Clinical manifestations and outcome of patients with primary amoebic meningoencephalitis in Pakistan. A single-center experience.

Primary amoebic meningoencephalitis (PAM) is a rapidly progressing central nervous system (CNS) infection caused by Naegleria fowleri, a free-living amoeba found in warm freshwater. The disease progression is very rapid, and the outcome is nearly always fatal. We aim to describe the disease course in patients admitted with PAM in a tertiary care center in Karachi, Pakistan between the periods of 2010 to 2021. A total of 39 patients were included in the study, 33 males (84.6%). The median age of the patients was 34 years. The most frequent presenting complaint was fever, which was found in 37 patients (94.9%) followed by headache in 28 patients (71.8%), nausea and vomiting in 27 patients (69.2%), and seizures in 10 patients (25.6%). Overall, 39 patients underwent lumbar puncture, 27 patients (69.2%) had a positive motile trophozoites on CSF wet preparation microscopy, 18 patients (46.2%) had a positive culture, and 10 patients had a positive PCR. CSF analysis resembled bacterial meningitis with elevated white blood cell counts with predominantly neutrophils (median, 3000 [range, 1350-7500] cells/µL), low glucose levels median, 14 [range, 1-92] mg/dL), and elevated protein levels (median, 344 [range, 289-405] mg/dL). Imaging results were abnormal in approximately three-fourths of the patients which included cerebral edema (66.7%), hydrocephalus (25.6%), and cerebral infarctions (12.8%). Only one patient survived. PAM is a fatal illness with limited treatment success. Early diagnosis and prompt initiation of treatment can improve the survival of the patients and reduce mortality.

A novel method to estimate the dead layer of HPGe detector for Monte Carlo FEPE computation.

In this study, a novel method to determine the surface and lateral dead layers of p-type HPGe detector is proposed to compute the full energy peak efficiency (FEPE). The method employed standard radioactive point sources 241Am, 133Ba and FEPE measurement at low energies to estimate the thickness of frontal and lateral dead layers. The method is simple to apply, requires only two standard radioactive sources to estimate the optimum thickness of frontal and lateral dead layers. The proposed method is validated by measuring the efficiency of various point sources and a volume source in the energy range from 59 to 1408 keV. The measured efficiencies agree to simulation with relative deviation less than 4.0% at each energy. The proposed detector model enables to calibrate the detector for environmental radioactivity measurement without standard volume sources.

Dosimetric properties of thermoluminescent NaCl pellets from Khewra Salt Mines, Pakistan.

Thermoluminescence (TL) and extended dosimetric characteristics of naturally occurring sodium chloride (NaCl) salt were studied. Pellets were prepared from mined crystalline salt obtained from Khewra Salt Mines, Pakistan and irradiated from 1 mGy to 10,000 mGy using cobalt-60 gamma source. The TL response showed two dominant peaks around 125°C and 230°C, respectively, at low doses, with an additional peak in between at doses beyond 300 mGy. A linear and supra-linear TL response was observed between 1 mGy-100 mGy and 100 mGy-10 Gy dose ranges, respectively. During the first 24 h post-irradiation, the TL intensity dropped by 20%. A maximum angular dependence of up to 50% was observed between 0° to 360°. For photon energies between 33 keV and 1.25 MeV significant energy dependence was observed for photons < 100 keV only. Sample sensitivity increased with dose a qualitatively similar behaviour to TLD-200. Effective atomic number (Zeff ) of the sample (14.6) was comparable to TLD-200 (16.3). No significant dose rate effects (deviation for a cobalt-60 source within 3.5%) on the TL sensitivity of the sample were found. The lowest detectable dose limit (LDDL) for salt sample was found to be 0.8 mGy whereas the sample reproducibility test showed a maximum of ±11% deviation from the first value.

Thermoluminescence (TL), kinetic parameters and dosimetric features of Pakistani limestone.

