Lipid packing defects and membrane charge control RAB GTPase recruitment

Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors, but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical‐shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C‐terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.      

Preeclampsia and Blood Pressure Trajectory during Pregnancy in Relation to Vitamin D Status

Every tenth pregnancy is affected by hypertension, one of the most common complications and leading causes of maternal death worldwide. Hypertensive disorders in pregnancy include pregnancy-induced hypertension and preeclampsia. The pathophysiology of the development of hypertension in pregnancy is unknown, but studies suggest an association with vitamin D status, measured as 25-hydroxyvitamin D (25(OH)D). The aim of this study was to investigate the association between gestational 25(OH)D concentration and preeclampsia, pregnancy-induced hypertension and blood pressure trajectory. This cohort study included 2000 women. Blood was collected at the first (T1) and third (T3) trimester (mean gestational weeks 10.8 and 33.4). Blood pressure at gestational weeks 10, 25, 32 and 37 as well as symptoms of preeclampsia and pregnancy-induced hypertension were retrieved from medical records. Serum 25(OH)D concentrations (LC-MS/MS) in T1 was not significantly associated with preeclampsia. However, both 25(OH)D in T3 and change in 25(OH)D from T1 to T3 were significantly and negatively associated with preeclampsia. Women with a change in 25(OH)D concentration of ≥30 nmol/L had an odds ratio of 0.22 (p = 0.002) for preeclampsia. T1 25(OH)D was positively related to T1 systolic (β = 0.03, p = 0.022) and T1 diastolic blood pressure (β = 0.02, p = 0.016), and to systolic (β = 0.02, p = 0.02) blood pressure trajectory during pregnancy, in adjusted analyses. There was no association between 25(OH)D and pregnancy-induced hypertension in adjusted analysis. In conclusion, an increase in 25(OH)D concentration during pregnancy of at least 30 nmol/L, regardless of vitamin D status in T1, was associated with a lower odds ratio for preeclampsia. Vitamin D status was significantly and positively associated with T1 blood pressure and gestational systolic blood pressure trajectory but not with pregnancy-induced hypertension.     

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light‐Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System.

QLF versus OCT imaging of enamel caries: a photonics assessment     

Decreased Proportion of Cytomegalovirus Specific CD8 T-Cells but No Signs of General Immunosenescence in Alzheimer’s Disease

Cytomegalovirus (CMV) has been suggested as a contributing force behind the impaired immune responsiveness in the elderly, with decreased numbers of naïve T-cells and an increased proportion of effector T-cells. Immunological impairment is also implicated as a part of the pathogenesis in Alzheimer’s disease (AD). The aim of this study was to investigate whether AD patients present with a different CMV-specific CD8 immune profile compared to non-demented controls. Blood samples from 50 AD patients and 50 age-matched controls were analysed for HLA-type, CMV serostatus and systemic inflammatory biomarkers. Using multi-colour flow cytometry, lymphocytes from peripheral blood mononuclear cells were analysed for CMV-specific CD8 immunity with MHC-I tetramers A01, A02, A24, B07, B08 and B35 and further classified using CD27, CD28, CD45RA and CCR7 antibodies. Among CMV seropositive subjects, patients with AD had significantly lower proportions of CMV-specific CD8 T-cells compared to controls, 1.16 % vs. 4.13 % (p=0.0057). Regardless of dementia status, CMV seropositive subjects presented with a lower proportion of naïve CD8 cells and a higher proportion of effector CD8 cells compared to seronegative subjects. Interestingly, patients with AD showed a decreased proportion of CMV-specific CD8 cells but no difference in general CD8 differentiation.     

Calcium alginate immobilized cells of clostridium acetobutylicum for solvent production

Conclusions Immobilized vegetative cells ofC. acetobutylicum has a similar product formation pattern when incubated in a simple glucose-salts solution as ordinary growing cells. If vegetative cells of the organism are immobilized in the solvent production phase, solvents are continuously produced on extended incubation.By immobi1izing spores of the organism the disturbance of the cells metabolic activity during the immobilization procedure was avoided. After the outgrowth of viable cells within the gel, the washed gel preparation retained at a high production capacity in the non-growth stage and the results indicate that continuous production might be fully possible. The butanol productivity was also found to be higher with immobilized cells than in a normal batch process.     

Expression in insect cells of active mature cruzipain from Trypanosoma cruzi,containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

A novel inhibitor rescues cerebellar defects in a zebrafish model of Down syndrome–associated kinase Dyrk1A overexpression

The highly conserved dual-specificity tyrosine phosphorylation–regulated kinase 1A (Dyrk1A) plays crucial roles during central nervous system development and homeostasis. Furthermore, its hyperactivity is considered responsible for some neurological defects in individuals with Down syndrome. We set out to establish a zebrafish model expressing human Dyrk1A that could be further used to characterize the interaction between Dyrk1A and neurological phenotypes. First, we revealed the prominent expression of dyrk1a homologs in cerebellar neurons in the zebrafish larval and adult brains. Overexpression of human dyrk1a in postmitotic cerebellar Purkinje neurons resulted in a structural misorganization of the Purkinje cells in cerebellar hemispheres and a compaction of this cell population. This impaired Purkinje cell organization was progressive, leading to an age-dependent dispersal of Purkinje neurons throughout the cerebellar molecular layer with larval swim deficits resulting in miscoordination of swimming and reduced exploratory behavior in aged adults. We also found that the structural misorganization of the larval Purkinje cell layer could be rescued by pharmacological treatment with Dyrk1A inhibitors. We further reveal the in vivo efficiency of a novel selective Dyrk1A inhibitor, KuFal194. These findings demonstrate that the zebrafish is a well-suited vertebrate organism to genetically model severe neurological diseases with single cell type specificity. Such models can be used to relate molecular malfunction to cellular deficits, impaired tissue formation, and organismal behavior and can also be used for pharmacological compound testing and validation.     

PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis

The p14^ARF protein is a well‐known regulator of p53‐dependent and p53‐independent tumor‐suppressive activities. In unstressed cells, p14^ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14^ARF undergoes an immediate redistribution to the nucleo‐ and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14^ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C‐terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14^ARF. In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14^ARF. Genotoxic stress causes augmented interaction between PRMT1 and p14^ARF, accompanied by arginine methylation of p14^ARF. PRMT1‐dependent NLS/NoLS methylation promotes the release of p14^ARF from NPM and nucleolar sequestration, subsequently leading to p53‐independent apoptosis. This PRMT1‐p14^ARF cooperation is cancer‐relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1‐mediated arginine methylation is an important trigger for p14^ARF’s stress‐induced tumor‐suppressive function.     

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.Subject terms: Macroautophagy, Macroautophagy