This study has evaluated urinary tract injuries and dysfunction after Radical Hysterectomy (RH) performed in patients with cervical cancer and has compared the cystometric parameters and urinary complications occurring in these patients with those occurring in patients who had undergone Simple Hysterectomy (SH).A prospective case-control study was conducted to evaluate urinary tract injuries (intra-operative and post-operative) and dysfunction in 50 patients undergoing RH for cervical cancer and to compare them with the same parameters in 50 patients who underwent SH for benign disease.Mean age in the RH group was 46.3 years and in the SH group was 50.1 (p = 0.63). There were no bladder and urethral injuries in either group of patients. There was one intra-operative ureteral injury in the RH patients but none in those who underwent SH. (p < 0.05). In the two weeks after surgery, 15% of RH patients and 11% of SH patients had experienced a urinary tract infection urinary tract infection (p = 0.61). Two week after surgery 62% of RH patients had no urinary symptoms, compared to 84% in the SH group who did (p < 0.02). Urinary residual volume, first urinary sensation and maximal bladder capacity were higher in the RH group, but this was not statistically significant. The only case of a urinary fistula appeared in a patient who received 5000 cGy radiation therapy pre-operatively, but this spontaneously healed after 3 weeks of catheterization.Intra-operative and post-operative urinary tract complications are comparable in patients undergoing RH and SH and an expert gynaecological oncologist might be able to further decrease complications. However, radiation therapy before surgery may increase the risk of complications. Although the incidence of lower urinary tract complications after RH has been reported with variable rates, up to one half of patients undergoing RH experience at least one lower urinary tract symptom that develops after surgery and at a variable period of time ,2. SeverIn this study, we prospectively evaluated intra-operative urinary tract injuries in addition to post-operative urinary tract dysfunction and infection at 2, 6, and 14 weeks following surgery. We also compared these findings with those at the same times in patients who underwent SH for benign disease.Between October 2000 and December 2002, 50 women who underwent RH and bilateral lymph node dissection (BLND) were considered eligible for inclusion in the study. All patients had squamous cell carcinoma of cervix (SCC Cx.) and were staged as being Stages I and II. The operations were performed by the same gynaecological surgeons, using the same standard technique (class III Piver & Rutledge). Pre-operative management was standardised for all patients. Preoperatively a detailed medical history, physical examination, routine laboratory tests, pelvic CT-scan , urine analysis, and urine culture were carried out.The exclusion criteria were; a history of voiding dysfunction, previous pelvic surgery, brain or spinal cord diseases, diabetes mellitus, and contraindications to urodynamic studies. The latter included a history of vesicoureteral reflux, hydronephrosis, frequent or recent urinary tract infection or urethral stricture. Patients received one pre-operative and three post-operative doses of a second generation cephalosporin (Cephazolin).The duration of surgery, amount of intra-operative haemorrhage and the occurrence of any organ injuries were recorded. A Foley's catheter was inserted at the time of surgery and was left in place for two weeks after surgery. The patient's urinary catheter was removed when their post-voiding residual volume was less than 75 ml.Water cystometry, urinalysis and urine culture were performed at 2, 6, 14 weeks after operation. The test for water cystometry was performed with the subject lying in a supine position. A 12F double-lumen catheter was introduced transurethrally into the bladder to withdraw residual urine. The pressure-volume relationship of the bladder was determined by filling the bladder with isotonic saline at a rate of 30–50 ml/min. The cystometry fill phase ceased when the patient experienced an urge to void urine, the first indication being leakage through the urethra, or a bladder volume of 600 ml (which ever occurred first). The volume at the termination of the fill-phase was designated as the maximum bladder capacity (MBC). We also assessed the bladder volume of each patient at their first desire to void (V desire, ml). Post-void residual urine volume (RU) was determined by transurethral catheterization after voiding had ceased. The presence or absence of any urinary symptoms was determined by both questionnaire and direct interview with the patient.Fifty patients with benign disease who had underwent SH, were evaluated at the same time periods in the same way for comparison with the RH group of patients. In the SH group of patients, the Foley's catheter was inserted for 24 hours after operation. Data were analysed by SPSS statistical software using the chi-square and Student's 't' test for data analysis.A and IIB stages, respectively. Patients who had stage IB2 or higher stages of cervical cancer received 4500–5000 cGy of irradiation pre-operatively. None of these patients received adjuvant radiation during the interval between surgery and performance of urodynamic studies.During this study, 50 patients with early stage cervical cancer and who underwent RH for cervical cancer and 50 patients who had undergone SH for benign disease were evaluated. Two patients in the RH group and 3 from the SH group were lost during the study. The mean ages and their BMIs (Body Mass Index) in two groups of patients were not statistically different. However, parity in the RH group was higher (p < 0.05) Table . In the In the SH group, the most common pathological conditions requiring hysterectomy were as follows; dysfunctional uterine bleeding (47.83%), uterine myoma (21.7%), ovarian cyst (10.8%), chronic pelvic pain (4.3%), adenomyosis (4.3%), endometrial cancer (4.35%), CIN (4.35%) and molar pregnancy (2.17%).1 cervical cancer) in the RH group had an intra-operative ureteral injury , and the ureteral anastomosis was carried out at that time. The urinary catheter and ureteral stent were removed four weeks after operation.The average blood loss and mean operative time for both groups are shown in table 2 cervical cancer) had received chemo-irradiation (5000 cGy) pre-operatively. She had a spontaneous urine leakage from the vagina approximately 2 months after surgery. A retrograde cystography revealed a minute vesico-vaginal fistula. After 3 weeks of continuous bladder drainage, the fistula resolved spontaneously and she had no urinary leakage at her follow-up visits.Another patient (with stage IbPost-operative positive urine culture and urinary symptoms (dysuria, frequency, nocturia and dribbling) are showed in table Modern surgical techniques have resulted in a decrease in the incidence of lower urinary tract complications occurring as a result of radical hysterectomy. In particular, in recent times, various surgical strategies have been developed to avoid damaging the inferior segment of the cardinal ligament as well as the terminal bundle in the uterosacral and pubocervicovesical ligaments. This has made it possible for patients' lower urinary functions to return more rapidly to their pre-operative states ,5. Howevet al, reported a 6.6% rate of intra-operative urinary tract injuries during radical surgery for cervical carcinoma [In a study by Zaino and colleagues intra-operative complications were reported to occur as being 4.5% urinary tract and 8.7% other organs  [arcinoma . In our et al, [Zaino et al,  reportedet al,  and thiset al, . In our et al,  but lesset al, . Also, Cet al, .et al reported that 67% of patients had impairment or absence of bladder sensation after a RH [et al, 84% of patients had an increased first desire to void and maximal capacity in the post-operative period [In our study, urinary tract dysfunctions that followed radical surgery were that 4% had an abnormal post-voiding residual volume at the first post-operative visit. The first voiding sensation at the third visit was 49% and stress urinary incontinence was 17%. However, maximal capacity remained abnormal in 65% of cases by 14 weeks after surgery. Ralph ter a RH . In the e period .Urinary symptoms in our study occurred in 20% 2 weeks after operation and which were higher in patients with pre-operative radiotherapy . Urinary symptoms remained high at the third post-operative visit, although they declined in patients who had undergone surgery alone.et al, (mean was 45 years) [In our study, the patients mean age; BMI, parity, operative time, and blood loss were higher in those undergoing RH. The mean age of our patients was higher than patients in the study by Vervest 5 years) . Also in5 years)  the patiIn recent years, several studies support the role of a gynaecological oncologist who is specifically trained in such aspects of care and who can obtain optimal cytoreductive surgery in patients with ovarian carcinoma ,13. TherThe author(s) declare that they have no competing interests.NB carried out the surgery and participated in drafting the manuscript.FG carried out follow-ups.HA participated in the design of the study and helped to draft the manuscript.HK helped in follow-ups and performed the statistical analyses.PH participated in the design of the study and helped to draft the manuscript.All authors read and approved the final manuscript.


