Adhesive Protein Gene Research Articles

BIOINFORMATIC ANALYSIS OF GENES OF ADHESIVE PROTEIN FOOT OF MUSSELS OF THE GENUS MYTILUS

Introduction. Today, it is relevant to identify and study the genes of adhesive proteins that contribute to the adhesion of marine mussels under water and play an important role in the industry for the commercialization of waterproof adhesives. The purpose of our research is based on bioinformatic analysis, because modeling is currently one of the advanced methods. Aim. The purpose of this work was to compare the nucleotide sequences of representatives of the genus Mytilus, which are flanked by primers Me 15 / Me 16, designed for the non-repetitive region of the mussel adhesive protein gene, as well as bioinformatic analysis of the complete amino acid sequence of the foot adhesive protein Mefp-1 coded by Fp1 gene for M. galloprovincialis. Methods. Nucleotide sequences were analyzed using BLAST (NCBI [https://www.ncbi.nlm.nih.gov]) and aligned by MAFFT [Madeira et al., 2019] methods. The phylogenetic tree was built in the MEGA program [Kumar et al., 2018] using the UPGMA method [Sneath, Sokal, 1973]. The physicochemical parameters of the adhesive protein for the amino acid sequence of M. galloprovincialis were calculated using the ProtParam software tool (ExPASy [https://web.expasy.org/protparam/]). Models of the three-dimensional structure of the M. galloprovincialis adhesive protein were built on the I-TASSER online platform [Yang, Zhang, 2015; Zhang et al., 2017] and with application of the AlphaFold program [https://alphafold.ebi.ac.uk/entry/Q27409]. The main results. The analysis of the nucleotide sequences of six representatives of the genus Mytilus within the «variable region» flanked by primers Me 15 / Me 16 showed the presence of deletions and a number of SNPs. The constructed dendrogram reflected the phylogenetic relationships of mussel species in the genus Mytilus, when comparing the nucleotide sequences of the genes of the adhesive protein of the mussel foot. An analysis of the primary and secondary structure of the adhesive protein of the mussel species M. galloprovincialis characteristic of the Black Sea region was carried out, and models of the three-dimensional structure of the adhesive protein for the specified species were built. Conclusions. The detected mutations in the studied «variable region» of the nucleotide sequences of the adhesive protein genes allow the molecular marker Me 15-16 to distinguish between four types of mussels: M. galloprovincialis, M. chilensis, M. edulis, M. trossulus. The obtained distribution of representatives of the genus Mytilus by means of cluster analysis is consistent with the literature data on the territorial distribution of these species. The molecular weight was calculated and models of the spatial structure of the gene of the foot adhesive protein of the mussel species M. galloprovincialis were constructed.

Comparison of two commercial methods for smooth-shelled mussels (Mytilus spp.) species identification

Seafood international trade has increased the labeling requirements in standards and regulations to include product information that enable traders and consumers to make informed choices. The European Union (EU) Regulation No. 1379/2013 imposes the declaration of an official commercial designation and scientific names for all the fishery and aquaculture products to be offered for sale to the final consumers. DNA analyses are used to enforce this regulation and to test authenticity in processed foods. We compared the performance of two mono-locus approaches for species identification (SI) in 61 Mytilus mussels: the high-resolution melting analysis of the polyphenolic adhesive protein gene and the partial sequencing of the histone H1C gene. The H1C sequences were analyzed with five different methods. Both approaches show discrepancies in the identification of putative hybrids (0.0 < κ < 0.687 and 0.0 < MCC < 0.724). Excluding putative hybrids, methods show substantial to perfect agreement (0.772 < κ < 1.0 and 0.783 < MCC < 1.0). This study highlights the need to use standardized molecular tools, as well as to use multi-locus methods for SI of Mytilus mussels in testing laboratories.

