Characterization of group B coxsackieviruses isolated from non-polio acute flaccid paralysis patients in Pakistan: vital assessment before polio eradication.

Pakistan is at the verge of polio eradication but isolation of non-polio enteroviruses (NPEVs) from acute flaccid paralysis (AFP) cases may result in serious or even fatal outcome. Many enteroviruses share similar symptoms and epidemiology as is the case with poliovirus and coxsackievirus (CV). The present study was designed to genetically characterize coxsackievirus B (CV-B) serotypes isolated from non-polio acute flaccid paralytic children, as well as to understand their probable role in paralysis. A total of 63 (20·1%) out of 313 stool samples during 2013 were found positive for NPEVs in rhabdomyosarcoma cells. Only 24 (38·0%) NPEVs were typed as CV-B by microneutralization assay and were further characterized by sequencing of the viral protein 1 (VP1) gene. Molecular phylogenetic analyses classified the study strains into six coxsackievirus B serotypes (coxsackievirus B1 to B6) with their respective prototype strains with evidence of epidemiological linkage and distinct clusters. Moreover, four major differences were found within the amino acid sequences of BC-loop in VP1 of CV-B strains. In conclusion, this study presented the molecular evolutionary genetic overview and distinct phylogenetic pattern of CV-B isolates from AFP cases in Pakistan, and explored the possible link between CV-B infections and AFP cases. Furthermore, our data reveal that these viruses might contribute to the incidence of paralysis in population and there is need of time to establish an enterovirus surveillance system for better understanding of epidemiological and virological characteristics of NPEV infections associated with AFP cases in the country.

Development, Characterization, and Evaluation of SLN-Loaded Thermoresponsive Hydrogel System of Topotecan as Biological Macromolecule for Colorectal Delivery.

BACKGROUND: Chemotherapeutic drugs cause severe toxicities if administered unprotected, without proper targeting, and controlled release. In this study, we developed topotecan- (TPT-) loaded solid lipid nanoparticles (SLNs) for their chemotherapeutic effect against colorectal cancer. The TPT-SLNs were further incorporated into a thermoresponsive hydrogel system (TRHS) (TPT-SLNs-TRHS) to ensure control release and reduce toxicity of the drug. Microemulsion technique and cold method were, respectively, used to develop TPT-SLNs and TPT-SLNs-TRHS. Particle size, polydispersive index (PDI), and incorporation efficiency (IE) of the TPT-SLNs were determined. Similarly, gelation time, gel strength, and bioadhesive force studies of the TPT-SLNs-TRHS were performed. Additionally, in vitro release and pharmacokinetic and antitumour evaluations of the formulation were done. RESULTS: TPT-SLNs have uniformly distributed particles with mean size in nanorange (174 nm) and IE of ~90%. TPT-SLNs-TRHS demonstrated suitable gelation properties upon administration into the rat's rectum. Moreover, drug release was exhibited in a control manner over an extended period of time for the incorporated TPT. Pharmacokinetic studies showed enhanced bioavailability of the TPT with improved plasma concentration and AUC. Further, it showed significantly enhanced antitumour effect in tumour-bearing mice as compared to the test formulations. CONCLUSION: It can be concluded that SLNs incorporated in TRHS could be a potential source of the antitumour drug delivery with better control of the drug release and no toxicity.

Study of low doses cisplatin synergistic effect on photodynamic outcome of aluminum phythalocyanine on soft tissue sarcoma (RD) cell line.

Photodynamic therapy (PDT) in combination with other treatment modality expects to overcome the drug resistance experienced in monotherapy. In this present work combination of chemo cum PDT is studied over the range of doses. It is found that treating cells/exposing cells to chemo drug (cisplatin, CDDP) and PDT individually results in minimal cell killing (∼7% and ∼16%) compared to the administration of chemo followed by PDT (∼50% cells were viable). These results showed that cell viability synergistically decreases in case of combination treatment as compared with individual treatment. Photodynamic therapy (PDT) in combination with CDDP chemotherapy expects to overcome the drug resistance experienced in monotherapy.

Effects of varying local temperature on the optical properties of cells in-vitro.

Increase in local temperature during light exposure of biological tissues plays an important role in determining the fate of most therapeutic modalities. Variations in the optical properties (absorption coefficient, scattering coefficient, anisotropy factor, optical depth etc.) of two cancer cell lines "Rhobdomyosarcoma and Cervical carcinoma" due to gradual increase in temperature were determine quantitatively with a double integrating sphere system. It was observed that all three coefficients showed decreasing tendency as the temperature increases for both the cell lines except for scattering coefficient of HeLa which remain constant within error limit. Anisotropy factor for both cell lines increased indicating temperature dependent subcellular density variations. Temperature dependent optical properties information may lead to precise dosimetry and could help clinicians for predicting the therapeutic modality outcome.

