Plodia interpunctella (Hübner) , is a serious insect pest of stored products, and its late-instar larvae diapause as pre-pupae. Diapause induction in P. interpunctella was investigated for four populations obtained from Modesto, California, U.S.A.; Vancouver, British Columbia, Canada; and two locations from Winnipeg, Manitoba, Canada. Insects were reared at 25° C and 16:8 L:D for 9 days. The larvae were then either continuously maintained under those conditions or transferred to 25° C 8:16 L:D, 20° C 16:8 L:D, or 20° C 8:16 L:D, and the percent diapause was recorded. In the experiment with four populations, the highest diapause frequency was observed at 20° C 8:16 L:D. The two Winnipeg populations had significantly higher frequency of diapause than the California populations, indicating the increased frequency of diapause in populations from higher latitudes.The Indianmeal moth, P. interpunctella was 0.39 in the first selection, and 0.82 in the second. This study has shown that both low temperature and short photoperiod are required to induce diapause in North American populations of P. interpunctella, and that selection can increase diapause in a few generations.In a second experiment, the Vancouver population was selected for diapause. Larvae were reared at 25° C 16:8 L:D for 9 days, then placed at 20° C 8:16 L:D for the rest of their development, and percent diapause was determined. Eggs laid by moths that completed diapause in this first  generation were used to obtain a second generation (F1), and the experiment was repeated as in the first generation. Selection increased the frequency of diapause to 91%, compared to 26% in the unselected population, after selecting over two generations. The narrow sense heritability of selection in  Plodia interpunctella (Hübner) , is an insect pest of stored products, and is found throughout the world ; Vancouver, British Columbia, Canada ; and two locations from Winnipeg, Manitoba, Canada  were used in this experiment. Insects were reared at 25° C, 16:8 L:D (using fluorescent lights), and 40–50% relative humidity, on a rearing medium prepared by mixing 1000 g cracked wheat, 40 g brewer's yeast , 50 g wheat germ, 2 g sorbic acid , 2 g methyl-p-hydroxybenzoate , 120 mL honey, 120 mL of glycerol, and 60 mL of water. To collect eggs, fifty adults comprised of both sexes were placed in an empty 900 mL glass bottle, covered with a nylon mesh, and placed upside down on a plastic Petri dish  were collected and tested for diapause induction as described for the parental generation. Eggs from the adults that completed diapause in the F1 (second) generation were used to start an F2 (third) generation. Frequency of diapause in this F2 generation was compared with that in unselected progeny derived from the original (Vancouver) population that was maintained continuously at 25° C 16:8 L:D. Frequency of diapause in selected and unselected groups was investigated using the methods previously described involving exposure to four sets of conditions: 25° C 16:8 L:D, 25° C 8:16 L:D, 20° C 16:8 L:D, or 20° C 8:16 L:D. As above, seven days after adult emergence ceased in each rearing jar, the rearing mediums were dissected in order to count the remaining insects.Bottles with 50 eggs from the Vancouver population were held in 200 g of the medium at 25° C 16:8 L:D for 14 days before they were transferred to 20° C 8:16 L:D, and held until emergence of non-diapausing adults was assumed to be completed. The emerged adults were counted and removed every 2–3 days. Ten days after the last adult emerged, the bottles were placed at 2.5° C in total darkness, and held for 6 weeks to terminate diapause of pre-pupae in the rearing medium . This inp = 0.05). In the selection experiment, the effects of selection on diapause frequency in the F1 and F2 generations were analyzed using ANOVA. An ANOVA was also performed to compare final diapause frequency in the selected population to final diapause frequency in the unselected population. This ANOVA was followed by Tukey's multiple range tests (p = 0.05). Narrow sense heritability was calculated by employing the diapause selection data in the following equation: Hn2 = P/S, where Hn2 = heritability in the narrow sense, R = response to selection, and S =selection differential  and populations . The interaction of treatment by population was also significant . In all the populations, maximum diapause frequency was observed at 20° C 8:16 L:D . Diapause frequency in Winnipeg 1 and Winnipeg 2 was significantly different from the California population . However, diapause frequency in Vancouver population was not significantly different from any of the other three populations .When only data from 20° C 8:16 L:D were analyzed, significant differences in the frequency of diapause were evident among populations that originated in different geographical locations . This could be due to infertile eggs, or mortality in the early instars that would not be detected during dissections of rearing mediums.1 to the F2 generation , 25° C 16:8 L:D, and 25° C 8:16 L:D. ANOVA could not be conducted in the last two treatment conditions because the diapause did not change from zero. These results showed that selection increased diapause in P. interpunctella over two generations.There was a significant change in percentage of adults emerging after cold treatment from the F2) (Narrow sense heritability (Hn2)  was esti2) , the res2) , resultiP. interpunctella fourth-instar larvae from 30° C to 20° C gave different diapause frequencies based on the photoperiod to which they were exposed. They found 0% diapause under continuous light, and 37% under complete darkness.In the experiment with four populations, diapause induction mostly took place at 20° C and 8:16 L:D. This result was also seen in the selected population, which had over 90% diapause in the same conditions, but no diapause at the other conditions. Bell et al.  showed tP. interpunctella, but requires the concurrent change in both factors. There have been some studies that used similar conditions to induce diapause in P. interpunctella, as tested in the current study. Plodia interpunctella larvae reared at 25° C, 16:8 L:D were induced to diapause when transferred to 20° C 8:16 L:D. The percentage that entered diapause varied with the age at which the larvae were transferred . In contrast, three out of four populations  increased diapause frequency when both the temperature and photoperiod were decreased. Although not significantly different, the fourth population  also had a trend to increase diapause frequency due to a similar change in temperature and photoperiod. This shows that the decrease in either photoperiod or temperature alone does not trigger diapause frequency in nsferred . In a lansferred  tested tThe two populations from a higher latitude (Winnipeg 1 and Winnipeg 2) had greater diapause frequency than the population from a lower latitude . The population from Vancouver was not significantly higher than California, but showed similar diapause frequency as the Winnipeg populations. This is in agreement with Bell et al. , which sP. interpunctella. Mbata . When larvae were reared at 20° C with 11:13 L:D, most entered diapause. By selecting non-diapause individuals over several generations, the diapause frequency dropped to 0.4% when reared at 20° C with 11:13 L:D. In contrast, selecting for diapausing individuals maintained a population, with almost all individuals entering diapause at 20° C with 11:13 L:D. Similar to Tsuji  (Holtzer et al. 1976; Drosophila melanogaster, selection for ten generations from a mass-bred population maintained in the lab for upward direction resulted in the heritability of 0.12 (P. interpunctella is not fixed, but can widely vary between generations.The narrow sense heritability of selection in  of 0.12 . Our stuP. interpunctella. This has implications for control, as diapause has shown to increase cold tolerance (P. interpunctella populations from southern latitudes or heated warehouses could quickly adapt to locations with cool temperatures and short photoperiods. Therefore, renewed attention needs to be paid to diapause phenomenon in P. interpunctella in order to determine diapause capacity in field populations, and to test the possibility of manipulating temperature and light in storage conditions in order to prevent diapause, and perhaps render insects more susceptible to control.This study has shown that both low temperature and short photoperiod, simulation of autumn conditions, are required to induce diapause in North American populations of olerance , so inseolerance . This st


Introduction. The ability to reliably differentiate neoplastic from nonneoplastic specimen and ascertain the tumour grade of diffusely infiltrating gliomas (DIGs) is often challenging. Aims and Objective. To evaluate utility of image morphometry in identifying DIG areas and to predict tumour grade. Materials and Methods. Image morphometry was used to analyze the following nuclear features of 30 DIGs and 10 controls (CG): major axis of nucleus (MAJX), minor axis of nucleus (MINX), nuclear area (NA), nuclear perimeter (NP), nuclear roundness (NR), nuclear density (ND), and percentage of total nuclear area (%TNA). Results. Statistically significant differences in all parameters, except NR, were observed between all groups, with strong positive correlation with tumour grade (r > 0.7). The mean values were maximum for HGG and minimum for CG. For NR, the difference between CG/HGG was statistically significant, unlike CG/LGG and LGG/HGG. It was observed that NA distributions for CG were nearly Gaussian type with smaller range, while gliomas displayed erratic pattern with larger range. NA and NP exhibited strong positive correlation with ND. Conclusion. Image morphometry has immense potential in being a powerful tool to distinguish normal from neoplastic tissue and also to differentiate LGG from HGG cases, especially in tiny stereotactic biopsies. The global annual incidence of primary malignant central nervous system (CNS) tumors is around 37 per million for male and 26 per million for female , 2, withIn the past several years, it has been well established that quite a few clinical and histopathological parameters are helpful in predicting the clinical outcome of cancer patients. Currently, well-established techniques like morphometry, stereology, static image, and flow cytometry are routinely used in diagnostic quantitative pathology. The potential significance of these techniques includes the objective distinction between benign, borderline, and malignant lesions; the objective grading of invasive tumors; and the prediction of prognosis and therapeutic response. Computer-assisted image analysis is a new powerful tool for high-precision measurement of different facets of tumor cells to achieve similar goals –7. To daIn the present study, mean nuclear density (ND) and percentage of total nuclear area (%TNA) were also evaluated to analyze their utility in the gliomas. These two parameters are related to the growth rate of the tumor. The main objectives of this study were to correlate morphometrically measured MAJX, MINX, NA, NP, NR, ND, and %TNA with WHO grade of diffusely infiltrating gliomas (DIG); and to assess their ability to differentiate normal from neoplastic areas.The present study included biopsies from thirty cases of DIGs that were managed at this tertiary care institute during 2009–2011. The spectrum of cases included (a) 14 cases of low-grade gliomas  and (b) 16 cases of high-grade gliomas . Brain tissue from ten autopsy cases   without The mean age at surgery was 39.1 years (range: 9–62 years) for LGG and 54.6 years (range: 27–79 years) for HGG. There was only one case in the pediatric age (<18 years) group (low grade astrocytoma).