Multiplex real-time RT-PCR assay for transfusion transmitted viruses in sero-negative allogeneic blood donors: an experience from Southern Pakistan.

BACKGROUND: Blood transfusion safety commences with healthy donor recruitment. The threat of transfusion transmitted infections is greatly minimized by serological tools but not entirely eliminated. Recently, nucleic-acid testing for blood donor screening has virtually eliminated this jeopardy. METHODS: This prospective study was conducted from February 2015 to February 2016. Samples from seronegative donors were run on multiplex assay (Cobas, S-201 system platform, Roche) in a batch of six [MP-NAT]. In case of reactive pool, tests were run on every individual sample [IDNAT]. RESULTS: Of 16957 donors, 16836 (99.2%) were replacement donors and the remaining 121 (0.7%) were voluntary donors, with a mean age of 29.09 ± 7.04 years. After serologic screening of all 16957 donors, 955 (5.6%) were found to be reactive; 291(1.71%) were reactive for hepatitis-B surface antigen, 361 (2.12%) for antibody to hepatitis C virus (anti-HCV), 14 (0.08%) for antibody to human immunodeficiency virus, 287 (1.69%) for syphilis and 2 (0.01%) for malaria. 14 (0.08%) NAT reactive donors were identified after testing the 16002 seronegative donors, with an overall NAT yield of one reactivity out of 1143 blood donations; 10 donors for HBV-DNA (HBV NAT yield-1:1600) and remaining 4 for HCV-RNA (HCV-NAT yield-1:4000). None were HIV positive. CONCLUSION: NAT has improved the safety attributes in blood products. Although the positivity rate for NAT testing is low but in view of the high prevalence of transfusion transmitted infections in our country, we recommend the parallel use of both serology and NAT screening of all donated blood.

Production and characterization of novel self-assembling biosurfactants from Aspergillus flavus.

AIM: This work was conducted to produce, purify and characterize biosurfactants from Aspergillus flavus AF612 isolated from citrus fruit. METHODS AND RESULTS: Biosurfactant named 'Uzmaq' was isolated from A. flavus AF612. The chemical characterization of the biosurfactant was conducted. Biosurfactant Uzmaq produced by A. flavus, was composed of methoxy phenyl oxime glycosides. Two molecular forms of the biosurfactant, Uzmaq-A and Uzmaq-B were isolated. Biological properties (antifungal activity) were evaluated. The fractions of the biosurfactant were isolated and their surface properties were analysed. Uzmaq-A and Uzmaq-B had critical micelle concentration (CMC) around 170 and 80 mg l(-1) , and lowered surface tension of water up to 20 and 25 m Nm(-1) respectively. The biosurfactants were stable at pH 3-12 and temperature up to 80°C. Growth and biosurfactant production kinetics were also analysed. CONCLUSION: Novel biosurfactant Uzmaq was produced from A. flavus, which was composed of methoxy phenyl oxime glycosides. The surface activity of Uzmaq was better than the maximum values of synthetic chemical surfactants. The biosurfactant showed antifungal activity and self-assembling properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus flavus AF612 can be used for commercial production of Uzmaq that may be employed for controlled drug release applications and bioremediation.

CF-LIBS based elemental analysis of Saussurea simpsoniana medicinal plant: a study on roots, seeds, and leaves.

