Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

Surgical Experience Affects the Outcome of Central Venous Access Catheter Implantation in Children: A Retrospective Cohort Study.

INTRODUCTION: Surgical complications occur in up to third of children, limiting the benefits of tunneled central venous catheters (tCVCs) in children. We aimed to identify risk factors for complications related to catheter implantation. METHODS: All children and adolescents undergoing tCVC implantation at a single center over a period of 9 years were analyzed. Infection, thrombosis, dislocation, and catheter dysfunction were defined as complications. Both patient-related (ie, age, sex, vessel characteristics, revision surgery) and surgical factors (ie, sex of surgeon, surgical experience) were analyzed for their association with complications. RESULTS: A total of 1024 catheters were inserted, 887 ports and 137 broviac catheters. In terms of patient-related factors, Broviac catheters, and nononcological patients had a higher complication rate. The use of the internal jugular vein and revision surgery was associated with significantly increased complications in patients with port catheters. Experience of the surgeon correlated with various outcome parameters. Implantation performed by an attending were associated with lower complication rates in comparison to those performed by residents. Within the resident group, insertions performed by experienced residents had more complications compared with those performed by residents during their first years. CONCLUSION: The study suggests that the outcome of tCVCs insertion is affected by the type of catheter used, the utilized vessel and above all by surgical experience. Residents had significantly increased complication rates in comparison to board-certified surgeons and amongst resident's outcome got worse with increasing experience of the residents. The presence of an experienced attending did not compensate for this effect. To improve the outcome of tCVCs, strategies like direct feedback after every procedure to achieve proficiency should be implanted in residency programs.

Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR.

The most widely used approach for defining gene function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene expression in many organisms. However, new biotechnological tools--specifically CRISPR-based technologies--have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: "Which technology should I use in my experiment?" This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools.

Case Report: 2-Year-Old Boy with Grade 4 Avulsion Injury of the Hand Caused by a Conveyor Belt System. / Case report: 2-jähriger Junge mit viertgradiger Avulsionsverletzung der Hand durch Einklemmung in ein Förderband.

In the western world, mutilating hand injuries such as amputations and severe avulsions are more common in the working population than in children (Pomares G et al. Orthop Traumatol Surg Res 2018; 104: 273­276). An epidemiologic retrospective study from France identified 1715 traumatic upper-limb amputations over a 10-years period with the majority of cases involving middle-aged men (Pomares G et al. Orthop Traumatol Surg Res 2018; 104: 273­276). Systematic literature search using Pubmed on conveyor belt system injuries found few articles. A recent study analyses occupational traumatic injuries in offshore seafood processors in Alaska. Processing equipment and machinery were among the leading causes of injuries, accounting for 28% of traumata (Syron et al. J Safety Res 2018; 66: 169­178). Tiwari et al. describe a collective of six children who sustained motorized machine belt entrapment injuries (Tiwari P et al. Indian Pediatr 2020; 57: 66­68). Overall mortality and paraplegia rate were 33.3% each. Those accidents mainly occured in rural areas in India. The children were caught in the belt by their clothes while their parents were working nearby. Five patients were pulled through their torso, those with involved head were dead at the time of arrival. Only one had only his limbs involved. If a conveyor belt involves the hand, severe avulsion or crush injuries result. The outcome of reconstructive surgery in hand avulsion injuries depends mainly on the initial degree of injury (Kay et al. J Hand Surg 1989; 14: 204­213). Finger avulsion injuries are classified according to Kay's classification. In class I injuries, tissue perfusion is still normal. Kay II injuries already show inadequate blood flow but no fracture is present. In class III injuries, perfusion is restricted and there is either an associated fracture or a joint injury. Kay IV injuries cover complete digital deglovings or amputations (Kay et al. J Hand Surg 1989; 14: 204­213). Generally, replantations for sharp injuries show higher success rates than for avulsion or crush injuries (Goodman et al. J Hand Surg Am 2017;42:456­463). In general, the age of pediatric patients is critical to the feasibility and success rate of finger replantations and reconstructive hand surgery.

A rapid and convenient sample preparation method for the analysis of cannabinoids in oral fluid samples.

