Two-year, prospective cohort data from the Japan epidemiological research of occupation-related back pain study in urban settings were used for this analysis.To examine the association between aggravated low back pain and psychosocial factors among Japanese workers with mild low back pain.Although psychosocial factors are strongly indicated as yellow flags of low back pain (LBP) leading to disability, the association between aggravated LBP and psychosocial factors has not been well assessed in Japanese workers.At baseline, 5,310 participants responded to a self-administered questionnaire including questions about individual characteristics, ergonomic work demands, and work-related psychosocial factors (response rate: 86.5%), with 3,811 respondents completing the 1-year follow-up questionnaire. The target outcome was aggravation of mild LBP into persistent LBP during the follow-up period. Incidence was calculated for the participants with mild LBP during the past year at baseline. Logistic regression was used to explore risk factors associated with persistent LBP.Of 1,675 participants who had mild LBP during the preceding year, 43 (2.6%) developed persistent LBP during the follow-up year. Multivariate analyses adjusted for individual factors and an ergonomic factor found statistically significant or almost significant associations of the following psychosocial factors with persistent LBP: interpersonal stress at work [adjusted odds ratio (OR): 1.96 and 95% confidence interval (95%CI): 1.00–3.82], job satisfaction , depression , somatic symptoms , support from supervisors , previous sick-leave due to LBP  and family history of LBP with disability .Psychosocial factors are important risk factors for persistent LBP in urban Japanese workers. It may be necessary to take psychosocial factors into account, along with physical work demands, to reduce LBP related disability. Low back pain (LBP) is a common musculoskeletal occupational health problem in industrialized countries and was found to be the leading specific cause of years lived with disability Many epidemiological studies of LBP have been conducted worldwide for decades. Psychosocial factors such as low job satisfaction, depression, or the tendency to somatize have been strongly indicated as ‘yellow flags’ for LBP leading to disability, as have ergonomic factors such as physical work demands Previously, we reported potential risk factors for new onset of back pain disability in Japanese workers enrolled in a prospective cohort study in urban settings The present study was designed to ascertain whether various psychosocial factors are associated with aggravating mild LBP into persistent LBP in workers with a 1-year history of mild LBP, using data from the previously reported cohort study; the findings of this further data analysis are reported here. This study was part of a series of clinical research projects conducted by the Japan Labor, Health and Welfare Organization related to 13 fields of occupational injuries and illnesses, including musculoskeletal disorders, mental health, and cancer. The research projects were conducted to help resolve occupational health issues and to disseminate the findings.Data were extracted from a prospective cohort of the “The Japan epidemiological research of Occupation-related Back pain (JOB)” study. Participants were recruited from 16 workplaces in various occupational fields, located in or near Tokyo. The major occupational groups at these workplaces were office workers, nurses, sales/marketing personnel, and manufacturing engineers. Each participating organization was asked to distribute a self-administrated questionnaire to their workers, along with a cover letter from the study administration office. Respondents were asked to return their completed questionnaires by post, including their names and mailing addresses, which were used to send follow-up questionnaires directly from the study administration office. A total of 6,140 baseline questionnaires were distributed during September 2005 and February 2006, and 5,310 completed questionnaires were returned (response rate: 86.5%).The baseline questionnaire included questions about the severity of the respondent's LBP and various individual and work-related factors. LBP severity was evaluated by the respondents themselves, who were asked to quantify the severity into one of four grades: grade 0, no LBP; grade 1, LBP not interfering with work; grade 2, LBP interfering with work; and grade 3, LBP interfering with work and leading to sick leave. The grades were determined with reference to Von Korff's grading method The follow-up questionnaire was distributed 1 year after the baseline questionnaire was administered. Of the 5,310 participants who completed the baseline questionnaire, 3,811 successfully completed and returned the follow-up questionnaire, resulting in a follow-up rate of 71.8%. The follow-up questionnaire included questions relating to LBP, such as severity of LBP during the past year, length of sick-leave due to LBP, whether medical care was sought, pain duration, and onset pattern. LBP severity was assessed by the respondents themselves, using the same categories as those of the baseline questionnaire.Ethical approval for the study was provided by the review board of the Japan Labour, Health and Welfare Organization. Informed consent was obtained in writing from all participants.The outcome of interest was occurrence of persistent LBP during the 1-year follow-up period. In this study, persistent LBP was categorized as LBP interfering with work (grade 2 or grade 3), with disability lasting for longer than 3 months. Incidence was calculated for the participants who reported mild LBP (grade 1) during the past year at baseline. Participants were excluded from the analysis if they met any of the following criteria: a job change for reasons other than LBP; LBP due to a traffic accident; or LBP caused by a tumor, including metastasis, infection or fracture.In addition to the compilation of simple, descriptive statistics, univariate and multivariate logistic regression analyses were used to explore risk factors associated with persistent LBP. Associations found by logistic regression analysis were summarized as odds ratios (ORs) with 95% confidence intervals (CIs). For the assessment of potential risk factors, crude ORs initially were estimated. Next, factors with P-values<0.1 were adjusted for individual factors, and also adjusted for individual factors and an ergonomic factor, in order to explore their potential risk factors. Factors with adjusted ORs that were statistically significant were considered to be potential risk factors. The following factors were used as adjusting factors because they are considered to be representative of individual and ergonomic factors: age, sex, obesity, smoking habits, education, and manual handling of objects 2 (SD 3.4 kg/m2). Of these participants, 1,165 (68.2%) were categorized as non-manual laborers; 147 (8.6%) as manual handlers of < 20-kg objects; 338 (19.8%) as manual handlers of ≥ 20-kg objects or as caregivers; and 58 (3.4%) were lacking job description data. In each category, the most common occupations were office work in the non-manual laborer category; manufacturing/engineering in the manual handler of < 20-kg objects category; and nurse in the manual handler of ≥ 20-kg objects or caregiver category.Of the 3,811 participants who responded to the 1-year follow-up questionnaires, 1,675 (excluding 43 who did not answer the question on LBP severity on their follow-up questionnaire) reported mild LBP during the past year at baseline and met the selection criteria. The mean age was 43.1 years (SD 10.1 years) and 1,342 (78.6%) were male. The mean BMI was 23.1 kg/mThe baseline characteristics of the 3,811 participants who provided follow-up data appeared to be not much different from those who did not. The mean (SD) ages were 42.9 (10.1) years and 38.0 (10.2) years, respectively, and the majority were male in both groups . Those who completed the study had a mean (SD) BMI of 23.1 (3.3) while the values for dropouts were 22.9 (4.1). In the follow-up group (vs. the drop-out group), 78.6% (vs. 75.5%) were categorized as manually handling < 20-kg objects or not manually handling any objects in their work, 17.8% (vs. 18.9%) manually handled ≥ 20-kg objects or were working as caregivers, and data were lacking for 3.6% (vs. 5.6%). In both groups, the most common occupational fields in the categories of “manual handling of < 20-kg objects or not manually handling any objects”, and “manual handling of ≥ 20-kg objects or working as a caregiver” were office worker and nurse, respectively.Of the 1,675 eligible participants, 43 (2.6%) reported persistent LBP within the 1-year follow-up period. Of the 43 participants reporting persistent LBP, 76.7% had pain that persisted for longer than 6 months.Crude ORs for persistent LBP, their 95% CIs, and P-values are shown in Potential risk factors for people with LBP that could aggravate the condition and cause too much disability to work were explored in a cohort of urban Japanese workers. The incidence of persistent LBP developing from mild LBP was 2.6%. ORs adjusted for individual factors and an ergonomic factor  showed that low job satisfaction, lack of support from supervisors, interpersonal stress at work, depression, somatic symptoms, and a family history of LBP with disability were significant risk factors, and previous sick leave a nearly significant