An ER‐resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

Nicotinic acetylcholine receptors (nAChRs) are homo‐ or heteropentameric ligand‐gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell‐surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA‐2 or NRA‐4 (n icotinic r eceptor a ssociated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype‐specific agonists of these nAChRs is altered differently, as demonstrated by whole‐cell voltage‐clamp of dissected adult muscle, when applying exogenous agonists or after photo‐evoked, channelrhodopsin‐2 (ChR2) mediated acetylcholine (ACh) release, as well as in single‐channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra‐2 mutants. Cell‐surface expression of different subunits of the ‘levamisole‐sensitive’ nAChR (L‐AChR) is differentially affected in the absence of NRA‐2 or NRA‐4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.     

Inhibitors of hepatitis C virus NS3/4A: α-Ketoamide based macrocyclic inhibitors

A novel series of hepatitis C virus (HCV) NS3/4A protease inhibitors bearing a P2-P4 macrocycle and a P1-P1′ α-ketoamide serine trap is reported. The NS3 protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. The optimization of both the macrocycle and the warhead portions led to the discovery of compounds 8b and 8g with a good activity both in the enzyme as well as in the cell based (replicon) assays with favorable PK profile in a preclinical species.     

An Adequately Robust Early TNF-α Response Is a Hallmark of Survival Following Trauma/Hemorrhage

Background

Trauma/hemorrhagic shock (T/HS) results in cytokine-mediated acute inflammation that is generally considered detrimental.

Methodology/Principal Findings

Paradoxically, plasma levels of the early inflammatory cytokine TNF-α (but not IL-6, IL-10, or NO₂ ^-/NO₃ ^-) were significantly elevated within 6 h post-admission in 19 human trauma survivors vs. 4 non-survivors. Moreover, plasma TNF-α was inversely correlated with Marshall Score, an index of organ dysfunction, both in the 23 patients taken together and in the survivor cohort. Accordingly, we hypothesized that if an early, robust pro-inflammatory response were to be a marker of an appropriate response to injury, then individuals exhibiting such a response would be predisposed to survive. We tested this hypothesis in swine subjected to various experimental paradigms of T/HS. Twenty-three anesthetized pigs were subjected to T/HS (12 HS-only and 11 HS + Thoracotomy; mean arterial pressure of 30 mmHg for 45–90 min) along with surgery-only controls. Plasma obtained at pre-surgery, baseline post-surgery, beginning of HS, and every 15 min thereafter until 75 min (in the HS only group) or 90 min (in the HS + Thoracotomy group) was assayed for TNF-α, IL-6, IL-10, and NO₂ ^-/NO₃ ^-. Mean post-surgery±HS TNF-α levels were significantly higher in the survivors vs. non-survivors, while non-survivors exhibited no measurable change in TNF-α levels over the same interval.

Conclusions/Significance

Contrary to the current dogma, survival in the setting of severe, acute T/HS appears to be associated with an immediate increase in serum TNF-α. It is currently unclear if this response was the cause of this protection, a marker of survival, or both. This abstract won a Young Investigator Travel Award at the SHOCK 2008 meeting in Cologne, Germany.     

The Drosophila Kinesin-13, KLP59D,Impacts Pacman- and Flux-based Chromosome Movement

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole–associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D''s impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.     

Radiation therapy with curative intention in men with de novo metastatic prostate carcinoma: shoot‘em all!

