Safety data regarding exposure to artemisinin-based combination therapy in pregnancy are limited. This prospective cohort study conducted in Zambia evaluated the safety of artemether-lumefantrine (AL) in pregnant women with malaria.Pregnant women attending antenatal clinics were assigned to groups based on the drug used to treat their most recent malaria episode . Safety was assessed using standard and pregnancy-specific parameters. Post-delivery follow-up was six weeks for mothers and 12 months for live births. Primary outcome was perinatal mortality .Data from 1,001 pregnant women  and 933 newborns  showed: perinatal mortality , comprised of early neonatal mortality (each group 2.3%), stillbirths ; preterm deliveries ; and gestational age-adjusted low birth weight . Infant birth defect incidence was 1.8% AL and 1.6% SP, excluding umbilical hernia. Abortions prior to antenatal care could not be determined: abortion occurred in 4.5% of women treated with AL during their first trimester; none were reported in the 133 women exposed to SP and/or quinine during their first trimester. Overall development  was similar in both groups.These data suggest that exposure to AL in pregnancy, including first trimester, is not associated with particular safety risks in terms of perinatal mortality, malformations, or developmental impairment. However, more data are required on AL use during the first trimester. Plasmodium falciparum resistance  from the AL plus 6 from the SP group) received AL treatment during the first trimester, primarily for treatment of the index episode Table .A total of 159 foetuses were exposed to AL during the first trimester, either AL only (n = 150) or AL plus SP (n = 9) Table . Six aboPerinatal mortality was very similar whether first trimester exposure was to AL alone, or to SP and/or quinine, or if no anti-malarial therapy had been received Table . Among wBirth weight  showed no notable differences between the groups.The incidence of malformations in babies born to mothers who received AL only in the first trimester was 6.9% (9/130); eight of these nine cases were umbilical hernias, and the remaining baby was reported as having small genital labia, lanugo, and a small nose  of infants from the AL group achieved a score of "good" or "excellent" at 14 weeks, and 100% (78/78) achieved the same scores at 12 months.A summary of common AEs is presented in Table There were six maternal deaths during the study; one death (0.2%) in the AL group (anaemia secondary to spontaneous abortion), and five deaths (1.0%) in the SP group . Mean haemoglobin levels 4 weeks after enrolment and at delivery were 12.0 and 11.6 g/dL in the AL group, and 11.9 and 11.7 g/dL in the SP group, respectively.The incidence of non-fatal SAEs was similar (AL 21.8% [108/495] vs. SP 23.3% [118/506]). The most frequent non-fatal SAEs were related to pregnancy and birth, specifically premature delivery , stillbirth, cephalo-pelvic disproportion, and abortion. Other SAEs included infections , of which there were three cases of severe malaria in the SP group.in utero.WHO treatment guidelines exclude artemisinin compounds for use in the first trimester of pregnancy unless there is no alternative, or the mother's life is at stake ,7. PreclThe observed rate of perinatal mortality was 4.2% in the AL group and 5.0% in the SP group, which is comparable with other data from malaria endemic areas -27. TherWomen with first trimester AL exposures did not have a greater perinatal mortality rate compared with SP exposure. Infant neurodevelopmental assessments were also similar, irrespective of drug exposure in first trimester. However, there were seven abortions in 159 foetuses exposed to AL in their first trimester, and no abortions in 135 foetuses exposed to SP and/or quinine in their first trimester. In four AL-exposed abortions, the women had risk factors for abortion: two women had three malaria episodes within the first trimester, one woman had syphilis diagnosed 1 week post-AL exposure, and one woman was given salbutamol for threatened abortion concomitantly with AL therapy for malaria. Furthermore, there was no clear relationship between the time at which AL exposure occurred and the occurrence of abortion; except in one case, the abortion occurred long after anti-malarial drug ingestion. Women were mainly enrolled after their first trimester and, therefore, this study could not determine the potential number of abortions that occurred during the first trimester. This may explain the lower abortion rate observed, compared with the 12-16% spontaneous abortion rate documented ,29. It iThe frequency of birth defects was low and the SAC did not consider that any reported defects (excluding the cases of trisomy) could be described as major. The most frequent finding was umbilical hernia  and all cases had resolved within 12 months. Umbilical hernia has been reported in rats exposed to artemisinins during gestation ,31. It iWith new data on more than 300 women exposed to AL (referring to index episodes) during the second and third trimester, this study substantially adds to data on the safety of artemisinins in the later stages of pregnancy in African women ,36, confThe study had both strengths and limitations. Firstly, it was undertaken in the context of care provided at antenatal clinics in Zambia, and represents the demographic, infectious disease, and drug exposure profile of pregnant women coming for routine care. The acquisition of drug safety data in pregnancy via clinical trials is associated with various difficulties, risks and costs . RegulatGestational age was assessed by midwives either via LMP (the primary method indicated in the protocol) or through use of the Dubowitz test. The Dubowitz test has been shown to be a reliable method for assessment of gestational age in African neonates . HoweverA low incidence of major birth defects could either reflect a lower risk or weaknesses in identifying these risks . On balaWith increasing resistance to SP, establishing the margin of safety of ACT in pregnancy is critical. Results from this cohort study confirm that exposure to AL during later pregnancy is not associated with increased safety risks in terms of perinatal mortality, malformations, or infant neurodevelopment, but they also suggest that more safety data are required on first trimester exposure to AL and consequently for ACT in general.ACT: artemisinin-based combination therapy; AE: adverse event; AL: artemether-lumefantrine; CI: confidence intervals; IPT: intermittent preventive treatment; LMP: last menstrual period; SAC: Study Advisory Committee; SAE: serious adverse event; SP: sulphadoxine-pyrimethamine; WHO: World Health Organization.CM, RM, LP, MS, EM, EN, MCh, and FMS received payments to attend meetings related to the study. VW, RS, and MCo are all full time employees of Novartis Pharma and VW and MCo hold stock ownership therein. MG is a full time employee of the WHO. For the duration of the study and manuscript preparation period, MV was a full time employee of Novartis Pharma and holds stock ownership therein. For the duration of the study, IR worked as a full-time consultant for the WHO.The authors of this manuscript were involved in study design, data interpretation, and the writing of the report, or in a combination of these activities. All authors had full access to study data and held equal final responsibility for the decision to submit this report for publication. All authors contributed either to the design of the study or assisted with data interpretation. CM and MV coordinated the study and supervised enrolment and patient follow-up. VW, RS, MV and CM participated in data entry, collection, and analysis of data. All authors participated in the preparation of the manuscript and approved the final version.