π·nuclear area)/(nuclear perimeter)2] × 100 in percentage. Thus, for a perfectly round nucleus its value is maximum, that is, equal to 100. A nucleus having irregular shape had smaller value of NR.Morphometric analysis was performed on hematoxylin and eosin (H&E)-stained histological sections of 5-micron thickness of formalin fixed paraffin-embedded CNS tissue, having optimal histological detail. A computerized digital photomicrograph system  was used for image analysis. The measuring scale of the image analysis software was properly calibrated. For each sample, five high power fields (400x), having maximum cellularity, were recorded for the study. For each case, 100 nuclei, clearly separated from others and relatively larger in size, were chosen to evaluate nuclear shape and size. The nuclei were outlined using a mouse attached to the computer and then separated from others prior to the determination of their nuclear parameters using the software . After mCounting of nucleus was done in the five recorded high power fields from which the mean nuclear density (ND) was calculated. The percentage of total nuclear area (%TNA) was determined for the five high power fields for each sample using the software, and their mean was calculated.t-test was performed to evaluate the difference in MAJX, MINX, NA, NP, NR, ND, and %TNA for each pair of the groups, and the P values were determined. Data was reported as mean, standard deviation of mean, and range of mean for these parameters. The statistical correlations of the analyzed morphometric parameters with tumor grades of all samples were investigated; correlations of ND with NA and NP were also studied. Pearson correlation coefficient (“r”) and P  value were calculated, and regression line was drawn in correlation studies.The various parameters used to analyze were MAJX, MINX, NA, NP, and NR for each nucleus of every sample of the three groups. Mean ND and %TNA were determined for each sample. The mean values of these parameters with standard deviation (SD) and range were calculated for the three groups: CG, LGG, and HGG. Student's The distribution of the measured area of nucleus for five samples of each group is also shown along with the calculated Gaussian distribution of nuclear area using mean and SD of the respective group. r > 0.70), whereas NR demonstrated mild negative correlation with grade (r = −0.42). Figures P  values to predict the difference of each pair of these three groups for all the parameters are also presented in this table. The mean values of MAJX, MINX, NA, NP, NR, ND, and %TNA of the three groups were represented in Figures The mean values of nuclear parameters and age at the time of surgery with standard deviation (SD) and range for controls, LGG, and HGG are presented in P < 0.0004) and that NR in HGG was significantly smaller than the controls (P = 0.0125). Again, between two tumor groups, age, MAJX, MINX, NA, NP, ND, and %TNA in HGG were significantly greater than LGG (P < 0.05), unlike NR (P = 0.4011).It was noted that the nuclear parameters: mean MAJX, MINX, NA, NP, ND, and %TNA, of the two tumor groups, LGG and HGG were significantly greater than the controls (µm (range: 6.23 µm–8.07 µm), mean MINX: 4.62 µm (range: 4.18 µm–5.30 µm), mean NA: 23.44 µm2 (range: 19.42 µm2–30.33 µm2), mean NP: 21.54 µm (range: 18.84 µm–25.33 µm), and mean NR: 65.55 (range: 59.85–73.48). ND was 1107 mm−2 (range: 849 mm−2–1282 mm−2), while mean %TNA was 2.32 (range: 1.65–3.33).The mean age of the CG was 46.3 years (range: 40–53 years). Nuclei, in the control tissues, were evenly distributed, with mean MAJX: 6.96 µm (range: 7.51 µm–10.30 µm); mean MINX was 6.34 µm (range: 4.98 µm–7.56 µm); and mean NA was 42.62 µm2 (range: 27.15 µm2–55.23 µm2); mean NP was 29.79 µm (range: 23.34 µm–32.74 µm); and mean NR was 61.53 (range: 48.99–70.43). ND was 3232 mm−2 (range: 1456 mm−2–5633 mm−2), and mean %TNA was 9.98 (range: 3.63–17.35). MAJX, MINX, NA, NP, ND, and %TNA in LGG were significantly larger than controls (P < 0.0004) but significantly smaller than the HGG (P < 0.05). Age of patients was also found significantly lower in LGG as compared to HGG (P = 0.0116).The mean age of LGGs was 39.1 years (range: 9–62 years). In LGG, the following was noted mean MAJX was 9.41 µm (range: 8.90 µm–12.87 µm); mean MINX was 7.11 µm (range: 4.66 µm–8.70 µm); mean NA was 55.22 µm2 (range: 30.26 µm2–76.90 µm2); mean NP was 34.24 µm (range: 28.92 µm–41.91 µm); and mean NR was 59.78 (range: 48.94–70.40). ND was 4350 mm−2 (range: 1941 mm−2–6360 mm−2) and mean %TNA was 15.88 (range: 6.64–35.33). MAJX, MINX, NA, NP, ND, and %TNA in LGG were significantly larger than controls (P < 0.0001), and LGG (P < 0.02). In HGG, the mean age was also found significantly higher than LGG (P = 0.0116), and mean NR was significantly lower than controls (P = 0.0125).The mean age of the HGG was 54.6 years (range: 27–79 years). In this group the following was noted: mean MAJX was 10.73 Figures r = 0.72) than NA (0.70).Figures Nuclear morphometric features have a potential role in differentiating between the different grades of gliomas and predicting patient survival –8. Nieder > 0.78) could be observed, which is translated as follows: the bigger the size of the nucleus, the higher is its probability of being a glioma of higher grade. Of these, NP and MAJX displayed the best correlation (r = 0.83).In the present study, we observed that in cases of CNS lesions, a panel of nuclear parameters evaluated by image morphometry was not only able to reliably distinguish normal from neoplastic areas, but also to indicate consistently the grade of tumour. Nuclei of astrocytic cells in the control groups had the smallest value of MAJX, MINX, NA, NP, and ND and %TNA, while neoplastic astrocytes of HGG measured the largest. In cases of parameters related to size of the nucleus, namely, MAJX, MINX, NA, and NP, a strong positive correlation with tumour grades (r > 0.74), thus confirming that higher grade tumors are indeed more susceptible to a rapid rate of growth. The faster growth rate in higher-grade tumor leads to an increase in the number density of nucleus, which is histologically seen as closely packed tumor cells with higher percentage of tumour area occupied by dysplastic nuclei. ND and %TNA, parameters related to the rate of growth of a tumor, showed strong positive correlation with tumour grade (α) that enhances the microvascular networks in the tumour [Therefore, it can be hypothesized that the nuclei of the tumour cells enlarge in an effort to increase their surface area so as to enhance the diffusion rate to maintain their required metabolic function. In support of this, it was observed that NP directly related to the surface area of a nucleus in three-dimensional cases, showed the best correlation with the tumour grade and nuclear density.In the current study, NA and NP exhibited strong positive correlation with ND, with NP being better. It is accepted that in high-grade tumors the nutritional and oxygen demands per unit volume of tissue are higher, which it tries to compensate by stimulating various proangiogenic factors like hypoxia inducible factor (HIF-1e tumour . Howeverr = −0.42). Hence, the nuclear size and parameters related to tumor growth have more importance in comparison to the parameters related to nuclear shape, for assessment of tumour grade by a quantitative morphometric method. Owing to its high nuclear density, all the nuclei of a closely packed high-grade tumour may not receive sufficient nutrients. In an effort to compensate there is enhancement in the rate of diffusion, which the tumour cell performs by escalating its surface area per unit nuclear mass, a feature that is seen histologically by the severe pleomorphism in shape of the cells .  Owing to this, the value of NR showed negative correlation with grades. The nuclear roundness, which was related to shape of the nuclei, showed a mild negative correlation with tumour grade . The nuclear enlargement showed strong correlation with nuclear density in gliomas. The nuclear area distribution pattern may also be gainfully utilized for identification of different grades of gliomas. These features have potentially immense utility in diagnostically challenging scenarios, like intraoperative cytological specimen, and in the analysis of tiny specimen as in stereotactic biopsies. In conclusion, our study showed a definitive correlation between various nuclear morphometric parameters with tumor grade in diffusely infiltrating gliomas. The nuclear morphometric study can be gainfully utilized not only to distinguish normal from neoplastic tissue, but also to differentiate between the various grades of gliomas. It also serves as an excellent model to comprehend various facets of tumour and tumour cell physiology. The present study also revealed that the tumor grade had a better correlation with nuclear enlargement than with nuclear roundness, where MAJX and NP showed the best correlation with grades (