Thermoluminescence (TL), kinetic parameters and dosimetric features of Pakistani limestone (CaCO3) is reported in this study. Both compositional and structural analyses reveal that the material has a crystalline nature with rhombohedral structure and non-uniform crystallite size having major content of CaCO3. A powdered limestone sample of 30 mg is found to be the optimized weight for TL and other dosimetric studies. After irradiating the samples with a test dose of 100 Gy using a ß source three composite glow peaks termed as P1, P2 and P3 are visible at 100, 230 and 330 °C respectively using a linear heating rate of 1 °C/s during the TL readout. The Coefficient of Variation (COV) is found to be about 4%. Kinetic parameters (i.e., frequency factor (f), activation energy (E), and the kinetic order (b)) are estimated using both first and second Order of kinetics using an in-house Computerized Glow Curve Deconvolution (GCD) software. The figure-of-merit (FOM) is found to be 2.12%. The distribution of continuum traps with activation energy in the range of 0.77-2.59 eV is observed in the kinetic parameter analysis of the glow peaks of the sample. The TL response in the dose range of 1-5 Gy (not reported previously) and linearity in the dose response in the dose range of 1-10 Gy is observed in samples of Pakistani limestone. The Minimum Detectable Dose (MDD) is 1.01 Gy clearly resembling the experimentally linear fitted results. After a fading study for a period of thirty days, only the first peak i.e., P1 majorly fades while no major change is observed in the amplitude of peaks P2 and P3. In addition, P1 is the main contributor fading by 92% within the first 24 h of irradiation while P2 fades by 30 %. However, P3 shows stability with a very minor fading of 0.05% within 24 h of irradiation. This study concludes that Pakistani limestone can be further assessed as a potential radiation dosimeter for various applications.

Computerized glow curve deconvolution (CGCD): A comparison using asymptotic vs rational approximation in thermoluminescence kinetic models.

In this study an open-source tool GCD Analyzer, based on Microsoft® Excel, for Computerized Glow Curve Deconvolution analysis (CGCD) of thermoluminescence (TL) glow peak has been developed using a more accurate rational approximation. It is capable of deconvolution of glow curves having discrete or continuous trap distribution and can be used for routine as well as emergency radiation dosimetric analysis. This tool has a unique feature of TL glow curve analysis by combining discrete and continuous energy distributions for crystalline, amorphous, and mixed materials. To obtain best values of trap parameters like activation energy (E), frequency factor (s), order of kinetics (b), GCD Analyzer has the capability of analyzing glow curve using selectable individual peak of various physical kinetic models i.e. FOK, SOK, GOK, MOK, and Continuous Traps Distribution (CTD) with subtraction of optional background signal. The residual graph gives a clear visual understanding of the Figure of Merit (F.O.M). A comparison of asymptotic and rational approximation to the built-in second order exponential integral function E2(E/KT) for E/KT < 600 is also presented. The results are verified by deconvolution of test synthetic glow curves with F.O.M up to 0.0005%, experimental glow curve for CTD with F.O.M of 0.9781%, and in the case of GLOWCANIN project glow curves, the F.O.M is comparable to the least values achieved by inter-comparison participants.

Assessment of Medication Dosage Adjustment in Hospitalized Patients With Chronic Kidney Disease.

Background Inappropriate medication dosing can cause adverse drug reactions or ineffective therapy due to declined renal function in patients with renal insufficiency. This necessitates proper renal dose adjustment. This study was proposed to evaluate medication dosage adjustment in hospitalized chronic kidney disease (CKD) patients. Methods This study included all CKD patients hospitalized between May 1, 2019, and April 25, 2020, at the Institute of Kidney Disease, Peshawar, Pakistan. The estimated glomerular filtration rate was calculated using the Modification of Diet in Renal Disease formula, and dose appropriateness was established by evaluating practice with relevant reference books. Results Of the total 1,537 CKD patients, 231 (15.03%) had evidence of dosing error, which was considered for final analysis. Overall, 1,549 drugs were prescribed; 480 (30.99%) drugs required dose adjustment, of which 196 (40.42%) were adjusted properly and the remaining 286 (59.58%) were unadjusted. The most common unadjusted drugs were meropenem, cefepime, ciprofloxacin, and rosuvastatin, whereas captopril, aspirin, bisoprolol, pregabalin, and levofloxacin had the highest percentage of adjusted drugs. On multivariate logistic regression, the number of drugs requiring dosing adjustments and obstructive nephropathy were found to be statistically significant factors that increased the likelihood of the medication dosing errors: a unit increase in the number of drugs requiring dose adjustment increases 5.241 times the likelihood of dosing error. Similarly, the presence of obstructive nephropathy (OR: 0.383; 95% Cl: 0.153-0.960; p = 0.041) was found to be significantly associated with dosing error after adjustment for potential confounding factors. Conclusion The dosing of more than half of the prescribed drugs that required adjustment in CKD patients was not adjusted, which showed that medication dosing errors were high. This highlights the importance of medication prescription according to guidelines in these patients to improve the outcomes of pharmacotherapy.

