Prior to the 2009 H1N1 Influenza pandemic, public health authorities in Canada and elsewhere prepared for the future outbreak, partly guided by an ethical framework developed within the Canadian Program of Research on Ethics in a Pandemic (CanPREP). We developed a telephone-based survey based on that framework, which was delivered across Canada in late 2008. In June, 2009, the WHO declared pandemic Phase 6 status and from the subsequent October (2009) until May 2010, the CanPREP team fielded a second (revised) survey, collecting another 1,000 opinions from Canadians during a period of pre-pandemic anticipation and peri-pandemic experience.Surveys were administered by telephone with random sampling achieved via random digit dialing. Eligible participants were adults, 18 years or older, with per province stratification approximating provincial percentages of national population. Descriptive results were tabulated and logistic regression analyses used to assess whether demographic factors were significantly associated with outcomes, and to identify divergences (between the pre-pandemic and intra-pandemic surveys).N = 1,029 interviews were completed from 1,986 households, yielding a gross response rate of 52% (AAPOR Standard Definition 3). Over 90% of subjects indicated the most important goal of pandemic influenza preparations was saving lives, with 41% indicating that saving lives solely in Canada was the highest priority and 50% indicating saving lives globally was the highest priority. About 90% of respondents supported the obligation of health care workers to report to work and face influenza pandemic risks excepting those with serious health conditions which that increased risks. Strong majorities favoured stocking adequate protective antiviral dosages for all Canadians (92%) and, if effective, influenza vaccinations (95%). Over 70% agreed Canada should provide international assistance to poorer countries for pandemic preparation, even if resources for Canadians were reduced.Results suggest Canadians trust public health officials to make difficult decisions, providing emphasis is maintained on reciprocity and respect for individual rights. Canadians also support international obligations to help poorer countries and associated efforts to save lives outside the country, even if intra-national efforts are reduced. In April 2009, a novel H1N1 influenza virus emerged from Mexico . MargareDespite considerable concern about H1N1 in 2009, its impact in terms of morbidity and mortality was relatively mild, although significant societal and health care system disruptions were observed -8. In OcStand on Guard for Thee: Ethical Considerations in Preparedness Planning for Pandemic Influenza (SOGFT) , marital status [married (including common law) vs. unmarried ], education [<college/university vs. ≥ college/university] and employment [employed (full time and part time) vs. unemployed ].A total of 3,188 randomized telephone numbers were drawn, of which 1,986 numbers were estimated to be households. Households included all numbers where successful interviews occurred, plus all households where refusals or callbacks were noted, as well as an assumption that the majority of the "never answered” numbers were households. N=1,029 interviews were completed from 1,986 households yielding a gross response rate of 52% (AAPOR Standard Definition 3). However, at telephone numbers where an identifiably eligible respondent answered, the response rate was 88%. The greatest number of repeated calls needed to get a single completion was 46, and approximately 10% of the interviews were completed on a 14th or subsequent call attempt.Table Responses to Survey 1 and 2 were compared, using a criterion of fluctuation greater than 10 percentage points to highlight survey items which may reflect substantial changes in public opinion. This value was selected to make the most substantial opinion shifts stand out more readily for analysis, given the large number of survey items. However, it must be noted that changes smaller than 10 percentage points could be significant, particularly for items where responses might predictably be highly homogenous under routine conditions. For example, in response to the question regarding the main purpose of a Canadian Pandemic Plan, the most frequent responses were ‘saving as many lives as possible in Canada” (36% in Survey 2 vs. 41% in Survey 1) and “saving as many lives as possible, globally” (40% in Survey 2 vs. 50% in Survey 1). While other response options to this question were somewhat more frequently endorsed in Survey 2 compared to Survey 1, they were still infrequently endorsed compared to the two options concerning saving lives.As seen in Additional file In terms of global obligations to help poorer countries during a pandemic, even if the result was a reduction of resources for Canadians, a majority were in favor (58.6% agreed and 38.4% disagreed) although a smaller majority than in the first survey (70% agreed and 18% disagreed in survey 1). In both surveys, an overwhelming majority favored provision of adequate amounts of antiviral medications to every Canadian .not have to report to work . In survey 2, males were more likely to think that governments should reserve the right to conscript health care workers during an H1N1 flu pandemic  a finding that again can be compared with survey 1, where females were more likely to not agree  with government conscription of health care workers.In terms of the relevant regression analyses, those over the age of 53 years  and males  were more likely to think health care workers who don’t report to work during an H1N1 flu pandemic should face loss of employment or professional license. It is notable that this gender difference was also observed in survey 1, where females were more likely to agree that health care workers with dependents should disagree with provision of international assistance and those unemployed  and living in rural communities  were both more likely to disagree specifically with Canadian donation of more than 10% of total resources to poorer countries. Finally, in survey 2 those with <college/university education  were more likely to feel that adequate amounts of antiviral medication should be provided to every Canadian. If anti-viral medication supplies were insufficient, those unmarried , <college/university education , and the unemployed  were all more likely to feel there should be an equal chance  of receiving the medication as opposed to a priority-based distribution. In survey 1, participants with < college/university education were significantly less likely  to support government-set priorities regarding distribution of antiviral medications.In terms of global obligations, respondents with ≥ college/university education  and without children  were more likely to think Canadians should help poorer countries even if it reduced resources for Canadians, while those who were unmarried , with <college/university education , and the unemployed  were all more likely to think all Canadian needs should be taken care of before sending resources to poorer countries. In survey 1, unemployed individuals  were more likely to Once again, the groups rated as having highest priority for hospital treatment/resources remained stable between surveys  although their relative order of priority changed in survey 2  compared to survey 1 . The differences in prioritization responses varied by less than 5 percentage points. The same pattern was evident with respect to access to antiviral drugs, with top priorities accorded to health care workers and children in both surveys. However, adults with chronic diseases were rated the third highest priority in survey 2, advancing ahead of public safety/social service workers and adults with dependents that had been rated higher in survey 1.About 19.7% of respondents felt becoming infected with H1N1 would have the most impact on them, followed by confronting a dysfunctional health system (17.9%) and loss of salary (17.5%). Approximately 76.3% of respondents felt schools and daycare facilities should be closed to prevent H1N1 infections. A strong majority of respondents felt it was acceptable to close entertainment venues (76.9%), require people to work from home (85.8%), close schools and daycares (77.3%), and limit non-urgent hospital visits/services (87.3%). Nearly half of respondents (46.8%) thought it was acceptable to shut down or limit public transportation to reduce H1N1 infection. In terms of regression results, the only significant finding was that males were more likely to think that schools and daycare facilities should be closed to prevent H1N1 flu infection .About 74% of respondents felt health care workers or other essential service workers had a duty to be immunized against H1N1, but this decreased to 56.7% if rare side effects were discovered to be associated with the vaccine. Seventy-seven percent felt that those injured by vaccine side effects should be compensated by the vaccine manufacturers, while 60% felt compensation should come from the government. Respondents stated they had the most trust in nationally selected scientific advisory boards (31.8%) and in the federal government (19.7%) to oversee a vaccine-injury compensation program. In considering a trade-off between safety testing and timely delivery of vaccines, 47.1% felt there should be no compromise in safety, while 25% felt there could be minor reductions in safety testing, and 23.6% felt there could be moderate reductions in safety testing. Altogether, 57% of respondents stated they would obtain a H1N1 vaccine during the fall months. In regression findings, those with <college/university education  and the unemployed  were both more likely to think that HCWs should be immunized against H1N1. The unemployed  also were more likely to think HCWs should be immunized even if rare vaccine related side effects were discovered. Those with ≥ college/university education  and those over 53 years of age  were more likely to state that they would obtain an H1N1 vaccine.increased ‘a great deal’ if adequate resources were directed to those most in need (62.2%). Respondents felt accurate communications (33%) and adequate resources provided to those most in need (25.1%) would be the most important observations in increasing their trust in public authorities.Respondents indicated they mainly accessed H1N1 information from the chief public health officer (83.4%) and their physician (94.8%), further indicating trust for information obtained from physician experts (35%) and the Public Health Agency of Canada (23.1%). Respondents also stated that receiving inaccurate information (55.7%), discovering that a specific group of Canadians were receiving less than a fair share of resources (51.4%), or that wealthier people were receiving special privileges (73.4%) were situations that would decrease respondents’ trust in public authorities by ‘a great deal’,. Trust in public authorities would be In terms of H1N1 information access, females were more likely to get information from their physician  while those ≥ college/university education , employed , younger (between 18–52 years)  and female  were more likely to access government websites. Additionally, those aged 53 and older  were more likely to access information from newspapers, while those younger than 53 were more likely to access Google news  and YouTube . Females , <college/university education , and those aged 18–52  were more likely to gain information from Facebook or MySpace.during the pandemic as contrasted with Survey 1 responses obtained when the pandemic was anticipated (but undefined). As a result, these data represent an important vantage point for pre-pandemic and peri-pandemic comparisons.These results demonstrate the complexity involved in understanding the public’s view of ethical issues in pandemic planning. While there is strong agreement and stability in many domains, there are important variations in opinion across the two time points. As Survey 2 was administered during the second wave of H1N1 in Canada, it reflects perceptions Most opinions of respondents were relatively stable in comparison to the previous survey. Canadians believe the main goal of a pandemic response is to reduce