Background and purpose Although total elbow arthroplasty (TEA) is a recognized procedure for the treatment of the painful arthritic elbow, the choice of implant is still obscure. We evaluated the survival of different TEA designs and factors associated with survival using data from a nationwide arthroplasty register.Methods 1,457 primary TEAs for rheumatoid elbow destruction were performed during 1982 to 2006 in one hospital specialized in the treatment of rheumatoid arthritis (n = 776) and in 19 other hospitals (n = 681). The mean age of the patients was 59 years and 87% of the TEAs were performed in women. We selected different contemporary TEA designs, each used in more than 40 operations including the Souter-Strathclyde (n = 912), i.B.P./Kudo (n = 218), Coonrad-Morrey (n = 164), and NESimplavit/Norway (n = 63) to assess their individual survival rates. Kaplan-Meier analysis and the Cox regression model were used for survival analysis.Results The most frequent reason for revision was aseptic loosening (47%). We found no differences in survival rates between different TEA designs. We did, however, find a 1.5-fold (95% CI: 1.1–2.1) elevated risk of revision in unspecialized hospitals as compared to the one hospital specialized in treatment of rheumatoid arthritis. In the Souter-Strathclyde subgroup, there was a reduced risk of revision  in TEAs implanted over 1994–2006 as compared to those implanted earlier (1982–1993). The 10-year survivorship for the whole TEA cohort was 83% (95% CI: 81–86), which agrees with earlier reports.Interpretation The influence of implant choice on the survival of TEA is minor compared to hip and knee arthroplasties. Inferior survival rates of the TEAs performed in the unspecialized hospitals demonstrates the importance of proper indications, surgical technique, and postoperative follow-up, and endorses the need for centralization of these operations at specialized units. The elbow is involved in two-thirds of all patients with rheumatoid arthritis (RA), and, in joints with severe destruction, total elbow arthroplasty (TEA) is often indicated . There aRecent studies have indicated that observational reviews may give results that are similar to those of randomized trials . HoweverThe present study was initiated in order to examine—at a nationwide level—the survival of different TEA designs and the factors affecting survival in patients with RA, by using the data from the Finnish Arthroplasty Register.This study was based on information recorded in the Finnish Arthroplasty Register  relatingIn Finland, the majority of the 1,457 TEAs for RA have been performed in a foundation-based hospital specialized in the treatment of RA . NineteeInclusion criteria In order to assess the survival of different TEA designs, we selected only those designs that had been used in more than 40 operations during the study period . In addiTo meet our inclusion criteria, the following implants were selected: NESimplavit , formerly Norway elbow ; Kudo and i.B.P. , Souter-Strathclyde , and Coonrad-Morrey  . All theThe endpoint for survival was defined as revision involving either one component or the whole implant . Kaplan-Meier survival data were used to construct the survival probabilities of implants at 4, 7, 10, and 15 years. Survival data obtained in the Kaplan-Meier analysis were compared by the log-rank test. The Cox multiple-regression model was used to study differences between groups and to adjust for potential confounding factors. In all models, the confounding factors were age and gender. The factors studied with the Cox model were as follows: TEA designs, hospital type , and implantation period. All models included adjustment for differences in age and gender.Survivorships of the other TEA designs were compared with that of the Souter-Strathclyde TEA (reference design). As the Souter-Strathclyde was the only design that had been used over 1982–1993 , the effOf the 1,457 TEA operations, 1,262 (87%) were performed in women patients. At the time of the operation, the mean age of the patients was 59 (18–89) years. 793 (54%) of all the TEAs were performed on the right elbow. The mean annual insidence of TEA was 1.3 per 100,000 inhabitants.Over the whole study period, 9 different TEA designs were used, 6 of them in 4 groups with more than 40 operations . All impIn the Souter-Strathclyde subgroup, the Cox regression model (adjusted for age and sex) showed a 1.5-fold (95% CI: 1.1–2.2) increased risk of revision for the non-specialized hospitals as compared to the specialized hospital (p = 0.02) .We found no differences in survival rates between different TEA designs over the whole study period, using either the Kaplan-Meier analysis or the Cox regression model (with or without adjustment for age and sex) .In the Cox regression analysis of the Souter-Strathclyde subgroup, there was a significantly reduced risk of revision  in TEAs implanted in 1994–2006 as compared to those implanted earlier, i.e. 1982–1993 (p = 0.001) .Age and sex did not have any statistically significant effect on survivorship in the Cox multiple regression model (unadjusted and adjusted for implant design)—either when all TEAs were analyzed or when analyzing the subgroup of patients treated with the Souter-Strathclyde total elbow replacement.During the period 1982–2006, 201 revisions were reported. Thus, the 10-year survivorship for the whole TEA cohort was 83% (95% CI: 81–86). The most common reason for revision was aseptic loosening . This was followed by prosthesis dislocation , periprosthetic fracture , infection , fracture of the prosthesis , and malalignment of the prosthesis . Other, miscellaneous reasons (including exchange of the liner) accounted for 4% of the revisions (n = 9).To the best of our knowledge, there have been no previous reports on results of TEA conducted at a nationwide level. The mean annual incidence of TEA was 1.3 per 100,000. We found no differences in survival between different TEA designs or concepts, based on the data recorded in the Finnish Arthroplasty Register. The most significant finding in our study was the better TEA survival when performed in a hospital that specialized in the treatment of rheumatoid arthritis.We are aware that the current register-based study had certain limitations. For example, we were not able to report any subjective outcome measurements, e.g. Mayo Elbow Performance Score or disease-specific quality of life measurements. Moreover, it is not possible to conduct radiographic analyses in the large number of register-based patients. Furthermore, when rheumatoid patients are involved, a register-based study may have the pitfall that some of the patients diagnosed as having RA may actually be affected by juvenile arthritis or other subtypes of chronic arthritis. Little is known about TEA in different subtypes of chronic arthritis .The primary indication for TEA is a painful arthritic elbow with Larsen grade-IV or grade-V  rheumatiTraditionally, the TEA designs have been divided into 3 categories: fully constrained rigid-hinge design, semi-constrained hinge design, and unconstrained unlinked design. Fully constrained designs have a lack of flexibility in the coronal plane and in rotation, resulting in high shear stresses and early loosening rates, and these model are no longer in use . UnconstIn the present study, we pooled the results of the Kudo prosthesis and its latest version (i.B.P.), and we also pooled the data for the NESimplavit and its former brand, the Norway prosthesis. In spite of this, we could not find statistically significant differences between survival rates of the different designs. In their comparative study of 3 implants using any revision as the endpoint, Little and coworkers Several studies have found an association between the hospital volume and adverse events in the context of total hip arthroplasty (THA), but no such studies on TEA have been published. Both surgeon volume and hospital volume have been suggested to be the best indicators of orthopedic adverse events in patients undergoing THA . In a syThe inferior survival rates in the Souter-Strathclyde TEAs implanted in the early years of the Finnish Arthroplasty Register have improved over the years. Possible reasons for this include better surgical technique with triceps-sparing approaches, better cementing technique and equipment, and a postoperative regime that allows the triceps to heal. Also, it is possible that the elbows that underwent earlier operations may have undergone more severe destruction from RA, and were thus less optimal for implant arthroplasty.According to our results, it appears that success of reconstruction of a non-weight bearing joint such as the elbow by means of TEA is not as affected by implant choice to the same extent as hip  and knee


In 1979 approximately 2,000 people were exposed to polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) due to ingestion of contaminated cooking oil in Taiwan. Although a previous study has shown delayed developmental milestones and poorer neurocognitive functioning in children born to exposed mothers, it is unclear whether neurocognitive functioning was impaired in people who were directly exposed to the PCBs and PDCFs.The objective of this study was to compare neurocognitive functioning in people exposed to PCBs and PCDFs with that of unexposed sex- and age-matched neighbors.We conducted a retrospective cohort study among exposed and unexposed subjects ≥60 years of age using prospective outcome measurements. We evaluated neurocognitive tests including cognition, memory modalities, learning, motor and sensory function, mood, and daily activity.In total, 162 (59%) exposed and 151 (55%) reference subjects completed this study. In exposed men, all test results were similar to the reference group; however, exposed women had reduced functioning in attention and digit span (ADS), visual memory span (VMS), and verbal memory recalls (VMR), especially learning ability. We also found a borderline reduction in the Mini-Mental State Examination. The digit symbol, motor, sensory, depression , and activity of daily life were not different between the exposed and reference groups. A significant dose–response relationship was found for VMR, ADS, and VMS.Our study showed dose-dependent neurocognitive deficits in certain aspects of attention, visual memory, and learning ability in women previously exposed to PCBs and PCDFs, but not in exposed men. Polychlorinated biphenyls (PCBs) are toxic chemicals that have been widely used throughout the world . PCB conIn rats, PCBs have been reported to have a profound effect on the nervous system. PCBs have also been shown to exert neurobehavioral effects on the second generation, as well as in those who were directly exposed. In a study by We conducted a cohort study with prospective outcome measurements among members of an existing cohort from the 1979 exposure in midcentral Taiwan (the Yucheng cohort), using their sex- and age-matched neighbors as a reference group. This study was approved by the Committee for Human Research at Cheng-Kung University Medical College, and all subjects provided written informed consent. The details of the Yucheng cohort and the