Thermoluminescence study of pellets prepared using NaCl from Khewra Salt Mines in Pakistan.

In this study, the thermoluminescence characteristics of naturally occurring salt (NaCl) were assessed for the development of a radiation dosimeter. For this purpose, mined crystalline samples of salt were procured directly from Khewra salt mines in Pakistan. The samples were hand crushed, sieved, and compressed to pellets comparable in size to standard TLD chips, and irradiated to gamma radiation doses in the range of 5 mGy and 5000 mGy. Thermoluminescence (TL) response showed three main peaks in the glow curve around 115-130 °C, 150-170 °C, and 220-240 °C. A linear TL response was observed for the dose range of 5-100 mGy. The TL response became supra-linear for the dose ranges of 100-1000 mGy and 1000-5000 mGy. The Tm-Tstop method was applied to identify the overlapping peaks of the glow curve. Computerized glow curve deconvolution (CGCD) was then employed for the characterization of electron trap parameters such as frequency factor (s), activation energy (E), and the kinetic order (b), using General Order (GO) kinetics. The figure-of-merit (FOM) was found to be 1.08%, 0.94%, 0.77%, and 0.75%, at 500 mGy, 1 Gy, 2 Gy, and 5 Gy, respectively. The TL intensity faded by 20% within the first 24 h after irradiation and finally stabilized after two weeks. In addition, structural, morphological, and elemental analyses, were also performed using various analytical techniques. X-ray diffraction (XRD) showed that the salt crystallizes in a face-centered cubic structure. Scanning electron microscope (SEM) micrographs indicated that the crystallites are closely packed and cubic-shaped with non-uniform size, and mostly found in the agglomerated form. Similarly, the elemental analysis confirmed the presence of impurities such as Mg, Sr, S, K, O, and Ca, in the samples. The present study concludes that the pellets made from salt samples from Khewra mines have a potential for use as radiation dosimeters.

Reorganising for COVID-19 Pandemic: A Review of Structural Modifications by the Department of Internal Medicine in Low to Middle-income Countries.

Scientists classified the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a novel coronavirus on January 7, 2020. The World Health Organization (WHO) acknowledged the SARS-CoV-2 outburst a public health emergency of international concern on January 30, 2020. Since its origin, this virus has disrupted the best healthcare systems, economies, and strained financial resources; and for underdeveloped countries' healthcare systems, the virus has become a crisis. To tackle the potential hazards from this virus, our Department restructured the services that not only helped us to cope with the pandemic, but also provided an example to copy for other contemporary institutes. This article aims to describe the measures and structural changes undertaken by the Internal Medicine Department, Aga Khan University Hospital (AKUH), Karachi, Pakistan. Key Words: COVID-19, Internal medicine, Structural modifications, Pandemic.

Predatory functional response and fitness parameters of Orius strigicollis Poppius when fed Bemisia tabaci and Trialeurodes vaporariorum as determined by age-stage, two-sex life table.

BACKGROUND: The polyphagous predatory bug O. strigicollis is an active predator used to control thrips and aphids. The whitefly species Bemisia tabaci and Trialeurodes vaporariorum are voracious pests of different economic agricultural crops and vegetables. METHOD: In this study, the Holling disc equation and the age-stage, two-sex life table technique were used to investigate the functional response and biological traits of third instar nymphs and adult female O. strigicollis when presented third instar nymphs of both whitefly species as prey. RESULTS: The results showed a type II functional response for each life stage of O. strigicollis when fed each whitefly species. The calculated prey handling time for different O. strigicollis life stages were shorter when fed T. vaporariorum than when fed B. tabaci nymphs. In contrast, the nymphal development of O. strigicollis was significantly shorter when fed B. tabaci than T. vaporariorum nymphs. Additionally, the total pre-oviposition period of adult females was statistically shorter when fed B. tabaci nymphs than T. vaporariorum nymphs. Furthermore, the survival rates and total fecundity of O. strigicollis were higher when fed B. tabaci than T. vaporariorum. There were no significant differences in any population parameters of O. strigicollis when fed either whitefly species. These results show that O. strigicollis could survive and maintain its populations on both species of whitefly and could therefore serve as a biological control agent in integrated pest management (IPM).