HIV/AIDS is the most dramatic epidemic of the century that has claimed over two decade more than 3 million deaths. Sub Saharan Africa is heavily affected and accounts for nearly 70% of all cases. Despite awareness campaigns, prevention measures and more recently promotion of anti viral regimens, the prevalence of cases and deaths is still rising and the prevalence of systematic condom use remains low, especially in rural areas. This study identifies barriers to condom use based on the Health Belief Model (HBM) in Benin, West Africa.The study was a cross-sectional survey conducted from June to July 2002. Two hundred fifty one (251) individuals were interviewed using a structured questionnaire adapted from a standardized WHO/GAP questionnaire. A logistic regression was used to identify factors associated with condom use.In spite of satisfactory knowledge on HIV/AIDS transmission, participants are still at high risk of contracting the infection. Sixty three (63) percents of the interviewees reported being able to recognize infected people, and condom use during the last occasional intercourse was declared by only 36.8% of males and 47.5% of females. Based on the HBM, failure to use condom was related to its perceived lack of efficacy [OR = 9.76 (3.71–30.0)] and perceived quality [OR = 3.61 (1.31–9.91)].This study identifies perceived efficacy (incomplete protective effect) and perceived utilization-related problem (any reported problem using condoms) as the main barriers to condom use. Hence, preventions strategies based on increasing perceived risk, perceived severity or adequate knowledge about HIV/AIDS may not be sufficient to induce condom use. These data will be useful in designing and improving HIV/AIDS prevention outreach programs in Sub Saharan Africa. One of the current challenging tasks faced by health professionals and scientists worldwide is the prevention and control of HIV/AIDS. This disease claims yearly a huge toll of deaths, productivity and economic losses, especially in sub-Saharan Africa where the population is already weakened by poverty, malaria and tuberculosis ,2. CurtaBenin is a West-African country with a population of 6.2 million people. The male/female sex ratio is 0.96, 48.5% of the population is under age 15, the rate of literacy is nearly 30.0 % and farming as the main occupation [The sample size was calculated based on results from the Benin 2001 Demographic and Health Survey . A stratData were stored in a file using Epi info 2000  and thenThe age distribution of the study population was similar to that of the Benin population . Table 1Table The overall condom use in this population was low 34%). Table 4%. TableHowever, it is interesting to note that whilst females declared ever using condom less often than males, they declared having used the condom during the last occasional intercourse more often. In particular, even though 73% of females declared never using the condom, 47% declared having used it during the last occasional intercourse. None of these differences were significant but they clearly indicated contradictory tendencies and answers to the questionnaire. It may be that among females who ever used condom, they use it more frequently than males. Finally, the median number of sexual partners during the last 12 months varied by gender , by education (2 for some versus 1 for none) and by marital status (2 for single versus 1 for other) but not by age groups.high perceived risk of contracting HIV infection among interviewees: 94% considered themselves as vulnerable to HIV/AIDS. This proportion was higher in females compared to males. Similarly there was a high perceived severity of HIV/AIDS: 99% of females compared to 87% of males perceived HIV/AIDS as a severe and deadly disease. Conversely, there was a relatively low perceived efficacy of condom as a protective measure: only 37% of the interviewees perceived condom as an effective mean in protecting from getting HIV infection. We identified several socio-cultural barriers to behavioural change namely reported problems using condom (88% of the interviewees), the alleged capability to physically recognize an HIV infected person and the denial all together of the disease (only 19% participants believe HIV/AIDS exists). Also, cultural practices such as polygamy (20% of the study population), poverty, the belief that there is a cure for the disease (74%) and religion (9 % of non favorable reaction towards condom are among declared Christians) were all not favorable to HIV infection control.Overall, there was a Table This study was the first ever to use the Health Belief Model (HBM) to assess cultural behaviour in rural Benin towards condom use and HIV/AIDS. The HBM was reported to be one of the most widely used behavioural frameworks for more than five decades but has been criticized for its inability to efficiently predict people's behaviour . There iOur results showed there is a high awareness on AIDS in general and that women knew more about the modes of transmission of HIV/AIDS and its impacts than men. Conversely, women were more likely to feel that they could identify HIV-infected individuals from their symptoms. In addition, females were less likely to declare using condom in general even though a higher proportion declared having used condom during the last occasional sexual intercourse. This finding is disturbing and could be explained by the difference in perception of the question "do you use condoms?" It is difficult to judge what the true answer is but it is likely that rare events are better reported, and thus women may be more prone to recall the use of condom than men during occasional intercourse given that they declared on average fewer sexual partners. It is also possible that among women who do use condom, they will use it more regularly than men.Our measure of perceived vulnerability might not be sensitive enough to capture differences in perceived risks. In fact, all women and most men felt they were at risk of acquiring the infection, yet only a small proportion were using condoms. Another explanation may be that perceived risk is not a driving force in behavioural change in this subset of the population. This is an illustration of the complexity of modeling human behaviour and can thus make a case for further cultural-specific HIV-behavioural research. When only considering the percentage of condom use by gender, females appear to be at a higher risk of acquiring HIV even though they appeared to know more about transmission routes and prevention methods. This might be due to the well established difficulty facing women in negotiating the terms of sexual intercourse. In fact, gender inequality is associated with poverty, condom with distrust and sexual economic exchange is not perceived as prostitution . All theDespite a relatively acceptable knowledge of modes of transmission and prevention methods, only a few of participants declared using condoms, which is an indication that a relatively good knowledge about HIV/AIDS, even though necessary, may not be a key factor in behavioural change in fighting HIV epidemic in the study population. These findings also indicate that programmes which aim only at increasing awareness and knowledge may not succeed.Using the HBM to analyze the determinants of behavioural change in our study population, we can conclude that there is a high-perceived vulnerability and perceived severity, and yet this does not encourage condom use. An important proportion of participants do not believe in the efficacy of condoms and there are barriers to the use of condoms.Our results are comparable to that found in a similar study in the USA  and in aOne limitation of our study was that for ethical reasons, subjects less than 15 years old were excluded even though some may have already been sexually active. Also, there was a potential selection bias by not having equal number of interviewers by gender, which resulted in an over-sampling of males. Our results would be biased if the reason for poor recruitment of women was linked to their behaviours, which is not likely to be the case. There were three males interviewers for one female , and interviewer/participants must be from same gender. For the purposes of the analysis we assumed that reported knowledge and behavioural risk factors are independent. Finally there is no evidence for the validity or reliability for the original WHO questionnaire, however its use allows for comparability of results across settings.Condom use, in our study population, depends on its perceived quality and perceived efficacy. There is an indication that behavioral communication change strategies based on increasing perceived risk or vulnerability of the population or based on fear factor by increasing perceived severity of HIV/AIDS are less likely to be deterrent towards condom use and require more researches. HIV outreach programs must target more barriers of condoms use. Condom outreach programmes should be defined at community level and must be defined in association with the community, using problem-solving techniques and selecting the most relevant targets, based on their importance and changeability . Data frThe author(s) declare that they have no competing interests.SHH conceived of the study, designed the protocol, carried out and supervised the field work and data collection, performed and interpreted the statistical analysis and wrote the manuscript. HC participated in analysis and interpretation of the data, and in the writing of the manuscript. NJH contributed in earlier analysis of the data and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Since it has been reported that inhibition of proteasome activities may prevent cancer, the effects of proteasome inhibitors on arachidonic acid release from cells and on prostaglandin I2 production in rat liver cells is inhibited by actinomycin D.The proteasome inhibitors, epoxomicin, lactacystin and carbobenzoxy-leucyl-leucyl-leucinal, stimulate the release of arachidonic acid from rat glial, human colon carcinoma, human breast carcinoma and the rat liver cells. They also stimulate basal and induced prostacycin production in the rat liver cells. The stimulated arachidonic acid release and basal prostaglandin IStimulation of arachidonic acid release and arachidonic acid metabolism may be associated with some of the biologic effects observed after proteasome inhibition, e.g. prevention of tumor growth, induction of apoptosis, stimulation of bone formation. The proteasome degrades many cellular proteins, several with regulatory functions. It is not surprising that proteasome inhibitors affect many biologic processes  includin++-dependent proteases, calpain, papain, chymotrypsin, trypsin and cathepsin are not affected by epoxomicin even at a 50 μM concentration [Epoxomicin, an α'-β'-epoxyketone, appears to be the most selective proteasome inhibitor. Based on its anti-tumor activity, this product was originally isolated from an actinomycetes strain C-996-17 . It inhi++-activated proteases [The β-lactone, lactacystin, is relatively selective but can inhibit cathepsin A . Peptideroteases . The peproteases ,9.2) production in rat liver cells [2 production in neuronal cells [I have shown that inhibition of proteolysis by phenylmethylsulphonyl fluoride, the peptide aldehydes carbobenzoxy-leucyl-leucyl-norvalinal and carbobenzoxy-leucyl-leucyl-leucinal (ZLLL) and lactacystin stimulate induced prostaglandin  [2, the main product of COX-mediated synthesis, can be quantitatively estimated. The rat liver cells express COX-2 both constitutively and after induction [2 synthesis during incubation of cells with epoxomicin is shown in Fig. Of the cells examined  the prostanoid metabolic profile has been described only for C-9 rat liver cells (95% is PGId PGF2α) . At the nduction . The eff2 production by epoxomicin and TPA-induced PGI2 production by epoxomicin and lactacystin as a function of dose is shown in Fig. 2 production significantly . Epoxomicin also stimulates the release of AA from rat glial, human colon carcinoma and human breast carcinoma cells  may be related to the cancer prevention [AA resulting from proteasome inhibition has many intrinsic biologic properties [reviewed in ]. Some oevention -24. It ievention .3H] AA (91.8 Ci/mmol) was obtained from NEN Life Science Products, Inc. (Boston, MA, USA). Epoxomicin, lactacystin and ZLLL were purchased from Biomol (Plymouth Meeting, PA, USA). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA). Rat liver cells incubating with lactacystin (5.4 μM) or ZLLL (1.0 μM) for 6-h have been tested for viability by a tetrazolium-based assay and found not to be toxic [The rat liver (C-9 cell line) and human breast carcinoma (the BT-20 cell line) were purchased from the American Type Culture Collection (Manassas, VA, USA). The rat liver glial cells (C-6 cell line) was obtained from Dr. Elaine Lai of the Department of Biology, Brandeis University and the human colon carcinoma (the HT-29 cell line) was obtained from Dr. Basil Rigas, American Health Foundation, Valhalla, NY, USA. They were maintained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum. [be toxic .5 cells/35 mm dish. The freshly seeded cultures were incubated for 24-h to allow for cell attachment. After decantation of MEM containing the fetal bovine serum, 1.0 ml fresh MEM containing 10% fetal bovine serum and [3H] AA (0.2 μCi/ml) were added and the cells incubated for another 24-h. The cells were washed 4 times with MEM and incubated for various periods of time with 1.0 ml of MEM containing 1.0 mg BSA/ml (MEM/BSA) and different concentrations of each compound. The culture fluids were then decanted, centrifuged at 2000 × g for 10 min, and 200 μl of the supernate counted for radioactivity. Radioactivity recovered in the washes before the incubation was compared to input radioactivity to calculate the % radioactivity incorporated into the cells [2 production, 1.0 ml of MEM supplemented with 10% fetal bovine serum, void of [3H]AA, was added after the first 24-h incubation. The cells were incubated for another 24-h, washed three times with MEM, then incubated with the compounds in MEM/BSA for various periods of time. The culture fluids were decanted and analyzed for 6-keto-PGF1α, the stable hydrolytic product of PGI2, by radioimmunoassay [Two days prior to experiments, the rat liver cells were treated with 0.25% trypsin-EDTA and, after addition of minimal essential media (MEM) containing 10% fetal calf serum, the floating cells were seeded onto 35 mm culture dishes. The plating densities varied from 0.1 to 0.5 × 10he cells . For PGIunoassay .3H] AA release is presented as a percentage of the radioactivity incorporated by the cells. Except for the time-course experiments which used duplicate dishes, three to five culture dishes were used for each experimental point. The data are expressed as mean values ± SEM. The data were evaluated statistically by the unpaired Student's t-test. A P value < 0.05 was considered significant.The [


Relatively few U.S.-based studies in chronic kidney disease have focused on Asian/Pacific Islanders. Clinical reports suggest that Asian/Pacific Islanders are more likely to be affected by IgA nephropathy (IgAN), and that the severity of disease is increased in these populations.To explore whether these observations are borne out in a multi-ethnic, tertiary care renal pathology practice, we examined clinical and pathologic data on 298 patients with primary glomerular lesions  at the University of California San Francisco Medical Center from November 1994 through May 2001. Pathologic assessment of native kidney biopsies with IgAN was conducted using Haas' classification system.Among individuals with IgAN (N = 149), 89 (60%) were male, 57 (38%) white, 53 (36%) Asian/Pacific Islander, 29 (19%) Hispanic, 4 (3%) African American and 6 (4%) were of other or unknown ethnicity. The mean age was 37 ± 14 years and median serum creatinine 1.7 mg/dL. Sixty-six patients (44%) exhibited nephrotic range proteinuria at the time of kidney biopsy. The distributions of age, gender, mean serum creatinine, and presence or absence of nephrotic proteinuria and/or hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups. Of the 124 native kidney biopsies with IgAN, 10 (8%) cases were classified into Haas subclass I, 12 (10%) subclass II, 23 (18%) subclass III, 30 (25%) subclass IV, and 49 (40%) subclass V. The distribution of Haas subclass did not differ significantly by race/ethnicity. In comparison, among the random sample of patients with non-IgAN glomerular lesions (N = 149), 77 (52%) patients were male, 51 (34%) white, 42 (28%) Asian/Pacific Islander, 25 (17%) Hispanic, and 30 (20%) were African American.With the caveats of referral and biopsy biases, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgAN. IgA nephropathy (IgAN) is the most common form of glomerulonephritis (GN) worldwide . ApproxiClinical reports suggest that individuals of Asian/Pacific Islander heritage are more likely to be affected by IgAN than whites, African Americans, and persons of Hispanic descent. Reports from U.S. centers have generally compared results of white and African American IgAN patients, with little or no available information on U.S. patients of Asian/Pacific Islander heritage. Studies from Japan and China have reported that more individuals with ESRD in these countries had IgAN, implying that IgAN may have a more severe disease course in certain Asian populations ,7.To explore whether IgAN was more common and severe among Asian/Pacific Islanders in our population, we examined clinical and pathologic data on 149 patients with IgAN and a random sample of 149 patients with other primary glomerular lesions  at the University of California San Francisco (UCSF) Medical Center.The records of 183 percutaneous native and transplant kidney biopsies with a diagnosis of IgAN received between November 1994 and May 2001 at the Renal Pathology Laboratory at UCSF were reviewed. Baseline demographic and clinical data included age, gender, race or ethnicity, history of kidney transplant, date of biopsy, and serum creatinine concentration at the approximate time of biopsy. In addition, the presence or absence of heavy proteinuria (≥ 3.0 g/day, with or without the nephrotic syndrome) and the presence or absence of hypertension at the time of biopsy were recorded. Major ethnic groups included white, Asian/Pacific Islander, Hispanic, African American, and other/unknown. Ethnicity was determined using information from patient health insurance forms and history provided at the time of biopsy. Any case in which Henoch-Schönlein purpura, systemic lupus erythematosus or chronic liver disease were considered likely diagnoses were excluded, as were cases of IgAN superimposed on a systemic disease involving the kidney (e.g., diabetic nephropathy). Two examiners unaware of the clinical data independently reviewed the biopsies. Biopsies displaying fewer than six glomeruli by light microscopy or insufficient immunofluorescence staining, as defined below, were also excluded. Fifteen biopsies were additionally excluded due to incomplete recovery of microscopic slides from files. Biopsies from 149 patients, including 25 kidney transplant recipients, satisfied the criteria for inclusion and provided the basis for the IgAN analytic sample.Aside from IgAN, the most commonly diagnosed primary glomerular lesions at our institution over the same time period were focal segmental glomerulosclerosis (N = 314), membranous nephropathy (N = 197) and minimal change disease (N = 147). To establish baseline race/ethnicity prevalences for our region and referral population, we collected demographic data on a computer-generated random sample of individuals with non-IgAN glomerular disease (N = 149), stratified by kidney transplant.Pathologic assessment of the IgAN native kidney biopsies was performed based on Haas' IgA nephropathy classification system . All cas2 test for categorical variables, and analysis of variance  or Kruskal-Wallis test for continuous variables. Two-tailed P-values <0.05 were considered statistically significant. SAS version 8.2 was used for all statistical analyses (SAS Institute, Cary, NC, USA).Demographic and clinical data are reported as mean ± standard deviation, medians with interquartile ranges, and proportions with 95% confidence limits. Inter-ethnicity comparisons were performed using the Cochrane-Mantel-Haenzsel χPatient clinical characteristics for the IgAN group are summarized in Table P = 0.64). Sixty-six patients (44%) exhibited heavy (≥ 3 g/d) proteinuria, and 74 (50%) had documented hypertension (systolic blood pressure ≥ 140 or diastolic blood pressure ≥ 90 mm Hg) at the time of kidney biopsy. The fractions of patients with heavy proteinuria and hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups.The median serum creatinine (SCr) of the IgAN cohort was 1.7 mg/dL (interquartile range 1.1–3.4 mg/dL). The median SCr of the African American group was signficantly higher (5.0 mg/dL) than the other ethnic groups; however, these calculations were based on a small sample size (N = 4) due to the low prevalence (3%) of African Americans with in our IgAN cohort. Median serum creatinine concentrations were not significantly different among white, Hispanic, and Asian/Pacific Islander groups (Of the 124 native kidney biopsies, the majority of cases (64%) fell into Haas subclasses IV or V, which are known independent predictors of progressive disease and poor renal outcomes . Only 22P = 0.006) compared to patients with IgAN. In addition, the distribution of race/ethnicity differed significantly between the two groups (P < 0.001). This association of IgAN and distribution of race/ethnicity persisted even when stratified by kidney transplant (P < 0.001 for native kidney comparison, P = 0.006 for kidney transplant recipient comparison).Table In a biopsy series of 244 patients with IgAN, Haas found fewer African Americans (in a major urban setting), similar to that noted in other U.S.-based studies of IgAN -11. WhilIn our study, the fraction of biopsies in subclasses I and II (18%) was similar to that observed by Haas (23%). However, we observed a higher proportion of biopsies in subclasses IV and V (64% vs. 31%), and a lower proportion of biopsies in subclass III (19% vs. 45%) compared with Haas, possibly reflecting a temporal trend towards a higher biopsy threshold along with intergrader measurement bias.In a study reviewing the pattern of glomerulonephritis in Singapore over the past two decades, Woo and colleagues reported that IgAN was the most common primary GN occurring in Singapore  . In our In contrast, based on smaller biopsy series, a striking variation in prevalence rates of IgAN has been reported from Europe and South America. In the UK, for example, Ballardie and colleagues noted comparatively low prevalence rates of IgAN in a predominantly white population (Manchester, England) in the early 1970's. In the subsequent 15-year period, however, these investigators reported a phenomenal rise in the observed incidence of IgAN , which the investigators felt more likely reflected a higher frequency of detection rather than true rise in disease incidence. Similar prevalence rates have also been documented from isolated white populations in Finland and southern Italy ,17. In cThese differences may be partially attributed to increased screening and disparities in the indication for kidney biopsy. In Japan and South Korea, for example, school-aged children undergo annual screening for urinary abnormalities; kidney biopsy is subsequently recommended for children with evidence of proteinuria or hematuria . More coH. parainfluenzae as a causative agent of IgAN in Japanese children and adults. Such claims are supported by studies showing the glomerular deposition of outer membrane H. parainfluenzae antigens and greater levels of plasma IgA1 antibody against OMHP in Japanese patients with IgAN  [H. parainfluenzae colonization and/or infection has yet to be established.Although the etiology of IgAN remains unknown, there exists a strong suspicion for an environmental antigen trigger combined with a genetic susceptibility factor. Along these lines, several