LEM4/ANKLE-2 deficiency impairs post-mitotic re-localization of BAF,LAP2α and LaminA to the nucleus,causes nuclear envelope instability in telophase and leads to hyperploidy in HeLa cells

The human LEM-domain protein family is involved in fundamental aspects of nuclear biology. The LEM-domain interacts with the barrier-to-autointegration factor (BAF), which itself binds DNA. LEM-domain proteins LAP2, emerin and MAN1 are proteins of the inner nuclear membrane; they have important functions: maintaining the integrity of the nuclear lamina and regulating gene expression at the nuclear periphery.LEM4/ANKLE-2 has been proposed to participate in nuclear envelope reassembly after mitosis and to mediate dephosphorylation of BAF through binding to phosphatase PP2A. Here, we used CRISPR/Cas9 to create several cell lines deficient in LEM4/ANKLE-2. By using time-lapse video microscopy, we show that absence of this protein severely compromises the post mitotic re-association of the nuclear proteins BAF, LAP2α and LaminA to chromosomes. These defects give rise to a strong mechanical instability of the nuclear envelope in telophase and to a chromosomal instability leading to increased number of hyperploid cells. Reintroducing LEM4/ANKLE-2 in the cells by transfection could efficiently restore the telophase association of BAF and LAP2α to the chromosomes. This rescue phenotype was abolished for N- or C-terminally truncated mutants that had lost the capacity to bind PP2A. We demonstrate also that, in addition to binding to PP2A, LEM4/ANKLE-2 binds BAF through its LEM-domain, providing further evidence for a generic function of this domain as a principal interactor of BAF.     

Electroconvulsive therapy for manic state with mixed and psychotic features in a teenager with bipolar disorder and comorbid episodic obsessive–compulsive disorder: a case report

Background

Comorbidity of bipolar disorder and obsessive–compulsive disorder is common in adolescence. Obsessive–compulsive disorder symptoms may be episodic and secondary to alterations in mood, and display specific features. Management of pediatric bipolar disorder-obsessive–compulsive disorder is challenging, as pharmacotherapy of obsessive–compulsive disorder may induce or exacerbate manic episodes and there is limited evidence of treatment efficacy. Electroconvulsive therapy is sparsely used in children and adolescents, but is documented to be a safe and efficacious intervention in adults with bipolar disorder. In view of the severity of symptoms in juvenile mania, studies on treatment strategies are warranted. We report a case of an adolescent with bipolar disorder-obsessive–compulsive disorder who was successfully treated with electroconvulsive therapy during an episode of severe mania.

Case presentation

A 16-year-old girl of Middle East origin first presented to us with depressed mood, irritability, and increased obsessive–compulsive disorder symptoms, which were initially interpreted in the context of acute stress secondary to migration. She had been diagnosed with bipolar disorder and obsessive–compulsive disorder in her previous home country, but had difficulties in accounting for earlier psychiatric history. During hospitalization her mood switched to a manic state with mixed and psychotic features, at times showing aggression toward others. Interruption in her lithium treatment for a short period and possibly the introduction of an atypical antipsychotic could in part have been triggering factors. After 8 weeks of in-patient care and psychotropic drug trials, electroconvulsive therapy was initiated and administered every second or third day for 4 weeks, with marked positive response. No apparent side effects were reported.

Conclusions

This case demonstrates the need for a detailed medical history, taking special note of periodicity and character of obsessive–compulsive disorder symptoms, in adolescents with mood disorders. When treating culturally diverse patients, extra consideration should be taken. Special concerns in the pharmacological treatment to avoid the patient’s condition from worsening must be addressed, including giving priority to mood stabilization before obsessive–compulsive disorder symptoms. There are potential benefits in considering electroconvulsive therapy in young patients with severe mania where first-line treatment options have failed.

     

Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies.

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.     