matched unexposed subjects have been detailed previously . ExposurIn 1980–1982, serum PCB levels were analyzed in approximately 80% of Yucheng individuals using packed column, electron-capture gas chromatography and the Webb–McCall method adapted to a computerized data system; a Japanese PCB mixture (Kanechlor 500) was used as a reference standard .For neurocognitive evaluation, we used a standardized neurobehavioral battery consisting of 10 tests, including intelligence, verbal, learning and memory, visual-constructive and organizational skills, sensory tactile function, and motor performance . All tesWe used five trials of the verbal memory recall (VMR) to assess learning and short-term memory. Each trial was composed of 10 items of different categories. Thirty minutes after finishing five trials, a delayed recall test was given without warning. Learning capacity was calculated by subtracting the score of the first trial from that of the last trial, with the total amount of the differences defined as learning ability. The purpose of and the capacity tested by the VMR test were similar to those of the California Verbal Learning Test CVLT;  by SchanWe invited registered nurses who were receiving continuing education in central Taiwan to administer the neuropsychological tests for this study, and 20 responded. The nurses were trained by a clinical psychologist (N.W.G.) and a neurologist (K.C.L.), and training included 12 hr of lectures followed by 8 hr of practice. Before the pilot study, the potential interviewers took a written test. Pilot test sessions were carried out on 20 subjects not included in the study. Ten trainees failed to fulfill the required standards and thus were not included as interviewers in this study. For the home visit and neurocognitive tests, each interviewer was assigned matched subjects (one Yucheng subject and one reference subject) on the same day of testing. The interviewers were blinded to the subjects’ exposure status.All test results were reviewed for possible poor performance or recording error before data processing by a clinical psychologist (N.W.G.). Subjects who did not complete all testing items were excluded from the analysis. Therefore, data for 162 exposed subjects (59%) and 151 reference subjects (55%) were analyzed. Although unexposed neighbors were matched to the exposed individuals by age and sex, the analysis was performed in an unmatched manner .t-test and chi-square test to determine differences in the demographics between exposed and reference groups. Multiple regression analyses were used to adjust for age and education. The adjusted values were then compared between exposed and reference groups. Because of a large difference of neurocognitive effects due to exposure across sexes, the analyses were performed separately for men and women. Yucheng individuals were grouped into high-, medium-, and low-exposure groups according to serum PCB levels measured in 1980–1982. We used a test for linear trend to examine dose response by coding unexposed as 0, those with 1980–1982 serum PCB levels of < 35.0 ppb as 1, those with PCB levels of 35.0–95.9 ppb as 2, and those with PCB levels ≥96.0 ppb as 3. A linear regression was performed using age- and education-adjusted neurocognitive scores as dependent variables and the exposure group as an independent variable. All tests for significance were two-sided.A total score was calculated for each neurocognitive test . A higher score indicated a better result for all tests, except for the Finger-Tapping serial and GDS-S. We used the Student’s The field work for this study was conducted from July 2002 to January 2004. The average time for neurocognitive testing was 72.9 ± 22.1 min; this was similar between exposed and reference groups. Test–retest was performed 4 months after the first test in a random sampling of 20 participants, with reliability of 0.85 for MMSE, 0.66 for DS, 0.69 for ADS, 0.73 for VMS, and 0.77 for VMS delayed recall. Most of the tests were inversely related to age and directly related to education; thus scores were adjusted for age and years of education. Smoking, alcohol use, BMI, and other covariates were not related to the test scores. In men, exposure to PCBs/PCDFs did not significantly affect any of the testing scores. In contrast, exposed women scored lower than the reference group in MMSE, VMR, ADS, and VMS, but their scores were similar to those of the reference group in DS, finger tapping, sensory scores, depression scores, and ADL . We alsoWe examined the dose–response relationship by dividing the Yucheng women into low- (< 35 ppb), intermediate- (35–95 ppb), and high-exposure (> 95 ppb) groups according to the 1980–1982 PCB levels. We found significant linear trends in the reduction in VMR, ADS, and VMS when reference, low-, intermediate-, and high-exposure groups were compared .The present study provides evidence that previous exposure to PCBs and PCDFs in adulthood caused neurocognitive declines, particularly in learning and memory, 25 years after exposure. Such damage was found in women who were, on average, in their late sixties. Despite the limited sample size, we found reduced capability in memory and learning in a dose-dependent manner in Yucheng women.The damage to the central nervous system observed in this study leads to our postulation that exposure to PCBs and PCDFs had an influence on the vulnerable deep-seated hippocampus but not on the the durable executive or sensory parts of the cortical brain. Although different tasks are involved, the ADS, VMR, and VMS all require immediate and short-term working memory, which appeared to be affected by PCBs/PCDFs in the Yucheng women. Our data show a relevant significance of working memory deficit rather than executive motor and sensorial performance.To date, there has been only one publication about cognitive function in PCB-exposed elderly .The studAge and education are well known to affect neurocognitive functioning . In the Complete neuropsychological tests, such as the Wechsler Adult Intelligence Scale-Revised WAIS-R; , the HalDepression can potentially affect neurocognitive functioning. We examined the effects of the GDS-S score on neurocognitive test performance and found no significant effects. In addition, Yucheng men and women did not have higher GDS-S scores than the reference group.Few studies have examined neurocognitive effects of human adult exposure to PCBs and related chemicals. Because we focused on people ≥60 years of age, some of the Yucheng subjects with the highest exposure may have died before the neurocognitive outcomes were assessed, and these subjects may have been most affected with respect to neurocognitive functioning. Therefore, in the present study, we may have underestimated the overall neurocognitive effects that Yucheng exposure could have caused.In previous studies of the Yucheng cohort, neurocognitive effects were found among children prenatally exposed to PCBs and PCDFs , likely In the present study, we found sex-related damage of memory and learning among Yucheng women. Although the mean serum PCB level in Yucheng women  was higher than that in Yucheng men (67.4 ± 62.3 ppb), the difference was not statistically significant, and probably did not account for the sex-difference in neurocognitive effects of individuals. This is further evidenced in the Yucheng women exposed to intermediate levels (35–95 ppb); the mean serum level (59.7 ppb) was lower than that in average Yucheng men, but the neurocognitive effects were greater. In a study of randomly selected Swedish people, In summary, among humans previously exposed to relatively high levels of PCBs and PCDFs, we identified neurocognitive hazards, especially memory and learning impairments in women.


Retroperitoneal cysts are uncommon, with an estimated incidence of 1/5750 to 1/250,000.A male patient was admitted with an abdominal pain, jaundice and fever. Clinical examination and investigations confirmed an idiopathic benign retroperitoneal cyst. He underwent surgery and was discharged after making good recovery.Retroperitoneal cysts are very rare, and most of the time they are discovered incidentally. Patients may be asymptomatic or present with abdominal pain, referred pain to the legs or weight loss. Imaging may help diagnose these lesions, but surgery is the keystone in confirming the diagnosis. This case is very rare and very educational as it highlights an unusual presentation of a benign retroperitoneal cyst. In our patient, the course of the disease was unique as the patient presented with jaundice. Retroperitoneal cysts (RPCs) are uncommon with an estimated incidence of 1/5750 to 1/250,000 [3/mm3), bilirubin (103 mg/dl) and ESR (75 mm/h). All other blood tests were normal. Ultrasound and CT scans of the abdomen revealed fluid collection/mass measuring 14 cm in the right hepato-renal space. The mass was separated from the liver, pancreas and the right kidney, but there was lack of definition of the right suprarenal gland. Because the patient was jaundiced, we thought that draining the cyst would relieve the pressure on the biliary system and resolve jaundice. About 150 ml brown-coloured fluid was aspirated from the mass under CT guidance and a sample was sent for histology. Cytological examination of the sample reported presence of amorphous material with occasional histiocytic cells. There was no evidence of malignancy. The patient was discharged in December 2003 after making good recovery.A 51 year-old man was admitted on the surgical ward in November 2003 for a right-sided abdominal pain which had been present for three days. Clinically, he was jaundiced with a high temperature. Blood tests showed white cell count : Urogenital; (b): Mesocolic; (c): Cysts arising in cell inclusions; (d): Traumatic; (e): Parasitic and (f): Lymphatic ,2. Only There are no pathognomonic signs or symptoms for RPCs, and in approximately one third of patients, the cyst is found incidentally ,5. Two tCT is ideal for assessing RPCs because it provides discrete sectional images of the organs and retoperitoneal compartments, and in some case, familiarity with the most relevant radiologic features, in combination with clinical information, allows adequate lesion characterization . MulleriSymptomatic cysts should be enucleated or excised, while preserving the surrounding vital structures. At times, the cyst can be marsupialised or drained if surgical enucleation is difficult or the cyst is infected . HoweverCysts arising within the retroperitoneum outside the major organs within that compartment are very rare. Approximately one third of patients with retroperitoneal cysts are asymptomatic and the cyst is found incidentally. CT may help diagnose these lesions, but surgery remains the keystone in determining the diagnosis.The author(s) declare that they have no competing interests.A A: Reviewed literature and wrote the manuscript, H M: Contributed to the concept of the manuscript, P D: Evaluated histopathology, J A: Searched literature, A B: Operated on the patient. All authors have read and approved the manuscript.Written informed consent was obtained from the patient for publication of this report and any accompanying images.