influenza-related mortality, with reduction of global mortality an important dimension of this objective. Respondents believed the H1N1 pandemic would have a significant impact and were willing to tolerate societal disruptions to increase safety, with a majority ready to accept closures of schools and daycare facilities to prevent infections although only a minority were prepared to shut down public transit. Canadians also acknowledged the difficulties that healthcare professionals (HCPs) face in personal health decisions, while still demanding high standards of care and professional practice responsibility. For example, Canadians strongly agreed that health care workers should face risks and provide care during an H1N1 pandemic (before and during pandemic conditions), but this view was tempered by support for excusing those with health conditions that put them at greater risk, or those with domestic (dependent) care obligations. This is further balanced by a consistent favoring of HCPs in prioritizing them for health-related resources, although it is impossible to determine whether this prioritization originates from a desire to maximize health care resources in a time of need, or a desire to ensure fairness or justice for HCPs. In trust-related questions, HCPs are seen as the most trusted purveyors of health information and the ones with the highest credibility to make difficult decisions regarding intensive care access. There is, nonetheless, substantial disagreement about the acceptability of sanctions for health care workers failing to provide care during a pandemic and the acceptability of conscription as a means of ensuring sufficient health care workers are available for pandemic response.Respondents remain supportive of public health social-distancing measures, with the exception of stopping public transit. Respondents also trust public health officials as sources of information in pandemics which may seem surprising given the confusion around messaging in the H1N1 vaccine roll-out, particularly concerning the unclear or changing information regarding vaccination priority groups during the 2009 H1N1 pandemic.Priority setting remains a controversial area as seen in the reluctance of a significant majority to identify categories of lowest priority. Future qualitative analyses from focus group data collected in association with this project  will permit greater insight into such responses. With current data, some of the underlying meanings of responses remain ambiguous. It is possible that participants are making a clear decision to not identify low priority groups based on a considered opinion that all Canadian subgroups are of relatively high importance, such that it may not be possible for or acceptable to respondents to determine priorities that may undermine valued notions of equality and fairness. However, it is also possible that participants are simply uncomfortable identifying low priority groups to surveyors as a matter of social desirability. The two highest priority populations shifted between survey 1 and 2, but only with respect to whether health care workers or children were the first priority. Both surveys provide potentially important insights into Canadian perceptions on the obligations of health care workers, the acceptability of public health measures, priority setting, communication and public trust, and indicate a number of avenues for further exploration with implications for infectious disease response planning and the day-to-day operations of our healthcare system.The strengths of this survey include the use of random digit dialing to access a large, representative Canadian sample and Time 1-Time 2 comparisons of baseline survey items. A carefully planned theoretical framework\underlies the survey structure. Nonetheless, there are limitations related to the overrepresentation of females and the large proportion of respondents self-identifying as white  and middle-aged. These demographics may better reflect the population with land-based telephone lines in Canada versus those with cellular phones; hence another limitation is the absence of a protocol for accessing cellular numbers. It is also worth noting that aside from the 2009 pandemic, other world-changing events occurred, or were occurring, between Surveys 1 & 2, including the initiation of world-wide banking and economic crises. As the data is based on a quantitative protocol, we can only hypothesize about the reasons for responses, but qualitative analyses, currently underway as another component of the overall CanPREP project, will soon supplement survey findings.increased ‘a great deal’ if adequate resources were directed to those most in need (62.2%). In summary, surveying the public on bioethical issues related to pandemic prevention and management is important. Increasing emphasis on public consultations will allow policymakers and public health authorities to target activities towards identified priorities, using modes of communication that address the loci of trust held by the broadest proportion of the population. Such targeted consultations can allow for better responses to the particular needs of so-called ‘vulnerable’ groups such as senior citizens, First Nations, and/or low-SES populations.Despite the understandable response fluctuations apparently related to sampling occurring during pandemic conditions, there was observable stability in the responses to survey 2 compared to the first (pre-pandemic) survey. In terms of new items, strong support was found for the duty or obligation of health care workers to be immunized against H1N1 and for vaccine manufacturers (77%) or government (60%) compensations, if immunization resulted in injurious side effects. Compensation could be overseen preferably by nationally selected scientific advisory boards (31.8%) and/or the federal government (19.7%), although opinion was divided on the question. Public trust appeared closely associated with accurate information and equitable resource-sharing. Trust in public authorities would be decreased ‘a great deal’ with discoveries that a specific group of Canadians were receiving less than a fair share of resources (51.4%), and wealthier people receiving special privileges (73.4%); it would be The authors declare that they have no competing interests.PR, KW, JLG, CST, JXN, ARJ, and REGU drafted survey items. CG, PR, and REGU conducted statistical analyses. CG, PR, DP, KW, JLG, CST, JXN, ARJ, and REGU interpreted statistical analyses. PR, DP, KW, JLG, CG, CST, JXN, ARJ, and REGU drafted the manuscript. PR, KW, CST, and REGU determined methodology. PR, KW, CST, and REGU supervised telephone survey administration. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/271/prepubComparison of Selected Responses to Identical Items in Survey 1 and Survey 2.Click here for fileFull Results per Question (Survey 2).Click here for file


Angiostrongylus costaricensis (AC) in surgical specimens. However, microscopic evaluation is time consuming and often fails in identifying the parasite. We tested whether PCR might help in the diagnosis of AA by identifying parasite DNA in formalin-fixed paraffin-embedded (FFPE) tissue. We used primers based on DNA from Angiostrongilus cantonensis. Four groups of FFPE intestinal tissue were tested: (1) confirmed cases (n = 20), in which AC structures were present in the target tissue; (2) presumptive cases (n = 20), containing changes secondary to AC infection in the absence of AC structures; (3) negative controls (n = 3), consisting of normal colonic tissue; and (4) tissue affected by other parasitoses (n = 7), including strongyloidiasis, ascaridiasis, schistosomiasis, and enterobiasis. Most lesions of confirmed cases were located in small and/or large bowel (90%), as compared with presumptive cases, in which 70% of lesions were in appendix (P = 0.0002). When confronted with cases of other parasitoses, PCR showed sensitivity of 55%, specificity of 100% and positive predictive value of 100%. In presumptive cases PCR was positive in 4 (20%). All specimens from negative controls and other parasitoses were negative. In conclusion, the PCR technique showed intermediate sensitivity and optimal specificity, being clinically relevant when positive for abdominal angiostrongyliasis. It allowed a 20% gain in diagnosis of presumptive cases. PCR might help in the diagnosis of abdominal angiostrongyliasis, particularly when the pathologists are not experienced with such disease.To date the diagnosis of abdominal angiostrongyliasis (AA) depends on the histological identification of  Angiostrongylus costaricensis is an intra-arterial nematode harboring wild rodents as a definitive host, and mollusks as intermediate hosts A definitive diagnosis of AA is made through pathological examination of surgical specimens. For histological evaluation it is necessary to include the specimens in paraffin block, in an attempt to find parasitic forms, which demands multiple histological sections and examiner expertise. However, in most cases the finding of parasitic structures such as worms, larvae and eggs is difficult, leading to a presumptive diagnosis or a false negative reading. The presumptive diagnosis is made in the presence of severe eosinophilic infiltration, granulomatous reaction and eosinophilic vasculitis in the absence of parasitic structures Ascaris lumbricoides, Strongyloides stercoralis and Schistosoma mansoniAscaris sp Trypanosoma cruziNeospora caninumLeishmania spp Angiostrongylus costaricensis in paraffin blocks from surgical specimens has not been tested A. costaricensis DNA in samples from infected patients.Immunodiagnostic tests in serum have been developed, but show limitations due to cross-reaction with other parasite-related antibodies, such as This study was conducted according with rules of the Helsinki Declaration and was approved by the Ethical Committee of Pontifícia Universidade Católica do Rio Grande do Sul, Brazil (protocol no 10/00174). The referred ethics committee waived the need for consent for utilization of tissue samples.Angiostrongylus costaricensis located in intestinal tissue, including worms, eggs and larvae. After identification of a parasitic structure in a standard Hematoxylin and eosin staining (H&E) section, the corresponding paraffin block (5×5×5 mm) was cut with a scalpel. Cases presenting histological findings suggestive of AA, i.e. eosinophilic infiltration, granulomatous reaction and eosinophilic vasculitis, in the absence of parasitic structures were designated presumptive cases and the tissue for PCR was collected in the same manner as the confirmed cases including tissue with the aforementioned reactive changes. Negative controls corresponded to normal colonic tissue obtained from surgical specimens of patients with adenocarcinoma, whereas cases with other parasitoses included patients surgically treated for intestinal infection by Enterobius vermicularis, Strongyloides stercoralis, Schistosoma mansoni and Ascaris lumbricoides. FFPE tissue from these patients was processed in the same manner as confirmed cases. We also analyzed demographic and clinical features taken from medical records, as well as gross and histological findings. For the latter we graded the eosinophilic infiltrate and granulomatous reaction as mild, moderate and severe.We used formalin-fixed paraffin-embedded (FFPE) tissue samples from 4 groups of patients archived in our pathology Institute: confirmed cases of AA without other parasitoses (n = 20), presumptive cases of AA (n = 20), negative controls (n = 3), and cases with other parasitic diseases (n = 7). All samples were obtained from an archive of formalin-fixed paraffin-embedded (FFPE) tissue, corresponding to the period between 1996 and 2012. Confirmed cases contained structures of Angiostrongylus costaricensis was extracted from 10 mm thick FFPE tissue. This procedure was repeated three times, generating 3 sub-samples for each case. A number of sections were treated with xylene for deparaffinization and approximately 25 mg of tissue were obtained. This was centrifuged and washed twice with 100% ethanol, and dried at room temperature. DNeasy Tissue kit (Qiagen) was employed for DNA extraction. The primers were designed based on published sequences from mRNA of