Lokale og systemiske forsvarsresponser hos skogtrær mot patogen sopp : transkripsjonsstudier avdekker forskjeller

Forest trees dominate the earth surface and has special place in the economy and ecology of the planet. Due to the long life span of woody trees they are challenged by the abiotic and biotic factors. Over the long history of existence trees have evolved defensive systems that still secure their longevity and dominance. Gymnosperms such as conifers are dominating the temperate forest together with the deciduous angiosperm trees and successfully defended their existence for millions of years. Norway spruce (Picea abies) is an important conifer species, and was selected to explore further its defensive system at the molecular level. Angiosperms are most diverse group of plants at present and contain many important tree species such as aspen (Populus tremula) and other Populus species. Populus having a sequenced genome guided us to aspen as our candidate angiosperm to study its defence responses at the transcriptome level. These most challenging pathogens for these species are the biotrophic and necrotrophic fungi. The resistance level within Norway spruce and Populus species show variation towards both biotrophic and necrotrophic fungi supporting that there is a genetic basis and variation in these genes for resistance. The necrotrophic fungus Heterobasidion parviporum as able to colonize Norway spruce and is responsible for great economic losses. Up to twenty present of the spruce trees in Norway show decay caused by this fungus. In Norway spruce trees with high level of resistance to H. parviporum transcript marker genes such as Chitinase class IV and peroxidases are induced rapidly both locally and systemically at early stages after pathogen infection while in more susceptible plants the response is slower. There are also differences in how these are induced between bark and sapwood. The transcripts level of some genes such a PaChi4 and PaPX3 increased more in the sapwood while the genes like PaPAL2 and PaHCT1 were more upregulated in the bark as a host response to infection. These results suggest that the systemic signalling of defence response may also occur through sapwood. In addition, the local defence responses to necrotroph infection, wounding and methyl jasmonate (MeJ) were found to show similarities. The use of aspen offered a great opportunity to study different aspects including defence responses at the transcriptome level using microarrays. This study addressed the defence responses of a susceptible (23) and relatively resistant (72) SwAsp aspen clone after wounding, and inoculation with a necrotroph (Ceratocystis sp. NFLI 2004-466/501) and a biotroph (Melampsora magnusiana) fungus by looking at the expression of a large part of the expressed genes in this species. Differences between the clones were evident at the transcripts level; the healthy un-inoculated clones showed 552 constitutive genes that were expressed differentially. Differential gene regulation in response to the pathogen attack was also evident between the two aspen clones after biotroph and necrotroph inoculation. In systemic leaves from the susceptible clone no differential regulation of genes found to biotroph while only 7genes in response to the necrotroph at 24 hour post infection. In contrast, 156 genes were differentially expressed in the more resistant clone to biotroph and 283 to necrotroph infection at the same time using microarrays. We also found that a larger portion of differentially regulated genes were upregulated in response to the biotroph while in contrast a majority of genes were downregulated in response to the necrotroph. qRT-PCR validation of selected genes supported that the systemic induction in leaves of clone 72 was higher after biotroph, necrotroph and wounding than in the susceptible clone 23. The regulation of putative defence genes were also followed in the same two aspen clones to find the local and systemic defence response after necrotroph and wounding of the stem. An aggressive and newly discovered necrotroph Ceratocystis sp. from Norway was used to inoculate both clones. In general, clone23 showed as strong defence induction and was higher than in clone 72 at the early stages as a local response to infection. However, clone72 showed systemic response in leaves to the necrotroph and more so to wounding, while clone 23 showed little or no systemic inductions to wounding and necrotroph. The necrotroph was highly aggressive and the results suggest that it has the ability to suppress part of the host defence signalling seen towards wounding. These results also suggest that clone 72 has a fully functional local and systemic defence signalling, while clone 23 is deficient or delayed in its systemic response to wounding and necrotroph. We also followed the defence induction markers, 21 NB-LRR Resistance gene-like homologues and five microRNA (miRNA) putatively targeting NB-LRRs in a relatively resistant Norway spruce clone. Ramets of the clone was wounded and inoculated with the necrotrophic fungus Ceratocystis polonica. The markers showed increase both locally and systemically indicative of a rapid and efficient host response and we also saw local and systemic changes in NB-LRR and miRNA transcript levels. However the transcriptional changes for the NB-LRRs and miRNAs followed were in general small, partly supporting the notion that Resistance-like genes