Cannabis is the world's most used illegal drug. The main psychoactive component of cannabis is Δ9-tetrahydrocannabinol (THC). To aid the identification of cannabis-impaired individuals, a simple but effective workflow for reliable quantification of THC and its metabolites in oral fluid samples collected with the Greiner Bio-One Saliva Collection System is presented. Sampling involves rinsing the oral cavity with an extraction solution containing a citrate buffer stimulating salivary flow. Sample processing targeted the cannabinoid fraction interacting with proteins and other insoluble constituents that can be separated by centrifugation. Approximately 50% of the total amount of cannabinoids included in the oral fluid was recovered from the obtained pellet by extraction with acetonitrile. Liquid chromatography-tandem mass spectrometry was used for cannabinoid quantification. Fitness of the developed workflow for application in forensic and clinical cannabis testing was demonstrated by evaluating multiple performance parameters, including selectivity, linearity, limits of quantification (LOQs), accuracy, precision, matrix effects, extraction recoveries, process efficiencies and stability. Furthermore, sensitivity and specificity of the developed oral fluid-based cannabis test was demonstrated by analysing 195 samples collected either from opioid addicts or persons suspected of driving under the influence of drugs. The accuracy of identifying a person with the presence of THC in blood was found to be 97.9%.

Clinical outcome, quality of life, and mental health in long-gap esophageal atresia: comparison of gastric sleeve pull-up and delayed primary anastomosis.

INTRODUCTION: Pediatric surgeons have yet to reach a consensus whether a gastric sleeve pull-up or delayed primary anastomosis for the treatment of esophageal atresia (EA), especially of the long-gap type (LGEA) should be performed. Thus, the aim of this study was to evaluate clinical outcome, quality of life (QoL), and mental health of patients with EA and their parents. METHODS: Clinical outcomes of all children treated with EA from 2007 to 2021 were collected and parents of affected children were asked to participate in questionnaires regarding their Quality of Life (QoL) and their child's Health-Related Quality of Life (HRQoL), as well as mental health. RESULTS: A total of 98 EA patients were included in the study. For analysis, the cohort was divided into two groups: (1) primary versus (2) secondary anastomosis, while the secondary anastomosis group was subdivided into (a) delayed primary anastomosis and (b) gastric sleeve pull-up and compared with each other. When comparing the secondary anastomosis group, significant differences were found between the delayed primary anastomosis and gastric sleeve pull-up group; the duration of anesthesia during anastomosis surgery (478.54 vs 328.82 min, p < 0.001), endoscopic dilatation rate (100% vs 69%, p = 0.03), cumulative time spent in intensive care (42.31 vs 94.75 days, p = 0.03) and the mortality rate (0% vs 31%, p = 0.03). HRQoL and mental health did not differ between any of the groups. CONCLUSION: Delayed primary anastomosis or gastric sleeve pull-up appear to be similar in patients with long-gap esophageal atresia in many key aspects like leakage rate, strictures, re-fistula, tracheomalacia, recurrent infections, thrive or reflux. Moreover, HrQoL was comparable in patients with (a) gastric sleeve pull-up and (b) delayed primary anastomosis. Future studies should focus on the long-term results of either preservation or replacement of the esophagus in children.

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches.

The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.

[Impact and Temporal Evolution of the Corona Pandemic on Pediatric Emergency Care]. / Auswirkungen der Corona Pandemie auf die Notfallversorgung im Kindesalter und die zeitliche Entwicklung im Verlauf der Pandemie.

HINTERGRUND: Die COVID-19 Pandemie hat zu massiven gesellschaftlichen und wirtschaftlichen Einschränkungen geführt. Im Kindesalter wurden elektive Vorsorgeuntersuchung oftmals nicht wahrgenommen und es zeigte sich eine Reduktion der Zahl der Notfallvorstellungen. MATERIAL UND METHODEN: In einer retrospektiven Studie erfolgte eine quantitative Auswertung aller Notfallpatienten der 5 Kindernotaufnahmen Hamburgs und der Kinder- und Jugendarztpraxen der Jahre 2019 und 2020. Zusätzlich erfolgte die detaillierte Analyse der Notfallbehandlungen des Altonaer Kinderkrankenhauses während der ersten Phase der Pandemie im Vergleich zum Vorjahr. Zusammenhänge zu den Eckpunkten der Pandemieentwicklung wurden analysiert. Die Berechnung signifikanter Unterschiede in Bezug auf die demographischen Daten und Krankheitsbilder erfolgte mittels Chi- Quadrat und t-Test. ERGEBNISSE: In allen 5 pädiatrischen Notaufnahmen Hamburgs zeigte sich eine nahezu gleichförmige Abnahme der Patientenvorstellungen während der Pandemie, ähnlich in den Kinder- und Jugendarztpraxen. Die Zahl der Behandlungen verhielt sich dabei nicht streng gegenläufig zu den Neuinfektionen, sondern korreliert eher mit den Mobilitätsdaten und entsprach somit den gesamtgesellschaftlichen Veränderungen. Während der Pandemie stieg der Anteil häuslicher Unfälle an der Gesamtzahl der Vorstellungen signifikant an. Die Analyse der Diagnosen zeigte unter anderem eine relative Abnahme von (viralen) Infektionskrankheiten. Nicht behandlungspflichtige Erkrankungen führten in der Pandemie seltener zur Vorstellung. Diese Phänomene entlasteten einerseits die Notaufnahmen, bergen aber auch die Gefahr, dass Erkrankungen durch Eltern falsch eingeschätzt werden und eine notwendige ärztliche Vorstellung somit zu spät erfolgt.

