To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with infective endocarditis (IE) in internal medicine; and to compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA.Fifty consecutive patients with IE underwent ANCA testing. The medical records of these patients were reviewed.P = 0.02); and more frequently: weight loss (P = 0.017) and renal impairment (P = 0.08), lower levels of C-reactive protein (P = 0.0009) and serum albumin (P = 0.0032), involvement of both aortic and mitral valves (P = 0.009), and longer hospital stay (P = 0.016). Under multivariate analysis, significant factors for ANCA-associated IE were: longer hospital stay (P = 0.004), lower level of serum albumin (P = 0.02), and multiple valve involvement (P = 0.04). Mortality rate was 25% in ANCA patients; death was because of IE complications in all these patients.Of the 50 patients with IE, 12 exhibited ANCA (24%). ANCA-positive patients with IE exhibited: longer duration between the onset of first symptoms and IE diagnosis . Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was due to IE complications in all patients, our data suggest that aggressive therapy may be required to improve such patients’ outcome. Our aims were to: determine the prevalence of ANCA in patients with IE; and compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA.Interestingly, ANCA have further been reported during the course of various infections, such as: viral , bacterial , and informed consent was obtained from all patients.From January 2010 to December 2014, 162 consecutive patients with IE were seen in the Department of Internal Medicine at the university of Rouen medical center. The definite diagnosis of IE was based on the modified Duke criteria.First, the medical records of patients with IE were reviewed for patients general characteristics at diagnosis: age and sex; comorbidities, such as arterial hypertension (cutoff 140/90 mm Hg), diabetes mellitus and cancer; previous medical history of: intravenous drug abuse, predisposing valvular disease , IE, endocavitary device, including pacemaker, cardioverter-defibrillator, left ventricular assist device, prosthetic material, that is, prosthetic heart valve, intravenous graft material, prosthetic joint, bone plate/screw, orthopedic rod; and immunosuppressive therapy for >30 days at time of IE diagnosis, including steroids, cytotoxic drugs, antitumor necrosis factor α, and rituximab.Constitutional symptoms: fever ≥38 °C, chills, asthenia, weight loss13 heart murmurs; vascular features, including arterial emboli, septic pulmonary infarctus, mycotic aneurysms, purpura, and Janeway lesions; rheumatologic signs: arthralgia, myalgia; and immunologic features, that are Osler nodes and renal impairment. In these patients, renal involvement was dichotomized into: acute renal failure and progressive renal failure. Kidney biopsies were designated as having the following patterns: mesangial proliferative glomerulonephritis (GN), focal necrotizing and crescentic GN, diffuse necrotizing and crescentic GN, focal proliferative or diffuse proliferative GN, membranoproliferative GN, thrombotic microangiopathy, and acute tubular injury; immunofluorescence pattern was determined, using fluorescein-tagged polyclonal rabbit antihuman antibodies to: C3, IgA, IgG, IgM, fibrinogen, and κ/λ-light chains. The outcome of renal involvement was determined as follows: complete resolution, characterized by normalization of serum creatinine values; persistent renal impairment characterized by persistent increase of serum creatinine 0.2 mg/L above baseline values; and end-stage renal involvement, requiring renal dialysis.14Systemic manifestations, especially: congestive heart failure defined according to the New York Heart Association classification system,All the patients had undergone the same routine clinical evaluation to investigate IE, as follows:15; and complications of IE such as paravalvular abscess and valvular perforation.Second, all patients had undergone transthoracic and/or transoesophageal echocardiography to detect: valvular impairment: aortic, mitral, tricuspid; localization and size of vegetations. Site of IE acquisition was defined following International collaboration on endocarditisThird, the medical records of patients with IE were reviewed for laboratory characteristics at diagnosis of IE: C-reactive protein (mg/L), hemoglobin level (g/dL), leukocytosis (G/L), creatininemia (μmol/L), serum albumin (g/L); urinanalysis: hematuria, proteinuria; microbiological data: number of positive blood cultures, identification of pathogen microorganism.15 Prosthetic valve IE was determined as an infection, involving: valve prothesis, reconstructed native heart valves whether a mechanical prosthesis and/or a bioprosthetic xenograft, stented or unstented, and/or a repaired native valve with implantation of an annular ring.16Infective endocarditis features also included the timing of initial symptoms and patients’ hospitalization. Thus, IE was classified as: community-acquired IE, when IE was diagnosed at the time of admission or ≤48 hours of admission; health care-associated IE, when IE was diagnosed within 48 hours of admission in outpatients as follows: intravenous therapy, wound care or specialized nursing care at home ≤30 days before IE onset; attendance at hospital and/or intravenous chemotherapy ≤30 days before IE onset; hospitalization for ≥2 days in the 3 months days before IE onset; and residence in a long-term care facility; and nosocomial IE, when IE developed in patients hospitalized for >48 hours.Antimicrobial therapyImmunosuppressive therapy, including steroids and cytotoxic drugsAnd surgical procedures of IE complicationsFurthermore, all patients with IE received specific therapy. Data regarding patients’ therapy were reviewed for treatment used at any time during the course of IE, that is:In addition, all patients underwent follow-up to determine the outcome of IE as follows: resolution; onset of complications; and in-hospital mortality (because of IE or to another cause than IE). Finally, the duration of hospital stay was also checked for all patients.20 Values above 20 IU/mL were considered positive.Antineutrophil cytoplasmic antibodies testing was performed using indirect immunofluorescence assay in ethanol-, formalin-, and methanol-fixed neutrophils. The ANCA staining pattern [cytoplasmic (c-ANCA) and perinuclear (p-ANCA)] was also determined by indirect immunofluorescence assays . Furthermore, PR3 and MPO-ANCAs were detected by commercially available enzyme-linked immunosorbent assay (ELISA) .We compared various features between the group of IE patients with and without ANCA, that is, median age, sex, comorbidities, previous medical history, constitutional, and systemic manifestations; biochemical findings; characteristics of IE; outcome of IE; and median duration of hospital stay.Statistical analyses were conducted to assess the features of ANCA-associated IE. Patients were divided into 2 groups consisting of IE patients with and without ANCA.P values less than 0.05 were considered significant in all performed tests.For group comparison involving binary data, we used the Fisher exact test. Comparisons involving continuous data were performed using: Student test when distribution of variables was normal and Mann–Whitney test in other patients. The results were reported as the odds ratio (OR) and 95% confidence interval (95% CI); P values <0.1, we further proceeded with multiple logistic regression, using backward stepwise selection, to calculate multivariate OR (95% CI); the used level of significance was P < 0.05.Finally, as regards variables with In addition to the current study, we conducted a literature review of the Medline database (1966–2015) , reviewing articles of all languages, and we compared data from the current report with previously published data.Among the 162 consecutive patients with IE, findings of serum samples for ANCA were available for 50 patients. Indeed, from July 2013 to December 2014, all patients with IE (N = 50) systematically underwent ANCA testing.Altogether, these 50 latter patients with IE were included in the study. Nevertheless, the characteristics of patients who did not undergo ANCA testing (from January 2010 to June 2013) were not different from those of patients who had ANCA testing .Using ANCA staining pattern, patients exhibited c-ANCA (N = 6) and p-ANCA (N = 5).Regarding ELISA test, patients had: PR3-ANCA (N = 4), MPO-ANCA (N = 1), PR3 + MPO-ANCA (N = 2). One of them exhibited high ELISA PR3 level without immunofluorescence positivity.P = 0.58), sex (P = 0.71); comorbidities: arterial hypertension (P = 0.11), diabetes mellitus (P = 0.74), and cancer (P = 1); previous history of: intravenous drug abuse (P = 1), predisposing condition on native valve (P = 0.70), IE (P = 1), pacemaker or cardioverter-defibrillator (P = 1), prosthetic heart valve (P = 0.47), and prosthetic joint (P = 1) (Table P = 0.24) between both groups of patients.We failed to show any statistically significant differences between ANCA-positive and negative patients with IE regarding: age (1) Table . FurtherP = 0.02). Furthermore, ANCA-positive patients more often exhibited weight loss at IE diagnosis (P = 0.017).The median duration between the onset of first symptoms and IE diagnosis was longer in ANCA-positive patients , chills (P = 0.66), asthenia (P = 1); arterial emboli (P = 0.51), heart failure (P = 1), heart murmurs (P = 0.16); arthralgia (P = 0.42), myalgia (P = 0.65); purpura (P = 0.34), Janeway lesions (P = 1), Osler nodes (P = 0.43); spondylitis (P = 0.62); neurologic involvement (P = 0.49), including cerebral emboli (25% versus 18%), cerebral hemorrhage (4% versus 8%), cerebral mycotic aneurysms (8% versus 0%), and amyloid-like angiopathy (0% versus 3%); splenic infarction (0.14); and pulmonary abscess (P = 1) , although not significantly so , hemoglobin (P = 0.02), and serum albumin (P = 0.0032).As shown in Table P = 1). The door-of-entry of IE was found in half of our ANCA-positive patients, as follows: colonic polyp (N = 2), intravenous drug abuse (N = 1), tooth extraction (N = 1), cardiac catheterization (N = 1), and chronic supra-pubic catheter (N = 1).Patients with and without ANCA did not differ for prevalence of community-acquired IE, health care-associated IE or nosocomial IE (P = 0.009). The prevalence of vegetations on echocardiography (P = 1) as well as the size of vegetations (P = 0.98) were not different between IE patients with and without ANCA ; valvular perforation (P = 1) and cardiac dysfunction, as shown by similar median value of left ventricular ejection fraction .On echocardiography, there was also no significant difference between patients with and without ANCA regarding the following IE complications: paravalvular abscess (P = 0.73), Streptococcus spp. (P = 0.73), Enterococcus spp. (P = 