Loss of Jagged1 in mature endothelial cells causes vascular dysfunction with alterations in smooth muscle phenotypes

BackgroundNotch signaling is an evolutionarily conserved pathway that functions via direct cell-cell contact. The Notch ligand Jagged1 (Jag1) has been extensively studied in vascular development, particularly for its role in smooth muscle cell maturation. Endothelial cell-expressed Jag1 is essential for blood vessel formation by signaling to nascent vascular smooth muscle cells and promoting their differentiation. Given the established importance of Jag1 in endothelial cell/smooth muscle crosstalk during development, we sought to determine the extent of this communication in the adult vasculature for blood vessel function and homeostasis. MethodsWe conditionally deleted Jag1 in endothelial cells of adult mice and examined the phenotypic consequences on smooth muscle cells of the vasculature. ResultsOur results show that genetic loss of Jag1 in endothelial cells has a significant impact on Notch signaling and vascular smooth muscle function in mature blood vessels. Endothelial cell-specific deletion of Jag1 causes a concomitant loss of JAG1 and NOTCH3 expression in vascular smooth muscle cells, resulting in a transition to a less differentiated state. Aortic vascular smooth muscle cells isolated from the endothelial cell-specific Jag1 deficient mice retain an altered phenotype in culture with fixed changes in gene expression and reduced Notch signaling. Utilizing comparative RNA-sequence analysis, we found that Jag1 deficiency preferentially affects extracellular matrix and adhesion protein gene expression. Vasoreactivity studies revealed a reduced contractile response and impaired agonist-induced relaxation in endothelial cell Jag1-deficient aortas compared to controls. ConclusionsThese data are the first to demonstrate that Jag1 in adult endothelial cells is required for the regulation and homeostasis of smooth muscle cell function in arterial vessels partially through the autoregulation of Notch signaling and cell matrix/adhesion components in smooth muscle cells.

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein gene of Babesia gibsoni isolates in dogs in South India.

Babesia gibsoni, the causative agent of canine piroplasmosis, is a tick-borne intraerythrocytic protozoan parasite predominantly reported in Asian countries. The present study aimed at genotypic characterization of B. gibsoni isolates prevalent in dogs in Kerala, a southern state of India. Blood samples were collected from 272 dogs in Kerala and B. gibsoni infection was detected by microscopy and polymerase chain reaction (PCR). Molecular confirmation of B. gibsoni parasites was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR detected a higher percentage of dogs (40.44%) positive for B. gibsoni infection than microscopy where 15.81% dogs were detected positive for infection. Genetic characterization of B. gibsoni isolates (n=11) prevalent in dogs in the state of Kerala was carried out by PCR amplification and sequencing of the 855bp thrombospondin-related adhesive protein (TRAP) gene fragment. Phylogenetic analysis of the B. gibsoni TRAP (BgTRAP) gene revealed that B. gibsoni isolates from Kerala formed a distinct cluster with the isolates from north India and Bangladesh, away from other East Asian isolates. Nucleotide analysis of the tandem repeats of BgTRAP gene showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh but not by the isolates of East Asian countries. The results of the present study further confirmed that B. gibsoni parasites in a distinct genetic clade are endemic in dogs in India and Bangladesh. However, elaborate studies are required for better understanding of the genetic diversity of B. gibsoni.

Glomerular endothelial cell-podocyte stresses and crosstalk in structurally normal kidney transplants

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

SYBR-Green real-time PCR assay with melting curve analysis for the rapid identification of Mytilus species in food samples

Mytilus spp. mussels are one of the most commonly cultivated species in the European Union, being M. galloprovincialis, M. edulis and M. chilensis the main species marketed here. Therefore, the identification of this seafood species is critical for labelling, traceability and food safety purposes. In the present study, 174 samples have been employed for the development of an alternative fast, simple, reliable and cost-effective methodology for the identification of Mytilus spp. mussels commercialized in Europe. This real-time PCR assay with a SYBR Green post-PCR melting curve analysis targeting the polyphenolic adhesive protein gene developed was successfully applied to fresh, frozen and canned mussels, showing 100% of specificity, sensitivity and precision. Besides, results have demonstrated the applicability of the assay in different platforms without special software requirements. Therefore, the methodology described in this study is useful for the authentication of mussel samples and could be easily applied as a fast routine food control laboratory test.

