Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca²⁺dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.     

A systems-level approach for metabolic engineering of yeast cell factories

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.     

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.     

Approximate maximum likelihood estimation for stochastic chemical kinetics

Recent experimental imaging techniques are able to tag and count molecular populations in a living cell. From these data mathematical models are inferred and calibrated. If small populations are present, discrete-state stochastic models are widely-used to describe the discreteness and randomness of molecular interactions. Based on time-series data of the molecular populations, the corresponding stochastic reaction rate constants can be estimated. This procedure is computationally very challenging, since the underlying stochastic process has to be solved for different parameters in order to obtain optimal estimates. Here, we focus on the maximum likelihood method and estimate rate constants, initial populations and parameters representing measurement errors.     

High initiation and long duration of breastfeeding despite absence of early skin-to-skin contact in Karen refugees on the Thai-Myanmar border: a mixed methods study

Background

Early skin-to-skin contact (SSC) after birth is recommended as part of the United Nations Children’s Fund (UNICEF) baby friendly health initiative to promote optimum breastfeeding. This paper reports rates of breastfeeding initiation and duration in a low resource environment, where early SSC is not practised, and explores views of pregnant women and midwives surrounding breastfeeding and swaddling.

Methods

Data from records from a single hospital on the Thai-Myanmar border where refugee women gave birth during a one-year period (2010) were used to determine breastfeeding initiation rates and the time of the first breastfeed, and duration of breastfeeding of the previous alive child in multigravidae. Focus group discussions (FGD) were conducted to obtain information from pregnant women attending antenatal care about their intended or previous duration of breastfeeding and views on breastfeeding. Interviews with local midwives explored reasons for high rates of breastfeeding in this setting and the practice of newborn swaddling.

Results

Of 1404 live births in 2010 in Maela refugee camp there were 982 evaluable mother-newborn pairs, including 80 infants born before 37 weeks gestation. Initiation of breastfeeding within the first hour after birth and exclusive breastfeeding at discharge in term mother-newborn pairs was 91.2% (823/902) and 99.3% (896/902); and before 37 weeks gestation, 48.8% (39/80) and 98.8% (79/80). Reported duration of previous breastfeeding was 19 (range 2 to 72) months.During FGD all primigravidae (n?=?17) intended to breastfeed and all multigravidae (n?=?33) had previously breastfed; expected or previous duration of feeding was for more than one year or longer. The major theme identified during FGD was breastfeeding is “good”. Women stated their intention to breastfeed with certainty. This certainty was echoed during the interviews with midwifery staff. SSC requires a delay in early swaddling that in Karen people, with animistic beliefs, could risk loss of the spirit of the newborn or attract malevolent spirits.

Conclusions

In a population with a strong culture of breastfeeding and robust breastfeeding practices, high rates of initiation and duration of breastfeeding were found despite a lack of early skin-to-skin contact. Local preferences, traditions and practices that protect, support and maintain high rates of breastfeeding should be promoted.

     

Vaccination against Feline Panleukopenia: implications from a field study in kittens

ABSTRACT: BACKGROUND: Feline Panleukopenia (FPL) is a serious disease of cats that can be prevented by vaccination. Kittens are routinely vaccinated repeatedly during their first months of life. By this time maternally derived antibodies (MDA) can interfere with successful vaccination and inhibit the development of active immunity. The efficacy of primary vaccination under field conditions was questioned by frequent reports to the Paul-Ehrlich-Institut on outbreaks of FPL in vaccinated breeding catteries. We therefore initiated a field study to investigate the development of immunity in kittens during primary vaccination against FPL. 64 kittens from 16 litters were vaccinated against FPL at the age of 8, 12 and 16 weeks using three commercial polyvalent vaccines. Blood samples were taken before each vaccination and at the age of 20 weeks. Sera were tested for antibodies against feline panleukopenia virus (FPV) by hemagglutination inhibition test and serum neutralisation assay in two independent diagnostic laboratories. RESULTS: There was a good correlation between the results obtained in different laboratories and with different methods. Despite triple vaccination 36.7% of the kittens did not seroconvert. Even very low titres of maternally derived antibodies (MDA) apparently inhibited the development of active immunity. The majority of kittens displayed significant titres of MDA at 8 and 12 weeks of age; in some animals MDA titres that interfered with vaccination were still detected at 20 weeks of age. Interestingly, the vaccines tested differed significantly in their ability to overcome low levels of maternal immunity. CONCLUSIONS: In the given situation it is recommended to quantify antibodies against FPV in the serum of the queen or of the kittens before primary vaccination of kittens. The beginning of primary vaccination should be delayed until MDA titres have declined. Unprotected kittens that have been identified serologically should be revaccinated.     

Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14⁺⁺CD16⁺ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14⁺⁺CD16⁺ monocyte subset.     

Current Status of Artemisinin-Resistant falciparum Malaria in South Asia: A Randomized Controlled Artesunate Monotherapy Trial in Bangladesh

Objective

Recent reports indicate that first cases of genuine artemisinin resistance have already emerged along the Thai-Cambodian border. The main objective of this trial was to track the potential emergence of artemisinin resistance in Bangladesh, which in terms of drug resistance forms a gateway to the Indian subcontinent.

Methods

We conducted an open-label, randomized, controlled 42-day clinical trial in Southeastern Bangladesh to investigate the potential spread of clinical artemisinin resistance from Southeast Asia. A total of 126 uncomplicated falciparum malaria patients were randomized to one of 3 treatment arms (artesunate monotherapy with 2 or 4 mg/kg/day once daily or quinine plus doxycycline TID for 7 days). Only cases fulfilling a stringent set of criteria were considered as being artemisinin-resistant.

Findings

The 28-day and 42-day cure rates in the artesunate monotherapy (2 and 4 mg/kg) and quinine/doxycyline arms were 97.8% (95% confidence interval, CI: 87.8–99.8%), 100% (95% CI: 91.1–100%), and 100% (95% CI: 83.4–100%), respectively. One case of re-infection was seen in the artesunate high dose arm, and a single case of recrudescence was observed in the low dose group on day 26. No differences in median parasite and fever clearance times were found between the 2 artesunate arms (29.8 h and 17.9 h vs. 29.5 h and 19.1 h). Not a single case fulfilled our criteria of artemisinin resistance. Parasite clearance times were considerably shorter and ex vivo results indicate significantly higher susceptibility (50% inhibitory concentration for dihydroartemisinin was 1.10 nM; 95% CI: 0.95–1.28 nM) to artemisinins as compared to SE-Asia.

Conclusion

There is currently no indication that artemisinin resistance has reached Bangladesh. However, the fact that resistance has recently been reported from nearby Myanmar indicates an urgent need for close monitoring of artemisinin resistance in the region.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00639873","term_id":"NCT00639873"}}NCT00639873.     

Long-term immunity against actual poxviral HLA ligands as identified by differential stable isotope labeling

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.     

Activation of human alveolar macrophages via P2 receptors: coupling to intracellular Ca2+ increases and cytokine secretion

Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X(1), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(13), and P2Y(14). We also showed that extracellular ATP induced Ca(2+) transients and increased IL-1beta secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-alpha, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.