Genomic characterization of Puccinia triticina using molecular marker technology / Caracterização genômica de Puccinia triticina usando tecnologia de marcadores moleculares

Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores. To study the genetic structure of the leaf rust population 20 RAPD primers were evaluated on 15 isolates samples collected in Pakistan. A total of 105 RAPD fragments were amplified with an average of 7 fragments per primer. The number of amplified fragments varied from 1 to 12. GL Decamer L-07 and GL Decamer L-01 amplified the highest number of bands (twelve) and primer GL Decamer A-03 amplified the lowest number of bands i.e one. Results showed that almost all investigated isolates were genetically different that confirms high genetic diversity within the leaf rust population. Rust spores can follow the migration pattern in short and long distances to neighbor areas. Results indicated that the greatest variability was revealed by 74.9% of genetic differentiation within leaf rust populations. These results suggested that each population was not completely identical and high gene flow has occurred among the leaf rust population of different areas. The highest differentiation and genetic distance among the Pakistani leaf rust populations were detected between the leaf rust population in NARC isolate (NARC-4) and AARI-11and the highest similarity was observed between NARC isolates (NARC-4) and (NARC-5). The present study showed the leaf rust population in Pakistan is highly dynamic and variable.

A ferrugem da folha, causada por Puccinia triticina, é a ferrugem mais comum do trigo. O fungo é um parasita obrigatório, capaz de produzir urediniósporos infecciosos. Para estudar a estrutura genética da população de ferrugem da folha, 20 primers RAPD foram avaliados em 15 amostras de isolados coletadas no Paquistão. Um total de 105 fragmentos RAPD foram amplificados com uma média de 7 fragmentos por primer. O número de fragmentos amplificados variou de 1 a 12. GL Decamer L-07 e GL Decamer L-01 amplificaram o maior número de bandas (doze), e o primer GL Decamer A-03 amplificou o menor número de bandas, ou seja, um. Os resultados mostraram que quase todos os isolados investigados eram geneticamente diferentes, o que confirma a alta diversidade genética na população de ferrugem da folha. Os esporos de ferrugem podem seguir o padrão de migração em distâncias curtas e longas para áreas vizinhas. Os resultados indicaram que a maior variabilidade foi revelada por 74,9% da diferenciação genética nas populações de ferrugem. Esses resultados sugeriram que cada população não era completamente idêntica e um alto fluxo gênico ocorreu entre a população de ferrugem da folha de diferentes áreas. A maior diferenciação e distância genética entre as populações de ferrugem da folha do Paquistão foram detectadas entre a população de ferrugem da folha no isolado NARC (NARC-4) e AARI-11 e a maior similaridade foi observada entre os isolados NARC (NARC-4) e (NARC-5). O presente estudo mostrou que a população de ferrugem da folha no Paquistão é altamente dinâmica e variável.

Alternanthera yellow vein virus (AYVV); a betasatellite independent begomovirus infecting Sonchus palustris in Pakistan.

Satellites associated begomoviruses are the most diverse group of plant viruses in tropical and subtropical regions. In Pakistan, during field surveys in 2019-2020, Sonchus palustris (a weed plant) was observed showing begomovirus symptoms i.e., vein yellowing and mosaic patterns on leaves. Rolling circle amplification from total isolated DNA of symptomatic leaves was performed to amplify circular viral genomes. Subsequent cloning and sequencing showed that a new strain of Alternanthera yellow vein virus (AlYVV) is associated with vein yellowing disease of S. palustris. The identity percentage analysis through BLAST search and SDT analysis showed that the new strain is 94-98% identical to AlYVV isolates reported from Pakistan, India and China. In phylogenetic tree, it clustered with AlYVV-[PK:E prostrata:15-KX710155], AlYVV-[PK:E prostrata:13]-KX906697] and AlYVV-[PK:E prostrata:11]-KX906694] previously reported from Pakistan. There was no detectable level of betasatellite or any other satellite molecule in the samples studied here. Phylogenetic analysis of Rep and CP genes of AlYVV with corresponding genes of closely related viruses circulating in Southeast Asia showed intra-specific recombination involving both complementary and virion sense region of virus. Relaxed clock and Bayesian Skyline Plot analysis based on CP gene sequences indicated slight higher substitution rates (4.75 x 10-3 substitutions/nucleotide/year). In the Indian subcontinent satellite-associated monopartite begomoviruses predominately infect crops and non-crop plants. But AlYVV is found infecting mostly non-crop plants independent of satellite molecules. We hypothesize here that AlYVV evolved as a true monopartite begomovirus in the Indian sub-continent and could be a great threat to introduced crops under suitable conditions. Such studies are crucial to understand probable future epidemics of begomoviruses in the region.