Angiostrongylus cantonensis  5′-CTCGGCTTAATCTTTGCGAC-3′) and Angio F1 For (5′ AACGAGCGGCAGTAGAAAAA-3′), using AmpliTaq Gold PCR Master Mix, on the following conditions: 94°C for 4 min, 35 cycles of 94°C for 1 min, 58°C for 2 min and 72°C for 10 minutes in the thermocycler FTC405. For verification of amplicon with the expected product of 232 bp we used the horizontal electrophoresis in agarose gel with ethidium bromide. DNA bands visualization was performed in transluminator. To quantify the DNA extraction we used a Kit Qubit fluorometer, Invitrogen. For the positive control we used 2 worms of Angiostrongylus costaricensis in FFPE, prepared as we did with clinical samples.DNA of t test and Fisher exact test were used when appropriate, with the help of Graph Pad Prism version 4. WinPepi software was used to calculate PCR performance. A P-value <0.05 was assumed as indicative of statistical significance.We used mean ± SD for quantitative data and frequency and percentage for qualitative ones. Student's Most patients with confirmed AA were adults (25th percentile: 21 years), and about two-thirds were men . PresumpAmong 20 confirmed cases, 8 compromised the small bowel , 6 were localized in the large bowel , 4 compromised ileum and ceccum , and 2 cases presented as acute appendicitis. Perforations were found in the 3 cases with intestinal infarction. Out of 20 presumptive cases, 14 presented as acute appendicitis, 4 compromised the large bowel , and 2 cases with infarction were localized in the small bowel. One of these presented perforation.Intra-arterial worms were found in 13 of the confirmed cases . The remA. costaricensis was positive in areas containing worms, eggs or larvae in a total of 11 (55%) out of 20 confirmed cases  out of 20 , in areaWhen confronted to cases with other parasitoses , PCR tecThe diagnosis of AA is often difficult due to the scarce number of parasites in surgical specimens as well as limited pathological expertise. A definitive diagnosis of AA is based on the histopathological identification of parasitic structures such as worms, eggs and larvae in tissue. This process is time-consuming and costly due to the need of a high number of histological sections. When parasitic structures are not found, a presumptive diagnosis of AA may be assumed in cases of suggestive intestinal lesions characterized by a combination of eosinophilic infiltration, granulomatous reaction and eosinophilic vasculitis Angiostrongylus cantonensis was able to identify genetic material of Angiostrongylus costaricensis in paraffin-embedded tissue of patients with AA, including specimens stored for more than 10 years; (2) Although the sensitivity of PCR for detection of the disease in confirmed cases was intermediate (55%), its specificity was optimal (100%), since no case including negative controls and other parasitoses showed a positive test; (3) Despite the absence of parasitic structures in presumptive cases, PCR was positive in 20% of them, decreasing the rate of false negative findings seen with histology alone.In this large surgical series of AA, the main findings of our study were: (1) PCR technique using primers of Angiostrongylus for confirmation of AA in human cases. Such DNA sequences were designed in our laboratory Angiostrongylus in cerebrospinal fluid of cases with A. cantonensis related eosinophilic meningitis Angiostrongylus were useful in diagnosing cases of either A. cantonensis and A. costaricensis, urther studies for DNA sequencing of specific Angiostrongylus species are still warranted in order to increase diagnostic accuracy of this technique. However, such limitation does not rule out the utility of PCR in the clinical practice, since anatomical and clinical presentation of AA caused by A. costaricensis and eosinophilic meningitis related to A. cantonensis dictate the final diagnosis. PCR was able to detect parasitic DNA from old FFPE specimens, despite potential tissue degradation inherent to storage time. This finding raises the possibility of using PCR for diagnostic revision of old and unknown cases of archived tissue The PCR technique here employed used two DNA sequences of the genus Angiostrongylus DNA in confirmed cases. In the clinical practice, it means that the test is particularly useful when positive, given its high specificity  and high positive predictive value. Moreover, in presumptive cases the PCR was able to indicate the diagnosis of AA in 20% of patients. This could be explained by advanced destruction of parasitic structures in retention sites after strong immunologic response PCR showed intermediate sensitivity and optimal specificity for detection of Nevertheless, the pathologist must be aware that a negative result in highly suspicious cases does not rule out that the patient may still be infected. Further studies in prospective cases are needed to confirm the accuracy of PCR in the diagnostic workup of AA.In our series, confirmed cases of AA occurred mainly in adult men, in contrast with series from Costa Rica, where infants were mostly described Angiostrongylus costaricensis were based on sequences from Angiostrongylus cantonensis adult worms. One could question whether these primers would be appropriate for detection of Angiostrongylus costaricensis in stages other than adult worms, i.e. eggs and larvae. Nevertheless, we emphasize that primers used in this study were able to identify genetic material of Angiostrongylus costaricensis either from adult worms, larvae and eggs in the analysis of human tissue. Future studies using primers properly developed for different stages of Angiostrongylus costaricensis are important for confirmation of PCR performance.We acknowledge limitations in our study. The primers used for identification of Angiostrongylus in the largest series of human cases surgically treated for AA. The employed PCR used two DNA sequences of Angiostrongylus genus and resulted positive in the analysis of FFPE tissue either for specimens with parasite structures or granulomatous reaction. The successful utilization of PCR here reported points to the need of further studies to establish the diagnostic performance of this technique in a prospective study based on routine pathology.In conclusion, we report the molecular identification of


The etiology of congenital heart defect (CHD) is commonly believed to involve the interaction of multiple environmental and genetic factors. This study aimed to explore the joint effects of the ABCB1 gene C3435T polymorphism and maternal periconceptional toxicants exposure on the CHD risk in a Han Chinese population.An age and gender matched case-control study with standardized data collection involving 201 pairs was conducted. Periconceptional toxicants exposure was obtained through a structured questionnaire. A job exposure matrix (JEM) was used for toxicants exposure assessment. Genotyping of the ABCB1 C3435T polymorphism was performed by sequencing. Logistic regression analysis was performed to assess the joint effects of the ABCB1 gene C3435T polymorphism and toxicants exposure on the risk of CHD. Placenta tissues and umbilical cords were collected to investigate the impact of C3435T polymorphism on the transcription and translation activities of ABCB1 gene.Maternal periconceptional exposures to phthalates  and alkylphenolic compounds  were associated with a higher incidence of CHDs in general. More cases were carriers of the ABCB1 CC/CT genotypes . Children carrying the CC/CT genotype and periconceptionally exposed to phthalates and alkylphenolic compounds suffered almost 3.5-fold increased risk of having CHD than non-exposed children with TT genotype , and the OR changed to 4.4 for septal defects . The ABCB1 mRNA expression of the TT genotype was significantly higher than that of the CC genotype (P = 0.03). Compared with TT genotype, lower P-glycoprotein expression was observed for the CC/CT genotypes.The C3435T polymorphism in the ABCB1 gene of fetus increases the risks of CHD in a Han Chinese population when the mothers are exposed to phthalates and alkylphenolic compounds during the periconceptional period, particularly for septal defects. Worldwide congenital heart defect (CHD) is one of the most common birth defects, occurring in 7 to 8 per 1000 live births in China. After years of study, the etiology of CHD is still unclear, but is commonly believed to involve the interaction of multiple environmental and genetic factors There is increasing evidence that the placenta expresses a range of transporters capable of controlling the transplacental disposition of many toxicant agents and thereby playing a crucial role in fetal protection against maternal toxins Specially locating in the maternal-facing apical membrane of the syncytiotrophoblast, together with its vectorial transport capacity, P-gp has the capacity to actively efflux a wide range of toxicants back to the maternal circulation, thus preventing potentially harmful compounds from entering the fetal compartment To date, evidence has suggested that several single nucleotide polymorphisms (SNPs) in ABCB1 gene were associated with altered P-gp expression and transport activity in placenta Informed written consent was obtained from the parents, who were on the behalf of their child as well; about their participation in the study after the nature of this study has been fully explained to them. Informed written consent was also obtained from the mothers when we collected the placenta tissue and umbilical cords. The study was approved by the University Committee on Human Subjects at Sichuan University.An age and gender matched case-control study (1∶1) was conducted from March to December in 2012. 216 children with CHD and 216 children without any congenital malformations or chromosomal abnormalities were enrolled. All children were recruited from the Department of Pediatric Cardiology in West China Second University Hospital, Sichuan University and born in Sichuan province and their birth mothers were local residents. When a new case was confirmed, we downloaded all information on eligible children from the hospital information system, and then the corresponding control was randomly selected from system matching with age and gender. The diagnoses of all cases were confirmed by cardiac catheterization and the diagnoses of all controls were checked by experienced pediatric cardiologists according to medical records. To minimize recall bias of exposure by the mother to the greatest extent, all cases and controls recruited were younger than 3 years old. We excluded those whose birth mother was absent or rejected to be interviewed. Additionally, cases with a family history of CHD and mothers with pre-gestational diabetes were also excluded, as these are strong risk factors for CHD. After the exclusion, 201 pairs were used for analysis. The included CHD phenotypes were perimembranous septal defect , atrial septal defect , pulmonary valve stenosis , Tetralogy of Fallot , pulmonary atresia with ventricular septal defect , anomalous pulmonary venous connection , atrioventricular septal defect , transposition of the great arteries ,and truncus arteriosus (n = 1).itrogen and stored at −80°C until use. Moreover, the maternal and neonatal clinical data were also collected, including maternal age, maternal ethnicity, health conditions, pregnancy complications, any medication consumptions, and neonatal birth outcomes .Thirty-five human placentas and umbilical cords were consecutively obtained from mothers who delivered at the Department of Gynecology and Obstetrics in West China Second University Hospital, Sichuan University by a well-trained nurse. The tissues were immediately frozen in liquid nOnly placentas and umbilical cords from healthy Han Chinese mothers with uncomplicated pregnancy and infants with a gestational duration between 38–41 weeks and a birth weight between 2500–4000 g were included. Those from mothers with chronic diseases, pregnancy complications , and any medication use were excluded.Structured questionnaires with pre-coded and open-ended questions were used for data collection. All questionnaires were pre-tested and revised accordingly. The questionnaires survey was conducted when the children were admitted to the hospital and their birth parents were interviewed