 42.63% higher likelihood of acquiring autoimmunity for patients who had suffered from COVID-19. This estimate was similar for common autoimmune diseases, such as Hashimoto thyroiditis, rheumatoid arthritis, or Sjögren syndrome. The highest IRR was observed for autoimmune diseases of the vasculitis group. Patients with a more severe course of COVID-19 were at a greater risk for incident autoimmune disease. CONCLUSIONS: SARS-CoV-2 infection is associated with an increased risk of developing new-onset autoimmune diseases after the acute phase of infection. Key Points • In the 3 to 15 months after acute infection, patients who had suffered from COVID-19 had a 43% (95% CI: 37-48%) higher likelihood of developing a first-onset autoimmune disease, meaning an absolute increase in incidence of 4.50 per 1000 person-years over the control group. • COVID-19 showed the strongest association with vascular autoimmune diseases.

Development of a risk score to identify patients at high risk for a severe course of COVID-19.

Aim: We aimed to develop a risk score to calculate a person's individual risk for a severe COVID-19 course (POINTED score) to support prioritization of especially vulnerable patients for a (booster) vaccination. Subject and methods: This cohort study was based on German claims data and included 623,363 individuals with a COVID-19 diagnosis in 2020. The outcome was COVID-19 related treatment in an intensive care unit, mechanical ventilation, or death after a COVID-19 infection. Data were split into a training and a test sample. Poisson regression models with robust standard errors including 35 predefined risk factors were calculated. Coefficients were rescaled with a min-max normalization to derive numeric score values between 0 and 20 for each risk factor. The scores' discriminatory ability was evaluated by calculating the area under the curve (AUC). Results: Besides age, down syndrome and hematologic cancer with therapy, immunosuppressive therapy, and other neurological conditions were the risk factors with the highest risk for a severe COVID-19 course. The AUC of the POINTED score was 0.889, indicating very good predictive validity. Conclusion: The POINTED score is a valid tool to calculate a person's risk for a severe COVID-19 course. Supplementary Information: The online version contains supplementary material available at 10.1007/s10389-023-01884-7.

Management of Children and Adolescents with Chest Trauma in Pediatric and Non-Pediatric Departments-A Claims Data Analysis.

BACKGROUND: To investigate the management of children and adolescents with isolated and combined chest trauma in pediatric (PD) and non-pediatric departments (non-PD). METHODS: Anonymized claims data were provided by two large German statutory health insurance funds, covering 6.3 million clients over a 10-year period (2010-2019). Data were extracted for patients who had an inpatient ICD diagnosis of section S20-S29 (injuries to the thorax) and were ≤18 years of age. Demographic and clinical data were analyzed. RESULTS: A total of 4064 children and adolescents with chest trauma were included (mean age 12.0 ± 5.0 years; 55% male). In 1928 cases (47.4%), treatment was provided at PD. Patients admitted to PD underwent CT imaging less frequently (8.1%; non-PD: 23.1%; p < 0.0001). Children with a chest drain treated at university/maximum care hospitals (UM) showed more injuries involving multiple body regions compared with non-UM (25.8% vs. 4.5%; p = 0.0061) without a difference in the length of hospital stay. CONCLUSION: Children and adolescents with chest trauma are treated almost equally often in pediatric and adult departments. CT is significantly less frequently used in pediatric departments. Patients with a chest drain treated at a UM showed more concomitant injuries without a longer hospital stay. However, the clinical validity of this finding is questionable.

Post-COVID-19-associated morbidity in children, adolescents, and adults: A matched cohort study including more than 157,000 individuals with COVID-19 in Germany.