Genomic characterization of Puccinia triticina using molecular marker technology.

Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores. To study the genetic structure of the leaf rust population 20 RAPD primers were evaluated on 15 isolates samples collected in Pakistan. A total of 105 RAPD fragments were amplified with an average of 7 fragments per primer. The number of amplified fragments varied from 1 to 12. GL Decamer L-07 and GL Decamer L-01 amplified the highest number of bands (twelve) and primer GL Decamer A-03 amplified the lowest number of bands i.e one. Results showed that almost all investigated isolates were genetically different that confirms high genetic diversity within the leaf rust population. Rust spores can follow the migration pattern in short and long distances to neighbor areas. Results indicated that the greatest variability was revealed by 74.9% of genetic differentiation within leaf rust populations. These results suggested that each population was not completely identical and high gene flow has occurred among the leaf rust population of different areas. The highest differentiation and genetic distance among the Pakistani leaf rust populations were detected between the leaf rust population in NARC isolate (NARC-4) and AARI-11and the highest similarity was observed between NARC isolates (NARC-4) and (NARC-5). The present study showed the leaf rust population in Pakistan is highly dynamic and variable.

Cultural Adaptation and Linguistic Validation of the Beliefs about Medicines Questionnaire in Malaysia.

BACKGROUND: Low rate of adherence was found strongly associated with patients' beliefs across the studies about chronic diseases with hypertension. A crucial move is needed to bridge the gap between appropriate assessment tools and local hypertensive patients' medication adherence. OBJECTIVE: To produce a translated version in Malay language of Beliefs about Medicines Questionnaire (BMQ) that was "conceptually equivalent" to the original English version for use in local clinical practice and research. METHODS: The forward translation process was conducted by two independent professional translators and back translation was done by two other independent translators. A reliability analysis was conducted on 238 conveniently selected hypertensive patients. Intraclass correlation coefficient (ICC) was used to assess test-retest reliability for the randomly selected 40 patients in a period of 2 weeks. Discriminant validity was tested through Necessity-Concerns differential, BMQ subscales, and other parameters. RESULTS: The overall Cronbach alpha for the internal consistency was good (0.860). The subscales of the BMQ demonstrated adequate internal consistency, with Cronbach alpha value of 0.759 for Specific-Necessity, 0.762 for Specific Concern, 0.624 for General-Overuse, and 0.756 for General-Harm. The ICC was excellent (0.922). Discriminant validity revealed that BMQ Specific-Necessity score was significantly inversely correlated with the systolic blood pressure level. Systolic and diastolic blood pressure levels (P = 0.038; P = 0.05) were reported to be significantly correlated with the Necessity-Concerns differential, with Necessity score equal or exceeding Concerns score. CONCLUSIONS: The Malay-translated version of BMQ is a reliable and valid tool to assess patient belief about medication, especially medication adherence among the hypertensive patients in Malaysia.

Using pictograms to assist caregivers in liquid medication administration: a systematic review.

WHAT IS KNOWN AND OBJECTIVE: It has been reported that more than 80% of out-of-hospital medication errors among the young children involve liquid formulations. The usefulness of pictorial aids to improve communication of medication instructions has not been extensively investigated for child health. The objective of this study was to determine the effectiveness of pictorial aids used to assist caregivers in the administration of liquid medications. METHODS: MEDLINE, CINAHL, PsycINFO, ScienceDirect, Scopus and the Cochrane Library were searched for articles published up to February 2015. Studies that used pictorial aids with liquid medications and measured at least one of the following outcomes were included: dosing accuracy, comprehension of medication instructions, recall of information and adherence of caregivers. Two authors independently selected studies, extracted data and assessed methodological quality of studies using the Cochrane Collaboration's tool. RESULTS AND DISCUSSION: Five experimental studies (four hospital based and one community based) with a total of 962 participants were included. A wide range of liquid formulations were studied, including both prescription and over-the-counter medications. The existing findings suggest that pictographic interventions reduced dosing errors, enhanced comprehension and recall of medication instructions and improved adherence of caregivers. Incorporating pictorial aids into verbal medication counselling or text-based instructions was more beneficial than using the single approach alone. Mixed results were identified for the relationship between health literacy of caregivers and effectiveness of pictorial aids. WHAT IS NEW AND CONCLUSION: The evidence remains limited due to the small number of studies found and variations in methodological quality. This review suggests that pictorial aids might be potential interventions, but more high-quality studies are needed to support the routine use of any pictogram-based materials with liquid medications in the clinical settings.

