In vitro expression and inhibition of procoagulant activity produced by bovine alveolar macrophages and peripheral blood cells

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.Abbreviations BAL bronchoalveolar lavage - LPS lipopolysaccharide - cDNA cloned deoxyribonucleic acid - cAMP cyclic adenosine monophosphate - GAPDH glyceraldehyde phosphate dehydrogenase - mRNA messenger ribonucleic acid - TF tissue factor - TNF tumour necrosis factor - DPBS Dulbecco's phosphate-buffered saline     

Molecular characterisation of Theileria equi and risk factors associated with the occurrence of theileriosis in horses of Punjab (Pakistan)

Theileria equi (T. equi) is an obligate intra- and extra-erythrocytic parasite that causes equine theileriosis (ET) in equids. Equine theileriosis is considered a notifiable disease of global significance, a major constraint to the international movement of horses, and endemic in many countries. This disease may be difficult to diagnose, as it can produce variable and nonspecific clinical signs. A cross-sectional study was designed for the molecular characterisation of T. equi and to investigate the associated risk factors of ET accompanied by its consequences on haematological and sero-biochemical parameters. A convenience sampling of 500 blood samples were collected from ET suspect horses from January to December 2017. PCR was performed on all blood samples targeting the 18S rRNA gene of T. equi followed by sequencing; 9% animals tested positive with confirmed sequences. The isolates of this study showed high homology with Cuban, Russian and Brazilian isolates of T. equi (accession numbers KY111762.2 , MG551915.1 and KY952237.1 , respectively). Based on multivariate analysis, the principal risk factors consisted of absence of dogs on the premises and presence of tick infestation. The haemato-biochemical parameters showed a decrease in granulocytes and erythrocytes, and an increase in lymphocytes, monocytes, mean corpuscular volume, mean corpuscular haemoglobin, mean platelet volume, glucose, phosphorus and aspartate aminotransferase in positive horses. This is the first study which identified ET in Punjab (Pakistan) using molecular techniques and risk factors together with the haemato-biochemical variations in horses.     

Simultaneous use of plant growth promoting rhizobacterium and nitrogenous fertilizers may help in promoting growth,yield, and nutritional quality of okra

This study was conducted to evaluate the simultaneous use of plant growth promoting rhizobacterium (QS1) and different combinations of urea-nitrogen and biogas slurry-nitrogen for improving growth, yield, and nutritional quality of field grown okra. The isolate QS1 was identified as Bacillus sp. QS1 based on its ribosomal ribonucleic acid (rRNA) sequence analysis. Results revealed that among the combinations, the application of 50% urea-nitrogen + 50% biogas slurry-nitrogen was optimal for improving crop performance. This combination significantly (p ≤ 0.05) promoted okra growth viz. plant height (69.63–80.03 cm), no of branches per plant (14–18), shoot fresh weight (86.66–136.25 g/plant), root fresh weight (22.5–26.58 g/plant), and fruit yield (8.5–13.5 kg/plot) compared to the plants receiving 100% urea-nitrogen. The interactive effective of this nitrogen combination and QS1 inoculation produced the highest growth and yield response. Similarly, the same treatment improved nitrogen, phosphorus, potassium, and protein contents in shoot and fruit of okra compared to other treatments.     

Thermal,swelling and stability kinetics of chitosan based semi-interpenetrating network hydrogels

The present study is focused on studying the swelling kinetics, thermal and aqueous stabilities, and determination of various forms of water in the chitosan (CS) and polyacrylonitrile (PAN) blend and semi-interpenetrating polymer network (sIPN). CS/PAN blend hydrogel films were prepared by solution casting technique. The blend film with optimum swelling properties was selected for the synthesis of sIPN. CS in the blend was crosslinked with the vapors of Glutaraldehyde (GTA) to prepare sIPN. The fabricated CS/PAN blend and sIPN hydrogels films were characterized with Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA) and field emission scanning electron microscope (FESEM). The kinetics of swelling, bound and unbound waters and aqueous stability were determined experimentally. FESEM showed good miscibility between CS and PAN, FTIR showed no chemical interaction between CS and PAN; however, it did show a doublet for the sIPN, TGA showed improved thermal stability and swelling kinetic followed second order kinetics. The degree of swelling of the sIPN hydrogels samples at room temperature varied from ~2200 % (with a fair degree of stability (~30 %)) to ~1000 % (with high degree of aqueous stability (43 %)) with increase in the crosslinking time. The calculated unbound water (W_UB) max., for the blend was 52.3 % whereas for the bound (W_B) the max., was 41.9 %. However, for sIPN hydrogel films, the W_UB water decreased (max. 21.0 %) where as the W_B increased (max. 52.0 %). The decrease in W_UB and increase in the W_B is attributed to the formation of a compact structure and increase in the contact area between the water and polymers in sIPN hydrogels due to the induction of new water contacting point in these hydrogel films, respectively.     