face to face by a well trained interviewer, who was blinded to the participant’s group assignment. The questionnaires were double checked to assure its completeness and consistency. All the participants’ birth parents were retrospectively interviewed in a similar manner during the period of the study.The contents of the questionnaire included:Personal characteristics: child’s name, gender, race, birth date, address, parents’ education, and yearly income per person.Conditions of participant’s birth: gestational weeks, birth length, birth weight, birth asphyxia or hypoxia, and family history of CHD or other congenital malformations.Characteristics of parents: age at pregnancy, pre-pregnancy weight and height, occupational exposure history, disease history, and lifestyle .Conditions of pregnancy: reproductive history, artificial fertilization or not, folic acid supplementation, diseases, medication use during pregnancy, and other potential environmental toxicants exposure .In the questionnaire, each mother was asked to report whether she worked in a full-time or part-time job for at least 1 month in duration during the periconceptional period. If she did report working, she was asked a series of questions about each job she held, including the job title, main tasks, and duties. Beginning and ending dates for each job were collected, as well as the work hours per day and work days per week. We assessed occupational exposure to chemicals by applying a job exposure matrix (JEM), with a focus on endocrine-disrupting chemicals Environmental exposure only included living in a newly decorated house for at least 1 month during the periconceptional period. Two studies have confirmed that higher concentrations of phthalates and alkylphenolic compounds were present in newly decorated apartments Given the relatively small number of exposed parents in any particular environmental and occupational category, statistical analysis for toxicant exposure was performed including both environmental and occupational exposure.−1),0.5 ul forward primer ,0.5 ul reverse primer, and 30 ng of genomic DNA in a total volume of 25 ul. PCR conditions were 34 cycles of 30 sec at 95°C, 30 sec at 63°C, and 1 min at 72°C, preceded by an initial denaturation of 5 min at 95°C, and followed by a final extension of 7 min at 72°C. PCR products were purified using a Watson DNA Purification Kit  and were directly sequenced on an ABI 3730  according to the manufacturer’s protocol.DNA was extracted from leucocytes of the whole blood and from the umbilical cord by a commercial SE blood DNA isolation kit  and a commercial tissue DNA isolation kit  respectively. A 110-bp fragment was amplified using 0.25 ul Taq DNA polymerase (Takara), 2.5 ul 10×PCR buffer,1 ul dNTP and subjected to qualitative and quantitative measurements using a spectrophotometer . 1 ug of total extracted RNA was reverse transcribed to cDNA using Prime Script RT reagent kit with gDNA eraser  according to the manufacturer’s instructions.−1), 0.3 ul reverse primer (10 pmol lL–1), 3.4 ul nuclease-free H2O, and 1 ul cDNA in a total volume of 10 ul. PCR conditions were 39 cycles of 30 sec at 95°C, 30 sec at 60°C, and 1 min at 72°C, preceded by an initial denaturation of 3 min at 95°C, and followed by a continuous melt curve from 65–95°C. All the primer sets were tested to ensure the efficiency of amplification over a range of template concentrations. All samples were amplified in triplicates. Expression level of ABCB1 transcript was represented by the mean of triple tests. The relative expressions of ABCB1 mRNA were normalized to expression of GAPDH using 2−△△Ct method.Real-time RT-PCR was performed with cDNA templates and SsoFast EvaGreen Supermixture . Briefly, sequences were amplified using 5 ul reaction mixture, 0.3 ul forward primer (10 pmolLThe primer sequences used were as follows:5′- TCCTGGAGCGGTTCTACGAC-3′ (ABCB1-F),5′-GCTGCAGTCAAACAGGATGG-3′(ABCB1-R),5′-GAAGGTGAAGGTCGGAGTC-3′(GAPDH-F),5′-GAAGATGGTGATGGGATTTC-3′(GAPDH-R).100 mg placenta tissues were homogenized in buffer containing 1 ml RIPA and 10 ul complete protease inhibitor cocktail . The homogenate was then centrifuged at 12,000 g for 5 min at 4°C. Protein concentration was determined using enhanced BCA protein assay kit  according to the manufacturer’s protocol. Total protein (30 ug/lane) was separated on 8% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked in 5% BSA in Phosphate-buffered saline containing 0.1% Tween-20 (PBST). Thereafter, blocked membranes were incubated with overnight at 4°C with monoclonal primary antibody for P-gp  and a-tublin . Following extensive washing with PBST, membranes were incubated with a 1∶10000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibodies . After extensive washing with PBST, protein-antibody complexes were visualized by the enhanced chemiluminescene detection system.2), normal (18.5–23.9 Kg/m2), overweight (24.0–27.9 Kg/m2) and obese (≥28 Kg/m2), in accordance with the criteria specially formulated for Chinese All materials were coded and data was doubly inputted using EPIDATA 3.1 version and all analysis were conducted with SPSS 17.0 version. For data analysis, BMI was calculated as weight divided by height (m) squared and it was divided into 4 categories: underweight  and 95% confidence intervals (CIs) for the associations between maternal toxicants exposure and CHDs. The ORs were adjusted for the following potential confounding factors: maternal age at births, maternal education level, BMI, gravity, history of previous abortions, periconceptional use of folic acid, medication use, settlement, and paternal toxicants exposure.In the genetic study, the chi-square test was used to test for deviation from Hardy–Weinberg equilibrium and to compare allelic and genotypic frequencies between the groups. For SNP alleles, we calculated three categories of p values for the different probable function of the mutant allele as follows: dominant, recessive, and co-dominant. The results showed that maternal exposure to phthalates  and alkylphenolic compounds  was associated with a higher incidence of CHDs in general. In light of these findings, we defined the “toxicant exposure” as maternal periconceptionally exposure to phathalates and/or alkyophenolic compounds in the present study. In the end, logistic regression analysis was performed to assess the joint effects of the ABCB1 gene C3435T polymorphism and “toxicants exposure” on the risk of CHDs. We coded separate categories for the risk of genotype in combination with periconceptional toxicants exposure. ABCB1 TT carriers without periconceptional toxicants exposure were expected to have the lowest risk and therefore considered as the reference category. Whereas the highest risk group consisted of ABCB1 CC/CT carriers with periconceptional toxicants exposure. In addition, the ORs were also adjusted for confounding factors mentioned above. Moreover, the genotype distribution and allele frequencies between cases and controls restricted to “toxicants exposure” were also calculated.For the ABCB1 mRNA expression in the placenta, data was expressed as as mean±SEM. Differences among three genotypes were determined by one-way ANOVA followed by a Student-Newman-Keuls multiple comparison post hoc tests.Power of this study was calculated by Quanto v1.2.4 Sociodemographic and lifestyle characteristics of mothers, fathers, and children are illustrated in The genotype distributions and allele frequencies of C3435T were listed in The results of the interaction analysis of ABCB1 gene C3435T in conjunction with the maternal phthalates and/or alkylphenolic compounds exposure were shown in The genotype distribution of C3435T polymorphism for the 35 umbilical cords were listed as follows: CC: 10(28.6%), CT: 20(57.1%), TT: 5(14.3%). After matched with maternal age for TT group (27–33 years old), 6 placentas in CC genotype group and 7 placentas in CT genotype group were recruited for mRNA and protein analyses. Clinical data of mothers and neonates stratified by C3435T genotype were illustrated in Results from our hospital-based study add to the literature proving that periconceptionally occupational exposure of the mothers to phthalates and alkylphenolic compounds increases the risk of CHDs, especially for septal defects, even after adjustment for confounding factors. Consistent with most of the reported results, the association in our study is weak. However, it provides supportive evidence that children have different susceptibilities to the adverse effects of toxic agents, thereby implying the inheritance of some particular genotypes might modulate the effect.Highly expressed early in fetal life and placenta, ABCB1 appears to be the most predominant transporter in the human placenta 3H]-paclitaxel into vesicles prepared from placental brush border membranes The C3435T SNP does not locate in any coding or regulatory regions of the ABCB1 gene; and consensus on the effect of this SNP on P-gp expression and transport activity in the placenta remains obscure at present. A study conducted by Rahi M and collegues in 2008 revealed that T alleles were associated with a higher placental P-gp protein level compared with the C alleles, but this difference did not have any consequences on P-gp-mediated placental transfer of saquinavir Some limitations must be considered when the results of this study are interpreted. Firstly, occupational exposure assessment in this study was based on a JEM, which was originally developed for jobs in Western Europe A limitation of the JEM is that it can not reflect intensity, or frequency of exposure. We tried to reduce the misclassification by assessing exposure based on both job titles and description of the work tasks. The outcome of the matrix, however, must be interpreted cautiously as exposure probabilities are only a crude measure of exposure.Secondly, our study only included the newly house decoration as an environmental exposure. Other background exposures were not considered, the reasons are as follows: firstly, we admitted that for many chemicals, most people experience a certain level of exposure through diet, environment, or widely used consumer products (such as air pollution), however, the level of exposure to chemicals within occupations scored by the JEM is generally much higher than background exposure through diet and environment, and two studies have confirmed that higher concentration of phthalates and alkylphenolic compounds was present in newly decorated apartments Thirdly, for phthalates and alkylphenolic compounds, it is still unknown whether they are P-gp substrates. Thus the detrimental effect of P-gp might be over- or underestimated. However, accumulating evidence suggests that P-gp is able to transport an extremely wide variety of chemically and structurally compounds. P-gp substrates are usually organic molecules ranging in size from about 200 Da to almost 1900 Da Recall bias is inevitably one of the pitfalls of case-control designs because of the time delay from delivery to interview. However, evidence is emerging that its role is rather small, and almost no differential in case-control studies focusing on congenital malformations The last but not the least, an undoubted limitation of our study was the modest size of our population and the results of the separate CHD phenotypes should be further investigated in much larger data sets.In spite of the limitations, we had tried our best to avoid the bias to a great extent and our results were adjusted by potential confounding factors. Furthermore, the preliminary results of the functional study also sustained the associations found in our study. Thus this significant association could not solely explained by recall bias or confounding by indications. As highlights of the present study, for the first time, our results suggested that maternal periconceptional exposures to phthalates and/or alkylphenolic compounds increase the risk of CHD/septal defects and fetal genotype with respect to ABCB1 gene is important in determining fetal susceptibility to CHDs. In conclusion, it is rather better to avoid exposures to phthalates and/or alkylphenolic compounds during the periconceptional period, especially for the fetal carriers of CC/CT genotypes of the ABCB1 gene.


