Analysis on risk factors of clopidogrel resistance in patients with ischemic stroke / 药学实践杂志

Objective To investigate the risk factors of drug resistance in patients with ischemic stroke by clopidogrel therapy and provide references for promoting clinical individualized drug therapy. Methods A total of 202 inpatients diagnosed with ischemic stroke were admitted and given dual anti-treatment (aspirin+clopidogrel). CYP2C19 genotype was detected by microarray hybridization during hospitalization, and CYP2C19 gene polymorphisms were classified into fast metabolism group, medium metabolism group and slow metabolism group according to the type of drug metabolism. Patients were tested for platelet inhibition induced by adenosine diphosphate (ADP) according to thromboelastographic (TEG) on 7~14 d of drug administration. ADP <30% was classified as clopidogrel drug resistance group and ADP ≥30% as non-resistance group. Logistic regression analysis was used to study the risk factors for the development of clopidogrel resistance. Results Among 202 patients with ischemic stroke, 87 were in the resistant group and 115 in the non-resistant group. The proportion of patients with clopidogrel resistance combined with diabetes and the level of white blood cell count were higher than that in the non-resistant group, and the differences were statistically significant (P<0.05).The proportion of patients with clopidogrel resistance in the CYP2C19 intermediate metabolism group was significantly higher than that in the fast metabolism group, and the rate of platelet inhibition was also significantly lower than that in the fast metabolism group, all with statistically significant differences (P<0.05). Conclusion Combined diabetes mellitus, high white blood cell count levels and CYP2C19 mid-metabolic phenotype are independent risk factors for the development of clopidogrel resistance in patients with ischemic stroke.

Expression features of cytochrome C oxidase subunit Ⅰ, BCL2 interacting protein 3 and interleukin-1β in MRL/lpr lupus mice liver tissue / 中华风湿病学杂志

Objective:To explore the expression features of cytochrome C oxidase subunit Ⅰ (MT-CO1), BCL2 interacting protein 3 (BNIP3) and interleukin (IL)-1β in the liver of MRL/lpr lupus mice.Methods:The mRNA and protein levels of MT-CO1, BNIP3, IL-1β, p16 and p21 in lupus mice and control mice were detected by polymerase chain reaction (PCR) and Western blot, the IL-1β expression site were detected by hematoxylin and eosin (HE) staining and immunohistochemical method, and themalondialdehyde (MDA) was detected by colorimetry. Hepatocytes and macrophages were stimulated with lipopolysaccharide (LPS), while hepatocytes were also cultured with supernatants obtained after macrophages stimulated with LPS, and the mRNA and protein levels of MT-CO1, BNIP3 and LC3B, as well as p16 and p21 expression, were determined by qPCR and Western blot. The expression of mitochondrial reactive oxygen species (mtROS) was detected by immunofluorescence. One way Analysis of Variance (ANOVA) was used to compare the mean of each group, and LSD method was used to compare the means of multiple samples, and Tamhane's T2 method was used to compare the means of multiple samples when the variance was uniform. Results:The results of PCR showed that the mRNA levels of MT-CO1 and BNIP3 in the liver tissue of the lupus group (0.14±0.04; 0.16±0.05) were significantly lower than those of the control group (0.11±0.04; 0.16±0.06), and the differences were statistically significant ( t=7.16, P<0.001; t=4.54, P<0.001). The expression levels of IL-1β, p16 and p21 in the lupus group (2.06±0.69; 0.37±0.14; 0.16±0.06) were significantly higher than those of the control group (0.23±0.06; 0.25±0.08; 0.11±0.04) ( t=9.58, P<0.001; t=24.35, P<0.001; t=22.36, P<0.001). The results of Western blot were consistent with those of PCR. HE staining showed lymphocyte infiltration in the liver tissue of lupus mice, and immunohistochemistry showed IL-1β in the liver tissue of lupus mice. The positive cells were mainly concentrated in the sinusoids, and the expression of hepatic parenchymal cells was not rearkable. The content of MDA in liver tissue of the lupus group (0.19±0.10) was higher than that of the control group (0.17±0.09), and the difference was statistically significant ( t=4.33, P=0.005). LPS directly stimulated AML12 hepatocytes (0.069±0.028; 0.17±0.07). The PCR results showed that compared with the control group (0.176±0.072; 0.08±0.03), the expression of MT-CO1, and BNIP3 were not significantly different ( t=1.01, P=0.337; t=0.88, P=0.399). The expression of IL-1β was significantly higher when incubated with the supernatants of LPS stimulated macrophages (0.28±0.09) compared than that of the control group (0.15±0.05) ( t=28.26, P<0.001). The results of PCR showed that the mRNA levels of MT-CO1 and BNIP3 in the LPS stimulated group (0.046±0.026; 0.17±0.05) were significantly lower than those in the control group (0.143±0.083; 0.18±0.06), and the differences were statistically significant ( t=7.52, P<0.001; t=4.24, P<0.001), The expression of p16 and p21 in LPS stimulated group (0.29±0.09; 0.27±0.09) were significantly higher than those in the control group (0.18±0.06; 0.22±0.07) ( t=13.54, P<0.001; t=8.69, P<0.001). The results of Western blot were consistent with those of PCR. Immunofluorescence showed that the fluorescence intensity of mtROS in LPS stimulated group (0.25±0.10) was higher than that in the control group (0.08±0.03), and the difference was statistically significant ( t= 4.86, P<0.001). Conclusion:Immune-mediated inflammation in the liver tissue of lupus mice can stimulate liver parenchymal cells to cause intracellular mitochondrial dysfunction. However, the mechanism of liver organ damage in lupus mice is not limited to the immune-mediated inflammation of immune active cells, but also include parenchymal cell mitochondrial dysfunction.