Alterations of tension-dependent ATP utilization in a transgenic rat model of hypertrophic cardiomyopathy

Although it is established that familial hypertrophic cardiomyopathy (FHC) is caused by mutations in several sarcomeric proteins, including cardiac troponin T (TnT), its pathogenesis is still not completely understood. Previously, we established a transgenic rat model of FHC expressing a human TnT molecule with a truncation mutation (DEL-TnT). This study investigated whether contractile dysfunction and electrical vulnerability observed in DEL-TnT rats might be due to alterations of intracellular Ca(2+) homeostasis, myofibrillar Ca(2+) sensitivity, and/or myofibrillar ATP utilization. Simultaneous measurements of the force of contraction and intracellular Ca(2+) transients were performed in right ventricular trabeculae of DEL-TnT hearts at 0.25 and 1.0 Hz. Rats expressing wild-type human TnT as well as nontransgenic rats served as controls. In addition, calcium-dependent ATPase activity and tension development were investigated in skinned cardiac muscle fibers. Force of contraction was significantly decreased in DEL-TnT compared with nontransgenic rats and TnT. Time parameters of Ca(2+) transients were unchanged at 0.25 Hz but prolonged at 1.0 Hz in DEL-TnT. The amplitude of the fura-2 transient was similar in all groups investigated, whereas diastolic and systolic fura-2 ratios were found elevated in rats expressing nontruncated human troponin T. In DEL-TnT rats, myofibrillar Ca(2+)-dependent tension development as well as Ca(2+) sensitivity of tension were significantly decreased, whereas tension-dependent ATP consumption ("tension cost") was markedly increased. Thus, a C-terminal truncation of the cardiac TnT molecule impairs the force-generating capacity of the cycling cross-bridges resulting in increased tension-dependent ATP utilization. Taken together, our data support the hypothesis of energy compromise as a contributing factor in the pathogenesis of FHC.     

Phosphodiesterase type 5 inhibitor sildenafil citrate does not potentiate the vasodilative properties of nebivolol in rat aorta

BACKGROUND: Sildenafil citrate (SIL) is contraindicated in patients with coronary heart disease who are treated with nitric oxide (NO) donators such as organic nitrates, as it potentiates NO-mediated vasodilation. The present study investigated whether SIL also affects the vasodilatory effects of nebivolol (NEB), a selective beta1-adrenoceptor blocker with an additional, endothelium-dependent NO-liberating property, in comparison to the combination SIL/glycerol trinitrate (GTN). METHODS AND RESULTS: Experiments were performed in isolated vessel rings of rat aorta (Wistar rats, 8-12 weeks), which had been pre-contracted with phenylephrine (10(-5) M). Isometric tension was measured by a force transducer, and cumulative concentration-response curves were obtained for each drug. The rank order of vasodilatory potency as measured by the concentration needed to achieve 50% relaxation (EC50) was GTN (0.08 microM) > SIL (1.25 microM) > or = NEB (3.5 microM). In the presence of both therapeutic (1 nM) and high (1 microM) concentrations of SIL, vasodilation of GTN was potentiated as indicated by a significant increase in vasodilatory potency (EC50 GTN + low SIL: 0.019 microM, EC50 GTN + high SIL: 0.002 microM; both P < 0.01 vs. GTN). In contrast, SIL did not potentiate the vasodilatory effect of NEB (EC50 NEB + low SIL: 5.01 microM, EC50 NEB + high SIL: 3.2 microM; n.s. vs. NEB). CONCLUSIONS: These data demonstrate that SIL does not potentiate NEB-induced vasodilation in vitro. These findings indicate that the interaction between SIL and NO-donators/organic nitrates does not apply to the NO-liberating properties of NEB. Our findings suggest that SIL may safely be used in hypertensive patients treated with NEB.     

Whole-genome QTL analysis for MAGIC

Key message

An efficient whole genome method of QTL analysis is presented for Multi-parent advanced generation integrated crosses.

Abstract

Multi-parent advanced generation inter-cross (MAGIC) populations have been developed for mice and several plant species and are useful for the genetic dissection of complex traits. The analysis of quantitative trait loci (QTL) in these populations presents some additional challenges compared with traditional mapping approaches. In particular, pedigree and marker information need to be integrated and founder genetic data needs to be incorporated into the analysis. Here, we present a method for QTL analysis that utilizes the probability of inheriting founder alleles across the whole genome simultaneously, either for intervals or markers. The probabilities can be found using three-point or Hidden Markov Model (HMM) methods. This whole-genome approach is evaluated in a simulation study and it is shown to be a powerful method of analysis. The HMM probabilities lead to low rates of false positives and low bias of estimated QTL effect sizes. An implementation of the approach is available as an R package. In addition, we illustrate the approach using a bread wheat MAGIC population.     