risk factor, for development of persistent from mild LBP. Our results indicate that these psychosocial factors are important in urban Japanese workers who have made the transition from mild to persistent LBP.In this study, the definition of persistent LBP was disability longer than 3 months, and the index for disability was LBP interfering with work, with or without sick leave. In Western countries, ‘absence from work' is often used as an outcome measurement for disability. The number of participants who were absent due to LBP (grade 3) was relatively small. Our previous international epidemiological study showed that taking sick leave due to musculoskeletal disorders, mostly LBP, appears to be less common among Japanese workers than British workers Among the five factors from the BJSQ , low job satisfaction, little support from supervisors, and interpersonal stress at work tend to relate to each other, and depression and somatic symptoms tend to relate to each other. The first three factors  could be considered stressful conditions that directly and negatively affect the individual, and the latter two factors  as symptoms of both physical and mental stress. Generally, the symptoms of somatization are headaches, neck and shoulder discomfort, dizziness, palpitations or shortness of breath, diarrhea or constipation, and back pain, and these symptoms are triggered by emotional discomfort and psychosocial distress A family history of persistent LBP was also suggested as a psychosocial risk factor in this analysis. Second-hand experience of LBP among people with whom a worker is in very close contact  may make it easier to imagine how mild LBP transforms to persistent LBP. Previous research has revealed that some people can share another person's physical pain experience, in both emotional and sensory components, by just observing the other person's pain Psychosocial intervention has been reported to improve overall well-being, as well as reducing distress and physical complaints, in patients with LBP in Western countries Limitations of the current study should be mentioned. One is the fact that the majority of the subjects were males, and that a broad range of Japanese occupations was not represented. The study cohort was not a representative sample of the entire Japanese workers in urban areas; therefore, the generalizability of the findings may be limited. Secondly, although cognitive and emotional aspects of back pain are known to influence disability aggravation, some important psychosocial factors, such as the attitudes of health care providers, and catastrophizing and fear-avoidance beliefs, were not included in this analysis. This was because appropriate questionnaires were not available in the Japanese language. Future studies should include additional self-reported outcome measures, such as results of the Fear-Avoidance Belief Questionnaire (FABQ) Psychosocial factors are one of the most important risk factors for making the transition to persistent LBP from mild LBP in urban Japanese workers. In the future, preventive strategies for reducing persistent LBP in the workplace should deal not only with physical work demands, which is already well-understood, but potentially should incorporate psychosocial management techniques as well.Table S1Crude odds ratios of the baseline factors for persistent low back pain (LBP) with work disability. OR: odds ratio, CI: confidence interval, BMI: body mass index, LBP: low back pain. a Obesity: BMI of ≥ 25 is defined as obesity in Japan. b Smoking habits: Brinkmann index of ≥ 400 was defined as heavy smoker, calculated from the total number of cigarettes smoked per day multiplied by duration of smoking in years c Working hours: ≥ 60 hours per week was assumed to be uncontrolled overtime. d Bending, twisting, lifting, and pushing: ≥ half of the day was considered frequent. e Hours of desk work: longer than 6 hours per day was considered as static posture. f Work-related stress factors assessed with the brief job stress questionnaire: not feeling stressed, feeling stressed: the 5 original responses were reclassified into “not feeling stressed”, where low, slightly low and moderate were combined, and “feeling stressed”, where slightly high and high were combined. g Monotonous task: feelings of monotony or boredom at work.(DOC)Click here for additional data file.


Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disease. Presentation of the disease such as primarily fever, hepatosplenomegaly, and cytopenia, which are the results of functional degradation in cytotoxic T-lymphocytes and natural killer cells, activation of macrophages and T-lymphocytes, over production of proinflammatory cytokines, and hemophagocytosis. In all, 5 genetic loci have been identified in FHL, and all known affected genes encode critical components of the granule exocytosis pathway, which is essential for the release of cytotoxic granules and proteases that are necessary for targeted cell death. Herein we present an FHL patient with a severe clinical course and a very rare perforin gene mutation. The patient was homozygous for A665G mutation. However, the child died in a short period of time. Prenatal diagnosis was performed in the family and the fetus was found to be heterozygous for the mutation. Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disease. The clinical presentation of FHL includes primarily fever, hepatomegaly, and cytopenia, which are the results of functional degradation in cytotoxic T-lymphocytes and natural killer (NK)-cells, activation of macrophages and T-lymphocytes, excessive production of proinflammatory cytokines, and hemophagocytosis ,2,3. AccFive loci causing FHL have been identified, as well as the underlying genetic defect for 4 of them. FHL1 is a locus first described in 1999 following linkage analysis in 2 Pakistani families, although the gene remains unknown. FHL2 encodes mutations in the perforin (PFR1) gene. Mutations in the UNC13D gene (FHL3) interfere with the role of the encoded protein Munc 13-4 in cytolytic granule exocytosis and FHL4 states the mutations in the STX11 gene and production of syntaxin 11, which also plays a role in cytotoxic granule release. Recently, mutations in STXB2, which encode syntaxin binding protein 2 Munc 18-2) and cause impaired protein expression and impaired NK-cell cytotoxic granule exocytosis, were also described (FHL5) -2 and ca,11,12. PA 3.5-month-old male infant presented with abdominal protuberance, fever, and cough, and was admitted to hospital. One week earlier he developed a fever with cough, was diagnosed as lower respiratory tract infection, and was given antibiotic treatment at another hospital. The parents are first-degree cousins. The initial physical examination performed at our hospital showed the following signs: jaundice, grunting, intercostal retraction, and marked hepatosplenomegaly. No one else in the patient’s family had a similar history. 9 L–1; absolute neutrophil count: 752 mm–3; hemoglobin: 4.8 g dL–1; platelet count: 15 x 109 L–1. Further laboratory test results include elevated transaminases , elevated lactate dehydrogenase (1984 u L–1), and elevated serum ferritin . Serum triglycerides was 207 mg mL–1, serum total bilirubin was 6.14 mg dL–1, serum direct bilirubin was 4.78 mg dL–1, serum uric acid was 30.9 mg dL–1, and fibrinogen was 50 mg dL–1. Arterial blood gas analysis was consistent with metabolic acidosis. Due to respiratory depression the patient was mechanically ventilated with multi-component transfusion support. He was given antibiotic treatment  during his hospitalization. Complete blood count findings were as follows: white blood cell count: 4.7x10This patient underwent bone marrow aspiration, which showed profuse hemophagocytosis. The clinical and laboratory findings, and consanguineous parents strongly suggested FLH which lead to the commencement of the HLH 2004 protocol . The patPostmortem splenic examination showed profuse hemophagocytosis. While alive the patient was in extremely poor clinical condition and was intubated; therefore, investigation of the central nervous system could not be performed, but postmortem cerebrospinal fluid examination showed mononuclear cell infiltration. Written informed consent for abdominal autopsy was obtained from the patient’s family. The bone marrow core biopsy showed normal cellular components and abundant hemophagocytic macrophage infiltration based on CD68 immunohistochemical staining. Liver smear showed extramedullary hematopoiesis and microvesicular steatosis, as well as massive hemophagocytic macrophage infiltration via CD68, particularly in portal regions and in some sinusoidal regions. An increase in sinusoidal macrophages was noted in lymph nodes dissected from the peripancreatic region and mesocolon. Adrenal medulla showed massive CD68-positive macrophage infiltration. Focal histiocytic cellular groups were observed in kidney specimens. Post-mortem genetic testing showed homozygosity for A665G PRF 1 mutation. The patient was the parents’ first child—the result of the mother’s first pregnancy. As the parents were consanguineous, the family was provided genetic counseling about subsequent pregnancies. One year later the mother was again pregnant and the fetus was determined to be a carrier for the same mutation; however, spontaneous abortion terminated the pregnancy.When the immune system is triggered by an infectious agent histiocytes (macrophages and dendritic cells), NKcells, and cytotoxic T-lymphocytes (CTLs) are activated, mutually stimulating each other via receptor interaction, as well as secretion of a variety of inflammatory cytokines and chemokines. In immune-competent individuals this concerted action leads to of the death of infected cells, removal of antigen, and termination of the immune response. In cases of inherited or acquired NK-cell and CTL defects this process is impaired, infected cells do not undergo apoptosis, and persistently high cytokine levels lead to the clinical picture of HLH .The cytotoxic function of NK-cells and CTLs is mediated by release of cytolytic granules, which contain perforin, granzymes, and other serine-specific proteases, into the immunological synapse that forms between the effector and the target cell. Cytolytic granules must travel to the contact site, dock and fuse with the plasma membrane, and release their contents . All knoLee et al. previously described a case where one allel has the same mutation as our case while the mutation of the other allel could not be found . RecentlIn addition to the diagnosis of FHL, an understanding of its genetic or acquired nature is also of great value, especially in families with consanguineous marriage and a history of early onset of FHL in their offspring. The family of the presented case had the same characteristics. Prenatal diagnosis was of special importance to the presented case’s family because he was their first child and they planned to have another baby. Currently, the most important problem is variation in FHL mutations according to country— almost 50% of which have not yet been determined. This was reported to be as 50% in Turkey and 70% in Germany .Due to the fact that FHL is quite common in Turkey, identification of the molecular pathologies in FHL patients is critical. Okur reported that 9 of 37 Turkish FHL patients (24%) were FHL type-2 based on linkage analysis. Genomic sequencing of the entire coding region of the perforin gene led to the identification of 5 different pathologic changes in these patients. In all, 3 patients (33%) carried the same nonsense W374X mutation in exon 3, and the 6 remaining patients (67%) had 4 different missense variations . Among these missense changes, G149S S was detected in 2 patients, V50M in 1 patient, and A91V in 2 patients . In contIn the Turkish population perforin gene mutation is the most commonly detected mutation in the 4 genes known to be responsible for HLH. Among perforin gene mutations, W374X is known as the Turkish mutation and is common in Turkey; this mutation, which has a severe clinical outcome, is thought to have first emerged in Southeast Anatolia . Clinica–1 [–1). HLH should be ruled out in any previously healthy child that abruptly develops fever, hepatosplenomegaly, cytopenia, and jaundice. The aim of this case presentation was to increase pediatricians’ awareness of HLH. HLH is a life-threatening disease that may be difficult to distinguish from severe sepsis. A simple clinical approach may be helpful in diagnosing HLH. As such, autopsy studies suggest that HLH may be under recognized in intensive care unit patients ,23,24. S–1 . The preChemo-immunotherapy based on the HLH-2004 protocol includes corticosteroids, epipodophyllotoxins, and cyclosporine, and succeeds in controlling the disease in the majority of patients; however, cure is rarely achieved. Most patients suffer an early death unless they undergo hematopoietic stem cell transplantation (HSCT), which is the only curative approach available to date . In concConflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


AbstractChrysomelinae have been chromosomally analyzed showing a wide range of diploid numbers from 2n = 12 to 2n = 50, and four types of male sex-chromosome systems. with the parachute-like ones Xyp and XYp clearly prevailing (79.0%), but with the XO well represented too (19.75%). The modal haploid number for chrysomelines is n = 12 (34.2%) although it is not probably the presumed most plesiomorph for the whole subfamily, because in tribe Timarchini the modal number is n = 10 (53.6%) and in subtribe Chrysomelina n = 17 (65.7%). Some well sampled genera, such as Timarcha, Chrysolina and Cyrtonus, are variable in diploid numbers, whereas others, like Chrysomela, Paropsisterna, Oreina and Leptinotarsa, are conservative and these differences are discussed. The main shifts in the chromosomal evolution of Chrysomelinae seems to be centric fissions and pericentric inversions but other changes as centric fusions are also clearly demonstrated. The biarmed chromosome shape is the prevalent condition, as found in most Coleoptera, although a fair number of species hold a few uniarmed chromosomes at least. A significant negative correlation between the haploid numbers and the asymmetry in size of karyotypes (r = -0.74) has been found from a large sample of 63 checked species of ten different genera. Therefore, the increases in haploid number are generally associated with a higher karyotype symmetry.Nearly 260 taxa and chromosomal races of subfamily  Chrysomelinae is a large cosmopolitan taxon of nearly 2000 species , have been reported in so few species that they should not be discussed in the frame of this contribution.Other cytogenetic findings of an even much finer resolution, such as those on genome size, C and/or Ag-banding, and fluorescent Chrysomelinae, the number and names of its tribes and subtribes differ strikingly among them. Thus, very recently, Timarchini and Chrysomelini, the former with one and the latter with four subtribes, and Gonioctenini with two. We shall follow here a mixed criterion taken from these latter authors with a few changes according with my own opinions, thereby, we assume the tribes Timarchini and Chrysomelini, the former with the subtribe Timarchina only, and the latter with five subtribes: Entomoscelina , to 2n = 50 and 24 + Xyp in the European Chrysolina rufoaenea . These shifts in number are almost always due to structural chromosome rearrangements, because only a few polyploidy parthenotes have been recognized to date, all of them restricted to the genus Calligrapha, , of a non-chiasmate nature, is clearly prevailing in the subfamily (79.0%) as shown in p), held together by a non-nucleolar argyrophilic substance  = 35 modal chromosome number and 17 + XO meioformula  karyotype and meioformula in half of the twelve sampled genera.On the contrary, in subtribe ectively , shared Chrysolina with SD = 8.67 in 72 sampled taxa and chromosomal races, Timarcha with SD = 4.33 in 42 taxa, and Cyrtonus with SD = 6.33 in 15 taxa, whereas other genera have zero or a low diversity such as Paropsisterna with SD = 0 in 10 taxa, Chrysomela with SD = 0 in 9 taxa, Oreina with SD = 1.15 in 12 taxa, and Leptinotarsa with SD = 2.77 in 16 taxa. The differences between “variable” and “conservative” genera in their chromosome numbers, were tenPageBreaktatively explained according with the ability for dispersal of flying vs. flightless species genera, and the number of host-plant families they are able to feed, being both characters in a presumed relationship with the size of local populations and thereby with the chances of fixation for new chromosomal shifts ; on the contrary, Paropsisterna, Chrysomela and Leptinotarsa have flying species, the first two feeding on a unique plant family each and the third on three, but Oreina has some species completely unable to fly and others flying very seldom, in spite of being chromosomally “conservative” as the previous three genera (SD ≤ 4.0), and feeding on two plant families only  of their male diploid chromosome numbers, for example l shifts . Under ties only . Hence, The chromosomes may show a huge variable morphology in size and shape, some species have karyotypes made of very few chromosomes of a large size while others have karyotypes of many small chromosomes and there are not evidences of any advantages of ones over others, although minute chromosomes are more easily lost at meiosis if a chiasma fails to be formed, and very large acro- or telocentric chromosomes can be cut across before they have been properly separated at anaphase . Anyway,Calligrapha , all with 2n (♂) = 35, have seven, four and three, respectively  = 47 and lacking any of them  of each chromosome relative length with respect to the averaged % length taken from the total complement length (TCL) .For the sake of simplicity we should only consider here the asymmetry vs. symmetry in chromosome size but not in shape. The karyotypes of th (TCL) . Here agTimarcha , 1976, aligrapha , Chrysolrysolina , Oreina   by p or XYp imply a translocation between an autosome and the X-chromosome, with the loss or fusion of the PageBreaky-chromosome. The karyotype with the lowest number reported to date in chrysomelines, that of Doryphora quadrisignata, with 5 + neo XY meioformula  using a ribosomal DNA probe (Chrysolina (Stichoptera) kuesteri with 2n = 22 chromosomes .These cytogenetic results are reported in oformula , has proNA probe , and c) omosomes , can be