are typically expressed at low and constitutive levels. Comparing the host responses between these two tree species, the results suggest that the defence responses to pathogens and wounding in the gymnosperm Norway spruce and the angiosperm aspen show similarities despite their 300 million years of evolutionary separation. In both species we saw up regulation of defence related genes (such as Class IV chitinases) that are also upregulated in response to necroptrophic pathogens.Skogstrær er dominerende planter som har stor økonomisk og økologisk betydning. Fordi trær har et langt livsløp utsettes de for betydelige abiotiske og biotiske stressfaktorer. Trær har utviklet effektive systemer for å motstå forskjellige former for stress som gjør at de kan overleve og trives på samme sted over lange tidsrom. Gran (Picea abies) er det viktigste treslaget i Norge og ble valgt ut til videre molekylære studier av dens forsvarssystemer mot skadesopp. Løvtrærne er mer diverse enn bartrærne, men blant løvtrærne finnes mange viktige arter i de nordlige boreale skoger, blant annet osp (Populus tremula) og andre Populus arter. For Populus er genomet sekvensert, noe som ledet oss til å bruke osp i det molekylære arbeidet på transkriptom nivå. De mest utfordrende skadegjørere for trær er biotrofe og nekrotrofe sopper, men det er forskjeller i graden av resistens mot disse skadegjørerne innen treslagene noe som viser at det er en genetisk basis for dette og at det er variasjon i genene for motstandsdyktighet. Granrotkjuka (Heterobasidion parviporum) er en nekrotrof sopp som effektivt angriper gran og gir store økonomiske tap. Over 20 % av grantrærne i Norge er angrepet av rotkjuke ved slutthogst. I grantrær med høy grad av resistens mot granrotkjuke ser man at gener slik som kitinaser (PaChi4) og peroksidaser (PaPX3) induseres raskt både lokalt og systemisk ved et soppangrep, mens mer mottagelige trær viser en forsinket respons. Det er også forskjeller mellom hvordan disse induseres i levende bark og yteved. Genuttrykket av PaChi4 og PaPX3 øker mest i yteved, mens gener som PaPAL2 og PaHCT1 oppreguleres mest i bark etter en infeksjon. Disse resultatene antyder at det også er systemiske forsvarsresponser i ved. I tillegg fant man at den lokale forsvarsresponsen mot det nekrotrofe patogenet viser likhet med responsen til skade og metyljasmonat (MeJ). Bruk av osp har muligjort studier av forsvarsresponser på transkriptom nivå ved bruk av mikromatriser. Studiene av osp ble utført på SwAsp kloner (23 og 72) med forskjeller i resistens mot den biotrofe soppen Melampsora magnusiana, men i tillegg ble responsen mot skade og en nylig oppdaget nekrotrof blåvedsopp Ceratocystis sp. (NFLI 2004-466/0501) studert. Det var store forskjeller molekylært mellom de to ospeklonene. I de friske klonene var 552 gener forskjellige, noe som reflekterer deres forskjellige genetiske bakgrunn. Forskjellen var også stor i hvordan disse reagerte på biotrof og nekrotrof sopp. Den mottagelige klonen (23) viste ingen systemisk induksjon av forsvarsresponser i blader 24 timer etter angrep med biotrof sopp, mens kun syv gener varierte i sitt uttrykk mot den nekrotrofe soppen. I skarp kontrast til dette ble 156 gener regulert i den mer resistente ospeklonen (72) etter behandling med den biotrofe soppen, og hele 283 gener ble regulert etter inokulering med den nekrotrofe soppen. En større andel gener ble oppregulert i den sterke klonen etter angrep av den biotrofe soppen enn mot den nekrotrofe. Senere qRT-PCR validering på utvalgte gener støttet disse resultatene og viste at den systemiske responsen i blader var mye sterkere i den mer resistente klonen (72) enn i den mer mottagelige klonen (23). Reguleringen av antatte forsvarsgener ble så fulgt i de to samme ospeklonene for å finne likheter og forskjeller mellom den lokale og systemiske responsen til den nekrotrofe soppen og til skade i bark på stammen. Den nekrotrofe Ceratocystis sp. ble brukt til å inokulere begge kloner. Klone 23 viste seg å gi lite systemisk rerspons, men like strek eller sterkere lokal respons mot den nekrotrofe soppen som klon 72. Den systemiske responsen mot skade var sterkere i klon 72 enn den var mot den nekrotrofe soppen. Nekrotrofen viste aggressiv vekst i begge klonene, og resultatene antyder at soppen har evnen til å undertrykke forsvarsresponsen i klon 72, mens det systemiske signalsystemet for forsvar mot skade og soppangrep ser ut til å være defekt eller tregt i klon 23. Vi fulgte også transkripsjons nivået av utvalgte markører for indusert forsvar, 21 NB-LRR resistenslignende gener samt fem microRNA (miRNA) i en gran klone med relativt høyt resistens nivå etter inokulering med blåvedsopp (Ceratocystis polonica) og etter skade. Forsvarsmarkørene ble indusert lokalt og systemisk noe som tyder på at klonens forsvarssystemer ble effektivt slått på, vi så også endringer i NB-LRR og miRNA uttrykket lokalt og systemisk. Transkripsjonsforandringene for NB-LRR og miRNA var imidlertid generelt små, noe som delvis støtter det at resistensgener uttrykkes på et lavt og konstitutivt nivå. Når man sammenligner de observerte forsvarsresponser mot nekrotrof sopp og skade mellom gran som er et bartre og osp som er et løvtre ser man likheter, selv om de har vært evolusjonært separert i mer enn 300 millioner år. For eksempel fant vi oppregulering av beslektede gener (som Klasse IV kitinaser) som oppreguleres raskt i respons mot nekrotrof sopp i begge treartene

