Molecular tests revealed influenza D viruses of D/OK lineage widely circulating in farmed animal species in Guangdong Province, southern China. In particular, we found high levels of influenza D virus infection in goats and pigs. We also detected viral RNA in serum specimens and feces of animals with certain severe diseases. The recently discovered influenza D virus is thought to cause respiratory diseases primarily in cattle and to a lesser extent in pigs  have been confirmed  in asymptomatic animals tested  for dairy cattle, 7.3% (4/55) for native yellow cattle, and 36.8% (7/19) for pigs. Rates in 324 total serum samples were 7.8% (15/193) for dairy cattle, 5.9% (3/51) for buffalo, and 33.8% (27/80) for goats. The influenza D virus–positive rate was also high  in swine lung samples. In contrast, we found no or low prevalence  with previously described sequences from China ,–.––,,When first discovered, influenza D virus was reported in diseased pigs in the United States ––, which i–,,––Previous studies have shown that influenza D viruses are mainly found in respiratory tract samples  observed in asymptomatic animals (p<0.05), which suggests a potential correlation between the disease severity and presence of influenza D virus. For influenza D virus found in rectal swabs, it might be that animals have swallowed the virus. Another possibility is that, similar to influenza A and B viruses, influenza D virus can replicate within the intestinal tract  co-circulate in North America and Europe (,,In addition, we found different minor genetic variants circulating on the same farm , indicatIn summary, our study investigating the infection status of influenza D virus in different farmed animal species in Guangdong Province provides novel insights into the epidemiology and evolution of this virus. In particular, we document the molecular evidence for influenza D virus infection in goats and buffalo. RT-PCR method and subcloning protocol used in study of influenza D viruses in cattle, goats, buffalo, and pigs, Guangdong Province and neighboring provinces, China.


AbstractNitzschia, Gomphonema, Pinnularia, Navicula, Sellaphora and Eunotia. About a third of the taxa found could not be identified to the species level. From those unidentified morphodemes, two are described as new species, namely Brachysiraaltepetlensis and Sellaphoraqueretana. Furthermore, Eolimnarhombica is transferred to Sellaphora. Canonical Correspondence Analysis (CCA) revealed that specific conductivity and pH were the main environmental factors driving the community composition observed. Three groups of samples were identified after the CCA: 1) characterized by acidic waters and low conductivity; 2) with circumneutral waters, low specific conductivity and high temperature and phosphorous concentrations; and 3) characterized by circumneutral waters, high conductivity and low nitrogen concentrations. The indicator value method , based on the relative abundance and relative frequency of the most abundant taxa was calculated based on the groups observed in the CCA, identifying the characteristic taxa for each of the three groups.The Lerma-Chapala Basin, in Central Mexico, is geologically heterogeneous, climatically diverse and boasts high biodiversity, lying within two Biodiversity Hotspots, namely Mesoamerica and the Madrean Pine–Oak Woodlands. Epilithon and water samples were collected in the basin from 14 sampling sites three times each, two sampling campaigns during the rainy season and one in the dry season. A total of 274 infrageneric taxa in 48 genera were recorded. The taxonomic composition observed was dominated by taxa from the genera  Lotic environments, i.e. streams, are unidirectional flows of water. They are characterized by a broad spatial  and temporal  heterogeneity, which determines the specialized biota that inhabit them . Stream Even though there is mounting evidence of the applied use of diatoms as indicators of environmental change in lotic environments , diatom per se but also on community structure and bioindication. Despite the research done, the diatom diversity of the region seems to be low due to clustering of taxa into species complexes and force–fitting into already described taxa.Diatom studies of lotic environments from Mexico have been mostly focused on the center of the country: Antigua River Basin ; Balsas The studies conducted in the Lerma-Chapala Basin have been focused on the polluted Lerma River and some of its main tributaries ; February 2014, dry season (sampling campaign II); and September 2014, rainy season (sampling campaign III); resulting in 42 water and epilithon samples. Each epilithon sample was collected from five cobbles across a transversal section of the stream, brushing with a disposable toothbrush ten square centimeters of epilithic growth from each of the five cobbles to make a composite sample, fixed in 70% alcohol. In situ measurements of pH, water temperature, specific conductivity and total dissolved solids were recorded using a Hanna multi–sensor . Dissolved oxygen was recorded with an YSI–85 oxygen meter . Dissolved oxygen saturation percentages were calculated from dissolved oxygen data according to correcting factors for elevation and water temperature. Specific conductivity values were corrected to 25°C. Water velocity was recorded with a Global Water FP111 velocity meter . At each sampling site, a 500 ml sample of water was filtered through 0.22 μm and 0.45 μm filter membranes  and collected in sterile polypropylene