Areca nut (commonly known as betel nut) chewing has been shown to be associated with metabolic syndrome and cardiovascular disease (CVD). The mechanism by which betel nut ingestion could lead to development of CVD is not precisely known; however, dyslipidemia, hyperhomocysteinemia, hypertriglyceridemia and inflammation could be some of the potential risk factors. This study was undertaken to investigate the effects of two dosages of betel nut on homocysteinemia, inflammation and some of the components of metabolic syndrome, such as hypertriglyceridemia, low HDL-cholesterol, obesity and fasting hyperglycemia in a rat model.Thirty-six adult female Sprague Dawley rats, aged 10–12 weeks were divided into three equal groups. Group-1 served as the control group (n = 12) and received water, whereas groups 2 and 3 were given water suspension of betel nut orally in two dosages, 30 mg and 60 mg, respectively for a period of 5 weeks. At the end of the fifth week, the animals were weighed and sacrificed, blood was collected and liver, kidney, spleen and stomach were removed for histological examination.12 and N-acetyl-β-D-glucosaminidase (NAG) – a marker of inflammation.Plasma/serum was analyzed for glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, homocysteine, folate, vitamin BWhen the mean concentration values of 3 groups were compared using one way ANOVA followed by Tukey’s HSD-test, there was a significant increase in the concentration of total cholesterol (p = 0.04) in the group receiving 30 mg/day betel nut compared to the control group. However, administration of a higher dose of betel nut (60 mg/day) had no significant effect on the serum concentrations of glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, and NAG. Histological examination of spleen revealed a dose-dependent extramedullary hematopoiesis. No other remarkable change in the tissues  was observed.12 and homocysteine were not found to be significantly different in all the groups. Betel nut ingestion had no effect on the mean body weights of rats.Mean serum/plasma levels of folate, vitamin BLow dosage of betel nut is found to be associated with hypercholesterolemia. However, betel nut ingestion is not associated with hyperhomocysteinemia, hypertriglyceridemia, hyperglycemia, inflammation and increase in body weight in a rat model. Using a 3-mL syringe and an oral gavage needle, 1 mL of betel nut suspension was delivered in the lower end of the buccal cavity of the rat.One of the objectives of the study was to investigate the effect of betel nut on weight gain in adolescent rats. Since the maximum weight gain in adolescent female Sprague Dawley rats is up to a period of 4–5 weeks, it was 0C until analysis. The study had been approved by the Ethics Committee for Research on Animals, Aga Khan University.At the end of 5 weeks of treatment, the animals were weighed and anesthetized after being subjected to fasting for 12 hours. Blood (3–4 mL) was collected from the heart of each rat by cardiac puncture and transferred in equal volumes to an EDTA tube and another tube without any anti-coagulant. The rats were sacrificed by cervical dislocation while they were under the influence of anesthesia. Liver, Kits for fasting serum glucose, total cholesterol, high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol and triglycerides were obtained from Roche Diagnostics, USA. Serum sample (200 μL) was analyzed on Cobas C III analyzer . The minimum limits of measurement for serum glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides were 2.0 mg/dL, 9.7 mg/dL, 3.0 mg/dL, 3.9 mg/dL and 8.9 mg/dL, respectively.Serum folate and vitamin B12 were analyzed by radioassays,18. BothLiver, kidney, spleen and stomach immersed in 10% buffered formalin for 24 hours were processed in a routine fashion and embedded in paraffin. Serial sections (3-5μm) were cut and stained with Hematoxylin and Eosin (H & E). Slides were then examined under light microscope by a histopathologist for any morphological changes. Extramedullary erythropoiesis in splenic sections of rats from all three groups was done in a semi quantitative method. Foci of extramedullary erythropoiesis were measured in millimeter using microstage and ocular scale on Olympus BX51 microscope. The sums of all foci showing extramedullary erythropoiesis were then compared in three groups.12 and plasma homocysteine. Levels of lipids, glucose, ALT, NAG, folate, vitamin B12 and homocysteine in the three groups of rats were analyzed using one way ANOVA because the assumptions of normality and equality of variance were met. Significant association by groups was then followed by Tukey’s HSD test for multiple pair-wise comparisons. The differences in mean body weights of rats over a period of 5 weeks by experimental groups were compared using repeated measures ANOVA. A p-value < 0.05 was considered significant. Post-hoc power of the study was determined using PASS 2008 software .Descriptive results pertaining to the experimental groups are reported as mean ± SD for body weight, lipid profile, serum glucose, serum ALT, serum NAG , serum folate, serum vitamin B12 and plasma homocysteine for the three groups were observed [TableTableMean body weights of Sprague Dawley rats in each of the three groups  at the start of the experiment were nearly the same  was more pronounced in the group receiving 60 mg of betel nut per day FigureA and1B Nearly 600 million people all around the world indulge in betel nut chewing. This haOur results in a rat model are novel in the sense that we observed significantly high levels of serum cholesterol at a dose of 30 mg betel nut powder per day compared to a higher dose of the betel nut. This suggests that very high consumption of betel nut might not be associated with dyslipidemia due to the inhibitory effect of high doses of betel nut on cholesterol absorption. This miA recently published study has shown a significant association between betel nut chewing and systemic inflammation suggesting that it might be playing a role in the development of systemic diseases at a later stage. Plasma Betel nut chewing has also been found to be associated with increased risk of developing diabetes mellitus. However12 and plasma homocysteine remained similar across the 3 groups  to 99.2% (triglycerides). However, power turned out to be sufficient (85%) for the significant variable  in this study. We are also mindful of the fact that betel nut is normally chewed by humans. However, in this study, in order to ensure complete ingestion of the nut in two different dosages, we administered a water suspension of betel nut in the buccal cavities of rats. This indeed was a limitation; yet, our focus in this study was on determining the systemic effects of betel nut via gut absorption. Furthermore, the fact that levels of serum cholesterol in rats receiving a high dose of betel nut (60 mg/day) were lower than expected could be attributed to the inhibition of two enzymes involved in cholesterol absorption, such as pancreatic cholesterol esterase and intestinal acyl-CoA:cholesterol acyltransferase, as reported earlier in a rat model. One posThe results of the investigation revealed that hypercholesterolemia was caused by a relatively small dose (30 mg/day) of betel nut in a rat model. This metabolic change was not accompanied by any significant alteration in inflammation, serum levels of HDL-cholesterol, LDL-cholesterol, triglycerides, ALT, fasting glucose, homocysteinemia and body weight. However, the findings suggest that even small doses of betel nut could have detrimental effects. Further studies on a relatively narrowed down range of dosages of betel nut would be required to find out the precise dose at which the deleterious effects of this nut begin to manifest. It is imperative that betel nut chewing must be investigated in populations which are at a greater risk of developing CVD.The authors declare that they have no competing interests.MPI, NM conceived and designed the study, interpreted the data and drafted the manuscript. NM and GH performed the experiments and analyzed the data. SP performed the histological studies, interpreted the data and helped in drafting the manuscript. IA performed the post-hoc power analysis of the study and helped in the interpretation of the data. All the authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/12/94/prepub


In this review, the relationship between angiotensin-converting enzymes and fertile ability is summarized, and a new procedure for the treatment of infertility is discussed.According to the World Health Organization, infertility, associated with metabolic syndrome, has become a global issue with a 10%–20% incidence worldwide. An accumulating body of evidence has shown that the renin–angiotensin system is involved in the fertility problems observed in some populations. Moreover, alterations in the expression of angiotensin-converting enzyme-1, angiotensin-converting enzyme-2, and angiotensin-converting enzyme-3 might be one of the most important mechanisms underlying both female and male infertility. However, as a pseudogene in humans, further studies are needed to explore whether the  MetS is a highly prevalent condition currently considered to be a constellation of metabolic abnormalities, including blood pressure elevation, abdominal obesity, impaired glucose metabolism and hyperglycemia associated with insulin resistance (IR) –3. Recen sterile . Despite fertile ,14. The  fertile –17. Some fertile ,19. This2.It is well acknowledged that the traditional RAS contains a system of finely tuned agonists and antagonists that balance blood pressure . In recetAce1-knockout male mice are sterile, whereas sAce1-deficient male mice are fertile [ACE1 is well recognized not only for its pivotal regulatory activities in cardiovascular homeostasis , but als fertile . Moreove fertile –43.ACE2 gene, which was recently cloned, has an expression pattern that is restricted to endothelial cells in the heart and