The purpose of this study was to evaluate the exposure to arsenic in preventive maintenance (PM) engineers in a semiconductor industry by detecting speciated inorganic arsenic metabolites in the urine.The exposed group included 8 PM engineers from the clean process area and 13 PM engineers from the ion implantation process area; the non-exposed group consisted of 14 office workers from another company who were not occupationally exposed to arsenic. A spot urine specimen was collected from each participant for the detection and measurement of speciated inorganic arsenic metabolites. Metabolites were separated by high performance liquid chromatography-inductively coupled plasma spectrometry-mass spectrometry.3+), pentavalent arsenic (As5+), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and total inorganic arsenic metabolites (As3+ + As5+ + MMA + DMA), respectively, in clean process PM engineers. In ion implantation process PM engineers, the concentrations were 1.74 g/L, 0.39 g/L, 3.08 g/L, 23.17 g/L, 28.92 g/L for As3+, As5+, MMA, DMA, and total inorganic arsenic metabolites, respectively. Levels of urinary As3+, As5+, MMA, and total inorganic arsenic metabolites in clean process PM engineers were significantly higher than that in the non-exposed group. Urinary As3+ and As5+ levels in ion implantation process PM engineers were significantly higher than that in non-exposed group.Urinary arsenic metabolite concentrations were 1.73 g/L, 0.76 g/L, 3.45 g/L, 43.65 g/L, and 51.32 g/L for trivalent arsenic (AsLevels of urinary arsenic metabolites in PM engineers from the clean process and ion implantation process areas were higher than that in office workers. For a complete assessment of arsenic exposure in the semiconductor industry, further studies are needed. The production of semiconductors continues to increase, as semiconductors are essential in the manufacturing of electronics. In Korea, the semiconductor industry has grown rapidly over the last decade, and semiconductors produced in Korea comprise a large share of the world market. Semiconductor manufacturing requires some toxic materials, which poses a risk of exposure to workers in the industry. The semiconductor production process can be divided into 2 sequential sub processes: wafer fabrication and chip assembly.The wafer fabrication process is a multiple-step sequence of photolithographic and chemical processing steps including diffusion, photolithography, etching, ion implant, and metal deposition, during which electronic circuits are created on a wafer. During the diffusion process, dopants diffuse and an oxide film is formed on the wafer. In the photolithography process, the circuit pattern is formed. In the etching process, unnecessary parts of the pattern are removed. In the ion implantation process, impurities such as arsenic or phosphorus ions are implanted in order to impart conductivity to the wafer. Finally, in the metal deposition process, the parts of the circuits are connected electrically by depositing a conductive metal on the silicon wafer to create the integrated circuits . Arsine Before and after each step of wafer fabrication, there is another step called clean process where the wafers are cleaned using a solution of hydrofluoric acid, sulfuric acid, phosphoric acid, hydrogen peroxide, nitric acid, or isopropyl alcohol. During the clean process after ion implantation, arsenic ion can be dissolved in the cleaning solution. Therefore, workers of the clean process can also be exposed to arsenic.Usually, workers in semiconductor manufacturing are categorized into operators and preventive maintenance (PM) engineers. The operators, who handle the wafers, are exposed to relatively low concentrations of chemicals. The PM Engineers are responsible for the processes and the equipment; hence, they frequently take apart the equipment and clean the inner parts. Thus, they are likely to be exposed to high concentrations of the various chemicals .Arsenic is abundant on the earth, but exposure to arsenic has adverse health effects. Acute arsenic toxicity affects many organ systems including the gastrointestinal, dermal, nervous, hematopoietic, and cardiovascular systems. Long-term exposure to non-lethal doses of arsenic has been found to induce peripheral neuropathy, anemia, cardiac and peripheral vascular disease, anemia, hyperkeratosis, and hyperpigmentation of the skin . Inorgan5+) can be reduced to trivalent arsenite (As3+) in the blood, which is taken up mainly by the liver [3+ is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) by oxidative methylation and reduction. In humans, these metabolic processes do not go to completion, and inorganic arsenic along with mono- and di-methylated metabolites are excreted in the urine [In the blood, pentavalent arsenate , except night shift workers and off-duty workers, participated in this study. The non-exposed group consisted of 14 office workers from another company who were not occupationally exposed to arsenic. Duty of the PM engineers of clean process was checking the equipment of the clean process where the wafers are cleaned before and after each step of wafer fabrication such as photolithography process or ion implantation process. PM engineers of ion implantation process did the similar job on the ion implantation equipment. All participants were men. Preliminary explanation for this study was presented to all the participants. Only workers who signed informed consent documents participated in the study. The participants were interviewed and data on smoking status, consumption of seafood  during the previous 3 days, and job description were recorded. At the end of a working weekday morning, a spot urine specimen was collected from each participant.3+, As5+, MMA, and DMA, respectively. In the analysis of urinary speciated arsenic metabolites from the study participants, there were 5 cases with As5+ below 0.197 g/L, which is the MDL. In 6 cases where the metabolite was not detected (ND), a value of half the MDL was assigned.A blank specimen, standard material for a calibration curve, and urine specimens for analysis  were placed in ICP-MS vials, after filtering through a cellulose acetate (0.2 m) filter. Filtered specimens were analyzed by HPLC-ICP-MS  . Urinary speciated inorganic arsenic metabolites were transformed to a logarithmic scale. Multiple regression analysis was conducted with the groups  as independent variables with adjustment for the effects of smoking and recent seafood consumption, which are well-known confounders of urinary arsenic values from occupational exposure. A p value of <0.05 was considered significant. Statistical analyses were conducted using SAS 9.2 .3+, As5+, MMA, and DMA). It shows no statistical significant differences on speciated urinary inorganic arsenic metabolites  among each group , 38.46% of ion implantation PM engineers (5 of 13), and 35.71% of the non-exposed group (5 of 14) were current smokers. Five clean process PM engineers had consumed seafood during last 3 days, and 2 clean process PM engineers and 6 office workers had ever had seafood. Neither smoking nor seafood consumption had a significant effect on the level of urinary speciated inorganic arsenic metabolites . The geometric mean of As5+ for clean process PM, ion implantation PM and office workers were 0.76 μg/L, 0.39 μg/L, 0.18 μg/L, respectively, which also showed significant difference (p < 0.05). MMA values showed a similar trend, i.e., 3.45 μg/L, 3.08 μg/L and 2.310 μg/L for clean process PM, ion implantation PM and office workers, respectively (p < 0.05). As3+ and As5+ value, summed up to represent inorganic arsenic level, were 2.58 μg/L, 2.23 μg/L and 1.17 μg/L for clean process PM, ion implantation PM and office workers, respectively, which shows significant difference (p < 0.05). Summed value of inorganic arsenic and MMA were 6.11 μg/L, 5.34 μg/L and 3.37 μg/L for clean process PM, ion implantation PM and office workers, respectively (p < 0.05). On the contrary, DMA values were statistically insignificant (p 0.08). They were 43.65 μg/L, 23.17 μg/L and 23.43 μg/L for clean process PM, ion implantation PM and office workers, respectively. Total inorganic arsenic metabolites values, which is the summation of As3+, As5+, MMA and DMA, were 52.32 μg/L, 28.92 μg/L and 27.57 μg/L for clean process PM, ion implantation PM and office workers, respectively, which showed statistically insignificant value (p 0.06). The GM of urinary As3+, As5+, and MMA in the clean process PM engineers were the highest, followed by that in the ion implantation PM engineers and the non-exposed group. The GM of the sum of DMA and inorganic arsenic metabolites was the highest in clean process PM engineers, followed by that in the non-exposed group and ion implantation PM engineers.The results of the analysis of urinary speciated arsenic metabolite values are shown in Table 3+, As5+, MMA, the sum of inorganic arsenic , the sum of inorganic arsenic and MMA, and the total inorganic arsenic metabolites were higher in clean process PM engineers than in non-exposed group. The concentrations of As3+, As5+, the sum of inorganic arsenic (As3+ + As5+), and the sum of inorganic arsenic and MMA were also higher in ion implantation PM engineers than in the non-exposed group , and the sum of inorganic arsenic and MMA were higher in ion implantation PM engineers than in the non-exposed group.The workers in semiconductor manufacturing plants can be exposed to various toxic substances. Among them, the arsenic used in the ion implantation process as arsine gas is one of the most significant problems. In this study, multiple regression analysis revealed that As3 if the operation is appropriately maintained by engineering controls such as exhaust ventilation, enclosure, and shielding of the operation equipment [Previous studies showed that PM engineers who handle the normal processes of the fabrication operation are exposed to arsenic levels that are substantially lower than the TLV of 10 μg/mquipment . Howeverquipment ,18. In aquipment . The resquipment .3,+, As5+, MMA, DMA, and total urinary inorganic arsenic metabolites, respectively. Further, both the concentration of MMA and its percentage of the total urinary inorganic arsenic metabolites were significantly higher in PM engineers than in the non-exposed group [In another Taiwanese study that monitored arsenic exposure in ion implanter PM engineers, the levels of total urinary inorganic arsenic metabolites in PM engineers were 1.7 g/L, 1.4 µg/L, 6.2 µg/L, 20.2 µg/L, 29.5 µg/L for Ased group .3+, As5+, MMA, DMA, and total arsenic metabolites  were 2.19 g/L, 0.82 g/L, 3.86 g/L, 