1), Gram-negative bacillus (P = 0.32), or absence of micro-organism (P = 0.58) (Table P = 0.15), although not significantly so.In addition, we failed to find differences between patients with and without ANCA regarding the causative pathogen microorganism of IE, that is, Staphylococcus spp. (8) Table . FinallyP = 0.69) and combined therapy of antimicrobial agents and surgery procedures . Surgical therapy consisted of: debridement associated with valvuloplasty (33% versus 43%) and heart valve replacement (67% versus 57%). Furthermore, 2 patients in ANCA-positive group also received immunosuppressive therapy [bolus of intravenous corticosteroids: 750 mg (N = 1) and 1 g N = 1)] because of initial diagnosis of systemic vasculitis. By contrast, no misdiagnosis was made in ANCA-negative group (P = 0.054).Therapy of IE did not differ between patients with and without ANCA for: antimicrobial therapy alone . Death was directly because of IE complications: in the 3 patients with ANCA (25%): multiple organ failure (N = 2), end-stage renal failure (N = 1); and in the 9 patients without ANCA (24%): cerebral hemorrhage (N = 3), heart failure (N = 2), respiratory distress (N = 1), cardiopulmonary failure (N = 1), multiple organ failure (N = 2), and sudden death (N = 1). Finally, the median duration of hospital stay was higher in ANCA-positive patients .Furthermore, in-hospital mortality was similar between IE patients with and without ANCA  and multiple valve  involvement  were significant factors for IE associated with ANCA.Longer duration of symptoms before diagnosis ; and PR3 ANCA compared with MPO-ANCA and PR3 + MPO-ANCA (63% versus 17% versus 10%)  have peptides homologous to cPR3; and may trigger an autoantibody response.63 It has been speculated that complementary peptides to PR3 could be beneficial to pathogens expressing cPR3 by binding to/neutralizing the antimicrobial properties of PR3 and MPO64; molecular mimicry between epitopes of self-antigens and antibodies to microorganisms.47 Lysosome-associated membrane protein-2 (LAMP-2) is a glycosylated type 2 membrane protein expressed on the membrane of neutrophil intracellular vesicles, containing PR3 and MPO.65 Anti-LAMP-2 ANCA cross react with the adhesive bacterial fimbrian protein Fim-H, which is detected in many Gram-negative bacteria; thus, previous investigators have suggested that anti-LAMP-2 ANCA may be because of molecular mimicry following Gram-negative bacteria (with Fim-H)47; neutrophil extracellular traps formation may result in the synthesis, in neutrophils, of antimicrobial proteins such as PR3 and MPO.47 It seems to play an important role in bacterial infection  control;60 and Toll-like receptors. The bacterial DNA hypomethylated motifs (ligands for Toll-like receptor 9) have been found to lead to the in vitro production of ANCA by B-lymphocytes.47Mechanisms of ANCA-induction are still unclear in patients with IE. Previous authors have postulated that ANCA formation in IE may result from: autoantigen complementarity has been suggested one of the mechanisms breaking tolerance to ANCA antigens.From a practical point of view, the identification of features of ANCA-associated IE appears to be crucial, to improve both early diagnosis and management in these patients. Nevertheless, to our knowledge, no predictive factors for ANCA-associated IE have been clearly defined to date.61 ; in these patients, the median age at IE diagnosis was 53.5 years61 In this instance, IE patients with ANCA, compared with those without, more frequently presented: deterioration of general status with weight loss; and renal impairment (58% versus 26%). In 29 IE-associated biopsy proven GN, 8 patients (28%) had ANCA. Unfortunately, these investigators did not compare the clinical and histologic features of GN between IE patients with and without ANCA.7 In our 3 ANCA-positive patients with biopsy proven GN, patients exhibited diffuse proliferative GN (N = 2) and necrotizing/crescentic GN (N = 1). Our findings suggest that IE patients with ANCA should undergo systematic investigations to detect underlying life-threatening renal complications, although no definite conclusion can be drawn from our data. Our literature review, in fact, identified 24 case reports of documented ANCA-positive patients with IE exhibiting renal impairment.61 Twenty-one of these latter patients underwent renal biopsy showing: extracapillary GN with immune deposits (N = 5), pauci-immune GN (N = 4), segmental and focal necrotizing GN (N = 4), endocapillary GN with immune deposits (N = 3), interstitial nephritis (N = 1), chronic sclerotic GN (N = 1), both pauci-immune GN and interstitial nephritis (N = 2), and both endocapillary GN and interstitial nephritis (N = 1).61 Altogether, our data suggest that whether patients with ANCA-associated IE, develop commonly renal impairment, renal damage are nonspecific in these patients. Some damage could mimic ANCA-associated vasculitis renal attempts (pauci immune GN). On the contrary, in ANCA-negative IE, other authors reported that the most common biopsy finding was crescentic GN followed by endocapillary proliferative GN.67 Interestingly, in our experience, most ANCA-positive patients with IE had favorable outcome of renal impairment (N = 4/7). Thus, we suggest that renal biopsy may be performed in the population of ANCA-positive patients with IE, who do not exhibit improvement of renal function tests after initiation of appropriate antibiotic therapy, to detect other underlying condition responsible for renal dysfunction.In our review, we further found that the main clinical manifestations of ANCA-associated IE were: asthenia (84%), fever (69%), weight loss 58%), and renal impairment (71%) , renal impairment, and ANCA has resulted in the diagnosis at later stage of IE. In our patients, weight loss, asthenia, and low fever (≤38.5 °C) were indeed the only symptoms of IE in up to 17% of patients. It is thus crucial to maintain a low threshold for investigation of IE in these ANCA-positive patients to avoid underdiagnosis. Also, the onset of a new heart murmur is an important clinical sign for the diagnosis of IE at earlier stage in these patients.69 Comparing previous and current reports of ANCA-associated IE and granulomatosis with polyangiitis,69 we have interestingly observed the following distinct features between both groups of patients, that is: pulmonary  and articular signs  were less common in ANCA-associated IE; fever (≥38 °C)  and weight loss were more frequent  in ANCA-associated IE; and up to 25% of our ANCA-associated IE patients presented splenic infarction, whereas splenic infarction is an uncommon complication of ANCA-associated vasculitis.73 On the contrary, we failed to find difference regarding renal involvement, cutaneous symptoms, or neurologic involvement between ANCA-positive patients with IE and patients with granulomatosis and polyangiitis  in ANCA-positive patients.49 This discrepancy may be explained by the predominance of S aureus, which has increasingly been described in America and Europe.75As with IE in the general population, the main causative pathogen microorganisms in ANCA-positive patients with IE were Staphylococci and Streptococci.A potential door-of-entry of IE was found in only 50% of our ANCA-positive patients; these data may be explained by the fact that most of ANCA-positive patients had community-acquired IE (83%); of note, no patient had health-care-associated IE and 17% of patients had nosocomial IE. In this instance, the prevalence of community-acquired IE and nosocomial IE was similar in both groups of patients.58 In this instance, we did not experience cases of ANCA-positive patients with IE related to Bartonella spp. On the contrary, in our literature review, we have noted an overrepresentation of IE related to Bartonella (mainly Bartonella henselae and Bartonella quintana) in ANCA-positive patients59. Indeed, Aslangul et al34 reported ANCA positivity in up to 60% of patients with Bartonella IE. These authors have postulated that Bartonella effects in endothelium with recruitment of macrophages and leukocytes associated with delayed leukocytes apoptosis may lead to ANCA production.76 These data suggest that ANCA-positive patients with IE, exhibiting negative blood cultures, should undergo Bartonella serology and/or research by polymerase chain reaction in heart valves (in those requiring cardiac surgery).Culture-negative IE accounted for as high as 17% of our ANCA-positive patients. Our findings confirm previous authors’ data, reporting culture-negative IE in 12% of such patients.61  or aortic (39%) valvular vegetations, and both mitral and aortic vegetation (18%).59 Thus, in our ANCA-positive patients, we suggest that diagnosis of IE at later stage and immunosuppression (related to older age and marked weight loss) may have resulted into increased bacterial mitral and aortic colonization. Our data are relevant as they suggest that the presence of multiple valve involvement may be a predictive marker of IE rather than systemic vasculitis in ANCA-positive patients. Because of the small number of ANCA-positive patients with IE, further investigations, however, are warranted to confirm our findings.Interestingly, Chirinos et al52 it is possible that this discrepancy may be related to: older age and higher prevalence of Staphylococcus infection in our cohort; and low rate of surgery (25%) in our patient, as early surgery is believed to be an effective treatment to decrease mortality in patients with IE.79 In the current study, only in-hospital follow-up, however, was available for our patients. Thus, we cannot exclude that IE patients with ANCA have a decreased late survival; in fact, because of multiple valve involvement, probability of valvular surgery may be higher at long-term follow-up in these patients, leading to increased morbidity and mortality of valvular surgery.22In addition, another finding in our study was that hospital stay was longer in our ANCA-positive patients with IE. Our data may be, in part, explained by the fact that these patients had diagnosis of IE at later stage, resulting in more severe forms of IE, including multiple valve involvement, renal impairment, and deterioration of general status with marked weight loss. Furthermore, the overall in-hospital mortality rate was 25% in our ANCA-positive patients with IE, which is higher than the rate of 10% reported in the literature;In conclusion, our study identifies a high prevalence of ANCA in unselected patients with IE in internal medicine (24%). Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was because of IE complications in all patients, our data suggest that aggressive therapy, including valvular surgery, may be required to improve such patients’ outcome; although, only randomized trials would specify whether valvular surgery may improve the prognosis of such patients.