kidney, epithelial cells in the distal tubule of the kidney, and adult Leydig cells in the testis [N-terminal catalytic domain of ACE1  mutant ACE1 and PI-PLC, which implies that tACE1 plays a crucial role in fertilization through this activity [et al. [et al. [et al. [et al. [Many studies have reported the implication of tACE1 in capacitation. Mammalian spermatozoa must undergo a maturation process known as capacitation and a morphological change called an acrosome reaction before successful fertilization. During capacitation, sperm membranes are modified by the epididymal proteins located on their surface, and this is a crucial step to ensure successful sperm–egg interactions ,68. Durireaction ,71. Beforeaction . In addiactivity ,74. Howeactivity ,76. Leis [et al.  utilized [et al.  and Kond [et al.  might ow [et al. .4.2.et al. further demonstrated the strong influence of ACE2 in the male reproductive system by showing that humans with severe spermatogenesis impairment have lower levels of ACE2, Ang-(1–7), and Mas compared with fertile subjects [Ace1−/− male mice, which display significantly reduced fertility, both male and female Ace2-null mice are fertile [In the male reproductive tract, ACE2 is selectively expressed by adult Leydig cells in the testis. In addition, the ACE2-producing Ang-(1–7) and its receptor Mas have also been detected in the testis, and these are mainly located in the interstitial compartment and cytoplasm of the Leydig cells . Reis etsubjects . Because fertile , which s fertile . Therefo4.3.Izumo−/− males are infertile despite their normal mating behavior, ejaculation, and sperm motility [Ace3−/− mice are healthy and fertile and exhibit only slight mislocalization of IZUMO1-positive sperm compare with control mice [Similarly to tACE1, tACE3 is an IZUMO1-associated protein . IZUMO1,motility . Howeverrol mice . These r5.Ace1 are related to the risk factors for PCOS. Jia and his team proposed that Ace1 insertion/deletion (I/D) polymorphisms are associated with an increased risk for PCOS [Ace1 DD genotype is related to increased IR in women with PCOS [Besides the impaired glucose metabolism, dyslipidaemia and hypertension of MetS, sterility of women and men is also associated with MetS. In females, an aberrant ovarian RAS can result in the development of several gynecological diseases such as PCOS, the patients of which are more vulnerable to MetS . PCOS isfor PCOS . The D afor PCOS . A studyith PCOS ,84. Moreith PCOS . Accordiith PCOS .et al. found that type and grade of obesity correlated with the genotypes of the ACE1 gene I/D polymorphism in subjects with coronary heart disease of MetS [In males, hypogonadism, erectile dysfunction and psychological disturbances are also often comorbid with MetS ,2. Sever of MetS . There i of MetS ,91. Furt of MetS ,93.6.Drugs inhibiting the RAS have shown benefits against multiple components of the MetS, indirectly ameliorate the reproductive health. Accumulating data indicate that ACE1 is a potential contributor to IR, which plays a crucial role in the pathogenesis of PCOS. Thus, treatment with the ACEI temocapril has been employed for females with IR, and this treatment improves their insulin sensitivity, which results in a favorable maternal and fetal outcome ,94. IR aDespite the controversial study results, there are data supporting the use of ACEIs as effective drugs for the management of infertile men with idiopathic oligospermia, because of its beneficial effect on the sperm number, motility and morphology ,98. ACEITo date, all of the drugs that target the RAS, including ACEIs and antagonists of angiotensin receptors, aim to decrease the RAS function. ACE2 may serve as a novel therapeutic component of the RAS that, if activated, could treat hypertension, IR and obesity of the MetS and other relative comorbid disease, such as infertility. A further understanding of the relationship between the ACEs and the sterility with or without MetS at specific cells may be an effective single therapy against infertility. In addition, dietary manipulations and sustainable strategies for weight loss benefit body composition and improve insulin regulation, which may ultimately treat specific features of MetS and improve the fertility.7.Ace1/Ace2, Ace1/Ace3, and Ace2/Ace3 double mutants has not been addressed. If these double-mutant mouse models are generated, the association between ACEs and the cause of infertility could be elucidated more clearly. Moreover, ACEIs have become first-line drugs for the management of PCOS-related IR in infertile females and idiopathic oligozoosperm in males, although some controversial results have been observed. Thus, the aforementioned findings require confirmation in larger multicenter studies.As shown in this review, infertility is associated with MetS, risk factors of which might impair the reproduction. Moreover, there is no doubt that ACE1 and ACE2 have gained recognition as significant regulators of the physiology and pathology of the reproductive system. The sperm–egg fusion process is associated with the ADAMs-associated protein ACE1 and IZUMO1-interacting protein ACE3. However, the fertility of


From December 1999 to April 2001, the greater Edmonton region had 61 cases of invasive meningococcal infection, two fatal. The outbreak was due to Neisseria meningitidis serogroup C, electrophoretic type 15, serotype 2a. Analysis of the strains showed that 50 of 56 culture-confirmed cases were due to a single clone and close relatives of this clone. This strain had not been previously identified in the province of Alberta dating back to January 1997 Neisseria meningitidis causes outbreaks of disease resulting in severe illness and death. These outbreaks occur in persons in their teens and early twenties; however, in some outbreaks, the very young (<2 years of age) are also severely affected . PersonsWe report an outbreak of a serogroup C clone of N. meningitidis in the Edmonton region of Alberta, Canada; the serogroup had a unique restriction fragment length polymorphism (RFLP) pattern as determined by pulsed-field gel electrophoresis (PFGE).The Edmonton region has a mixed metropolitan and rural population totaling 827,507 . From JaIn relation to clinical outcome, 43 (70.5%) of the 61 patients fully recovered; 2 (3.3%) died  ; 4 6.6%. The conA vaccination campaign that targeted persons ages 2 to 19 was undertaken in the region from February 14 to 28, 2000, using polysaccharide quadravalent meningococcal vaccine; 168,000 children were immunized. Because of a continuing higher-than-expected number of cases, the vaccine was again offered in October 2000 . This tiElectrophoretic typing, serotyping, and serosubtyping performed by the National Microbiology Laboratory, Population and Public Health Branch of Health Canada, Winnipeg, Canada, showed that all serogroup C strains belonged to electrophoretic type (ET)15, serotype 2a . ET15 enSerogrouping, ET, and serosubtyping provided accurate characterization of the circulating strains in the Edmonton region; however, they failed to determine if the outbreak was clonal or if the increased cases were due to unrelated N. meningitidis strains. To determine this, RFLP analysis via PFGE was used with minor modifications . BacteriRFLP analysis showed 15 distinguishable patterns . Five ofIn conclusion, the Edmonton region in the province of Alberta, Canada, had an outbreak of N. meningitidis caused by a clone unique to this region. This clone was associated with increased deaths and can readily spread beyond defined geographic boundaries. Other provincial and state laboratories need to be able to recognize this clone should it appear in their area.


Biphasic cuirass ventilation (BCV) assists ventilation by applying intermittent or continuous negative pressure to the thorax. BCV has been reported to improve lung function in various respiratory failures. However, to determine the therapeutic effect of BCV is difficult, because it is too difficult to include animal experiments. Therefore it is important to compile amounts of clinical cases for discussion. We have tried to find a way of developing BCV in critical care.2, pCO2, tidal volume, P/F ratio, respiratory index, A-aDO2, shunt ratio, dead space ventilation rate, and chest X-ray. We also performed a questionnaire study about BCV which focused on physicians and nurses working in the ICU.This is a retrospective, nonrandomized study. Before and after BCV, we compared pO2O, positive pressure +7 cmH2O, I:E ratio 1:1). It improved the tidal volume, P/F ratio, shunt ratio in all cases during BCV (P < 0.05). Skin damage caused by the cuirass was observed in one case. According to the questionnaire survey, they had some problems about the durability of the urethane of the cuirass, too close to a thin body or deformation. Some of them had no confidence because of unfamiliarity with the machine.From April 2008 to May 2010, BCV was performed by applying RTX  for 18 patients admitted to the ICU, National Hospital Organization Nagasaki Medical Center. All of them had acute respiratory failure, and 15 of them were intubated and mechanically ventilated. Thirteen were men, and the mean age was 68 years (1 to 82 years). One case could not continue the treatment due to discomfort of wearing the cuirass. We used the control mode (negative pressure -21 cmHWe conclude that BCV is also useful for respiratory care in the ICU. Further confirmation is needed regarding problems such as the criteria to start and terminate BCV.