hypotheses have been proposed to account for the reportedly higher prevalence of IgAN in Asian/Pacific Islanders. With respect to potential dietary antigens, Wakai et al. found that high intake of rice and n-6 polyunsaturated fatty acids (PUFA) were associated with an increased risk of IgAN . Recent iseases) ,23. WhetThe presence of either hypertension or proteinuria ≥ 3.0 g/24 hrs at the time of diagnosis significantly correlated with worsened renal survival in IgAN, even when controlling for serum creatinine at the time of kidney biopsy [Despite ongoing investigative efforts, scant data are available regarding genetic markers that may predispose individuals to progressive disease from IgAN. Recent immunogenetic studies have suggested a potential role for the T-cell receptor (TCR) in the development of immune-mediated diseases. Deenitchina and colleagues found that genetic polymorphism of the TCR constant alpha chain was associated with progression of CKD in a cohort of Japanese patients with IgAN. Although promising, such polymorphisms of the TCR gene have yet to be evaluated in large, prospective studies or by genetic analysis of familial IgAN .Our results contest the assertion that IgAN follows a more severe course in individuals of Asian/Pacific Islander descent. One reason for the similar disease severity of IgAN in our study population may stem in part from the large subpopulation of Filipino patients comprising our Asian/Pacific Islander cohort. It is unclear whether certain subpopulations of Asian/Pacific Islanders, including Filipinos, exhibit IgAN prevalence rates similar to those documented by Koyama and Woo. Anecdotal reports from Thailand and India documenting prevalence rates of 4–9% suggest that IgAN may not have the same epidemiology among all southeast Asians . DespiteThere are several important limitations to this report. As with any single-center biopsy series, we may have been underpowered to detect a clinically significant difference due to the limited sample size (type II error). In addition, racial admixture may have also confounded the results, as we were unable to subclassify patients in the Asian/Pacific Islander group or account for the growing population of bi- or multi-ethnic individuals in our population. Furthermore, due to the study's case control design, and breadth of our referral base , we were unable to control for the criteria for kidney biopsy. As a result, a biopsy bias may have confounded our results. In other words, Asian/Pacific Islander patients in our referral base with mild to moderate proteinuria and/or hematuria might have been given a presumptive diagnosis of IgAN without nephrology referral or confirmatory kidney biopsy. With regard to disease prevalence, these potential referral and biopsy biases based on race/ethnicity are largely conservative in nature, and would have biased our results towards the null. Finally, we have included data from a modest-sized IgAN transplant population N = 25), the donor demographics of which were unavailable at the time of the study. However, the association of race/ethnicity and distribution of glomerular lesion persisted, even when stratified by kidney transplant, and thus our overall conclusions remained the same. In addition, a small European study of donor-recipient pairs (average follow-up 7 years) has shown that when a donor kidney with asymptomatic IgA deposits is transplanted into a recipient with ESRD secondary to a disease other than IgAN, the IgA immune deposits in the donor kidney are rapidly removed [, the donIn conclusion, with the caveats of referral bias and biopsy bias, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgA nephropathy.None declared.YH designed the study, collected and analyzed the data, and drafted the manuscript. GC supervised the study design, analyzed the data, and edited the manuscript. EF graded the IgAN histopathologic slides. JO collected, reviewed and graded all histopathologic data, and edited the manuscript. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


The study aimed to investigate the role of these nanoparticles on the release of the pro-inflammatory cytokine TNF-α and IL-1α gene expression. We also investigated the role of intracellular calcium signalling events and oxidative stress in control of these cytokines and the effect of the particles on the functioning of the cell cytoskeleton. We showed that there was an increase in intracellular calcium concentration in J774 cells on treatment with PM10 particles which could be significantly reduced with concomitant treatment with the calcium antagonists verapamil, the intracellular calcium chelator BAPTA-AM but not with the antioxidant nacystelyn or the calmodulin inhibitor W-7. In human monocytes, PM10 stimulated an increase in intracellular calcium which was reduced by verapamil, BAPTA-AM and nacystelyn. TNF-α release was increased with particle treatment in human monocytes and reduced by only verapamil and BAPTA-AM. IL-1α gene expression was increased with particle treatment and reduced by all of the inhibitors. There was increased F-actin staining in J774 cells after treatment with PM10 particles, which was significantly reduced to control levels with all the antagonists tested. The present study has shown that PM10 particles may exert their pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of pro-inflammatory cytokines. Impaired motility and phagocytic ability as shown by changes in the F-actin cytoskeleton is likely to play a key role in particle clearance from the lung.The effects of PM Much ofrticles) -5 are a rticles) ,6,7. Ratrticles) -11.The alveolar macrophage plays an important role in particle-mediated inflammation by phagocytosing particles and release of pro-inflammatory mediators such as the cytokine tumor necrosis factor-alpha (TNF-α) . The sigIn order for macrophages to migrate and phagocytose foreign material, an intact functional cytoskeleton is necessary. The cytoskeleton is sensitive to ROS and oxidative stress, due to the presence of thiol groups located on the actin microfilaments. On oxidation, these filaments cross-link, leading to reduced cell motility, impaired phagocytosis and hence clearance of foreign material from the lung. The cytoskeleton mediates several basic cell functions: chemotaxis, migration, phagocytosis, phagosome-lysosome fusion, and intracellular signalling -21. SeveThe pro-inflammatory cytokine interleukin 1 (IL-1) is not normally produced by the cells of healthy individuals, exceptions being skin keratinocytes, some epithelial cells and some cells of the central nervous system. In response to inflammatory stimuli, however, there is a dramatic increase in the production of IL-1 by macrophages and other cell types . There a10 particles produce cytokine release and cytokine gene expression in macrophages by a process which involves calcium signalling and reactive oxygen species (ROS). Furthermore, we hypothesise that other effects of PM10, such as alterations in the cytoskeleton, are also mediated via signalling processes involving both ROS and calcium.Our hypothesis in this study was that PM10 samples was co-ordinated by Casella Stanger, London, England. Particles were collected onto TEOM filters in Marylebone Road, London, a site which had particularly high levels of traffic and therefore high levels of primary, combustion-derived nanoparticles. Ultrafine carbon black (UfCB) was obtained from Degussa (Printex 90), the average particle size was 14 nm. The characteristics and details of UfCB particles have been published previously [Collection of PMeviously .10 filter was placed into a bijou bottle and 0.5 ml phosphate buffered saline (PBS) added. The bottle was vortexed for 4 minutes to remove the particles from the filter and the resulting suspension transferred to a clean bijou bottle. The mass of particles was assessed by densitometry. As standards, a series of dilutions of UfCB particles were made, ranging from 15.625 μg/ml to 1 mg/ml in saline, sonicated for 5 minutes, and 75 μl of each concentration was added into triplicate groups of wells in a 96-well plate. Seventy-five microlitres of PM10 sample were added into a separate triplicate group of wells. The samples and standards were then read on a plate reader at 340 nm and the mass of particles calculated from a linear regression of the UfCB standards.A single PM5/ml in RPMI plus 5% FCS. Sterile 10 mm glass cover slips were placed in each well of a 24-well plate and 1 ml of cell suspension added to each well. Cells were incubated at 37°C for 24 hours prior to particle treatment.The mouse macrophage cell line J774.A1 (a kind gift from Dr W Muller GSF, Gauting, Germany) was routinely cultured in RPMI medium (Sigma) containing 5% foetal calf serum (FCS) and Penicillin/Streptomycin. Cells were cultured until confluency was reached and then scraped from the surface of the flasks using a cell scraper. The cells were counted and adjusted to 5 × 106 cells/ml and 1 ml added to each well of a 24 well plate. For calcium imaging, cells were also set up in 6-well plates containing a 26 mm diameter sterile glass coverslip. The cells were incubated for 1 hour at 37°C, the medium removed and replaced with RPMI plus 10% autologous serum and incubated for 48 hours at 37°C. After the second incubation, the medium was replaced and the cells incubated for a further 72 hours prior to treatment.Human peripheral blood mononuclear cells were prepared according to the protocol of Dransfield et al, . In brie10 particles were diluted to give a final concentrations ranging from 5 μg/ml to 40 μg/ml in RPMI medium without serum and the suspension was sonicated for 5 minutes to disperse the particles. Cells which had been set up as described above, were washed twice with sterile PBS and 250 μl of particle suspension added to appropriate wells. UfCB particles were quantified as described for the PM10 and set up in parallel with PM10 particles at similar mass concentrations with J774 cells to investigate TNF-α release. One well received medium only (-ve control) and one received 250 μl of 1 μg/ml LPS (+ve control). The calcium antagonists were added concomitantly with the particles to give final concentrations of verapamil (100 μM), BAPTA-AM (50 μM), W-7 (250 μM), trolox (25 μM), and nacystelyn (5 mM). The cells were then incubated at 37°C for 4 hours and the supernatants removed and stored at -80°C until required. The cells cultured on 10 mm cover slips were fixed by the addition of 3% formaldehyde.PM6 cells/ml in RPMI plus 10% FCS and incubated at 37°C until required for the assay. One millilitre of cell suspension was transferred to an Eppendorf tube, centrifuged at 145 g for 2 minutes, the medium removed, the cell pellet resuspended in 1 ml PBS and again centrifuged at 145 g for 2 minutes. The PBS was removed and cells resuspended in serum-free RPMI medium containing 23 mM Hepes buffer. Cells were loaded with 1 μg/μl Fura 2-AM (Sigma) in DMSO, 2 μl/ml cell suspension, the tube wrapped in foil and incubated in a shaking water bath for 20 minutes at 34°C. After incubation, the tube was centrifuged at 145 g for 2 minutes at 4°C, the medium removed and replaced with 1.5 ml fresh RPMI without serum. The Fura 2-AM-loaded cells were transferred to a quartz cuvette with stirrer and placed immediately into a fluorimeter with heated block and basal fluorescence measurements obtained over a 100 second period. The fluorimeter was set up with to give excitation wavelengths of 340 nm and 380 nm, emission 510 nm and excitation and emission slit widths set at 5 nm. During the experiments, the cuvette temperature was kept constant at 37°C. After 100 seconds, 10 μl appropriate treatment in RPMI medium was added to the cuvette and the experiment allowed to run for a further 1700 seconds. Treatments consisted of PM10 to give a final concentration of 10 μg/ml with and without the calcium antagonists at the concentrations described above. Twenty microlitres of 5% Triton solution were added to the cuvette to lyse the cells to give the maximum fluorescence (Rmax) and the experiment continued for 500 seconds. To give the minimum