44.33 g/L, and 51.21 g/L for exposed workers, respectively, and exposed workers had significantly higher concentrations of MMA, DMA, and total arsenic metabolites than non-exposed workers [In the study of Hu et al. (2006) that evaluated the effects of arsenic exposure among semiconductor workers, the mean urinary concentrations of As workers ,19, the 3+, As5+, MMA, DMA, and total arsenic metabolites  were 0.6 µg/L, 0.2 µg/L, 0.7 µg/L, 4.9 µg/L, and 6.4 µg/L, respectively, for exposed workers in the semiconductor manufacturing industry [In a British study that monitored concentrations of urinary arsenic compounds, the mean urinary concentrations of Asindustry . These lIn our study, the levels of urinary arsenic metabolites in clean process PM engineers were higher than those in ion implantation process PM engineers and the non-exposed group. In an experimental study conducted by Ungers et al. (1985), silicon wafers were found to emit inorganic arsenic following ion implantation. Data collected during this experiment demonstrated that arsenic is released over a 3.5-hour period following implantation and that the total amount of arsenic emitted may approach 6.0 g per 100 wafers processed within 4 hours after implantation [3+, As5+, MMA, and DMA) was 54 g/L, which exceeded the BEL [Urinary arsenic is determined in a secondary confirmative test in special medical examinations in Korea, with the limit being 220 g/L . An uppe the BEL , which i the BEL ; both va the BEL .3+, As5) is more useful for assessing occupational exposure to arsenic, rather than total urinary inorganic arsenic metabolites [3+, As5) was significantly different between groups. Because seafood consumption in Asian countries is higher than in Western countries, Hata et al. (1995), on the basis of their findings in the general population in Japan, recommended excluding DMA when assessing occupational exposure to arsenic [3+, As5+, and MMA (excluding DMA), and found a significant difference between the exposed and the non-exposed groups. The DMA concentration, however, was not significantly different between groups, even after adjusting for confounders such as smoking and seafood intake.Shellfish are rich in DMA, which when ingested is not metabolized, but directly excreted in the urine . Arsenosabolites . In this arsenic . We analThis study has certain limitations. First, the study results are based on a single urine sample from each participant; hence, we could not establish the reproducibility and accuracy of the measurements. A second limitation concerns control of the seafood intake. To exclude the arsenic intake from seafood, we strongly recommended participants to consume seafood for at least 2 days, but it was not strictly followed. This might have affected the interpretation of the levels of DMA. Finally, the non-exposed group should have been selected from the same community or workplace to control for environmental arsenic exposure (such as from water or air pollution), but we could not find suitable candidates among the workers’ colleagues.This study aimed at assessing exposure to arsenic in semiconductor manufacturing workers, specifically in PM engineers of the clean process and the ion implantation process areas using urinary inorganic arsenic metabolite levels. Prior studies assessing arsenic exposure in the semiconductor industry focused mainly on ion implantation process workers, in whom the level is known to be negligible . In thisThe authors do not have any competing interests issues in this study.KWB analyzed the data and described the whole paper. YLW and HSI analyzed the metabolites of arsenics. YIH and DHK analyzed the data and participated all the discussion. EAK organized this study, analyzed the data and discussed all the results. All authors read and approved the final manuscript.


Background. CDRI 97/78 has shown efficacy in animal models of falciparum malaria. The present study is the first in-human phase I trial in healthy volunteers. Methods. The study was conducted in 50 healthy volunteers in a single, ascending dose, randomized, placebo-controlled, double blind design. The dose ranges evaluated were from 80 mg to 700 mg. Volunteers were assessed for clinical, biochemical, haematological, radiographic, and electrocardiographic parameters for any adverse events in an in-house facility. After evaluation of safety study results, another cohort of 16 participants were administered a single oral dose of 200 mg of the drug and a detailed pharmacokinetic analysis was undertaken. Results. The compound was found to be well tolerated. MTD was not reached. The few adverse events noted were of grade 2 severity, not requiring intervention and not showing any dose response relationship. The laboratory and electrocardiographic parameters showed statistically significant differences, but all were within the predefined normal range. These parameters were not associated with symptoms/signs and hence regarded as clinically irrelevant. Mean values of T1/2, MRT, and AUC∞0− of the active metabolite 97/63 were 11.85 ± 1.94 h, 13.77 ± 2.05 h, and 878.74 ± 133.15 ng·h/mL, respectively Conclusion. The novel 1,2,4 trioxane CDRI 97/78 is safe and will be an asset in malarial therapy if results are replicated in multiple dose studies and benefit is shown in confirmatory trials. P. falciparum is one of the important causative strains of malaria. Falciparum malaria, which was traditionally effectively treated by chloroquine, now commonly shows resistance to the drug  propyl] ester. It has powerful antimalarial activity against chloroquine resistant in vivo metabolite 97/63. The concentrations of 97/63 and 97/78 can be measured by validated LC-MS/MS method [Firstly 97/63 was synthesized but, due to its poor bioavailability, it was resynthesized as a hemisuccinate derivative and coded as 97/78. Upon administration of 97/78 it gets converted into its activeS method . The pre in vivo metabolite 97/63 after oral administrations of 97/78.The aim of this study was to calculate the maximum tolerated dose of the compound in healthy human volunteers along with evaluation of PK profiles of 97/78 and itsThe study was approved by the Institute's Ethics Committee. The single ascending dose study and the pharmacokinetic study are registered with Central Trial Registry of India . Normal healthy male volunteers in the age group of 18–45 years were invited to join the trial. A detailed informed consent process of the volunteers was undertaken and screening of the volunteers was done after obtaining informed consent. They were recruited through contacts and hospital employees were excluded. The health status of the volunteers was determined by taking medical history, physical examination, routine haematology , clinical biochemistry , serum glutamic pyruvic transaminase (SGPT), serum bilirubin, serum creatinine, blood urea, serum electrolytes, random blood sugar, total protein, and serum lipid profile), and urine and stool analysis (routine examination and microscopy). The volunteers were asked to report to the study site after 12 hours of fasting for the blood tests. Chest X-ray and 12-lead ECG were also taken. The participants were also screened for human immunodeficiency virus (HIV) and hepatitis B.The volunteers were excluded if there was a history of having donated blood in the past 3 months, HIV or HBsAg positivity, history of any drug intake in the past 15 days, history of receiving any enzyme-inducing agent in the preceding 15 days, history of participating in any investigational new drug (IND) study in the previous 6 months, history of participating in any non-IND study in the preceding 3 months, history of allergy or hypersensitivity to any medication, history of peptic disorder, chronic smoker >6 cigarettes/beedies per day, alcoholic , history of any drug abuse, tobacco user in any form, history of any chronic disease and taking medication, and history of taking nonallopathic drug in the past 6 months for any illness or history of any major illness in the past .The procedure for recruiting participants for single dose PK study was similar to the above. The PK study was undertaken in 16 separate volunteers following the evaluation of safety data for phase I study and evaluation of pharmacodynamics study in animalsThis was a single ascending dose, placebo-controlled, randomized, double blind trial. Randomization was carried out by computer generated random number sequence. Randomization was carried out for the drug and the placebo in a 3 : 1 ratio for each dose level. The drugs were dispensed in sealed bottles with the randomization code written on them. Randomization codes were provided in sealed opaque envelopes with the protocol specification that the codes may be opened by the investigator only in case of a serious or severe adverse event.The starting dose was 80 mg; calculation of starting dose was based on the basis of maximum tolerated dose of 100 mg/kg obtained in rats. This was used to obtain a dose of 96 mg for a 60 kg man. The formulation which was available for a dose below this was of 80 mg and hence taking these factors into consideration a starting dose of 80 mg was chosen. The following dose levels were evaluated: 80, 160, 320, 400, 500, 600, and 700 mg. The starting dose was calculated by allometric scaling from animals. At each dose level, six volunteers received CDRI 97/78 and two received placebo except at 700 mg where only two volunteers were given this dose. At each dose level, volunteers were assessed before proceeding to the next dose. The decision to dose only 2 volunteers was taken, after analysis of data of 600 mg, wherein no remarkable adverse events were noted.The decision to stop the dose escalation was based on the appearance of the dose limiting toxicity (DLT) which was defined as the dose at which any of the following appeared: severe nausea, vomiting, heartburn, headache, remarkable change in vital parameters such as blood pressure, heart rate, respiratory rate or oxygen saturation of blood, changes in QT interval, or any event that in the opinion of the investigators required stopping of the trial.Maximum tolerated dose was defined as the dose before which any of the components of DLT appeared at a significantly higher rate as compared to placebo. In case this was not achieved, it was planned not to dose volunteers beyond 700 mg.The volunteers after giving informed consent were screened. The screening process was done latest within 1 month of admission for dosing. The volunteers who fulfilled the inclusion/exclusion criteria were admitted in the CPU the night before drug administration. Standardized dinner was served at 9 pm the night prior to dosing. On day 0, baseline blood sample for biochemical and haematological examination and stool and urine samples for routine and microscopy were taken. They received the study drug, according to the randomization sequence, under supervision. The empty packaging was returned to the container. The volunteers received drug as per randomization. Breakfast was given half an hour after drug administration. Subsequently, standardized lunch and dinner were served to the volunteers.For pharmacokinetic (PK) study, eligible participants were admitted and housed in the clinical pharmacology unit an evening prior to the PK study. A standard dinner was served and a fasting condition for at least 10 hrs prior to dosing and 4 hrs after dosing was ensured. A peripheral forearm vein was cannulated for sampling for the PK analysis. 