Identification of predictive biomarkers of disease state in transition dairy cows.

In dairy cows, periparturient disease states, such as metritis, mastitis, and laminitis, are leading to increasingly significant economic losses for the dairy industry. Treatments for these pathologies are often expensive, ineffective, or not cost-efficient, leading to production losses, high veterinary bills, or early culling of the cows. Early diagnosis or detection of these conditions before they manifest themselves could lower their incidence, level of morbidity, and the associated economic losses. In an effort to identify predictive biomarkers for postpartum or periparturient disease states in dairy cows, we undertook a cross-sectional and longitudinal metabolomics study to look at plasma metabolite levels of dairy cows during the transition period, before and after becoming ill with postpartum diseases. Specifically we employed a targeted quantitative metabolomics approach that uses direct flow injection mass spectrometry to track the metabolite changes in 120 different plasma metabolites. Blood plasma samples were collected from 12 dairy cows at 4 time points during the transition period (-4 and -1 wk before and 1 and 4 wk after parturition). Out of the 12 cows studied, 6 developed multiple periparturient disorders in the postcalving period, whereas the other 6 remained healthy during the entire experimental period. Multivariate data analysis (principal component analysis and partial least squares discriminant analysis) revealed a clear separation between healthy controls and diseased cows at all 4 time points. This analysis allowed us to identify several metabolites most responsible for separating the 2 groups, especially before parturition and the start of any postpartum disease. Three metabolites, carnitine, propionyl carnitine, and lysophosphatidylcholine acyl C14:0, were significantly elevated in diseased cows as compared with healthy controls as early as 4 wk before parturition, whereas 2 metabolites, phosphatidylcholine acyl-alkyl C42:4 and phosphatidylcholine diacyl C42:6, could be used to discriminate healthy controls from diseased cows 1 wk before parturition. A 3-metabolite plasma biomarker profile was developed that could predict which cows would develop periparturient diseases, up to 4 wk before clinical symptoms appearing, with a sensitivity of 87% and a specificity of 85%. This is the first report showing that periparturient diseases can be predicted in dairy cattle before their development using a multimetabolite biomarker model. Further research is warranted to validate these potential predictive biomarkers.

A metabolomics approach to uncover the effects of grain diets on rumen health in dairy cows.

Dairy cows fed high-grain diets during early lactation have a high incidence of metabolic disorders. However, the precise mechanism(s) of how grain feeding causes disease is not clear. In an effort to understand how this diet transition alters the rumen environment and potentially leads to certain metabolic disorders in dairy cattle, we undertook a comprehensive, quantitative metabolomic analysis of rumen fluid samples from dairy cows fed 4 different diets. Using a combination of proton nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry, and direct flow injection tandem mass spectroscopy, we identified and quantified 93 metabolites in rumen samples taken from 8 dairy cows fed graded amounts of barley grain (i.e., 0, 15, 30, and 45% of diet dry matter). We also studied temporal changes in the rumen by studying metabolite concentration differences between the first day and the last day of each diet phase following the diet adaptation period. Multivariate analysis showed that rumen metabolites arising from the diet containing 45% barley grain were clearly different from those containing 0, 15, and 30% barley grain. Likewise, a clear separation of the metabolic composition of the ruminal fluid was evident at the beginning and at the end of each diet phase-contrary to the belief that 11 d are suitable for the adaptation of cows to high-grain diets. High-grain diets (>30%) resulted in increased rumen fluid concentrations of several toxic, inflammatory, and unnatural compounds including putrescine, methylamines, ethanolamine, and short-chain fatty acids. Perturbations in several amino acids (phenylalanine, ornithine, lysine, leucine, arginine, valine, and phenylacetylglycine) were also evident. The present study confirms and greatly extends earlier observations on dietary effects on rumen fluid composition and shows that the use of multiple metabolomic platforms permits a far more detailed understanding of metabolic causes and effects. These results may improve our understanding of diet-related rumen metabolism and the influence of grain on the overall health of dairy cattle.

Drug attitude and adherence: a qualitative insight of patients with hypertension.