PCR detection and genetic characterization of piroplasms from dogs in Myanmar, and a possible role of dogs as reservoirs for Theileria parasites infecting cattle, water buffaloes, and goats

Canine vector-borne pathogens can act as zoonotic agents in humans; however, it poorly understood whether dogs play a role as reservoirs of vector-borne parasites in livestock animals. Here, we report the unexpected detection of 18S rRNA gene (rDNA) sequences of five ruminant Theileria species from the peripheral blood of dogs in Myanmar, in addition to those of two canine Babesia species. Using novel BTH primers capable of amplifying the 18S rDNA of Babesia, Theileria, and Hepatozoon spp., approximately 1,500 bp nested PCR products were detected in 19% (17/91) of local or imported dog breeds in different regions of Myanmar. Among the sequences of the 17 PCR products, ten were determined as Theileria 18S rDNA, including three as Theileria orientalis, three as Theileria buffeli, two as Theileria cf. velifera, one as Theileria luwenshuni, and one as Theileria sp. Most of these sequences showed higher identities with Theileria sequences determined in previous studies of cattle, water buffaloes, and goats in Myanmar. Six PCR products were identified as Babesia vogeli and one sample was determined as Babesia gibsoni. Furthermore, we obtained approximately 900 bp thrombospondin-related adhesive protein (TRAP) gene fragments from three dog blood DNA samples. Phylogenetic analysis of the TRAP gene showed that B. gibsoni parasites in Myanmar were considerably related to isolates from China, Korea, Japan, and Taiwan, but clearly separated from those from Bangladesh and India. These results provide new insights into a possible role of dogs in maintaining and spreading tick-borne pathogens among livestock and canine populations in Myanmar.

The Effects of Negative Pressure on the Gene Expression of Motility Related Proteins in Boar Spermatozoa during Liquid Storage at 17oC

Background: The present study was aimed to investigate the effects of negative pressure applied before storage on the gene expression of motility related proteins in boar spermatozoa.Methods: Boar semen samples were collected and pooled and diluted with Modena solution containing 0.4% (w/v) of bovine serum albumin. Negative pressure was applied for 2-5 min using a vacuum pump with a barometer. The pressure applied was 0 (Control), -0.02 MPa (P2), -0.04 MPa (P4) and -0.08 MPa (P8). The expression of AQN-1, AQN-3, AWN, PSP-I, PSP-II gene in boar spermatozoa was evaluated.Result: Application of -0.04 MPa improved the sperm motility compared with the other groups. In conclusion, our results confirmed thatnegative pressure preservation at 17oC had an effect on the expression of boar sperm adhesion protein gene. The relative expression of AWN0PSP-I and PSP-II genes in boar sperm were lower under -0.04 MPa and -0.08 MPa pressure, which was beneficial to protect spermatozoa motility.

Genetic diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum on Bioko Island, Equatorial Guinea and global comparative analysis

BackgroundThrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described.Methods153 blood spot samples from Bioko malaria patients were collected during 2016–2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools.ResultsA total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN–dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure.ConclusionsEvidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Prevalence Determination of m. Hominis and m. Genitalium in the Semen Samples in the Northeast of Iran Using Culture and Multiplex Polymerase Chain Reaction.