bottles for chemical analysis. Samples were kept cold and in the dark before laboratory analysis. The subsequent chemical laboratory analyses were adapted from Standard Methods for the Examination of Water and Wastewater and analyzed using a DR 3900 laboratory Spectrophotometer  (2––N), nitrate nitrogen (NO3––N), ammonium nitrogen (NH4+–N), soluble reactive phosphorus  and total alkalinity (as CaCO3). Dissolved inorganic nitrogen (DIN) was calculated as the sum of the three inorganic nitrogen forms in water .olorado) : nitriteQBR from its Catalan abbreviation) was calculated in order to evaluate the riparian habitat quality  in order to account for diatom diversity, using a Zeiss Axioscope microscope with Differential Interference Contrast equipped with an AXIOAM MRc camera. In order to estimate the relative abundance of the taxa, a minimum of 500 valves per sample were counted and identified with the 100x immersion oil objective. Aliquots of cleaned sample material for scanning electron microscopy observations were mounted on stubs, sputter-coated with gold-palladium and observed under a Hitachi FE 8010 scanning electron microscope (SEM) operated at 1.0 kV. Samples and slides are stored at the Diatom Collection of the Botanical Garden and Botanical Museum Berlin–Dahlem, Freie Universität Berlin. Diatoms were identified to the lowest taxonomical level possible using monographs as well as papers for particular taxa  before the epithet indicate that it could be that taxon but the taxonomic identity is still uncertain, ‘aff.’ (affinis) that it has some similarity to the taxon but it is not conspecific and ‘sp.’ (species) was used when the taxon showed no similarity with any known species after the literature review.Data analysis. Only taxa with relative abundance ≥1% were included in the statistical analyses, resulting in 105 diatom taxa. Diatom abundances were transformed using Hellinger’s transformation, which is suited to large abundance datasets with lots of low counts and zeros (nd zeros .10 (x+1) because they had skew distributions. Distribution tests were run in Statistica 8.0.From the initial dataset composed of 42 samples, only 39 were used for the analysis of running waters, i.e. those streams with water velocity records in at least one of the sampling campaigns; the three samples of site 10 were omitted since no water velocity was recorded in this spring-fed creek at any of the three sampling campaigns, with 10 cm/s being the detection limit of the water velocity meter. All the environmental variables, except for temperature, pH and water velocity were transformed using logDCA) was used to estimate gradient lengths. The first four axes showed lengths of 5.7, 3, 2.3 and 2.2, suggesting a strong unimodal response, meaning that a method based on unimodal models like Canonical Correspondence Analysis (CCA) would be appropriate for subsequent ordination. CCA was run to identify variation in species composition that can be determined by environmental variables. Since not all the environmental variables influence diatom distributions independently, CCA with forward selection and unrestricted Montecarlo permutation tests was used . All ordinations were done using CANOCO 4.5 for Windows (ter Braak and Ṧmilauer 2002), with downweighting of rare species in all cases.Multivariate analyses were performed to explore gradients in diatom composition and its relation to environmental factors. Detrended Correspondence Analysis  (CCA. This method combines the specificity (relative abundance) and fidelity (relative frequency) of a species to a given group. The indicator value of a species is given in percentage, reaching its maximum when all the individuals of a species are present at all the sites of a single group. Species with high indicator values >50% are considered to be good indicators; species with values between 25–50% might be regarded as detector species of change, therefore detector species can be present in more than one group . The Shannon-Wiener diversity index and Pielou evenness index were calculated as in CCA.The indicator value method   was usedne group . IndVal PC–ORD 4  with untSpecies composition and taxonomy. A total of 196 taxa (species and varieties) were found while performing the counts to determine relative abundances. Seventy-eight additional taxa were observed by scanning the whole slides looking for rare taxa, bringing the total diversity to 274 taxa belonging to 48 genera , Gomphonema (26 taxa), Pinnularia (21 taxa), Navicula (19 taxa), Sellaphora (18 taxa) and Eunotia (16 taxa). About a third of the diversity found, 94 taxa, did not fit completely into already described species. Most of the taxa were found in relatively low abundances while further scanning the slides under the LM after the enumeration of 500 valves; when scanning samples under the SEM, some of those rare unidentified taxa were found but in several cases not. When the taxa were found under the SEM, not enough valves were observed for reliable identification. This is why only two new species from those 94 unidentified taxa are here described as new, one belonging to the genus Brachysira and the other to Sellaphora. Furthermore, one Eolimna species is transferred to Sellaphora, this species sharing the same morphology of areolae as the Sellaphora species here described as new.A high specific taxa richness was found among the genera Taxon classificationPlantaeNaviculalesBrachysiraceaeD.Mora, R.Jahn & N.Abarcasp. nov.urn:lsid:ipni.org:names:B 40 0042006; Figure