Assessment of thyroid function in pesticide formulators.

Thirty male pesticide formulators exposed to the dust and liquid formulation of endosulfan, quinalphos, chlorpyriphos, monocrotophos, lindane, parathion, phorate, and fenvalerate and 20 comparable control subjects from the same area of study were examined for the evaluation of thyroid function tests. The level of TSH was elevated (about 28%) in pesticide formulators as compared to a control group, but the increase was statistically insignificant. Based on the individual TSH measurement, 3 of 30 formulators had isolated elevated levels of TSH and seem to have acquired sub-clinical hypothyroidism; five had TSH values slightly elevated to the upper boarder line (4.03 muIU/ml); and the majority of formulators (N= 22) had TSH values in the normal range varying from 1.29 to 3.9 muIU/ml. Total T3 was suppressed significantly (P< 0.01) in formulators, while marginal decrease (about 7%) was noticed in T4 level. This study indicated thyroid function impairment in few pesticide formulators.

A simple chemical method for the oxidation of beta-hydroxybutyrate; application of the method for the estimation of ketone bodies.

A simple one-step method employing potassium persulphate as an oxidising agent in presence of catalyst, Ag2+, for the oxidation of beta-hydroxybutyrate (beta-OHB) to acetoacetate (AcAc) has been developed and standardized. Under the condition of assay, beta-OHB (0.079-0.395 microM) was quantitatively transformed to AcAc. The reaction linearity was observed from 0.079 to 0.634 microM. Optimum conditions were: pH, 6.2; temp., 40 degrees C; persulphate saturation, 40% and catalyst, 1.82 mM. Under the experimental condition, no reversal of inhibition caused by chloride (22.96 mM) was observed at Ag+ concentrations (9.09 and 18.18 mM), while higher conc. of Ag+ (27.27 mM) caused significant reversal of inhibition (about 60%). The maximum reversal of inhibition was achieved at Ag+ (36.36 mM). The level of ketone bodies, when estimated by the present method, was greatly enhanced during starvation period and about 2- and 12-fold higher level of ketone bodies was observed (compared to control) in rats fasted for 24 and 48 hr respectively.

Spectral evidence for the oxidation of beta-hydroxybutyrate to acetoacetate: diminution of spectra by glucose and chloride.

Chemical oxidation of beta-hydroxybutyrate (beta-OHB) to acetoacetate (AcAc) has been carried out by a simple and new method employing potassium persulphate as an oxidising agent. Under the conditions of assay, beta-OHB (0.079-0.395 microM) was instantaneously oxidised to AcAc and the authenticity of the oxidised product was proved by absorption spectroscopy. A common absorption maxima at about 446 nm was observed in all the spectra recorded for the product (AcAc-complex) obtained after the oxidation of beta-OHB (0.079-0.395 microM) to AcAc followed by coupling with diazotized p-nitroaniline. This absorption maxima was almost equal to that obtained for AcAc-complex using AcAc as reference standard. It implies that AcAc formed by the chemical oxidation of beta-OHB is identically similar to the AcAc used as reference standard for the study. This fact was further strengthened when absorption spectra, recorded either individually or in combination (mixed-type), exhibited a single peak with a common absorption maxima at about 446 nm. Absorption spectra was found to be partially diminished by glucose (1.77 microM) and chloride (17.1 microM), while almost complete diminution of absorption spectra was observed at higher concentration of glucose (8.88 microM) and chloride (51.3 microM).

Epidemiology and phylogenetic analysis of VP7 and VP4 genes of rotaviruses circulating in Rawalpindi, Pakistan during 2010.

Human group A rotaviruses (RVAs) possess a large genetic diversity and new RVA strains and G/P genotype combinations are been identified frequently. Only a few studies reporting the distribution and co-circulation of RVA G and P genotypes are available for Pakistan. This hospital based study showed a RVA prevalence rate of 23.8%, which is similar to RVA detection rates estimated in other Eastern Mediterranean countries. During 2010, the following RVA strains were found to co-circulate: G1P[8] and G2P[4] (both 24.3%), G1P[6] (12.1%), G9P[8] (10.8%), G9P[6] (5.4%), G12P[6] (6.7%), G6P[1] (2.7%) and mixed infections (6.7%). Sequence analyses of selected G1, G2, G9 and G12 RVA strains revealed a close evolutionary relationship with typical human RVA strains. Sequence identities among the Pakistani VP7 RVA genes encoding G1, G2, G9 and G12 ranged between 91.5-98.7%, 99.6-98.9%, 97.7-99.5% and 99.2-99.9%, respectively. Analysis of the VP4 genes revealed co-prevalence of distinct lineages of the P[8] genotype. P[6] and P[4] showed a close relationship with typical human RVA strains detected in several Asian countries. The two G6P[1] RVA strains were closely related to typical bovine RVA strains, suggesting one or multiple interspecies transmission events. Our data provide important baseline data on the burden of RVA disease and genotype distribution in Rawalpindi, Pakistan, which is important with respect to vaccine introduction in national immunization programs.