Infertility has recently become a growing social and economic world problem. Genital mycoplasmas, such as Mycoplasma hominis and M. genitalium, are most frequently associated with several adverse effects on men&amp;rsquo;s fertility. The present study aimed to determine the prevalence of M. hominis and M. genitalium in the semen samples in thenortheast of Iran. During thiscross-sectional study from February to May, 2018, 100 semen samples were collected from 100 infertile men in Mashhad, Khorasan Razavi province, northeast of Iran. The presence of M. hominis and M. genitalium was detected by cultivation, polymerase chain reaction (PCR), and Multiplex PCR assays. The colony of mycoplasma was confirmed by Diene&amp;rsquo;s stain; moreover, arginine hydrolysis, glucose, and urea utilization were evaluated. The following semen indices were analyzed according to World Health Organization guidelines for semen analysis: color, volume, appearance, liquefaction, viscosity, concentration, pH, leukocyte concentration, progressive motility, morphological normality, motile sperm concentration, functional sperm concentration, sperm motility index, and functional sperm. The gene of 16SrRNA (GPO1&amp;amp; MGSO primers) was used as the target gene of the Mycoplasma genus in PCR assay. Multiplex-PCR was performed with a specific primer for conserved regions in the 16SrRNA gene for M. hominis (RNAH1&amp;amp; RNAH2 primers) and the 140-kDa Adhesion Protein Gene for M. genitalium (MG1 &amp;amp; MG2 primers).According to the results,9 (9%) samples were PCR-positive for Mycoplasma spp , while there were 7 (7%) cases isolated by cultivation. M. hominis was detected in 8 (8%) samples by Multiplex PCR, while there was no evidence for M. genitalium. The mean age scores of all infertile and infected men were obtained at 31 and 30 years, respectively. The study could not show any statistical correlation between mycoplasma infection and abnormal semen parameters. The heterogeneity of mycoplasma prevalence in the reports can be ascribed to differences in geographic areas, the sensitivity of the identification method, condition of the group (fertile/infertile), sample size, and operator proficiency. Various results have been reported in numerous studies conducted on the relationship between mycoplasma infection and abnormal semen parameters.

Natural distribution of pure and hybrid Mytilus sp. along the south Mediterranean and North-east Atlantic coasts and sensitivity of D-larvae stages to temperature increases and metal pollution

The distribution of the Mediterranean mussel Mytilus galloprovincialis extends more and more northwards in the Atlantic. Crossings are frequently observed with the blue mussel Mytilus edulis along the French and English coasts. The aim of this study is firstlyto identify the co-presence of M. galloprovincialis, M. edulis, and their hybrids in different sites of the Mediterranean and Atlantic coasts, and to provide insights for the thermal tolerance and toxicant susceptibility of Mytilus edulis, Mytilus galloprovincialis and their hybrids. Mussels were collected from the shore at 20 sampling sitesin Europe and Tunisia and identified using Me 15/16 primers targeting the adhesive protein gene sequence. Samples were screened for the presence of Mytilus edulis, Mytilus galloprovincialis, and hybrids alleles using PCR. To get more information on hybrids sensitivities to temperature and metals, freshly fertilized eggs of the two species and their hybrids were reared at four temperatures 18, 20, 22, and 24 °C and exposed to concentrations of Cu, Ag, and a mixture of both metals. Arrests of development and malformations were recorded after 48 h of exposure. The genotypic identification of the two species on 20 sites of the Mediterranean and Atlantic coasts carried out during this study confirms the presence of pure and hybrid species of mussel. Our results highlighted that hybrid larvae from a female of M. galloprovincialis are significantly more tolerant to temperature increases than pure larvae of M. galloprovincialis and pure and hybrid larvae of M. edulis. No significant interspecies-differences of sensitivity were noted for metal exposure alone. However, a co-exposure of larvae to both metal and high temperature highlighted the higher tolerance of hybrid larvae from a female of M. galloprovincialis to both stresses. The overall results could allow the prediction of the future evolution of mussel populations facing environmental changes.

Measuring of IgG2c isotype instead of IgG2a in immunized C57BL/6 mice with Plasmodium vivax TRAP as a subunit vaccine candidate in order to correct interpretation of Th1 versus Th2 immune response

Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.