Outcome variables were measured during two ad libitum baseline days, a four-day intervention of either decreased TWI (HIGH) or increased TWI (LOW), and one ad libitum recovery day. Results: During the four-day intervention, HIGH and LOW experienced differences in thirst (p = 0.002); also, a statistically significant change of AVP occurred , with no effect (TWI or day) on aldosterone and serum osmolality. Urine osmolality and volume distinguished HIGH from LOW (p = 0.002) when they consumed similar 24 h TWI.Background: Surprisingly little is known about the physiological and perceptual differences of women who consume different volumes of water each day. The purposes of this investigation were to (a) analyze blood osmolality, arginine vasopressin (AVP), and aldosterone; (b) assess the responses of physiological, thirst, and hydration indices; and (c) compare the responses of individuals with high and low total water intake  when consuming similar volumes of water each day and when their habitual total water intake was modified. Methods: In a single-blind controlled experiment, we measured the 24 h total water intake  of 120 young women. Those who consumed the highest (HIGH, 3.2 ± 0.6 L·day The me, etc.) . The dire, etc.) ,5. Howevontinuum ; if this. For this reason, controversy has arisen surrounding scientific findings related to purported benefits of consuming larger volumes of fluid on a daily basis. Although avoidance of low water intake has been linked to such benefits as improved mood [Humans’ capability to function normally across a wide range of daily fluid intakes has resulted in the scientific community’s ability to suggest daily water intake only as “adequate intake” as opposed to the “recommended intake” associated with less tightly regulated nutrients ,7. Thus,ved mood ,16, imprved mood ,18, and ved mood ,20, the ved mood . This, cved mood ,23.et al. randomized participants to three days of either water restriction (1 L∙day−1), or to water loading (6.8 L∙day−1) followed by three moderate intake days, and then switched treatments. This type of intervention is valuable in terms of characterizing general responses to fluid extremes. However, it does not account for potential differences due to the participants’ previous habitual water intake. Prior research that has accounted for habitual water intake prior to a change in water intake has demonstrated that urinary markers of hydration respond similarly between groups [i.e., hormonal concentrations and perception of thirst) have yet to be characterized. Thus, it is important to further illustrate the perceptual and physiological responses to changes in water intake within the normal physiological range after accounting for previous fluid intake.The investigations mentioned above would seem to suggest that there is no difference. However, many previous studies that would support this thought have altered water intake after assuming all participants are equal when consuming their habitual intake. For example, the intricate work of Shore n groups ,25, howe−1) and high  volumes of water during daily activities. The purposes of this research were to (a) analyze blood osmolality, AVP, and aldosterone; (b) assess the perceptual responses of thirst; and (c) compare the responses of HIGH and LOW, when consuming similar volumes of water each day and when their habitual total water intake (TWI) was modified. We hypothesized that during the four-day intervention that AVP concentration  and urinary hydration indices would change significantly in both HIGH and LOW. We also hypothesized that, when consuming a similar 24 h TWI, the LOW and HIGH groups would be similar with regard to all measured variables. If these hypotheses were upheld, then it could be confirmed that the only difference between HIGH and LOW are the concentration and volume of their urine. However, observed differences in response to water intake change would constitute evidence of physiological adaptation to either chronic high or low water intake.The present investigation was designed to clarify differences between free-living healthy women who consume low  provided written informed consent to participate. This study was conducted according to the guidelines of the Declaration of Helsinki and all procedures involving human subjects were reviewed and approved by the University of Connecticut Institutional Review Board for human research (Protocol H10-144). The results presented here are a follow-up analysis of an experiment that has resulted in other publications ,26 but iWe identified the habitual daily water intake of 120 healthy young women; all were oral contraceptive users. Participants were instructed to maintain their regular diet over five consecutive days, while recording all food and fluids consumed (days 1–5). Participants returned to the laboratory on days 2–6 between 0530 h and 0800 h to review the previous 24 h diet record with a nutritional consultant. Daily beverage volume  was calculated directly from participant diet records, and 24 h TWI (including moisture in foods) was computed via commercial software .n = 14) habitually consumed the largest 24 h TWI volume (3.2 ± 0.6 L·day−1) and the second  habitually consumed the smallest daily TWI volume . These women, verified again as oral contraceptive users, provided separate written consent to participate in modified TWI. Selected information was withheld from the participants, including their TWI rank and group status, to avoid influencing or biasing normal fluid consumption during the baseline days.The purpose of the experimental intervention was to compare fluid-electrolyte regulatory hormones and thirst ratings in two distinct subsamples of the original 120 women. The first subsample , four days of modified fluid intake , and one recovery day when each group returned to ad libitum fluid intake . During the treatment (days 3–6), the TWI of each group was modified to approximately match the habitual mean TWI of the other group. Although both groups of 14 women were observed during periods of large and small water intake, the group names HIGH and LOW are used in this manuscript to represent habitual baseline TWI.During the experimental phase of the investigation, women were observed on seven consecutive days of the experimental intervention: two days of baseline Participants reported to the laboratory between 05:30 h and 09:00 h. Arrival time did not differ more than 1 h on all experimental days. Day 1 was individualized for each participant, occurring on the self-reported first placebo day of her oral contraceptive pill pack; this allowed testing to occur when plasma estrogen and progesterone concentrations were relatively low. Beginning on the morning of Day 1, subjects collected consecutive 24 h urine samples that were returned during each subsequent morning laboratory visit. On the morning of days 1–8, one blood sample was drawn from an antecubital vein via single venipuncture, and was collected in serum and plasma EDTA tubes . Filled serum tubes were allowed to clot at room temperature. Next, all blood samples were centrifuged at 5000 rpm for 15 min at −4 °C . Serum osmolality was measured immediately following centrifugation. The remaining serum and all plasma samples were separated into aliquots and frozen at −80 °C until additional assays were performed within three months, as described below. All 24 h urine collections (days 1–7) were analyzed for total volume by mass , and urine osmolality by freezing point depression .i.e., days 1–2, 2–3, 3–4, 4–5, 5–6, 6–7, and 7–8). Body mass , blood variables, and perceived thirst were recorded during each of eight morning visits, on days 1–8. Thus, three baseline measures were obtained on these acute variables  because the third measurement occurred on the morning of day 3, immediately prior to the start of modified fluid intake. All variables were finalized on the morning of day 8 because they involved response to the recovery day (day 7). Thirst was assessed with a 9-point Likert scale [Some variables were measured across seven continuous 24 h periods, and others were evaluated on eight consecutive mornings. Specifically, TWI and urine collections were evaluated from one morning to the next, during seven 24 h periods  that was provided each morning in plastic bottles. This water contained a low mineral content . Due to additional moisture in solid foods, the TWI per 24 h was greater than these volumes.During the intervention (days 3–6), participants were instructed to maintain normal solid food choices, but to consume water as their only fluid. Members of HIGH were instructed to reduce their fluid intake to 1.3 L·day−1. Purification and concentration of samples twenty-fold, in accordance with the manufacturer’s instructions, was performed to bring the AVP concentrations into a detectable range. The intra-assay coefficient of variation (CV) was 7.4% and the inter-assay CV was 8.1%. The assay wavelength was read at 405 nm on a tunable microplate reader . Serum aldosterone was analyzed by Enzyme-linked immunosorbent assay  in accordance with the manufacturer’s instructions, with a sensitivity of 15.0 pg·mL−1, an intra-assay CV of 9.2%, and inter-assay CV of 6.2%. The assay wavelength was read at 450 nm on the microplate reader described above.Plasma arginine vasopressin (AVP) was analyzed by enzyme immunoassay , with sensitivity of 23.0 pg·mLn = (Zα/2·σ/E)2 where z = critical value (1.96), σ = expected standard deviation (1.03 L) based on the above reference, and E = the allowed margin of error (0.20 L). Given this equation, the calculated minimum sample size was 102. For the intervention, a critical sample size of four participants was calculated, based on previous observations of AVP during water deprivation [1 = 0.5 pmol·L−1, μ2 = 3.0 pmol·L−1, σ = 1.0, and β = 0.9.The initial sample size of 120 women was chosen in an effort to achieve a sample mean similar to that of previous investigations  and to arivation , with μ1post-hoc independent sample t-tests were used to compare HIGH and LOW groups at specific TWI. When main effects of fluid volume or an interaction effect (TWI × group) was noted, post-hoc paired-sample t-tests were used to compare days 1–2 versus days 3–6 within HIGH and LOW.Commercially-available software  was utilized to perform all statistical analyses. Two-way (TWI × group) repeated measure analysis of variance (ANOVA) was used to test each dependent variable during the fluid modification period. In this instance, TWI refers to the fluid volume that either HIGH or LOW consumed during the treatment. For example, HIGH consumed a larger fluid volume during days 1–2, but LOW consumed a larger fluid volume during days 3–6. Therefore, the first time point entered in the statistical analysis represented the mean values from days 1–2 for HIGH, and days 3–6 for LOW. Accordingly, the second time point corresponded to the mean value of days when both groups were consuming a smaller TWI . Days 7–8 were excluded from this analysis. Sphericity was assumed for within-subject effects unless Mauchley’s test of sphericity was violated, in which case the Greenhouse-Geisser correction was used. When an effect of group was noted with ANOVA, i.e., days 1–2 versus days 3–6) as a covariate during analyses of covariance (ANCOVA).The volume of fluid prescribed to HIGH on days 3–6 resulted in a higher TWI than the volume consumed by LOW on days 1–2. Therefore, dependent variable ANOVAs that would be directly influenced by this discrepancy  were corrected using the absolute value of TWI change , four during the treatment , and one during recovery (day 8) to describe the time course of responses to abrupt changes in TWI. When a main effect of time was detected, simple contrasts were used to compare individual data points to the baseline mean.Fluid regulatory hormone concentrations were analyzed separately for HIGH and LOW via one way repeated measures ANOVA across eight time points: three during Lastly, the relationship between AVP and perception of thirst values was compared between groups via individual regression analysis. Each participant provided two values to the analysis, a mean value during small fluid intake and a mean value during large fluid intake. The individual lines of regression were then compared for similarities of slope and y-intercept .−1) frequency distribution (mean of five days) of the original sample of 120 women, during screening. Although this distribution (mean ± SD for TWI was 2.3 ± 0.8 L·day−1) is moderately, positively skewed  and is slightly leptokurtic (excess kurtosis 0.33), neither characteristic influenced the selection of test participants. Based on ad libitum TWI, the 14 women in HIGH  were selected because they habitually consumed a large 24 h TWI volume (3.2 ± 0.6 L·day−1). The 14 women in LOW  were selected because they habitually consumed a small daily TWI volume .Measurements across five days allowed investigators to determine the TWI of 120 healthy young women, with the intent to compare these findings to published recommendations of daily adequate intake of water for women ,7. Figur−1), and LOW consumed 219% more than baseline TWI (3.5 ± 0.1 L·day−1). Baseline TWI volumes did not differ from those observed during the screening period. A main effect of TWI (L·day−1) was observed as HIGH and LOW modified their daily TWI  = 33.08, p < 0.001). A significant interaction effect was observed for the absolute volume of fluid intake during smaller and larger volumes of fluid intake  = 14.25, p = 0.001). Post-hoc analyses and mean values for each stage of the treatment revealed different TWI volumes for HIGH and LOW, when each was consuming a smaller volume of water  = 6.06, p = 0.02) and urine osmolality decreased  = 4.35, p = 0.04) as TWI increased within both groups  = 11.94, p = 0.002), and urine osmolality  = 8.76, p = 0.007). No significant effect of TWI or group on serum osmolality existed throughout the entire investigation: HIGH days 1–3, 293 ± 3 mOsm·kg−1; days 4–7, 294 ± 2 mOsm·kg−1, LOW days 1–3, 295 ± 4 mOsm·kg−1; days 4–7, 293 ± 2 mOsm·kg−1.Urine volume increased  = 98.43, p < 0.001). Plasma AVP also was analyzed over time and a significant effect of day  = 21.39, p < 0.001) and LOW  = 18.87, p < 0.001); only AVP concentrations during the treatment (days 3–6) differed from baseline time points. During recovery , AVP concentrations were similar to those observed during baseline, for both groups. No between-group differences were observed when groups were consuming similar total water volumes (A significant main effect of TWI on plasma AVP concentration existed (t of day  was obse volumes A.