The study is aimed to explore the perceptions and experiences of hypertensive patients toward medication use and adherence. The study was qualitative in nature conducted at Sandamen Provisional Hospital of Quetta city, Pakistan; a public hospital catering to the health needs of about 40% of the population. A qualitative approach was used to gain an in-depth knowledge of the issues. Sixteen patients were interviewed, and the saturation point was achieved after the 14(th) interview. All interviews were audio-taped, transcribed verbatim, and were then analyzed for thematic contents by the standard content analysis framework. Thematic content analysis yielded five major themes. (1) Perceived benefits and risks of medications, (2) physician's interaction with patients, (3) perception toward traditional remedies, (4) layman concept toward medications, and (5) beliefs toward hypertension and its control. The majority of the patients carried specific unrealistic beliefs regarding the long-term use of medication; yet these beliefs were heavily accepted and practiced by the society. The study indicated a number of key themes that can be used in changing the beliefs and experiences of hypertensive patients. Physician's attitude, patient's past experiences, and knowledge related to hypertension were noted as major contributing factors thus resulting in nonadherence to therapy prescribed.

Modulation of botulinum toxin-induced changes in neuromuscular function with antibodies directed against recombinant polypeptides or fragments.

Botulinum toxin is an agent that is typically encountered in two settings: as an agent that can cause disease (e.g. botulism), and as an agent that can be used to treat disease (i.e., a variety of neurologic disorders). In both cases it would be advantageous to develop a sound understanding of the mechanisms by which antibodies neutralize the toxin. In the present study, recombinant antigens were used to generate antibodies against the carboxyterminal half of the toxin heavy chain (HC50), the entire toxin light chain (LC), and the HA17, HA35 and HA70 components of the progenitor toxin complex. These antibodies were then evaluated for their respective abilities to alter botulinum toxin-induced changes in locomotor behavior in mice. The botulinum toxin type A complex was shown to produce dose-dependent depression of locomotor behavior within the dose range of 0.3-0.7 mouse LD50 units. At a dose of 0.5 LD50, the toxin typically reduced running behavior by 90% or more, and full recovery was not observed for approximately 4 weeks. Mice that were actively or passively vaccinated against the HC50 polypeptide were resistant to toxin action, presumably because the antibodies occluded the toxin binding domain. Interestingly, mice that were actively or passively vaccinated against LC were also resistant to toxin action. This effect may have been due to steric hindrance of the binding process. There was no scenario in which anti-HA antibodies altered the effects of toxin on locomotor behavior. This absence of effect was likely due to the fact that HAs and neurotoxin in the progenitor toxin complex spontaneously dissociate in physiologic media.

Tracing disease gene(s) in non-syndromic clefts of orofacial region: HLA haplotypic linkage by analyzing the microsatellite markers: MIB, C1_2_5, C1_4_1, and C1_2_A.

BACKGROUND: Cleft lip with or without cleft palate (CL/P) is the most frequent craniofacial malformation seen in man. The etiology of CL/P is complex involving both genetic and epigenetic (environmental) factors, and the genes play an almost deterministic role in the normal development of craniofacial structures. This study was aimed at ascertaining the association of HLA microsatellites in CL/P patients. MATERIALS AND METHODS: Case DNA was obtained from 76 patients (40M and 36 F, average age 7.8 years, range 1-16 years). Unaffected individuals from the same geographical area without population mixing included as controls (n=154, 76 M and 78 F, average age 8.2 years, range 2-17 years). All DNA samples were purified from peripheral blood by standard techniques. RESULTS: Four microsatellites were compared in this case-control study. C1_2_5 locus was the most polymorphic marker with 15 observed alleles while C1_4_1 had the least number of alleles. Three of the four markers viz MIB,C1_4_1 and C1_2_5 showed a significant association of microsatellite alleles with CL/P. Five alleles (MIB_326,332,350; C1_4_1 - 213 and C1_2_5-204) were seen with an increased frequency among the test samples, whereas two alleles (C1-4_1_217, and C1_2_5_196) had an increased frequency among the control samples. One allele (C1-4-1-209) had an increased frequency in patient group but was not observed in the controls. CONCLUSION: The role of HLA complex in the pathogenesis of CL/P is speculative and has not been established so far. The result of this study shows that a few alleles have an increased frequency of expression in the diseased group which suggests that these alleles may predispose the individuals to clefting. This finding may be beneficial to aid in early diagnosis and plan intervention strategies.

Applying patient centered approach in management of pulmonary tuberculosis: A case report from Malaysia.

