Novel scabies mite serpins inhibit the three pathways of the human complement system

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.     

Morphology, biophysical properties and protein-mediated fusion of archaeosomes

As variance from standard phospholipids of eubacteria and eukaryotes, archaebacterial diether phospholipids contain branched alcohol chains (phytanol) linked to glycerol exclusively with ether bonds. Giant vesicles (GVs) constituted of different species of archaebacterial diether phospholipids and glycolipids (archaeosomes) were prepared by electroformation and observed under a phase contrast and/or fluorescence microscope. Archaebacterial lipids and different mixtures of archaebacterial and standard lipids formed GVs which were analysed for size, yield and ability to adhere to each other due to the mediating effects of certain plasma proteins. GVs constituted of different proportions of archaeal or standard phosphatidylcholine were compared. In nonarchaebacterial GVs (in form of multilamellar lipid vesicles, MLVs) the main transition was detected at T_m = 34. 2°C with an enthalpy of ΔH = 0.68 kcal/mol, whereas in archaebacterial GVs (MLVs) we did not observe the main phase transition in the range between 10 and 70°C. GVs constituted of archaebacterial lipids were subject to attractive interaction mediated by beta 2 glycoprotein I and by heparin. The adhesion constant of beta 2 glycoprotein I – mediated adhesion determined from adhesion angle between adhered GVs was in the range of 10⁻⁸ J/m². In the course of protein mediated adhesion, lateral segregation of the membrane components and presence of thin tubular membranous structures were observed. The ability of archaebacterial diether lipids to combine with standard lipids in bilayers and their compatibility with adhesion-mediating molecules offer further evidence that archaebacterial lipids are appropriate for the design of drug carriers.     

Reproductive factors and serum uric acid levels in females from the general population: the KORA F4 study

Objective

Hyperuricemia is associated with an increased risk of metabolic and cardiovascular diseases. There are pronounced sex differences in the levels of uric acid. It is largely unknown whether or not reproductive parameters which induce hormonal changes are responsible for this. We examined if there are associations between reproductive parameters and uric acid levels in a female population-based sample.

Methods

In this cross-sectional analysis, data of 1530 women aged 32 to 81 years participating in the KORA F4 study, conducted between 2006 and 2008 in Southern Germany were used. Reproductive parameters were obtained by standardized interviews. Uric acid levels were tested by the uricase method. The whole study sample and stratified in pre- and postmenopausal women was analyzed.

Results

Menopausal status and earlier age at menarche were associated with higher serum uric acid levels (age-adjusted: p-values 0.003, <0.001 respectively; after multivariable adjustment, including BMI: p-values 0.002, 0.036). A history of oral contraceptive use showed an association with uric acid levels only after multivariable adjustment (p-value 0.009). Hot flushes showed an association with uric acid levels only after age-adjustment (p-value 0.038), but lost significance after adding other confounders. Other reproductive factors, including parity, current or ever use of hormone replacement therapy, current use of oral contraceptives, hysterectomy, bilateral oophorectomy, or depressive mood related to menopausal transition were not associated with uric acid levels.

Conclusions

Postmenopausal status, earlier age at menarche and a history of oral contraceptive use were independently associated with higher serum uric acid concentrations in women from the general population. Further studies, especially longitudinal population-based studies investigating the relationship of female reproductive parameters with uric acid levels are necessary to confirm our findings.     

Breast cancer risk and 6q22.33: combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2

Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR)?=?1.03, 95% CI 1.00-1.06, p?=?0.023). There was evidence for heterogeneity in the ORs among studies (I(2)?=?49.3%; p?=?<0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR?=?0.89, 95%CI 0.80-1.00, p?=?0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.     

Variable neuronal participation in stereotypic motor programs

To what extent are motor networks underlying rhythmic behaviors rigidly hard-wired versus fluid and dynamic entities? Do the members of motor networks change from moment-to-moment or from motor program episode-to-episode? These are questions that can only be addressed in systems where it is possible to monitor the spiking activity of networks of neurons during the production of motor programs. We used large-scale voltage-sensitive dye (VSD) imaging followed by Independent Component Analysis spike-sorting to examine the extent to which the neuronal network underlying the escape swim behavior of Tritonia diomedea is hard-wired versus fluid from a moment-to-moment perspective. We found that while most neurons were dedicated to the swim network, a small but significant proportion of neurons participated in a surprisingly variable manner. These neurons joined the swim motor program late, left early, burst only on some cycles or skipped cycles of the motor program. We confirmed that this variable neuronal participation was not due to effects of the VSD by finding such neurons with intracellular recording in dye-free saline. Further, these neurons markedly varied their level of participation in the network from swim episode-to-episode. The generality of such unreliably bursting neurons was confirmed by their presence in the rhythmic escape networks of two other molluscan species, Tritonia festiva and Aplysia californica. Our observations support a view that neuronal networks, even those underlying rhythmic and stereotyped motor programs, may be more variable in structure than widely appreciated.     

Polyubiquitin Is Required for Growth, Development and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae

Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.     

The h-Current in the Substantia Nigra pars Compacta Neurons: A Re-examination

The properties of the hyperpolarization-activated cation current (I_h) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the I_h kinetics showed that, at 37°C and minimizing the disturbance of the intracellular milieu with perforated patch, this current actually activates at potentials more positive than what is generally indicated, with a half-activation potential of −77.05 mV and with a significant level of opening already at rest, thereby substantially contributing to the control of membrane potential, and ultimately playing a relevant function in the regulation of the cell excitability. The implications of the known influence of intracellular cAMP levels on I_h amplitude and kinetics were examined. The direct application of neurotransmitters (DA, 5-HT and noradrenaline) physiologically released onto SNc neurons and known to act on metabotropic receptors coupled to the cAMP pathway modify the I_h amplitude. Here, we show that direct activation of dopaminergic and of 5-HT receptors results in I_h inhibition of SNc DA cells, whereas noradrenaline has the opposite effect. Together, these data suggest that the modulation of I_h by endogenously released neurotransmitters acting on metabotropic receptors –mainly but not exclusively linked to the cAMP pathway- could contribute significantly to the control of SNc neuron excitability.     

Model-guided fieldwork: practical guidelines for multidisciplinary research on wildlife ecological and epidemiological dynamics

Infectious disease ecology has recently raised its public profile beyond the scientific community due to the major threats that wildlife infections pose to biological conservation, animal welfare, human health and food security. As we start unravelling the full extent of emerging infectious diseases, there is an urgent need to facilitate multidisciplinary research in this area. Even though research in ecology has always had a strong theoretical component, cultural and technical hurdles often hamper direct collaboration between theoreticians and empiricists. Building upon our collective experience of multidisciplinary research and teaching in this area, we propose practical guidelines to help with effective integration among mathematical modelling, fieldwork and laboratory work. Modelling tools can be used at all steps of a field-based research programme, from the formulation of working hypotheses to field study design and data analysis. We illustrate our model-guided fieldwork framework with two case studies we have been conducting on wildlife infectious diseases: plague transmission in prairie dogs and lyssavirus dynamics in American and African bats. These demonstrate that mechanistic models, if properly integrated in research programmes, can provide a framework for holistic approaches to complex biological systems.     

Development of quantitative metabolomics for <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.