Surveillance of molecular markers of antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Federally Administered Tribal Area (FATA), Pakistan

This molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified

An Unbiased Lipid Phenotyping Approach To Study the Genetic Determinants of Lipids and Their Association with Coronary Heart Disease Risk Factors.

Direct infusion high-resolution mass spectrometry (DIHRMS) is a novel, high-throughput approach to rapidly and accurately profile hundreds of lipids in human serum without prior chromatography, facilitating in-depth lipid phenotyping for large epidemiological studies to reveal the detailed associations of individual lipids with coronary heart disease (CHD) risk factors. Intact lipid profiling by DIHRMS was performed on 5662 serum samples from healthy participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). We developed a novel semi-targeted peak-picking algorithm to detect mass-to-charge ratios in positive and negative ionization modes. We analyzed lipid partial correlations, assessed the association of lipid principal components with established CHD risk factors and genetic variants, and examined differences between lipids for a common genetic polymorphism. The DIHRMS method provided information on 360 lipids (including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids), with a median coefficient of variation of 11.6% (range: 5.4-51.9). The lipids were highly correlated and exhibited a range of associations with clinical chemistry biomarkers and lifestyle factors. This platform can provide many novel insights into the effects of physiology and lifestyle on lipid metabolism, genetic determinants of lipids, and the relationship between individual lipids and CHD risk factors

Prevalence and distribution of human Plasmodium infection in Pakistan

Background: Both Plasmodium vivax and Plasmodium falciparum are prevalent in Pakistan, yet up-to-date data on the epidemiology of malaria in Pakistan are not available. This study was undertaken to determine the current prevalence and distribution of Plasmodium species across the country. Methods: A malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were reconfirmed by polymerase chain reaction (PCR). Confirmed parasite-positive samples were subjected to species-specific PCR capable of detecting four species of human malaria. Results: Of the 707 PCR-positive samples, 128 (18%) were P. falciparum, 536 (76%) were P. vivax, and 43 (6%) were mixed P. falciparum and P. vivax. Ninety-four microscopy-positive samples were PCR-negative, and Plasmodium malariae and Plasmodium ovale were not detected. Prevalence of P. vivax ranged from 2.4 % in Punjab Province to 10.8 % in Sindh Province and prevalence of P. falciparum ranged from 0.1 % in Islamabad to 3.8 % in Balochistan. Conclusions: Plasmodium infections in Pakistan are largely attributed to P. vivax but P. falciparum and mixed species infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high

Effects of a high-dose 24-h infusion of tranexamic acid on death and thromboembolic events in patients with acute gastrointestinal bleeding (HALT-IT): an international randomised, double-blind, placebo-controlled trial

Background: Tranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding. Methods: We did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0·9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0·9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124. Findings: Between July 4, 2013, and June 21, 2019, we randomly allocated 12 009 patients to receive tranexamic acid (5994, 49·9%) or matching placebo (6015, 50·1%), of whom 11 952 (99·5%) received the first dose of the allocated treatment. Death due to bleeding within 5 days of randomisation occurred in 222 (4%) of 5956 patients in the tranexamic acid group and in 226 (4%) of 5981 patients in the placebo group (risk ratio [RR] 0·99, 95% CI 0·82–1·18). Arterial thromboembolic events (myocardial infarction or stroke) were similar in the tranexamic acid group and placebo group (42 [0·7%] of 5952 vs 46 [0·8%] of 5977; 0·92; 0·60 to 1·39). Venous thromboembolic events (deep vein thrombosis or pulmonary embolism) were higher in tranexamic acid group than in the placebo group (48 [0·8%] of 5952 vs 26 [0·4%] of 5977; RR 1·85; 95% CI 1·15 to 2·98). Interpretation: We found that tranexamic acid did not reduce death from gastrointestinal bleeding. On the basis of our results, tranexamic acid should not be used for the treatment of gastrointestinal bleeding outside the context of a randomised trial