Chemical Constituents of Lagenaria siceraria Mesocarp and Its Xanthine Oxidase and Alpha-Amylase Inhibitory Activities

The present study was conducted to identify natural products in the bioactive fractions of Lagenaria siceraria mesocarp. Column chromatography and gas chromatography-mass spectrometry (GC-MS) was used for separation and identification of phytochemicals. Methanolic extract and its fractions displayed alpha-amylase and xanthine oxidase inhibitory activities comparable to standard drugs. GC-MS analysis of the fractions and column eluates revealed the presence of a variety of chemical compounds, including 4-(methoxymethyl)phenol, 2,2?-methylenebis{6(1,1-dimethylethyl)-4-ethyl}phenol, methyl 2-hydroxy-3-phenylpropanoate, oxacyclododecane-2,8-dione, N-(2-(4-hydroxyphenyl)ethyl] acetamide, 2,3-Epoxycarane, 4-(propan-2-yl)benzaldehyde, octadec-1-ene, 4a-methyl-1-methylidene-1,2,3,4,4a,9,10,10a-octahydrophenanthrene, 2,6-di(propan-2-yl)naphthalene, methyl 3-(4-hydroxy-3,5-dimethylphenyl)propanoate, 4,8a-dimethyl-6-(prop-1-en-2-yl)-3,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one 1,3-dihydro-2-benzofuran, methyl N-hydroxybenzenecarboximidoate, linoleic acid, and palmitic acid. Many of these compounds are being reported for the first time from this plant, the presence of which may explain, at least partly, its medicinal efficacy. The fruit possesses significant anti-enzymatic properties against alpha-amylase and xanthine oxidase, and the most active compounds of mesocarp of the fruit appear in ethyl acetate fraction of its methanolic extract.     

Challenges for the sustainable use of water and land resources under a changing climate and increasing salinization in the Jizzakh irrigation zone of Uzbekistan

Jizzakh Province in Uzbekistan is one of the largest irrigated areas in Central Asia without natural drainage.In combination with aridity,climate change and extensive irrigation practices,this has led to the widespread salinization of agricultural land.The aim of this study was to identify opportunities to improve the reclamation status of the irrigated area and how best to effectively use the water resources in Jizzakh Province based on investigations conducted between 1995 and 2016.A database of field measurements of groundwater levels,mineralization and soil salinity conducted by the provincial Hydro-Geological Reclamation Expeditions was used in the study.The total groundwater mineralization was determined using a portable electric conductometer(Progress 1T)and the chloride concentration was determined using the Mohr method.The soil salinity analyses were conducted by applying two different methods:(1)the extraction and assessment of the soluble salt content,and(2)using an SM-138 conductivity sensor applied to a 1:1 mixture of soil sample and water.The analyses of the monitoring results and the salt balance in the"irrigation water–soil–drainage water"system clearly demonstrated that the condition of the irrigated land in the province was not significantly improved.Under these conditions,the stability of crop yields is achieved mainly through the use of large volumes of fertilizer.However,excess amounts of mineral fertilizers can also cause the salinization of soils.The average groundwater salinization value in most of the irrigated land(75.3%)fluctuated between 1.1 and 5.0 g/L,while the values were less than 1.0 g/L in 13.1%of the land and in the range of 5.1–10.0 g/L in 10.5%of the land.During the period of 1995–2016 the salinization level of the irrigated land in Jizzakh Province increased slightly and the area could be divided into the following classes:no salinity(17.7%of the total area),low salinity(51.3%),moderate salinity(29.0%),and high salinity(2.0%).Detailed studies of the salt balance in irrigated land,the impact of climate change,increased fertilizer use,and repeated remediation leaching on the groundwater level and mineralization should be conducted in the future,due to the possibility of accelerated salinization,fertility decline,and reduced yields of agricultural crops.     