Determination of fitness traits of Orius strigicollis Poppius (Hemiptera: Anthocoridae) on Pectinophora gossypiella (Lepidoptera: Gelechiidae) using two-sex life table analysis.

BACKGROUND: Pink bollworm (Pectinophora gossypiella) is a destructive insect pest of cotton crops in China and globally, which is actively predated on by Orius strigicollis. Studies on the fitness or survival of O. strigicollis fed on P. gossypiella at different temperatures have not been reported. The fitness of O. strigicollis may be well explained using two-sex life table parameters. Thus, the present study provides important insights for the effective biocontrol of P. gossypiella. METHODOLOGY: Considering the importance of fitness parameters and biocontrol, the present study explores the feeding potential and age-stage, two-sex life table traits of O. strigicollis on P. gossypiella eggs at different temperatures (24, 28 and 32 °C) in the laboratory. RESULTS: The intrinsic rate of increase (r) was higher at 28 °C (0.14 d-1) than at 24 °C (0.0052 d-1) and 32 °C (0.12 d-1). Similarly, the net reproductive rate (R 0) was higher at 28 °C (17.63 offspring) than at 24 °C (1.13 offspring) and 32 °C (10.23 offspring). This concluded that the maximum feeding potential and growth capacity of O. strigicollis could be attained at 28 °C when fed on P. gossypiella eggs. O. strigicollis adults preferred to feed on P. gossypiella eggs compared with first instar larvae. Based on these results, the present study suggests that O. strigicollis represents a promising biological control agent against P. gossypiella eggs in cotton fields.

Temperature-Dependent Functional Response of Harmonia axyridis (Coleoptera: Coccinellidae) on the Eggs of Spodoptera litura (Lepidoptera: Noctuidae) in Laboratory.

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is a major pest of several economically important crops with worldwide distribution. Use of insecticides is the principal strategy for its management, which has subsequently led to insecticide resistance and control failures. Functional response of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) at larval and adult stages was evaluated in this study, using S. litura eggs as the prey at various temperatures varying between 15 and 35 °C. Based on logistic model findings, linear parameters of various predatory stages of H. axyridis at various temperatures were significantly negative, which indicate a type II functional response. The theoretical maximum number (T/Th) of eggs consumed increased with increasing temperature for all predatory stages. According to the random predator equation, the coefficients of attack rate increased and that of handling time decreased as the temperature increased. The 4th instar and adult stages were superior candidates for biocontrol of the target prey, typically at higher temperatures. The maximum attack rate (0.546 ± 0.058 h) and lowest handling time (0.189 ± 0.004 h-1) were exhibited by the females at 30 and 35 °C, respectively, whereas these parameters were inferior for early instars. These findings clearly depict that the 4th instar and adult predators are efficient egg consumers and can serve as potential suppressors of S. litura field populations. The limitations of the predictions formulated by functional response trials are also discussed.

Lethal and Sublethal Effects of Chlorpyrifos on Biological Traits and Feeding of the Aphidophagous Predator Harmonia axyridis.

Except of pest control, insecticides have shown adverse effects on natural enemies as well. Thus, risk assessment of pesticides for biological control agents is critical for effective use in integrated pest management (IPM) schemes. In the present study, the lethal and sublethal effects of chlorpyrifos, a commonly used insecticide that may negatively affect biological control agents, were evaluated on a non-target predator, the Asian ladybeetle Harmonia axyridis. Previous studies have reported on lethal concentrations, but the effects of sublethal concentrations remain unclear. Lethal and sublethal concentrations of chlorpyrifos were applied to third instar larvae of H. axyridis, and different growth and developmental parameters were measured. Treatment with LC10 (4.62 mg a.i. L-1) significantly shortened the developmental period of third instar larvae, whereas it significantly prolonged those of fourth instar larvae and pupa. Treatment with LC30 (9.59 mg a.i. L-1) significantly increased the larval and pupal developmental period compared with that of the control, whereas feeding potential, female fecundity, and adult longevity significantly decreased after LC10 and LC30 treatment. The pre-oviposition period significantly increased compared with that of the control. Population growth parameters, the finite (λ) and intrinsic rate of increase (r) and the net reproductive rate (R0), decreased following exposure to sublethal concentrations of chlorpyrifos. According to the results, the use of chlorpyrifos in IPM schemes requires further research because even sublethal concentrations of this insecticide were harmful to H. axyridis population growth.

