Differential sorting and Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway

Sorting signals for apically destined proteins are highly diverse and can be present within the luminal, membrane-associated, and cytoplasmic domains of these proteins. A subset of apical proteins partition into detergent-resistant membranes, and the association of these proteins with glycolipid-enriched microdomains or lipid rafts may be important for their proper targeting. Recently, we observed that raft-associated and raft-independent apical proteins take different routes to the apical surface of polarized Madin-Darby canine kidney cells (Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G., and Weisz, O. A. (2007) EMBO J. 26, 3737-3748). Here we reconstituted in vitro the export of raft-associated and raft-independent markers staged intracellularly at 19 degrees C. Surprisingly, whereas release of the raft-associated protein influenza hemagglutinin was dependent on the addition of an ATP-regenerating system and cytosol, release of a yellow fluorescent protein (YFP)-tagged raft-independent protein (the 75-kDa neurotrophin receptor; YFP-p75) was efficient even in the absence of these constituents. Subsequent studies suggested that YFP-p75 is released from the trans-Golgi network in fragile tubules that do not withstand isolation procedures. Moreover, immunofluorescence analysis revealed that hemagglutinin and YFP-p75 segregate into distinct subdomains of the Golgi complex at 19 degrees C. Our data suggest that raft-associated and raft-independent proteins accumulate at distinct intracellular sites upon low temperature staging, and that upon warming, they exit these compartments in transport carriers that have very different membrane characteristics and morphologies.     

Citizen science and wildlife biology: Synergies and challenges

Citizen science (CS) has evolved over the past decades as a working method involving interested citizens in scientific research, for example by reporting observations, taking measurements or analysing data. In the past, research on animal behaviour has been benefitting from contributions of citizen scientists mainly in the field of ornithology but the full potential of CS in ecological and behavioural sciences is surely still untapped. Here, we present case studies that successfully applied CS to research projects in wildlife biology and discuss potentials and challenges experienced. Our case studies cover a broad range of opportunities: large‐scale CS projects with interactive online tools on bird song dialects, engagement of stakeholders as citizen scientists to reduce human–wildlife conflicts, involvement of students of primary and secondary schools in CS projects as well as collaboration with the media leading to successful recruitment of citizen scientists. Each case study provides a short overview of the scientific questions and how they were approached to showcase the potentials and challenges of CS in wildlife biology. Based on the experience of the case studies, we highlight how CS may support research in wildlife biology and emphasise the value of fostering communication in CS to improve recruitment of participants and to facilitate learning and mutual trust among different groups of interest (e.g., researchers, stakeholders, students). We further show how specific training for the participants may be needed to obtain reliable data. We consider CS as a suitable tool to enhance research in wildlife biology through the application of open science procedures (i.e., open access to articles and the data on publicly available repositories) to support transparency and sharing experiences.     

Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane

Albeit previous experiments suggest potential anti-inflammatory effect of exogenous methane (CH₄) in various organs, the mechanism of its bioactivity is not entirely understood. We aimed to investigate the potential mitochondrial effects and the underlying mechanisms of CH₄ in rat cardiomyocytes and mitochondria under simulated ischaemia/reperfusion (sI/R) conditions. Three-day-old cultured cardiomyocytes were treated with 2.2% CH₄-artificial air mixture during 2-hour-long reoxygenation following 4-hour-long anoxia (sI/R and sI/R + CH₄, n = 6-6), with normoxic groups serving as controls (SH and SH + CH₄; n = 6-6). Mitochondrial functions were investigated with high-resolution respirometry, and mitochondrial membrane injury was detected by cytochrome c release and apoptotic characteristics by using TUNEL staining. CH₄ admixture had no effect on complex II (CII)-linked respiration under normoxia but significantly decreased the complex I (CI)-linked oxygen consumption. Nevertheless, addition of CH₄ in the sI/R + CH4 group significantly reduced the respiratory activity of CII in contrast to CI and the CH₄ treatment diminished mitochondrial H₂O₂ production. Substrate-induced changes to membrane potential were partially preserved by CH₄, and additionally, cytochrome c release and apoptosis of cardiomyocytes were reduced in the CH₄-treated group. In conclusion, the addition of CH₄ decreases mitochondrial ROS generation via blockade of electron transport at CI and reduces anoxia-reoxygenation-induced mitochondrial dysfunction and cardiomyocyte injury in vitro.     