Effect of solar particle event radiation on gastrointestinal tract bacterial translocation and immune activation

Space flight conditions within the protection of Earth's gravitational field have been shown to alter immune responses, which could lead to potentially detrimental pathology. An additional risk of extended space travel outside the Earth's gravitational field is the effect of solar particle event (SPE) radiation exposure on the immune system. Organisms that could lead to infection include endogenous, latent viruses, colonizing pathogenics, and commensals, as well as exogenous microbes present in the spacecraft or other astronauts. In this report, the effect of SPE-like radiation on containment of commensal bacteria and the innate immune response induced by its breakdown was investigated at the radiation energies, doses and dose rates expected during an extravehicular excursion outside the Earth's gravitational field. A transient increase in serum lipopolysaccharide was observed 1 day after irradiation and was accompanied by an increase in acute-phase reactants and circulating proinflammatory cytokines, indicating immune activation. Baseline levels were reestablished by 5 days postirradiation. These findings suggest that astronauts exposed to SPE radiation could have impaired containment of colonizing bacteria and associated immune activation.     

Abacavir Induced T Cell Reactivity from Drug Na?ve Individuals Shares Features of Allo-Immune Responses

Abacavir hypersensitivity is a severe hypersensitivity reaction which occurs exclusively in carriers of the HLA-B*57∶01 allele. In vitro culture of PBMC with abacavir results in the outgrowth of abacavir-reacting CD8⁺ T cells, which release IFNγ and are cytotoxic. How this immune response is induced and what is recognized by these T cells is still a matter of debate. We analyzed the conditions required to develop an abacavir-dependent T cell response in vitro. The abacavir reactivity was independent of co-stimulatory signals, as neither DC maturation nor release of inflammatory cytokines were observed upon abacavir exposure. Abacavir induced T cells arose in the absence of professional APC and stemmed from naïve and memory compartments. These features are reminiscent of allo-reactivity. Screening for allo-reactivity revealed that about 5% of generated T cell clones (n = 136) from three donors were allo-reactive exclusively to the related HLA-B*58∶01. The addition of peptides which can bind to the HLA-B*57∶01-abacavir complex and to HLA-B*58∶01 during the induction phase increased the proportion of HLA-B*58∶01 allo-reactive T cell clones from 5% to 42%. In conclusion, abacavir can alter the HLA-B*57∶01-peptide complex in a way that mimics an allo-allele (‘altered self-allele’) and create the potential for robust T cell responses.     

Detection of Puumala Hantavirus Antigen in Human Intestine during Acute Hantavirus Infection

Background

Puumala virus (PUUV) is the most important hantavirus species in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute renal injury (AKI) with thrombocytopenia and frequently gastrointestinal symptoms.

Methods

456 patients with serologically and clinically confirmed NE were investigated at time of follow-up in a single clinic. The course of the NE was investigated using medical reports. We identified patients who had endoscopy with intestinal biopsy during acute phase of NE. Histopathological, immunohistochemical and molecular analyses of the biopsies were performed.

Results

Thirteen patients underwent colonoscopy or gastroscopy for abdominal pain, diarrhea, nausea and vomiting during acute phase of NE. Immunohistochemistry (IHC) revealed PUUV nucleocapsid antigen in 11 biopsies from 8 patients; 14 biopsies from 5 patients were negative for PUUV nucleocapsid antigen. IHC localized PUUV nucleocapsid antigen in endothelial cells of capillaries or larger vessels in the lamina propria. Rate of AKI was not higher and severity of AKI was not different in the PUUV-positive compared to the PUUV-negative group. All IHC positive biopsies were positive for PUUV RNA using RT-PCR. Phylogenetic reconstruction revealed clustering of all PUUV strains from this study with viruses previously detected from the South-West of Germany. Long-term outcome was favorable in both groups.

Conclusions

In patients with NE, PUUV nucleocapsid antigen and PUUV RNA was detected frequently in the intestine. This finding could explain frequent GI-symptoms in NE patients, thus demonstration of a more generalized PUUV infection. The RT-PCR was an effective and sensitive method to detect PUUV RNA in FFPE tissues. Therefore, it can be used as a diagnostic and phylogenetic approach also for archival materials. AKI was not more often present in patients with PUUV-positive IHC. This last finding should be investigated in larger numbers of patients with PUUV infection.