Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum)

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl₂ buffer (pH 8.2) with a flow rate of 0.5?mL min^?1. The molecular weight and purity of ～23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg^?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.     

Bleeding disorders in the tribe: result of consanguineous in breeding

Objective

To determine the frequency and clinical features of bleeding disorders in the tribe as a result of consanguineous marriages.

Design

Cross Sectional Study

Introduction

Countries in which consanguinity is a normal practice, these rare autosomal recessive disorders run in close families and tribes. Here we describe a family, living in village Ali Murad Chandio, District Badin, labeled as haemophilia.

Patients & Methods

Our team visited the village & developed the pedigree of the whole extended family, up to seven generations. Performa was filled by incorporating patients, family history of bleeding, signs & symptoms, and bleeding from any site. From them 144 individuals were screened with CBC, bleeding time, platelet aggregation studies & RiCoF. While for PT, APTT, VWF assay and Factor VIII assay, samples were kept frozen at -70 degrees C until tested.

Results

The family tree of the seven generations comprises of 533 individuals, 63 subjects died over a period of 20 years and 470 were alive. Out of all those 144 subjects were selected on the basis of the bleeding history. Among them 98(68.1%) were diagnosed to have a bleeding disorder; 44.9% patients were male and 55.1% patients were female. Median age of all the patients was 20.81, range (4 months- 80 yrs). The results of bleeding have shown that majority had gum bleeding, epistaxis and menorrhagia. Most common bleeding disorder was Von Willebrand disease and Platelet functional disorders.

Conclusion

Consanguineous marriages keep all the beneficial and adversely affecting recessive genes within the family; in homozygous states. These genes express themselves and result in life threatening diseases. Awareness, education & genetic counseling will be needed to prevent the spread of such common occurrence of these bleeding disorders in the community.     

NO synthase-generated NO acts downstream of auxin in regulating Fe-deficiency-induced root branching that enhances Fe-deficiency tolerance in tomato plants

In response to Fe-deficiency, various dicots increase their root branching which contributes to the enhancement of ferric-chelate reductase activity. Whether this Fe-deficiency-induced response eventually enhances the ability of the plant to tolerate Fe-deficiency or not is still unclear and evidence is also scarce about the signals triggering it. In this study, it was found that the SPAD-chlorophyll meter values of newly developed leaves of four tomato (Solanum lycocarpum) lines, namely line227/1 and Roza and their two reciprocal F(1) hybrid lines, were positively correlated with their root branching under Fe-deficient conditions. It indicates that Fe-deficiency-induced root branching is critical for plant tolerance to Fe-deficiency. In another tomato line, Micro-Tom, the increased root branching in Fe-deficient plants was accompanied by the elevation of endogenous auxin and nitric oxide (NO) levels, and was suppressed either by the auxin transport inhibitors NPA and TIBA or the NO scavenger cPTIO. On the other hand, root branching in Fe-sufficient plants was induced either by the auxin analogues NAA and 2,4-D or the NO donors NONOate or SNP. Further, in Fe-deficient plants, NONOate restored the NPA-terminated root branching, but NAA did not affect the cPTIO-terminated root branching. Fe-deficiency-induced root branching was inhibited by the NO-synthase (NOS) inhibitor L-NAME, but was not affected by the nitrate reductase (NR) inhibitor NH(4)(+), tungstate or glycine. Taking all of these findings together, a novel function and signalling pathway of Fe-deficiency-induced root branching is presented where NOS-generated rather than NR-generated NO acts downstream of auxin in regulating this Fe-deficiency-induced response, which enhances the plant tolerance to Fe-deficiency.     

Inter-observer agreement on subjects' race and race-informative characteristics

Health and socioeconomic disparities tend to be experienced along racial and ethnic lines, but investigators are not sure how individuals are assigned to groups, or how consistent this process is. To address these issues, 1,919 orthodontic patient records were examined by at least two observers who estimated each individual's race and the characteristics that influenced each estimate. Agreement regarding race is high for African and European Americans, but not as high for Asian, Hispanic, and Native Americans. The indicator observers most often agreed upon as important in estimating group membership is name, especially for Asian and Hispanic Americans. The observers, who were almost all European American, most often agreed that skin color is an important indicator of race only when they also agreed the subject was European American. This suggests that in a diverse community, light skin color is associated with a particular group, while a range of darker shades can be associated with members of any other group. This research supports comparable studies showing that race estimations in medical records are likely reliable for African and European Americans, but are less so for other groups. Further, these results show that skin color is not consistently the primary indicator of an individual's race, but that other characteristics such as facial features add significant information.     

Re-description and molecular characterisation of Choricotyle australiensis Roubal,Armitage & Rohde, 1983 (Monogenea: Diclidophoridae) infecting Chrysophrys auratus (Forster) (Perciformes: Sparidae)

Choricotyle australiensis Roubal, Armitage & Rohde, 1983, a diclidophorid monogenean species, is redescribed and genetically characterised using the partial nuclear 28S ribosomal RNA gene (28S rRNA) and a fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences for specimens collected from Chrysophrys auratus (Forster) off Australia and New Zealand. Previous studies have either provided morphological or genetic results, whereas this study combines morphological and advanced molecular methods. A total of 70 Ch. auratus were examined with 22 individuals of C. australiensis recovered from the gills (overall prevalence of 23%). This study has provided the first evidence for the exploration of mitochondrial cox1 region for C. australiensis. Comparison of the newly generated sequences with other available data supported the distinction of C. australiensis among diclidophorid Furhmann, 1928 species thus confirming its taxonomic status.

     

A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician’s skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as “Patho-tb”, for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen’s kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 10³ cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.     

Optimization of conformational stability and catalytic efficiency in chondroitinase ABC Ι by protein engineering methods

Chondroitinase ABC Ι can promote the recovery of spinal cord injuries by depolimerization of glycosaminoglycans. However, low thermal stability is one of the limitations regarding its clinical application. In order to increase the conformational stability of the enzyme, Leu⁶⁷⁹ at the starting point of a short helix located at the C‐terminal domain of the protein was replaced by serine (L679S mutant) and aspartic acid (L679D mutant). Theoretical and spectroscopic studies showed that the stability of enzyme increased upon mutation. Based on the activity measurements, the catalytic efficiency of L679S was improved in comparison with the wild‐type protein; while that of L679D (a more stabilized protein) was not changed. According to the structural and kinetic data, we proposed a model in which a higher conformational stability results in a slower rate of the formation of the open conformation. On the other hand, a higher flexibility slows down the rate of the formation and holding of the closed conformation. Therefore, the L679S mutant, which is structurally stable relative to the wild‐type protein and is destabilized compared to the L679D mutant, exhibited the best catalytic efficiency. However, it was also found that the L679D mutant was more suitable for long‐term storage of the enzyme.