BackgroundAbout 5% of prostate cancer cases are metastatic at diagnoses. Radiotherapy of both primary tumor and secondary lesions can be, in addition to systemic treatments, a radical alternative for selected patients.Materials and methodsPatients with de novo prostate carcinoma with bone or lymph node metastases were retrospectively reviewed. All patients received moderate hypofractionated IMRT/VMAT up to 63 Gy in 21 daily fractions of 3 Gy to prostate and metastases with neoadjuvant and concurrent androgen deprivation therapy (ADT). According to known advances some patients also received abiraterone, enzalutamide, or docetaxel.ResultsBetween 2015–2020, we attended 26 prostate cancer patients (median age 69.5 years, range 52–84) with simultaneous oligometastases [mean 2.1 metastases, median 1.5 metastases (range 1–6)]. Eighteen patients (69%) presented lymph node metastases, 4 (15.5%) bone metastases and 4 (15.5%) both lymph node and bone metastases. With a median follow-up of 15.5 months (range 3–65 months), 16 patients (62%) are alive and tumor free while 10 (38%) are alive with tumor. Four patients (17%) developed tumor progression, out of irradiated area in all cases, with a median time to progression of 43.5 months (range 27–56 months). Actuarial progression-free survival (PFS) rates at 12 and 24 months were 94.1% and 84.7%, respectively. No grade > 2 acute or late complications were recorded.ConclusionsSimultaneous directed radical hypofractionated radiation therapy for prostate and metastases is feasible, well tolerated and achieves an acceptable PFS rate. However, further studies with longer follow-up are necessary to definitively address these observations.     

Cardiomyogenic differentiation of human bone marrow mesenchymal cells: Role of cardiac extract from neonatal rat cardiomyocytes

Bone marrow mesenchymal stromal cells (BM-MSCs) with regenerative potential have been identified in heart. Whether these cells become new cardiac lineage cells by phenomena of transdifferentiation or fusion is also being investigated. Although, these mechanisms give cardiomyocytes, it has to be considered that MSCs transplantation could carry out ossification and calcification processes. An alternative might be the use of myocytes; however, the problem is the arrythmia. For those reasons, is that we investigated how to obtain cardiomyocyte-like cells from human MSCs (hMSCs). The aim of the present work was to evaluate a nuclear reprogramming of the hMSCs by a neonatal rat cardiomyocytes extract (EX) using Streptolysin O (SLO) treatment. hMSCs treated with 57.5 ng/ml SLO presented ball-like, stick-like and myotube-like morphology. In the absence of cardiomyogenic stimuli, hMSCs expressed markers of cardiac phenotype-like sarcomeric α-actinin, connexin-43 and GATA-4. However, when hMSCs were treated with SLO+EX or 10 μM of 5-azacytidine (5-AZA), the expression of these markers were significantly increased and furthermore, expressed SERCA-2, cardiac Troponin I, β-MyHC, desmin, MLC-2a and MLC-2v thus showing the phenotype of mature cardiomyocytes. PCR analysis showed that cardiomyocyte-related genes, such as β1-adrenergic receptor (β1-AR), MLC-2a and cardiac Troponin T, were expressed after SLO+EX treatment like with 5-AZA. We concluded that the extract of neonatal rat cardiomyocytes could promote a nuclear modification of hMSCs to cardiomyogenic-like cells differentiation. Since the 5-AZA treatment appears to be genotoxic and taking into account the obtained results, the nuclear reprogramming by cell extract may be an approach leading to the identification of soluble factors that drives the reprogramming.     

Foxp3+ cell infiltration and granzyme B+/Foxp3+ cell ratio are associated with outcome in neoadjuvant chemotherapy-treated ovarian carcinoma