DNA barcoding revels first records of three rare coleopteran genera in Northern lakes of Egypt / O código de barras de DNA revela os primeiros registros de três gêneros raros de coleópteros nos lagos do norte do Egito

Abstract One aquatic coleopteran species from family Dytiscidae and two aquatic coleopteran genera from family Hydrophilidae were recorded in the summer period and represent first records in the Egyptian lakes. Beetles were collected from two northern lakes, Lake Idku and Lake Burullus. They were identified by morphological characteristics as well as the mtDNA barcoding method. A molecular phylogenetic approach was used to determine the genetic identity of the collected samples based on the mitochondrial cytochrome oxidase I (COI). Prodaticus servillianus (Dytiscidae) from Egypt showed no significant difference in the COI region and they are highly similar to P. servillianus from Madagascar. The phylogenetic analysis revealed that the other two coleopteran genera belong to family Hydrophilidae. Based on COI only, there is no clear evidence for their genetic identity at the species level. So, we defined them to the closest taxon and denoted them as Cymbiodyta type A and B. The results indicated that resolving the molecular identity of the aquatic beetles from northern lakes of Egypt need more considerations in the field of biological conservation. We concluded that utilization of COI as a barcoding region for identifying some coleopteran species is not sufficient and additional molecular markers are required to uncover the molecular taxonomy at deep levels.

Resumo Uma espécie de coleópteros aquático da família Dytiscidae e dois gêneros de coleópteros aquáticos da família Hydrophilidae foram registrados no período de verão e representam os primeiros registros nos lagos egípcios. Os besouros foram coletados em dois lagos do norte, o lago Idku e o lago Burullus, e identificados por características morfológicas e pelo método de código de barras mtDNA. Uma abordagem filogenética molecular foi usada para determinar a identidade genética das amostras coletadas com base no citocromo oxidase I mitocondrial (COI). Prodaticus servillianus (Dytiscidae) do Egito não mostrou diferença significativa na região COI e é altamente semelhante a P. servillianus de Madagascar. A análise filogenética revelou que os outros dois gêneros de coleópteros pertencem à família Hydrophilidae. Com base apenas no COI, não há evidências claras de sua identidade genética no nível da espécie. Assim, nós os agrupamos no táxon mais próximo e os denominamos Cymbiodyta tipo A e B. Os resultados indicaram que a identidade molecular dos besouros aquáticos dos lagos do norte do Egito precisa de mais considerações no campo da conservação biológica. Concluímos que a utilização de COI como região de código de barras para identificar algumas espécies de coleópteros não é suficiente, sendo necessários marcadores moleculares adicionais para descobrir a taxonomia molecular em níveis profundos.

Accediendo al pasado: uso de especímenes de colección como fuentes de información genética para el género Bombus (Hymenoptera: Apidae)