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.D.S. is supported by grants from the National Institutes of Health, the Fogarty International, the Wellcome Trust, the British Heart Foundation, and Pfizer. P.N. is supported by the John S. LaDue Memorial Fellowship in Cardiology from Harvard Medical School. H.-H.W. is supported by a grant from the Samsung Medical Center, Korea (SMO116163). S.K. is supported by the Ofer and Shelly Nemirovsky MGH Research Scholar Award and by grants from the National Institutes of Health (R01HL107816), the Donovan Family Foundation, and Fondation Leducq. Exome sequencing was supported by a grant from the NHGRI (5U54HG003067-11) to S.G. and E.S.L. D.G.M. is supported by a grant from the National Institutes of Health (R01GM104371). J.D. holds a British Heart Foundation Chair, European Research Council Senior Investigator Award, and NIHR Senior Investigator Award. The Cardiovascular Epidemiology Unit at the University of Cambridge, which supported the field work and genotyping of PROMIS, is funded by the UK Medical Research Council, British Heart Foundation, and NIHR Cambridge Biomedical Research Centre ... Fieldwork in the PROMIS study has been supported through funds available to investigators at the Center for Non-Communicable Diseases, Pakistan and the University of Cambridge, UK

Effectiveness of Female Heads’ Leadership Styles in School Improvement at Secondary Level

The study’s primary goal was to investigate the effectiveness of female heads’ leadership styles in school improvement at the secondary level. The study's objectives were to: 1) assess the female head teachers' leadership styles at the secondary level; 2) investigate the effectiveness of female head teachers' leadership styles in improving the school; and 3) compare head teachers' opinions based on demographics, such as professional qualification and locality. This study used a descriptive survey approach to meet its goals. The researcher used the census method to select the sample because the teacher population in the Tehsils of Okara, Depalpur, and Renala Khurd varied. Finally, 112 female head teachers of secondary schools were selected for the sample. Self-structured questionnaire comprising 60 items were used to investigate the effectiveness of female heads' leadership styles in school improvement at the secondary level. The study revealed that female head teachers’ performance in handling the six administrative tasks was rated as good overall by the teachers. The principals also rated them above average on all administrative tasks

ENVIRONMENTAL FACTORS AFFECTING WEANING WEIGHT IN LOHI SHEEP

Data on 3984 lambing records of 1285 Lohi ewes kept at the Livestock Production Research Institute, Bahadurnagar, Okara for he period 1960-90 were analyzed by using Harvey’s Mixed Model Least Squares and Maximum Likelihood Computer Program. The purpose was to estimate the magnitude of various environmental sources of variation influencing weaning weight in this breed of sheep. The least squares mean for 120-day adjusted weaning weight was 23.09  0.13 kg. The trait was significantly (P<0.01) influenced by the year and season of birth, type of birth and the sex of the lamb born

Effects of Dried Aloe Vera Gel and Diclofenac on Sodium and Potassium Homeostasis: An Experimental Study on Hypertensive Rats

Background: Anti-inflammatory role of Aloe vera gel is well established. Diclofenac is extensively used for acute and chronic inflammation. The present study was conducted to compare dried Aloe vera gel and diclofenac effects on sodium and potassium balance in hypertensive rats. Material and Methods: This experimental study was conducted at Sargodha Medical College from May to November 2016. Twenty-four healthy male Sprague Dawley rats 7-8 weeks of age were included in study. Any unhealthy-looking rat was excluded from the study. Rats were equally and randomly divided into four groups Normal control (group A), Model control (group B), Aloe vera (group C) &amp; Diclofenac (group D). Hypertension was induced by a 20 % sucrose diet in all groups except group A in 8 weeks’ time. Group B, C &amp; D received distilled water and Aloe vera dried gel 400 mg/kg &amp; diclofenac powder 12 mg/kg body weight respectively orally between 8 to 10 weeks. Serum and urine analysis was performed for hematocrit, sodium, and potassium concentrations at zero, eight and ten weeks. Twenty-four-hour urinary sodium excretion was calculated. Data was analyzed using Graph Pad Prism version 6. Result: After 2-week administration of aloe vera and diclofenac powder, serum potassium significantly decreased in Group C (p &lt;0.001) while increased in Group B and D (p &lt;0.001) as compared to group A. Urinary sodium concentration and excretion increased significantly in Group C (p &lt;0.01) as compared to Group A whereas result of Group D was insignificant. No significant change in serum sodium and hematocrit of any group was observed. Conclusion: Aloe vera causes less sodium retention than diclofenac but decreases serum potassium contrary to the effect of diclofenac in hypertensive rats