Preoperative neoadjuvant chemotherapy (NAC) can significantly reduce tumour burden in patients with primarily unresectable chemosensitive tumours, allowing a more complete cytoreduction during debulking surgery and facilitating evaluation of tumour chemosensitivity, identification of appropriate treatment options and improvement of intervention protocols. In this study, we investigate, using immunohistochemistry, the impact of platinum/taxane-based NAC (NAC) on tumour-infiltrating lymphocytes (TILs) in advanced epithelial ovarian cancer (EOC) and their relationship with clinical outcome. All patients had clinical response, as shown by ascites volume and CA125 levels compared to pre-treatment findings. NAC intervention significantly increased CD4⁺, CD8⁺ and granzyme B⁺ infiltration while Foxp3⁺ accumulation remained unaffected. TILs were prognostically neutral for both progression-free survival (PFS) and overall survival (OS) before NAC. In contrast, after NAC, elevated granzyme B⁺ infiltration displayed a tendency for improved PFS (log-rank 0.064). Further, low Foxp3⁺ cell density was associated with longer PFS, as compared with strong Foxp3⁺ infiltration (median 20.94 vs. 11.24 months; log-rank 0.0001) and with improved OS (median 30.75 vs. 16.04 months, respectively; log-rank 0.056), demonstrating clear prognostic significance for PFS. In addition, high granzyme B⁺/Foxp3⁺ ratio post-NAC strongly correlated with improved PFS compared to low granzyme B⁺/Foxp3⁺ cell ratio (median 17.88 vs. 11.24 months, respectively), and showed to be a favourable prognostic factor for PFS (log-rank 0.014). Our findings indicate that NAC elicited an immunologic profile in which low immunosuppressive Foxp3⁺ infiltration and elevated numbers of activated granzyme B⁺ cells were significantly associated with EOC-specific PFS, suggesting a contribution of immunologic effects to improved clinical outcome.     

Quantitatively defining the conservation status of Natura 2000 forest habitats and improving management options for enhancing biodiversity

The main goal of Natura 2000 network is to guarantee the favourable conservation status of habitats and species ensuring European biodiversity. As a result, certain forest areas have been included in this network listed as 9230-Quercus pyrenaica habitat and 9340-Quercus ilex subsp. rotundifolia forest habitat. These areas were previously used for firewood extraction or livestock grazing and browsing. Nowadays these habitats are coppice forests with asexual regeneration, which is far from the desired conservation status. Traditional timber harvesting plans do not take account of the new objectives required for these Natura sites, which attempt to ensure biodiversity and recreational uses instead of simply focusing on timber production. This paper proposes a flexible methodology (applied to the study area “Dehesa Boyal” in ávila, Spain) for managing Natura 2000 forest sites by stands for sustainable forest management and the new requirements. The methodology has two phases. The first, “Division of the forest area into stands”, defines homogeneous patches of vegetation distinct in species composition, physiognomic structure and future management. The second, “Conservation status assessment of stands”, quantifies the conservation status of each previously classified stand considering a series of factors such as: functional health, restoration, floral richness and structure. A total value integrating the conservation status of stands is then calculated for the habitat. Both phases use Geographic Information System tools for managing information and visualizing results. The proposed methodology provides forest managers with a good knowledge of the territory and subsequently enables them to take appropriate conservation measures to maintain biodiversity.     

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Background

Chloroquine (CQ) or sulfadoxine-pyrimethamine (SP) monotherapy for Plasmodium falciparum often leads to therapeutic failure in Indonesia. Combining CQ with other drugs, like SP, may provide an affordable, available and effective option where artemisinin-combined therapies (ACT) are not licensed or are unavailable.

Methods

This study compared CQ (n = 29 subjects) versus CQ + SP (with or without primaquine; n = 88) for clinical and parasitological cure of uncomplicated falciparum malaria in the Menoreh Hills region of southern Central Java, Indonesia. Gametocyte clearance rates were measured with (n = 56 subjects) and without (n = 61) a single 45 mg dose of primaquine (PQ).

Results

After 28 days, 58% of subjects receiving CQ had cleared parasitaemia and remained aparasitaemic, compared to 94% receiving CQ combined with SP (p < 0.001). Msp-2 genotyping permitted reinfection-adjusted cure rates for CQ and CQ combined with SP, 70% and 99%, respectively (p = 0.0006).

Conclusion

Primaquine exerted no apparent affect on cure of asexual stage parasitaemia, but clearly accelerated clearance of gametocytes. CQ combined with SP was safe and well-tolerated with superior efficacy over CQ for P. falciparum parasitaemia in this study.     

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.