Editorial Note. Letters to the Editor are peer reviewed to ensure that the arguments are reasonable and clearly expressed. However, letters may express a particular opinion rather than a balanced interpretation. Authors of papers commented on are invited to reply, but neither the journal nor peer reviewers should be assumed to support the arguments made.We have read with interest the recent work by the University of Wuppertal group  using maet al., 2013) for Holland lactose of Dtot = 5.2 (0.4)% and Dresp = 0.9 (0.1)% are fully consistent with those of the UNC group , with Dtot = 5.1 (0.9)% and Dresp = 1.3 (0.5)% for the same material.) In the technique comparison, Part 1, of the Wuppertal study, only three Venturi measurements were made for each powder, and no ranges or statistics were reported. In the pharmaceutical, Part 2, of their study, five Venturi measurements were made for each powder, and standard deviations were reported, permitting some analysis of possible error. Finally, we observed an empirical correlation between respirable and total dustiness, as measured with the Venturi device, to hold for a wide range of powders  RSDs ; the RSDs (n = 9) for the five materials tested in the original UNC study (n = 5) for the Wuppertal pharmaceutical measurements (Wupp-Pharm) are systematically higher and become increasingly poor at lower dustiness values.With the Venturi device, we measured total and respirable dustiness for 27 materials  for the paired dustiness techniques.Finally, we commend the attempt to correlate these four different dustiness methods. As we previously discussed , we cons4, 2), there may be an intrinsic difference in dustiness due to the Reynolds number of dispersion. This is the subject of current investigation at NIOSH. We note that weak correlations were obtained between the EN 15051 rotating drum and EN 15051 continuous drop measurements, both gravity driven techniques at low Reynolds numbers, for nine industrial minerals (


Recent toxicological and epidemiological studies have shown associations between particulate matter (PM) and adverse health effects, but which PM components are most influential is less well known.2.5)] concentrations and daily mortality in two U.S. cities—Seattle, Washington, and Detroit, Michigan.In this study, we used time-series analyses to determine the associations between daily fine PM [PM ≤ 2.5 μm in aerodynamic diameter (PM2.5 filters for the years of 2002–2004 and analyzed trace elements using X-ray fluorescence and black carbon using light reflectance as a surrogate measure of elemental carbon. We used Poisson regression and distributed lag models to estimate excess deaths for all causes and for cardiovascular and respiratory diseases adjusting for time-varying covariates. We computed the excess risks for interquartile range increases of each pollutant at lags of 0 through 3 days for both warm and cold seasons.We obtained daily PM2.5 components and gaseous pollutants associated with mortality in Detroit were most associated with warm season secondary aerosols and traffic markers. In Seattle, the component species most closely associated with mortality included those for cold season traffic and other combustion sources, such as residual oil and wood burning.The cardiovascular and respiratory mortality series exhibited different source and seasonal patterns in each city. The PM2.5 on daily mortality vary with source, season, and locale, consistent with the hypothesis that PM composition has an appreciable influence on the health effects attributable to PM.The effects of PM Evidence from epidemiological studies has accumulated to support associations between PM and adverse health effects e.g., . A number) e.g., .2.5 mass alone might not be able to explain the health outcomes , organic carbon, and sulfate], and the toxicity of each of these chemical components and their mixtures may vary. These different components originate from various sources, such as traffic-related emissions, biomass combustion, residual oil burning, and resuspended dust. Metals that are components of ambient PM2.5, and especially the transition metals, have been cited as likely toxic components. The focus has often been on iron (Fe), vanadium (V), nickel (Ni), chromium, copper, and zinc (Zn) based on their ability to generate reactive oxygen species in biological tissues. The National Research Council (NRC) has highlighted the importance of investigating characteristics and chemical components of PM that contribute to their toxicity  initiated by the U.S. Environmental Protection Agency (EPA) in 2000 has provided new opportunities for studies on PM components’ health effects . Howeverl cities . Direct 2.5 mass  or to th2.5 mass . In the Detroit’s air quality is heavily influenced by a variety of industries, including motor vehicle factories, refinery operations, power plants, and metalworking plants. Accordingly, a number of air pollution–health effect studies have been conducted in Detroit . Seattle2.5 samples for the 3-year period 2002 through 2004 were collected on 47-mm Teflon filters using samplers operating at a flow rate of 16.7 L/min. These filters were from two monitoring sites: Detroit, Michigan , and Seattle, Washington  and were weighed pre- and postsampling to determine daily fine particle mass concentration. We obtained these filters from the Washington Department of Ecology (Seattle data) and Department of Environmental Quality (Detroit data) and subsequently sent them to the Research Triangle Institute  for analysis of trace element concentrations using energy-dispersive X-ray fluorescence (XRF). Black carbon, as a surrogate index of EC in the filter samples, was determined via reflectometry at New York University Department of Environmental Medicine .Two sets of 24-hr PMa priori, based on previous studies , Fe, potassium (K), sodium (Na), Ni, sulfur (S), silicon (Si), V, Zn, and EC. Source types were selected  studies , as well3), and nitrogen dioxide (NO2) from the We obtained daily concentrations of carbon monoxide (CO), ozone  nationwide multiple cause-of-death files with date of death through the U.S. EPA’s special arrangement with NCHS to support research . Using c ICD-10; , we aggrWe retrieved daily average temperature and dew point data for the Detroit Metro Airport and the Seattle-Tacoma International Airport from the 2.5 total mass showed little evidence of a strong association with health effects beyond 3 days  per calendar year  to control for seasonality and long-term trends , a smootd 3 days . In presWe also employed a constrained  distributed lag model using dlnm (version 1.2.4), an R package developed by 2.5 and most components  had somewhat higher concentrations in the warm season. Na, CO, and NO2 levels were higher in the cold season. In Seattle, PM2.5, Fe, K, Zn, EC, CO, and NO2 showed higher levels in the cold season, whereas the levels of Al, Na, Ni, S, Si, and V were higher in the warm season.The risk estimates at individual lag days often showed positive associations at multiple lag days, especially at lag 0 through lag 2 days. Thus, the results from the distributed lag models exhibited patterns of associations generally similar to those from the individual lag analysis. Therefore, for clarity, we present the results from the distributed lag models here but also describe some aspects of individual lag results [see Supplemental Material (doi:10.1289/ehp.1002613)] that were lost in the distributed lag results.2.5, S, and O3 for all-cause and cardiovascular mortality in the warm season  were significantly positively associated with all-cause or cardiovascular mortality in the cold season .In Seattle, in contrast to Detroit, we observed no significant association in the warm season for any mortality category , and muld season . We obseThe estimated risks were not sensitive to alternative degrees of freedom per year . We observed no indication of systematic difference in risk estimates . Likewise, we examined the sensitivity of risk estimates to adjustment of degrees of freedom for temperature and delayed temperature, using 4, 5, and 6 df, as opposed to 3 df used in the original model, and found that the risk estimates were robust to the change in degrees of freedom to fit temperature effects (data not shown). The fitted temperature terms showed generally positive slopes for the same-day temperature and negative slopes for the average of 1- to 3-day-lag temperature for both cities for all-cause and cardiovascular deaths (data not shown).2 at lag 3 days in the cold season in Seattle, and at lag 1 day in the warm season in Detroit), the air pollutants’ associations with “other cause” deaths were, in general, much lower than those for cardiovascular and respiratory deaths (data not shown).In the analysis of “other cause” of deaths, although we observed a few significant associations  is at the current National Ambient Air Quality Standard (NAAQS) (2.5 concentration in Seattle (9.7 g/m3) would be in compliance with even the lowest concentration limit being considered as a revised annual average for PM2.5 (11–13 g/m3)  in Seattle, although further analyses are needed to shed light on the association between a low level of PM2.5 and health effects. The PM2.5 components and gaseous pollutants most closely associated with cardiovascular mortality in Seattle were K, S, Si, Zn, EC, NO2, and CO. Thus, reduction of cardiovascular mortality risk in Seattle may need to focus on residual oil combustion, the major source of S in this city; biomass burning, the major source of K; metal processing industries and incinerators, important sources of Zn; and motor vehicles, the major source of EC, NO2, and CO. The PM components and gaseous pollutants associated with daily mortality in Detroit (in the warm season only) were S and O3. Thus, reduction of cardiovascular and respiratory mortality risk in Detroit may need to focus most on traffic emissions and power plants, which are sources of sulfate and O3 precursors.It is also noteworthy that the annual average PM (NAAQS) , whereas13 g/m3) . Because2.5 components and gaseous pollutants appeared only in the warm season, when there is more infiltration of outdoor air into occupied spaces. Detroit and Seattle showed similar temperature effects: Warm weather had positive associations with same-day health effects and negative associations with delayed health effects. However, the mortality showed opposite seasonal patterns in these cities, although the pollutant concentration variations did not indicate obvious seasonal patterns in either of the two cities. In Seattle, where the annual range of temperature was smaller, we observed significant associations between mortality and air pollutants in the cold season, when some pollutants had higher concentrations, for example K, EC, and CO, and others had lower concentrations, such as Al, S, and Si. The mortality effects exhibited differing seasonal patterns in these