−1).The mean serum aldosterone concentration in morning serum samples exhibited no main effect of TWI or group, and no significant interaction as a result of the treatment  = 18.70, p = 0.001) was observed. Also, a main effect of group  = 8.03, p = 0.014) also was observed, with HIGH consistently rating thirst greater than LOW, regardless of TWI. The relationships between plasma AVP concentration and thirst rating appear in F = 2.98, p = 0.10, R2 = 0.11) and LOW  = 3.44, p = 0.08, R2 = 0.13). The regression lines of best fit in this figure have equivalent slopes (t(48) = 0.327, p = 0.74), but the y-intercepts are significantly different (t(49) = 3.694, p < 0.001).The mean rating of thirst was compared when HIGH and LOW consumed similar TWI during the baseline and treatment periods B. A mainad libitum total water consumption of women, within a range  [−1) [−1, or 0.3 L·day−1 less than LOW (3.5 L·day−1). In this instance HIGH excreted approximately 0.3 L·day−1 less of slightly more concentrated urine. The main difference between the groups appeared in perception of thirst. Thirst was elevated in HIGH at all times, even after accounting for the small differences in fluid intake between groups at each prescriptive level . Together these findings tell us that in free-living conditions, habitual high and low drinkers both maintain fluid balance. However, their perception of thirst may be the driving force behind their habitual intake. Thus, it appears that these groups are physiologically similar in terms of their management of large and small volumes of water.The present investigation furthers our understanding of the complex effects of habitual high and low −1) [−1) . Both HIad libitum. The present database confirms their two fundamental observations regarding the general population: (a) habitual water intake is a determinant of urinary volume and concentration ; and (b) urine volume and osmolality are well-suited to track subtle differences in the hydration process which are modulated by TWI. The present investigation also extends the work of Perrier et al. in the following ways. Although AVP was determined to be a defining factor between HIGH and LOW at rest [i.e., amount of salt consumption, inclusion of alcohol with meals) influence fluid-electrolyte balance and renal function in the general population [Similar to three publications by Perrier and colleagues ,24,32, t at rest , the res at rest . Secondl at rest , and culpulation .post-hoc analysis revealed that HIGH was consuming significantly more water than LOW (2.0 ± 0.2 versus 1.6 ± 0.4 L). It is unclear if the elevated thirst in HIGH is a “secondary”  drinking behavior that results from hedonic compensation [Lastly, circulating AVP and its association with thirst due to their mutual response to osmoreceptors located within organum vasculosum of the lamina terminalis and the hypothalamus has been well established ,36, to tensation , or if tensation . Both ofensation .−1 and the threshold for stimulation of thirst can range as far as 289–299 mOsm∙kg−1 across individuals [i.e., HIGH) from those with a high thirst threshold . Conversely, we may have also separated those from a low AVP threshold  from those with a high AVP threshold . It is not possible given the current experimental design to confirm this. However, because both groups were confirmed to be in fluid balance, it would appear that they differ on the mechanism on which they rely most. Body water is conserved in HIGH through increased dependence on water intake and LOW maintains homeostasis due to increased water conservation.Regardless, it has previously been observed that the plasma osmolality threshold for AVP secretion can range as far as 276 to 291 mOsm∙kgividuals ,41. Thusividuals , may preDespite the above mentioned strengths, the methodology employed does have limitations that constrain full interpretation of the results. First, the slight difference in fluid volumes between groups at each prescriptive level was not purposeful. All efforts during the subject identification phase of the investigation were made to establish a single volume that would bring each group to the habitual TWI of the other group during the intervention. However, small differences between food, sodium, and fluid intake during observation and the experimental phases, as well as differences in water content of food may have contributed to dissimilarities between groups. Follow-up investigations should employ water and food prescription during both “habitual” water intake phases and “modified” water intake phases to limit any small discrepancy. Second, the levels of AVP observed are slightly higher than those reported in the literature, with few samples falling below the detectable limit . We attr−1) and the 24 h TWI distribution (−1) and EFSA [−1). Secondly, women at the extremes of normal daily fluid consumption (HIGH and LOW) are physiologically and perceptually distinct during their habitual drinking behavior (The following four statements summarize our findings. Firstly, the mean 24 h TWI of 120 healthy young women in the present investigation (2.3 ± 0.8 L·dayribution  support and EFSA  (2.0 L·dbehavior . Thirdlybehavior . Fourthlbehavior . Within


At all sites, low-density, dry season extension were more variable than high-density, wet season extension and on average, was lower at two of the three sites. We applied multi-variate linear regressions that revealed high-density, wet season band extension to inversely correlate most significantly to temperature . In contrast, low-density, dry season band extension was more variable and correlated most significantly with dry season chlorophyll-a (Chl-a) . Additionally, we find that corals at the site with highest dry season Chl-a have the highest dry season extension lengths. Our findings indicate that relative mixing of fresh and salt water in the wet season and primary productivity in the dry season, and their influences on aragonite saturation, are likely to impact interannual coral extension variability in marginal environments.Southern China hosts coral communities in marginal environments that are characterized by low linear extension rates, low coral cover and/or no reef formation, thus providing natural laboratories to study coral communities with below average growth rates. Here we compare the annual linear extension rates over 10 years (range 1.2 to 11.4 mm yr Marginal coral communities, classified by low coral cover and/or no reef formation, generally have multiple limiting environmental conditions, including low seawater temperatures, low pH, high nutrient concentrations, low light, and high turbidity Most studies of marginal corals have quantified the relationships between coral growth and only one or two environmental parameters, such as temperature, salinity, turbidity, or pH Porites sp. from three different regions within Hong Kong  seasonally ranging from 0.20 to 35 µg L−1−1) Hong Kong , Pak Lap Tsai  and Crescent Island   . The thrA pneumatic hand-held drill connected to a modified, high-pressure air compressor was used to collect cores of 5 cm in diameter and 30–80 cm in length. Permission to collect corals was granted by the Agriculture, Fisheries, and Conservation Department of the Government of Hong Kong, Special Administrative Region of China. Core holes were filled with a mixture of silt and coarse sand and plugged with cement. Samples HK005 and HK006 were collected whole as they were too small to drill. Samples were cleaned with deionised water and dried under the sun. The cores were labelled and cut longitudinally along the maximum growth axis. Slabs of ∼10 mm thickness were obtained using a diamond-coated trim saw. The slabs were rinsed with deionised water 10 times, submerged in an ultrasonic bath three times for 10 minutes, and then dried at 60°C for 24 hours Each coral slab was X-radiographed at the Biomedical Imaging Center in Hong Kong with the following settings: film focus distance = 100 cm; distance between machine and coral = 110 cm, exposure = 0.16 sec, voltage = 48 kV, current = 20 mA. Linear extension (growth) was measured along the primary, axial growth axis where the polyps were perpendicular to the line of slicing http://www.epd.gov.hk/epd/english/environmentinhk/water/marine_quality/mwq_home.html). EPD data from 2000–2010 were extracted for three sites: SM18 , MM15 , MM3   . EPD datThe seven environmental variables temperature (T), salinity (S), dissolved oxygen (DO), turbidity, chlorophyll a (Chl-a), total inorganic nitrogen (TIN) and pH were used, as previous studies showed that these are most relevant for coral growth Physical parameters from EPD stations at Lamma Island-SM18, Pak Lap Tsai-MM15, and Crescent Island-MM3 from 2000–2009 were used to analyze relationships between seasonal extension rates and environmental conditions for the 10 year period. High- and low-density band lengths were correlated to all seven environmental conditions from both the wet and dry seasons to identify parameters independently correlating with the band length. Pearson correlations were used for both the complete data set and the data set excluded years of exceptional growth (>3 times a quartile) in situ measurements of T, S, and dissolved inorganic carbon (DIC) with pH measurements from coral sites to investigate relative changes to Ω et al. (2008) 3 = , and bicarbonate, HCO3−, neglecting the very minor contribution by H2CO3.The saturation state of aragonite (Ω) between the Pearl River Estuary and open ocean water in Hong Kong was estimated by using previously published 2, the dissociation constant of bicarbonate to carbonate was calculated as a function of T and S according to Emerson and Hedges (2008) 2 is in mol kg−1. K2 is equal to:Ksp, is calculated as a function of T and S according to Mucci (1983) sp is in mol2 kg−2.The solubility product of aragonite, KFinally, Ω is equal to:For T and DO, raw monthly data averaged into wet and dry seasons show offsets in the mean values between seasons at all sites . The pH −1 to 11.4 mm yr−1, with an average of 4.4 (±2.2) mm yr−1 , showed the lowest and least variable growth. HK005 exhibited the lowest average annual extension (2.2±0.6 mm yr−1) among all cores while HK006 from the same site averaged 3.7±1.7 mm yr−1. The Pak Lap Tsai cores  showed average growth rates of 3.7±1.2 and 4.8±1.0 mm yr−1, respectively. Linear extension was greatest in the Crescent Island cores (avg. of 4.9±2.1 and 7.6±2.3 mm yr−1), though lower than a previously published rate of 10 mm yr−1 from the northeastern region From 2000–2009, the six corals had annual extensions ranging from 1.2 mm yr mm yr−1 . Porites2 respectively, F-test p = 0.01). The Lamma Island and Pak Lap Tsai corals generally exhibited greater extension during high-density banding  than low-density banding  . In contrast, Crescent Island corals exhibited low-density growth that tended to be higher than high-density growth  . Crescent Island showed greater low-density variability than the other two sites , although Crescent Island’s high-density variability was not significantly different from that of Lamma Island and Pak Lap Tsai .Linear extension of the low-density, dry season bands (dark) was significantly more variable than that of the high-density, wet season bands (light)  with wet season T (inverse) and S . NeitherLow-density, dark band extension was significantly correlated (p<0.05) with wet season TIN (inverse) and dry season DO, Chl-a, and TIN (inverse) both with and without the growth outlier . Dry seaAragonite saturation (Ω) in the Pearl River Estuary and eastern open ocean waters was 3.3 and 4.1, respectively , Table 4Our coral extension rate results must take into account local variability, measurement errors and the possibility of confounding influences. First of all, the exact timing of growth banding in Hong Kong is unknown and changes in timing between colonies and between years would be expected Our assumption that coral banding was likely to follow seasonal temperature shifts appears to be sound, as the high-density (light) banding correlated only with wet season (warm T) parameters and the low-density (dark) banding correlated primarily with dry season (cold T) parameters, but also with wet season TIN  and 3. WCoral extension is commonly believed to be highly positively correlated with temperature −1, r = −0.58, p<0.001, n = 47, High-density growth is most significantly described by its inverse relationship with wet season temperature. Wet season temperature in Hong Kong can exceed 30°C, but averages ∼25°C. On average, wet season temperature was lower than for typical non-marginal reefs, and should not regularly lead to thermal stress et al. (2011) In addition to T and S, aragonite saturation (Ω) is likely to play a role in coral extension. While we have no direct measure of Ω, the literature provides an understanding of changes to Ω under varying conditions. Using data from Yuan In contrast to the wet season, when physical circulation appears to play a role in growth variability, dry season, low-density growth is most significantly described by variability in Chl-a . Chl-a, Algal blooms indicated by Chl-a will also likely have an impact on Ω. The detailed study of an algal bloom showed that the bloom decreased DIC and alkalinity, and increased pH In conclusion, corals living in marginal environments are already experiencing significant limits to growth. Our study indicates that in Hong Kong, these limits are due to relative estuarine influence in the wet season, and primary production or variable Ω in the dry season.