BACKGROUND: Long-term health sequelae of the Coronavirus Disease 2019 (COVID-19) are a major public health concern. However, evidence on post-acute COVID-19 syndrome (post-COVID-19) is still limited, particularly for children and adolescents. Utilizing comprehensive healthcare data on approximately 46% of the German population, we investigated post-COVID-19-associated morbidity in children/adolescents and adults. METHODS AND FINDINGS: We used routine data from German statutory health insurance organizations covering the period between January 1, 2019 and December 31, 2020. The base population included all individuals insured for at least 1 day in 2020. Based on documented diagnoses, we identified individuals with polymerase chain reaction (PCR)-confirmed COVID-19 through June 30, 2020. A control cohort was assigned using 1:5 exact matching on age and sex, and propensity score matching on preexisting medical conditions. The date of COVID-19 diagnosis was used as index date for both cohorts, which were followed for incident morbidity outcomes documented in the second quarter after index date or later.Overall, 96 prespecified outcomes were aggregated into 13 diagnosis/symptom complexes and 3 domains (physical health, mental health, and physical/mental overlap domain). We used Poisson regression to estimate incidence rate ratios (IRRs) with 95% confidence intervals (95% CIs). The study population included 11,950 children/adolescents (48.1% female, 67.2% aged between 0 and 11 years) and 145,184 adults (60.2% female, 51.1% aged between 18 and 49 years). The mean follow-up time was 236 days (standard deviation (SD) = 44 days, range = 121 to 339 days) in children/adolescents and 254 days (SD = 36 days, range = 93 to 340 days) in adults. COVID-19 and control cohort were well balanced regarding covariates. The specific outcomes with the highest IRR and an incidence rate (IR) of at least 1/100 person-years in the COVID-19 cohort in children and adolescents were malaise/fatigue/exhaustion (IRR: 2.28, 95% CI: 1.71 to 3.06, p < 0.01, IR COVID-19: 12.58, IR Control: 5.51), cough (IRR: 1.74, 95% CI: 1.48 to 2.04, p < 0.01, IR COVID-19: 36.56, IR Control: 21.06), and throat/chest pain (IRR: 1.72, 95% CI: 1.39 to 2.12, p < 0.01, IR COVID-19: 20.01, IR Control: 11.66). In adults, these included disturbances of smell and taste (IRR: 6.69, 95% CI: 5.88 to 7.60, p < 0.01, IR COVID-19: 12.42, IR Control: 1.86), fever (IRR: 3.33, 95% CI: 3.01 to 3.68, p < 0.01, IR COVID-19: 11.53, IR Control: 3.46), and dyspnea (IRR: 2.88, 95% CI: 2.74 to 3.02, p < 0.01, IR COVID-19: 43.91, IR Control: 15.27). For all health outcomes combined, IRs per 1,000 person-years in the COVID-19 cohort were significantly higher than those in the control cohort in both children/adolescents (IRR: 1.30, 95% CI: 1.25 to 1.35, p < 0.01, IR COVID-19: 436.91, IR Control: 335.98) and adults (IRR: 1.33, 95% CI: 1.31 to 1.34, p < 0.01, IR COVID-19: 615.82, IR Control: 464.15). The relative magnitude of increased documented morbidity was similar for the physical, mental, and physical/mental overlap domain. In the COVID-19 cohort, IRs were significantly higher in all 13 diagnosis/symptom complexes in adults and in 10 diagnosis/symptom complexes in children/adolescents. IRR estimates were similar for age groups 0 to 11 and 12 to 17. IRs in children/adolescents were consistently lower than those in adults. Limitations of our study include potentially unmeasured confounding and detection bias. CONCLUSIONS: In this retrospective matched cohort study, we observed significant new onset morbidity in children, adolescents, and adults across 13 prespecified diagnosis/symptom complexes, following COVID-19 infection. These findings expand the existing available evidence on post-COVID-19 conditions in younger age groups and confirm previous findings in adults. TRIAL REGISTRATION: ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT05074953.

Bioactivity assessment of natural compounds using machine learning models trained on target similarity between drugs.

Natural compounds constitute a rich resource of potential small molecule therapeutics. While experimental access to this resource is limited due to its vast diversity and difficulties in systematic purification, computational assessment of structural similarity with known therapeutic molecules offers a scalable approach. Here, we assessed functional similarity between natural compounds and approved drugs by combining multiple chemical similarity metrics and physicochemical properties using a machine-learning approach. We computed pairwise similarities between 1410 drugs for training classification models and used the drugs shared protein targets as class labels. The best performing models were random forest which gave an average area under the ROC of 0.9, Matthews correlation coefficient of 0.35, and F1 score of 0.33, suggesting that it captured the structure-activity relation well. The models were then used to predict protein targets of circa 11k natural compounds by comparing them with the drugs. This revealed therapeutic potential of several natural compounds, including those with support from previously published sources as well as those hitherto unexplored. We experimentally validated one of the predicted pair's activities, viz., Cox-1 inhibition by 5-methoxysalicylic acid, a molecule commonly found in tea, herbs and spices. In contrast, another natural compound, 4-isopropylbenzoic acid, with the highest similarity score when considering most weighted similarity metric but not picked by our models, did not inhibit Cox-1. Our results demonstrate the utility of a machine-learning approach combining multiple chemical features for uncovering protein binding potential of natural compounds.

Pediatric solid organ injury - frequency of abdominal imaging is determined by the treating department.