SEM stub), QMEX DIAT0001 (Slide).B 40 0042007 . Collected by Demetrio Mora on 07.09.2014.Paredones stream, on the outskirts of Paredones village, Dolores Hidalgo, Guanajuato, Mexico , wider at valve center (4.5–6 µm) and has less striae in 10 µm (27–30) , have more striae (36–40), the areolae are arranged in a way that they give the appearance of waves and each single areola is comparatively not as elongated as in B.altepetlensis , have less striae (26–32) and lack papillae in the interstriae ( (27–30) . The valetlensis . Brachyserstriae .Brachysiraneoexilis Lange-Bertalot species complex, but the type population of B.neoexilis has clear capitate apices and the larger specimens have a very slightly triundulate valve margins , closely resemble B.altepetlensis in valve outline but they clearly differ from specimens depicted in the type description of B.neoexilis (Brachysira found by Abarca-Mejía (2010) in a spring also in the Lerma-Chapala Basin, closely resemble B.altepetlensis in LM, but her identification was based on B.neoexilis.The valve dimensions as well as the striae density of the new species fall within the range of the  margins . All thecription  have subeoexilis . The speBrachysira species takes the name from the word “āltepētl” which means “water mountain” in Náhuatl language, that is how the surrounding mountains were used to be named by native people 500 years ago, at the time Spaniards first came to the region.this new B.altepetlensis only reached high relative abundances (>10%) in acidic waters (pH= 5.1–5.8) with low specific conductivity (42–53 μS/cm).apart from the type locality, this species was also found in four streams sampled for this study, namely Peña Colorada (site 4), San Martín (site 5), La Laborcilla 1 (site 7) and La Laborcilla 2 (site 8), all of these sites were characterized by low specific conductivity (≤ 100 μS/cm) and pH values going from acidic to slightly alcaline (5.1–7.9). But Taxon classificationPlantaeNaviculalesSellaphoraceaeD.Mora, N.Abarca & J.Carmonasp. nov.urn:lsid:ipni.org:names:B 40 0042009; Figure SEM stub), QMEX DIAT0003 (Slide).B 40 0042010 . Collected by Demetrio Mora on 18.09.2013.stream Los Ailes 1, close to the town San Pedro, Huimilpan, Querétaro, Mexico  C.E. Wetzel, Ector, Van de Vijver, Compère & D.G. Mann; the linear–elliptical forms of Sellaphoracrassulexigua (E. Reichardt) C.E. Wetzel & Ector; and that of Sellaphoranigri (De Notaris) C.E. Wetzel & Ector. But S.chistiakovae has uniseriate to irregularly biseriate striae  C.E. Wetzel & Ector, Sellaphorarhombica  D. Mora, N. Abarca & R. Jahn, comb. nov. (see new combination below) and Sellaphorathioense  C.E. Wetzel, Ector, Van de Vijver, Compère & D.G. Mann. But the valves of S.labernardieri are linear to linear–elliptical, slightly inflated at the center and have consistently more striae 10 µm  (Sellaphorarhombelliptica has more striae (25), which are uniseriate and the valves are rhomboelliptic (Sellaphorarhombica has similar number of striae (17–21) but the valve outline is rhombic to rhombic–lanceolate  with higher striae density (27–28)  . Sellaphelliptic . Sellaphnceolate . Sellaph (27–28) .Sellaphora species takes its name from the demonym of the Mexican state Querétaro, from where it was collected.this new so far only known from the type locality (sampling site 11 in this study) and from stream Laguna de Servín 2 (site 13) located 4 km away from the type location, in acidic waters (pH 5.9–6.2) with low conductivity (77–88 μS/cm).PageBreakPageBreakSellaphora species, Eolimnarhombica Gerd Moser, Lange–Bertalot & Metzeltin is transferred to Sellaphora:Based on morphological similarities with other small Taxon classificationPlantaeNaviculalesSellaphoraceae D.Mora, N.Abarca & R.Jahncomb. nov.urn:lsid:ipni.org:names:Eolimnarhombica Gerd Moser, Lange-Bertalot & Metzeltin, 1998, Bibliotheca Diatomologica, vol. 38, p. 156, pl. 23, figs 11–20.http://phycobank.org/100103QBR values are enlisted in Table DCA, total dissolved solids and total alkalinity were highly correlated with specific conductivity and therefore removed from the analysis. Dissolved oxygen and dissolved oxygen saturation percentage were also highly correlated, the latter being removed from further analysis. Dissolved inorganic nitrogen was also removed because it correlated strongly with nitrates. CCA with forward selection and unrestricted Monte Carlo permutations tests  identified temperature , pH , specific conductivity , soluble reactive phosphorous  and the Riparian Forest Quality Index  as the variables that significantly explained variation in the diatom data. The first two CCA axes accounted for 66.5 % of the cumulative variance of the species – environmental relation, both axes being significant (p=0.0010). The first CCA axis was strongly correlated with specific conductivity (inter-set correlation r= 0.93) and pH (r= 0.80). The second CCA axis was negatively correlated with QBR (r= -0.61) and positively correlated with temperature (r= 0.44).The physical and chemical composition of the water from the sampling sites, as well as CCA biplot three groups of samples were visualized . Other indicator taxa  for this group were Achnanthidiumaff.catenatum (J.Bílý & Marvan) Lange-Bertalot, Brachysiraaltepetlensis, Eunotia sp. 3, Fragilariaaustriaca (Grunow) Lange-Bertalot, Frustuliacrassinervia (Brébisson) Lange-Bertalot & Krammer and Gomphonemaexilissimum (Grunow) Lange-Bertalot & E. Reichardt  Mayama, Encyonemaminutum (Hilse) D.G. Mann, Mayamaeapermitis (Hustedt) Bruder & Medlin and Nitzschiapaleavar.tenuirostris Grunow, all these taxa with high and significant IndVals (>50%)  Table .QBR on average. The mean species richness was 17  Kociolek & Stoermer and Sellaphoraatomoides (Grunow) C.E. Wetzel & Van de Vijver.Samples from the third group correspond to well mineralized waters with the highest pH values on average, and also the lowest nitrogen concentrations. The sites in this group scored the higher values for the 17 Table . This grCCA plot  from 59 samples analyzed from three substrates; 178 taxa by Segura-García (2012) from 66 epilithon samples analyzed; 173 taxa by PageBreakThis kind of comparison is difficult to make since it depends on the number of samples analyzed, the timing of the samplings, the physical and chemical composition of the waters, the number of substrates sampled and the taxonomic effort with which the diatom valves were analyzed  plus seemingly endemic regionals . So far PageBreakstreams from headwaters to the midlands and also from the plains, resulting in different riparian communities, reflected in the QBR index values obtained; c) streams ranging from perennial to temporary; d) heterogeneity of physical and chemical composition of the water. Environmental heterogeneity of habitats has been proposed in other studies as a determinant of species richness and distribution .Another hypothesis that could explain the high species richness found in our study is the heterogeneity of environmental conditions of the study areas: a) the sampling campaigns were done in both rainy and dry seasons; b) varied geomorphologies of the Craticulasubminuscula, Gomphonemaparvulum, Naviculaveneta, Nitzschiaamphibia, N.capitellata, N.palea and Sellaphorapupula  De Candolle, Paralemaneamexicana (Kützing) Vis & Sheath and Sirodotiasuecica Kylin, species typically found in headwater mountain streams of temperate regions  apparently did not respond to those seasonal fluctuations  was observed on the three groups of sampling sites observed after the tuations . This isurbation . On the urbation . The timurbation . In ordeurbation . Since durbation , it shouCCA, there are several similarities with previous reports on the ecological preferences of these taxa. Some species were found in all three groups but with varying relative abundances, so only those with the largest abundances were taken as the representative for a group.Regarding the characteristic species of the three groups visualized after the Brachysira, Eunotia and Frustulia are well regarded as characteristic from acidic and electrolyte poor waters  C.E. Wetzel & Ector, Cyclotellameneghiniana Kützing, Fistuliferasaprophila  Lange-Bertalot and Navicularostellata Kützing  D.G. Mann , Naviculareichardtiana, Nitzschiainconspicua, Planothidiumvictori  Lange-Bertalot sensu lato), Reimeriasinuata and Sellaphoraatomoides (former Eolimnatantula (Hustedt) Lange-Bertalot) (Amphorapediculus (Kützing) Grunow, Epithemiaadnata (Kützing) Brébisson, Epithemiasorex Kützing, Gomphonemapumilum (Grunow) E. Reichardt & Lange-Bertalot, Halamphoramontana (Krasske) Levkov and Naviculagregaria Donkin  . Other ta Donkin . When lonitrogen .This work contributed to increase the knowledge of the diatom flora from the Lerma-Chapala Basin, Central Mexico, providing a diversity baseline and evidence of its distinctiveness from the floras of other areas in Mexico, with a large proportion of unidentified taxa to be described as new. The studied diatom communities are subjected to moderate environmental disturbance, representing a transition between warm and cold waters, with ionic composition, temperature and the quality of the riparian forest being the main factors defining the community composition observed. The next approach to investigate the diatom diversity of the region would be by means of environmental DNA metabarcoding in combination with the development of a taxonomic reference database, in order to highlight the complementary aspect of classical taxonomy and eDNA metabarcoding, i.e. the importance of the reciprocal illumination .PageBreak


OBJECTIVES: Combined coronary artery bypass grafting and valve surgery requires a prolonged period of cardioplegic arrest (CA) predisposing to myocardial injury and postoperative cardiac-specific complications. The aim of this trial was to reduce the CA time in patients undergoing combined coronary artery bypass grafting and valve surgery and assess if this was associated with less myocardial injury and related complications.METHODS: Participants were randomized to (i) coronary artery bypass grafting performed on the beating heart with cardiopulmonary bypass support followed by CA for the valve procedure (hybrid) or (ii) both procedures under CA . To assess complications related to myocardial injury, we used the composite of death, myocardial infarction, arrhythmia, need for pacing or inotropes for >12 h. To assess myocardial injury, we used serial plasma troponin T and markers of metabolic stress in myocardial biopsies.RESULTS: Hundred and sixty patients  were randomized. Mean age was 66.5 years and 74% were male. Valve procedures included aortic (61.8%) and mitral (33.1%) alone or in combination (5.1%). CA time was 16% lower in the hybrid group . Complications related to myocardial injury occurred in 131/160 patients , odds ratio 1.24, 95% CI 0.54–2.86, P = 0.61. Release of troponin T was similar between groups . Adenosine monophosphate was 28% lower in the hybrid group .CONCLUSIONS: The hybrid procedure reduced the CA