We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified. Over the past 12 years, projects involving structural genomics have generated structural data for ~12,000 proteins within the Protein Data Bank (PDB) , glycolyAlthough protein-ligand interactions are fundamental to most biochemical reactions, structural information concerning these binding sites is still inadequate. Once ligand-binding sites can be predicted from structural data, putative functions can be assigned to these proteins. More complete annotation of protein function will benefit both basic science and the pharmaceutical industry. Mutations or deletions within these ligand-binding domains often alter biochemical reactions and are the root causes of many diseases. This makes binding sites attractive targets for drug therapies, including anticancer chemotherapy. In recent years computational methods have been used to identify ligand-binding sites within proteins. These methods include empirical approaches , supportThe structural genomics initiative resolves 20 new protein structures each week, and more than 60,000 structures have been deposited into PDB. The functional surfaces of proteins, which interact with cofactors, tend to be more structurally conserved than internal structures . ResidueTo characterize the structural environment of NAD-/FAD-binding sites, we compared binding-site residues to whole-protein residues. The three-dimensional structure of the NAD/FAD molecule was divided into three moieties according to function. Within the spherical environment of NAD, the adenosine-binding site typically contained glycine, isoleucine, tyrosine, and aspartic acid residues; the phosphate-binding site contained glycine, isoleucine, serine, threonine, methionine, phenylalanine, tyrosine, tryptophan, arginine, and histidine residues; and the nicotinamide-binding site contained serine, threonine, cysteine, phenylalanine, asparagine, tyrosine, tryptophan, histidine, and asparagine residues. For FAD, adenosine was bound by glycine, valine, cysteine, and tryptophan; phosphate was bound by glycine, serine, and arginine; and flavin was bound by cysteine, methionine, phenylalanine, tyrosine, tryptophan, and histidine. The residue types whose ratio of binding-site residues frequency to whole-protein residues frequency was greater than 1.2 were listed above. As such, the binding residues were primarily polar residues, containing charged groups, amide groups, and nucleophilic groups .We also characterized the types of atoms that were within 3.5 Å of the three moieties of each NAD/FAD ligand . NicotinWe chose two criteria to evaluate the performance of our binding-site predictions: performance at less than 5% FPR and the Matthews correlation coefficient (MCC). We used a combination of features that included the number of aligned residues, RMSD, BLOSUM, and DSSP. Using a 5% FPR threshold, NAD-binding sites were predicted with an accuracy of 93.46%, a sensitivity of 67.09%, and an MCC of 0.52. Under these same conditions, FAD-binding-site predictions yielded 93.59% accuracy, 68.43% sensitivity, and an MCC of 0.54 . When MCWe next compared our results with other prediction methodologies. For these comparisons we chose two published methods that use similar criteria for analyzing these kinds of ligand-protein complexes , 11. TheFigures For the FAD-binding proteins, our method accurately predicted chain A of deoxyribodipyrimidine photolyase (PDB ID:1OWL)  which coSmall molecular cofactors (ligands) are essential for cells to perform numerous biological functions. NAD and FAD, for example, bind to proteins that play critical roles in energy transfer, energy storage, and signal transduction, to name just a few. To understand the mechanism by which these ligands affect protein function, it is important to identify ligand-binding residues within relevant proteins. The experimental identification of these interacting residues is so difficult; however, that computational methods to accomplish this task are in high demand.Here we developed a structure comparison method that uses both sequence and structure information to predict NAD-/FAD-binding residues within proteins. This approach also provides valuable information concerning the microenvironment of the protein-ligand interaction. The composition of NAD-/FAD-binding residues that we identified here is generally similar to previous studies , 11. Intα carbons from local structure alignments and using a secondary structure substitution matrix [We extracted structures of proteins bound to NAD or FAD from PDB and constructed a database of NAD-/FAD-binding residue templates. Residues that were defined as binding residues by the ligand-binding database BioLiP  were incn matrix  accordinn matrix . ResidueWe adopted the same datasets with previous research , 11. AllS with the binding template T. The structural unit consists of a triplet formed by the N–Cα–C atoms within a given residue. S denotes the query protein of length m, and T denotes the template of n residues. The query protein S of length m and the template T of n residues can therefore be expressed in terms of triplets as S = {σ1, σ2,…, σm} and T = {τ1, τ2,…, τn}, where σi = , τj = , and p and q are PDB coordinates for each atom.We used the fragment transformation method to align NAD-/FAD-binding residues. Each residue was treated as an individual unit and was used to align the query protein m × n was then constructed for the residues of S and T asMij is a rigid-body transformation matrix that transforms the triplet σi to τj .A matrix of dimensions Dklij is the Cartesian distance between the target τl and the transformed triplet Mijσk, providing a measure of how similarly the triplet pairs  and  are oriented. This allows clustering of triplet fragments using the single-linkage algorithm [σi, τj) and , Dklij < D0, i ≠ k and j ≠ l, then the triplets are clustered. Let G1 and G2 be two clusters, with the first containing  and  and the second containing  and . If Dk′l′ij < D0, then G1 and G2 are merged to form a new cluster G3, where G3 = G1 ∪ G2. These procedures are performed iteratively until no new clusters can be formed. For each final cluster Gμ, we can obtain the transformation matrix Mk,lμ and aligned substructure pair Sμ = ⋃σk∈Gμσk and Tμ = ⋃τl∈Gμτl, where Gμ has the minimum Cartesian distance when using Mk,lμ.lgorithm  as folloi, the binding score Ci is defined asεμ is the number of triplets of Sμ . The alignment scores CμR, CμB, and CμD are defined asSμ, Tμ) is the RMSD of all Cα atoms between Sμ and Tμ, BLOSUM  is the sequence alignment score between Sμ and Tμ calculated using the BLOSUM62 [Tμ, Tμ) is the maximum sequence alignment score of Tμ, DSSP  represents the secondary structure alignment score based on a construction substitution matrix [Sμ and Tμ, and DSSP  is the maximum secondary structure alignment score of Tμ. The value of RMSD  should be <3 Å.For each residue BLOSUM62  substitun matrix  using thn matrix  between i, we predict a geometric center Θiω of the ligand by Θiω = Mk,lμ−1Lω, where Lω is the geometric center of the binding template type ω in template T. ω represents the three moieties of NAD/FAD: nicotinamide, adenosine, and phosphate for NAD; flavin, adenosine, and phosphate for FAD. The binding score Ck is added to Ci if the distance between Θiω and Θkω′ is between 3 and 9 Å, and ω ≠ ω′. Finally, the normalized binding score ZiC is calculated asSDC denote the mean and standard deviation, respectively, of the binding score Ci.For each residue The accuracy of predicting NAD-/FAD-binding sites wasdefined as the number of true positives and true negatives and was evaluated using a leave-one-out approach. Accuracy (ACC), the true positive rate (TPR), and the false positive rate (FPR) were calculated using true positive (TP), true negative (TN), false positive (FP), and false negative (FN) values as follows:


Caffeine is consumed by more than 85% of adults and little is known about its role on erectile dysfunction (ED) in population-based studies. We investigated the association of caffeine intake and caffeinated beverages with ED, and whether these associations vary among comorbidities for ED.Data were analyzed for 3724 men (≥20 years old) who participated in the National Health and Nutrition Examination Survey (NHANES). ED was assessed by a single question during a self-paced, computer-assisted self-interview. We analyzed 24-h dietary recall data to estimate caffeine intake (mg/day). Multivariable logistic regression analyses using appropriate sampling weights were conducted.rd (85-170 mg/day) and 4th (171-303 mg/day) quintiles of caffeine intake were less likely to report ED compared to men in the lowest 1st quintile (0-7 mg/day) , but no evidence for a trend. Similarly, among overweight/obese and hypertensive men, there was an inverse association between higher quintiles of caffeine intake and ED compared to men in the lowest 1st quintile, P≤0.05 for each quintile. However, only among men without diabetes we found a similar inverse association (trendP = 0.01).We found that men in the 3Caffeine intake reduced the odds of prevalent ED, especially an intake equivalent to approximately 2-3 daily cups of coffee (170-375 mg/day). This reduction was also observed among overweight/obese and hypertensive, but not among diabetic men. Yet, these associations are warranted to be investigated in prospective studies. In the US, the prevalence of erectile dysfunction (ED) in men aged ≥20 years is 18.4% suggesting that more than 18 million are affected : at age Cardiovascular risk factors like physical inactivity, alcohol consumption and smoking have been suggested to increase the risk of ED , 8. Yet,The prevalence of overweight/obesity, hypertension and diabetes is increasing dramatically among US men, and previous studies have linked them with ED –20. Yet,The National Health and Nutrition Examination Survey (NHANES) is a program of studies undertaken by the National Center for Health Statistics (NCHS) of the United States (US) Centers for Disease Control and Prevention (CDC) to assess the health and nutritional status of adults and children in the US. Continuous NHANES used a multistage, stratified and clustered probability sampling in which Mexican-Americans, non-Hispanic blacks, and the elderly were oversampled to ensure adequate samples sizes and to represent the total US civilian, non-institutionalized population . For the“Many men experience problems with sexual intercourse. How would you describe your ability to get and keep an erection adequate for satisfactory intercourse? The following answers