In the county of Scania, Sweden, antibiotic use among small children is among the highest in the country. The aim of this study was to investigate the associations between antibiotic use among 8-month-old children in Malmö and characteristics of the child as well as parental sociodemographic characteristics, lifestyle factors, and psychosocial support.The study was a population-based cross-sectional survey. The study population consisted of children who visited the Child Health Care (CHC) centres in Malmö for their 8-month health checkup during 2003–2006 and whose parents answered a self-administered questionnaire (n = 7266 children). The questionnaire was distributed to parents of children registered with the CHC and invited for an 8-month checkup during the study period.The odds of using antibiotics increased as parental educational level decreased. Using high educational level as a reference group, low maternal educational level was associated with an increased antibiotic use for the child, odds ratio (OR) = 1.61 (95% CI: 1.34–1.93). Furthermore, children whose parents were born outside Sweden showed higher antibiotic use, OR = 1.43 (95% CI: 1.24–1.65), in comparison with children whose parents were born in Sweden. Exposure to environmental smoking, parental experience of economic stress, and a low level of emotional support increased the odds for antibiotic use. Boys had higher odds of use of antibiotics than girls, OR = 1.40 (95% CI: 1.25–1.57). Having a low birth weight, having an allergy and having siblings also increased the odds for early antibiotic use, while breastfeeding seemed to have a protective role.There were clear associations between parental factors such as sociodemographic, psychosocial and lifestyle factors and antibiotic use at this early stage of life. Several characteristics of the child also affected the use of antibiotics. A survey of 25 European countries in 2003 revealed that Greece and France had the highest out-patient use of antibiotics, while Sweden had a relatively low use compared to the other countries . In addiSince there is a correlation between high antibiotic use and the development of resistance ,5; a redThe aim of this study was to investigate the associations between antibiotic use among 8-month-old children in Malmö, Sweden, and characteristics of the child as well as parental sociodemographic characteristics, lifestyle factors, and psychosocial support.This study was conducted in Malmö, the third largest city in Sweden. According to statistics provided on the Malmö city website , the citThe study was a population-based cross-sectional survey. The study population consisted of children who visited the Child Health Care (CHC) centres in Malmö for their 8-month checkup during 2003–2006 and whose parents answered a self-administered questionnaire (n = 7266 children). The CHC centres in Sweden are a well-established organisation, with responsibility for reducing mortality, morbidity, and disability in newborn and younger children. Another purpose is to educate parents so they can make the most of their child's developmental opportunities. Through regular visits to the child health nurses, each child's weight and physical and developmental health are closely followed, and vaccinations are given until the child is 5–6 years old. The CHC focus is prevention; visits are voluntary and the consultations are free of charge. As many as 99% of children aged 0–6 participate in the programme .The data on the children in the present study was derived from a self-administered questionnaire distributed to parents of children who were registered with the CHC and invited for an 8-month checkup during the study period. The questionnaire was distributed by the pediatric nurses working at the CHC centres.The questionnaire contained approximately 30 questions about the child's family situation, as well as the parents' education, occupation, country of birth, and financial and emotional security. It also included questions about exposure to environmental smoking, allergies, breastfeeding, and antibiotic consumption. The questionnaire was validated and tested for reliability, and translated into five different languages: Albanian, Arabic, English, Serbo-Croatian, and Somali . In addiAntibiotic use was assessed by the question: "Has the child been treated with any form of antibiotics during the child's first eight months of life". The parent could answer yes or no, and also fill in the number of times.Parents' country of birth was divided into: both parents born in Sweden, one parent born in Sweden, and both parents born outside Sweden. Maternal and paternal educational level was based on years of schooling and divided into 9 years and less, 10–12 years, and more than 12 years of education. Employment status was classified into: parental leave (being home with the child), working, and other . Maternal smoking during pregnancy was divided into yes and no. Exposure to environmental smoking was divided into no  and yes .Economic stress was assessed with the question "How many times during the past year did you not have money enough to afford the food or the clothes you and your family need?", with answers being classified into yes  and no . Emotional support was assessed with the question "Do you have someone that can give you proper personal support to cope with life's stress and problems?", with answers being classified into high emotional support (definitely yes or probably yes) and low emotional support (not for certain or no).Gender was divided into boy or girl. Low birth weight was classified as < 2500 g, and normal birth weight as ≥ 2500 g. Position among siblings in the family was dichotomised into firstborn versus secondborn or later. Breastfeeding was classified into the following categories: 0 months, 0.1–3 months, 3.1–6 months, and 6.1–8 months of breastfeeding. Still being breastfed at 8 months was classified as yes or no. Allergies were categorised by the answer to the question "Does the child have allergies?" to which the parent could answer yes or no.Characteristics of the child were also assessed. Odds ratios (OR) and 95% confidence intervals (95% CI) were used to analyse the associations between antibiotic use and the various child and parental characteristics. Multiple logistic regression analyses were performed in order to adjust the estimated OR for the influence of confounding factors. Model 1 included year and gender; model 2 included year, gender, and parents' country of birth; model 3 included year, gender, parents' country of birth, and maternal educational level; and model 4 included year, gender, parents' country of birth, maternal educational level, and having had recurrent infections. Statistical analyses were performed with version 12.0.1 of SPSS for Windows. The study was approved by the Regional Ethical committee, Lund University.The study included 7266 8-month-old children over the four years of the study period (2003–2006). Table Table Table This population-based study of 8-month-old children in the city of Malmö, Sweden, showed that the use of antibiotics up to this young age was increased among children whose parents were born outside of Sweden, had a low educational level, experienced economic stress, or had low emotional support. Although exposure to environmental smoking was associated with an increased antibiotic use in the crude model, this association was attenuated and turned non-significant after adjustment for potential confounders. Breastfeeding seemed to have a protective role against antibiotic treatment. Furthermore, the odds of antibiotic use were significantly increased by being a boy, having a low birth weight, having siblings, and having an allergy.In our study, antibiotic consumption was significantly higher among children whose parents were less well educated. Similar results have been shown in other studies ,10. The Certain methodological issues need to be addressed. All parents with 8-month-old children were invited to participate in the study by answering the questionnaire. The total number of children whose parents answered the questionnaire during the four-year study period was 7266, or about two thirds of all those who received the questionnaire. We have no information about the country of birth in the non-participating families, but according to Rosvall et al. , the nonOur results showed that the use of antibiotics among 8-month-old children in Malmö, Sweden, was influenced by several factors including parental sociodemographic factors, lifestyle factors, psychosocial support, as well as child-related factors. The parental characteristics associated with higher antibiotic use were low educational level, being born outside Sweden, economic stress, and having low emotional support. The child-related characteristics associated with higher antibiotic use were male gender, low birth weight, having siblings, having an allergy, and not being breastfed at all.The authors declare that they have no competing interests.EM, AW and MR have contributed to the conception of the work, the analysis of the data, the interpretation and the discussion of the results, the drafting, writing, and revision of the content. JM and A-CB have contributed to the interpretation and the discussion of the results. All authors read and approved the final manuscript. MM has contributed with help of the statistical work behind this article.The pre-publication history for this paper can be accessed here:


Doppler ultrasonography is an important tool for evaluating hepatic portal hemodynamics. However, no study in dogs of different body weights, in the range encountered in routine clinical veterinary practice, has been reported. It can be difficult to obtain an ideal insonation angle when evaluating the main portal vein, so evaluation of the right portal vein branch has been described in humans as an alternative. The aim of this study was to analyze, through Doppler ultrasonography, the hemodynamics in the right portal vein branch in dogs of different body weights.Thirty normal dogs were divided in three groups by weight, in order to establish normal values for mean velocity, flow volume and portal congestion index of the right portal vein branch by means of Doppler ultrasonography.In all dogs ideal insonation angles were obtained for the right portal vein branch. The average velocity was similar in the three groups, but the portal congestion index and the flow volume differed, showing that the weight of the dog can influence these values.Doppler ultrasonography for the evaluation of flow in the right branch of the portal vein could be a viable alternative, or complement, to examining the main vessel segment. This is especially so in those animals in which an ideal insonation angle for examination of the main portal vein is hard to obtain. In addition, the weight of the dog must be considered for the correct evaluation of the portal system hemodynamics, particularly for portal blood flow and the congestion index. Doppler ultrasonography is a safe and effective technique for evaluation of portal vein hemodynamics in dogs. Mean velocity (Vmean), average portal blood flow volume (PBFV) and congestion index (CI) are important indicators in hepatic evaluation, mainly to detect alterations such as chronic hepatic diseases that lead to portal hypertension . HemodynKnowledge of the normal values for such variables is essential to recognize and diagnose alterations that may occur in hepatic disease. Since dogs of different breeds, sizes and weights are routinely assessed, it is important to know whether these normal values differ between dogs according to size.In the literature, there are only reports on values for mean velocity, flow volume and portal congestion index of the portal vein in healthy, medium-sized dogs ,3; no stporta hepatis region is described in both dogs and humans [Another important factor, the insonation angle, must be considered for Doppler evaluation of portal hemodynamics. This is the angle between the ultrasonographic waves and the studied vessel. An ideal angle of 0 degrees occurs when the flow and ultrasound waves are parallel, but this is hard to obtain. A large number of authors have reported that this angle must be kept below 60°, in order to measure flow velocity with a minimal margin of error ,3-5. Difd humans ,3,5,6. HThe aim of this study was to make Doppler flow measurements from the right intra-hepatic branch of the portal vein in order to establish the normal values for mean velocity, flow volume and portal congestion index in healthy dogs; and to detect variations between groups with different body weights.Thirty healthy dogs, males and females, of several breeds obtained from the Veterinary Hospital, College of Veterinary Medicine and Animal Science-FMVZ, São Paulo State University-UNESP, Botucatu, São Paulo State, Brazil, were evaluated. These animals were considered healthy based on physical, hematological and biochemical analyses, as well as on abdominal ultrasonography. Initially there were 42 animals, but seven dogs could not be examined due to the breathing or the behaviour of the animal, and five dogs had abnormal values of clinical laboratory tests. The animals were divided into three groups, with ten dogs in each, according to weight range: Group A, dogs weighed ≤ 10 kg; Group B, dogs weighed 10.1-20 kg; and Group C, dogs weighed ≥ 20.1 kg.A triplex scan ultrasonographic device  was used. It had two multi-frequency transducers, convex from 3.5 to 5 MHz and linear from 6 to 10 MHz.® 75 mg/ml, Hipolabor Farmacêutica LTDA, Borges Sabará-MG, Brazil), orally, at a dose of 4 drops/kg, three times from the beginning of the fasting period to 20 minutes before the ultrasonographic evaluation. Dimethicone is an antiflatulent and was used to avoid intestinal gas formation. No sedatives were used.Dogs were deprived of food for 12 hours before the test and received dimethicone, A: portal vein area; D: portal vein diameter; π: 3.14Color and spectral Doppler examination was carried out in the right branch of the portal vein to evaluate blood flow direction and velocity within the vessel Fig. . To acceThe experimental design was completely randomized, with three groups and ten replicates per group. For each animal, all measurements had three replicates, from which a mean was calculated. Based on the obtained data, portal CI and average portal blood flow volume (PBFV) were assessed.To calculate CI, the following formula was used :A: portal vein area; Vmean: portal vein flow mean velocityTo calculate PBFV (ml/min/kg), the following formula was used (5):w: body weightIt was necessary to change the unit of the Vmean (cm/sec to cm/min).Data were analyzed through an F-test and means compared by Tukey's test, at 5% significance.This study included dogs of various body conformation, breed and size. In all evaluated animals, blood flow evaluation was possible in the portal right branch it was easily found, and the insonation angle was always lower than 60° .Flow velocity was similar among all three groups (P > 0.05). Portal blood flow volume was significantly (P < 0.05) greater in Group A, relative to Group C, whereas the results of Groups B and C were similar. With respect to the portal CI, Group A had significantly P < 0.01) lower values, relative to the other two groups . Carried out the ultrasonography study, analysed the data, and drafted the manuscript. MJM Supervisor of the Master Degree Thesis (Raquel Sartor). MJM participated in the analysis of the data and in the draft of the manuscript. RKT carried out the blood analysis and the interpretation of the results. MFA auxiliary on the data collect. All authors read and approved the final manuscript.