fluorescence value (Rmin), 15 μl of 0.5 M EGTA in 3 M Tris buffer were added to the cuvette. The experiment was terminated after a further 500 seconds. The ratio of the fluorescence measurements at excitation wavelengths of 340 and 380 nm were converted to calcium concentration values using the method of Grynkiewicz et al, [J774.A1 cells were cultured and removed from flasks as described above. Cells were pooled into a single tube, adjusted to 4.5 × 10z et al, .The supernatants previously prepared were assayed for TNF-α protein content using a commercially available human TNF-α kit (Biosource) or mouse TNF-α kit (R&D Systems) according to the manufacturer's instructions. Briefly, each well of a 96-well plate was coated overnight with capture antibody, before washing with PBS containing 0.05% tween, and then adding test supernatant to the appropriate wells in triplicate groups. After incubation for 2 hours at room temperature, the wells were washed, a detection antibody added and incubated for a further hour at room temperature. The wells were then washed with PBS/tween before addition of Horseradish peroxidase (HRP)-conjugated streptavidin and incubated for 45 minutes at room temperature. Finally, the colour was developed by adding peroxidase substrate to each well, before reading the absorbance at 450 nm using a Dynatec plate reader.The experiments described above were also used to generate cells for total RNA extraction. After removal of the supernatant, 400 μl Tri reagent (Sigma) was added to each well. The lysed cells were then scraped from the surface of the plate using a cell scraper and transferred to Eppendorf tubes. Two hundred microlitres of chloroform were added to each Eppendorf, vortexed for 15 seconds and allowed to stand at room temperature for 15 minutes. The resulting mixture was centrifuged at 12000 g for 15 minutes at 4°C. The colourless upper phase was transferred to a fresh Eppendorf, before adding 450 μl isopropanol. The mixed samples were allowed to stand for a further 10 minutes at room temperature. Again the tubes were centrifuged at 12000 g for 10 minutes at 4°C, the supernatant removed and the RNA pellet washed in 1 ml of 75% ethanol. The resulting samples were then vortexed briefly, centrifuged at 7500 g for 5 minutes at 4°C and the RNA pellet air-dried for 10 minutes. The RNA was then suspended in 50 μl diethylpyrocarbonate (DEPC)-treated water and stored at -70°C until required for quantification and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).The RT-PCR procedure was carried out using the Promega Access Kit. Briefly, a master mix of the kit reagents was prepared according to the manufacturers instructions. Ten microlitres of RNA at 0.03 μg/ml was added to 40 μl of the master mix, containing 10 μl of the appropriate human primers Glyceraldehyde Phosphate Dehydrogenase (GAPDH) or IL-1-α (MWG AG Biotech, Ebersberg, Germany). Tubes were placed in a thermal cycler which was programmed for the following temperatures and times. Following an initial 45 minute incubation at 48°, samples were cycled as follows: 94°C for 2 minutes, 95°C for 30 seconds, 60°C for 1 minute, 68°C for 2 minutes. This cycle was repeated 25 times for GAPDH and 30 times for IL-1 alpha. To conclude, the sample was incubated at 68°C for 7 minutes and then cooled to 4°C. The resulting RT-PCR products were separated by electrophoresis using a 2% agarose gel containing 1 μg/ml ethidium bromide and viewed under UV light. The RT-PCR bands were quantified by densitometry using Syngene software and the IL-1α band intensity was expressed as a percentage of the corresponding GAPDH band. These results were then expressed as a percentage of the untreated control.5 cells/ ml and incubated at 37°C, 5% CO2 for 1 hour before washing with 1 ml PBS. Prior to particle treatment and digital enhanced video microscopy (Roper scientific), cells were loaded with the calcium-sensitive dye, Fura 2-AM (2 μg/ml in RPMI) (Sigma) for 30 minutes at 37°C. The coverslips were then washed with PBS, assembled into the microscope holder and 400 μl RPMI medium without phenol red (Sigma) added. The fluorescence ratio was observed (excitation 340 and 380 nm, emission 510 nm) at a magnification of 63× (Zeiss Axiovert microscope). Images were captured every 2 seconds by a Coolsnap fx Photometrics (Roper Scientific) camera controlled by Metafluor software. After 100 seconds particle treatment was added to the cells  contained in phenol red-free RPMI medium.Human mononuclear cells were isolated from blood as previously described followed by adhesion onto 26 mm glass coverslips contained in 6-well plates. Cells were seeded in RPMI medium containing 0.1% BSA and penicillin/streptomycin at a density of 5 × 10The cells cultured on cover slips and fixed with formaldehyde were washed three times with PBS and permeablised with 0.1%Triton for 4 minutes. Cells were then washed three times with PBS and the F-actin stained using 33 ng/ml Phalloidin-FITC (Sigma) in PBS for 30 minutes at room temperature. Cells were washed three times with PBS before staining with propidium iodide (10 μg/ml in PBS) for 5 seconds. Cells were further washed three times with PBS before being mounted on glass slides using Citifluor mounting medium. The cells were then examined using an Axiofluor fluorescence microscope. Images were captured and quantified using Metamorph software . Seven fields of view were captured from each treatment and the images deconvolved using the image software. The staining intensity of each cell was then measured using the analysis software.Data from all of the experiments were analysed using analysis of variance with Tukey or Fishers multiple comparison test. Significance was set at p < 0.05.10 on J774 murine macrophages was investigated at final concentrations of 5, 10 or 25 μg/ml PM10. Treatment of the cells with these particles produced a dose-dependent increase in cytosolic calcium concentration [Ca2+]c up to a concentration of 10 μg/ml. At 25 μg/ml the [Ca2+]c decreased to a value similar to the 5 μg/ml particle concentration. . The [Ca2+]c following concomitant treatment of cells with particles and calcium antagonists was reduced . In our previous studies [The effects of PM. Figure . Subsequ studies . There wd figure . Both th studies ,35 we de10 also induced a significant increase in cytosolic calcium in the primary human monocytes (p < 0.05). The dose of 10 μg/ml final concentration was chosen as the dose at which a significant increase in [Ca2+]c had previously been observed .PMd figure . At timease in Ca+c had pr10 and UfCB particles on TNF-α release by J774 macrophages is shown in figure 10 particles caused significantly more TNF-α release as the same mass of UfCB particles by the mouse macrophage cell line.A comparison of the gram for gram dose effect of PM10 is shown in figure 10 induced TNF-α release with W-7 (W), Trolox (T) or Nacystelyn (N) at any particle dose tested.The release of TNF-α protein by human monocytes after treatment with varying concentrations of PM10 for 4 hours produced a significant increase in IL-1α mRNA content compared with unstimulated cells (p < 0.05) .Treatment of human peripheral blood monocytes with 10 μg/ml PMs p < 0.0 , although the increase in the fluorescence intensity of the PM10-treated cells was modest (a 5% difference).The fluorescence intensity of cells stained for F-actin after treatment with PM10 are considered to drive these effects [10 particles on oxidative stress- and calcium-related cytokine regulation in human monocytes and on the cytoskeleton in mouse J774 cells.There is evidence that increases in particulate air pollution correlate with increased morbidity and mortality from respiratory and cardiovascular causes ,36-38 an effects ,40. The 2+]c changes lead to production of the proinflammatory cytokine TNF-α [10 particles can also stimulate entry of extracellular calcium into both J774 macrophages and human macrophage derived monocytes, and that this process is inhibited by a calcium channel blocker suggesting that the PM10, in a similar fashion to UfCB induces opening of plasma membrane calcium channels leading to a calcium influx. The results obtained using the antioxidant nacystelyn were confusing. In the J774 macrophages nacystelyn was unable to inhibit PM10 induced increases in cytosolic calcium concentration, whereas the same antioxidant was very effective in the human monocyte derived macrophages. This difference could be due to a species difference or a comparison between a cell line and primary cells. A number of cell lines have been demonstrated to exhibit aberrant calcium signalling pathways. Our previous studies using human macrophages suggest that ultrafine particle-induced increases in cytosolic calcium can be mediated by ROS [10 are ultrafine, it is conceivable that much of the calcium increase is ROS mediated, at least in part. However, PM10 also contains other substances, such as metals, that could influence this pathway. Metals would in fact be expected to increase the ROS production by the PM10 particles [We have previously shown that ultrafine or nanoparticles enhanced the calcium influx into cells of a monocytic cell line (MM6) ,34 and tne TNF-α . We demod by ROS  and sincarticles .10 (10 μg/ml) that induces calcium elevation also stimulates significant increases in both TNF-α protein release and IL-1α mRNA production by macrophages. The calcium channel blocker verapamil and the intracellular calcium chelator BAPTA-AM reduced the calcium increase, TNF-α protein release and IL-1 mRNA expression by human monocytes when stimulated with PM10 particles. This is strong evidence to suggest that influx of extracellular calcium plays a key role in upregulating the proinflammatory response induced by PM10 that could lead to disease. However, the calmodulin inhibitor W-7 had little effect on TNF-α release, while it did inhibit IL-1 mRNA expression. The antioxidants also had variable abilities to block cytokine expression, inhibiting IL-1 mRNA production but not TNF-α protein release. These differences could be explained either by divergent pathways controlling expression of the two cytokines, or that TNF-α protein was measured in comparison to IL-1 mRNA. However, clearly both calcium and ROS are important in the regulation of IL-1α mRNA expression while only calcium is important in controlling TNF-α expression in macrophages exposed to PM10.The present study clearly shows that the same dose of PM10 is far more potent that UfCB in terms of its ability to induce TNF-α protein release. This is likely to be due to other components, such as metals and organic compounds other than the carbon core, within PM10 that can promote inflammation. It is also possible that components such as the UF particles and metals could interact to enhance toxicity as has been shown for ROS production in vitro and inflammation in vivro [10 particles, therefore it is unlikely that cytokine release, changes in intracellular calcium, and IL-1α gene expression can be explained solely by endotoxin.These studies indicate that on an equal mass basis PMin vivro . Our pre10 generates ROS [10 induced increase in F-actin staining in this study demonstrates that particle-derived ROS impact on the macrophage cytoskeleton. Our previous studies also demonstrate that Uf particle-induced ROS play a role in elevating the cytosolic calcium concentration of macrophages leading to increased TNF-α production [10 exposure. As has been shown by other workers [10 particles increased the F-actin fluorescence signal in cells stained with FITC-labelled phalloidin, although changes in the distribution of actin filaments was not apparent from microscope analysis there appeared to be more cortical staining. In accord with the role of calcium and ROS in the induction of IL-1 expression, both of these factors appeared to play an important role in modulating the F-actin cytoskeleton.As explained previously, the cytoskeleton is the scaffold of cells, and in the case of motile cells such as macrophages it is responsible for controlling movement. Disruption of the cytoskeleton, particularly via oxidative stress, is thought to disrupt cellular structure and hence function . We haveates ROS . The abioduction . The res workers ,45, in b workers  which ma10 particles may exert their increased pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of proinflammatory cytokines. An additional consideration is the effects of particles on the cytoskeleton of the cell. Impaired cellular motility and phagocytic ability is likely to play a key role in particle clearance from the lung, thus perpetuating the effects of PM10. The role of calcium and ROS in other cellular responses are under investigation.The present study has shown that PM