Should the processus vaginalis sac be opened or closed in pediatric herniotomy? A two-center cohort study.

BACKGROUND: Inguinal hernia repair is one of the most common operations in children. To improve outcome, several techniques are used. However, it has not been established if the open or the closed hernia sac preparation technique is superior in (premature) neonates and older children. METHODS: Retrospective study including all cases of inguinal hernia repair in children at two large centers. Demographic data and outcome parameters including procedure time and intra as well as postoperative complications were evaluated. To compare open vs. closed hernia sack preparation, cases with secondary open preparation were excluded and propensity score matching was performed. Regression analysis was used to determine factors affecting operative time and recurrence rate. RESULTS: In total 2476 cases of inguinal hernia repair were identified. After exclusion of direct hernias as well as revision cases, 2257 cases were analyzed. Overall mean operative time was 25.8 min. Intraoperative complications occurred in 0.1% and. postoperative complications occurred in 3.0% of all cases, the most frequent postoperative complication being recurrence (1.7%). Closed preparation technique resulted in significantly faster procedure time and lower recurrence rates in premature neonates and older children compared to the open hernia sac preparation technique. Operative technique, prematurity, gender and training of the surgeon are highly associated with operative time, whereas operative technique is the main factor affecting recurrence rate. CONCLUSIONS: It appears that closed hernia sack preparation is superior to open regarding speed and recurrence. This was true for premature neonates, neonates and older children. All other outcome parameters including intra- and postoperative complications were similar. Thus, we recommend to use the closed preparation technique whenever possible.

T cell cytokines in the diagnostic of early-onset sepsis.

BACKGROUND: Early-onset sepsis (EOS) remains a substantial cause of morbidity and mortality among neonates. Yet, currently available biological parameters have not proven to be accurate enough to predict EOS reliably. This study aimed to determine serum concentrations of 13 cytokines in umbilical cord blood and evaluate their diagnostic value for EOS. METHODS: A prospective single-center study that included analysis of umbilical cord blood of term and preterm neonates who were born from March 2017 to November 2017. Using ELISA analysis, 13 cytokines were simultaneously quantified and correlated with the development of EOS. RESULTS: Four hundred and seventy-four neonates were included, of which seven met the criteria for culture-positive EOS. Interleukin (IL)-6 (p < 0.001), IL-9 (p = 0.003), and IL-21 (p < 0.001) were significantly increased in neonates with EOS compared to controls. Sensitivity and specificity for IL-6, IL-9, and IL-21 at the defined cut-off points were 85.7 and 77.3%, 71.4 and 62.5%, and 71.4 and 52.0%, respectively. CONCLUSIONS: In neonates with EOS, IL-9 and IL-21 are significantly elevated and may be employed in the diagnostic of EOS. However, diagnostic accuracy remains lower than with IL-6. Values of 13 T cell cytokines may be used as reference values for future studies in neonates. IMPACT: Interleukin-9 (IL-9) and interleukin-21 (IL-21) are significantly elevated in neonates with early-onset sepsis. IL-9 and IL-21 have been shown to play a specific role in neonatal sepsis. Neonatal reference values were generated for several cytokines. IL-9 and IL-21 might be attractive biomarkers for neonatal sepsis in future. This study is likely to promote further research in this area. Values of several T cell cytokines may be used as reference values for future studies in neonates.

Safety, pharmacodynamic, and pharmacokinetic characterization of vericiguat: results from six phase I studies in healthy subjects.