Setaria italica) and Common millet (Panicum miliaceum) are the oldest domesticated dry farming crops in Eurasia. Identifying these two millets in the archaeobotanical remains are still problematic, especially because the millet grains preserve only when charred. Phytoliths analysis provides a viable method for identifying this important crop. However, to date, the identification of millet phytoliths has been questionable, because very little study has been done on their morphometry and taxonomy. Particularly, no clear diagnostic feature has been used to distinguish between Foxtail millet and Common millet. Here we examined the anatomy and silicon structure patterns in the glumes, lemmas, and paleas from the inflorescence bracts in 27 modern plants of Foxtail millet, Common millet, and closely related grasses, using light microscopy with phase-contrast and microscopic interferometer. Our research shows that five key diagnostic characteristics in phytolith morphology can be used to distinguish Foxtail millet from Common millet based on the presence of cross-shaped type, regularly arranged papillae, Ω-undulated type, endings structures of epidermal long cell, and surface ridgy line sculpture in the former species. We have established identification criteria that, when used together, give the only reliable way of distinguishing between Foxtail millet and Common millet species based on their phytoliths characteristics, thus making a methodological contribution to phytolith research. Our findings also have important implications in the fields of plant taxonomy, agricultural archaeology, and the culture history of ancient civilizations.Foxtail millet ( Phytoliths are microscopic silica bodies that precipitate in or between cells of living plant tissues, and are widespread in all types of plants and all their different organs and structures, from roots to culms to inflorescences Setaria italica) and Common millet , belong to Panicoideae of Poaceae, are considered to be dry farming cereals that form the oldest cultivated foods known to humans Foxtail millet  and Common millet (P. miliaceum).The inflorescence bracts in Poaceae are characterized by phytoliths with specific morphological characteristics, hence their taxonomic significance S. italica L. Beauv. and twelve species of P. miliaceum L.; wild species include two species of P. bisulcatum Thunb., S. plicata (Lam.) T. Cooke, S. glauca (Linn.) Beauv., S. viridis (L.) Beauv., and Echinochloa crusgalli (L.) Beauv. Of these 27, four species were sampled from annotated folders at the National Crop Gene Bank of China, Chinese Academy of Agricultural Sciences (CAAS); six species were sampled from the Culture Museum of Cishan, Wuan, Hebei Province, China; fourteen species were sampled from Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, China; and three species were sampled from East China Normal University, Shanghai, China. The folders contained samples of field collections by many investigators. For passport data on the plants, see We examined modern phytoliths from twenty-seven species of domesticated and wild Paniceae. Domesticated species include nine species of Setaria and Panicum, the palea of first floret is atrophied to a very small and membranous organ and sometimes becomes lost in the spikelet. Thus, in this study, we used “palea” for the “palea of second floret”. The five parts of spikelet were prepared in the following manner.In this study, we dissected the spikelet of modern plants into five parts, including lower glume, upper glume, lower lemma (lemma of sterile floret), upper lemma, and palea (i) Each part of spikelet was cleaned with distilled water in a water bath to remove adhering particles. (ii) All samples were placed in 20 ml of saturated nitric acid for over 12 h to oxidize organic materials completely. (iii) The solutions were centrifuged at 2000 r.p.m. for 10 min, decanted and rinsed twice with distilled water, and then rinsed with 95% ethanol until the supernatants were clear. (iv) The phytolith sediments were transferred to storage vials. The residual subsamples were mounted onto microscopic slides in Canada Balsam medium for photomicrography and in liquid medium for counting, measuring, and line drawing. (v) Light photomicrography  at 400× magnification was used to determine their anatomy and silicon structure patterns in the glumes, lemmas, and paleas. (vi) Phytolith parameters were measured using computer-assisted image analysis.Micromorphological characters of the lower lemma and glumes are generally similar for each millet sample based on our observation and statistic of all samples. Silicification always occurs in the short cells (silica cells), and occasionally occur in some of the long cells, micro-hairs, macro-hairs, and stomata in the lower lemma and glumes of both Foxtail millet and Common millet.The shape of the silica bodies formed in the short cells of Foxtail millet is different from Common millet . Cross-sS. italica, and the bilobe-shaped type is formed in those of P. miliaceum.Other silicified cells, including long cells, micro-hairs, macro-hairs, and stomata, are without any characteristic shape, and not easily identified in phytoliths. This suggests that the cross-shaped type is formed in the lower lemma and glumes of S. italica  from center toward the base and top part of the upper lemma  toward the base and top of the palea  or η  sinuous margins that join the margins across the cells. The different Ω-undulated regular patterns occur at different parts by gradual change in a general way from base and top (Ω I), to side (ΩII), to center (Ω III) of the lemmas and palea in Foxtail millet . SimilarS. italica, and η-undulated  in P. miliaceum.This suggests that the surface undulated patterns of epidermal long cell walls in the upper lemma and palea can be used to distinguish between Foxtail millet and Common millet. The epidermal long cell walls are Ω-undulated  in Based on our observation and statistics of endings structures of epidermal long cells, we found that three important parameters can be used to characterize the morphological variations of structures of epidermal long cells in the upper lemma and palea : (1) W =S. italica. The average width of endings interdigitation of dendriform epidermal long cells is about 4.37±0.89 µm (N = 2774)  in the Cross finger type of P. miliaceum than that in S. italica.   . The R-value is higher  in P. miliaceum than in S. italica  ( = 2774)  , outer epidermis, hypoderm fibres, vascular bundle, and occasional silicification of internal spongy mesophyll in the transection of lemma and palea S. italica, which is characterized by having an adnate silicon extracellular sheet and outer epidermis, forming a very heavy silicon layer  (P. miliaceum.The phylogenetic relationship of Eurasian ηI type)  that is S. italica) is presumed to have originated from S. viridis (green foxtail), a ubiquitous weed from the Eurasian continent S. viridis, S. plicata (Lam.) T. Cooke. S. viridis  for discriminating between OryzaAvena sativa and A. strigosaThe inflorescence bracts are well known sites of heavy silicification in cereals, due to the hot and arid habitat of the cereals, conditions that promote intensive transpiration and water loss and lead to the close formation of phytoliths in inflorescence bract cell Foxtail millet and Common millet are vitally important food crops for people living in the Far East and even in the entire Eurasian continent prior to the popularity of rice and wheat S. italica and P. miliaceum. This character is as significant in the taxonomy of each genus as other quantitatively important characters.Because of this, we had to dissect the spikelet of modern plants into five parts, including lower glume, upper glume, lower lemma (lemma of sterile floret), upper lemma, and palea, to examine the variation of anatomy and silicon structure patterns in different parts, from base to center to apex, and margins, cover with whole glumes, lemmas, and paleas. Our observation and statistics of the micromorphology throughout each glume, lemma, and palea reveal well-regulated variations in phytoliths between Foxtail millet and common millet, particularly regarding the presence or absence of papillae, undulated patterns of epidermal long cell, and surface ridgy line sculpture. Based on a large sample, the average width of endings interdigitation of dendriform epidermal long cells examined in a large number is found to be consistently and significantly different between the two species examined and therefore can be considered a diagnostic feature to distinguish between S. italica, whereas Bilobe-shaped type is formed in those of P. miliaceum. (ii) Regularly arranged papillae on the surface of the upper lemma and palea are peculiar to S. italica. (iii) The epidermal long cell walls are Ω-undulated  in S. italica, and η-undulated  in P. miliaceum. (iv) Cross wavy type  occurs in the upper lemma and palea of S. italica, whereas Cross finger type  is formed in those of P. miliaceum. The R value (ratio of the width of endings interdigitation to the amplitude of undulations) is higher  in P. miliaceum than in S. italica . (v) Surface ridgy line sculpture of the upper lemma is also important for the identification of S. italica, which is characterized by having an adnate silicon extracellular sheet and outer epidermis, forming a very heavy silicon layer that is a reliable feature in distinguishing them from P. miliaceum. These five diagnostic characteristics used together give the only reliable way of distinguishing Foxtail millet from Common millet. A species-specific identification of phytoliths is possible for S. italica and P. miliaceum because they have typically well-defined silica skeletons that are distinguishable from those in P. bisulcatum, S. viridis, and S. plicata, which have no such demonstrable patterns.By all accounts, our research indicates that five key diagnostic characteristics in phytolith morphology could be used to distinguish Foxtail millet from Common millet : (i) CroThe results of this study have revealed distinct differences between Foxtail millet and Common millet. Nevertheless, several caveats exist in the morphological characteristics of phytoliths, which should be mentioned. A number of factors may influence the within-individual variations in phytolith morphology, including the stage of plant maturity It is not the focus of this paper to discuss in detail how phytoliths can be used to distinguish millets from other related grasses. Although the phytolith production patterns revealed in our preliminary research give encouraging results that may point the way to distinguishing the millets from related grasses in China, more research is needed, especially the study of more wild species and landraces of domesticated millet species. To be of practical use to investigators, further morphometric analysis of a wide variety of millet species is required. Future work could use this methodology to develop classification paradigms, and to gain an understanding of the effect of domestication and polyploidization on phytolith morphometries.In discussions of the origin of early agriculture, Foxtail millet and Common millet have received particular attention, since they were the dominant traditional crops in the Far East and even in the entire Eurasia. However, until now, the identification and taxonomic distinction between Foxtail millet and Common millet in archaeobotanical remains had been problematic, especially because the crop grains preserve only when charred, or where their preservation is poor.S. italica and P. miliaceum, respectively; (ii) Papillae on the upper lemma and pales are peculiar to S. italica; (iii) The epidermal long cell walls are Ω-undulated in S. italica, and η-undulated in P. miliaceum; (iv) The endings structures of epidermal long cells are Cross wavy type in S. italica, and Cross finger type in P. miliaceum; (v) Surface ridgy line sculpture of the upper lemma are peculiar to S. italica. Collectively, these five diagnostic characteristics provide the only reliable way of distinguishing Foxtail millet from Common millet. A species-specific identification of phytolith is possible for S. italica and P. miliaceum because they have typically well-defined silica skeletons that are distinguishable from those in P. bisulcatum, S. viridis, and S. plicata, which have no such demonstrable patterns.This is the first study of the variation of the anatomy and silicon structure patterns in the glumes, lemmas, and paleas occurring among 27 modern millets and related grass species collected from different regions in China. We found that five key diagnostic characteristics in phytolith morphology could be used to distinguish between Foxtail millet and Common millet, as follows. (i) Cross-shaped and Bilobe-shaped are formed in This practical protocols, if supported by additional studies of phytoliths derived from more millets and related grass species, can be give the only reliable way of separating the Common millet, Foxtail millet, and other related grass species based on their phytoliths.