Study of CdTe detection efficiency for medical applications using Geant4-based stochastic simulations.

The determination of detection efficiency and peak-to-total ratios has been performed for rectangular CdTe detectors for various x-ray and low-energy γ-ray source configurations including parallel beams, point, and cylindrical sources. The dependence of efficiency values on axial and off-axial distances, detector thickness and area, and source dimensions has been studied. The detector model developed in this work has been validated by comparing the Monte Carlo simulated values of detector efficiency for a parallel incident beam with the available published data and good agreement has been found with discrepancies remaining within 2% throughout the energy range. Geant4 simulations show nearly 100% photopeak and total efficiency with peak-to-total ratios approaching a maximum value of 1.0 for photons in the 4-70 keV energy range. Similar high values of detection efficiency have been obtained for brachytherapy I-125 seed sources having cylindrical geometries which indicates the suitability of CdTe detectors for the calibration of sources used in therapy. The logistic power curve was found excellent for empirically fitting the photopeak efficiency variations with axial displacement of the I-125 brachy source in the horizontal configuration. Geant4 simulations clearly show that small thicknesses, of the order of 0.5 mm, of CdTe material are sufficient for attaining almost 100% detection efficiency for low-energy photons having energies up to 100 keV.

Estimation of peripheral dose from Co60 beam in water phantom measured in Secondary Standard Dosimetry Laboratory, Pakistan.

BACKGROUND: Peripheral or scatter dose harms neighbouring normal tissues during administration of dose to cancerous tissues, therefore, knowledge of peripheral dose is an important consideration in radiotherapy. AIM: In present study, absorbed dose measurements in a water phantom were performed for three field sizes, 7 × 7 cm2, 10 × 10 cm2 and 15 × 15 cm2. MATERIALS AND METHODS: For each field size, dose was measured at six depths below the front surface of the water phantom; 2.5-15 cm with an interval of 2.5 cm. Measurements were made at equal transverse distances along the horizontal axis, from 1 cm to 6 cm, on both sides of the central beam axis and normalized with central axis dose of each field. All measurements were made at the source to surface distance of 100 cm. RESULTS: Variation of peripheral dose with lateral distance was analysed and an appropriate parametric equation for each field size and depth was constructed. CONCLUSIONS: The peripheral radiation dose showed a strong dependence on field size and distance from field boundary.

Synthesis of Gallic-Acid-1-Phenyl-1H-[1,2,3]Triazol-4-yl Methyl Esters as Effective Antioxidants.

Using a click chemistry approach, a series of gallic-acid-1-phenyl-1H-[1,2,3]triazol-4-ylmethyl esters was synthesized to develop more effective antioxidants. The results of DPPH screening indicate that few of the synthesized analogs display better antioxidant effect compared to the standards. Among all, compounds, 9 and 20 displayed highest DPPH radical scavenging effect with IC50 values as low as 6.4±0.2 and 7.9±0.4 µM respectively, compared to the standard ascorbic acid (IC50=12±0.8 µM) and gallic acid (IC50=9.0±0.6 µM). Compound 10 also displayed a potent antioxidant effect with IC50 of 10.80±0.4 µM. This study provides an important aspect with regard to the use of these gallic-acid based synthetic antioxidants in food industry as dietary supplements.

Isolation and characterization of alborixin from Streptomyces scabrisporus: A potent cytotoxic agent against human colon (HCT-116) cancer cells.