two cities. In Detroit, significant associations between mortality and PMAir-conditioned buildings may keep people indoors from being exposed to high levels of outdoor air pollutants. According to the American Housing Survey 2005, the2.5 components is likely to lead to random measurement error and the potential for downwardly biased effect estimates that may vary with PM source. Therefore, one of our concerns is a monitor’s representation of regional, subregional, and local air pollution exposures for the population in these two metropolitan areas. Some investigators have also questioned whether the observed associations are plausible given these findings. However, It is important to note the limitations of our study. First, the use of a single location in each city to represent the concentrations of PMAnother limitation on the interpretation of our results is that our definitions of cardiovascular and respiratory mortalities reduce the specificity of the estimated associations, compared with more narrowly defined categories. However, the countervailing benefit of this approach is the increased power to detect associations between PM components and health outcomes. As in many other studies, we had to strike a balance between statistical power and specificity of the outcome. A third limitation is that we conducted this research for only two cities with daily speciation data. More states should follow these two states’ lead in retaining daily sample filters to allow distributed lag modeling and to improve the power of future analyses.2.5 speciation data for Detroit and Seattle, we were able to examine the distribution of mortality effects over time, characterize the short-term health effects of PM2.5 more thoroughly, and potentially provide insights for better targeted regulation to reduce source emissions. Second, this study indicates sources of PM air pollution that could be targeted as part of a comprehensive air quality control strategy. Ambient PM2.5 is produced by numerous emission sources, and individual PM2.5 components identified as having associations with health outcomes may be acting as markers for other components or a set of components with similar sources. For example, EC results from combustion of fossil fuels, but also from the combustion of biomass and from industry (2.5 components and gaseous pollutants associated with cardiovascular mortality in Detroit appear to be associated with secondary aerosols and traffic markers in the warm season, whereas in Seattle, the PM components and gaseous pollutants associated with cardiovascular mortality include traffic and other combustion sources in the cold season, such as residual oil burning, incinerators, and wood smoke.Our results add to those previously reported in two important ways. First, in our study, with 3 years of daily PMindustry . Our res2.5 and its elemental components, as well as gaseous pollutants. We found some inconsistent results: although we observed slightly more daily deaths in the cold season than in the warm season in both cities, the daily mortality associations in these cities showed opposite seasonal patterns, whereas the pollutants did not indicate obvious seasonal concentration patterns in either city. It is generally accepted that weather conditions affect mortality rates, and it is difficult to rule out the possibility that there are other causes of mortality. Our results were quite different depending on the location, so we could not draw a definitive conclusion as to whether this is due to differences in the composition of PM2.5 and other air pollutants, differences in weather, or some other variables.In summary, we analyzed data from Detroit and Seattle to assess the associations between daily mortality and PM2.5 components, can and should be developed. The range of chemical components and sources linked to various health responses supports the hypothesis that the toxicity of various components may vary. Although some sources, such as vehicular traffic and power plants, have been indicated as important contributors in this and other published analyses, understanding the health effect contributions of various pollutants and their sources still needs further investigation in more cities with daily speciation data.Most important, our study found a major contrast in seasonal pattern of association between air pollutants and daily mortality in two major U.S. cities. Along with the previous works of others, our results provide substantial evidence that a more refined ambient air quality control strategy, based on the effects of PM


Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response. Discovered in 1989, hepatitis C virus infects 3% of the world population . The theFlaviviridae family. Members of the Flaviviridae family share a common genomic organization. Their short positive-strand RNA genome encodes a single polyprotein, which serves as a precursor for the structural and non-structural proteins. In the case of HCV, the 3000-aminoacid-long polyprotein is cleaved into 10 mature products: the structural proteins core, E1 and E2, and the non-structural (NS) proteins p7, NS2-3-4A-4B-5A-5B  has led to our understanding of the glycoprotein synthesis, folding, topological and functional organization. The mode and characteristics of their incorporation onto the infectious virion remain however incompletely understood. Converging lines of evidence indicate that HCV glycoproteins are structurally different from the flavivirus homologs. E1 and E2 are highly glycosylated  and do nAs for many RNA viruses, HCV proteins are synthesized sequentially from a common polyprotein precursor . The strInterestingly, cleavages at the E2/p7 and p7/NS2 junctions are delayed so that both E2-p7 and E2-p7-NS2 precursors can be detected in infected cells . These uBoth E1 and E2 glycoproteins are type I transmembrane proteins with a large N-terminal ectodomain facing the ER lumen and a C-terminal single-membrane spanning transmembrane domain (TMD) . Curioustrans and in the absence of any other HCV protein, the two proteins heterodimerize in the ER [trans heterodimerize and are incorporated in the virion in the context of the HCVpp [trans-complementated HCV particles (HCVTCP) [Although the oligomeric status of the global HCV envelope protein complex might fluctuate during the HCV replication cycle ,23, in pn the ER ,32). Alshe HCVpp  and tran(HCVTCP) . This in(HCVTCP) ,35. Inte(HCVTCP) ,23,36,37The main heterodimerization interface is composed by E1 and E2 TMDs ,42,43,44Noteworthy, in heterologous expression systems, a fraction of intracellular E1E2 proteins form non‑functional disulfide-linked aggregates. These can be discriminated from the E1E2 heterodimers by their association with ER chaperones, their sensitivity to protease digestion, their non-reactivity with specific conformation-sensitive antibodies or by SDS-PAGE in non-reducing conditions ,35,49. Ten bloc by an oligosaccharyltransferase from its dolichol anchor onto the nascent polypeptide. A complex sequence of glycan trimming, protein folding and glycan modification follows and can continue in the Golgi apparatus. Hence, N-glycosylation offers a wide range of variations on the basis of a core oligosaccharide [While the presence of O-glycans on HCV envelope glycoprotein remains disputed, their N‑glycosylation has been extensively studied ,51,52,53ccharide  .Flaviviridae, a virus family whose every single crystallized fusion protein until 2013 belonged to the class II [Bunyaviridae, a virus family whose glycoprotein was recently ascribed to the class II fusion fold [Noteworthy, all these studies were based on the postulate that HCV had a class II fusion protein, similarly to alpha- and flaviviruses . This hyclass II . More spion fold . Lastly,Flaviviridae family, and closer to HCV than the flaviviruses, have a different fusion strategy questioned the previous postulate. Finally, even more recently, Kong et al. solved the crystal structure of E2 glycoprotein core domain in complex with a neutralizing antibody [et al. [However, recent arguments have now challenged this hypothesis. First, HCV virion morphology does not comply with the criteria predicting a class II fusion mechanism. Indeed, HCV particles are pleomorphic with no clear outer glycoprotein scaffold ,15,16 whantibody . Notewor [et al. , with on [et al. ,80,81. AImmediately after the polyprotein translation, all HCV proteins are associated, directly or indirectly with the ER membrane via diverse membrane anchors . E1 and The dynamics of virus assembly is challenging to track. New developments of live-cell-compatible detection methods for HCV core protein , RNANew tagged versions of E1 or E2 proteins, compatible with live-cell microscopy, need to be developed to get a dynamic insight into HCV envelopment and release. Ideally, tagging E1 or E2 proteins could be combined with the existing core, HCV RNA or apoE tracking systems to follow the gathering of the pieces of the puzzle for HCV assembly and release. The development of super resolution fluorescence microscopy, with approaches already validated on other small animal viruses such as HIV , might aTCP and HCVcc. The HCVpp system relies on the incorporation of HCV envelope glycoproteins on a retroviral backbone, containing a retroviral mini-genome that encodes a reporter protein [Systems to study HCV assembly and glycoprotein functions have been reviewed in details elsewhere ,94. Brie protein ,89,90. I protein . This sy protein . However protein ,36. NeveSince 2005, HCVcc are the best compromise between robustness and authenticity . Indeed,TCP model is a variation of the HCVcc system, particularly convenient to look at HCV assembly and entry [trans of the remaining viral proteins, which are necessary to enable the production of a viral progeny. The proteins provided in trans are expressed independently of the HCV subgenome replication, and therefore, one can genetically manipulate their genes, while avoiding potential side effects on HCV RNA replication and polyprotein processing. HCVTCP are single-round infectious particles. They are useful tools to investigate E1E2 incorporation in an authentic viral particle, but also to generate authentic virions [Finally, the HCVnd entry ,107,108. virions  harborin virions . A striking and unique feature of HCV biology is the virion association with lipoproteins. The unusually low density of serum-derived particles was the first hint for this interaction . HCV deThe infectious density profile of HCVcc is also broad and shifted towards light fractions as compared to the peak of RNA and core ,117,118.Purified infectious HCVcc have been observed by electron microscopy, eventually combined with immunogold labeling ,15,16 Fa. These et al. and supported by electron microscopy observation of patient-derived serum samples [These intriguing observations closely resemble the recently proposed structural model of HCV lipoviroparticle . Such