Introducción: Recientemente ha tomado relevancia el uso de especímenes de museo como fuente de información genética para desarrollar estudios que resuelven preguntas taxonómicas, ecológicas, demográficas y evolutivas a diversas escalas temporales y geográficas. Sin embargo, material genético obtenido a partir de ejemplares depositados en colecciones biológicas es poco usado, debido al deterioro natural del ADN preservado en dichos ejemplares, de manera que la obtención de material genético de calidad es demandante en términos de tiempo y dinero. Objetivo: Usando material de museo, identificar una secuencia mini-barcode que pueda ser empleada en la determinación taxonómica, y que a su vez suministre información que permita la estimación de relaciones filogenéticas de especies del género Bombus. Métodos: Se estandarizó el protocolo de extracción de ADN a partir de la extremidad mesotoracica derecha y/o una muestra de músculo torácico de 96 especímenes depositados en la colección LABUN entre 7 y 38 años atrás. Las diferentes combinaciones de oligonucleótidos evaluadas permitieron amplificar fragmentos de 152 a 407 pares de bases (pb) del gen mitocondrial Cytochrome Oxidase I (COI). Usando como plantilla un grupo de 31 secuencias amplificadas a partir de especímenes recolectados recientemente, los fragmentos obtenidos de los especímenes del museo fueron ensamblados y analizados en un marco filogenético. Además, se realizó un análisis de red de haplotipos para evaluar en detalle las relaciones entre los haplotipos mitocondriales resultantes. Resultados: Se determinó un mayor éxito de extracción de ADN a partir de muestras de extremidad depositadas a partir del año 1982.Entretanto, la amplificación exitosa de fragmentos de más de 300 pares de bases (pb) se logró principalmente en muestras depositadas en fechas posteriores a 1999, lo que indica una mayor integridad del material genético recuperado de individuos de 19 años de recolección en adelante. Aunque todos los fragmentos evaluados pueden ser empleados como mini-barcode, solo con uno se obtiene una topología similar a la observada con el fragmento completo. Se detectó una gran variacion genética, particularmente al interior de las especies Bombus atratus y B. funebris, en las que se reveló una clara estructura filogeográfica. Conclusiones: Se obtuvieron nuevas secuencias de códigos de barras mediante extracción de ADN y protocolo de amplificación de muestras de museos. Además, se generó nueva información sobre la variabilidad genética intraespecífica, detectando la presencia de haplotipos mitocondriales únicos que podrían constituir Unidades Significativas Evolutivas sujetas a conservación. Dicha información es de vital importancia para formular estrategias de conservación para estos polinizadores en Colombia.

Introduction: The use of museum specimens as a source of genetic information to develop studies that resolve taxonomic, ecological, demographic, and evolutionary questions at various temporal and geographic scales, has recently become relevant. However, genetic material obtained from specimens deposited in biological collections is not used frequently due to the natural deterioration of the DNA preserved in these specimens. Getting quality genetic material is demanding in terms of time and money. Objective: By using museum material,to identify a mini-barcode sequence that can be used in the taxonomic determination and provides information that allows the estimation of phylogenetic relationships of species of the genus Bombus. Methods: The DNA extraction protocol for museum samples was standardized using the mesothoracic right leg and / or a sample of thoracic muscle of 96 specimens deposited in the LABUN collection between 7 and 38 years ago. Different combinations of oligonucleotides allowed to amplify fragments from 152 to 407 base pairs (bp) of the mitochondrial gene Cytochrome Oxidase I (COI). Using as a template a group of 31 sequences amplified from recently collected specimens, the fragments obtained from the museum specimens were assembled and analyzed in a phylogenetic framework. Additionally, a haplotype network analysis was performed in order to evaluate in detail the relationships between the resulting mitochondrial haplotypes. Results: The greatest success of DNA extraction was achieved from limb samples deposited since the year 1982 on. Meanwhile, successful amplification of fragments longer than 300 base pairs (bp) was achieved mostly in samples deposited on dates after 1999, which indicates greater integrity of the genetic material recovered from individuals of 19 years of collection and onwards. Although all the fragments evaluated can be used as mini-barcode, only with one primer pair, it was possible to obtain a topology similar to that observed with the complete fragment. A large genetic variation was detected, particularly within the Bombus atratus and B. funebris species, in which a clear phylogeographic structure was revealed. Conclusions: New barcode sequences were obtained through DNA extraction and amplification protocol from museum samples. Furthermore, new information on intraspecific genetic variability was generated, detecting the presence of unique mitochondrial haplotypes that could constitute management units subject of conservation. Such information is of vital importance to formulate conservation strategies for these pollinators in Colombia.

Molecular phylogeny and evolution of Pulmonata (Mollusca: Gastropoda) on the basis of mitochondrial (16S, COI) and nuclear markers (18S, 28S): an overview

The phylogenetic relationships among the major groups of Pulmonata were studied by the information derived from a concatenated dataset consisting of mitochondrial (16S and COI) and nuclear (18S and 28S) markers. Heterobranchia are recovered as monophyletic. Euthyneura as paraphyletic due to the emergence of taxa from Opisthobranchia and lower Heterobranchia. The major groups of Pulmonata, namely Stylommatophora, Veronicellidae, Onchidiidae, Otinoidea, Siphonarioidea and Hygrophila are recovered as monophyletic. Monophyly of Basommatophora was not confirmed due to the variable position of Siphonarioidea and Amphiboloidea. Evolutionary divergence times for different taxa were also estimated using a relaxed molecular clock method in Bayesian evolutionary analysis by sampling trees (BEAST). The common ancestor of Heterobranchia and Caenogastropoda was originated in the Silurian period and the common ancestors of Euthyneura and Pulmonata were originated in the Carboniferous and lower Triassic periods, respectively.