The ethyl acetate extract from the fermentation broth of an actinomycete strain, identified as Streptomyces scabrisporus isolated from soil of Kashmir Himalayas - India, exhibited significant cytotoxic activity against a panel of human cancer cell lines. The active fraction subjected to column chromatography led to the isolation of pharmacologically potent anticancer compound whose structure was established to be alborixin on the basis of spectral data analysis. The compound exhibited antiproliferative activity against panel of cell lines N2a, MCF-7, MiaPaca-2, PC-3, HCT-116, MDA-MB-231, HL-60 and A-549 cells with IC50 of 9.7, 15.4, 7.2, 8.1, 3.2, 9.7, 7.5 and 11.5 µM respectively. Alborixin displayed the maximum cytotoxic activity against HCT-116 human colon carcinoma cells and therefore further studies were carried on this cell line. Alborixin decreased the clonogenic potential of HCT-116 cells in a dose dependent manner. It induced apoptotic cell death in HCT116 cells that were confirmed by Flow cytometric analysis of Annexin V/PI staining and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by mitochondrial membrane potential loss, decreasing the expression profile of anti-apoptotic protein Bcl-2, whereas it augments cleavage of caspase-3 and PARP-1, activates caspase-8 and 9 with concomitant increase in expression of proapoptotic protein Bax in a dose dependent manner. These results indicate that alborixin obtained from Streptomyces scabrisporus IIIM55 induces apoptotic cell death in colon cancer cells HCT-116 and can be further evaluated for its potential as an anticancer agent.

Molecular gate keepers succumb to gene aberrations in colorectal cancer in Kashmiri population, revealing a high incidence area.

BACKGROUND/AIM: Colorectal cancer (CRC) is one of the leading malignancies worldwide and has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The aim of this study was to identify p53 and K-ras gene mutations in CRC patients in a Kashmiri population, and to assess whether these mutations are linked with clinicopathological parameters. MATERIALS AND METHODS: Paired tumor and normal tissue samples from a consecutive series of 53 patients undergoing resective surgery for CRC were prospectively studied for p53 and K-ras gene mutations by PCR/single strand conformation polymorphism (SSCP). RESULTS: Less than half (45%, 19/42) of the patients presented mutations in the p53 gene. Twenty eight mutations were found in the p53 gene, which comprised of 23 substitutions (17 transitions + 6 transversions), and five insertions. The 23 substitutions constituted 18 missense mutations, two nonsense mutations, and three silent mutations. Of the 28 mutations (7.14%) observed in this study, 2 were not previously reported for CRC samples and were identified as novel p53 mutations. A few patients (22.64%, 12/53) presented with mutations in K-ras, constituting 13 missense mutations, out of which 11 were G-->A transitions, one was a G-->C transversion, and one a G-->T transversion. More than half (61.5%) of the mutations occurred in codon 12 whereas a few (38.5%) occurred in codon 13. One tumor contained missense mutations in both codons. Comparison of the mutation profiles of our patients with those of other ethnic populations and regions reflected both differences and similarities, indicating co-exposure to a unique set of risk factors. CONCLUSION: Mutations of the p53 and K-ras genes are some of the most common genetic changes in the development of human CRC. The high frequency of p53 gene mutations implicates p53 as a predominant factor for CRC in the high-risk ethnic Kashmiri population.

6Li atom percentage determination by atomic absorption-emission spectrometry using a natural lithium hollow cathode lamp.

A simple method has been developed for the determination of 6Li atom % using combined atomic emission-absorption spectrometry employing a commonly available natural lithium hollow cathode lamp. Unlike in previous practice, there is no need for specially fabricated and high cost 6Li and 7Li monoisotopic lamps in this method. The method requires adjustment of total lithium contents of the sample, i.e., 6Li + 7Li, to 2 microg x mL(-1) based upon atomic emission spectroscopy (AES) (C(aes)) against a 2 microg x mL(-1) natural lithium standard. The concentration of the sample was then analyzed by atomic absorption spectroscopy (AAS) measurements (C(aas)). The difference between the concentration measured by AES and AAS, i.e., C(aes)-C(aas), was calculated. The magnitude of the difference was found to be a function of 6Li fraction in the sample. A calibration curve was constructed by plotting 6Li atom % versus [(C(aes)-C(aas))/C(aes)] x 100. 6Li atom % of an unknown sample can be evaluated by putting its [(C(aes)-C(aas))/C(aes)] x 100 value in the calibration curve. The method is fast, convenient, and precise.