The plant Saussurea Simpsoniana, which has been used in traditional medicine for its biocompatibility and abundant nutrients, offers a wide range of remedies. Local communities effectively utilize medicines derived from the plant's roots to treat various ailments such as bronchitis, rheumatic pain, and abdominal and nervous disorders. In this study, we present an elemental analysis of the chemical composition (wt%) of this medicinal plant using the laser-induced breakdown spectroscopy (LIBS) technique. In the air atmosphere, an Nd:YAG (Q-switched) laser operating at a wavelength of 532 nm is utilized to create plasma on the sample's surface. This laser has a maximum pulse energy of approximately 400 mJ and a pulse duration of 5 ns. A set of six miniature spectrometers, covering the wavelength range of 220-970 nm, was utilized to capture and record the optical emissions emitted by the plasma. The qualitative analysis of LIBS revealed the presence of 13 major and minor elements, including Al, Ba, C, Ca, Fe, H, K, Li, Mg, Na, Si, Sr, and Ti. Quantitative analysis was performed using calibration-free laser-induced breakdown spectroscopy (CF-LIBS), ensuring local thermodynamical equilibrium (LTE) and optically thin plasma condition by considering plasma excitation temperature and electron number density. In addition, a comparison was made between the results obtained from CF-LIBS and those acquired through energy-dispersive X-ray spectroscopy (EDX) analysis.

Public health surveillance at a mass gathering: urs of Baba Farid, Pakpattan district, Punjab, Pakistan, December 2010.

The objective of this study was to identify health related problems encountered during an annual mass gathering in Pakpattan, Pakistan, the anniversary of the death (urs) of Baba Farid, and to make recommendations for planning and prevention activities. A surveillance system was established to capture health related data for the event. A list of reportable diseases was developed. Data were collected pre-, during, and post-event by health care workers trained for the purpose. A total of 5918 people reported to the 15 health care facilities providing services during the event; 58% of consultations were because of communicable diseases, 21% of which were respiratory tract infections and 26% gastrointestinal illness. Injuries accounted for 31% of cases and noncommunicable diseases for 11%. Prevalence of reportable disease during the event showed sizeable increases. No major disease outbreak was observed.

Nanostructured materials for water desalination.

Desalination of seawater and brackish water is becoming an increasingly important means to address the scarcity of fresh water resources in the world. Decreasing the energy requirements and infrastructure costs of existing desalination technologies remains a challenge. By enabling the manipulation of matter and control of transport at nanometer length scales, the emergence of nanotechnology offers new opportunities to advance water desalination technologies. This review focuses on nanostructured materials that are directly involved in the separation of water from salt as opposed to mitigating issues such as fouling. We discuss separation mechanisms and novel transport phenomena in materials including zeolites, carbon nanotubes, and graphene with potential applications to reverse osmosis, capacitive deionization, and multi-stage flash, among others. Such nanostructured materials can potentially enable the development of next-generation desalination systems with increased efficiency and capacity.

Grain Size and Interface Dependence of Bias Stress Stability of n-Type Organic Field Effect Transistors.

The effect of grain size and interface dependence of bias stress stability of C60-based n-type organic field effect transistors (OFETs) has been studied. It has been realized that, with increasing grain size of C60, the bias stress induced threshold voltage shift can be controlled and this effect is mainly attributed to the mechanism of charge trapping at grain boundaries. It is further studied that the growth of C60 on the surface of parylene at elevated substrate temperature leads to the creation of radicals at the interface between the active layer and the gate dielectric. These radicals help to improve the bias stress stability of C60-based n-type OFETs. For achieving the bias stress stability, we have presented a procedure of creation of radicals at the interface between C60 and parylene in single gate OFETs instead of dual gate OFETs.

A hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin.

A simple hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin is described. The assay is based on the use, as indicator cells, of sheep red blood cells coated with monospecific antibody against the hemoglobin variant, another complement fixing multispecific anti-hemoglobin antibody as developing serum and an antibody against red cell ghosts which can lyse only the red cells of the species being studied and not the indicator sheep red cells. The test red cells are mixed with the indicator sheep red cells, the developing antiserum and the anti-ghost antibody. The mixture is transferred into thin chambers prepared with 2 microscope slides and incubated at room temperature (25 degrees C). The indicator sheep red cells around each test red cell carrying abnormal or mutant hemoglobin are lysed resulting in the formation of a plaque. Instead of anti-hemoglobin-coated sheep red blood cells, protein A-coated sheep red blood cells may be used as indicator cells provided the developing antiserum is monospecific for the hemoglobin variant. When sheep red blood cells coated with an antibody specific for DBA/2J mouse hemoglobin were used in the assay, only red cells from DBA/2J mice (carrying d hemoglobin) formed plaques while C57BL/6J red cells (carrying s hemoglobin) did not.

