Mps1 regulates kinetochore-microtubule attachment stability via the ska complex to ensure error-free chromosome segregation

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation

Das komplexe Beckentrauma: Matching des Beckenregisters DGU mit dem TraumaRegister DGU®

BACKGROUND Complex pelvic traumas, i.e., pelvic fractures accompanied by pelvic soft tissue injuries, still have an unacceptably high mortality rate of about 18 %. PATIENTS AND METHODS We retrospectively evaluated an intersection set of data from the TraumaRegister DGU® and the German Pelvic Injury Register from 2004-2009. Patients with complex and noncomplex pelvic traumas were compared regarding their vital parameters, emergency management, stay in the ICU, and outcome. RESULTS From a total of 344 patients with pelvic injuries, 21 % of patients had a complex and 79 % a noncomplex trauma. Complex traumas were significantly less likely to survive (16.7 % vs. 5.9 %). Whereas vital parameters and emergency treatment in the preclinical setting did not differ substantially, patients with complex traumas were more often in shock and showed acute traumatic coagulopathy on hospital arrival, which resulted in more fluid volumes and transfusions when compared to patients with noncomplex traumas. Furthermore, patients with complex traumas had more complications and longer ICU stays. CONCLUSION Prevention of exsanguination and complications like multiple organ dysfunction syndrome still pose a major challenge in the management of complex pelvic traumas

A Rodent Model for Dirofilaria Immitis, Canine Heartworm: Parasite Growth, Development, and Drug Sensitivity in NSG Mice

Heartworm disease, caused by Dirofilaria immitis, remains a significant threat to canines and felines. The development of parasites resistant to macrocyclic lactones (ML) has created a significant challenge to the control of the infection. The goal of this study was to determine if mice lacking a functional immune response would be susceptible to D. immitis. Immunodeficient NSG mice were susceptible to the infection, sustaining parasites for at least 15 weeks, with infective third-stage larvae molting and developing into the late fourth-stage larvae. Proteomic analysis of host responses to the infection revealed a complex pattern of changes after infection, with at least some of the responses directed at reducing immune control mechanisms that remain in NSG mice. NSG mice were infected with isolates of D. immitis that were either susceptible or resistant to MLs, as a population. The susceptible isolate was killed by ivermectin whereas the resistant isolate had improved survivability, while both isolates were affected by moxidectin. It was concluded that D. immitis survives in NSG mice for at least 15 weeks. NSG mice provide an ideal model for monitoring host responses to the infection and for testing parasites in vivo for susceptibility to direct chemotherapeutic activity of new agents

Ex vivo correction of selenoprotein N deficiency in rigid spine muscular dystrophy caused by a mutation in the selenocysteine codon

Premature termination of translation due to nonsense mutations is a frequent cause of inherited diseases. Therefore, many efforts were invested in the development of strategies or compounds to selectively suppress this default. Selenoproteins are interesting candidates considering the idiosyncrasy of the amino acid selenocysteine (Sec) insertion mechanism. Here, we focused our studies on SEPN1, a selenoprotein gene whose mutations entail genetic disorders resulting in different forms of muscular diseases. Selective correction of a nonsense mutation at the Sec codon (UGA to UAA) was undertaken with a corrector tRNASec that was engineered to harbor a compensatory mutation in the anticodon. We demonstrated that its expression restored synthesis of a full-length selenoprotein N both in HeLa cells and in skin fibroblasts from a patient carrying the mutated Sec codon. Readthrough of the UAA codon was effectively dependent on the Sec insertion machinery, therefore being highly selective for this gene and unlikely to generate off-target effects. In addition, we observed that expression of the corrector tRNASec stabilized the mutated SEPN1 transcript that was otherwise more subject to degradation. In conclusion, our data provide interesting evidence that premature termination of translation due to nonsense mutations is amenable to correction, in the context of the specialized selenoprotein synthesis mechanism

A guideline for placement of an infra-acetabular screw based on anatomic landmarks via an intra-pelvic approach