In the last decade, intramedullary limb lengthening has become a viable alternative to traditional external systems. We retrospectively analyzed the use of an intramedullary motorized nail (Fitbone) in a consecutive series of 32 patients.During the period September 2006 to December 2008, 32 consecutive patients with a median age of 17 (IQR: 15–19) years were treated with a fully implantable, motorized intramedullary lengthening device (Fitbone). The median leg length discrepancy was 35 (IQR: 30–44) mm at the femur (n = 21) and 28 (IQR: 25–30) mm at the tibia (n = 11).Leg lengthening was successful in 30 of 32 cases, with no residual relevant discrepancy (± 5 mm). No intraoperative complications were observed. The consolidation index was significantly different (p = 0.04) between femoral lengthening (mean 35 days/cm) and tibial lengthening (mean 48 days/cm) but did not depend on age older/younger than 16 or previous operations at the affected site. 3 problems, 3 obstacles, and 4 complications  were encountered in 8 patients, 5 of which were implant-associated.This technique even allows correction in patients with multiplanar deformities. Compared to external devices, intramedullary systems provide comfort and reduce complication rates, give improved cosmetic results, and lead to fast rehabilitation since percutaneous, transmuscular fixation is prevented. This results in reasonable overall treatment costs despite the relatively high costs of implants. Operative leg length equalization includes techniques such as contralateral epiphysiodesis during growth, or shortening and lengthening procedures. In the last decade, lower complication rates and greater patient comfort  have madHere we present the indications and limitations, and the results and complications of the Fitbone nail. We identified possible factors that may influence the clinical outcome. In addition, we did a comparative cost analysis of different treatment methods.During the period September 2006 to December 2008, 32 consecutive patients with a median age of 16.9 (IQR: 15.3–19.4) years were treated with a fully implantable, motorized intramedullary lengthening device (the Fitbone Sliding Active Actuator (SAA) nail and the Telescope Active Actuator (TAA) nail; WITTENSTEIN Intens GmbH, Igersheim, Germany) . The medThe underlying pathologies and the number of operations at the affected site are listed in The indication for surgery was based on a minimum LLD of more than 2 cm, clinical/functional symptoms, and ability to independently handle the lengthening device. Lengthening for cosmetic reasons only was not accepted.The SAA, which is restricted to femoral use, is mostly implanted through an antegrade approach. As the SAA features a slotted sliding hole in its middle part, this device allows lengthening as well as bone transport. TAA nails are available for both the femur and the tibia. They are implanted through a retrograde approach at the femur and an antegrade approach at the tibia. The femoral TAA is a straight nail; the tibial one is available as either a straight nail or one with a proximal Herzog angulation. In contrast to the SAA, lengthening with the TAA is accomplished by a telescoping mechanism. Various sizes, different designs, and even custom-made implants of both nail types are available.Preoperatively, standardized anteroposterior and lateral long standing radiographs of both legs were taken and a preoperative planning was performed, based on the protocol for retrograde corrective planning described by Percutaneous osteotomies were performed using a 4.0-mm drill and a chisel for completion, which causes minimal thermal damage to the bone and preserves the periosteal sleeve, for optimal callus formation.Every patient received detailed oral and written instructions on the use of the device as well as an individual schedule with timing, rate, and total amount of lengthening. We aimed at starting distraction 5 days postoperatively at a distraction rate of 1.3 mm/day in femurs and starting 7 days postoperatively at 1.0 mm/day in tibias.All patients were counseled regularly regarding the device and were followed radiographically on a biweekly basis during and at 4-week intervals after distraction until full consolidation. The range of motion of hip, knee, and ankle joints was recorded in addition to pain , wound status, and complications. All patients underwent an intensive rehabilitation program under the guidance of physiotherapists not any less frequently than twice a week, at least until consolidation.Consolidation was defined as corticalization of at least 3 sides of the callus on the biplane radiographs, with subsequent transition to full weight bearing. Insufficient callus formation was defined as a consolidation period more than 3 times longer than the distraction period (femur) or more than 4 times longer (tibia).Every single lengthening period  and the appropriate indices  were calLeg lengthening was considered successful in cases with no residual relevant discrepancy of ± 5 mm. For deformity correction in the frontal plane, the mechanical axis deviation (MAD) was evaluated preoperatively and intraoperatively with respect to the contralateral side and/or normal physiological conditions . IntraopComplications were differentiated as problems (grade 1), obstacles (grade 2), and minor or major complications (grade 3) .The calculation of total costs for a Fitbone treatment was based on the current local reimbursement rates for a public patient, 1 segment affected, implantation and explantation, hospital stay of 3 weeks in total (implantation and explantation), and physiotherapy twice a week for 1 year. Likewise, overall costs for leg length equalization by means of orthotic devices were calculated by a local orthopedic technician assuming a non-private patient with a leg length discrepancy of more than 2 cm requiring shoe lifts and necessary additional insoles twice a year for 60 years. In addition, implant costs for the Taylor Spatial Frame were taken from the manufacturer's price list.The median follow-up time was 16 months . By the end of the observation period, all but 2 nails had been explanted after a median of 14 months .Data are given as means, medians, and interquartile range (IQR). Comparisons between patient subgroups were assessed using Fisher's exact test for binary endpoints and by the Mann-Whitney U-test for continuous endpoints. A p-value of ≤ 0.05 was taken to indicate statistical significance. Statistical analysis was performed using SPSS software version 17.0.Leg length equalization was successful in 30 of 32 cases (± 5mm). 2 showed a remaining LLD of 7 mm and 10 mm, which was clinically irrelevant. At the femur, the target length was achieved with a standard deviation of ± 6.1%. Regarding the tibia, the equalization was accomplished with a standard deviation of ± 7.3%.Distraction was started at an average of 6 (3–12) days postoperatively with a median distraction index of 1.0 (IQR: 0.9–1.2) mm/day and a median distraction period of 30 (IQR: 28–42) days. The median hospital stay was 12 (IQR: 10–14) days.There was no statistically significant difference in the maturation index in terms of the influencing variables examined.The median maturation period was 75 (IQR: 60–106) days for femurs (n = 21) and 96 days (IQR: 66–112) for tibias (n = 11). The median maturation index was 22 days/cm (IQR: 16–30) for femurs and 36 (IQR: 22–40) days/cm for tibias.The median consolidation index was 35 (IQR: 27–44) days/cm for the femur and 48 (IQR: 34–63) days/cm for the tibia. A statistically significant difference between femurs and tibias was seen, but not regarding other influencing variables.The median preoperative mechanical axis deviation (MAD) in patients with a varus axis (n = 8) was 13 (6–50) mm medially to the center of the knee joint, whereas the median postoperative MAD was 4 mm medially . In patients with a valgus axis (n = 9), the median preoperative MAD was 13 (5–54) mm laterally as compared to a postoperative median of 0 mm .In patients with additional rotatory and/or sagittal corrective osteotomies (n = 5), a bilaterally equal range of motion of the hip and knee joint was seen immediately postoperatively .The functional outcome was excellent in 26 patients, good in 5, and fair in 1. All but one had regained or even improved upon their preoperative range of motion of the hip, knee, and ankle joint (n = 8) .No intraoperative complications and no postoperative bone or soft tissue infections occurred. We observed 3 problems, 3 obstacles, and 4 complications  in 8 patients . InsuffiIn 1 case with a history of lengthening by external fixation (patient 12), a progressive knee extension-contracture required manual mobilization under anesthesia 5 months after the end of distraction.One motor stopped because of overstraining of the mechanical implant by full weight bearing early on the patient's own authority, during the initial distraction period and after discharge from the hospital. It had to be exchanged, but this did not affect the final result. In 3 cases we observed running back of the telescopic part of the nail during the initial consolidation phase, which was twice associated with loosening of locking bolts. Another bolt loosened without loss of length. The persisting equinus of the foot in 1 patient (patient 24) was successfully corrected by a combined calcaneus and talus osteotomy using the Taylor Spatial frame.Total costs of approximately 100,000 euros were calculated to equalize a leg length discrepancy of more than 2 cm by means of orthotic devices. For a Fitbone treatment, total costs of approximately 38,000 euros were calculated (see Patients and methods). Pure implant costs amount to approximately 11,000 euros for the Fitbone nail, and to 7,500 euros for a standard Taylor Spatial Frame.The use of mechanically driven intramedullary nails for limb lengthening and deformity correction has been reported repeatedly . The ISKSince most peer-reviewed literature on intramedullary lengthening involves a small number of patients , we consUnlike the application of external fixators, a solid intramedullary device requires defined anatomical preconditions that have to be adhered to, both during the preoperative planning and intraoperatively. However, for the accomplishment of corrections in 3 dimensions, reaming of the intramedullary cavity with straight and rigid reamers (not flexible ones) is as a prerequisite. Only the use of rigid reamers, which do not follow the line of minor resistance, allows to carry out the correlations anticipated during preoperative planning. In this context, adequate medullar dimensions respecting a minimal nail diameter of 11 mm at its shaft are one of the most important limitations of the Fitbone TAA. Further restrictions derive from its short overall length and a mandatory osteotomy level 7–11 cm from the joint line to ensure stable interlocking conditions. Marked angular deformities