Senescence induced structural reorganization of thylakoid membranes in Cucumis sativus cotyledons; LHC II involvement in reorganization of thylakoid membranes.

We report the formation and appearance of loosely stacked extended grana like structures along with plastoglobuli in the chloroplasts isolated from 27-day old senescing cucumber cotyledons. The origin and the nature of these extended grana structures have not been elucidated earlier. We isolated Photosystem I complexes from 6-day-old control and 27-day-old senescing cotyledons. The chlorophyll a/b ratio of the isolated Photosystem I complex obtained from 6-day cotyledons was 5-5.5 as against a ratio of 2.9 was found in Photosystem I complexes obtained from 27-day-old senescing cotyledons. We also found that the presence of LHC II in the Photosystem I complexes isolated from 27-day cotyledonary chloroplasts. The presence of LHC II in Photosystem I complexes in senescing and not in control samples, clearly suggest the detachment and diffusion of LHC II complexes from stacked grana region to Photosystem I enriched stroma lamellar region thereby, forming loose disorganized extended grana structures seen in the transmission electron microscope. Furthermore, we show that under in vitro condition the senescing cotyledon chloroplasts exhibited lower extent of light induced phosphorylation of LHC II than the control samples suggesting a possible irreversible phosphorylation and diffusion of LHC II in vivo during the progress of senescence in Cucumis cotyledons. From these findings, we suggest that the senescence induced phosphorylation of LHC II and its migration towards Photosystem I may be a programmed one some how causing the destruction of the thylakoid membrane. The released membrane components may be stored in the plastoglobuli prior to their mobilization to the younger plant parts.

The induction of neurotrophin and TRK receptor mRNA expression during early avian embryogenesis.

Nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, designated neurotrophins, are a family of neurotrophic factors, having important functions in the survival of embryonic and adult neuronal subpopulations. Through the trk family of receptors, these neurotrophins utilize phosphotyrosine-mediated signal transduction. We have used RT-PCR to detect the expression of mRNA for the above neurotrophins and their respective receptors, namely trkA, trkB and trkC in embryonic stages 1-8 of chicken development. While trkA and trkC mRNAs were expressed from stage 1 onwards, NGF and NT-3 mRNAs were expressed only at stages 3 and 5, respectively. In contrast, BDNF mRNA was expressed at stage 1, being the only neurotrophin expressed prior to expression of its respective receptor trkB. However, the latter was not expressed until stage 8. These results indicate an earlier expression of some but not all trk proto-oncogenes, suggesting that the two different receptor mRNAs expressed i.e. trkA and trkC in conjunction with BDNF, at stage 1, may act in aspects of very early embryonic development, such as gastrulation. Thereafter, mRNAs for trkB, NGF and NT-3 are expressed reflecting their later action in early embryonic development.

Development of in vivo somatic mutation system using antibody against hemoglobin: preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells.

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin "single" (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin "diffuse" (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit anti-sera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with a d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4 X 10(-6). Thus, the system is sensitive enough to detect d leads to s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at beta 20 position and/or a substitution of threonine by alanine at beta 139 position.

Seroprevalence of HIV, HBV, and syphilis and associated risk behaviours in male transvestites (Hijras) in Karachi, Pakistan.

In Pakistan, male sex workers are predominantly transvestites and transsexuals known as Hijras. In 1998 in Karachi, Pakistan, we studied the seroprevalence of HIV, HBsAg and syphilis and associated risk factors in Hijras. Study subjects were verbally administered a structured questionnaire that determined risk behaviours for sexually transmitted and blood-borne diseases and knowledge of AIDS/STDs. After pre-test counselling, verbal consent was taken for serological testing. The results were provided on a one-on-one post-test counselling session. Three hundred male transvestites were approached; all agreed to answer the questionnaire, 208 consented to blood testing. Of 300, 81% acknowledged commercial sex with men. Of 208, prevalence of syphilis was 37%; HBsAg 3.4%; HIV 0%. The prevalence of HIV and hepatitis B virus (HBV) is low in transvestite sex workers but that of syphilis is high. Intervention programmes implemented at this stage can have an impact on HIV and STD prevention.