Background: Due to demographic changes, more and more fracture patterns involving anterior acetabular structures occur. The infra-acetabular screw is seen a useful tool to increase stability in fixation of the acetabular cup. However, the exact position of this screw in relation to anatomic landmarks which are intra-operatively palpable via an intra-pelvic approach has not yet been determined. Methods: This biomorphometric experimental study references the ideal screw position of an infra-acetabular screw to anatomic landmarks palpable via an intra-pelvic approach. Therefore, we created a computer tomography-based 3D-model of 40 patients (20 women, 20 men) who received a computer tomography (CT) scan of the pelvis for any other reason than an acetabular fracture. Results: The entry point of an ideal infra-acetabular was of high constancy. At mean, this point was 10.2 mm caudal and 10.4 mm medial of the ilio-pubic/ilio-pectineal eminence. This reference is independent of age, gender, or physical dimensions. However, we found gender-dependent differences for the angulation and the length of the screw. Conclusions: This study provides a comprehensive guideline to determine the ideal entry point for an infra-acetabular screw via an intra-pelvic approach. The entry point is located 10.2 mm caudal and 10.4 mm medial of the ilio-pubic/ilio-pectineal eminence

Einfluss des Beckenregisters der DGU auf die Versorgung von Beckenringfrakturen

Fractures of the pelvic ring are comparatively rare with an incidence of 2-8 % of all fractures depending on the study in question. The severity of pelvic ring fractures can be very different ranging from simple and mostly "harmless" type A fractures up to life-threatening complex type C fractures. Although it was previously postulated that high-energy trauma was necessary to induce a pelvic ring fracture, over the past decades it became more and more evident, not least from data in the pelvic trauma registry of the German Society for Trauma Surgery (DGU), that low-energy minor trauma can also cause pelvic ring fractures of osteoporotic bone and in a rapidly increasing population of geriatric patients insufficiency fractures of the pelvic ring are nowadays observed with no preceding trauma.Even in large trauma centers the number of patients with pelvic ring fractures is mostly insufficient to perform valid and sufficiently powerful monocentric studies on epidemiological, diagnostic or therapeutic issues. For this reason, in 1991 the first and still the only registry worldwide for the documentation and evaluation of pelvic ring fractures was introduced by the Working Group Pelvis (AG Becken) of the DGU. Originally, the main objectives of the documentation were epidemiological and diagnostic issues; however, in the course of time it developed into an increasingly expanding dataset with comprehensive parameters on injury patterns, operative and conservative therapy regimens and short-term and long-term outcome of patients. Originally starting with 10 institutions, in the meantime more than 30 hospitals in Germany and other European countries participate in the documentation of data. In the third phase of the registry alone, which was started in 2004, data from approximately 15,000 patients with pelvic ring and acetabular fractures were documented. In addition to the scientific impact of the pelvic trauma registry, which is reflected in the numerous national and international publications, the dramatically changing epidemiology of pelvic ring fractures, further developments in diagnostics and the changes in operative procedures over time could be demonstrated. Last but not least the now well-established diagnostic and therapeutic algorithms for pelvic ring fractures, which could be derived from the information collated in registry studies, reflect the clinical impact of the registry.Zusammenfassung Die Inzidenz von Beckenringfrakturen ist mit einem Anteil von je nach Studienlage 2–8 % aller Frakturen vergleichsweise gering. Das Erscheinungsbild einer Beckenringfraktur ist sehr vielfältig und reicht von einfachen und meist „harmlosen“ Typ-A-Verletzungen bis hin zu lebensbedrohlichen komplexen Typ-C-Verletzungen. Während in der Vergangenheit postuliert wurde, dass ein hohes Energieniveau erforderlich ist, um den Beckenring zu frakturieren, wurde in den letzten Jahren nicht zuletzt durch Daten des Beckenregisters der DGU evident, dass bei osteoporotischem Knochen oft auch ein Bagatelltrauma zu einer Beckenringfraktur führen kann. Es kristallisierte sich heraus, dass bei einem rasant wachsenden geriatrischen Patientenkollektiv sogar ohne vorangegangenes Trauma Insuffizienzfrakturen des Beckenrings zu beobachten sind. Auch an großen Traumazentren ist die Anzahl der Patienten mit Beckenringfrakturen vielfach nicht ausreichend, um durch Monocenterstudien valide Aussagen über epidemiologische, diagnostische und therapeutische Entwicklungen treffen zu können. Aus diesem Grunde wurde bereits 1991 von der AG Becken der DGU das weltweit erste und bis heute einzige Register zur Dokumentation und Evaluation von Beckenverletzungen ins Leben gerufen. Standen anfänglich v. a. epidemiologische und diagnostische Fragestellungen im Vordergrund der Dokumentation, entwickelte sich im Laufe der Zeit ein zunehmend wachsender Datensatz mit umfassenden Parametern zu Verletzungsmustern, operativen und konservativen Therapieregimen sowie dem kurz- und langfristigen Outcome der Patienten. Während das Beckenregister ursprünglich in 10 Einrichtungen gestartet wurde, nehmen mittlerweile über 30 Kliniken auch außerhalb Deutschlands an der Datendokumentation teil. Allein in der dritten Phase der Registerarbeit konnten seit 2004 an die 15.000 Patienten mit Becken- und Azetabulumfrakturen in die Datenbank eingeschlossen werden. Neben dem wissenschaftlichen Einfluss des Beckenregisters, der sich in zahlreichen nationalen und internationalen Publikationen widerspiegelt, konnten durch die Datenerhebungen die sich dramatisch wandelnde Epidemiologie von Beckenringverletzungen, Weiterentwicklungen in der Diagnostik sowie die sich über den Zeitverlauf ändernden operativen Therapieverfahren aufgezeigt werden. Nicht zuletzt die aus der Datenlage abgeleiteten und heute etablierten Diagnostik- und Therapiealgorithmen bei Beckenringfrakturen spiegeln den klinischen Wert des Beckenregisters wider