Genome-wide association analysis for stripe rust resistance in spring wheat(Triticum aestivum L.) germplasm

Stripe rust is a continuous threat to wheat crop all over the world. It causes considerable yield losses in wheat crop every year. Continuous deployment of adult plant resistance(APR) genes in newly developing wheat cultivars is the most judicious strategy to combat this disease. Herein, we dissected the genetics underpinning stripe rust resistance in Pakistani wheat germplasm. An association panel of 94 spring wheat genotypes was phenotyped for two years to score the infestation of stripe rust on each accession and was scanned with 203 polymorphic SSRs. Based on D' measure, linkage disequilibrium(LD) exhibited between loci distant up to 45 c M. Marker-trait associations(MTAs) were determined using mixed linear model(MLM). Total 31 quantitative trait loci(QTLs) were observed on all 21 wheat chromosomes. Twelve QTLs were newly discovered as well as 19 QTLs and 35 previously reported Yr genes were validated in Pakistani wheat germplasm. The major QTLs were QYr.uaf.2 AL and QYr.uaf.3 BS(PVE, 11.9%). Dissection of genes from the newly observed QTLs can provide new APR genes to improve genetic resources for APR resistance in wheat crop.     

The effect of dry conditions on subsequent leakage and rotting of fungal sclerotia

Dried sclerotia of Sclerotium delphinii rotted in moist soil whereas those of Sclerotium cepivorum. Botrytis cinerea and Botrytis tulipae did not. A number of fungi invaded dried sclerotia of S. delphinii in soil, the principal coloniser found in the first sampling being Trichodermu hamatum. Leakage of ¹⁴C compounds from dried labelled sclerotia placed in water was rapid and was little affected by variation in leakage temperature from 1 to 25°C or by prolonging the drying period beyond a day. Leakage from dried sclerotia which were allowed to imbibe water through a small part of their surface was much reduced. Sclerotia which were redried after leakage leaked again when returned to water but with all four fungi the first of three leakage cycles gave the highest ¹⁴C levels. Loss in dry weight in the first leakage cycle was greater with S. delphinii than with the other three fungi and this may be related to the poor survival of dried sclerotia of S. delphinii in moist soil. Substances lost during leakage appear to originate from within sclerotial hyphae rather than from the hyphal free space.     

Involvement of CD28/CTLA4-B7 costimulatory pathway in the development of lymphadenopathy and splenomegaly in MRL/lpr mice

MRL/lpr mouse is an established animal model which develops autoimmune diseases including glomerulonephritis, sialoadenitis, hepatitis and inflammatory lung disease. Additionally, it has been reported that lpr strains uniquely accumulate CD3+ CD4- CD8- B220+ (double negative, DN) T cells in lymphoid organs leading to lymphadenopathy and splenomegaly. To investigate the role of CD28/CTLA4-B7 pathway in the development of lymphadenopathy and splenomegaly, MRL/lpr mice were treated with soluble form of CTLA4 molecules, CTLA4IgG, which efficiently blocks this pathway. It was demonstrated that (i) the development of DN T cells was independent of the CD28/CTLA4-B7 pathway, (ii) the CD28/CTLA4-B7 pathway was required for the development of lymphadenopathy and splenomegaly, (iii) the CD28/CTLA4-B7 pathway was important for the accumulation of various cell populations in the lymph node and spleen, (iv) composition of the accumulating cell populations was not altered by CTLA4IgG treatment, and (v) activation of conventional T cells and IL-4 production from conventional T cells were the CD28/CTLA4-B7 pathway dependent. Thus, we concluded that the CD28/CTLA4-B7 pathway was required for the development of full-blown lymphadenopathy and splenomegaly in MRL/lpr mice.     

The Sepsis-Coagulant Axis: A Review

Activation of coagulation is a normal component of the acute inflammatory response. Inflammatory cytokines initiate coagulation events locally at sites of inflammation by converting endothelium from an antithrombotic surface to a prothrombotic surface; by stimulating tissue factor production, which activates both the extrinsic and intrinsic coagulation systems; and by stimulating production of platelet-activating factors. The fibrinolytic system is initially activated but is subsequently inhibited. This results in a marked imbalance in coagulation and fibrinolysis resulting in a net procoagulant state. When thrombin generation and platelet activation exceed the body's capacity to inactivate or remove these factors, disseminated intravascular coagulation (DIC) results. DIC directly contributes to multiple organ failure and death associated with sepsis. Presently available treatments (ie, heparin and aspirin) are relatively ineffective in treating DIC; however, newer, more potent drugs may soon be available for clinical use.