were provided, “would you say that you are… always or almost always able, usually able, sometimes able, or never able? For the purpose of this study, we defined and dichotomized positive ED from the answers “sometime able” or “never able” to keep an erection, and subsequently negative ED was derived from the answers “almost always able” or “usually able” to maintain an erection . Yet, we did not find a trend in this association (Ptrend = 0.19).In 2), hypertensive and diabetic men , normal blood pressure, and diabetes. However, among overweight/obese and hypertensive men, we found that men in the 2nd (8–84 mg/day), 3rd (85–170 mg/day), 4th (171–303 mg/day) and 5th (304–700 mg/day) quintiles of total caffeine intake had a reduced prevalence of ED than men in the lowest quintile (0–7 mg/day) (P ≤ 0.05 for each quintile)  and interaction effect (Pinteraction ≥ 0.05) for any of the associations. Among men without diabetes (Ptrend = 0.01). Yet, the interaction effect did not reach statistical significance (Pinteraction = 0.65). The association of caffeine intake with ED was also analyzed in age categories , however, no statistical significant associations were found (data not shown).In stratified analyses, we investigated the associations of total caffeine intake with ED among overweight/obese BMI ≥ 25 kg/m5 kg/m2, men Figs . We did diabetes , we founWe also analyzed the association of caffeinated beverages with ED and whether these associations varied by comorbidities for ED. The results are presented in an Online Supplement  Tables. In the present study, our nationally representative sample of men showed that total caffeine intake was associated with a reduced likelihood to report ED in multivariable analyses. This reduced prevalence of ED was mainly observed when the amount of caffeine intake was between 170 and 375 mg/day, which is approximately equivalent to 2–3 cups of coffee per day. In addition, total caffeine intake seemed to reduce the odds of ED among men who were overweight/obese, hypertensive and non-diabetic. A few caffeinated beverages were inversely associated with ED, yet they were not consistent when stratified by comorbidities for ED.Our results are consistent with two population-based studies that showed an inverse association between caffeine intake and ED , 10. TheHowever, our findings differ from two previous studies that showed no association between caffeinated drinks , coffee consumption (prospective) and ED , 12. ComWe did not find a significant inverse association between caffeine intake and ED among diabetic men. In an animal study, we previously showed that caffeine consumption improved the erectile function of diabetic rats by up-regulating cavernous cGMP . DiabeteInterestingly, our findings showed a lower prevalence of ED among men with caffeine intake, especially when this is equivalent to 2–3 daily cups of coffee (170–375 mg/day). Previously, a similar caffeine intake amount was associated with beneficial effects on cardiometabolic factors and cardiovascular health . In addiStrengths of this study is the large sample size with a representative sample of the US men population, and the validated dietary recall methodology from NHANES that allows for measurement of caffeine intake from drinks , 24 and In general we found a reduced likelihood to report ED among men with caffeine intake, especially with 2–3 daily cups of coffee, which is approximately 170–375 mg/day. Interestingly, we found differences in the inverse associations of caffeine intake and caffeinated beverages among overweight/obese, non-diabetic and hypertensive men.S1 TableβErectile dysfunction was defined as “sometimes” or “never” able to maintain an erection for satisfactory sexual intercourse. †Model 1- Adjusted for age only. ‡Model 2- Adjusted for age, vigorous and moderate physical activity, smoking status, education, race/ethnicity, obesity (BMI ≥ 30 kg/m2), total water intake (plain and tap), total energy (continuous), alcohol (continuous). £ Approximately 170–375 mg/day of caffeine intake is equivalent to 2–3 cups of coffee. aP ≤ 0.05 bP ≤ 0.01(DOC)Click here for additional data file.S2 TableβErectile dysfunction was defined as “sometimes” or “never” able to maintain an erection for satisfactory sexual intercourse. ‡Adjusted for age, vigorous and moderate physical activity, smoking status, education, race/ethnicity, total water intake (plain and tap), total energy (continuous), alcohol (continuous). £Approximately 170–375 mg/day of caffeine intake is equivalent to 2–3 cups of coffee. aP ≤ 0.05 bP ≤ 0.01(DOC)Click here for additional data file.S3 TableβErectile dysfunction was defined as “sometimes” or “never” able to maintain an erection for satisfactory sexual intercourse. ‡Adjusted for age, vigorous and moderate physical activity, smoking status, education, race/ethnicity, obesity (BMI ≥ 30 kg/m2), total water intake (plain and tap), total energy (continuous), alcohol (continuous). £Approximately 170–375 mg/day of caffeine intake is equivalent to 2–3 cups of coffee. aP ≤ 0.05 bP ≤ 0.01(DOC)Click here for additional data file.S4 TableβErectile dysfunction was defined as “sometimes” or “never” able to maintain an erection for satisfactory sexual intercourse. ‡ Adjusted for age, vigorous and moderate physical activity, smoking status, education, race/ethnicity, obesity (BMI ≥ 30 kg/m2), total water intake (plain and tap), total energy (continuous), alcohol (continuous). £Approximately 170–375 mg/day of caffeine intake is equivalent to 2–3 cups of coffee. aP ≤ 0.05 bP ≤ 0.01(DOC)Click here for additional data file.


AbstractMegacormusgranosus is described for the first time and the female redescribed. A homology scheme proposed recently is applied to hemispermatophore structures. The specimens were collected in an oak forest from Pico de Orizaba Volcano at an average altitude of 2340 m. All adult males were collected by pitfall traps, whereas all adult females and both sex immatures were collected using Berlese funnels, suggesting that males are comparatively more mobile within the leaf litter layer, probably due to mating season.The male of  PageBreakMegacormus  is comprised of four species, Megacormusgranosus ; Megacormussegmentatus Pocock, 1900; Megacormusgertschi Díaz Nájera, 1966 and Megacormusgrubbsi Sissom, 1994. All species of Megacormus are restricted to the slopes of the Sierra Madre Oriental and the costal lowlands of the Gulf of Mexico, ranging in altitude from 300 m to over 2300 m. They prefer habitats with high relative humidity such as oak, oak-pine and evergreen tropical forest, and are found in these communities within the leaf litter, outcrop crevices, decaying logs or under rocks. Their distribution includes the states of Hidalgo, Oaxaca, Puebla, Querétaro, San Luis Potosí, Tamaulipas, and Veracruz . In this contribution we extend that terminology and homologies to the scorpion family Euscorpiidae Laurie, 1896, when possible, applying it to the hemispermatophore of Megacormusgranosus.The unsatisfactory working terminology and concomitant absence of homologies in the hemispermatophore of the Order mologies . Recentlmologies , 2014 comologies , upon whAll scorpions were collected by sifting leaf litter processed with Berlese funnels and pitfall traps. Specimens are deposited in the Laboratorio de Aracnología de la Facultad de Ciencias, Universidad Nacional Autónoma de México (UNAM), Mexico. Measurements in mm were taken with an ocular micrometer. Illustrations were produced with a Nikon SMZ 1000 dissecting stereomicroscope and a Nikon Eclipse E200 with a camera lucida. Photomicrographs were taken with a digital camera Nikon DS-U3 under LED illumination and Ultraviolet (UV) light using a 20W Techno Lite® bulb mounted in a desktop stand lamp. Hemispermatophores were digested with pancreatine , clearedEuscorpius, the putative sister group of Megacormus. Pedipalp chela dentition is modified from Nomenclature follows 19°8'17.4"N, 97°12'16.2"W, altitude 2,300 m. Plot II 19°8'30.2"N, 97°12'21.5"W, altitude 2388 m. Three expeditions were conducted, two in May 21–30 th and October 4–14th 2012 and the third in February 15–24th 2013.Specimens were collected in a 15–20-year old oak forest near the boundary of the Pico de Orizaba National Park ca. two kilometers southwest from Atotonilco de Calcahualco, Veracruz. Two plots, of one hectare each, were established with the following central coordinates. Plot I PageBreakTaxon classificationAnimaliaScorpionesEuscorpiidaeScorpiogranosusChactasgranosus : Karsch 1879: 111.Megacormusgranosus : MEXICO: Holotype female, depository unknown , or very deep (♀). Coxa IV two and half times longer than coxa II. Coxae I–IV surfaces with scattered granules and margins densely granular; coxa II, prolateral subproximal margin with three oblique slit-like structures, adjacent to a moderate (♀) or low (♂) granular protuberance; coxae II–IV, prolateral carinae strongly granular  or six (♀) pairs of short and translucent macrosetae; sclerites free longitudinally, anterior margin fused on distal two thirds (♂) or fused longitudinally by a loose pleura folding into a valve covering the genital opening (♀). Genital papillae present, protruding posteriorly (♂) or absent (♀)  or isosceles trapezoidal (♀). Marginal and median lamellae nearly fused into one piece with a fine, shallow furrow (♂), or completely fused, indistinguishable furrow (♀). Fulcra absent. Pectinal teeth: three to four (♂) or one to four (♀). Pectines relatively short, fused lamella aligned with midpoint of coxa IV  or absent (♀)  Figure . SternitMetasoma: Length 0.9 times greater than mesosomal length  and 11 (5%), caught 18 and 9 specimens respectively. These yields are consistent with low population density of this species; adult males are particularly rare. The habitat of, and behavior exhibited by, this species as well as its cryptic morphology  are congruent with a humiculous ecomorphotype .Megacormusgranosus type material is either unknown or lost. It is important to investigate the whereabouts of Gervais’ unique specimen to verify the holotype, or failing this, to designate a neotype. Workable keys to the species of Megacormus are provided in Megacormus is under revision by O.F. Francke (per. comm.).The catalog of the scorpions of the world  indicatePageBreakPageBreakMegacormusgranosus hemispermatophore presented in Figures acc, figure 8). According to our findings, the accessory lobes are the termini of both the dorsal and ventral trough margins with a spiculate process  and predicted by other studies (The illustrations of the  Figures , 27 and  Figures . Further studies .


