Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease

Background aimsAllogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear.MethodsIn this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients.ResultsIn co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism.ConclusionsIn summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.     

In silico epitope-based vaccine design against influenza a neuraminidase protein: Computational analysis established on B- and T-cell epitope predictions

ObjectiveInfluenza A virus belongs to the most studied virus and its mutant initiates epidemic and pandemics outbreaks. Inoculation is the significant foundation to diminish the risk of infection. To prevent an incidence of influenza from the transmission, various practical approaches require more advancement and progress. More efforts and research must take in front to enhance vaccine efficacy.MethodsThe present research emphasizes the development and expansion of a universal vaccine for the influenza virus. Research focuses on vaccine design with high efficacy. In this study, numerous computational approaches were used, covering a wide range of elements and ideas in bioinformatics methodology. Various B and T-cell epitopic peptides derived from the Neuraminidase protein N1 are recognized by these approaches. With the implementation of numerous obtained databases and bioinformatics tools, the different immune framework methods of the conserved sequences of N1 neuraminidase were analyzed. NCBI databases were employed to retrieve amino acid sequences. The antigenic nature of the neuraminidase sequence was achieved by the VaxiJen server and Kolaskar and Tongaonkar method. After screening of various B and T cell epitopes, one efficient peptide each from B cell epitope and T cell epitopes was assessed for their antigenic determinant vaccine efficacy. Identical two B cell epitopes were recognized from the N1 protein when analyzed using B-cell epitope prediction servers. The detailed examination of amino acid sequences for interpretation of B and T cell epitopes was achieved with the help of the ABCPred and Immune Epitope Database.ResultsComputational immunology via immunoinformatic study exhibited RPNDKTG as having its high conservancy efficiency and demonstrated as a good antigenic, accessible surface hydrophilic B-cell epitope. Among T cell epitope analysis, YVNISNTNF was selected for being a conserved epitope. T cell epitope was also analyzed for its allergenicity and cytotoxicity evaluation. YVNISNTNF epitope was found to be a non-allergen and not toxic for cells as well. This T-cell epitope with maximum world populace coverages was scrutinized for its association with the HLA-DRB1*0401 molecule. Results from docking simulation analyses showed YVNISNTNF having lower binding energy, the radius of gyration (Rg), RMSD values, and RMSE values which make the protein structure more stable and increase its ability to become an epitopic peptide for influenza virus vaccination.ConclusionsWe propose that this epitope analysis may be successfully used as a measurement tool for the robustness of an antigen–antibody reaction between mutant strains in the annual design of the influenza vaccine.     

Respiratory chain composition and activity in some thermotolerant streptomycetes

The cytochrome composition of membrane preparations from 3 thermotolerant species of Streptomyces was determined from difference spectra. All contained b, c and a-type haemoproteins and in addition, 2 species (Streptomyces thermoviolaceus and Streptomyces thermoflavus) possessed cytochrome oxidase d. A CO-reacting b cytochrome similar to cytochrome oxidase o could also be detected in all 3 organisms. Thermotolerance of the respiratory chains showed no correlation with cytochrome composition. In general, Streptomyces thermonitrificans had the most thermostable activities, whilst those of S. thermoviolaceus were the most thermolabile. Growth temperature exerted no qualitative effects on respiratory chain composition but did affect the thermostability of some substrate-supported oxidase activities.     

The Association of Phytoplasma with Stunting, Leaf Necrosis and Witches' Broom Symptoms in Magnolia Plants