Identification of Panax ginseng and Panax quinquefolius by Mitochondrial Cox Ⅱ Intron / 中国实验方剂学杂志

Objective:To develop a simple and accurate method for molecular authentication of Panax ginseng and P. quinquefolius.Method:The mitochondrial cox Ⅱ sequences of P. ginseng and P. quinquefolius were amplified by polymerase chain reaction（PCR）with universal primers. PCR products of the two species were sequenced in both directions, and sequence alignments were conducted for intron length polymorphisms exploitation. Multiplex PCR was established for the identification of P. ginseng and P. quinquefolius with their specific primers，which were designed respectively based on their insertion sequences. And the limit of detection of the multiplex PCR was also determined.Result:The insertion/deletion sequences were exploited in mitochondrial cox Ⅱ. Under the established multiplex PCR assay，P. ginseng generated a 729 bp specific band, while P. quinquefolius yielded a 141 bp specific amplicon，and the mixture of the two species yielded both 729 bp and 141 bp fragments. The established multiplex PCR assay could detect 0.1% of intentional adulteration of P. quinquefolius into P. ginseng, with down to 0.001 ng of genomic DNA.Conclusion:The established multiplex PCR assay can accurately identify P. ginseng and P. quinquefolius from different sources, without the optimization of reaction system and the introduction of additional mismatches，so as to provide a new molecular marker method for identifying botanical origin of P. ginseng and P. quinquefolius.

Avistamientos de aves y análisis bioinformático en entornos rurales con estudiantes de la Institución educativa Ferralarada en Choachí (Colombia) / Bird sightings and bioinformatic analysis in rural settings with students from the educational institution Ferralarada in Choachí (Colombia)

Objetivo: La ejecución de esta investigación presenta dos objetivos principales: primero. Realizar avistamientos de aves en entornos rurales; con estudiantes de primaria, por medio del dibujo como herramienta descriptiva. Segundo. Desarrollar una metodología sencilla para realizar un análisis bioinformático rápido, utilizando el criterio de parsimonia. Materiales y métodos: El presente trabajo se desarrolló con la comunidad educativa de la Institución Educativa Departamental Técnica Agropecuaria Ferralarada; ubicada en el municipio de Choachí, Colombia. Como respuesta a la necesidad de continuar los procesos pedagógicos de la institución en materia ambiental y a los retos actuales de la educación presentados por la emergencia sanitaria del COVID-19. Se realizó una serie de actividades experimentales, que surgen de las aves como elemento motivacional. Resultados. Las actividades realizadas por los docentes de primaria a propósito de los avistamientos, han generado registros de dibujo y escritos, los cuales expresan las representaciones que tienen los estudiantes a propósito de su entorno natural. Se construyo un árbol filogenético basado en el criterio de parsimonia utilizando secuencias del gen CO1 extraídas de Genbank, y analizadas con los programas ClustalX, Nona y Asado. Conclusión: Las actividades ejecutadas a propósito de los avistamientos, han resultado ser viables y de fácil desarrollo con la comunidad educativa. La propuesta bioinformática se configura como una herramienta de análisis sencillo con validez procedimental, sin embargo, requiere avances conceptuales frente al manejo de programas y plataformas, así como disponibilidad de recursos tecnológicos para su implementación.

Aim: The execution of this proposal has two main aim: 1. Perform bird watching in rural settings; with primary school students, using drawing as a descriptive tool. 2. Develop a simple methodology to perform a rapid bioinformatic analysis, using the parsimony criterion. Materials and methods: This work was developed with the educational community of the Institución Educativa Departamental Técnica Agropecuaria Ferralarada; located in the municipality of Choachí, Colombia. In response to the need to continue the pedagogical processes of the institution in environmental matters and to the current challenges of education presented by the health emergency of COVID-19. A series of experimental activities were carried out, arising from birds as a motivational element. Results. The activities carried out by the primary school teachers regarding the sightings have generated drawing and written records, which express the representations that students have of their natural environment. A phylogenetic tree was built based on the parsimony criterion using sequences of the CO1 gene extracted from Genbank, and analyzed with the ClustalX, Nona and Asado programs. Conclusion: The activities carried out in connection with the sightings have turned out to be viable and easy to develop with the educational community. The bioinformatics proposal is configured as a simple analysis tool with procedural validity, however, it requires conceptual advances in the management of programs and platforms, as well as the availability of technological resources for its implementation.

Haplotype diversity in medically important red scorpion (Scorpiones: Buthidae: Hottentotta tamulus) from India

The medically important Indian red scorpion, Hottentotta tamulus, is one of the most poisonous scorpions of Indian subcontinent. We studied the haplotype diversity in eight populations of H. tamulus based on mitochondrial cytochrome oxidase subunit I (COI) partial gene sequence. Analyses revealed 22 haplotypes with a haplotype diversity of 0.941 and nucleotide diversity of 0.023. For the first two codon positions both transition and transversion types of substitutions were equally likely and the test for neutrality was not rejected. However, codon substitution pattern indicated that the gene has experienced purifying selection. Model-based clustering method indicated that the eight populations form three groups that correspond to high, moderate and low rainfall areas, indicating that there is biogeographical separation of haplotypes. Populations from three groups formed distinct clades in maximum likelihood analysis and median joining genetic network and were statistically supported by low within group and high among group variation in analyses of molecular variance. We provide the first account of haplotype diversity in Indian red scorpions and their biogeographical separation.