time but myocardial injury outcomes were not superior to conventional approach.TRIAL REGISTRATION: ISRCTN65770930. The number of patients requiring surgery to treat concomitant valve and coronary disease is rapidly increasing . BetweenA multicentre parallel-group randomized controlled trial (ISRCTN 65770930).Adults aged between 16 and 85 years with severe coronary disease and aortic and/or mitral valve disease (confirmed by transthoracic echocardiography and angiogram) were eligible. History of diabetes, malignancy, debilitating neurological disease, ongoing sepsis or endocarditis, or preoperative creatinine >160 µm/l, or need for emergency or salvage procedures were exclusion criteria.The study was conducted at the Bristol Heart Institute, Bristol (UK), Rabindranath Tagore International Institute, Kolkata and the SAL Hospital, Ahmedabad (India) by surgeons willing to operate using either methods. The University Hospital Bristol NHS Foundation Trust sponsored the trial in the UK. In India, the study was sponsored by the 2 host institutions. The trial was approved by the Southmead Research Ethics Committee (ref. CS/2006/2267) in the UK and by local hospital Ethics Committees in India.conventional surgery with both coronary and valve surgery being performed during CPB and CA; or (ii) hybrid surgery with coronary surgery carried out first on the beating heart with CPB support, followed by CA to undertake valve surgery.Participants were randomly allocated to either (i) + and Mg2+ added. Final K+ and Mg2+ concentrations were 20 and 4 mM, respectively. Antegrade cardioplegia was delivered under pressure (100–150 mmHg) directly into the coronary ostia or the aortic root, while retrograde cardioplegia was delivered under pressure (20–30 mmHg) directly into the coronary sinus at 20 min intervals. Following surgery, patients were admitted to the cardiac intensive care unit and managed by blind intensivists. Decisions regarding need for inotropic support, pacing and mode of ventilation were based on established routine clinical care  if distributions are skewed. Categorical data are summarized as number (percentage). Outcomes were compared using logistic (binary variables), Cox proportional hazards , or linear mixed model (continuous variables measured at multiple time points) regression, with conventional surgery as the reference group. Model validity was checked using standard methods; if a model fitted poorly, transformations were explored. Outcomes analysed on a logarithmic scale were transformed back to the original scale after analysis and results presented as geometric mean ratios (GMR). See the Between 19 March 2008 and 01 July 2012, 165 patients gave written consent and were randomized; 5 were withdrawn prior to surgery, leaving 160 patients who received surgery and were included in analysis population  in the conventional surgery group and 67/80 83.8%) in the hybrid group [odds ratio (OR) 1.24, 95% confidence interval (CI) 0.54–2.86, .8% in thP = 0.20, overall estimate of difference between the 2 groups GMR 1.04, 95% CI 0.87–1.24, P = 0.68, Fig. P = 0.48), although higher levels of cTnT release were observed in India compared to Bristol  and the conventional group 0.72 . There were no differences between the groups with respect to adenosine triphosphate (ATP), ATP/adenosine diphosphate, ATP/adenosine monophosphate with mean differences of −0.21, 95% CI −0.94 to 0.52, P = 0.57; 0.03, 95% CI −0.28 to 0.34, P = 0.83 and 0.73, 95% CI −0.45 to 1.91, P = 0.21, respectively. Similarly, there was no difference for lactate . CPB time was 7% longer in the hybrid group . Operation time was 4% longer in the hybrid group (P = 0.2). Descriptive only key secondary clinical outcome including pulmonary, infective, renal and neurological complications are shown in Table Operative details are shown in Table P = 0.65), transfusion requirement , blood loss , combined non-cardiac subsystem organ complication , serious adverse events, intubation time, cardiac intensive care unit length of stay and hospital stay being also similar between the 2 groups  in the hybrid group this difference had no effect on measures of myocardial injury including the selected composite as well as the release of cTnT and markers of metabolic stress.The trial confirms that the hybrid procedure is surgically feasible and reproducible; 12 surgeons participated across the 3 centres and there was only 1 (1.2%) cross-over from hybrid to conventional surgery. It also suggests that the hybrid procedure is safe; health outcomes were similar to the conventional technique overall and across the 3 centres, although the incidence of mortality was higher in the Indian centres (15.2%) compared to the UK centre (4.9%), probably reflecting the geographical differences in patient characteristics, health outcome, and service provision. The health outcome of the Bristol centre is in keeping with current North American and European standards populations , 24.Shortening of CA time by 9 min was insufficient to demonstrate a clinical benefit. The saving of only 9 min was probably triggered by a high proportion of single or double-vessel coronary disease recruited (74%), leading to 78% of patients receiving 1 or 2 bypass grafts. Indeed, the amount of CA time saved increased with the number of bypass grafts performed , but no subanalysis was undertaken to assess if longer CA time savings were associated with myocardial injury in keeping with the predefined statistical analysis plan.et al. [The concept of