Neisseria species, and phylogenetic trees, to investigate whether genotypic clusters can be resolved among these recombinogenic bacteria and, if so, the extent to which they correspond to named species.It is a matter of ongoing debate whether a universal species concept is possible for bacteria. Indeed, it is not clear whether closely related isolates of bacteria typically form discrete genotypic clusters that can be assigned as species. The most challenging test of whether species can be clearly delineated is provided by analysis of large populations of closely-related, highly recombinogenic, bacteria that colonise the same body site. We have used concatenated sequences of seven house-keeping loci from 770 strains of 11 named N. meningitidis, N. lactamica and N. gonorrhoeae. A few isolates arose from the branch that separated N. meningitidis from N. lactamica leading us to describe these species as 'fuzzy'.Alleles at individual loci were widely distributed among the named species but this distorting effect of recombination was largely buffered by using concatenated sequences, which resolved clusters corresponding to the three species most numerous in the sample, Neisseria where individual loci are inadequate for the task. This approach should be applied by taxonomists to large samples of other groups of closely-related bacteria, and especially to those where species delineation has historically been difficult, to determine whether genotypic clusters can be delineated, and to guide the definition of species.A multilocus approach using large samples of closely related isolates delineates species even in the highly recombinogenic human  The definition of bacterial species, and a concept of species applicable to all bacteria, are problems that have long exercised systematists and microbiologists -4. WhileMolecular approaches to assigning bacteria to species began with the introduction of DNA-DNA hybridization, which allowed an objective assessment of the extent of sequence similarity among a set of genomes, and remains the systematicist's gold standard, defining bacterial species as those isolates whose genomes show at least 70% hybridization under standardized conditions . HoweverIndividual isolates of a named species differ in gene content  and the A multilocus approach has recently been applied to small numbers of isolates of several relatively distantly related named species of enterobacteria , and othNeisseria into genotypic clusters. We chose this example because Neisseria are naturally transformable, are among the most recombinogenic bacteria, and there is good evidence for relatively frequent localised recombination between the named Neisseria species [Neisseria isolates, but the tree based on the concatenated sequences effectively resolves the three major named species within the sample, although the boundaries are fuzzy due to the presence of a small number of intermediate genotypes.In this study we have evaluated the ability of seven individual house-keeping gene sequences, and of the concatenated sequences of these genes, to resolve a large sample of human pathogenic and commensal  species ,16 throuNeisseria MLST database, which includes several thousand sequence types (STs) of N. meningitidis and smaller numbers assigned to several other named human Neisseria species [N. meningitidis were compared with all STs assigned to the other human Neisseria species. The sequences of the seven gene fragments were concatenated in-frame and a tree was constructed (using third codon position sites) using Mr Bayes [N. gonorrhoeae, and all but two of the 171 STs of N. lactamica, descend from single well-supported nodes . The great majority of N. meningitidis also formed a single well-resolved cluster, but a few arise from the branch leading to the N. lactamica isolates. Very similar clustering of these three species was observed using other sets of 500 N. meningitidis STs from the database, and in a neighbour-joining tree constructed using all STs in the Neisseria MLST database (data not shown). The high levels of recombination in the Neisseria make the fine structure of the tree meaningless   for epid species  on the bMr Bayes . Figure N. gonorrhoeae, N. meningitidis and N. lactamica, their boundaries are not perfectly defined and a number of isolates are placed on the branch between N. lactamica and N. meningitidis, representing intermediate genotypes.Analysis of the individual gene trees shows that these fail to resolve the named species and highlights many examples where interspecies recombination has resulted in anomalous clustering Figure . The cleNeisseria species do not cluster clearly. A significant separation is observed between two subtrees  in the MLST database, using Neighbour-Joining, minimum evolution and UPGMA tree-building approaches (data not shown).The small numbers of STs assigned to other human Current molecular definitions of species use rules or cut-off values (e.g. ≥ 70% DNA-DNA hybridization) and rarely take account of the genotypic diversity within and between populations . A more N. meningitidis and N. lactamica  may be a Figure . The conN. meningitidis, N. lactamica and N. gonorrhoeae are reasonably secure; the two N. lactamica that clustered highly anomalously probably represent species mis-identification. The most critical test of the multilocus approach is the ability to resolve N. lactamica from N. meningitidis since these colonise the same body site, the nasopharynx. Resolution of these named species was remarkably good, although the boundaries between N. lactamica and N. meningitidis are somewhat fuzzy, due to the existence of intermediate forms. This is to be expected as recombinogenic bacteria have mosaic genomes, resulting from the occasional replacement of chromosomal segments with those from related populations. Thus, in any large dataset, there may be isolates in which one or more of the loci used in a multilocus approach to species definition will have been recently introduced from a related population. Single unusually divergent replacements, or replacements at more than one of the multiple loci, may place isolates away from the majority of isolates of the species. However, only seven STs in Figure N. meningitidis or N. lactamica), and there was no overlap between these two named species (i.e. a region containing isolates identified as both species interspersed with one another).Network based methods  using the multilocus approach with large datasets, should clarify whether they fall into distinct clusters, or whether the difficulties in defining species by phenotypic methods reflect an underlying genetic reality in which resolved clusters are not evident.Sorting the human commensal es names . We gainIf necessary, further resolution between apparent clusters may be attempted by increasing the numbers of loci sequenced. Provided that the alleles at these loci show a degree of specificity to a given species cluster, then the resolution of that cluster will be enhanced. If this cannot be demonstrated, then it is likely that the isolates under test do not genuinely form separate populations, and should not be considered to be distinct species. This approach lends itself to "electronic taxonomy", in which systematic classification may be evermore finely elucidated through the accumulation of online sequence databases.The work described here obviously begs the question of what forces or mechanisms could generate such separation among recombining bacteria. We offer a simple model for recombining organisms as follows: consider two populations freely recombining within themselves and with each other. New mutations arising in one population will readily spread to the other, and to an observer they appear to form one cluster of related strains. If a barrier to recombination should be erected between them, such that isolates are much more likely to undergo recombination with their own population, then the rate of generation of new genotypes within each population may increase beyond the rate at which such genetic innovation is shared and the two populations begin to diverge. As the populations diverge, decreasing sequence identity will further impede recombination, thus reinforcing the effect of the original genetic barrier and creating a permanent separation ,25.N. gonorrhoeae. Unlike the other human Neisseria, which colonise the nasopharynx, the primary niche of the gonococcus is the genital tract, and it has been proposed that gonococci arose relatively recently due to the successful invasion of the genital tract by a nasopharyngeal Neisseria lineage [It is not difficult to suggest candidate mechanisms. Niche separation is one example, and almost certainly underlies the tight well-defined cluster of  lineage . Similar lineage , limitat lineage  and simiThe point at which such a group is described as a species is a matter more of human interest and attention than any intrinsic evolutionary process. The properties of the species clusters we observe will be determined by the diversification of those strains sharing the speciation loci (i.e those that determine gene flow). Because speciation is gradual, we should be able using estimates of recombination within and between groups derived from multilocus data, to define nascent species which if they continue to diversify in isolation, are expected to form distinct sequence clusters, ie species, in the future.The bacterial domain of life is not uniform. Instead we see clumps of similar strains that share many characteristics, and with an innate human urge to classify, we have defined these as species. This work shows that by applying a simple approach using sequence data from multiple core housekeeping loci, we can resolve those clusters, provided such clusters exist. However, these species clusters are not ideal entities with sharp and unambiguous boundaries; instead they come in multiple forms and their fringes, especially in recombinogenic bacteria, may be fuzzy and indistinct. A multilocus approach using large numbers of isolates will provide data that help us to develop theoretical models of how species emerge, and relate these to the observed population genetic structure of bacteria. This should be enormously helpful to taxonomists, whose foremost duty will remain to provide us with pragmatic species designations which attempt to reflect the underlying genetic reality.Neisseria MLST database [N. meningitidis (67 isolates of N. gonorrhoeae, 171 of N. lactamica, 5 of N. sicca, 3 of N. mucosa, 5 of N. cinerea, 7 of N. polysacchareae, 3 of N. flava, 4 of N. perflava, 4 of N. subflava and 1 isolate of N. flavescens) were concatenated as described below, and analysed together with the concatenated sequences of N. meningitidis strains with ST numbers from 1 to 500. Species definitions were as recorded at [Neisseria were also used to construct individual gene trees.The contents of the publicly accessible database ,18 were MLST loci were concatenated in-frame to form a 3267 bp sequence, of which only third position sites were used in subsequent analyses. To illustrate clustering in this dataset, a tree was constructed using Mr Bayes 3.0b4 . A startrRNA ribosomal RNAMLST Multi Locus Sequence TypingST Sequence TypeUPGMA Unweighted Pair Group Method with Arithmetic MeanBGS conceived of the study and drafted the manuscript, CF participated in study design and analysis of results, WPH designed the study, carried out the analyses and interpreted the results, and drafted the manuscript.