200 mg of the study drug was administered with 300 mL of water. The time of actual dose administration to the nearest minute was recorded. 2.5 mL of blood was collected from the indwelling cannula at 0 (before dose), 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 18, 24, 48, 72, 96, 120, and 144 hrs (after dose). 10 mL of additional blood was collected at 0 (before dose) and 24 hr (after dose) for laboratory parameters for safety assessment and PK control sample. A time window of ±2 minutes was permitted for collecting the post dose samples from the scheduled time for PK analysis.Plasma was separated from blood sample intended for PK analysis, within 1 hour of blood sampling, by centrifugation at 2000 rpm for 10 min at 4°C. Samples were stored at −80°C pending transportation. Heparinised blood samples were transported to the laboratory within 7 days of collection.The subjects were attached to a cardiac monitor and vital HR, BP, ECG, oxygen saturation, and RR were recorded. Corrected QT interval using Fridericia's formula was calculated. Adverse events were assessed at specific time points . Unscheduled monitoring was undertaken whenever it felt necessary. The volunteers were discharged after 24 hrs and they had to return at 48 hrs and on the 7th day for follow-up. At the end of the 7th day, they were said to have completed the study. Routine haematological and biochemical investigations were performed at 0 hr, 24 hrs after drug administration, and on day 7. The study was monitored by a Data Safety Monitoring Board.α-Arteether was used as an internal standard and all the analyses were performed as per the published method by Singh et al. 2009 [T1/2), maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), area under the curve (AUC∞0–), and mean residence time (MRT).Quantitative analyses of clinical phase ascending dose and single dose PKs samples were done at GLP lab of Pharmacokinetics and Metabolism Division, CDRI, Lucknow, India. al. 2009 . Acceptan (%). For comparison between the drug and placebo at each dose level, unpaired t-test was used for continuous variables and Fischer's exact test for categorical variables. For comparison between various dose levels, ANOVA was used. P value <0.05 was considered significant.Continuous data were presented as mean ± SD and categorical data were presented as P = 0.7), height (P = 0.5), weight (P = 0.5), and body mass index (P = 0.3) among participants in various treatment groups .Adverse events that occurred during the study were few  and largSedation was the most common adverse event reported across different groups . It was noted in 3 volunteers given 500 mg CDRI 97/78, 600 mg CDRI 97/78, and placebo about 2 hours after drug administration and lasted for 1-2 hours. It was grade 1-2 in severity, spontaneous and complete recovery was seen in all, and no action was required. The other adverse events noted were mild drowsiness, mild nausea, slight heaviness of head, bradycardia (56-57 bpm), visual disturbances, and itching .Severe adverse event, nausea and retching immediately after drug administration, was observed in one patient which lasted for 10 minutes; the patient also complained of dizziness and slight heaviness of head 1 hour after drug administration. On decoding, he was found to have received placebo. The symptoms resolved spontaneously; hence, no further action needed to be taken.P = 0.02), although clinically significant decline was not observed in any of the volunteers. Statistical significance was observed for between group comparison of systolic and diastolic blood pressures 2 hours after drug administration. The SBP for 80 mg, 160 mg, 320 mg, 400 mg, 500 mg, 600 mg, 700 mg, and placebo groups were 112.17 ± 6.24, 119.5 ± 5.17, 127.5 ± 6.38, 111.83 ± 7.91, 114.5 ± 8.34, 112.83 ± 6.18, 115.5 ± 2.12, and 116.33 ± 8.77, respectively (P = 0.01). The DBP for 80 mg, 160 mg, 320 mg, 400 mg, 500 mg, 600 mg, 700 mg, and placebo groups were 73 ± 5.21, 77.17 ± 8.06, 82.33 ± 4.68, 71.33 ± 8.19, 71.83 ± 7.81, 66.5 ± 6.53, 69 ± 8.48, and 73.75 ± 8.24 (P = 0.03). Respiratory rate was found to be significantly different between treatment groups 4, 6, and 8 hours after drug administration, although this was not clinically relevant.At baseline, the different treatment groups were similar in terms of clinical parameters . None of the treatment groups demonstrated a significant alteration in pulse rate, respiratory rate, and systolic blood pressure at any time point compared to baseline. However, there was a significant change in diastolic blood pressure in 600 and 700 mg CDRI 97/78 group .There was no statistically or clinically significant difference noted in the corrected QT interval within various treatment groups (P = 0.02). A statistically significant change in platelet counts was also observed in 320 mg CDRI 97/78 group /L, respectively; P = 0.04). All these values were well within normal limits. Other haematological parameters, which showed statistically significant difference, however, remained well within normal range, and did not demonstrate any dose response relationship, were PCV, total leukocyte and lymphocyte counts, RBC counts, and methemoglobin levels.Hemoglobin values demonstrated a statistically significant alteration during posttreatment period in the group receiving 400 mg CDRI 97/78  and higher doses (500 and 600 mg CDRI 97/78), although none of the groups was significantly different from placebo. All the values remained within normal range . The othThe groups demonstrated significant variation in serum sodium and serum potassium values at all time points of measurements. None of the volunteers had values outside the normal physiological ranges nor presented with any signs or symptoms of dyselectrolytemia, so these findings were considered clinically irrelevant.Tmax of active metabolite 97/63 was found to be 2.28 ± 0.27 h with Cmax of 143.93 ± 28.32. Mean values of T1/2, MRT, and AUC∞0– of 97/63 were 5.05 ± 0.57 h, 9.73 ± 1.06 h, and 845.22 ± 142.96 ng·h/mL, respectively. Active metabolite 97/63 was quantified from 0.5 h and up to 48 h in healthy volunteers with exception to two volunteers in whom it was observed up to 72 h. No additional metabolite peaks were observed during analysis except 97/63. In most of the volunteers, parent molecule 97/78 was below the limit of quantitation. Moreover, very low concentrations of 97/78 were observed in some volunteers, but these were not sufficient for generating the PK parameters.Following oral administration at 200 mg, compound 97/78 was rapidly absorbed, gets immediately converted into active metabolite 97/63, and is eliminated from the systemic circulation within 48 h except in two volunteers (72 h), as observed from plasma concentration-time profile . PK paraThis was a first-in-human study conducted on healthy male volunteers for a new antimalarial drug, CDRI 97/78. Overall, the drug was found to be safe.Few adverse events were reported which were of mild nature and were probable in causality. But the adverse events were neither clinically nor statistically significant. The 14 adverse events reported in this trial occurred in various treatment groups over a large time frame. Hence, it cannot be said definitely whether they were due to the drug or not. Moreover, similar adverse events were also seen in the placebo group. Even the statistically significant alteration in blood pressure was not associated with signs and symptoms of altered blood pressure and the values fell within normal ranges.For none of the adverse events, any intervention in the form of drug administration, fluid substitution, and resuscitation was required. All the reported events continued to be observed and resolved spontaneously.The severity of the adverse events was at best graded at 2. No serious adverse events were reported. The NCE under evaluation is a derivative of artemisinin compound which is rarely associated with any SAE. However, structural similarity is just an indicator for predicting adverse effect profile of a novel compound. A future possibility of such reactions coming into light always remains.Hence, the MTD could not be determined. Though determination of MTD was the main purpose of the study, this could not be achieved. This is a definite possibility in phase I studies as has been seen previously , 22. ForThere were some haematological and biochemical parameters which showed statistically significant values when compared to placebo but the differences were not clinically significant as most of them stayed within the normal laboratory range. Further, the absence of dose response relationship highlighted the fact that they were innocuous. Nonclinical toxicity data serves as a guide to the monitoring of adverse drug events once the studies are conducted in healthy volunteers. Though there were no specific concerns raised in nonclinical toxicity, a thorough evaluation of haematological and biochemical parameters was undertaken. Statistically significant differences should be interpreted with caution keeping in mind the clinical relevance , 24.Since structurally the compound was similar to artemisinin, which has been reported to prolong QTc interval , a carefIn conclusion, we can say that the new compound, CDRI 97/78, appears to be a safe and tolerable drug. Its safety can be further tested in multiple dose studies and, if found tolerable, can be a good therapeutic option for malaria, especially malaria resistant to available drugs.