Mussels (Mytilus spp.) products authentication: A case study on the Italian market confirms issues in species identification and arises concern on commercial names attribution

In November 2019, the FishLab was consulted by a wholesaler for solving an authentication issue concerning a batch of products labelled as “Chilean mussels” (Mytilus chilensis). Samples of the batch had been molecularly identified first as M. chilensis by an external private lab and, subsequently, as Choromytilus chorus through a second analysis entrusted to another external lab by the customer company. The analysis carried out at the FishLab only allowed to identify the samples to genus level (Mytilus spp.) by sequencing the mitochondrial COI gene. The amplification of the Polyphenolic Adhesive Protein (PAP) gene, a nuclear marker reported as informative for mussel, allowed to suppose the presence of M. chilensis and M. galloprovincialis based on the length of the obtained fragment. In parallel, an in silico analysis of the PAP sequences currently available on GenBank highlighted the lack of the typical mutation point in some sequences of M. galloprovincialis that were identical to those of M. chilensis. Based on a case study investigating mussel products suspected to be mislabelled, this work wants to confirm issues in molecular species identification of Mytilus spp. and highlight concerns on commercial names attribution. Results first confirmed previous scientific community opinion discouraging the use of mtDNA for Mytilus spp. identification due to both its unusual inheritance mechanism and introgression phenomena. The outcomes of the in silico analysis of the PAP sequences called into question the reliability of this marker in discriminating among M. chilensis and M. galloprovincialis. Therefore, also considering the uncertainties of the taxonomic status of these species, a proper re-evaluation process of this marker is needed. However, also the approach one species-one name, currently adopted by the Italian legislator, should be revised for solving labelling issues affecting the market.

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia

AbstractBone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in thrombopoiesis can lead to thrombocytopenia associated with increased bleeding tendency. Recently, the platelet disorder congenital autosomal-recessive small-platelet thrombocytopenia (CARST) was described; it is caused by mutations in the adhesion and degranulation-promoting adaptor protein (ADAP; synonym: FYB, SLAP130/120) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study, we used constitutive ADAP-deficient mice (Adap−/−) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that Adap−/− mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. Adap−/− platelets had a shorter life span than control platelets, and macrophage depletion, but not splenectomy, increased platelet counts in mutant mice to control levels. Whole-sternum 3-dimensional confocal imaging and intravital 2-photon microscopy revealed altered morphology of ADAP-deficient MKs with signs of fragmentation and ectopic release of (pro)platelet-like particles into the BM compartment. In addition, cultured BM-derived MKs lacking ADAP showed reduced spreading on extracellular matrix proteins as well as activation of β1 integrins, impaired podosome formation, and displayed defective polarization of the demarcation membrane system in vitro. MK-/platelet-specific ADAP-deficient mice (PF4-cre) also produced fewer and smaller-sized platelets and released platelets ectopically. These data demonstrate that the abnormal platelet production in the mutant mice is an MK-intrinsic defect. Taken together, these results point to an as-yet-unidentified role of ADAP in the process of MK polarization and platelet biogenesis.

Diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium knowlesi in Malaysia

BackgroundPlasmodium knowlesi a parasite of the macaques is currently the most common cause of human malaria in Malaysia. The thrombospondin-related adhesive protein (TRAP) gene is pre-erythrocytic stage antigen. It is a well-characterized vaccine candidate in Plasmodium vivax and Plasmodium falciparum, however, no study has been done in the orthologous gene of P. knowlesi. This study investigates nucleotide diversity, haplotypes, natural selection and population differentiation of full-length pktrap genes in clinical samples from Malaysia.MethodsForty full-length pktrap sequences from clinical isolates of Malaysia along with the reference H-strain were downloaded from published databases. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. McDonald–Kreitman test was conducted using P. vivax and Plasmodium coatneyi as ortholog sequence in DnaSP 5.10 software. Population genetic differentiation index (FST) of parasite populations was determined using Arlequin v3.5. Phylogenetic relationships between trap ortholog genes were determined using MEGA 5.0 software.ResultsComparison of 40 full-length pktrap sequences along with the H-strain identified 74 SNPs (53 non-synonymous and 21 synonymous substitutions) resulting in 29 haplotypes. Analysis of the full-length gene showed that the nucleotide diversity was lower compared to its nearest ortholog pvtrap. Domain-wise analysis indicated that the proline/asparagine rich region had higher nucleotide diversity compared to the von Willebrand factor domain and the thrombospondin-type-1 domain. McDonald–Kreitman test identified that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. knowlesi was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. knowlesi and P. vivax. The von Willebrand factor domain also indicated balancing selection using MK test, however, it did not give significant results when tested with P. coatneyi as an outgroup. Phylogenetic analysis of full-length genes identified three distinct sub-clusters of P. knowlesi, one originating from Peninsular Malaysia and two originating from Malaysian Borneo. High population differentiation values was observed within samples from Peninsular Malaysia and Malaysian Borneo.ConclusionsThis study is the first to report on the genetic diversity and natural selection of full-length pktrap. Low level of genetic diversity was found across the full-length gene of pktrap. Balancing selection of the von Willebrand factor domain indicated that TRAP could be a target in inducing immune response against P. knowlesi infections. However, higher number of samples would be necessary to further confirm the findings.

Clinical characteristics of infections caused by Mycoplasma pneumoniae P1 genotypes in children.

Mycoplasma pneumoniae (M. pneumoniae) isolates can be classified into two major genetic groups, P1 type 1 (MP1) and P1 type 2 (MP2), based on the DNA sequence of the P1 adhesion protein gene. The aim of our study was to determine if M. pneumoniae P1 genotype is associated with disease manifestation and severity of acute M. pneumoniae infection. We compared epidemiological and clinical data of children infected with either MP1 or MP2. In addition, we separately analysed data of patients presenting with individual manifestations of M. pneumoniae infection. Data of 356 patients infected with MP1 were compared with those of 126 patients infected with MP2. MP2-infected children presented with higher median baseline C-reactive protein levels and were admitted to the hospital more often. The distribution of P1 genotype varied among groups of patients with different manifestations of M. pneumoniae infection. MP2 was more common than MP1 among patients with neurological and cardiovascular manifestations, whereas MP1 was more prevalent in other manifestations. The results from our large cohort indicate that the two P1 subtypes may have different pathogenic potential and that infections with MP2 strains could be more virulent than those with MP1 strains.

Sequence analysis of the thrombospondin-related adhesive protein gene and heat shock protein 70 gene of Babesia gibsoni isolated from dogs in Nanjing, China

In this study, the thrombospondin-related adhesive protein (TRAP) gene and the heat shock protein 70 (Hsp70) gene of Babesia gibsoni isolated from the naturally infected dog in the Nanjing area were cloned and sequenced. Twenty blood samples were collected from the suspected cases of babesiosis at the animal hospital of Nanjing Agriculture University. Genomic DNA was extracted from the blood samples, and the 18S rDNAs were amplified by PCR to confirm Babesia infection. As a result, 10 cases of Babesia 18S rDNA gene amplification were positive in the 20 blood samples, confirming that the 10 cases were infected with canine Babesia. The TRAP and the Hsp70 gene fragments were amplified from all 10 positive cases. The four isolates, named NJN1, NJN2, NJN3 and NJN4, were sequenced and compared with other isolates in Asian. The similarity of TRAP and Hsp70 gene sequences among four isolates in Nanjing were above 99%. The Nanjing isolates were closely related to isolates from Taiwan and Japan. Indian isolates were different form Chinese and Japanese isolates, despite the very high similarity of the 18s rRNA genes sequence. These results suggest that the TRAP and Hsp70 genes have a reference value for the genetic diversity analysis of Babesia gibsoni.