We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish  luciferase gene under a clock gene promoter enables the study of molecular circadian rhythms in vivoGeneration of zebrafish that express the firefly  The circadian clock controls biological processes such as behavior, gene expression, and physiology in diverse organisms, ensuring that these processes to take place at appropriate times of the day. This is crucial for many organisms, such as plants, which must synchronize photosynthesis with day-night cycles. Animals also synchronize to environmental cycles because of more subtle but nevertheless important needs such as predator avoidance, food availability, and optimal temperatures for various processes. Circadian clocks in all species share the following properties: They persist even in constant environmental conditions with periods near 24 h; they can be reset by environmental stimuli such as light and temperature; and their periods are relatively constant at different temperatures.Drosophila, has been extremely valuable in identifying important players of the clockworks and their roles [Drosophila lead to molecular dissection of clock mechanisms in mammals mainly by testing whether homologs of Drosophila clock genes are involved in mammalian clocks. While this approach is informative, it harbors the risk of missing important factors that would have been found by forward genetic searches without preconceptions. Furthermore, gaps in our understanding of the clock mechanisms include factors responsible for the expression of positive transcription factors Clock and Bmal and mechanisms for clock protein turnover. In this regard, zebrafish is in a unique position as a vertebrate species in which large-scale forward genetic screens are convenient [Recently, remarkable progress has been made in elucidation of molecular mechanisms of circadian clocks in diverse organisms. The common feature of clocks is cycling gene expression due to intracellular transcriptional feedback loops ,2,3. Genir roles ,5,6. Idenvenient ,8. Furthnvenient ,10,11.zper4-luc promoter fusion construct have been studied successfully in the zebrafish PAC-2 cell line [A behavioral screening for circadian mutations has been successfully carried out in zebrafish . Howeverell line . While tluc gene is driven by the promoter of per3 [per3 mRNA has been shown to oscillate rhythmically in embryos as well as in a cell line [per3-luc was considered ideal for mutagenesis screening, because an in situ hybridization study showed that per3 mRNA cycles starting on day 1 postfertilization with or without any entraining signals [per3 mRNA present in the oocyte can set the phase of per3 mRNA rhythms in early embryos. This result, however, is not consistent with other studies involving development of circadian rhythms: Rhythms of melatonin production require a light-dark (LD) transition later than 20 h postfertilization [In this study, transgenic zebrafish were made in which cycling expression of the firefly  of per3 . This prell line ,22. For lization ; circadilization ; and rhylization .luc expression can be monitored in the per3-luc transgenic fish, embryos from the transgenic lines were monitored for bioluminescence from day 1 of development  from this region, it was found that the cDNA contains another exon 5′ to the first coding exon. By comparing BAC-end sequences from the construct to genomic sequences from CH211–138E4 as well as with the Ensembl Zebrafish whole genome shotgun assembly sequence version 4 (http://www.ensembl.org/Danio_rerio/, the construct was found to be approximately 72 kb long, and spans from 26 kb upstream of exon 1 to intron 19 of per3, and contains part of another gene 5′ to per3 (per3 promoter (unpublished data). In the modified BAC, the coding portion of the first coding exon was replaced by the luc and r)kanamycin resistance (Km genes (To develop a system in which circadian gene expression in zebrafish can be monitored in vivo, transgenic fish were generated in which the expression of the firefly er3 gene . The conne [per3 . By comp to per3 . One canr) genes . This raAfter screening 147 injected founders by PCR, five independent transgenic lines were found. Each positive founder was bred to a wild-type fish, and their progeny were individually tested for bioluminescence as 5–7-d-old larval fish. Larval fish with bioluminescence above background (more than 100 counts per second [cps]) were raised as transgenic F1 fish. Three of the five lines emitted bioluminescence above background. The level of bioluminescence varied depending on the line. The strongest-glowing line (#23) was used for this study unless otherwise stated. All the animals used in this study were the progeny of crosses between a transgenic line and the *AB wild-type strain. Therefore, these animals carried the transgene in hemizygous condition.per3-luc transgenic zebrafish is screening for mutations that affect bioluminescence rhythms. Since it is most convenient to screen the youngest possible animals, bioluminescence from transgenic embryos was monitored first. It was also expected that per3-luc-mediated bioluminescence in embryos should cycle from day 1 of development even in constant conditions, because per3 mRNA expression detected by in situ hybridization has been demonstrated to oscillate from day 1 postfertilization in constant conditions [A.M.; lights off at 10 P.M. CST) cycle on day 1, the majority of the animals showed no circadian rhythmicity of bioluminescence .One of the intended usages of the nditions . Therefonescence . Nevertheporting . ImportaSince the light signal on day 1 was not enough to elicit detectable rhythmicity of bioluminescence, an increasing number of LD cycles were given to embryos while they were monitored for bioluminescence see . LD cyclper3-luc rhythms. There was no systematic effect of the time of the last lights-off on free-running periods . As in the experiment shown in per3 and luc mRNA levels were much lower on day 3 compared to day 8 . In contrast, approximately 5-fold fluctuations of the transcripts were observed on day 8 (per3 and luc mRNA (see Discussion). Importantly, the peak phase of mRNA cycling on day 8 was 5–7 h advanced compared to the phase of bioluminescence cycling  of the fish were rhythmic with a 25.2 ± 0.7 h (mean ± standard deviation) period under this condition, and their rhythms persisted for 6 d, albeit with some damping (per3-luc rhythms were tested in LD (n = 179) compared to DD, although the difference was not significant (p > 0.1, G-test). The waveform of the rhythm in LD was different from that in DD: The ascending part of the wave that happens during the day was steeper in LD than in DD  was observed in all three independent experiments done. However, this does not mean that the low-amplitude per3 RNA oscillations that occur earlier during development can amplify into high-amplitude ones without any entrainment by LD cycles.The results presented here, namely the gradual development of rhythmicity and responsiveness to entraining stimuli during the first several days of development and requirement of light signal, is consistent with previous observations on rhythms of melatonin production, locomotor activity, and the cell cycle in larval zebrafish ,24,25. Ielopment . In our per3. Alternatively, cycling amplitude may increase because various cellular oscillations present become synchronized. However, it was not only the cycling amplitude that increased with age, but the overall levels of per3 mRNA also. Therefore, more cells may start expressing per3 with high-amplitude oscillations later during development. This rather simple scenario is in fact what happens in developing Drosophila; as soon as the central pacemaker Lateral Neurons start expressing the PERIOD protein, the molecular rhythm is entrainable, and so is the eventual behavioral rhythmicity [The increase of cycling amplitude over the course of several days of development in LD may happen within each cell that expresses thmicity ,41. In aper3 RNA. Again, clocks in some tissues may develop earlier than in other tissues. It is also possible that per3 may not be expressed in all the clock cells.It is worth mentioning that rhythms of melatonin release starts as early as day 2 postfertilization, and this roughly corresponds to the time when the light-sensitive pineal gland is formed . Anotherper3-driven luc expression is not known. Most animals seemed to hatch between the first minor developmental peak and the subsequent trough (unpublished data). However, hatching itself is unlikely to induce per3 expression in early embryos, because dechorionated per3-luc embryos showed developmental profiles of bioluminescence similar to those of nondechorionated siblings, albeit with an accelerated second rise of bioluminescence (unpublished data). Larval fish after the hatching period are supposed to have completed most of their morphogenesis and start swimming actively [per3 is important for rhythmic processes specific to hatched animals, such as behavioral rhythms [The biological significance of the developmental profile of actively . It may  rhythms .per3-driven bioluminescence largely reflected endogenous per3 expression. However, the amplitude of bioluminescence cycling was greatly reduced compared to that of per3 or luc RNA. In general, proteins synthesized from cycling mRNAs show amplitude reduction and phase delay due to protein stability [per-luc transformants of Drosophila, approximately 6-fold cycling of luc RNA was reduced to 3- to 4-fold bioluminescence rhythms [per1-luc mouse [per3-luc larval fish. This may mean that the luciferase protein is somehow more stable in larval fish than in flies or mice.The developmental and circadian profiles of tability . Consist rhythms . Similaruc mouse . HoweverIt should be noted that the luciferase protein itself is quite stable, but the enzymatic activity of this protein is unstable ,45. Therper3 and luc mRNA, measured by real-time qPCR. The difference between days 3 and 8 of development was much larger for luc than per3. Furthermore, the difference between the 22 °C and 28.5 °C groups was larger for luc than for per3. These differences may be due to positional effect of the insert in the particular transgenic line used in this study. However, differences in bioluminescence levels between the first few days and older fish were found in two other independent per3-luc lines (unpublished data). Therefore, it is more likely that these differences between the two mRNA species reflect a property of the transgene itself. Although the BAC transgene used in this study has approximately 26 kb of upstream sequences, there may be critical sequences missing from this transgene, such as the first coding exon. Alternatively, posttranscriptional modification could be responsible for the difference. Posttranscriptional control of clock gene mRNA expression has been documented elsewhere [Besides the difference between bioluminescence and mRNA, there were differences between lsewhere ,28,46,47per3 expression: It increased peak levels and cycling amplitudes of mRNA, and accelerated the developmental profile and development of responsiveness to the entraining stimuli. The latter two effects presumably are due simply to the fact that development is accelerated by higher temperatures. These results are not consistent with the report that development of a cell-cycle rhythm in larval fish was not affected by ambient temperature [per3 mRNA induced by increased temperature is mostly independent of developmental speed. The peak per3 mRNA level in the high-temperature group on day 6 was higher than that in the low-temperature group on day 10, when bioluminescence levels should have reached the plateau  cycle, and group-housed in plastic tanks in a Z-MOD holding system (Marine Biotech) with recirculating filtered water at about 28.5 °C. They were fed commercial flake food in the morning, baby brine shrimp at midday, and adult brine shrimp in the evening. Experimental protocols were approved by the Institutional Animal Care and Use Committee.Animals used in this study were derived from the University of Oregon *AB strain. Adults were kept under 14:10 LD ; then a HindIII-BamHI fragment containing luc and the SV40 polyA signal from pGL3-Basic (Promega, Madison, Wisconsin, United States) was cloned upstream of rKm into HindIII and BamHI sites of pBluescriptSK+; finally, a NotI site between rKm and the polyA signal was destroyed by digesting the clone with NotI and BamHI, followed by treatment with the Klenow fragment, and ligation of these blunted ends together.The luc coding sequences flanked by a 53-bp overhang corresponding to sequences just upstream of the initiation codon of per3. The reverse primer consists of a 50-bp overhang that corresponds to the intron sequences just downstream of the first coding exon, and 25 bp of sequence from the 3′ end of rKm as the primer.Using this gene cassette as a template, a PCR fragment flanked by approximately 