Integrative taxonomy of commercially important deep water penaeoid shrimps from India

The deep water penaeoid shrimp is an important commercial crustacean resource along the Indian coast. The molecular and morphological information of this group from the Indian coast is scarcely known. In this study, we investigated the identification and phylogenetic relationships of the deep water penaeoid shrimps using three mitochondrial (cytochrome oxidase subunit I (COI), cytochrome b, 16S rRNA) genes, which were compared with 54 morphological characters and further used to evaluate character evolution. Our study revealed remarkable molecular divergence (3.3–33.0%) in nine species from three genera of Solenoceridae, four species from three genera of Penaeidae and one species from Aristeidae using COI. Phylogenetic analysis using maximum likelihood and Bayesian approaches revealed that all species from these families are monophyletic. The present analysis revealed the existence of subgroups in the genus Solenocera suggesting the slow reduction of postrostral carina which corresponds to the increase in distributional depth during the evolutionary process which further indicates the origin of the genus in the continental shelf and extending up to the continental slope. In addition, we generated the DNA barcode database involving these species which can help further to investigate the detailed evolution and biogeography of these valuable crustacean resources.

Genetic diversity of Echinococcus vogeli in the western Brazilian Amazon

Human polycystic echinococcosis is a parasitic infection caused by the larval stage of Echinococcus vogeli, which occurs in rural areas of Central and South America. Until now, little information on the genetic variability of E. vogeli is available. Here, 32 samples from human-excised E. vogeli cysts had a 396-bp sequence of the mitochondrial cytochrome oxidase I (COI) gene sequenced and compared to another 17 COI sequences representing nine Echinococcus species. A Bayesian COI tree revealed that all E. vogeli sequences formed a monophyletic and well-supported clade with an E. vogeli reference sequence. The occurrence of geographically restricted E. vogeli COI haplotypes suggests retention of ancestral polymorphisms with little migration in Acre, Brazil.

Zeylanicobdella arugamensis, the marine leech from cultured crimson snapper (Lutjanus erythropterus), Jerejak Island, Penang, Malaysia

Objective To investigate the prevalence, phylogenetics and DNA barcoding of Zeylanicobdella arugamensis (Z. arugamensis) from crimson snapper (Lutjanus erythropterus), Jerejak Island, Penang, Malaysia. Methods Experiment was conducted with 200 fish specimens of cultured Lutjanus erythropterus from Jerejak Island, Penang, Peninsular Malaysia. The water temperature and length for each fish were measured prior to parasites examination. Next, the morphological identification of parasites was performed. Genomic DNA from parasites was extracted for further molecular analysis. After PCR amplification, phylogenetic tree was constructed. The lowest Bayesian information criterion scores showed that the most compatible model is Tajima and Nei. Finally, data sets of cytochrome oxidase subunit I gene sequence and trace file have been submitted to Barcode of Life Data System. Results The prevalence rate of Z. arugamensis was recorded to be 11.5%, and the intensity was 1.48. The low intensity was due to the water temperature recorded in this study (32.9–33.2 °C). All the individuals of Z. arugamensis recorded in this study showed a close relationship with species that were recorded in NCBI database (Z. arugamensis DQ414344, Aestabdella leiostomi DQ414305, Pterobdella amara DQ414334 and Cystobranchus meyeri DQ414315) but less relationship with Aestabdella abditovesiculata DQ414300. Finally, the DNA sequences submitted to Barcode of Life Data System in accordance to species have already obtained Barcode Index Number as BOLD: ACM3477. Conclusions This study has provided an overview of sequence divergence at cytochrome oxidase subunit I gene, DNA barcodes and parasite prevalence of Z. arugamensis.

Effects of Zhibai Dihuang Decoction on mitochondrial cytochrome oxidase in the spermatogenic cells of rats with ureaplasma urealyticum infection / 中华男科学杂志

Objective@#To explore the effects of Zhibai Dihuang Decoction (ZDD) on mitochondrial cytochrome oxidase (COX) in the spermatogenic cells of rats with ureaplasma urealyticum (UU) infection.@*METHODS@#From forty 4－5 months old SD rats, 30 were randomly selected for the establishment of the model of testicular UU infection by inoculating the bladder with UU suspension and the other 10 injected with normal saline as controls (group A). At 7 days after inoculation, the rat models of testicular UU infection were treated orally with normal saline (group B), ZDD at 1 g per kg of the body weight per day (group C), and azithromycin at 0.105 g per kg of the body weight per day (group D), respectively, once daily for 21 days. Then all the animals were sacrificed and the epididymal and testicular tissues collected for examination of sperm motility with the color sperm dynamic detection system, measurement of the COX activity with the immunohistochemical DAB method, and determination of the mRNA expressions of COXⅠ and COXⅡ by RT-PCR.@*RESULTS@#Compared with group A, group B showed significant decreases in such sperm parameters as grade a sperm (［1.03 ± 0.09］ vs ［0.07 ± 0.03］ %, P0.05), average path velocity (VAP) (［16.22 ± 1.52］ vs ［10.05 ± 1.80］ μm/s, P0.05), and all the parameters were significantly higher in group C than in D (P<0.05or P<0.01).@*CONCLUSIONS@#UU infection can reduce grades a and b sperm, linear, curvilinear and mean sperm velocities, and the mRNA expressions of COX Ⅰ and Ⅱ while ZDD can improve these parameters. The improvement of sperm motility may not be associated with the activity of COX, and the COX activity may be related to the mRNA expression of COX II but not that of COXⅠ.