During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING  is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.In this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING. Our understanding of the basic mechanisms underlying most biological processes has been transformed by the systematic application of mutational analysis. Traditionally, forward genetics, driven by the identification of mutant phenotypes, has been the most widely used approach. On the other hand, genome sequencing projects over the past few decades have identified numerous genes in key species, and the completion of these sequences produced a situation in which most of the genes are known, but most of their phenotypes are obscure. In this situation, reverse genetics, which provides targeted inactivation of genes identified by sequence analysis followed by phenotype analysis of the mutant, has become an important tool for many biologists. In mice, reverse genetics is usually carried out using homologous recombination in embryonic stem cells, which allow a precise mutation to be constructed in nearly any gene. However, embryonic stem cells are only available in a limited number of organisms. Thus, a general method that is applicable to many organisms would be in great demand, and several approaches have been tried.Arabidopsis thaliana  ethanesulfonic acid , and incubated at 27°C.Cell cultures were established from embryos as described previously . OLCAB-e3 cells were irradiated with γ-rays at the dose of 100 Gy and used for feeder cells. After irradiation, atm+/+ or atmS444X/S444X cells were cultured in 6-well plates in the presence of feeder cells (7 × 105 cells/well) and incubated at 27°C. Two weeks after irradiation, the cells were fixed with ethanol and stained with 0.006% crystal violet, the formed colonies were counted, and the survival rate was calculated.Cultured cells were exposed to γ-rays from a atr+/S1339X fishes were intercrossed and eggs were cultured at 27°C in a 10-cm plastic dish. After culture for 7, 10, 15, 20, 25, or 30 days, the genotypes of surviving eggs or fry (80-100 for each fraction) were determined by PCR or by sequencing.The gonads were dissected and fixed in Bouin's solution. Fixed tissues were embedded in paraffin blocks. Cross-sections were cut at a 5-μm thickness and stained with hematoxylin-eosin. Sections were examined and photographed under light microscopy.R qPCR kit (Toyobo). For atr and atm quantitative PCR, cDNA-specific primers were designed to amplify either the 5' or 3' region of the atm and atr transcripts . The sequences of all oligonucleotides that were used are listed in Additional file RNA was isolated from cultured cells established from mutant or wild-type fish using Sepasol RNAI Super  and reverse transcribed by using the ReverTra Aceatr phenotype. YKu and MT provided expertise on histological examinations of fish gonads and supervised their interpretation. HI and TTo secured funding for the experiments. TTo conceived the project; designed the experimental plan; and supervised the design, execution, and interpretation of all experiments.TI and YKa participated in the design and performance of all experiments described in this paper. In addition, TI wrote the first draft of the first manuscript. SO, JK, and HI participated in mutant screening with the HRM assay. AS and TTs performed histology, and TD participated in identification of the p53 exons 5 and 6.Figure S1: Location of PCR primers  used for screening of mutations in  Mutations are indicated with vertical arrows.Click here for fileTable S1: Primer sequences for screening of mutations.Click here for fileatr+/+, atr+/S1339X, or atrS1339X/S1339X fishes.Figure S2: Growth of  (A) Images of representative embryos and fry of each genotype are shown. Body length (B) and body height (C) of 92 fry were measured and plotted.Click here for fileTable S2: Primer sequences for genotyping, real-time PCR, and RT-PCR.Click here for file


Liver fibrosis progression is commonly found in patients with CHB. Liver biopsy is a gold standard for identifying the extent of liver fibrosis, but has many draw-backs. It is essential to construct a noninvasive model to predict the levels of risk for liver fibrosis. It would provide very useful information to help reduce the number of liver biopsies of CHB patients.339 chronic hepatitis B patients with HBsAg-positive were investigated retrospectively, and divided at random into 2 subsets with twice as many patients in the training set as in the validation set; 116 additional patients were consequently enrolled in the study as the testing set. A three-layer artificial neural network was developed using a Bayesian learning algorithm. Sensitivity and ROC analysis were performed to explain the importance of input variables and the performance of the neural network.There were 329 patients without significant fibrosis and 126 with significant fibrosis in the study. All markers except gender, HB, ALP and TP were found to be statistically significant factors associated with significant fibrosis. The sensitivity analysis showed that the most important factors in the predictive model were age, AST, platelet, and GGT, and the influence on the output variable among coal miners were 22.3-24.6%. The AUROC in 3 sets was 0.883, 0.884, and 0.920. In the testing set, for a decision threshold of 0.33, sensitivity and negative predictive values were 100% and all CHB patients with significant fibrosis would be identified.The artificial neural network model based on routine and serum markers would predict the risk for liver fibrosis with a high accuracy. 47.4% of CHB patients at a decision threshold of 0.33 would be free of liver biopsy and wouldn't be missed. Approximately 350 million people worldwide are chronically infected with hepatitis B virus (HBV), especially in many developing countries in Asia-3. LiverCurrently, liver biopsy, a widely used gold standard for the examination of the extent of liver fibrosis and HCC in patients with CHB, has many draw-backs, such as pneumothorax, pain, hemorrhage, or puncture of other viscera-10. In aThe complete blood count and liver function are routine tests on CHB patients with hepatitis B surface antigen (HBsAg-positive) in the clinical treatment. It is easy to measure those markers with low cost in some laboratories, even in the Community Hospitals. Moreover, the artificial neural network (ANN) is potentially more successful than a traditional statistical model in predicting clinical outcome ,20. It cThe present study was performed on HBsAg-positive patients. An artificial neural network model based on routine and serum markers was constructed to predict the risk for liver fibrosis. The objective of this study was to determine the feasibility of these indices of routine and serum markers to predict the risk for liver fibrosis. Furthermore, we aimed to similarly validate the probability value in order to identify the risk for liver fibrosis on CHB patients and classify high risk groups.In April 2008, all HBsAg-positive in-patients who had liver biopsy between January 2006 and March 2008 were retrospectively investigated at the Hospital of Infectious Diseases. Only patients without the following conditions were included for the study: presence of other causes of liver disease such as chronic hepatitis C (CHC), or hepatitis E (HE), etc., acute hepatitis, hepatocellular carcinoma, prior liver transplantation, insufficient liver tissue for staging of fibrosis, and incomplete data on complete blood count or some serum markers of fibrosis. Additionally, patients without routine and serum markers prior to drug treatment were also excluded. Within a week of liver biopsy, those markers were recorded. If more than one set of laboratory results were available, the set of results closest to the time of biopsy were used. Later, additional in-patients who met the aforementioned criteria were consequently enrolled in the study from April 2008 to July 2009.The enrolled patients were informed all procedures which conformed to the Helsinki Declaration. The study procedures were also approved by the Ethics Committee of the Infectious Diseases Hospital of Shenyang. The verbal informed consent for all patients was provided.Clinical chemistry tests were performed using 7150 Analyzer , and the complete blood count were measured on Hematology Analyzer .Liver biopsy was performed by automatic fare cut biopsy needle. Length and width of each sample were at least 10 mm and 1 mm, respectively. Liver sample of patients who were enrolled since April 2008 would contain 2 or more portal spaces. A single pathologist who had no clinical information of patients evaluated all biopsy results. The level of fibrosis was measured according to the METAVIR system, which had previously been applied in other reports on CHB,25. FibrAll statistical analysis was performed using MATLAB Neural Network Toolbox (2006) and SPSS Version 11.5 . Data are reported as median (minimum-maximum) unless otherwise stated. The relationship between variables and the presence of significant fibrosis was assessed. The Mann-Whitney U Test, or Kruskal-Wallis Test was used for continuous variables when appropriate. The Chi-square test was used for categorical variables, and Fisher's exact test when appropriate. Those variables found to be strongly correlated to the presence of significant fibrosis were used to build the ANN.Patients between January 2006 and March 2008 were randomly divided into 2 subsets: training set and validation set, with twice as many patients in training set as in validation set. Patients since April 2008 formed the testing set. The three-layer neural network model was built and trained using a Bayesian learning algorithm, which had 1 output neuron . To determine the optimal number of neurons in the intermediate layer, we randomly split all data into 5 subsets of equal size. For any given number of neurons, we trained the network on all but one subset, and tested it on the remaining one. We started from 6 neurons, and gradually increased the number of neurons. When there were more than 13 neurons, the performance of the trained neural network on output sample in the testing set began to deteriorate. Hence, the intermediate layer had 13 neurons in the current neural network model. The number of training epochs was set to 300, the learning rate was 0.05, and the training goal was set at 0.001.As the neural network can be overtrained to recognize specific