PURPOSE: To characterize the safety, pharmacodynamics, and pharmacokinetics (PK) of vericiguat in healthy males. METHODS: Six phase I studies were conducted in European, Chinese, and Japanese males. Subjects received oral vericiguat as a single dose (0.5-15.0 mg solution [for first-in-human study] or 1.25-10.0 mg immediate release [IR tablets]) or multiple doses (1.25-10.0 mg IR tablets once daily [QD] or 5.0 mg IR tablets twice daily for 7 consecutive days). Bioavailability and food effects on vericiguat PK (IR tablets) were also studied in European subjects. RESULTS: Overall, 255 of 265 randomized subjects completed their respective studies. There were no deaths or serious adverse events. Vericiguat was generally well tolerated at doses ≤ 10.0 mg. In the first-in-human study, the most frequent drug-related adverse events were headache and postural dizziness (experienced by five subjects each [7.2%]). Three of four subjects who received vericiguat 15.0 mg (oral solution, fasted) experienced orthostatic reactions. Vericiguat (≤ 10.0 mg, IR tablets) was rapidly absorbed (median time to reach maximum plasma concentration ≤ 2.5 h [fasted]) with a mean half-life of about 22.0 h (range 17.9-27.0 h for single and multiple doses). No evidence for deviation from dose proportionality or unexpected accumulation was observed. Administration of vericiguat 5.0 mg IR tablets with food increased bioavailability by 19% (estimated ratio 119% [90% confidence interval]: 108; 131]), reduced PK variability, and prolonged vericiguat absorption relative to the fasted state. CONCLUSION: In general, vericiguat was well tolerated. These results supported further clinical evaluation of vericiguat QD in patients with heart failure. REGISTRY NUMBERS: EudraCT: 2011-001627-21; EudraCT: 2012-000953-30.

Buprenorphine-cannabis interaction in patients undergoing opioid maintenance therapy.

Buprenorphine is a partial µ-opioid agonist widely used for opioid maintenance therapy (OMT). It is mainly metabolized to pharmacologically active norbuprenorphine by the cytochrome P450 (CYP) isozyme 3A4. This may give rise to drug-drug interactions under combinations with inhibitors or inducers of CYP3A4. Cannabis is a potential inhibitor of CYP3A4, and there is a large degree of concomitant cannabis use among OMT patients. We performed a retrospective analysis on liver healthy OMT patients substituted with buprenorphine, either with (n = 15) or without (n = 17) concomitant use of cannabis. Patients with additional illicit drugs or medications affecting CYP3A were excluded. Measured blood concentrations of buprenorphine and norbuprenorphine were compared between the two groups. Cannabis users and non-users received similar doses, but users had 2.7-fold higher concentrations of buprenorphine (p < 0.01) and 1.4-fold for norbuprenorphine (1.4-fold, p = 0.07). Moreover, the metabolite-to-parent drug ratio was 0.98 in non-users and 0.38 in users (p = 0.02). Female gender did not produce significant effects. These findings indicate that cannabis use decreases the formation of norbuprenorphine and elevates buprenorphine and norbuprenorphine concentrations in blood most probably by inhibition of CYP3A4. The pharmacokinetic interaction may give rise to enhanced or altered opioid activity and risk of intoxications. Physicians should inform patients about this risk and supervise cannabis users by regular control of buprenorphine blood levels, i.e., by therapeutic drug monitoring.

Fetoscopic techniques for prenatal covering of gastroschisis in an ovine model are technically demanding and do not lead to permanent anchoring on the fetus until the end of gestation.

INTRODUCTION: This is the cumulative technical report on the operative procedures and limitations of fetoscopic bag insertion, intestinal bag placement, and bag fixation to the fetus in a series of pilot studies in an ovine model for prenatal treatment of gastroschisis. MATERIAL AND METHODS: In 24 German blackhead sheep, a surgically created gastroschisis was managed by fetoscopic placement of the extruded intestines into a bag. The bag was then fastened onto the fetal abdominal wall. Different materials (sterile gloves, latex condoms, laparosopic retrieval bags) and different fixation techniques (laparoscopic staplers, interrupted and continuous sutures) have been examined. The fetuses were retrieved and evaluated at the end of gestation. RESULTS: Uterine bag insertion was successful in 15 of 24 (62.5%) and intestinal bag placement in 10 of 15 available fetuses (66.6%). The main factor limiting fetoscopic procedures was chorioamniotic separation (CAS). Sterilized condoms provided the most appropriate type of bags and the V-Loc™ running suture, the most expedient type of fixation, which was achieved in 9 of the 10 fetuses (complete = 2, partially = 7) by using a three port access (5 mm and 2 × 3 mm). All bags were encountered completely or partially dislocated from the fetus at the end of gestation. CONCLUSIONS: Fetoscopic intestinal bag placement and fixation in gastroschisis technically demanding. None of the evaluated techniques led to permanent anchorage of the bag to the fetus. The development of specially designed instruments, bags and fixation methods is required to optimize this approach.

Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation.

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B-Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.

Time- and dose-dependent inhibition of neutrophil extracellular trap formation by blocking of the interleukin-1 receptor.