Zeylanicobdella arugamensis, the marine leech from cultured crimson snapper (Lutjanus erythropterus), Jerejak Island, Penang, Malaysia

Objective:To investigate the prevalence,phylogenetics and DNA barcoding of Zeylanicobdella arugamensis (Z arugamensis) from crimson snapper (Lutjanus erythropterus),Jerejak Island,Penang,Malaysia.Methods:Experiment was conducted with 200 fish specimens of cultured Ltttjanus erythropterus from Jerejak Island,Penang,Peninsular Malaysia.The water temperature and length for each fish were measured prior to parasites examination.Next,the morphological identification of parasites was periormed.Genomic DNA from parasites was extracted for further molecular analysis,After PCR amplification,phylogenetic tree was constructed.The lowest Bayesian information criterion scores showed that the most compatible model is Tajima and Nei.Finally,data sets of cytochrome oxidase subunit Ⅰ gene sequence and trace file have been submitted to Barcode of Life Data System.Results:The prevalence rate of Z arugamensis was recorded to be 11.5％,and the intensity was 1.48.The low intensity was due to the water temperature recorded in this study (32.9-33.2 ℃).All the individuals of Z arugamensis recorded in this study showed a close relationship with species that were recorded in NCBI database (Z.arugamensis DQ414344,Aestabdella leiostomi DQ414305,Pterobdella amara DQ414334 and Cystobranchus meyeri DQ414315) but less relationship with Aestabdella abditovesiculata DQ414300.Finally,the DNA sequences submitted to Barcode of Life Data System in accordance to species have already obtained Barcode Index Number as BOLD:ACM3477.Conclusions:This study has provided an overview of sequence divergence at cytochrome oxidase subunit Ⅰ gene,DNA barcodes and parasite prevalence of Z arugamensis.

DNA barcoding revels first records of three rare coleopteran genera in Northern lakes of Egypt

Abstract One aquatic coleopteran species from family Dytiscidae and two aquatic coleopteran genera from family Hydrophilidae were recorded in the summer period and represent first records in the Egyptian lakes. Beetles were collected from two northern lakes, Lake Idku and Lake Burullus. They were identified by morphological characteristics as well as the mtDNA barcoding method. A molecular phylogenetic approach was used to determine the genetic identity of the collected samples based on the mitochondrial cytochrome oxidase I (COI). Prodaticus servillianus (Dytiscidae) from Egypt showed no significant difference in the COI region and they are highly similar to P. servillianus from Madagascar. The phylogenetic analysis revealed that the other two coleopteran genera belong to family Hydrophilidae. Based on COI only, there is no clear evidence for their genetic identity at the species level. So, we defined them to the closest taxon and denoted them as Cymbiodyta type A and B. The results indicated that resolving the molecular identity of the aquatic beetles from northern lakes of Egypt need more considerations in the field of biological conservation. We concluded that utilization of COI as a barcoding region for identifying some coleopteran species is not sufficient and additional molecular markers are required to uncover the molecular taxonomy at deep levels.

Resumo Uma espécie de coleópteros aquático da família Dytiscidae e dois gêneros de coleópteros aquáticos da família Hydrophilidae foram registrados no período de verão e representam os primeiros registros nos lagos egípcios. Os besouros foram coletados em dois lagos do norte, o lago Idku e o lago Burullus, e identificados por características morfológicas e pelo método de código de barras mtDNA. Uma abordagem filogenética molecular foi usada para determinar a identidade genética das amostras coletadas com base no citocromo oxidase I mitocondrial (COI). Prodaticus servillianus (Dytiscidae) do Egito não mostrou diferença significativa na região COI e é altamente semelhante a P. servillianus de Madagascar. A análise filogenética revelou que os outros dois gêneros de coleópteros pertencem à família Hydrophilidae. Com base apenas no COI, não há evidências claras de sua identidade genética no nível da espécie. Assim, nós os agrupamos no táxon mais próximo e os denominamos Cymbiodyta tipo A e B. Os resultados indicaram que a identidade molecular dos besouros aquáticos dos lagos do norte do Egito precisa de mais considerações no campo da conservação biológica. Concluímos que a utilização de COI como região de código de barras para identificar algumas espécies de coleópteros não é suficiente, sendo necessários marcadores moleculares adicionais para descobrir a taxonomia molecular em níveis profundos.