Tarsius) and scops owls (Otus), taxa united by numerous anatomical homoplasies, including foveate vision. A functional interpretation of these homoplasies predicts dietary convergence. We tested this prediction by analyzing stable isotope ratios that integrate dietary information. In Borneo and the Philippines, the stable carbon isotope compositions of Tarsius and Otus were indistinguishable, whereas the stable nitrogen isotope composition of Otus was marginally higher than that of Tarsius. Our results indicate that species in both genera consumed mainly ground-dwelling prey. Taken together, our findings support a functional interpretation of the many homoplasies shared by tarsiers and scops owls, including a retinal fovea. We suggest that the fovea might function similarly in tarsiers and scops owls by calibrating the auditory localization pathway. The integration of auditory localization and visual fixation during prey detection and acquisition might be critical at low light levels.The fovea is a declivity of the retinal surface associated with maximum visual acuity. Foveae are widespread across vertebrates, but among mammals they are restricted to haplorhine primates , which are primarily diurnal. Thus primates have long contributed to the view that foveae are functional adaptations to diurnality. The foveae of tarsiers, which are nocturnal, are widely interpreted as vestigial traits and therefore evidence of a diurnal ancestry. This enduring premise is central to adaptive hypotheses on the origins of anthropoid primates; however, the question of whether tarsier foveae are functionless anachronisms or nocturnal adaptations remains open. To explore this question, we compared the diets of tarsiers ( It is aligned with the visual axis of the eye and contains a disproportionately high density of photoreceptors. The optics of foveae are an enduring interest  and night monkeys (Aotus) – are nocturnal, and the former sits at a crucial position in the primate phylogenetic tree . Tarsiers are the basal crown haplorhine primate and their fovea has long informed hypotheses on the origins of anthropoid primates  opsin gene  failed to detect a fovea , Tarsius sirindhornae (Middle Miocene), and living tarsiers are most parsimoniously interpreted as evidence of continuous nocturnality for at least 45 million years , a tarsier-sized faunivore (90–140 g), suggest a comparable niche . Potential niche convergence has attracted attention due to the many homoplasies that unite tarsiers and scops owls, such as (i) hyperenlarged eyes that protrude from the orbit ; (ii) orbit-induced displacement of the olfactory tract, which itself is unusually long; (iii) a loss of ocular mobility that corresponds with increased cervical mobility ; (iv) acute directional hearing; (v) enlarged semicircular canals; and, (vi) derived feeding morphologies for perforating prey , has a fovea with three cone classes  and little owl (Athene noctua), can make chromatic discriminations at low light levels . For example, a fovea can be present  or absen classes . Strix and orbit . Furthert levels . The rett levels , a view A controlled experimental approach is preferable for testing foveal function; however, the mortality rate of captive tarsiers is unacceptably high . Accordi13C:12C or δ13C) and nitrogen (15N:14N or δ15N) isotopes in a two-dimensional “δ-space”  and -14‰ (range -12 to -16‰), respectively , whereas those in the understory are 13C-depleted  due to the recycling of CO2  relative to conspecifics in primary forest, whereas δ15N values did not differ between treeshrews and squirrels inhabiting different forest types.Variation in ic level . Thus in typical . In the itivores . These rlability  and distlability . This vaTarsius and Otus are predicted to have similar isotopic niches, or overlapping δ13C and δ15N values. Affirmation of this prediction would be consistent with functional interpretations of the many anatomical homoplasies shared between these two taxa, including the retinal fovea.Thus Otus lempiji, formerly O. bakkamoena lempiji; n = 8) and Philippine scops owls  and Philippine tarsiers . The specimens, all wild-caught adults, were chosen on the basis of maximum overlapping provenience . The majority of specimens are accessioned in the American Museum of Natural History, the Field Museum of Natural History, the Kinabalu National Park Museum, and the Universiti Malaysia Sabah Museum . These tarsiers (n = 12) were hand-caught and anaesthetized as part of a larger study of their sensory ecology  and weighed (500 ± 15 μg) into precombusted tin capsules.We sampled the contour feathers of Sunda scops owls  – 1) and R = 13C/12C or R = 15N/14N; reference standards are Vienna Pee Dee Belemnite (VPDB) for carbon and atmospheric N2 for nitrogen. Units are expressed as parts per thousand (‰). The dried samples were combusted and analyzed with a Thermo-Chemical Elemental Analyzer (TCEA) interfaced with a Delta Plus XP isotope ratio mass spectrometer  located in the Stable Isotope Laboratory, University of California, Santa Cruz. The analytical precision (±1 SD) for δ13C and δ15N was 0.3‰ and 0.05‰, respectively, based on four International Atomic Energy Agency (IAEA) acetanilide replicates.Isotope ratios are presented as 13C content of atmospheric CO2 due primarily to fossil fuel burning during the past 150 years , although the semivariograms are potentially uninformative due to the small number of samples spread over a relatively large spatial scale. Each δ15N value is therefore assumed to have statistical independence for assessing diet.Another confounding factor stems from geographic and temporal variation in soil N availability, both natural  and anthχ2 (multiple comparison) tests to assess whether the carbon and nitrogen isotope compositions differentiate sympatric taxa of Tarsius and Otus. For all normally distributed data, comparisons of significance were investigated using Welch’s Two Sample t-tests. The significance for all tests was set at α = 0.05.We performed all statistical tests in R version 2.14.1 . As someδ13C. The mean ± SD of all time-corrected samples was –23.76 ± 1.6‰ (range: –27.80‰ to –17.41‰). Within Borneo, the time-corrected δ13C values of O. lempiji (mean: -22.87 ± 1.7‰) were ca. 1.95‰ greater than those of T. bancanus (mean: –24.82 ± 0.2‰), but the difference did not reach statistical significance . Similarly, in the Philippines, the time-corrected δ13C values of O. megalotis (mean: –23.33 ± 2.5‰) were ca. 0.62‰ greater than those of T. syrichta (mean: –23.95 ± 1.1‰), but the difference did not reach significance . Intrageneric comparisons revealed differences between the two species of Tarsius  but not the two species of Otus .Appendix 1 summarizes the raw and time-dependent corrections to δ15N. The mean ± SD of all samples was 5.79 ± 2.2‰ (range: 2.39–11.37‰). Within Borneo, the δ15N values of O. lempiji (mean: 7.45 ± 1.7‰) were ca. 1.65‰ greater than those of T. bancanus (mean: 5.80 ± 1.8‰), but the difference did not reach statistical significance . The effect size of this analysis is sufficient to rule out a Type II error (Cohen’s d = 0.95). Within the Philippines, the δ15N values of O. megalotis (mean: 7.04 ± 2.3‰) were ca. 2.22‰ greater than those of T. syrichta (mean: 4.82 ± 1.9‰), indicating significant 15N-enrichment ; however, the samples from T. syrichta collected in 2010 exhibited systematically low δ15N values, perhaps due to recent anthropogenic changes to the landscape . Intrageneric comparisons revealed no differences between the two species of Tarsius  or the two species of Otus .Appendix 1 summarizes the raw values pe e.g., . When weFigure 5 also illustrates the larger food web by including the δ13C and δ15N values of a primary consumer, the frugivorous Müller’s gibbon , and a predator of vertebrates, the leopard cat . The isotopic differences (Δ) between these taxa  approximate a full trophic step, albeit a rather large one. 13C value of 2.4‰, but a smaller Δ15N value of 3.0‰ between herbivores and predators in Lambir National Park, Sarawak, Borneo. In any case, the magnitude of the isotopic difference (Δ) between O. megalotis and T. syrichta (Δ15N = 2.22) is much less than that between Felis and Hylobates (Δ15N = 4.93‰).In many respects, tarsiers are not owls, but almost , p. 953δ13C values of Otus and Tarsius were indistinguishable, whereas the δ15N values of Otus were often higher than those of Tarsius. The low and comparable δ13C values indicate use of the same stratum (the forest floor), a foraging pattern that agrees well with behavioral observations. The differences in δ15N values – a pattern that was a trend in Borneo and temporally variable in the Philippines – are potentially instructive because they indicate a subtle degree of prey partitioning. Yet the magnitude of the isotopic difference (Δ) between O. megalotis and T. syrichta (Δ15N = 2.22) is much less than that between Felis and Hylobates , suggesting limited partitioning of invertebrate and vertebrate prey (discussed below). However, 15N of 3.0‰ on the basis of a much larger data set from Sarawak. Taken together, our isotopic results demonstrate that Otus and Tarsius occupy similar dietary niches, although a trend toward 15N-enrichment among scops owls, particularly O. megalotis, suggests some prey partitioning.Our results demonstrate isotopic overlap: the T. bancanus and T. syrichta  represented <11% of 133 successful predation events. In Sulawesi, tarsiers seldom consume vertebrates  in favor of crickets, whereas T. syrichta exhibits the reverse pattern  coleopterans:Another possible explanation for the Otus lempiji) habitually hunts nocturnal insects attracted to cow dung or poultry droppings around houses. Some stomachs examined were crammed with cockroaches (Blattidae) and a particular type of black dung beetle (Scarabidae). The Sumatran (Minangklabau) name for this owl is kuas cirit ayam, which means ‘fowl’s-excrement owl”’  relative to diet  counterparts, and the relative 15N-enrichment of Otus could reflect a greater proportion of scatophagous coleopterans or nocturnal squamates, or both, in the diet. To discriminate the relative contributions of these putative food sources, it would be useful to collect food samples and perform a Bayesian multiple source isotopic mixing model  and minimized movement in response to models of an ochre-bellied boobook (Ninox ochracea) and spotted kestrel . Among adult tarsiers, the kestrel elicited the twin antipredator behaviors of mobbing and alarm calling during 47% of encounters, indicating that adults recognized it as a threat . In