shortening CA time to minimize myocardial injury is not new. Perrault et al.  suggesteet al. .The use of on-pump beating heart coronary surgery in patients with valve disease has been reported only in anecdotal studies , 27. OurThe impact of the hybrid procedure on the clinical components of the composite end-point suggests that this was a negative trial. We selected these components due to their direct relation with myocardial injury. This is in keeping with the evidence by others suggesting that a CA time >75 min is associated with a higher risk of cardiac death and major adverse cardiac events and that high levels of cTnT are associated with major adverse cardiac events , 28. Theet al. [et al. [In both groups, the median CA time was higher than the critical threshold of 75 min suggested by Liakopoulos et al. . Of note [et al.  suggest The study has limitations. There were some protocol deviations and 5 randomized patients were excluded from the analysis population. Including all valve pathologies provided an inclusive but potentially heterogeneous population. Follow-up was also limited and quality of life was not assessed. In addition, it may be argued that the 5 components of the composite differ in quantitative clinical importance i.e. a death or a large MI impact more negatively than an arrhythmic event or need for inotropes or for pacing. However, these components were selected as they are all qualitatively clinical reflections of myocardial injury, regardless of their quantitative impact.The hybrid procedure was surgically feasible, reproducible and safe. The statistically significant reduction in CA time of 9 min was not clinically meaningful as clinical and biochemical measures of myocardial injury were similar between groups. The hybrid procedure is not superior to conventional technique.EJCTS online.Supplementary DataClick here for additional data file.


Ficus species growing in a common garden in SW China. The leaf anatomical traits, stem wood density (WD), and stem anatomical and mechanical properties of these species were also measured. We analyzed leaf–stem allometric relationships and their associations with stem hydraulic ad mechanical properties using species-level data and phylogenetically independent contrasts. We found isometric relationship between leaf lamina area/mass and stem area/mass, suggesting that the biomass allocation to leaf was independent to stem size. However, allometric relationship between LA/mass and petiole mass was found, indicating large leaves invest a higher fractional of biomass in petiole than small ones. LI, i.e., leaf numbers per unit of stem mass, was negatively related with leaf and stem size. Species with larger terminal branches tend to have larger vessels and theoretical hydraulic conductivity, but lower WD and mechanical strength. The size of leaf lamina, petiole, and stem was correlated positively with stem theoretical hydraulic conductivity, but negatively with stem WD and mechanical strength. Our results suggest that leaf–stem allometry in Ficus species was shaped by the trade-off between stem hydraulic efficiency and mechanical stability, supporting a functional interpretation of the relationship between leaf and stem dimensions.Leaf–stem allometry is an important spectrum that linked to biomass allocation and life history strategy in plants, although the determinants and evolutionary significance of leaf–stem allometry remain poorly understood. Leaf and stem architectures – including stem area/mass, petiole area/mass, lamina area/mass, leaf number, specific leaf area (LA), and mass-based leafing intensity (LI) – were measured on the current-year branches for 28  Leaf surface area spans six orders of magnitude across terrestrial plants species , and theThis “Corner’s rules” has been tested empirically within and across species and environments . PositivThe leaf–stem size spectrums are often expected partly as a result of the mechanical and conductive demands of leaves , i.e., lPlant xylem plays three main functions, i.e., mechanical support, water conduction, and water and photosynthate storage . Vessel-Ficus species growing in a common garden in tropical China. We analyzed the allometric relationships among these traits, both across species and across phylogenetically independent contrasts (PICs). We asked the questions: (1) are the scaling relationships between leaf size and stem/petiole size isometry or allometry? (2) Can the adjustments of plant xylem structures along with leaf/stem dimensions explain scaling relationships between leaves and stem size. We tested the specific hypothesis that there is a trade-off between hydraulic and mechanical function of stems, and this trade-off has compensated effect on the twig–leaf size allometric relationship.In the study, we measured leaf, stem, and petiole dimensions  of terminal branches of 28 This study was conducted at Xishuangbanna Tropical Botanical Garden , Yunnan, Southwest China. The region is dominated by warm, wet air masses from the Indian Ocean in summer and continental air masses from temperate regions in winter. The mean annual temperature is 21.7°C, with the monthly mean temperature being 15.9°C during the coldest month (December) and 25.7°C during the warmest month (June). The annual rainfall is 1560 mm, with more than 80% occurring during the wet season from May to October.Ficus genus that commonly found in the rainforests of Xishuangbanna . The individual lamina area (mass) was calculated as the total area (mass) divided