50-bp homology arms was amplified. The primer sequences used for the PCR reaction were: forward, 5′-GGGTTGTGAATCAGATCTTCAGTAGAGGAGGACAGGAGATCTCACAGGGAATGGAAGACGCCAAAAACATAAAGAAAG-3′; reverse, 5′-GTGCAGATTAAGTCAAATTCCACATAAAAAAAGCCACATTTCAAGTGTACCGTTAATAATTCAGAAGAACTCGTC-3′. The forward primer contains 25 bp of sequence from the 5′ end of the luc sequences in the modified BAC clones were checked for PCR errors by sequencing. One PCR error that resulted in a Val 217 to Ala change in the luciferase protein sequence was found. However, this is a conservative change, and fish injected with this construct showed bioluminescence above background. Therefore, it was judged that luciferase encoded by this construct can still function.The PCR fragment was purified and electroporated into DH10B cells containing the BAC and the plasmid pBAD-αβγ  as a souper3-luc transgene was purified, linearized by NotI digestion, and injected into one- to two-cell-stage zebrafish embryos according to [The rding to  with min2, and 0.1 g of KCl [pH 7.0] in 2 l of ddH2O) aerated overnight and containing 0.5 mM D-luciferin potassium salt (Biosynth, Naperville, Illinois, United States) and 0.013% Amquel Instant Water Detoxifier . Once loaded with animals, four such plates were subjected to automatic monitoring of bioluminescence every 30 min by the Topcount multiplate scintillation counter (Perkin-Elmer) equipped with six detectors and plate stackers. The room temperature was set at 21–22 °C, and the machine at 24 °C. However, due to the heat created by the machine, temperature at the bottom of the stacker was 1–2 °C higher than the room temperature. In order to minimize high background counts under lighted conditions, each plate was dark-adapted for approximately 5 min before being counted for bioluminescence. Each well was counted for 4.8 s every 30 min. The plates were illuminated with two white fluorescent lamps, each facing the left or right side of the stacker. The approximate intensity of the light that reached the plates was 17–35 lux, depending on the position of the plates within the stacker.Embryos or larval fish were placed individually in every other well of a white 96-well Optiplate  with 200 μl of Holtfreter solution . This burst of luminescence is typically followed by a low background level of luminescence (< 50 cps). Furthermore, intermediate levels of spikes were also found in many plots just before the burst of high bioluminescence. Therefore, in order to eliminate data from dead fish, data that exceeded 5,000 cps, or those that went down below 50 cps, at any point of the analyzed portion of the data were first discarded. Then an averaged plot of the remaining data from each clutch of embryos was examined, and the highest count on days 8–9 was determined for experiments presented in Bioluminescence data from the Topcount were imported into Microsoft Excel 2000 by the Import and Analysis macro (kindly supplied by Steve Kay, Scripps Institute). In many of the experiments performed on the Topcount, some plates were placed in the machine several days earlier than others in order to monitor fish that experienced different numbers of LD cycles . TherefoPeriod and rhythmicity for each animal were determined by a macro  based on12–18 (Invitrogen) as the primer in a 25–40 μl reaction volume. Real-time PCR was performed in a 25 μl reaction volume containing a probe, forward and reverse primers, and qPCR Mastermix according to the manufacturer's instruction (Eurogentec, Seraing, Belgium). Each reaction was quadrupled in order to minimize pipetting errors. The primers and TaqMan MGB probes for per3 and luc were designed and synthesized by the Assays-by-Design Gene Expression service : per3 forward, 5′-GCCCTGGCAGCACCA-3′; per3 reverse 5′-GAAAGCTGGAGGACGAGGAA-3′; probe, 5′-6-FAM-CTAAGAGCTCAAAATCC-NFQ-3′; luc forward, 5′-GCAGGTGTCGCAGGTCTT-3′; luc reverse, 5′-GCGACGTAATCCACGATCTCTTTT-3′; probe, 5′-6-FAM-TCACCGGCGTCATCG-NFQ-3′. The ABI Sequence Detection System 7000 (Applied Biosystems) was programmed to perform the following protocol: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Total RNA was extracted from 9–42 embryos or larval fish raised in petri dishes using TRIzol reagent . The number of animals used for each extraction was recorded. Once extracted, total nucleic acid concentration was determined by a spectrophotometer. In order to prevent genomic DNA contamination, RNA samples were treated with Turbo DNA-free (Ambion, Austin, Texas, United States), and the concentration determined again by a spectrophotometer. Total RNA (0.5–1 μg) was subjected to cDNA synthesis by Superscript II Reverse Transcriptase (Invitrogen) using Oligo (dT)per3 or luc cDNA per animal was calculated by the standard-curve method [per3 and luc were compared between two different developmental stages as well as among different times of the day. It was also important to calculate the amount of each mRNA species per animal in order to compare these data to bioluminescence data. The amount of a specific control RNA, as well as the total RNA, may differ among fish of different ages, in which case RNA per animal cannot be calculated by the relative quantification method using a constitutive control. As a concentration standard, a single-stranded DNA oligonucleotide of known concentration was used for each gene. These oligonucleotides span from the 5′ end of the forward primer to the 5′ end of the reverse primer, and including 75 bp for per3 and 110 bp for luc . The standard concentration was varied from 102 to 107 copies per reaction in 10-fold increments. For every qPCR experiment, reactions for standards were performed in four replicates along with reactions for cDNA samples.In this study, relative amount of e method  rather tt-test was performed. The nonparametric Wilcoxon/Kruskal-Wallis test was performed on data that were not normally distributed even after various transformations (logarithmic, square root, and inverse) were tried. Where multiple tests were performed on a set of data, the experimentwise error rate (α) was adjusted by the Dunn-Sˇidák method [To test whether percentages of rhythmic fish among different experimental groups were equal, the G-test was performed using Microsoft Excel 2000 according to Sokal and Rohlf . If multk method .Protocol S1(27 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers of the sequences discussed in this paper are per 3 cDNA (NM_131584) and BAC clone CH211–138E4 (AL929204). The Ensembl (http://www.ensembl.org/Danio_rerio/) ID of the flanking gene of per 3 mentioned in The GenBank (


Microarrays can survey genome-wide expression patterns. Not only can these gene expression profiles be used to identify a few genes of interest, they are now being creatively applied for hypothesis generation and testing Microarrays are used to survey the expression of thousands of genes in a single experiment. Applied creatively, they can be used to test as well as generate new hypotheses. As the technology becomes more accessible, microarray analysis is finding applications in diverse areas of biology. Microarrays are simply a method for visualizing which genes are likely to be used in a particular tissue at a particular time under a particular set of conditions. The output of a microarray experiment is called a “gene expression profile.”Gene expression profiling has moved well beyond the simple goal of identifying a few genes of interest. The notion that this is the major objective of microarray studies has engendered the oft-repeated criticism that the approach only amounts to “fishing expeditions.” The sophistication of microarray analysis very much blurs the distinction between hypothesis testing and data gathering. Hypothesis generation is just as important as testing, and very often expression profiling provides the necessary shift in perspective that will fuel a new round of progress. In many gene expression profiling experiments, the hypotheses being addressed are genome-wide integrative ones rather than single-gene reductionist queries. In general, without a hypothesis only the most obvious features of a complex dataset will be seen, while clear formulation of the scientific question undoubtedly fuels better experimental design. And in some cases, the results of a microarray screen that was initially designed as an effort at cataloguing expression differences are so unexpected that they immediately suggest novel conclusions and areas of enquiry.All microarray experiments rely on the core principle that transcript abundance can be deduced by measuring the amount of hybridization of labeled RNA to a complementary probe. The idea of a microarray is simply to lay down a field of thousands of these probes in perhaps a 5 sq cm area, where each probe represents the complement of at least a part of a transcript that might be expressed in a tissue. Once the microarray is constructed, the target mRNA population is labeled, typically with a fluorescent dye, so that hybridization to the probe spot can be detected when scanned with a laser. The intensity of the signal produced by 1,000 molecules of a particular labeled transcript should be twice as bright as the signal produced by 500 molecules and, similarly, that produced by 10,000 molecules half as bright as one produced by 20,000 molecules. So a microarray is a massively parallel way to survey the expression of thousands of genes from different populations of cells. Trivially, if fluorescence is observed for a gene in one population but not another, the gene can be inferred to be on or off, respectively. With appropriate replication, normalization, and statistics, though, quantitative differences in abundance as small as 1.2-fold can readily be detected. The output of all microarray hybridizations is ultimately a series of numbers, which covers a range of almost four orders of magnitude, from perhaps one transcript per ten cells to a few thousand transcripts per cell .It is the comparison of gene expression profiles that is usually of most interest. This is because the visualization is done at the level of transcript abundance, but just seeing a transcript does not guarantee that the protein is produced or functional. If, however, a difference in transcript abundance is observed between two or more conditions, it is natural to infer that the difference might point to an interesting biological phenomenon.A general approach to performing gene expression profiling experiments is indicated as a flow diagram in The ability to survey transcript abundance across an ever-increasing range of conditions gives geneticists a fresh look at their cellular systems, in many cases providing a more holistic view of the biology, but at the same time feeding back into the classical hypothetico-deductive scientific framework. The technology has rapidly advanced beyond the simple application of fishing for candidate genes and now sees applications as diverse as clinical prediction, ecosystem monitoring, quantitative mapping, and dissection of evolutionary mechanisms.Two of the better-known examples of the interplay between microarray profiling and hypothesis testing are provided by the studies of Much excitement has been generated recently by the potential for clinical applications of gene expression profiling in relation to complex diseases such as cancer, diabetes, aging, and response to toxins. An early foray into this realm was provided by PLoS Biology by DeRisi and colleagues (Plasmodium falciparum might identify a handful of genes that are induced at critical times and hence might be novel drug targets. Employing very careful staging, a platform with low experimental noise, and appropriate statistical procedures, they discovered an extremely tight molecular lifecycle within the organism. Families of functionally related genes are induced as a unit, one after another, in a tightly orchestrated rhythm that testifies to incredible integration of the physiology of the parasite. They show that with microarray analysis it is possible to model the physiology and biochemistry of the pathways instead of just targeting a few genes.A good example of the ability of microarray analyses to simply surprise us is provided by the study reported in this issue of lleagues . They reIn the coming years, expect to see microarrays developed for an extremely diverse range of organisms and applied to an even wider range of questions, from parasitology to nutritional genomics. Consensus on a core set of statistical options will likely emerge, as will agreement on data quality standards. Applications will encompass defining gene function; inferring functional networks and pathways; understanding how variation is distributed among individuals, populations, and species; and developing clinical protocols relating to cancer prognosis and detection of toxin exposure. Similar profiling methods for proteins and metabolites will attract just as much attention as functional genomics, building on the foundations laid by genome sequencing.