cases in the training set and result in good performance in the training set but not in the testing set, the validation set was used to decide when to stop training in order to minimize the potential bias.Neural network models have been long criticized for being black box solutions primarily because of their inability to generate interpretable parameters for each input variable. To mitigate this problem, sensitivity analysis was adapted to explain their inference mechanism. In our The receive operating characteristic (ROC) methodology is a computational methodology which has a very important connection to the neural network applied to classification applications. An impoFrom January 2006 to March 2008, 396 patients who showed evidence of HBsAg-positive and liver biopsy were investigated. Among them, 57 were excluded from this study because of the following reasons: 24 had HCV or HIV co-infection, 4 had acute hepatitis, 7 had hepatocellular carcinoma, and 22 lacked complete data on laboratory tests. The demographic and clinical characteristics were similar between those 57 excluded patients and the rest of 339 patients fulfilling the entry criteria. From April 2008, 116 CHB patients were considered for enrollment. So, a total of 455 patients were included in this study in the end. Of overall subjects, 174 (38.2%) had no fibrosis (F0), 155 (34.1%) had portal fibrosis (F1), 70 (15.4%) had septal fibrosis (F2), 47 (10.3%) had numerous septa (F3), and 9 (2.0%) had cirrhosis (F4). Their mean age was 33.9 ± 11.7 years; 289 (63.5%) were male, and 166 (36.5%) were female. Table There were 226 subjects in the training set; 113 subjects in the validation set; 116 ubjects in the testing set. The variables used to construct the artificial neural network were compared in three sets , 0.884 , and 0.920 , which distorts the hepatic architecture. Liver fibrosis progression could commonly be found in chronic hepatitis B patients after an extensive period of time during which liver biochemical indices are found to be predominately or even persistently abnormal. Due to Age, platelet, AST, ALT, GGT, etc., as routine biochemical markers, had been well known predictors of significant liver fibrosis,29,30. IAs demonstrated by ROC curves, the predictive accuracy of the artificial neural network was reasonably high in the training, validation, and testing sets. The AUROC were 0.883, 0.884 and 0.920, respectively. In view with some noninvasive parameters directly or indirectly related to fibrogenesis, the most prevalent are the Fibrotest and the Actitest for necro-inflammator activity. They ar2-macroglobulin)+ 3.096 lg(age) + 2.254 lg(GGT) + 2.437 lg(HA). The AUROC of this model in the training and validation groups were 0.84 and 0.77, respectively. The logistic regression, a generalized linear model used for binomial regression, is used for prediction of the probability of occurrence of an event by fitting data to a logistic curve. The artificial neural networks, a non-linear statistical data modeling tool, can be used to model complex relationships between inputs and outputs. Therefore, the present study provided evidence that the three-layer neural network model based on routine and serum markers was superior to other indices or models for identification of individual with or without significant fibrosis.Hui AY constructed and validated a multivariate logistic regression model using body mass index, platelet, Alb, and TBIL level to predict advanced fibrosis, and the AUROC were 0.765-0.803. Zeng MDIn our study, there were 126 patients with significant liver fibrosis, which only accounted for 27.7% of 455 CHB patients with liver biopsy. In other words, 329 (72.3%) patients without significant liver fibrosis had undergone liver biopsy, and they could bear the damages from such an invasive procedure. From a more practical point of view, we wanted to reduce the number of liver biopsy procedures and also identify all CHB patients with significant fibrosis. Therefore, our study evaluated the influence of the different cutoff points on the accuracy of ROC. When we chose a high cutoff point, the number of CHB patients at high risk for significant liver fibrosis was few, and also fewer patients needed further liver biopsy or other examinations. But, there was a low sensitivity and many CHB patients with significant liver fibrosis could be missed. The purpose of predicting the state of liver fibrosis is to identify CHB patients with significant liver fibrosis or at high risk for liver fibrogenesis to prevent them from further liver fibrogenesis. Thus, we should choose a lower cutoff point to improve the sensitivity, and to reduce the number of missed CHB patients at high risk for liver fibrogenesis. So, we considered a probability value of 0.33 as a cutoff value. CHB patients with a probability value > 0.33 were considered with significant liver fibrosis or at high risk for liver fibrogenesis. In our study, all CHB patients with significant liver fibrosis would be identified. 47.4% (55/116) of the CHB patients would be free of liver biopsy and also wouldn't be missed.Our study was conducted in a specialized hospital for infectious diseases. All individuals investigated were in-patients, therefore not a completely random sample of all CHB patients. However, owing to the limitation of liver biopsy, we thought that the predictive model in this study might be applied to identify the risk of liver fibrosis as some studies showed .The accuracy of a test could vary with the definition of the target condition. In our The study highlighted the construction and assessment of an artificial neural network for identifying the risk for liver fibrosis. Age, platelet, AST, ALT, GGT, etc., as the input variables of the artificial neural network model, were widely available. The results of our study showed that the three-layer artificial neural networks could effectively identify the risk for liver fibrosis in CHB patients with positive HBsAg. It could improve patient compliance, and reduce the need for liver biopsy required prior to antiviral or other types of therapy.HBV: hepatitis B virus; CHB: chronic hepatitis B; HCC: hepatocellular; ANN: artificial neural network; HBsAg-positive: hepatitis B surface antigen; CHC: chronic hepatitis C; HE -hepatitis E; HCV: hepatitis C virus; HIV: human immunodeficiency virus; ROC: receive operating characteristic; AUROC: area under ROC; PPV: positive predictive values; NPV: negative predictive values; ECM: carcinoma extracellular matrix; WBC: white blood cell; RBC: red blood cell; Hb: hematoglobin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: γ-glutamyltransferase; ALP: alkaline phosphatase; ChE: Cholinesterase; TP: total protein; Alb: albumin; TBIL: total bilirubinThe authors declare that they have no competing interests.DW conducted the study, participated in the data collection and wrote the initial draft and revised the manuscripts. QW collected the preliminary data, and helped to draft the manuscript. FS participated in the study design and interpretation of the data. BL participated in data collection and gave inputs to the drafting of the manuscript. CL helped to coordinate the study and the drafting and revisions of the paper. All co-authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/251/prepub


The purpose of our study was to examine the association of prior outpatient use of statins and angiotensin converting enzyme (ACE) inhibitors on mortality for subjects ≥ 65 years of age hospitalized with acute COPD exacerbations.We conducted a retrospective national cohort study using Veterans Affairs administrative data including subjects ≥65 years of age hospitalized with a COPD exacerbation. Our primary analysis was a multilevel model with the dependent variable of 90-day mortality and hospital as a random effect, controlling for preexisting comorbid conditions, demographics, and other medications prescribed.We identified 11,212 subjects with a mean age of 74.0 years, 98% were male, and 12.4% of subjects died within 90-days of hospital presentation. In this cohort, 20.3% of subjects were using statins, 32.0% were using ACE inhibitors or angiotensin II receptor blockers (ARB). After adjusting for potential confounders, current statin use  and ACE inhibitor/ARB use  were significantly associated with decreased 90-day mortality.Use of statins and ACE inhibitors prior to admission is associated with decreased mortality in subjects hospitalized with a COPD exacerbation. Randomized controlled trials are needed to examine whether the use of these medications are protective for those patients with COPD exacerbations. Althoug overall .In COPD, airway pro-inflammatory cytokine levels have been demonstrated to be associated with increased airway obstruction and exaggerated airway inflammatory response ,5. In adThe study aims were to assess the association of the use of statins and ACE inhibitors on mortality in a population of largely male subjects ≥ 65 years of age hospitalized with acute COPD exacerbations after adjusting for other potential confounders using the extensive administrative databases of the Department of Veterans Affairs (VA).This study was conducted with VA inpatient and outpatient administrative data that was collected as part of a larger study of inappropriate prescribing practices in the elderly . The InsSubjects who were: a) aged 65 and older on October 1 1999, b) had at least one outpatient clinic visit during fiscal year (FY) 1999 (October 1 1998 – September 30 1999), c) were hospitalized during FY 2000 with a primary discharge diagnosis of acute exacerbation of COPD , d) and received at least one of the following respiratory medication(s) within 90-days of presentation  We excluded subjects with a history of asthma. If a subject was admitted more than once during the study period, only the first hospitalization was included. Unfortunately pulmonary function test data was not available as part of these databases to confirm the diagnosis of COPD. However a recent publication by Joo et al., using a similar methodology to define COPD demonstrated that >90% of subjects were Global Initiative on Chronic Obstructive Lung Disease class 3–4 .This study used data from the National Patient Care Database at the Austin Automation Center, pharmacy data from the VA Pharmacy Benefit Management group, and vital status data from the Beneficiary Identification Records Locator Subsystem death file and inpatient portion of the National