Performance of cross-linked enzyme crystals of engineered halohydrin dehalogenase HheG in different chemical reactor systems

Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β-substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross-linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross-linked with bis-maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space-time yield of 23.9 kg_product g_CLEC⁻¹ h⁻¹ L_{reactor volume}⁻¹ along with the highest achieved product enantiomeric excess [64%] for application in a packed-bed reactor. Additionally, lyophilization of those CLECs yielded a storage-stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature.     

A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei

Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells.     

Increased HEV Seroprevalence in Patients with Autoimmune Hepatitis

Background

Hepatitis E virus (HEV) infection takes a clinically silent, self-limited course in the far majority of cases. Chronic hepatitis E has been reported in some cohorts of immunocompromised individuals. The role of HEV infections in patients with autoimmune hepatitis (AIH) is unknown.

Methods

969 individuals were tested for anti-HEV antibodies (MP-diagnostics) including 208 patients with AIH, 537 healthy controls, 114 patients with another autoimmune disease, rheumatoid arthritis (RA), and 109 patients with chronic HCV- or HBV-infection (HBV/HCV). Patients with AIH, RA and HBV/HCV were tested for HEV RNA. HEV-specific proliferative T cell responses were investigated using CFSE staining and in vitro stimulation of PBMC with overlapping HEV peptides.

Results

HEV-antibodies tested more frequently positive in patients with AIH (n = 16; 7.7%) than in healthy controls (n = 11; 2.0%; p = 0.0002), patients with RA (n = 4; 3.5%; p = 0.13) or patients with HBV/HCV infection (n = 2; 2.8%; p = 0.03). HEV-specific T cell responses could be detected in all anti-HEV-positive AIH patients. One AIH patient receiving immunosuppression with cyclosporin and prednisolone and elevated ALT levels had acute hepatitis E but HEV viremia resolved after reducing immunosuppressive medication. None of the RA or HBV/HCV patients tested HEV RNA positive.

Conclusions

Patients with autoimmune hepatitis but not RA or HBV/HCV patients are more likely to test anti-HEV positive. HEV infection should been ruled out before the diagnosis of AIH is made. Testing for HEV RNA is also recommended in AIH patients not responding to immunosuppressive therapy.     

Vertical transmission of a phylogenetically complex microbial consortium in the viviparous sponge Ircinia felix

Many marine demosponges contain large amounts of phylogenetically complex yet highly sponge-specific microbial consortia within the mesohyl matrix, but little is known about how these microorganisms are acquired by their hosts. Settlement experiments were performed with the viviparous Caribbean demosponge Ircinia felix to investigate the role of larvae in the vertical transmission of the sponge-associated microbial community. Inspections by electron microscopy revealed large amounts of morphologically diverse microorganisms in the center of I. felix larvae, while the outer rim appeared to be devoid of microorganisms. In juveniles, microorganisms were found between densely packed sponge cells. Denaturing gradient gel electrophoresis (DGGE) was performed to compare the bacterial community profiles of adults, larvae, and juvenile sponges. Adults and larvae were highly similar in DGGE band numbers and banding patterns. Larvae released by the same adult individual contained highly similar DGGE banding patterns, whereas larvae released by different adult individuals showed slightly different DGGE banding patterns. Over 200 bands were excised, sequenced, and phylogenetically analyzed. The bacterial diversity of adult I. felix and its larvae was comparably high, while juveniles showed reduced diversity. In total, 13 vertically transmitted sequence clusters, hereafter termed "IF clusters," that contained sequences from both the adult sponge and offspring (larvae and/or juveniles) were found. The IF clusters belonged to at least four different eubacterial phyla and one possibly novel eubacterial lineage. In summary, it could be shown that in I. felix, vertical transmission of microorganisms through the larvae is an important mechanism for the establishment of the sponge-microbe association.     

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo. phosphatidylinositol 4,5-bisphosphate; cytoskeleton; membrane traffic; Arp2/3; actin comets