Genetics of the Rh-Hr locus in humans.

An analysis is attempted to throw more light on the inheritance of rhesus factor in humans and the Fisher-Race and Wiener's concepts are respectively evaluated. It is claimed that the Rh-Hr locus is not comprised of the recombination of C, D, E, c and e genes, much less the help of the auxiliary (d) which merely appears to be a conventional approach derived from an even principle of recombination for a polyhybrid cross. As for Wiener's rare or common mutants of Rh-Hr in humans and the emergence of different agglutinogens like a sort of genetic proliferation, no such parallel of inheritance has yet been found in experimental plants or animals. Probably the scarcity of certain genotypes is due to the rare chance of fertilization or embryonic development due to incompatibility between certain mutants of Rh-Hr genes, similar to ABO-Rh incompatibility.

Colorimetric assay of 2,2-bis(4-chlorophenyl)acetic acid (DDA) in urine.

2,2-Bis(4-chlorophenyl) acetic acid (DDA) was extracted from acidified urine at pH 2 by benzene. It was complexed by partitioning with crystal violet in acetic to yield a blue color which was quantitated at 610 nm. Its identity was confirmed by paper chromatographic separation. The DDA excretion in nine subjects ranged from 0.024 to 0.130 microgram/ml with a mean of 0.065 while that determined by gas chromatography ranged from 0.038 to 0.120 with a mean of 0.069.

DDT residues in human milk samples from Delhi, India.

PIP: Possible contamination of human milk through the excretion of DDT and its metabolites is a concern in India, where DDT is widely used as an insecticide. To assess this risk, milk samples collected from 60 lactating women admitted to hospitals in Delhi were quantitatively analyzed. 55 of the 60 milk samples showed evidence of residues of pp'DDE (mean, 0.176 + or - 0.382 ppm), op'DDT (mean, 0.046 + or - 0.011 ppm), and pp'DDT (mean, 0.122 + or - 0.434 ppm). The mean residue level in milk fat (mg/kg fat) was 7.280 + or - 23.240, 1.428 + or - 2.697, and 1.597 + or - 5.936, respectively. A large variation in individual values was observed. The finding that pp'DDE is the DDT metabolite excreted into human milk at the highest level is consistent with previous research. Daily intake of total DDT averaged 0.062 mg/kg of body weight--a value that is 12 times higher than the acceptable level of DDT (0.005 mg/kg/day) set by the World Health Organization. Although no harmful effects of DDT have been recorded to date in breastfed infants in India, preventive measures aimed at reducing the body burden of DDT in lactating women are urged.^ieng

Semen quality and reproductive hormones among welders -A preliminary study.

OBJECTIVES: Welders are exposed to intense heat, toxic metals and their oxides and toxic gases during welding, and some of these substances reported to have adverse effects on reproductive organs. Thus, the present study was planned to investigate the sperm quality as well as reproductive hormones among welders exposed to a complex mixture of pollutants. METHODS: Seventeen welders aged 43.8±12.0 (mean±SD) years with welding exposure of 1 to 10 years or more participated in this study. They were performing are and gas welding in a workshop. Blood (5-6 ml) and semen samples by masturbation were collected from them. Serum was separated and analyzed for reproductive hormones by RIA techniques. Semen was processed for microscopic examination. RESULTS: The results of this study indicated that sperm concentration was in the normal range (≥20 million/ml) in all seventeen welders, however, motility, morphology as well as Hypo Osmotic Swelling test (HOS) demonstrated impairment in few welders, indicating deterioration in sperm quality compared to the reported normal reference value. Compared with the normal value of hormones provided in diagnostic kits brochure, follicle stimulating hormone (FSH), leutinizing hormone (LH) and testosterone levels were in the normal range, except in two subjects who showed higher FSH and LH levels and one who showed a lower value of testosterone. CONCLUSIONS: This study suggests that welding might have had some adverse effects on sperm motility, morphology and physiologic function even though sperm concentration was in the normal range. Hormonal changes did not show any definite pattern, indicating a need for further study.