by leaf number. Individual petiole mass was calculated as the total petiole mass divided by leaf number. Leafing intensity (LI), a common developmental correlate of leaf size, was defined as leaf numbers per unit of stem mass (no. g-1).For each sampled branch, a current-year terminal (un-branched) shoot (here defined as “twig”) with no herbivore damage was selected for trait measurements. For each twig sample, leaf lamina, petiole, and stem were separated. Leaf numbers were counted and leaf area (LA) was measured with an area meter . Stem and petiole diameters were measured with a vernier caliper. Twig stem, all leaf laminas, and petioles from a twig were oven dried at 70°C to constant weight, and their dry masses were weighted. Specific leaf area (SLA) was expressed as LA per unit of dry mass (m-3) was determined by dividing the dry mass by the volume of the samples.After removing the bark and pith with a razor blade, the fresh wood (FW) are weighted and wood fresh volume was measured the water displacement method. Wood saturated weight (SW) was determined after submerging under water for 48 h for rehydration. Wood samples were then over-dried for at least 48 h at 70°C to determine the dry weight (DW). Stem saturated water content (SWC) was calculated as: SWC = (FW - DW)/(SW – DW) × 100%. WD . Since vessel is mostly elliptical, vessel diameter was calculated as D = [32(ab)3/(a2+b2)]1/4, where a and b are the major and minor axis dimensions, respectively (Dh) is calculated as Dh = [1/nΣD4]1/4. VD (no. mm-2) was calculated as number of vessels per unit of xylem area (mm-2). We calculated vessel fraction (VF) as the product of vessel area and VD. The theoretical specific hydraulic conductivity  was estimated according to the Hagen–Poiseuille equation: Ktheo = μρ/(128μAs)[ΣD4], where ρ is the density of water (998.2 kg m-3 at 20°C), μ is the viscosity of water (1.002 × 10-9 MPa s-1 at 20°C), and As is the cross-sectional area of sapwood . We used ImageJ softwareectively . Hydraul sapwood . The calconduits .The modulus of elasticity (MOE) of stem  was measured by three-point bending method with an INSTRON mechanical testing machine with a 5 kN load cell . Stem segments had length-to-diameter ratios of 25:1 to avoid share. Stem segments were placed on a steel frame apparatus at two supporting points at a precise span distance. Consecutive weights were added to a pannier suspended from the exact center of the stem segment. Flexural stiffness (EI) and structural Young’s modulus (MOE) were calculated from linear relationships resulting from observed deflections of stem segments after adding a sequence of weights.For each sampled branch, three to five full-developed leaves were sampled from twigs closed to those used for measuring twigs architecture. Hand transverse sections were made for leaves and then mounted on glass slides, and then these sections were examined and photographed at 10× magnifications with a light microscope . Total leaf thickness (LT), thickness of the upper epidermis , lower epidermis , palisade tissue , and spongy tissue  were measured with ImageJ software. Stomatal size (SS) was represented as the guard cell length and stomatal density (SD) was calculated as the number per unit LA.10-transformed to fit the normal distribution , α = log(γ), and X = log(x). The linear relationship is described by its slope β or scaling coefficient and its y-intercept α or allometric constant. The value of the slope determines the relationship is isometric (β = 1) or allometric (β ≠ 1). Standardized major axis (SMA) regression was used to estimate the parameters of the allometric equation, by using the R package “SMATR”  for the measured stem and leaf traits, with the method of Ficus species, by using the FactoMineR package . Petiole area (mass) was highly related to lamina area (mass), with the scaling slopes between petiole and leaf mass were significantly smaller than 1.0, indicating an allometric scaling relationship between petiole and leaf lamina . Leaf size showed a negative, isometric relationship with mass-based LI, with the best-fit common regression slopes ranged from -0.9 to -1.1 . A significant positive allometric relationship was found between lamina mass and lamina area, with common slopes (1.1–1.6) significantly departed from the value of 1.0  scaled positively with stem area (mass), with best-fit common regression slopes were not significantly different with slope 1.0 : i.e., (1) trade-off between stem hydraulic efficiency and mechanical strength; (2) trade-off between SLA and the thickness of all lamina tissues, (3) trade-off between leaf/stem size and LI. The first axis of MFA accounted for 44.79% of the total variance among the 24 traits, which loaded mainly the leaf architecture traits, i.e., stem area/mass, LA/mass, petiole area/mass, and mass-based LI . In other word, species with larger leaves have thicker stem and petiole, but lower mass-based LI. Meanwhile, the first axis also loaded positively the traits associated with stem hydraulic efficiency , but loaded negatively the traits linked to stem mechanical strength (WD and MOE). The second axis accounted for 24.85% of the variance, and mainly loaded SLA and traits related to lamina tissue thickness  and SS and SD  had lower VD, but higher VF and thus high theoretical hydraulic conductivity (Ktheo) . On the other hand, theoretical hydraulic conductivity (Ktheo) showed negative correlations with WD and MOE . Thus, higher xylem hydraulic efficiency can only be achieved