Aim. We aimed to determine spinopelvic balance in 8–19-year-old-people in order to assess pelvic and spinal parameters in sagittal view. Methods. Ninety-eight healthy students aged 8–19 years, who lived in the central parts of Tehran, were assessed. Demographic data, history of present and past diseases, height (cm), and weight (kg) were collected. Each subject was examined by an orthopedic surgeon and spinal radiographs in lateral view were obtained. Eight spinopelvic parameters were measured by 2 orthopedic spine surgeons. Results. Ninety-eight subjects, among which 48 were girls (49%) and 50 boys (51%), with a mean age of 13.6 ± 2.9 years (range: 8–19) were evaluated. Mean height and weight of children were 153.6 ± 15.6 cm and 49.9 ± 13.1 kgs, respectively. Mean TK, LL, TT, LT, and PI of subjects were 37.1 ± 9.9°, 39.6 ± 12.4°, 7.08 ± 4.9°, 12.0 ± 5.9°, and 45.37 ± 10.7°, respectively. Conclusion. Preoperation planning for spinal fusion surgeries via applying PI seems reasonable. Predicating “abnormal” to lordosis and kyphosis values alone without considering overall sagittal balance is incorrect. Mean of SS and TK in our population is slightly less than that in Caucasians. Various parameters have been introduced to describe sagittal alignment of the spine and pelvis. Sagittal spine and spinopelvic parameters are different in adults and children, but these parameters correlate with each other to maintain global balance in both groups. There is no proper description of sagittal spinopelvic balance parameters, characteristics, and relationships in children. Correct concept of normal spinopelvic balance of children would effectively help spinal surgeons assessing spinal deformities and proper planning for treatment. Human standing posture is the result of balance between spine and pelvis . ThoraciSubjects of our study were 98 healthy students (50 boys and 48 girls) aged 8–19 years, who live in one of the central parts of Tehran. The study was approved by the ethical committee of our university. Goals and design of the study in addition to X-ray exposure were fully explained to them, and those children and parents who accepted the principles of study were recruited. Demographic data, history of present and past diseases, height (cm), and weight (kg) of all children were recorded. Each subject was examined by an orthopedic surgeon 3rd author). Children with more than 1 cm difference in their legs' length, history of trauma, present or past pelvic or spinal pain, disorder or abnormality, deformity proven via Adam's test, or signs of hip disorder were not included. Entirely 106 subjects had these inclusion criteria. A long cassette (30 cm in 90 cm) was chosen; children were asked to place their right side closely to the cassette in relaxed standing position, with their shoulders being 90-degree flexed and elbows fully flexed so that their fingers touched their ipsilateral shoulder. X-ray source was placed at 120 cm distance from the cassette. If femoral head or 7th cervical vertebra was not clearly seen in the radiograph (eight subjects), the subject was excluded. Eight spinopelvic parameters were measured twice by 2 orthopedic spine surgeons on each radiograph of 98 subjects separately. None of them were aware of the other surgeon's measurements. Recorded values of each surgeon for each radiograph were compared, and in case of any inconsistency, the aforesaid values were recalculated by a 3rd orthopedic spine surgeon (4th author). Assessed landmarks were superior end plates of T1, L1, S1, center of C7 body, anterosuperior of T1, L1, anteroinferior of T12, L5, center of sacral plate, and center of femoral heads. If two femoral heads were seen, the midpoint of the connecting line was selected. As shown in Figures rd authorThoracic tilt (TT) is the angle between the vertical line and line drawn at the anterosuperior point of T1 body and anteroinferior point of T12 body. Lumbar tilt (LT) is the angle between the vertical line and a line drawn from anterosuperior point of T1 body and anteroinferior point of T12 body  and 50 boys (51%), with mean age of  13.6 ± 2.9  years (range: 8−19) were evaluated. Mean height and weight of children were  153.6 ± 15.6 cm and  49.9 ± 13.1 kgs.P  values are shown in Correlation matrix between dependent and independent parameters using Pearson's correlation and their related  Thoracic kyphosis was positively related to lumbar lordosis which means lumbar lordosis would increase as thoracic kyphosis increases. LT had linear positive relation with TK. Besides PI was significantly related to LL. This relationship was positive. However, PI shows significant inverse relation with LT and was not related to TT.Normal ranges of sagittal spinal parameters are incumbent for pre- and intraoperation planning of spinal fusion surgeries  to minimr = 0.56,  P  value < 0.001. r = 0.62,  P  value < 0.001), as similarly shown in the algorithm, and tight relationship with LL.Mac-Thiong et al.  found thr = 0.57,  P  value < 0.001), and LL and TK  unlike TT which was not significantly related to LT and LL. In addition LT was positively related to TK  and negatively related to SS , in linear correlation the following equation was obtained:There are some differences between relations in This equation is relatively similar to Mcthiong's equation LL = 0.5919 × PI + 29.461 , particuPreoperation planning for spinal fusion surgeries applying PI seems applicable. Predicating “abnormal” to lordosis and kyphosis values alone without considering global sagittal balance is incorrect. Mean of SS and TK in our population was slightly less than that in Caucasians.


Objective. To determine the correlation between maternal bile acid (BA) level and fetal pulmonary surfactant in rats and study the effects of BA on fetal lung in rat model of intrahepatic cholestasis of pregnancy.  Methods. Forty pregnant rats were treated with (A) 5.5 mg/kg BA, (B) 1.4 mg/kg BA, and (C) 1 ml physiological saline. Levels of total bile acid (TBA), ALT, AST, TBIL, DBIL, and SP-A were determined and the lungs of fetal rats were analyzed for pathological changes.  Results. Groups A and B intervened with BA showed significant higher level of TBA in both maternal and fetal serum, more mortality rate of fetal rats, more concentration of SP-A in fetal serum, and wider alveolus mesenchyme of fetal rats than the control Group C. Higher level of BA associated with increased fetal risk and lower numerical density of mitochondria in type II alveolar epithelial cells. The levels of TBA in maternal serum were found to have significant positive correlation with those in fetal serum and SP-A level but negatively with the area of alveolus and the numerical density of lamellar body.  Conclusions. The TBA level in maternal serum showed significant association with lung pathological changes in fetal rats. Intrahepatic cholestasis of pregnancy (ICP) is liver disease which could lead to premature birth, fetal distress and neonatal asphyxia, and increasing risk of fetal morbidity and mortality . The incRecently, the abnormalities of pulmonary surfactant system have been implicated in the pathogenesis of respiratory distress syndrome , 7. Howe In vitro experiments showed that chenodeoxycholic acids could also disrupt cellular membranes in the alveoli, increasing cationic permeability and intracellular Ca2+ concentration, leading to overloading of Ca2+ in cells, and causing injury to type II pneumonocyte [However, some bile acids have toxic effects on a variety of cells. For example, chenodeoxycholic and lithocholic acids are cytotoxic and have detergent effects.monocyte . Porembkmonocyte . Intratrmonocyte . Toxic emonocyte . Recentlmonocyte  and Qiximonocyte  by direcmonocyte , 19. In Forty Sprague-Dawley pregnant rats were randomly divided into three groups: Group A, Group B, and Group C. The rats provided by animal experiment center of Xiangya Hospital, Central South University, were weighed 250~270 g and aged at 150~180 days. From day 13 of gestation, rats in Group A and Group B were injected intraperitoneally with bile acid (cholic acid (CA), Sigma-Aldrich, USA) for 8 days consecutively. Group A (high dose group) was injected with 5.5 mg/kg bile acid. Group B (low dose group) was injected with 1.4 mg/kg bile acid. Group C  was injected intraperitoneally with Sodium Chloride (NS). On day 13 of gestation, 1 mL blood was drawn from tail vein of pregnant rats to determine the levels of TBA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL). On day 21 of gestation, cesarean section was performed with anesthesia by intraperitoneal injection of 10% hydral with dosage of 3 mL/kg and the number of fetal rats, the number of still birth, and fetal weight were recorded. 1 mL blood from abdominal vein of pregnant rats was drawn to measure the levels of TBA, ALT, AST, TBIL, and DBIL. The fetal rat serum was collected to determine the level of TBA and SP-A. Ten fetal rats were selected randomly and the lung tissue was completely removed with ophthalmic scissors. The tissue was cut in size of about 1 mm × 1 mm × 1 mm from the lower right lung and then put in 2.5% glutaraldehyde phosphate buffer to fix specimens used for the electron microscopy. The remaining lung tissue was fixed by 10% formalin to be used for light microscope analysis.