tr samples . In thiset al. [Alternatively, lipoproteins might be peripherally associated, transiently or permanently, with the canonical viral particles via interaction between apolipoproteins and HCV envelope lipids or proteins . The twoduction ,127,128.The first task for HCV assembly is to gather the virion components. These include the three structural proteins, C, E1 and E2, and the viral RNA. Lipoprotein components are also incorporated into the virion , but this will be dealt with in a different paragraph. As mentioned before, E1 and E2 are ER-resident proteins. The replication complexes, and therefore the nascent RNA, are also associated with rearranged ER membranes, often forming double-membrane vesicles protruding from the ER surface . HoweverHow and where exactly the three virion bricks are brought together for assembly is still a controversial issue. The junction between ER and LDs probably forms the site for virion assembly ,132. TheThis is getting even more complicated when trying to integrate the viral RNA into this schema. Four viral proteins have been shown to interact with HCV RNA: core, NS5A, the NS3/4A helicase domain and the polymerase NS5B . NS5A anin vitro [Although the glycoproteins are anchored in the ER membrane, the connection with the other virion components might be direct or indirect. An interaction between core and E1 has been reported in vitro ,143,144 in vitro ,145,146,in vitro ,139,146,in vitro ,145,146,in vitro . Moreovein vitro ,147. Altin vitro ,145,146,in vitro ,138 or tin vitro ,139. The link between structural and non-structural proteins made by p7 and NS2 has been extensively explored. It is supported by the position of their precursor in the polyprotein and by the necessary compatibility between the protein sequences. Thus, the capacity of an HCV chimera to produce high-titer virus depends on the compatibility between NS2 first TM segment and the structural proteins on one side, and between NS2 C-terminus and the non-structural proteins on the other side . FurtherOnce the virion components are gathered at the assembly site, virion morphogenesis takes place with genome encapsidation and capsid envelopment at the ER membrane. A panel of assays is available to pinpoint a block in this assembly sequence in the context of assembly-deficient virus mutants or host cell perturbations . Note thTCP systems, it is possible to distinguish the effect of E1E2 variation on virion assembly, from its effect on entry. First of all, the expression as part of the common polyprotein precursor is neither crucial for entry nor for assembly. Indeed, glycoproteins expressed in trans, in a single or two separate cistrons, are incorporated into infectious HCVpp [TCP [Thanks to the combination of HCVcc, HCVpp and HCVus HCVpp ,33 and HVpp [TCP ,109,158.Secondly and as expected, folding and glycosylation of E1 and E2, two tightly linked processes ,54 deterThirdly, E1E2 heterodimerization is a prerequisite for E1E2 to mediate entry , but itsMoreover, compatibility within E1E2 sequences and between E1E2 and other viral proteins determines the assembly success. By switching the genotype or subtype of one of the two glycoproteins in the HCVpp or HCVcc systems, several interfaces between E1 and E2 were described as important for virion entry or assembly ,28,30. IEnveloped viruses hijack a portion of cellular membrane for envelopment of the viral capsid. In the case of HCV, the virions bud in the ER lumen and are released through the secretory pathway. For budding to happen, curvature and scission of the host membrane are needed. Enveloped viruses have evolved three main strategies to induce the membrane curvature and initiate budding  Figure . In the It is still unclear which model applies to HCV budding, although several hints favor the preponderant role of the glycoproteins in the process. Although in heterologous expression systems, core protein can oligomerize and drive envelopment without the need for any other viral protein ,161, in Moreover, the incorporation of E1E2 as large covalent complexes at the virion surface  is anothOn a different note, the importance of the capsid in the budding process is undermined by the extreme cases found among HCV relatives. For example, CSFV , a pestivirus, can still assemble fully infectious virions after the deletion of its capsid-coding sequence . FurtherThe capacity of viral envelope glycoproteins to produce subviral particles can be a further indication for their ability to trigger budding. Importantly, such subviral particles, decorated with envelope proteins but lacking nucleocapsids, have been reported for flaviviruses ,165. In Finally, other motors could drive HCV budding . First oCompletion of virion budding requires scission of the virion from the host cell membrane. The ESCRT machinery is used by many viruses to fulfil this function  and mighin vitro and/or from humanized animals has been shown to display a lower density and higher specific infectivity compared to Huh-7.5-derived HCV [Several lines of evidence from different laboratories highlight that the lipoprotein machinery is crucial for HCV assembly ,171,172.ived HCV ,100. Comin vitro [Besides, how and when exactly lipoprotein and HCV syntheses converge to release a lipoviroparticle is unclear. A possible scenario is illustrated in in vitro , and prein vitro . ApoAI win vitro . Finallyin vitro ,177,178 in vitro ,178. GivThe complexity of the HCV outer coat with its incorporation of lipoprotein component is partly responsible for the extremely complicated entry sequence . LipoprFinally, glycoprotein determinants modulate the virion-lipoprotein association. This is particularly evident with E2 HVR1. Indeed, the deletion of HVR1 results in a decrease in the number of very-low-density particles  and in tIn order to establish and maintain lifelong chronic infections in immunocompetent hosts, HCV glycoproteins have likely been shaped to optimize entry efficiency and at the same time neutralization escape . Thus, iThe variability of the E1E2 sequence reflects the strong immune pressure exerted on these proteins and the remarkable flexibility to escape these antibodies while preserving essential functions for cell entry. The maturation of the glycoproteins, the way they are incorporated in the virion and the virion association with lipoproteins constitute additional factors that permit viral escape from humoral immunity. First of all, the presence of a dense glycan shield on both glycoproteins renders the whole glycoprotein complex globally less immunogenic and hides the receptor-binding sites ,80. E2 cThe influence of all these factors differ between HCVpp and HCVcc  ,36. ThisAlthough the intracellular unit for HCV glycoprotein is the E1E2 heterodimer, surprisingly, a majority of large (>400 kDa) covalent-bound heterodimers are incorporated on the virion . Importade novo between previously buried free cysteine residues that would become exposed on the glycoprotein surface upon a change in protein conformation. However, the motor driving this rearrangement of the disulfide bonds is unknown. The first hypothesis could involve the proximity or recruitment of a disulfide isomerase. The second scenario could be triggered by a local redox or electric charge perturbation. The discrepancy between intra- and extracellular E1E2 oligomerization status in the HCVcc system suggests a late rearrangement of the glycoproteins, possibly triggering the budding process see . The conThe nature of the covalent envelope complexes remains enigmatic. However, our most recent data suggest a hierarchical organization of E1E2, first in heterodimers, themselves bound into E1-driven trimers, which are finally clustered with disulfides . WhetherFlaviviridae family have evolved different solutions to solve this problem. Flaviviruses incoporate an immature form of the prM companion protein that needs to be cleaved for the fusion protein E to be entry competent. This cleavage is mediated by a furin protease in the secretory pathway [Viral envelope proteins should be efficiently packaged into the virion, in a way that they are competent for mediating viral fusion with naive cells, yet do not induce premature fusion with the producer cell upon virion secretion. Viruses from the  pathway . Pestivi pathway . Filoviridae [In the case of HCV, the glycoprotein maturation step seems to occur upon entry rather than assembly, since, contrary to flaviviruses, the secreted virions are still pH-resistant, and therefore not yet competent to mediate fusion . Only upoviridae , remainsIsomerisation of the disulfide bonds as observed for pestiviruses is an appealing possibility for HCV, which incorporates disulfide-linked glycoprotein complexes . It is tFinally, while HCV envelope proteins are pH-resistant once secreted, a study reported that this might not yet be the case during virion egress . This isThe way HCV glycoproteins interact with their host receptors and mediate virus entry has been reviewed by others . While EFlaviviridae have focused on the E2 homologs, but the role of E1 in HCV biology might deserve more attention. After all, it is still unclear which one of E1 or E2 protein mediates fusion or whether both glycoproteins cooperate for this process. Ultimately it is to be hoped that increased understanding of the function(s) of the E1 and E2 proteins combined with structural information will guide the way to develop immunogenic antigens that may be useful components of future vaccine formulations against hepatitis C. The recent acceleration into HCV assembly research has highlighted a complicated but concerted process involving every single HCV protein plus a still underestimated number of host factors. Integrating, in time and space, the numerous protein-protein interactions and assembly cofactors described is a future challenge to take up for a more comprehensive view on the HCV assembly complexes and their coordination. The difficulty to image assembly events has led to the optimization of indirect assays to evaluate the different steps of assembly, including virus envelopment and the incorporation of E1 and E2 glycoproteins. Proteomics analysis of assembly complexes and the development of new tools to track assembly cofactors in live cell might yield a broader picture. Larger‑scale purification of the virion-embedded glycoprotein complexes will help understanding the organization of the virion outer shell, how large complexes can be built from heterodimers and why they might need priming for entry. Similarly to assembly, HCV entry is extremely complex and the role of the multiple host factors must now be ordinated and correlated with virion progression in the host cell but also with changes in HCV envelope shell and glycoprotein complexes. As long as the structure of the E1E2 glycoprotein complex remains unknown, grey areas will shade our understanding of the HCV entry and assembly. Structural and functional studies on other