Epigenetic associations with kidney disease in individuals of African ancestry with APOL1 high-risk genotypes and HIV

Background. Apolipoprotein L1 (APOL1) high-risk variants are major determinants of chronic kidney disease (CKD) in people of African ancestry. Previous studies have identified epigenetic changes in relation to kidney function and CKD, but not in individuals with APOL1 high-risk genotypes. We conducted an epigenome-wide analysis of CKD and estimated glomerular filtration rate (eGFR) in in people of African ancestry and APOL1 high-risk genotypes with HIV. Methods. DNA methylation profiles from peripheral blood mononuclear cells of 119 individuals with APOL1 high-risk genotypes (mean age 48 years, 49% female, median CD4 count 515 cells/mm3, 90% HIV-1 RNA <200 copies/mL, 23% with CKD) were obtained by Illumina MethylationEPIC BeadChip. Differential methylation analysis of CKD considered technical and biological covariates. We also assessed associations with eGFR. Replication was pursued in three independent multi-ancestry cohorts with and without HIV. Results. DNA methylation levels at 14 regions were associated with CKD. The strongest signals were located in SCARB1, DNAJC5B and C4orf50. Seven of the 14 signals also associated with eGFR, and most showed evidence for a genetic basis. Four signals (in SCARB1, FRMD4A, CSRNP1 and RAB38) replicated in other cohorts, and 11 previously reported epigenetic signals for kidney function or CKD replicated in our cohort. We found no significant DNA methylation signals in, or near, the APOL1 promoter region. Conclusions. We report several novel as well as previously reported epigenetic associations with CKD and eGFR in individuals with HIV having APOL1 high-risk genotypes. Further investigation of pathways linking DNA methylation to APOL1 nephropathies is warranted

Genetic studies of paired metabolomes reveal enzymatic and transport processes at the interface of plasma and urine.

The kidneys operate at the interface of plasma and urine by clearing molecular waste products while retaining valuable solutes. Genetic studies of paired plasma and urine metabolomes may identify underlying processes. We conducted genome-wide studies of 1,916 plasma and urine metabolites and detected 1,299 significant associations. Associations with 40% of implicated metabolites would have been missed by studying plasma alone. We detected urine-specific findings that provide information about metabolite reabsorption in the kidney, such as aquaporin (AQP)-7-mediated glycerol transport, and different metabolomic footprints of kidney-expressed proteins in plasma and urine that are consistent with their localization and function, including the transporters NaDC3 (SLC13A3) and ASBT (SLC10A2). Shared genetic determinants of 7,073 metabolite-disease combinations represent a resource to better understand metabolic diseases and revealed connections of dipeptidase 1 with circulating digestive enzymes and with hypertension. Extending genetic studies of the metabolome beyond plasma yields unique insights into processes at the interface of body compartments

Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression

Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3′ untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level