A 24 year university student with history of productive cough was registered as sputum smear confirmed case of pulmonary tuberculosis. During treatment, patient suffered from itchiness associated with anti tuberculosis drugs and was treated with chlorpheniramine (4mg) tablet. Patient missed twenty eight doses of anti tuberculosis drugs in continuation phase claiming that he was very busy in his studies and assignments. Upon questioning he further explained that he was quite healthy after five months and unable to concentrate on his studies after taking prescribed medicines. His treatment was stopped based on clinical improvement, although he did not complete six months therapy. Two major reasons; false perception of being completely cured and side effects associated with anti TB drugs might be responsible for non adherence. Non sedative anti histamines like fexofenadine, citrizine or loratidine should be preferred over first generation anti histamines (chlorpheniramine) in patients with such lifestyle. Patient had not completed full course of chemotherapy, which is preliminary requirement for a case to be classified as "cure" and "treatment completed". Moreover, patient had not defaulted for two consecutive months. Therefore, according to WHO treatment outcome categories, this patient can neither be classified as "cure" or "treatment completed" nor as "defaulter". Further elaboration of WHO treatment outcome categories is required for adequate classification of patients with similar characteristics. Likelihood of non adherence can be significantly reduced by applying the WHO recommended "Patient Centered Approach" strategy. Close friend, class mate or family member can be selected as treatment supporter to ensure adherence to treatment.

Distribution and seasonal activity of mosquitoes in al Madinah Al Munwwrah, Saudi Arabia.

In this study, 2654 adults and mosquito larvae, which belong to 18 species and 4 genera, were collected: Aedes (2 spp.), Anopheles (7 spp.), Culex (8 spp.) and Culiseta (1 sp.). They were Aedes caspius, Ae. aegypti, Anopheles. azaniae, An. d'thali, An. multicolor, An. rhodesiensis, An. stephensi, An. Sub-pictus, An. turkhudi, Culex laticinctus, Cx. perexiguus, Cx. pipiens, Cx. quin-quefasciatus, Cx. simpsoni, Cx. theileri, Cx. tritaeniorhynchus, Cx. univittatus and Culiseta longiareolata. A total of 2270 mosquito larvae were collected, and Culex spp. were the most abundant, where 1629 (71.76%) larvae were collected, followed by 499 (21.98%) Anopheles spp., 94 (4.14%) Aedes spp. and 48 (2.12%) Culiseta longiareolata. Of, 384 adult mosquitoes collected Culex spp. were the most abundant and 328 (85.42%) were collected, followed by 22 (5.73%) Aedes spp., 19 (4.94%) Anopheles spp. and 15 (3.91%) Culiseta longiareolata. The physical properties of the water in the breeding sites of mosquito larvae showed that pH of water varied between 6.9 & 9.9, the total dissolved salts (TDS) varied between 378-9504 ppm and water temperature varied between 8.7 degrees C in winter to 29.9 degrees C in summer. There was no correlation between pH & TDS of water in breeding site and distribution of larvae. The population density started to increase in March, with a peak in August when temperature was 36 degress C. The activity started to decrease in October, and minimum activity was in January, when temperature was below 5 degrees C. The seasonal abundance of adult mosquitoes was not affected by rainfall. A. aegypti, vector of Dengue fever virus, Cx. tritaeniorhynchus, vector of Rift Valley fever and Cx. univittatus, vector of sindbis virus were reported for the first time in Al Madinah Al Munawwrah Region. These vectors constituted a major health problem, and every effort should be made for feasible control.

Characterization of BK channel splice variants using membrane potential dyes.

BACKGROUND AND PURPOSE: Large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that displays extensive pre-mRNA splicing. Here we exploited a membrane potential assay to investigate the sensitivity of different BK splice variants to elevations in intracellular free calcium and their inhibition by the BK channel blocker paxilline. EXPERIMENTAL APPROACH: Murine BK channel splice variants were expressed in human embryonic kidney 293 cells and their properties analysed in response to ionomycin-induced calcium influx in both fluorescent membrane potential (fluorescent-imaging plate reader) and patch clamp electrophysiological assays. The dose-dependent inhibition of distinct splice variants by the BK channel-specific blocker paxilline was also investigated. KEY RESULTS: Ionomycin-induced calcium influx induced a robust hyperpolarization of human embryonic kidney 293 cells expressing distinct BK channel splice variants: stress regulated exon (STREX), e22 and ZERO. Splice variant expression resulted in membrane hyperpolarization that displayed a rank order of potency in response to calcium influx of STREX > e22 > ZERO. The BK channel inhibitor paxilline exhibited very similar potency on all three splice variants with IC(50)s in membrane potential assays of 0.35 +/- 0.04, 0.37 +/- 0.03 and 0.70 +/- 0.02 micromol x L(-1) for STREX, ZERO and e22 respectively. CONCLUSIONS AND IMPLICATIONS: BK channel splice variants can be rapidly discriminated using membrane potential based assays, based on their sensitivity to calcium. BK channel splice variants are inhibited by the specific blocker paxilline with similar IC(50)s. Thus, paxilline may be used in functional assays to inhibit BK channel function, irrespective of the variant expressed.