other words, foveate vision may guide sound localization by verifying the accuracy of auditory orientation to a sound source  and the phenomenon of lunar philia (increased activity under moonlight) among spectral tarsiers (If instructed learning in the auditory localization pathway is at least partly dependent on foveate vision, then a unified representation of visual and auditory sensory stimuli was potentially a central factor in the enduring success of tarsiers . It mightarsiers . Taken tThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The implications of this data are discussed.The kidney displays specialized regions devoted to filtration, selective reabsorption, and electrolyte and metabolite trafficking. The polarized membrane pumps, channels, and transporters responsible for these functions have been exhaustively studied. Less examined are the contributions of spectrin and its adapter ankyrin to this exquisite functional topography, despite their established contributions in other tissues to cellular organization. We have examined in the rodent kidney the expression and distribution of all spectrins and ankyrins by qPCR, Western blotting, immunofluorescent and immuno electron microscopy. Four of the seven spectrins  are expressed in the kidney, as are two of the three ankyrins (G and B). The levels and distribution of these proteins vary widely over the nephron. αΙΙ/βΙΙ is the most abundant spectrin, found in glomerular endothelial cells; on the basolateral membrane and cytoplasmic vesicles in proximal tubule cells and in the thick ascending loop of Henle; and less so in the distal nephron. βΙΙΙ spectrin largely replaces βΙΙ spectrin in podocytes, Bowman’s capsule, and throughout the distal tubule and collecting ducts. βΙ spectrin is only marginally expressed; its low abundance hinders a reliable determination of its distribution. Ankyrin G is the most abundant ankyrin, found in capillary endothelial cells and all tubular segments. Ankyrin B populates Bowman’s capsule, podocytes, the ascending thick loop of Henle, and the distal convoluted tubule. Comparison to the distribution of renal protein 4.1 isoforms and various membrane proteins indicates a complex relationship between the spectrin scaffold, its adapters, and various membrane proteins. While some proteins ( Specialized regions along the nephron unit enable the kidney’s selective filtering and absorptive capacities. The polarization and distribution of plasma membrane transport proteins, receptors, ion channels, and adhesion molecules are critical, yet the molecular events effecting this organization remain incompletely understood.A spectrin-based scaffold is likely to be one component contributing to renal tubule organization. Although models based on the erythrocyte envision a spectrin, ankyrin, and protein 4.1 skeleton stabilizing a membrane by linking actin filaments to integral membrane proteins , this moEarlier studies clearly identified the presence of a non-erythroid spectrin, and its two major adapter proteins ankyrin and protein 4.1 in the kidney, and correlated these with the distribution of α1-Na,K-ATPase , 20, 21.C57/B6 mice were housed under pathogen-free conditions and maintained as a breeding colony. The Institutional Animal Care and Use Committee (IACUC) at Yale University, accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC), approved all mouse procedures.Standard procedures followed reagent manufactures directions. RNA was isolated from adult whole mouse kidney using Trizol regent (Invitrogen); cDNA was prepared with Superscript (Invitrogen) and quantified with a Nanodrop spectrophotometer. Gene specific PCR primers bridging consecutive exons  were ensAntibodies have been previously documented. Mouse monoclonal antibodies were: Anti βΙ spectrin . The right atrium was nicked with scissors and perfused by a syringe through the left ventricle. Blood was cleared from the animal by an initial bolus of ~20 ml PBS followed by ~50 ml 4% paraformaldehyde. Kidneys were harvested, post fixed for 24h, and sections paraffin embedded. Alternatively, kidneys for Western blot analysis were snap frozen and stored at -80°C.Immunostaining of paraffin sections was as before . DeparafImmediately after retrograde perfusion with paraformaldehyde specimens were frozen, sectioned, and incubated with primary antibody for 2h at RT followed by secondary antibody conjugated to HRP (Impress). At each step, unbound antibodies were removed by PBS wash. After post fixation with 1% glutaraldehyde in 0.1M sodium cacodylate, 2.5% sucrose, pH 7.4 for 1 h, bound antibody was labeled with 0.1% di-aminobenzidine (DAB), sections fixed in reduced osmium tetroxide, and dehydrated in a series of increasingly concentrated ethanol solutions. Specimens were viewed after Epon812 embedding.Whole kidneys in triplicate were homogenized with a glass Teflon tissue grinder in 3 ml of 0.32 M sucrose, 10 mM Tris-HCl, pH 7.4 containing Protease Arrest (1:100). Centrifugation at 1,000xg for 10 min. at 4°C removed cell debris. Equivalent amounts of supernatant protein were analyzed by SDS-PAGE on Novex 4%–12% bis-acrylamide Nu-PAGE gels. Proteins transferred to PVDF membrane were incubated with primary antibody for 2h at RT; washed in Tris-buffered saline, and incubated with HRP-conjugated secondary antibody for 1 h. Blots were visualized by ECL .RT-PCR with primer sets bridging constitutively expressed exons for all ten mammalian spectrin and ankyrin genes  revealedWestern blotting and immunofluorescent microscopy examined the steady-state protein composition of the different spectrins and ankyrins in the cortical and medullary portions of the kidney. Aquaporins 1 & 2  were used as aids to distinguish these regions. AQP1 is expressed in the PT and descending thin limb of Henle’s loop; as such, it is equally represented in both cortical and medullary tissues. AQP2 is preferentially expressed in the distal collecting tubule and collecting ducts , markingThe predominant spectrin in kidney was αII spectrin. This protein was uniformly distributed throughout the kidney and present in all cell types . βΙΙ speTwo forms of ankyrin B were detected, the canonical version at 220 kDa, and a shortened form at 150 kDa . SeveralThe distribution of the spectrins and ankyrins were compared to the distribution of well-characterized renal tubule proteins. Immunofluorescent microscopy data for five of these are presented: α1-Na,K-ATPase; AQP1; AQP2; calbindin1; and NKCC2 (sodium-potassium-chloride co-transporter). α1-Na,K,-ATPase is expressed at the basolateral membrane in all tubule cells, including proximal tubules, but is significantly more abundant in the distal collecting tubule and collecting ducts, with the exception of the intercalated cells of the distal collecting tubule where ankyrin and spectrin co-distribute with band 3 ). As notDouble immunofluorescence identified αΙΙ and βΙΙ spectrin in all tubules of the renal cortex . This stAs with βΙΙ spectrin, βΙΙΙ spectrin accumulated along both the plasma membrane and more prominently on cytoplasmic vesicular and canalicular structures deep within the cytoplasm of distal tubular and collecting duct cells , Fig 4. Co-staining with AQP1 confirmed βΙΙ spectrin expression in the cortical proximal tubule, where AQP1 (green) is confined to the apical brush border . Co-staiIn nervous tissue and muscle, homopolymeric forms of beta spectrin or beta-spectrin-like molecules that cross-react with beta-spectrin antibodies, unassociated with a paired alpha spectrin subunit have been detected , 40. SecAnkyrin G was widely expressed in all nephron segments, overlapping α1-Na,K,-ATPase at the lateral cell membrane; it was also generally present albeit at lesser abundance at the apical membrane and diffusely distributed within the cytoplasm . ImmunoEAnkyrin B was nearly absent in proximal tubules . It was Spectrins αΙΙ, βΙΙ, βΙΙΙ, and ankyrins B and G are all expressed in various components of the glomerulus. αΙΙ spectrin was ubiquitous in all glomerular cell types (not shown). The capillary endothelial cells stained with βΙΙ and βΙΙΙ spectrin and with ankyrin G . Podocytin vitro cell culture studies to normal kidney); 6) αII spectrin expression is ubiquitous but the expression of βΙΙ vs. βΙΙΙ spectrin is largely complementary; 7) different cell populations within the glomerulus display distinct spectrin/ankyrin compositions; and 8) there is no simple mapping of specific spectrins or ankyrins with any examined membrane protein . A summary of the localization of the spectrins and ankyrins correlated with other reported membrane proteins and protein 4.1 along the nephron unit is depicted in cartoon form  at least three (and possibly four) different spectrin genes and two ankyrin genes are expressed in the mature kidney; 2) alternative transcripts of at least two spectrin genes and the two ankyrin genes are also utilized in the kidney; 3) these gene products are stable, abundant, and localize to regionally distinct cell populations along the nephron unit; 4) all of the spectrins and ankyrins localize to both the basolateral and apical domains of renal tubule cells; 5) all spectrins and ankyrins associate with populations of cytoplasmic organelles and vesicular carriers (extending oon form .e.g. a sodium/calcium exchanger, an amino-acid transporter, or a voltage-gated sodium channel. Ankyrin interacts with a variety of renal ion transporters [The significance of these findings derives, as noted above, from the profound consequences that may follow inherited or experimental disruption of spectrin or ankyrin in other tissues . For exasporters , 47, 48.sporters  and recesporters , 49. It It is interesting to speculate what sorts of disorders of renal function might result from spectrin or ankyrin dysfunction. Hypertension is associated with loss-of-function mutations in genes that regulate normal renal salt reabsorption in the TALH and DCT including the Na-K-2Cl co-transporter gene; the inward rectifier K+ channel gene ROMK; and the Na-Cl co-transporter gene SLC12A3 –52. CandFinally, it is fair to question how spectrin and ankyrin might contribute to the organization and stability of so many membrane proteins. Classical concepts envision spectrin and ankyrin as skeletal proteins, their action one of direct linkage and tethering. This concept emerged from early studies of the red cell skeleton, and endures to this day. It is incomplete. Emerging data suggests a more complex role envisioning spectrin and ankyrin as true scaffold proteins, about which post-translational signaling pathways converge –60. The