Kinetics of alpha-amylase production in a batch and a fed-batch culture of Bacillus subtilis.

Analysis of the kinetics of alpha-amylase production in a batch and a fed-batch culture of Bacillus subtilis made it possible to derive a kinetic model of the process describing mutual interactions between growth and production. The specific growth rate is limited by the concentration of both corn-steep liquor and starch. Higher concentrations of reducing sugars in the medium also inhibit growth. The overall production of alpha-amylase is a result of an equilibrium between the rate of enzyme production and its degradation due to the effect of environment. The actual specific production rate is directly proportional to the specific growth rate (characterizing the physiological state of the culture) and is inhibited by higher concentrations of corn-steep liquor in the medium.

Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons.

Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the beta-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.

Age dependent alterations in photosystem II acceptor side in Cucumis sativus cotyledonary leaf thylakoids: analysis of binding characteristics of herbicide [14C]-atrazine.

Senescence induced temporal changes in photosystems can be conveniently studied in cotyledonary leaves. We monitored the protein, chlorophyll and electron transport activities in Cucumis sativus cv Poinsette cotyledonary leaves and observed that by 20th day, there was a 50%, 41% and 30-33% decline in the chlorophyll, protein and photosystem II activity respectively when compared to 6th day cotyledonary leaves taken as control. We investigated the changes in photosystem II activity (O2 evolution) as a function of light intensity. The photosystem II functional antenna decreased by 27% and the functional photosystem II units decreased by 30% in 20-day old cotyledonary leaf thylakoids. The herbicide [14C]-atrazine binding assay to monitor specific binding of the herbicide to the acceptor side of photosystem II reaction centre protein, D1, showed an increase in the affinity for atrazine towards D1 protein and decrease in the QB binding sites in 20th day leaf thylakoids when compared to 6th day leaf thylakoids. The western blot analysis also suggested a decrease in steady state levels of D1 protein in 20th day cotyledonary leaf thylakoids as compared to 6th day sample which is in agreement with [14C]-atrazine binding assay and light saturation kinetics.

Laboratory scale studies on removal of chromium from industrial wastes.

Chromium being one of the major toxic pollutants is discharged from electroplating and chrome tanning processes and is also found in the effluents of dyes, paint pigments, manufacturing units etc. Chromium exists in aqueous systems in both trivalent (Cr(3+)) and hexavalent (Cr(6+)) forms. The hexavalent form is carcinogenic and toxic to aquatic life, whereas Cr(3+) is however comparatively less toxic. This study was undertaken to investigate the total chromium removal from industrial effluents by chemical means in order to achieve the Pakistan NEQS level of 1 mg/L by the methods of reduction and precipitation. The study was conducted in four phases. In phase I, the optimum pH and cost effective reducing agent among the four popular commercial chemicals was selected. As a result, pH of 2 was found to be most suitable and sodium meta bisulfate was found to be the most cost effective reducing agent respectively. Phase II showed that lower dose of sodium meta bisulfate was sufficient to obtain 100% efficiency in reducing Cr(6+) to Cr(3+), and it was noted that reaction time had no significance in the whole process. A design curve for reduction process was established which can act as a tool for treatment of industrial effluents. Phase III studies indicated the best pH was 8.5 for precipitation of Cr(3+) to chromium hydroxide by using lime. An efficiency of 100% was achievable and a settling time of 30 minutes produced clear effluent. Finally in Phase IV actual waste samples from chrome tanning and electroplating industries, when precipitated at pH of 12 gave 100% efficiency at a settling time of 30 minutes and confined that chemical means of reduction and precipitation is a feasible and viable solution for treating chromium wastes from industries.