and a center of rotation and angulation far from the planned osteotomy are additional geometric obstacles, and may make segmental translation unavoidable . Axis alApplication of solid lengthening nails in children is limited by open growth plates. However, data on children treated for tumors and also unpublished observations of experienced surgeons (regarding limb lengthening) suggest that the growth potential may remain unaffected when smooth polished implants are inserted through the central portion of a physis .In keeping with the results of Without transfixation of skin and muscles, lengthening nails facilitate full joint motion in the first few days after surgery, whereas external fixators decrease the range of motion even before the lengthening process has started . Thus, rThe complication rates of external fixators range from 24% to 117%  and fromA complication rate of 12.5% in our series (4 of 32 patients) is in keeping with the findings of Technical complications or implant breakage rarely occur with Fitbone. In the series of 150 patients, The overall cost of lengthening with Fitbone was less than for conservative treatment, assuming the conditions mentioned earlier. Compared to external fixators such as the Taylor Spatial Frame, the Fitbone nail is somewhat more expensive. However, the much higher complication rate of external fixation—probably associated with additional hospital stays, medication , and physiotherapy sessions—warrant differentiation between pure implant costs and costs of treatment, in addition to “soft” factors such as cosmetics, patient satisfaction, and quality of life during and after the lengthening procedure .Apart from the explicit desire of the majority of our patients to have the implants removed after the lengthening procedure, we consider the removal mandatory in order to avoid difficulties in the future, such as removal of an ingrown nail or interference with arthroplastic devices later on. Furthermore, the analysis of each explanted device by the manufacturer contributes to the quality of management and promotes development and perfection of the nail.Our study was limited by the lack of a matched control group, treated with different lengthening devices. Furthermore, long-term results on lengthening with Fitbone nails are still not available.In summary, the outcome achieved in our patients is a respectable result, and there was a low complication rate. There was a significant difference in the consolidation index in the treatment of femurs and tibias. The high implant costs are compensated for by competitive treatment costs. Intramedullary leg lengthening is, however, a demanding procedure.


Senna italica, a member of the Fabaceae family , is widely used in South African traditional medicine to treat a number of disease conditions. Aqueous extracts of the plant are mainly used to treat sexually transmitted infections and intestinal complications. The roots of S. italica were ground to a fine powder and sequentially extracted with n-hexane, dichloromethane, acetone, and methanol using serial exhaustive extraction (SEE) method. Thin layer chromatography was used to analyse the phytochemical composition of the extracts and DPPH radical scavenging method to detect the presence of antioxidant compounds. The bioassay guided fractionation of the acetone fraction afforded an antioxidant compound with free radical scavenging activity. The isolated compound was subsequently identified as 3,4′,5-trihydroxystilbene (resveratrol). This study represents the first report of the stilbene resveratrol in S. italica. Dioscorea species and used to synthesise contraceptive agents [Catharanthus roseus as well as laxative agents from Cassia species [Plants have formed the basis of sophisticated traditional medicinal systems that have been in existence for thousands of years, and they continue to provide humanity with new remedies . The impe agents . Other e species . in vitro and in vivo test systems [The current advancement in science has made it possible for the isolation of compounds of medical importance from plants that are used in traditional medicinal practices. Different parts of plants such as leaves, roots, stems barks and rhizomes are often extracted using different solvents. In most cases, extracts have been shown to be biologically active both in the  systems . Some pl systems . HoweverSenna italica, a member of the Fabaceae family , is known for its therapeutic properties in African folk medicine. Some Senna species from Venda in South Africa are used to treat sexually transmitted infections (STIs) while others are considered to possess significant antibacterial activities [S. italica is indicated for the treatment of STIs [Senna revealed the presence of alkaloids, quinines, and anthraquinones. These types of compounds have been isolated from heartwood, seeds, root bark, roots, and leaves of the genus Senna [S. italica [S. italica on selected bacterial strains, total phenolic content as well as its effect on cancerous Jurkat T cell [S. italica was further investigated with the aim of identifying the compound(s) that exerted the observed biological activities. Antioxidant compounds are important in the prevention or treatment of various human diseases. Undeniably, reports abound that link free radical scavenging potential and other biological benefits of antioxidant compounds [Senna italica through bioassay-guided fractionation.tivities . In the  of STIs . A literus Senna . Other s italica . We havet T cell . Based oompounds . We hereSenna italica were collected from Bolahlakgomo village . A voucher specimen (UNIN11129) has been deposited in the Larry Leach herbarium of the University of Limpopo. The roots were dried at room temperature and subsequently milled to a fine powder using a grinder  Ltd., RSA). The milled material (700 g) was serial exhaustively extracted using n-hexane, dichloromethane (DCM), acetone and methanol (MeOH) as extractants. The extraction process was repeated three times with 2 litres of each solvent. The extracts were filtered through a Whatman no. 3 filter paper and the filtrates concentrated using a rotary evaporator . The concentrated extracts were then transferred into preweighed beakers, dried under a stream of cold air, and weighed to determine the resultant mass. The roots of The presence of antioxidant constituents in the various extracts was determined using the method of . Thin la3 : MeOH of increasing gradient polarity, starting with 100% CHCl3 to 100% MeOH. A 460 mg sample of acetone extract was fractionated  using column chromatography packed with silica gel 60 (63–200 µm). Following elution, the antioxidant constituents of the resultant fractions were qualitatively evaluated using the TLC-DPPH method described above. The fraction that eluted with 9 : 1 (CHCl3 : MeOH (9 : 1)) was found to contain the major compound with antioxidant activity. Hence, the fraction was chosen for further purification of the target compound.The acetone extract had constituents with antioxidant activity and was thus chosen for further fractionation with CHCltionated  into sev3 : EtAOC (1 : 1). The 9 : 1 (125 mg) was subjected to a silica gel 60 column chromatography using CHCl3 : MeOH (4 : 1) as the eluent. Fractions 13–18 were subjected to chromatography using CHCl3 : MeOH (9 : 1) as eluent. After recrystallisation, a 13 mg of the pure compound was obtained. The compound was dissolved either in methanol, chloroform, or dimethyl sulfoxide depending on its solubility for analysis. The structure of the isolated compound was elucidated from the data obtained from 1H- and 13C-NMR spectra.Several mobile phases were tested and fraction F9 : 1 was better resolved with CHCln-Hexane, for instance, extracts waxes, fats, and fixed oils while acetone extracts alkaloids, aglycones, and glycosides. On the other hand, methanol extracts sugars, amino acids, and glycosides while DCM will commonly extract alkaloids, aglycones, and volatile oils. For these reasons, plant material was extracted using solvent of varying polarity, namely, hexane, DCM, acetone, and methanol. An increase in the extracted material was observed as the polarity of the extracting solvent increased  followed by fraction 9 : 1 (125 mg), with fraction 0 : 1 giving the lowest yield (14 mg) as shown  gave a better separation of the active compound. The 9 : 1 fraction of the acetone extract was then subsequently eluted with CHCl3 : MeOH  through a silica gel column. Tubes containing similar compounds noted on TLC fingerprint were pooled together and reeluted with CHCl3 : MeOH  through a silica gel column. The reelution of the fraction obtained from the pooled tubes gave yield to a pure compound with a positive free radical scavenging activity evident by a yellow spot against a purple background on TLC following spraying with DPPH  and 6.86  and δ 6.55  and 6.16  showed the typical resveratrol skeleton [δ 6.89, 1H, d) and H-8  implied that the configuration of compound 1 was in a trans form configuration. In the 13C-NMR spectrum, three oxygenated aromatic carbons (δ 159.6 (×2) and 158.2) and eleven sp2 carbons (102.7~140.9) were shown. From these data and those presented in trans-resveratrol [Compound 1 was obtained as a white powder that was positive to FeClskeleton . The couveratrol , 13.S. italica. Plant parts such as roots, leaves, stems, and rhizomes possess a myriad of chemical constituents that are biologically active against various disease conditions . In thisS. italica. The phytochemical constituents of the extracts were best separated in the intermediate/polar mobile phase, ethylacetate/methanol/water (EMW: 40 : 5.4 : 5), which separates polar and neutral compounds. Acetone extracts showed the presence of antioxidant compounds which were better separated in the intermediate mobile phase (CEF). The free radical scavenging activity demonstrates the hydrogen donating ability on reaction to the stable free radical which results in the decolouration of the DPPH free radical from purple to yellow [Methanol extract had the highest yield. The high extract yield obtained with methanol extract could be related to the ability of the solvent to extract compounds of varying polarity . All solo yellow .Senna revealed the presence of alkaloids, quinines, and anthraquinones that are mainly polar-based compounds [cis and trans isomeric forms. Although the nutritional value and biological activity of resveratrol have been well documented [trans resveratrol from the roots of Senna italica belonging to the family Fabaceae. The biological effects of resveratrol together with its well-known antioxidant properties are beneficial for the prevention of several diseases. Findings in our previous study [S. italica with antioxidant, antibacterial, and antiproliferative activities while other studies have also confirmed the antiproliferative effect of resveratrol [A literature survey on the chemical constituents of the genus ompounds . Resveracumented , its olicumented , 20. Thius study  showed tveratrol . The preveratrol  and may trans-conformation (4′-hydroxystyryl moiety), is a significant prerequisite in its ability to inhibit cell proliferation [S. italica and the observed antioxidant effect could possibly give more credence to its traditional use in treating various ailments in the Limpopo Province of South Africa [Structural activity relationships of the antioxidant potential of resveratrol from other studies suggest that the hydroxyl group in 4′ position is not the sole determinant for antioxidant activity. It has also been suggested that the presence of 4′-OH together with its stereoisomer in the feration . The preh Africa , 6. Furt