Morphological, molecular characterization and taxonomic status of Triplophysa marmorata and Triplophysa kashmirensis (Cypriniformes: Nemacheilidae) from Kashmir valley, India

AbstractIn India the distribution of genus Triplophysa has been reported only in the upper drainage of the Indus River in Jammu and Kashmir and Lahul and Spiti area of Himachal Pradesh. There is no study on the taxonomic characterization of this genus from Kashmir Himalaya. Therefore the present study was aimed to characterize two important fish species Triplophysa marmorata and T. kashmirensis from Kashmir valley, by using morphometric and molecular tools. It is difficult to discriminate these two species due to the poor quality of original descriptions, and the lack of good reviews. Keeping this in view, a morphometric and molecular study was conducted. Morphometric data were analyzed by using univariate analysis of variance (ANOvA) and multivariate analyses (Principal component analysis) and mtDNA marker Cytochrome oxidase 1 was used for molecular support. Altogether, 22 morphometric characters were used and 15 characters were found significantly variable (P < 0.05). First two components of principal component analysis (PCA) i.e. PC1 and PC2 grouped these two species into separate clusters. The Cytochrome oxidase 1 analysis showed that the mean intraspecific nucleotide divergence (K2P) was 0.001 and interspecific nucleotide divergence was 0.007. Despite having low K2P divergence, these two species got separated into two distinct clades in both Neighbour joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree building methods. But the pattern of clade formation showed that these species were recently radiated from each other and may have the same ancestor. Furthermore, these two species were found closer to Nemacheilidae than to Balitoridae family in the phylogenetic analysis. The molecular divergence between these species was also supported by variance in morphometric data. This work may build the base for the revision of taxonomic identity of these two important fishes of genus Triplophysa. The present investigation formulated that, based on morphological and mtDNA COI sequences analysis, these two taxonomic Triplophysa species should be considered as valid. The results may further assist to enhance the knowledge of the ichthyologists in understanding the ichthyofauna of Kashmir valley and will help them in planning strategies for conservation and management of these less studied small indigenous species along their natural range of distribution. Rev. Biol. Trop. 64 (2): 473-482. Epub 2016 June 01.

ResumenEn la India, la distribución del género Triplophysa se ha reportado solo en la parte superior del río Indus en Jammu, Kashmir, Lahul y Spiti en el área de Himachal Pradesh. No existen publicaciones acerca de la caracterización taxonómica de este género en Kashmir Himalaya. Por lo tanto, en este estudio se caracterizaron dos especies del valle de Kashmir: Triplophysa marmorata y T. kashmirensi, mediante el uso de herramientas morfométricas y moleculares. Es difícil diferenciar entre estas dos species debido a las vagas descripciones originales y a la falta de buenas revisiones. Debido a esto se realizó un estudio morfométrico y molecular. Los datos morfométricos se analizaron mediante un ANOvA y un análisis de componentes principales y el marcador del gen de la citocromo oxidasa 1 se usó para apoyo molecular. En general, se usaron 22 caracteres morfométricos y 15 fueron significativos (P < 0.05). Los dos primeros componentes del análisis de components principales (PCA), PC1 y PC2, agruparon estas dos especies en clusters separados. El análisis con citocromo oxidasa 1 mostró que el promedio de divergencia del nucleótido intraespecífico (K2P) fue de 0.001 y la divergencia del nucleótido intraespecífico fue de 0.007. A pesar de la baja divergencia de K2P, estas dos species se separan en dos clados diferentes tanto por el método NJ como por el método UPGMA. Sin embargo, el patrón de formación del clado mostró que estas species radiaron recientemente una de la otra y que podrían tener un ancestro en común. Además, en el análisis filogenético estas dos especies se encontraron más cerca de la familia Nemacheilidae que de Balitoridae. La divergencia molecular entre estas dos especies también fue respaldada por la varianza en los datos morfométricos. Este estudio puede establecer la base para una revisión taxonómica de estos dos importantes peces del género Triplophysa. Esta investigación postuló que, basada en análisis morfológicos y de secuencia de ADNm COI, estas dos especies taxonómicas de Triplophysa deben considerarse válidas. Los resultados pueden contribuir a mejorar el conocimiento de los ictiólogos en la comprensión de la ictiofauna del valle de Kashmir y les ayudará a planear estrategias de conservación y manejo de estas dos pequeñas especies indígenas y poco estudiadas en su rango de distribución natural.