Genome-Wide Analysis of the First Sequenced Mycoplasma capricolum subsp. capripneumoniae Strain M1601.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is a common pathogen of goats that causes contagious caprine pleuropneumonia. We closed the gap and corrected rRNA operons in the draft genome of Mccp M1601: a strain isolated from an infected goat in a farm in Gansu, China. The genome size of M1601 is 1,016,707 bp with a GC content of 23.67%. We identified 915 genes (occupying 90.27% of the genome), of which 713 are protein-coding genes (excluding 163 pseudogenes). No genomic islands and complete insertion sequences were found in the genome. Putative determinants associated with the organism’s virulence were analyzed, and 26 genes (including one adhesion protein gene, two capsule synthesis gene clusters, two lipoproteins, hemolysin A, ClpB, and proteins involved in pyruvate metabolism and cation transport) were potential virulence factors. In addition, two transporter systems (ATP-binding cassette [ABC] transporters and phosphotransferase) and two secretion systems (Sec and signal recognition particle [SRP] pathways) were observed in the Mccp genome. Genome synteny analysis reveals a good collinear relationship between M1601 and Mccp type strain F38. Phylogenetic analysis based on 11 single-copy core genes of 31 Mycoplasma strains revealed good collinearity between M1601 and Mycoplasma capricolum subsp. capricolum (Mcc) and close relationship among Mycoplasma mycoides cluster strains. Our genome-wide analysis of Mccp M1601 provides helpful information on the pathogenic mechanisms and genetics of Mccp.

Molecular detection and genetic diversity of Babesia gibsoni in dogs in India

Babesia gibsoni is a tick borne intraerythrocytic protozoan parasite causing piroplasmosis in dogs and has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present communication is the first evidence on the genetic diversity of B. gibsoni of dogs in India. Blood samples were collected from 164 dogs in north and northeast states of India and 13 dogs (7.9%) were found positive for B. gibsoni infection by microscopic examination of blood smears. Molecular confirmation of these microscopic positive cases for B. gibsoni was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR for the 18S rRNA gene was also carried out on microscopically B. gibsoni negative samples that detected a higher percentage of dogs (28.6%) infected with B. gibsoni. Genetic diversity in B. gibsoni in India was determined by studying B. gibsoni thrombospondin-related adhesive protein (BgTRAP) gene fragments (855bp) in 19 isolates from four north and northeast states of India. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasite in India and Bangladesh formed a distinct cluster away from other Asian B. gibsoni isolates available from Japan, Taiwan and Korea. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in India and Bangladesh. Further studies are required for better understanding of the genetic diversity of B. gibsoni prevalent in India and in its neighbouring countries.

Efficient Expression of Mussel Adhesive Protein in &lt;i&gt;Pichia pastoris&lt;/i&gt; GS115 Promoted by Ectoine

Mussel adhesive protein has significant potential application in the field of medical adhesion. Genetic engineering method is gaining more and more attention, by which mussel adhesive protein can be heterologously expressed. In order to improve the expression efficiency of mussel adhesive protein with heterologous recombinant, it is reported that the compatible solutes Ectoine promoted to expression of adhesive protein on Pichia pastoris GS115. In this study, the adhesive protein gene msfp-1 from Mytilus sp. JHX-2002 was transformed into P. pastoris GS115. Inducement expression of adhesive protein Msfp-1 with various methanol concentrations was investigated. The promotion of Ectoine on the expression level of recombinant protein was studied. The results showed that adhesive protein Msfp-1 was induced with methanol on the recombinant GS115/msfp-1. The optimal concentration of methanol was 1% on heterologous expression. In the inducement expression phase of Msfp-1 with methanol, Ectoine could play a promotion role on expression of heterologous proteins. When the concentration of methanol was 1.5% and the addition of Ectoine was 1.5 mM, the expression of Msfp-1 was up to 2.1 g/L. Compared to fermentation broth without Ectoine, the expression was increased by 61.5%. Ectoine has an important promotion in the efficient expression of mussel adhesive protein on P. pastoris GS115.