Mutational spectrum of conserved regions of TP53 and PTEN genes in Kangri cancer (of the skin) in the Kashmiri population.

Kangri cancer is a unique, thermally induced squamous cell carcinoma (SCC) of the skin that develops due to persistent use of a Kangri (a brazier) by the Kashmiri people to combat the cold temperature during winter. Unlike classical UV-induced SCC of the skin, Kangri cancer appears on the legs and abdomen. Its common features are erythematous patches, recurrence and metastasis. In the absence of any molecular etiology, we made a preliminary attempt to estimate the nature and frequency of mutations in the TP53 and PTEN genes in Kangri cancer patients from Kashmir. PCR-SSCP analysis followed by direct sequencing revealed that TP53 mutations account for 40% (12/30) of sporadic Kangri cancer patients and that PTEN mutations account for only 6.6% (2/30). There were 16 mutations in TP53 exons 5 and 7, found in 12 patients. They consisted of 11 substitutions (7 transitions, 3 transversions and 1 double-base) and 5 insertions. The 11 substitutions represent 8 distinct missense mutations, 3 of which were silent mutations. The mutations detected in the PTEN gene consisted of one insertion and one C>T transition. This high percentage of TP53 mutations (especially A>G) showed a statistically significant association with age and positive lymph node status. Our results indicate that TP53 is a predominant target of chronic hyperthermia in the development of Kangri cancer in the moderate risk Kashmiri population. The differences in the TP53 mutation spectrum of UV-induced SCC of the skin and Kangri cancer are probably due to the nature of the respective environmental carcinogens. The study also suggests that TP53 may function as a potential molecular marker and prognostic tool, at least in a subset of sporadic Kangri tumors.

Polymorphic analysis of MHClinked Heat Shock Protein 70 genes: Their susceptibility and prognostic implication in Kangri cancer cases of Kashmiri population.

Kangri cancer is a unique thermally-induced squamous cell carcinoma (SCC) of skin that develops due to persistent use of Kangri (a brazier), used by Kashmiri people, to combat the chilling cold during winter months. We designed a large scale case-control study to characterize the frequency of two polymorphisms within the MHC class III-linked HSP70genes, Hsp70-2 and Hsp70-hom, in order to find any association of these genotypic variants for predisposition to and clinical outcome of Kangri cancer patients from Kashmir valley in North India. Polymerase Chain Reaction and restriction enzymes were utilized to characterize the frequency of two polymorphisms with in Hsp70-2 and Hsp70-hom genes in 118 Kangri carcinoma cases and 95 healthy controls from the same population of Kashmir. Association of high frequency allelic variants of Hsp70genes with various clinicopathological features of prognostic significance was assessed by Chi-square test using SPSS software. In this study, allelic frequency of Hsp70-2 A/G heterozygote (0.87) (P = 0.012) was found to be significantly high in Kangri cancer cases compared to control (0.736) with a Relative Risk of 2.45 fold. Conversely, the allelic frequency of Hsp70-2 A/A allele in homozygous condition was significantly low in Kangri cancer cases and worked out to be 0.084 (Vs 0.252 in control) with P is equal to 0.001, implicating it as a protective allele against Kangri cancer in subjects with this genotype. Similarly, significantly high frequency of 0.50 (Vs 0.29 in control) of Hsp70-homC/C allele was found in homozygous condition in Kangri cancer cases suggestive of a positive relative risk associated with this genotype (RR is equal to 2.47) (P is equal to 0.002). The overall allele frequency data analysis of Hsp70-2 and Hsp70-hom genes was significant (chi(2) is equal to 12.38, P is equal to 0.002; and chi(2) is equal to 12.21, P is equal to 0.002). The study also reveals considerable association of high frequency alleles of HSP70 genes, especially of Hsp70-2 A/G or G/G in Kangri tumors with clinico-pathological features of poor prognosis. These results indicate that the relative risk of Kangri cancer associated with Hsp70-2 and Hsp70- hom gene polymorphisms is confined to Hsp70-2 A/G or G/G and Hsp70homC/C haplotype in our population. The study, therefore, suggests Hsp70-2 A/G or G/G and Hsp70homC/C genotypes as potential susceptibility markers and independent prognostic indicators in Kangri carcinoma patients in Kashmiri population.