NPPA) by RNA sequencing in the same tissue.The atrium is the major site of ANP synthesis, which has been said to increase in heart failure as a result of increased production in the left ventricular (LV) myocardium. This is a key issue related to its diagnostic and prognostic capabilities. We aimed to evaluate protein levels of proANP and ANP and the enzymes that cleave the natriuretic peptides, corin and furin, in the LV tissue of heart transplant patients with dilated (DCM) and ischemic (ICM) cardiomyopathy compared with control donors (CNT). We also evaluate mRNA levels of ANP gene . Corin (but not furin) was elevated in the ICM group compared to CNT , and its level was inversely related with LV ejection fraction (LVEF) .Seventy-three human LV tissue samples from ICM (n = 30) and DCM (n = 33) patients and CNT (n = 10) were analyzed by Western blot and RNA sequencing. Comparing protein levels according to etiology, neither DCM nor ICM showed levels of proANP or ANP different from those of CNT. However, NPPA but not of proANP or ANP proteins in LV tissue, which may be due to posttranscripcional regulation of NPPA or different pathways for ANP secretion between the atrium and ventricle. Moreover, there are differences between DCM and ICM in corin levels, indicating that a different molecular mechanism may exist that converge in this syndrome. Further, LV concentration of corin is inversely related to LVEF in ICM.Patients present with elevated levels of  Heart failure (HF) is caused by any condition that reduces the efficiency of the myocardium through damage or overloading. Natriuretic peptides (NP) are a family of peptides that cause effects such as diuresis, natriuresis, vasodilation, and inhibition of aldosterone synthesis and renin secretion; playing an important role in regulating blood pressure and blood volume ANP and BNP are produced as a prohormones, proANP and proBNP, which are cleaved by 2 endoproteases (corin and furin) to form active ANP and BNP and inactive N-terminal molecules NPPA) by RNA sequencing (RNA-seq) in the LV tissue of HF patients undergoing heart transplantation compared with healthy controls. Furthermore, we measured protein levels of ANP and proANP, and its related enzymes corin and furin, in the same LV tissue.ANP is synthesized and secreted in the atria under normal conditions and by the ventricular myocardium during fetal development, hypertrophy, or HF All patients gave their written informed consent to participate in the study. The project was approved by the Biomedical Investigation Ethics Committee of “La Fe” University Hospital of Valencia, Spain. The investigation was conducted in accordance with the guidelines of the Declaration of Helsinki LV tissue samples were obtained from 73 subjects: 63 patients with HF (33 with non-ischemic DCM and 30 with ICM) undergoing cardiac transplantation and 10 control (CNT) samples from non-diseased donor hearts The clinical characteristics of patients are shown in LV samples were taken from a portion proximal to the apex of the left ventricle. The DCM, ICM and CNT samples were stored in 0.9% NaCl at 4°C for a mean time of 4.4±3 h after the loss of coronary circulation.Twenty-five milligrams of frozen left ventricle were homogenized in a total protein extraction buffer  with protease inhibitors (25 µg/mL aprotinin and 10 µg/mL leupeptin) in a FastPrep-24 homogenizer  with specifically designed Lysing Matrix D tubes. The homogenates were centrifuged, and the supernatant was aliquoted. The protein content of the aliquot was determined by Peterson's Modification Forty-five micrograms of each homogenate were separated by Bis-Tris Midi gel electrophoresis with 4–12% polyacrylamide in separate gels for proANP, ANP, corin, and 10% for furin. For the Western blot, the proteins were transferred from the gel to a PVDF membrane, using an iBlot Dry Blotting System . The membrane was blocked for the entire night at 4°C with 1% BSA in Tris-buffer solution containing 0.05% Tween 20. After blocking, membranes were incubated for 2 h with the primary antibody in the same buffer. The primary detection antibodies used were: a mouse monoclonal anti-human IgG against proANP and ANP  , and a mouse monoclonal anti-human IgG against corin  and a rabbit polyclonal anti-human IgG against furin  . A mouse monoclonal anti-human IgG against GAPDH antibody  was used as the loading control for each of the blots.The bands were visualized using an acid phosphatase-conjugated secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate  substrate system. Finally, bands were digitalized using an image analyzer  and quantified by the Gel Capture (v. 4.30) and the GelQuant Pro (v. 12.2) programs.Heart samples were homogenized with TRIzol agent in a TissueLysser LT . All RNA extractions were performed using a PureLink Kit according to the manufacturer's instructions . RNA was quantified using a NanoDrop1000 spectrophotometer  and the purity and integrity of RNA samples was measured using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip kit . All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers ≥9.PolyA-RNA was isolated form 25 micrograms of total RNA using the MicroPoly(A) Purist kit . Total PolyA-RNA samples were used to generate whole transcriptome libraries for sequencing on the SOLiD 5500XL platform, following the manufacturer's recommendation . No RNA-spike in controls was used. Amplified cDNA quality was analyzed by the Bioanalyzer 2100 DNA 1000 kit  and quantified using the Qubit 2.0 Fluorometer . The whole transcriptome libraries were used for making SOLiD templated beads following the SOLiD Templated Bead Preparation guide. Bead quality was estimated based on WFA  parameters. The samples were sequenced using the 50625 paired-end protocol, generating 75 nt+35 nt (Paired-End)+5 nt (Barcode) sequences. Quality data were measured using software SETS parameters .http://www.lifetechnologies.com/), version 1.3. It was using the standard Bioscope parameters of version 1.3, in paired ends and whole transcriptome analysis. For both reads, forwards and revers, the seed was the first 25 nucleotides with a maximum of 2 mismatches allows, The initial whole transcriptome paired-end reads obtained from sequencing were mapped against the latest version of the human genome (version GRchr37/hg19) using the Life Technologies mapping algorithm (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55296).Subsequently, isoforms and gene prediction were estimated using the cufflinks method Data are presented as mean value ± standard deviation for continuous variables and as percentage for discrete variables. The Kolmogorov-Smirnov test was used to analyze the distribution of the variables. Comparisons of clinical characteristics were achieved using Student's t-test for continuous variables and Fisher's exact test for discrete variables. Comparisons of tissue levels of proteins by Western blot were performed using Student's t-test, and Pearson's correlation coefficients were calculated to determine the relationships among levels of proteins and clinical characteristics. Significance was assumed as p<0.05. All statistical analyses were performed using SPSS software v. 20 for Windows .We analyzed 73 human hearts; heart tissue was explanted from 63 patients undergoing cardiac transplantation after being diagnosed with non-ischemic DCM (n = 33) or ICM (n = 30) cardiomyopathy, and CNT samples were obtained from 10 non-diseased donor hearts.Most of the patients were men (87%) and their mean age was 51±11 years. The patients had an NYHA functional classification of III–IV and had previously been diagnosed with significant comorbidities, including hypertension (34%), hypercholesterolemia (13%), and diabetes mellitus (32%). The CNT group mainly comprised men (73%) and had a mean age of 49±19 years. To analyze the pathophysiology of NP and enzymes in cardiac tissue, we used Western blot techniques to determine the protein levels of the immunoreactive forms of the NP  and the endoproteases (corin and furin) in dilated and ischemic human hearts. When we compared NP levels within the DCM group, we observed that tissue proANP levels were higher than ANP levels . In contrast, within the ICM group, tissue proANP levels were lower than ANP levels  .vs. 100±15, p = 0.728; 89±25 vs. 100±17, p = 0.160, respectively). Similarly, there were no significant differences in corin and furin between the HF patients and the CNT group: 104±30 vs. 100±7, p = 0.334 for corin; and 101±46 vs. 100±33, p = 0.971 for furin.When we compared the protein levels in HF patients and CNT, proANP and ANP did not show any significant differences . The same applied to furin levels  for DCM and ICM, respectively, compared to CNT; however, we noted that there was an increase in furin levels in ICM respect to the DCM group  , but not in DCM hearts  compared to CNT; besides the ICM group also showed higher levels compared to DCM group   . Corin s p<0.05) .NPPA was increased in both the DCM  and ICM  groups, compared to the CNT group  and 13 fold in ICM (p<0.0001).The mRNA differences between patients and CNT were determined by RNA-seq. NT group . In bothWe further determined whether there was any relationship among the levels of each of the proteins and between mRNA and protein levels. We found that corin and proANP were related in both etiologies, DCM  and ICM  . We alsoFinally, we determined whether there was any relationship in protein and mRNA levels with changes in LV function. For 27 out of 30 samples the LV function parameters were completely available in ICM. We found a significant inverse correlation between corin levels and LV ejection fraction (EF) in ICM  .NPPA levels by RNA-seq in the LV tissue of HF patients undergoing heart transplantation, compared with healthy controls. In addition, we measured protein levels of ANP and proANP, and its related enzymes corin and furin, in the same LV tissue.There are few studies of ANP in LV tissue; most of them explored the changes at the mRNA level This study offers several insights into the tissue expression of ANP forms in human hearts. We determined the proANP and ANP expression by Western blot techniques and we were able to find both molecular forms