Patient Care Database. Encrypted patient identifiers were used to link the information from each database for each subject.Demographic information  was obtained from inpatient and outpatient data. Missing race data were supplemented using self-reported race from the 1999 Large Health Survey of Veterans, a nationally representative survey of VA enrollees  . Race caComorbid conditions were obtained from inpatient and outpatient administrative data. Charlson's comorbidity score was used to assign a comorbidity score for preexisting comorbid conditions ,24. CharPharmacy data were obtained from the Pharmacy Benefits Management group databases. Subjects were considered a "current user" of a given medication if their last filled prescription included enough pills to last until the date of hospitalization assuming an 80% compliance rate, which is based on prior research of medication compliance ,26. MediTo further control for potential confounding by medications, a count of unique drugs in each of the following classes per patient was calculated for drugs refilled/filled within 90-days of presentation: cardiac medications , diabetic medications, and respiratory medications. Respiratory medications included short-acting bronchodilators , long-acting inhaled bronchodilators , and theophylline. Two dichotomized variables were created for corticosteroid use: one for inhaled steroids and another for oral steroids. Previous research has demonstrated that using the count of these medication classes is preferable to adjusting for the individual medications .In addition, we created a category of non-statin lipid lowering agents  filled within 90-days of hospital presentation so as to examine confounding in our models.Our primary outcome, 90-day mortality, was assessed using the Beneficiary Identification Records Locator Subsystem and the National Patient Care Database. Previous studies have demonstrated that after 1972 this methodology had a sensitivity of ~96% for veterans' deaths .Bivariate statistics were used to test the association of sociodemographic and clinical characteristics with all-cause 90-day mortality. Categorical variables were analyzed using the Chi-square test and continuous variables were analyzed using Student's t-test. Due to the number of statistical tests and the sample size of this database we set statistical significance at p ≤ 0.001.A propensity score technique was used to balance covariates associated with medication use between groups -31. We cTo analyze time-to-death for subjects by medication use (statin or ACE inhibitor/ARB) we used Cox proportional hazard models to estimate, and graph, the baseline survivor functions after adjusting for the respective propensity score.Our primary analysis employed generalized linear mixed-effect models with the patient's hospital as a random effect and 90-day mortality as the dependent variable. We created separate models with either ACE inhibitors or statin use as the independent variables of interest, and adjusting for the appropriate propensity score. In addition, as secondary analyses we created similar models with the dependent variable of 30-day mortality.We used a similar methodology to examine the association of non-statin lipid lowering drugs with 90-day mortality after adjusting for potential confounders using the appropriate propensity scores. We hypothesized that non-statin lipid lowering medications would not be associated with mortality since they have not been demonstrated to have immunomodulatory effects.In addition, to explore whether these associations with mortality were only for those with pre-existing vascular disease we repeated our analyses stratifying by the presence of specific comorbid conditions .All analyses were performed using STATA 9 .Of the patients who had an inpatient stay in 2000 n = 142,169), 11,212 subjects (7.9%) met our inclusion criteria. The mean age was 74.0 years with a standard deviation (SD) of 5.6 years, 42.0% of subjects were married, and 98.0% were male. In this cohort 12.8% were black, 82.3% were white, 2.7% were Hispanic, and 2.2% were other/unknown. In our cohort 6.5% of the subjects died within 30-days of presentation, and 12.4% died within 90-days of presentation. Table 42,169, 1In our cohort 20.4% (n = 2286) were on statins, 30.4% (n = 3404) were on ACE inhibitors, and 1.6% (n = 180) were on ARBs. Due to the small number of subjects using ARBs, for all analyses ACE inhibitor and ARB use was combined into one variable. In the bivariate analyses, both statin and ACE inhibitor use was significantly associated with decreased 90-day mortality. No other medication classes were significantly associated with mortality in our bivariate analyses.Figures In the multilevel models, after adjusting for the appropriate propensity score and admitting hospital, prior use of a statin (odds ratio (OR) 0.51, 95% confidence interval (CI) 0.40–0.64) and ACE inhibitor use  were significantly associated with decreased 90-day mortality. For the outcome of 30-day mortality we had similar findings with both statin  and ACE inhibitor use  associated with decreased 30-day mortality. In addition, to examine whether there were significant interactions between statins and ACE inhibitors use, we re-ran our model with medication variables and the individual potential confounders, rather than the propensity score. Our results were similar with both prior use of a statin  and prior use of an ACE inhibitor  significantly associated with decreased 90-day mortality, and there was no significant interaction between the ACE inhibitor and statin use.In addition, to examine whether use of both medication classes may be associated with improved survival beyond that observed in subjects using statins or ACE inhibitors alone, we re-ran our model with 3 medication variables  and the individual potential confounders, rather than the propensity scores. We found that all 3 groups were associated with decreased mortality: ACE inhibitors alone , statins alone , and those using both .To further examine whether these associations were only for pre-existing comorbid conditions for which statins and ACE inhibitors have been shown to be protective  or potentially protective (congestive heart failure) we repeated our primary multivariable analyses stratified by each of these conditions. Table In the multilevel model that examined non-statin lipid lowering medication use there was no significant association with mortality .We found that prior outpatient use of statins and ACE inhibitor was associated with decreased 90-day mortality for subjects ≥ 65 years of age hospitalized with acute COPD exacerbations. Further studies, including randomized control trials, are needed to examine the impact of statins and ACE inhibitors, both pre-hospitalization and acute, for subjects who are at risk for acute exacerbation of COPD and COPD-related mortality.Our study supports the recent findings that subjects with COPD, or hospitalized with acute exacerbation of COPD who were on statins and/or ACE inhibitors at admission had significantly improved outcomes -19. UnfoSoyseth and colleagues  examinedFrost and colleagues published a paper in Chest , which dMancini and colleagues  conducteAlthough our study was a large database analysis and subject to the recognized limitations of such studies, we carefully assembled our cohort from complete patient discharge data to avoid ascertainment bias. Our sample was predominantly men due to our use of VA administrative data, and it is possible that women may have differential responsiveness to these medications as compared to men. Unfortunately due to the lack of pulmonary function data in these databases we had to rely upon ICD-9 codes and medication use to define COPD. However, a recent publication by Joo et al. demonstrated that using a similar methodology that > 90% of subjects classified as having COPD were GOLD class 3–4 . In addiA strength of our study is that our cohort has the same access to medical care, and low to no-cost prescriptions due to the structure of the VA health care system . In addiIn conclusion, our study finds that prior outpatient use of statins and ACE inhibitors was associated with decreased mortality for subjects hospitalized with acute COPD exacerbations. These results add an additional potential benefit of statin and ACE inhibitor use to the already compelling data for their use in subjects with vascular disease and diabetes. Randomized trials are needed to confirm the magnitude of the impact of statin and ACE inhibitor use, either pre-hospitalization or acute, on subjects hospitalized with acute exacerbation of COPD and to elucidate the mechanism(s) by which they may work.ACE: Angiotensin converting enzyme; ARB: Angiotensin II receptor blocker; CI: Confidence interval; COPD: Chronic obstructive pulmonary disease; FY: Fiscal year; HR: Hazards ratio; MI: Myocardial infarction; OR: Odds ratio; VA: Department of Veterans Affairs.None of the authors, except for AA and MIR, have any conflicts of interests to disclose regarding this paper. AA has served on the speaker's bureaus of Pfizer, Boehringer Ingelheim, GlaxoSmithKline, Astra-Zeneca; Ortho-McNeil-Janssen, Johnson & Johnson; Bayer-Schering Pharma, Schering- Plough; in the advisory board of GlaxoSmithKline, Boehringer Ingelheim, Bayer-Schering Pharma, Schering- Plough, and Pfizer; and is the principal investigator on research grants that were awarded to the University of Texas Health Science Center at San Antonio by GlaxoSmithKline, Pfizer, and Lilly Pharma. MIR is a consultant for Ortho-McNeil-Janssen, Johnson & Johnson, and Pfizer; and on the speaker's bureau for Ortho-McNeil-Janssen, Johnson & Johnson, GlaxoSmithKline, Pfizer, Covidien and BARD, Inc.EMM originated and designed the study, was primarily responsible for analysis of the data, and preparation of the paper.LAC contributed to the design of the study, contributed to the analysis of the data, and preparation of the paper.MJP contributed to the design of the study, contributed to the analysis of the data, and preparation of the paper.MIR contributed to the design of the study and preparation of the paper.RMM contributed to the design of the study and preparation of the paper.BN contributed to the design of the study, contributed to the analysis of the data, and preparation of the paper.AA originated and designed the study, contributed to the analysis of the data, and preparation of the paper.All authors approved the final manuscript.