μL standard solution and sample solution were taken and chromatograms of the standard sample and experimental sample were obtained , and hematoxylin-eosin (HE) staining. Lung tissue samples were removed from fixed specimens fixed by 2.5% glutaraldehyde phosphate buffer and then by 1% osmic acid fixation, gradient acetone dehydration, Epon812 embedded agent, and ultrathin sections (thickness 60 nm), as uranium and lead citrate double staining, then observed under Hitachi H-7500 transmission electron microscope and photographed with Germany CCD camera system.Hitachi 7060 Automatic Biochemistry Analyzer was used to measure TBA, ALT, AST, TBIL, and DBIL and amniotic fluid TBA. The concentrations of SP-A in the fetal rat blood were investigated by ELISA . High efficiency liquid chromatography (HPLC) was applied to determine the contents of amniotic fluid phosphatidyl choline (PC), phosphatidyl inositol (PI), lysophosphatidyl choline (LPC), and sphingomyelin (SM), which were provided by Sigma-Aldrich . Pure grade methanol, phosphoric acid, chloroform and acetonitrile were purchased from Medicines Group Shanghai Chemical Reagent Co., Ltd. HPLC system was manufactured by Shimadzu Corporation. Hw-2000 color spectrum data station was provided by Nanjing Qianpu Software Corporation. The main procedures were described as follows: 5 mg of standard phospholipids product was accurately weighed and solved by 100 mL chloroform-methanol and mixed evenly; phospholipids in the taken samples were solved by 10 mL chloroform-methanol. Under the above color spectrum conditions, 20HE stained sections were put under the optical microscope to observe morphological development of fetal lung. 6 fields of vision were randomly selected from each slice and analyzed by the image analysis system to determine alveolar septum thickness and alveolar size . The alvt-test was used to compare between groups of multiple groups, χ2 test was used to compare the rates of different samples, and Spearman correlation analysis was applied to do correlation analysis.SPSS13.0 software was adopted to proceed statistical analysis, and the results were presented as μmol/L), Group B (3.32 ± 1.90 μmol/L), and Group C (3.53 ± 1.97 μmol/L), P > 0.05. No significant differences were found in TBA, ALT, AST, TBIL, and DBIL between Group A, Group B, and Group C before intervention, P > 0.05, as shown in There were no significant differences in TBA concentration of maternal serum between Group A (3.45 ± 1.74 μmol/L) was significantly higher than that of Group B (10.49 ± 3.22 μmol/L), P < 0.01; the level of TBA in Group B (3.98 ± 2.18 μmol/L) was significantly higher than that in Group C, P < 0.01, as shown in The level of TBA in maternal serum of Group A (21.09 ± 8.58 μmol/L was significantly higher than that in Group B (10.73 ± 4.76) μmol/L, P < 0.01; the TBA level in Group B was significantly higher than that in Group C (4.08 ± 2.40) μmol/L, P < 0.05. The concentration of SP-A in fetal serum of Group A (12.45 ± 4.67 ng/mL) was significantly higher than that in Group B (7.33 ± 2.54 ng/mL), P < 0.01; the concentration of SP-A in Group B was also significantly higher than that in Group C (2.71 ± 1.30 ng/mL), P < 0.01, as shown in r = 0.882, P < 0.01), and the level of SP-A is also significantly positively correlated with the level of TBA    < 0.01) .P < 0.01; the mortality rate of fetal rats in Group B was significantly higher than that in Group C (3.77%), P < 0.01, as shown in The mortality rate of fetal rats in Group A (18.35%) was higher than that in Group B (8.82%), μm2) was significantly smaller than that in Group B (717.46 ± 22.07 μm2) (P < 0.01); the area of alveolus of fetal rats in Group B was significantly smaller than that in Group C (900.13 ± 15.89 μm2), P < 0.01. The width of alveolus mesenchyme of fetal rats in Group A (70.21 ± 8.95 μm) was significantly thicker than that in Group B (32.21 ± 4.23 μm) (P < 0.01), while the width in Group B was thicker than that in Group C (23.51 ± 2.73 μm), P < 0.01, as shown in r = −0.794, P < 0.01) and SP-A   . In low  < 0.01) .In the experimental control group, type II alveolar epithelium had clear structure, with short microvillus on its surface; the nucleus was oval and the karyolemma was complete, with uniform distribution of chromatin. Abundant lamellar body could be observed in alveolar and type II intracytoplasm, which was in dark color and having clear lamellar structure . In low μm−3) was significantly lower than that of Group B (31.46 ± 3.45 μm−3), P < 0.01; the density between Group B and Group C had no statistical difference, P > 0.05. Comparison of the average volume of mitochondria between the two dose groups (23.22 ± 2.76 × 10−3 μm3and 21.27 ± 2.62 × 10−3 μm3) showed no significant difference. However, these two dose groups had significantly higher average volume of mitochondria than the control, P < 0.01.Comparison of mitochondria density and average volume of type II alveolar cells in fetal rats of four groups showed that the mitochondria density intype II alveolar cells ofGroup A (20.44 ± 4.99 μm−3  and 12.22 ± 2.89 × 10−3 μm3) was lower than Group B (74.46 ± 3.06 μm−3  and 23.27 ± 2.43 × 10−3 μm3). Group B was significantly lower than Group C (81.30 ± 6.56 μm−3 and 31.40 ± 2.92 × 10−3 μm−3), as shown in r = −0.701, P < 0.01).Comparison of the density and average volume of lamellar body in fetal rats between four groups showed that Group A  could cause increasing maternal serum bile acid level without increasing bilirubin level, thus excluding the toxic effects of bilirubin in this study.Bile acid could enter lung tissue by two ways in terms of amniotic fluid aspiration and blood circulation. Previous studies have showed that high concentration of bile acid gathered in alveolus cleaning solution in ICP neonates , but thePulmonary surfactant is secreted by type II alveolar epithelial cells, which can reduce tension in the surface of alveolar in order to maintain the alveolar stability. De Luca et al. showed that bile acids cause surfactant inactivation by enhancing the activity of secretory phospholipase A2 (sPLA2), a key enzyme in the lung injury pathway . HerraezInterestingly, immature fetal lung tissues with smaller alveolar space and thicker alveolar septal were found in high bile acid group. A large number of hemorrhagic lesions were also observed. The width of alveolus mesenchyme of fetal rats in bile acid group was thicker than that in control groups. There is negative correlation between the area of alveolus and TBA. The thinning of the alveolar space and the expansion of alveolar area are the morphologic sign of mature lungs . We obseIn addition, type II alveolar epithelial cells with swelling mitochondria were degenerated in high bile acid group, and lamellar body disappeared. A large number of red blood cells, proteins, cell debris falling, and edema fluid were filled in alveolar cavity. In low bile acid group, type II alveolar epithelial cells with decreasing microvilli on the surfaces and slight swelling mitochondria were observed. The density and average volume of lamellar body ofbile acid group were lower than those in control group. There is significant negative correlation between the density of mitochondria intype II alveolar cells and TBA. There is significant negative correlation between the density of lamellar body and SP-A. It may result from the damaging effects of bile acid on the lung epithelium. Lamellar body is a distinctive structure in type II alveolar epithelial cells. Because of the degeneration and necrosis of type II alveolar epithelial cells, the synthesis of pulmonary surfactant was hampered and affected by the perinatal prognosis.In summary, we found that, as an increasing bile acid in the maternal blood, the morphology of fetal lung tissue and alveolar-capillary membrane are disposed to be damaged. Some changes in the mitochondria of type II alveolar cells and lamellar body were observed in our study. The necrosis of type II alveolar cells and a decreasing of lamellar body may lead to hampering of the synthesis of pulmonary surfactant, which may impair normal breathe of fetus at birth and affect the perinatal prognosis.


Drynaria quercifolia fronds are used to treat inflammation as poultice on swellings and as antibacterial, hepatoprotective, and antipyretic agent. Herein, we have evaluated the antioedematous, antiproliferative, and analgesic properties of the ethanolic extract of fertile fronds of D. quercifolia (FF) by standard procedures. Oral administration of FF produced significant inhibition of carrageenan and histamine induced paw oedema in Wistar rats. FF significantly reduced both wet weight and dry weight of granuloma tissue which shows the inhibitory effect on exudative and proliferative phases of inflammation. FF significantly attenuated acute and delayed phases of formalin induced pain, acetic acid-induced writhing, capsaicin-induced nociception, and hot plate test in mice. Phytochemical analysis revealed the presence of coumarins, flavonoids, glycosides, phenolics, saponins, steroids, tannins, and terpenoids. Total phenolic content was 186 mg/g equivalent of gallic acid. The HPLC estimation showed flavanone glycoside naringin (1.2%) and its aglycone naringenin (0.02%). The presence of potent anti-inflammatory and analgesic principles in FF and their synergistic action may be the reason for the proposed therapeutic effects.Inflammation is a complex biological response of tissue cells to harmful stimuli including trauma, tissue necrosis, and infections which plays a key role in the pathophysiology of many deadly diseases. In ethnomedicine  Inflammation is a complex biological response of tissue cells to harmful stimuli including mechanical trauma, tissue necrosis, and infections which plays a key role in the pathophysiology of many deadly diseases including Alzheimer's disease, Type 2 diabetes, rheumatoid arthritis, liver cirrhosis, cancer, and neurological, pulmonary, and cardiovascular diseases. Although several modern drugs are used to treat these types of disorders, their prolonged use may cause severe adverse side effects . ConsequDrynaria quercifolia (L.) J. Smith (Polypodiaceae) is an epiphytic/epilithic medicinal pteridophyte, distributed widely in the Western Ghats, and locally called “Marappannakizhangu” or “Attukalkizhangu.” Fertile fronds are long stalked, large, 2–8 ft. long, pinnately lobed, and leathery or membranous in texture. The fertile fronds bear the sori, which are round and superficial. The fronds are emollient, pectoral, and expectorant, used as poultice on swelling, and possess antibacterial property [D. quercifolia is anthelmintic and tonic, used to treat chest, skin diseases, and loss of appetite [D. quercifolia using in vivo pharmacological methods.property . The whoappetite . It is aappetite  and antiappetite , 13. In Acetyl salicylic acid (aspirin), carrageenan, gallic acid, histamine, indomethacin, naringin, naringenin, and sodium carbonate were obtained from Sigma Chemicals Co., USA. Sodium salicylate was purchased from Hi-Media, India, and thiosol sodium from Neon Laboratory, India. All the other chemicals were of analytical and HPLC grade.D. quercifolia were collected from the Western Ghats and authenticated by plant taxonomist of the Institute. A voucher specimen had been deposited at the Herbarium of the Institute .The fertile fronds of The fertile fronds were washed