To investigate the use of abdominal CT scanning in the management of pediatric blunt abdominal trauma in pediatric and non-pediatric departments.In this observational cohort study, anonymized data were extracted from 2 large German statutory health insurances (∼5.9 million clients) in a 7-year period (2010-2016). All patients with inpatient International Classification of Diseases (ICD) codes S36.- and S37.- (injury of intra-abdominal organs; injury of urinary and pelvic organs) aged ≤18 years were included. Demographic and clinical data were analyzed by logistic regression analysis for associations with the use of abdominal CT.A total of 524 children with blunt abdominal trauma (mean age 11.0 ±â€Š5.2 years; 62.6% males) were included; 164 patients (31.3%) received abdominal CT-imaging. There were no significant differences in traumatic non-intraabdominal comorbidity patterns (injuries of external causes; injuries to the head or thorax). There was substantial variability in the rate of abdominal CT imaging among different medical disciplines ranging from 11.6% to 44.5%. Patients admitted to pediatric departments (Pediatrics and Pediatric Surgery) underwent abdominal CT imaging significantly less frequently (19.7%; N = 55) compared to patients treated in non-pediatric departments (General/Trauma Surgery: 44.5%; N = 109) irrespective of concomitant injuries. The estimated OR for the use of abdominal CT by General/Trauma Surgery was 6.2-fold higher (OR: 6.15 [95-%-CI:3.07-13.21]; P < .001) compared to Pediatric Surgery. Other risk factors associated with the use of abdominal CT were traumatic extra-abdominal comorbidities, increasing age, male gender, and admission to a university hospital.Abdominal CT imaging was significantly less frequently used in pediatric departments. The substantial variability of the abdominal CT rate among different medical disciplines and centers indicates a potential for reduction of CT imaging by implementation of evidence-based guidelines. Furthermore, our study underlines the need for centralization of pediatric trauma care in Germany not only to improve patient outcome but to avoid radiation-induced cancer mortality.

Frequency of neuroimaging for pediatric minor brain injury is determined by the primary treating medical department.

To investigate the use of neuroimaging in children and adolescents with minor brain injury in pediatric and non-pediatric departments.In this observational cohort study data were extracted from a large German statutory health insurance (AOK Plus Dresden ∼3.1 million clients) in a 7-year period (2010-2016). All patients with International Classification of Diseases (ICD) code S06.0 (concussion; minor brain injury; commotio cerebri) aged ≤ 18 years were included. Demographic and clinical data were analyzed by logistic regression analysis for associations with the use of CT and MRI (independent variables: gender, age, length of stay, pediatric vs non-pediatric department, university vs non-university hospital).A total of 14,805 children with minor brain injuries (mean age 6.0 ±â€Š5.6; 45.5% females) were included. Treatment was provided by different medical departments: Pediatrics (N = 8717; 59%), Pediatric Surgery (N = 3582, 24%), General Surgery (N = 2197, 15%), Orthopedic Trauma Surgery (N = 309, 2.1%). Patients admitted to pediatric departments (Pediatrics and Pediatric Surgery) underwent head CT-imaging significantly less frequently (3.8%) compared to patients treated in non-pediatric departments (18.5%; P < .001; General Surgery: 15.6%; Orthopedic Trauma Surgery: 39.2%). Logistic regression confirmed a significantly higher odds ratio (OR) for the use of cranial CT by the non-pediatric departments (OR: 3.2 [95-%-CI: 2.72-3.76]).CT was significantly less frequently used in pediatric departments. Educational efforts and quality improvement initiatives on physicians, especially in non-pediatric departments may be an effective approach to decreasing rates of CT after minor traumatic brain injuries.

Species-specific activity of antibacterial drug combinations.

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine1,2. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.

Reduction of radiochemotherapy-induced early oral mucositis by recombinant human keratinocyte growth factor (palifermin): experimental studies in mice.

PURPOSE: To study the effect of recombinant human keratinocyte growth factor (rHuKGF or palifermin) on oral mucositis induced by radiochemotherapy in a mouse model. METHODS AND MATERIALS: Cis-diamminedichloroplatinum (cisplatin) and/or 5-fluorouracil were given before single dose irradiation, combined with palifermin before or after the treatment, or both. Daily fractionated irradiation for 2 weeks was followed by graded test doses. With additional chemotherapy in Week 1, palifermin was given before radiotherapy and at the end of the first week, or additionally at the end of Week 2. Radiochemotherapy in Week 2 was combined with palifermin at the end of Weeks 1 and 2, Weeks 1, 2, and 3, or additionally before radiotherapy. Ulceration of mouse tongue mucosa was analyzed as the endpoint. RESULTS: The dose associated with ulcer induction in 50% of the mice (ED(50)) for single-dose irradiation was 11.5 +/- 0.7 Gy. Palifermin increased the ED(50) to about 19 Gy in all protocols tested. Similar values were observed when chemotherapy was added before irradiation. With fractionated irradiation, palifermin increased the ED(50) for test irradiation from 5.7 +/- 1.5 Gy to 12-15 Gy, depending on the administration protocol. With chemotherapy in Week 1, two palifermin injections had no significant effect, but a third injection increased the ED(50) to 13 Gy. With chemotherapy in Week 2, all palifermin protocols resulted in ED(50) values of 13-14 Gy. CONCLUSION: A marked increase in oral mucosal radiation tolerance by palifermin was found, which was preserved in combinations with chemotherapy using cisplatin and/or 5-fluorouracil.