In 1999–2000 a severe disease was observed on plants of four Magnolia spp. cultivated in a commercial nursery in Poland. Affected plants showed a progressive loss of vigour, were stunted, and had severely malformed leaves, leaf necrosis and witches' broom. Phytoplasma was detected in magnolias with severe symptoms and in dodder-inoculated Catharanthus roseus seedlings by nested polymerase chain reaction (PCR) assay with primer pair R16F1/R0 followed by universal (rA/fA) and group specific (R16(I)F1/R1) primer pairs which amplified a fragment of phytoplasma 16S rDNA. The PCR products (560 bp or 1.1 kb) of all samples used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes Alu I and Mse I produced the same profile which corresponded to that of an aster yellows phytoplasma reference strain. Phytoplasma DNA was detected throughout the growing season in roots, stems and young but not mature leaves. Electron microscope examination of the ultra-thin sections of the leaf and stem of diseased magnolias showed collapsed and degenerated sieve tube elements with wall thickening. The reduced lumen of these sieve elements contained numerous vesicles and membrane-bound structures, but no typical phytoplasma cells. This is the first report of aster yellows phytoplasma in magnolia identified by molecular assays.     

陆地棉品种抗黄萎病反应规律的研究

对我国自育的108个陆地棉品种的抗黄萎病性进行了研究。在黄萎病发病期内,对黄萎病发病情况进行连续调查,测定产量、考查产量因素并检测纤维品质。利用因子分析法对陆地棉抗黄萎病反应规律进行分析,得出不同时期的黄萎病病指主要与前后3～5个阶段抗病性有关。病情发展主要由4个主因子决定,且第1、2主因子具有较大的方差贡献率。第1主因子(F1)主要与品种7月26日至8月9日的黄萎病病指有关,第2主因子(F2)主要与品种8月20日至9月4日的黄萎病病指有关。利用因子分析结果将108个品种划分为4个类型,前期抗病性较好而后期发病较快的第Ⅰ类品种,其产量较低,单株结铃数、单铃重、衣分均低于其他3类;纤维品质均较差,纤维长度、整齐度、比强度和马克隆值均较其他3类差。前期和后期病指均较低、发病缓慢的第Ⅱ类则小区产量最高,纤维品质处于平均水平。第Ⅲ类品种前期发病较慢,中期发病较快,具有较高的小区产量,单铃重最高;纤维整齐度、比强度和伸长率好于其他3类品种;前期发病较快,中期发病平缓,后期仍具有较高病指的为第Ⅳ类品种,小区产量较低,单株产量、单株结铃数和衣分较高;其他性状处于中等水平。但研究表明,某一阶段具有的抗病性并不能完全代表品种的抗病性。     

Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n?=?136) was collected from malaria patients in Thailand (n?=?50, 2002–2005), Vietnam (n?=?39, 2004), Laos (n?=?15, 2007) and Cambodia (n?=?32, 2009). Amino acid sequences at codons 72–76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.     

Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-Å crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.     

Mitogenomic phylogeny of the Percichthyidae and Centrarchiformes (Percomorphaceae): comparison with recent nuclear gene-based studies and simultaneous analysis

Delineation of the fish family Percichthyidae (Percomorphaceae) has a long and convoluted history, with recent morphological-based studies restricting species members to South American and Australian freshwater and catadromous temperate perches. Four recent nuclear gene-based phylogenetic studies, however, found that the Percichthyidae was not monophyletic and was nested within a newly discovered inter-familial clade of Percomorphaceae, the Centrarchiformes, which comprises the Centrarchidae and 12 other families. Here, we reexamined the systematics of the Percichthyidae and Centrarchiformes based on new mitogenomic information. Our mitogenomic results are globally congruent with the recent nuclear gene-based studies although the overall amount of phylogenetic signal of the mitogenome is lower. They do not support the monophyly of the Percichthyidae, because the catadromous genus Percalates is not exclusively related to the freshwater percichthyids. The Percichthyidae (minus Percalates) and Percalates belong to a larger clade, equivalent to the Centrarchiformes, but their respective sister groups are unresolved. Because all recent analyses recover a monophyletic Centrarchiformes but with substantially different intra-relationships, we performed a simultaneous analysis for a character set combining the mitogenome and 19 nuclear genes previously published, for 22 centrarchiform taxa. This analysis furthermore indicates that the Centrarchiformes are divided into three lineages and the superfamily Cirrhitoidea is monophyletic as well as the temperate and freshwater centrarchiform perch-like fishes. It also clarifies some of the relationships within the freshwater Percichthyidae.