in the LV tissue of all groups. Therefore, we concluded that ANP forms are present in adult LV tissue, although the highest levels are found in fetal stages and decline during the progress of gestation in developing hearts NPPA, acting as a negative regulator of the ANP production The literature includes reports of increases in proANP mRNA in the left ventricle, something we can also observe with RNA-seq experiments. This technique provides a far more precise measurement of levels of transcripts and their isoforms than microarray technology Previous studies have established a correlation between the forms of plasma NP LV function parameters are closely related to the ventricular remodeling that occurs in HF progression. A long-term remodeling process becomes detrimental leading to a progressive cardiac decompensation From the clinical point of view, our findings allow us to better understand the pathophysiology of the ANP. Furthermore, it appears discarded that part of the higher ANP concentration in plasma in these patients could be due to an increased production of ANP in its left ventricular fibers, at least in the apical region.A limitation of this study is the inherent variability of the samples, given their origin from human hearts and the medical treatments that the patients were undergoing. Furthermore, we chose DCM patients who did not report any family history of the disease. Our study is confined to the LV apex and therefore, we cannot exclude an increased production of ANP in other regions of the left ventricle. However, we want to emphasize the importance of having carried out this study in a significant number of samples from explanted human hearts from DCM and ICM patients undergoing cardiac transplantation. This has allowed us to extract the region of tissue that we wanted to analyze, which could not have been possible if the study had been made using biopsied tissues. However, we do not have atrial tissue of these patients in our collection of samples.NPPA mRNA but not of proANP or ANP protein levels in human left ventricular apical tissue, which may be due to posttranscripcional regulation of NPPA or different pathways for ANP secretion between the atrium and ventricle. Moreover, we observed differences between the two etiologies in the tissue levels of corin, indicating that different molecular mechanism may exist that converge in this syndrome. Further, left ventricular levels of corin are inversely related to the ventricular ejection fraction of patients with ischemic cardiomyopathy.In summary, this study shows that patients with dilated and ischemic cardiomyopathy present elevated expression of Figure S1Correlation between pro-atrial natriuretic peptide and corin. The scatter plots showed that pro-atrial natriuretic peptide (proANP) was correlated with corin in dilated cardiomyopathy (A), and in ischemic cardiomyopathy (B). Values were normalized to GAPDH and finally to the CNT group.(TIF)Click here for additional data file.Figure S2Correlation of endoproteases. The scatter plots showed the correlations of furin and corin in dilated cardiomyopathy (A) and ischemic cardiomyopathy (B). Values were normalized to GAPDH and finally to the CNT group.(TIF)Click here for additional data file.Figure S3Correlation between atrial natriuretic peptide and corin in ischemic cardiomyopathy. Values were normalized to GAPDH and finally to the CNT group.(TIF)Click here for additional data file.Figure S4Correlation between corin and ejection fraction. Values were normalized to GAPDH and finally to the CNT group.(TIF)Click here for additional data file.Table S1Main statistical parameters of mapping step of analysis.(XLSX)Click here for additional data file.Table S2Percentage splicing reads of NPPA gene.(XLSX)Click here for additional data file.


Lygus hesperus Knight (Hemiptera: Miridae). This diet has been used for more than a decade. However, because it contains cooked chicken egg, and thus requires laborious preparation . This was significantly greater than diets D1, containing autoclaved dry chicken egg yolk based component (9027 ± 811 SE), D2, containing autoclaved chicken egg white based component (8311 ± 628 SE), and NI, which contained autoclaved chicken egg yolk + cooked egg diet (7890 ± 761 SE). Significant differences were observed in the weights of all developmental stages except for eggs and first instar nymphs. Higher rates of fertility, hatchability, and low mortality in nymphs during the first instar were also obtained in the modified D3 diet. The results clearly indicated that the D3 diet provided an opportunity to significantly reduce rearing cost by avoiding time-consuming issues with preparation of a cooked egg diet. This should result in an increase in production capacity and a reduction in production costs.The NI artificial diet is the only known successful diet for mass rearing the western tarnished plant bug, paration , this di Lygus hesperus Knight (Hemiptera: Miridae), is a destructive pest that attacks many important crops including alfalfa (Medicago sativa), beans (Phaseolus vulgaris), strawberries (Fragaria virginiana), peaches (Prunus persica), cotton (Gossypium hirsutum), and various other seed crops. Its impact is amplified by a remarkable ability to become resistant to pesticides  developed by Patana , with 15 × 25 cm openings cut into the bases and covered with bleached muslin fabric (www.theonlinefabricstore.com). The top was also cut and the 20 × 30 cm opening was replaced with bleached muslin fabric for smaller nymphs, and changed with 1.0 mm mesh fiberglass screen for larger nymphs and adults. The insects were reared in an environmental room with a photoperiod of 16:8 L:D, temperature of 27° C (± 1.5° C), and 55% relative humidity, ± 10%  diet preparation.The NI diet consisted of three groups , while the diet modifications  consisted of two groups  . The NI Effect of diet modification on the reproduction rates. To compare the mass fitness parameters for each diet, nymphs in a rearing cage were fed one of the treatment diets  while nymphs in the control were fed the standard NI diet. Approximately 3000 first-instar L. hesperus were placed in each cage in each of the three replications of the four treatments. The first day of oviposition by an emerged adult occurred after approximately 18 days of rearing. One diet parafilm pack per cage was used for L. hesperus feeding and one gel parafilm pack per cage was used for oviposition  was used to count and calculate egg production.position . Both dis coffea . Eggs weL. hesperus, three replicate cages of the D1, D2, D3, and NI diet treatments were set up with F1 progeny (first instar) using the same procedure described above. As the F1 progeny developed, 30 individuals per stage  were randomly taken from each replication of each treatment and individually weighed. A Santorium ISO 9001 microbalance (www.sartorius.com) was used to determined weight in each stage.To calculate the effect of the modified diets on the mean weight of various stages of p < 0.05). Regression analysis was used to determine correlations between diet modifications and egg production rate per day.Parametric statistics were used to compare means of egg production and weight of first and fifth instar, along with female and male adults. Nonparametric statistics for rates of fertility, hatchability, first instar mortality, and daily egg production per female were performed using SAS system software . Data foL. hesperus parents were reared on D1, the dry-yolk chicken egg-based diet. Mortality of nymphs fed the D1 diet increased every day as compared to mortality to the NI, D2, and D3 diets, in which mortality was consistently lower . Similar results were also obtained with two other important fitness measures; first instar mortality/day/gel pack was significantly higher  using the D1 diet as compared to the others three diets  using the D3 diet compared with NI diet, but not with the D1 and D2 diets.Mortality in the first instar (at the time of hatching) was much higher when ly lower . Howeverly lower . In geneee diets , and totL. hesperus are provided in F = 19.21; df = 3, 2; p = < 0.01), female , and male  stages, though no differences were found for eggs or first instar stages. Upon reaching the fifth instar L. hesperus females and males were always significantly larger in weight for individuals reared on the D3 diet. Daily egg production and greater live weight for female, male, and fifth instar stages obtained with the D3 diet resulted in greater biomass accumulation.Maximum weights for eggs, first and fifth instars, and female and male adults of L. hesperus. However, the D3 modified treatment was superior in biomass accumulation and fecundity to the regular NI diet (control). Egg production with the D3 diet (eggs/gel pack/day) was similar to that found by Cohen , who obtained higher live weight in females and males of L. lineolaris fed with the D3 diet. Chang (2009) reported greater pupal weight for the oriental fruit fly, Bactrocera dorsalis (Coquillet) fed with torula yeast after comparison with insects fed with Brewer's yeast and Korea yeast.It is important to note that the live weight was greater for fifth instar, along with female and male adults of  Streett  stated tL. hesperus and L. lineolaris. It is not an indispensable ingredient for completion of the life cycle of L. hesperus, as we demonstrated in our study with the dry yolk-based D1 diet. The use of egg yolk autoclaved with plant material (component B in D3) seemed to be essential to the nutritional and phagostimulatory quality of the complete diet as mentioned by Cohen , propionic acid , and sorbic acid  did not significantly affect biological fitness as compared with the control at any of the three concentrations. However, the highest total biomass, measured as dry weight in mg, was obtained when insects were reared with the NI diet prepared with 2000 and 3000 ppm of benzoic acid. Based on these studies, we replaced the formaldehyde (1 ml), propionic acid (1 ml), and acetic acid (2 ml) in the three modified NI diets with benzoic acid (2 g). Bacteria contamination in Lygus diet is uncommon; the 25 mg of streptomicycin added to D1, D2, and D3 diets was used as a preventive compound.Mold control in insect diets and colonies is one of the greatest challenges of insect rearing . The tem control . Antimic control  demonstrnd Cohen  evaluateL. hesperus. This species can be produced with higher biological fitness values than those reported for the existing standard NI diet.We modified the regular NI diet in order to obtain an inexpensive diet that required little labor to produce. This was achieved by cutting the cooking part (referred to as NI cooked egg diet component A), reducing antibiotics, avoiding the addition of formaldehyde, and by changing other ingredients such as preservatives and minerals. These modifications made the NI artificial diet less expensive (20% reduction) , less la