Besides performing phagocytosis and degranulation, neutrophils are capable of eliminating microorganisms by releasing neutrophil extracellular traps (NETs). NET formation was found to be associated with increased mortality in sepsis. During sepsis levels of interleukin 1ß (IL-1ß), a cytokine, increases significantly and also was associated with increased mortality. Blocking of the interleukin 1 (IL-1) receptor by anakinra leads to less NET formation in gout patients. However, NET formation is crucial during infection by trapping pathogens and thereby slowing the process. Total or early blocking of cascades leading to NETs may lead to aggravation of infection in otherwise mild cases. The dose- and time-dependent effect of the IL-1 receptor antagonist anakinra was tested on spontaneous, lipopolysaccharide (LPS)-induced and phorbol-12-myristate 13-acetate (PMA)-induced formation of NETs in vitro. Quantitative detection of NETs was performed for NETspecific proteins and cell-free DNA. Immunostained microscopy imaging was used for visualization. Our study shows a dose- and time-dependent inhibitory effect of anakinra that involves the change of intracellular calcium mobilization on the formation of NETs in vitro for PMA-stimulated neutrophils but not for LPS-stimulated neutrophils. It may be useful for treatment of sepsis as part of a multimodal treatment concept, but it seems that timing and dose need to be carefully chosen.

Challenges of LC-MS/MS ethyl glucuronide analysis in abstinence monitoring of liver transplant candidates.

Background Urinary ethyl glucuronide (EtG) has emerged as the biomarker of choice for alcohol abstinence monitoring in forensic toxicology and is now used in the listing decision process for liver transplantations (LTs) in the German transplant program. However, EtG analysis in this patient group is challenging due to severely impaired liver function, renal failure, co-morbidities and multidrug regimens. The aim of our study was to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based EtG analysis for a precise abstinence monitoring in transplant candidates. Methods EtG and ethyl sulfate (EtS) were analyzed by a commercial LC-MS/MS assay in 1787 spot urine samples of 807 patients (>85% from the Department of Hepatology) using a combination of quantifier and two qualifier mass transitions for each analyte. Influences of bacterial contamination, kidney and liver function were investigated. Results Two hundred and sixty-four urine samples had elevated (≥0.5 mg/L) EtG concentrations when only analyzing one quantifier mass transition. Eleven results (4.2%) were found to be false positive after combining three mass transitions for EtG quantification and verification with parallel analysis of EtS. Decreased kidney function was associated with a significantly higher rate of positive EtG samples. One of the false positive results was caused by bacterial metabolism. Conclusions Multimorbid pre-transplant patients have a high risk of individual analytical disturbances of EtG results obtained by LC-MS/MS. Therefore, EtG and EtS should always be measured by a combination of one quantifier and two qualifiers each and evaluated together.

How safe are our studies? Analysis of adverse events in Bayer First-in-Human trials from 2006 to 2016
.

PURPOSE: In regard to the current scientific discussion, this analysis aims to broaden the database for a risk evaluation of First-in-Human (FiH) trials with healthy volunteers. MATERIALS AND METHODS: Study documents of each FiH study conducted between 2006 and 2016 for Bayer Clinical Pharmacology Cardiovascular were reviewed for inclusion. Study types, treatments, dose steps, study population, number, incidence, and intensity of treatment-emergent adverse events (AEs) were cumulatively analyzed using descriptive statistics. A comparison to a previous similar analysis (period 2000 - 2005) was made. RESULTS: 22 out of 25 studies were included (20 small molecules, 2 biologics) investigating drugs for cardiovascular (9), hematological (7), pulmonary (3), kidney (2), and metabolic (1) diseases. The mean age of subjects was 34.2 years. 1,250 subjects received treatment (950 active, 300 placebo). 952 AEs occurred (0.76 AEs/treatment, 0.85 AEs/active treatment, 0.49 AEs/placebo treatment). 88.2% (840/952) of AEs were mild, 11.3% (108/952) moderate, and 0.4% (4/952) were severe. 0.4% (5/1250) of subjects had active drug- or procedure-related serious AEs. The most frequent AE was headache (12.9% (123/952)), the mostly affected system organ class was CNS (14.4% of all subjects). The relative risk for an AE was significantly higher under active drug compared to placebo (1.24, 95% LCL >1). The incidence of AEs increased with higher dose steps. A higher incidence of AEs (active and placebo) in recent compared to previous studies was observed. CONCLUSION: The risk of severe harm for healthy participants was low. The risk to experience any AE was higher under active drug compared to placebo. A trend change towards more frequent reporting of AEs in the recent studies was observed.

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.

BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells. RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA. CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.