Two new species of Mycodrosophila (Diptera, Drosophilidae) proposed by molecular and morphological approaches, with a key to American species

ABSTRACT There are approximately 130 species of MycodrosophilaOldenberg, 1914 worldwide, although only nine species were recorded in American countries so far, three of which are exclusively Nearctic, five exclusively Neotropical and one found in both biogeographic regions (Mycodrosophila projectans). Such a small number of American species is likely a consequence of collecting bias, which favors the capture of frugivorous drosophilids, and to the general absence of Neotropical Mycodrosophila studies in the last 50 years. Here, we describe two commonly sampled species of Mycodrosophila from the Amazonian and Pampa Brazilian biomes, which share morphological similarities with Mycodrosophila neoprojectans and M. projectans, respectively. We compared sequences of the mitochondrial gene cytochrome oxidase subunit I (COI), external morphology characteristics and male terminalia among these species. Based on a DNA barcoding approach coupled to morphological differences, we proposed the delimitation of two new species, Mycodrosophila hofmanni sp. nov. and Mycodrosophila valentae sp. nov. An updated key to identifying Neotropical and Nearctic Mycodrosophila species is also provided.

Utility of COX1 phylogenetics to differentiate between locally acquired and imported Plasmodium knowlesi infections in Singapore

<p><b>INTRODUCTION</b>Although there have been several phylogenetic studies on Plasmodium knowlesi (P. knowlesi), only cytochrome c oxidase subunit 1 (COX1) gene analysis has shown some geographical differentiation between the isolates of different countries.</p><p><b>METHODS</b>Phylogenetic analysis of locally acquired P. knowlesi infections, based on circumsporozoite, small subunit ribosomal ribonucleic acid (SSU rRNA), merozoite surface protein 1 and COX1 gene targets, was performed. The results were compared with the published sequences of regional isolates from Malaysia and Thailand.</p><p><b>RESULTS</b>Phylogenetic analysis of the circumsporozoite, SSU rRNA and merozoite surface protein 1 gene sequences for regional P. knowlesi isolates showed no obvious differentiation that could be attributed to their geographical origin. However, COX1 gene analysis showed that it was possible to differentiate between Singapore-acquired P. knowlesi infections and P. knowlesi infections from Peninsular Malaysia and Sarawak, Borneo, Malaysia.</p><p><b>CONCLUSION</b>The ability to differentiate between locally acquired P. knowlesi infections and imported P. knowlesi infections has important utility for the monitoring of P. knowlesi malaria control programmes in Singapore.</p>

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari:Ixodidae)

Objective: To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for con-firming and identifying the nymphal stages of I. granulatus. Methods: A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results: Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94%sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions: These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.

Effect of Heat Reinforcing Acupuncture Manipulation on Articular Synovium LDH, SDH and CCO in Rheumatoid Arthritis ;Rabbits / 上海针灸杂志

Objective To investigate the regulating effect of heat reinforcing acupuncture manipulation on body energy metabolism enzymes in rheumatoid arthritis (RA) and preliminarily explain the mechanism of heat-producing action of heat reinforcing acupuncture manipulation. Method Forty chinchilla rabbits were randomized into normal, model, equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups. A model of cold syndrome-type RA was made by ovalbumin induction and exposure to low temperature in the other four groups not including the normal group. From two days after successful model making, the normal and model groups were grabbed and fastened (bound) by the same way as for the acupuncture groups, 30 min once daily. The equal reinforcement and reduction group received even reinforcing-reducing method;the twirling reinforcement group, twirling reinforcement method;the heat reinforcing group, heat reinforcing acupuncture manipulation. The needle was manipulated for 1 min and retained for 30 min once daily, for a total of seven days. The RA rabbit knee joint circumference was measured and the inflammation score was recorded according to synovial histopathological sections before and after treatment. After the completion of intervention, the rabbits were sacrificed and the articular synovium was rapidly separated for frozen sections. Articular synovium lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and cytochrome oxidase (CCO) activities were measured by histochemical staining. Result After acupuncture intervention, the RA rabbit knee joint circumference was shortened in all the equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups, but the shortening effect on the RA rabbit knee joint circumference was better in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05);the inflammation score recorded according to the RA rabbit synovial histopathological sections was also decreased more in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05). Synovial LDH integral optical density, total positive area and area percentage were significantly higher in the model group than in the normal group (P<0.05), and significantly lower in the equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups than in the model group(P<0.05) and also in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05). Rabbit synovial SDH and CCO activities were significantly higher in the model group than in the normal group (P<0.05). Rabbit synovial SDH and CCO integral optical density, total positive area and area percentage were significantly higher in the equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups than in the model group (P<0.05) and also in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05). Conclusion Heat reinforcing acupuncture manipulation has a definite therapeutic effect on RA and can increase SDH and CCO activities to enhance aerobic metabolism and produce more local energy in a rabbit model of RA. That may be the mechanism by which heat reinforcing acupuncture manipulation produces heat.

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari: Ixodidae)

Objective To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for confirming and identifying the nymphal stages of I. granulatus. Methods A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94% sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.