thoroughly with tap water, cut to 1 cm cubes, shade-dried, and powdered. The powder (100 g) was extracted with 95% ethanol (1000 mL) for 24 h, at room temperature with constant stirring. The extract was filtered and the filtrate was concentrated at 30°C under reduced pressure in a rotary evaporator. The yield (w/w) of the crude extract was found to be 6%. The crude extract was suspended in 1% Tween-80 to required concentrations and used for the experiments and referred to as FF.ad libitum. All experiments involving animals were done according to NIH guidelines, after getting the approval of the Institute's Animal Ethics Committee.Wistar rats (150–250 g) and Swiss albino mice (25–30 g) of either sex were selected for the study, housed in poly acrylic cages , and maintained under standard laboratory conditions . They were fed commercial rat feed (Lipton India Ltd. Mumbai) and boiled water Rats were divided into 5 groups of 6 animals each. Group 1  received p.o., 1% Tween-80, 30 min prior to carrageenan injection, groups 2, 3, and 4 were given 125, 250, and 500 mg/kg of FF p.o., and group 5, the standard group, was given p.o., as aqueous solution of indomethacin (10 mg/kg), respectively, 30 min prior to carrageenan injection. The paw volume was measured plethysmographically just before and 3 h after carrageenan injection. The percentage inhibition of oedema was calculated for each group with respect to the vehicle treated control group .Anti-inflammatory activity of FF was assessed by the histamine-induced paw oedema method. Rats were divided into 5 groups of 6 animals each. Animals of all the groups were injected with 0.1 mL of 0.1% histamine in the right hind foot under the plantar aponeurosis. Group 1 animals  received p.o., 1% Tween-80, 30 min prior to histamine injection, groups 2, 3, and 4 were given 125, 250, and 500 mg/kg of FF p.o., respectively, 30 min prior to histamine injection, and group 5, the standard reference group, was given p.o., an aqueous solution of indomethacin (10 mg/kg), 30 min prior to histamine injection. The paw volume was measured using a plethysmometer just before and 1 h after histamine injection. The percentage inhibition of oedema was calculated for each group with respect to the vehicle treated control group .The effect of FF on granuloma formation was studied in Wistar rats. The dorsum of the rats was shaved and wiped with alcohol. A 1 cm midline incision was made in the interscapular region. Sterile cotton pellets (100 ± 1 mg) were inserted on either side of a midline incision (1 cm) made on the dorsum of rats and the incision was closed by interrupted sutures. FF  was administered p.o., daily for 7 consecutive days starting from the day of pellet implantation. Indomethacin  was given to the standard group while the control group received p.o., an equal quantity of 1% Tween-80. The animals were sacrificed on the 8th day in which the cotton pellets were removed, cleaned of extraneous tissue, weighed and dried in hot air oven at 80°C for 24 h .Mice were divided into 5 groups (6 mice/each group). All the groups received i.p., 0.5% aqueous solution of acetic acid. Group1, the acetic acid control (AC) group, received a single dose of 1% Tween-80 (0.5 mL) p.o., 20 min prior to the administration of acetic acid. Groups 2, 3, and 4 received p.o., 100, 200, and 300 mg/kg of FF whereas group 5, the standard control group, received p.o., acetyl salicylic acid (aspirin) (100 mg/kg), respectively, 20 min prior to the administration of acetic acid. The number of writhes per animal was recorded during the 20 min period, beginning 5 min after the injection of acetic acid .μL of 1% formalin was injected into the dorsal surface of the left hind paw. The time spent for licking the injected paw was recorded. Animals were observed for the 5 min after formalin (acute phase) or for 10 min starting at 20 min after formalin (delayed phase) [Mice were divided into 5 groups of 6 animals each. Group 1, the control group, received 1% Tween-80, p.o., groups 2, 3, and 4 received 100, 200, and 300 mg/kg FF extract p.o. whereas group 5, the standard group received sodium salicylate (100 mg/kg), 30 min prior to formalin injection. 20 d phase) .μL of capsaicin (1.6 μg/paw) was injected into the right paw. Capsaicin was dissolved in 5% ethanol, 95% phosphate buffer saline, and pH 7.4. Animals were observed individually for 5 min following capsaicin injection. The amount of time spent licking the injected paw was recorded and considered indicative of nociception [To provide direct evidence concerning the antinociceptive effect of FF on neurogenic nociception, FF  was tested against capsaicin-induced paw licking in mice. Morphine 7.5 mg/kg (s.c.) was used as the standard. The animals were treated with 1% Tween-80 (vehicle), FF, and morphine and placed in a glass box. After 30 min (which served as an adaptation period) 20 iception .Analgesia of FF was assessed using Eddy's hot plate , temperature 55 ± 1°C. Mice were divided into 6 groups of 6 animals each. Group 1, the control group, received 1% Tween-80, p.o., groups 2, 3, and 4 received 100, 200, and 300 mg/kg FF extract, p.o., and groups 5 and 6 the standard groups received sodium salicylate (100 mg/kg) and morphine (7.5 mg/kg). Mice were placed on hot plate maintained at 55 ± 1°C at 0, 15, 30, 60, 90, and 120 min after drug administration. The time taken by animals to lick the fore or hind paw or jump off the plate was taken as the reaction time(s); 15 s cutoff was used to prevent tissue damage [Six groups of 10 mice each were administered p.o., 500, 1000, 2000, 3000, 4000, and 5000 mg/kg, respectively, of FF, maintaining appropriate controls. All the animals were observed continuously for the first 3 h then 1 h intermittently up to 24 h for behavioral changes like convulsions, hyperactivity, sedation, grooming, loss of righting reflex, epilation, respiratory rate, food and water intake, state of fecal pellets, and mortality. The animals were observed for post treatment toxic symptoms daily for 7 days after treatment .FF was subjected to preliminary phytochemical analysis and TLC studies, as per the standard methods .μg/mL) prepared in ethanol. To the above standard solutions of gallic acid (1 mL each), 5 mL of Folin-Ciocalteu's reagent (1 : 10 dilution in distilled water) was added and, after 8 min, 4 mL of 7.5% sodium carbonate was added. These solutions were incubated at room temperature for 2 h and their absorbances at 765 nm were measured on UV-visible spectrometer . For the test solution, 1 mL of FF (10 mg in 10 mL EtOH) was mixed with 5 mL of Folin-Ciocalteu's reagent and 4 mL of 7.5% sodium carbonate was added after 8 min and incubated at room temperature for 2 h and its absorbance was also read at 765 nm. The gallic acid equivalent (GAE) was plotted and the total phenolic content of FF was calculated in terms of gallic acid equivalents by considering the dilution factor [The total phenolic content in FF was determined by Folin-Ciocalteu's method. A standard curve was prepared using different concentrations of gallic acid  driven by a UNIPOINT system software and the chromatographic separations were performed using a C18 Kromasil Column (250 × 4.6 mm), with a flow rate of 1 mL/m and a sample size of 15 μL. The mobile phase used was methanol, acetonitrile, and ammonium acetate buffer (5 : 11 : 4) and isocratic elution was performed. The sample was monitored with UV detection at 260 nm at the flow rate of 1 mL min−1 at ambient temperature. Retention time for naringin was found at 2.3 min and that for naringenin was at 2.6 min [The HPLC analysis of  2.6 min .P ≤ 0.01 [The results were expressed as mean ± standard deviation of mean (SD). Analysis of variance (ANOVA) was done to compare and analyse the data followed by Duncan's multiple range test. Effects were considered significant at P ≤ 0.01 .FF at all the doses  studied significantly inhibited the carrageenan-induced paw oedema in rats in a dose-dependent manner, 60.00%, 63.53%, and 71.76%, respectively. The standard group treated with indomethacin showed 88.24% inhibition of oedema formation .The amount of oedema produced was quantified 1 h after histamine injection. FF at the 3 doses  studied significantly inhibited the histamine induced paw oedema in rats, 53.33%, 62.22%, and 66.67%, respectively. The group treated with indomethacin (10 mg/kg) showed 68.89% inhibition .FF produced a significant dose-dependent inhibition of granuloma formation. It was almost equally potent as indomethacin in inhibiting both exudative and proliferative phases of granuloma formation. The formation of granuloma tissue around implanted cotton pellet is shown in Intraperitoneal injection of acetic acid produced 54.0 ± 2.0 writhes in the control group, 20 min after injection. The groups pretreated with FF  inhibited the number of writhes in a dose-dependent manner, 56.66%, 63.70%, 74.07%, respectively. Aspirin 100 mg/kg produced 88.33% inhibition of writhes .SS did not show any significant effect on the acute phase of the formalin test (40.48%), but FF at 100, 200, and 300 mg/kg reduced the nociception in this phase in a dose-dependent manner . In the delayed phase of formalin test, SS produced only 63.55% inhibition while FF reduced the nociception significantly that is, 68.94%, 76.28%, and 81.03% dose dependently .μg) evoked nociceptive paw licking behavior in mice that appeared maximal up to 5 min. A significant decrease in the duration of paw licking was observed in mice pretreated with FF at doses of 100, 200, and 300 mg/kg . The results show that FF produced marked and dose-related inhibition of the capsaicin-induced neurogenic pain in mice. Morphine (7.5 mg/kg) was found to be very effective (91.94%) against capsaicin-induced pain , the pungent analgesic substance obtained from hot red chilli peppers, is regarded as a valuable pharmacological tool for studying a subset of mammalian primary sensory C-fibers and Aγ afferent neurons including polymodal nociceptors and warm thermoreceptors [Oral administration of FF elicited a dose-dependent antinociceptive effect on the capsaicin-induced neurogenic paw-licking response. Capsaicin  has potent antioedematous and analgesic properties which substantiates its ethnomedicinal claims in the treatment of inflammation and pain.The results obtained from the present study reveal that