by the cost of reduced WD and mechanical strength. As compared with cross-species correlations, the correlations based on PICs were generally weaker, and in some case became non-significant .Figure 4). For example, stem area correlated positively with vessel lumen diameter (Dh), VF, and theoretical hydraulic conductivity (Ktheo), but negatively with VD, WD, and MOE. In other word, thicker stem is more efficient for water transport, but less efficient for mechanical stability. Moreover, significant positive correlations were found between stem theoretical hydraulic conductivity (Ktheo) and petiole area/mass and LA/mass for both cross-species means and PICs . However, stem WD was negatively correlated with stem, petiole, and leaf dimensions .Interestingly, wood hydraulic and mechanical traits varied with stem size of terminal branches . These results are consistent with the findings of Figures 1B,E and Table 1) implied that more biomass investment to petiole with a unit increase of lamina size.We found an isometric relationship between leaf size and stem size, indicating that the increase of total or individual LA/mass is generally proportional to the increase of stem area/mass , which demonstrates the generality of the leaf size/number trade-off as found by other studies   is largely determined by vessel diameter (Dh), because Ktheo increases linearly with VD but with the forth power of Dh, and because of the tight negative relationship between Dh and VD . Significant negative correlation between Ktheo and WD and MOE suggests a trade-off between water transport and mechanical support. Wood composed of closely spaced large vessels will have low density with high hydraulic conductivity, whereas wood composed of small vessels spaced widely within a fiber matrix will be dense with low hydraulic conductivity . These results indicated that higher conductive and lower density woody species grow more rapidly, not only in volume, but also in stem and total leaf mass. Species with higher hydraulic efficiency, expressed by larger Dh, VF, and Ktheo, should be able to transport more water and thus deploy a larger total LA per stem . Moreover, higher theoretical hydraulic conductivity and lower WD were associated with lower LI (leaf numbers per sapwood mass) , which implied that few larger leaves gave a better return for investment on the stem than many smaller leaves , indicating that bigger branches are less efficient for mechanical strength. The negative relations between leaf size and stem mechanical properties invoke an indirect correlation via plant hydraulics, i.e., species with lower WD have higher hydraulic conductivity per sapwood area as a result of having a higher proportion of stem cross-section taken up by vessel lumen (Figure 1; In addition to supplying leaves with water and nutrients, twigs also provide mechanical support for static loads and the drag forces exerted on leaves by winds . Howeverel lumen , which iel lumen . For theFicus species growing in a common garden of tropical China. However, large leaves tend to have a larger fractional biomass investment in petioles. Larger stems have wider vessel lumens in the xylem and achieve higher stem specific hydraulic conductivity, but the function of mechanical strength is diminished with less dense wood. The trade-off between hydraulic efficiency and mechanical strength in xylem may shape the leaf–stem allometric relationships. We provide functional interpretation of the relationships between leaves and stem dimensions.We provide evidence that leaf–stem size follows an isometric relationship across 28 Z-XF and P-LF carried out the experiment and collected the data. Z-XF and FS analyzed the data and wrote the first draft of the manuscript. FS, S-BZ, and G-YH contributed to data analysis, writing, and interpretation. All authors contributed critically to the drafts and approved the final version of the manuscript.The 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.


No significant differences in body composition and muscle strength improvements were found between groups. In young healthy men, insect protein supplementation did not improve adaptations to eight weeks of resistance training in comparison to carbohydrate supplementation. A high habitual protein intake in both Con and Pro may partly explain our observation of no superior effect of insect protein supplementation.During prolonged resistance training, protein supplementation is known to promote morphological changes; however, no previous training studies have tested the effect of insect protein isolate in a human trial. The aim of this study was to investigate the potential effect of insect protein as a dietary supplement to increase muscle hypertrophy and strength gains during prolonged resistance training in young men. Eighteen healthy young men performed resistance training four day/week for eight weeks. Subjects were block randomized into two groups consuming either an insect protein isolate or isocaloric carbohydrate supplementation within 1 h after training and pre-sleep on training days. Strength and body composition were measured before and after intervention to detect adaptions to the resistance training. Three-day weighed dietary records were completed before and during intervention. Fat- and bone- free mass (FBFM) improved significantly in both groups )), control group (Con):   The United Nation’s (UN’s) Food and Agriculture Organization (FAO) estimates that global food production must increase by >70% by 2050 to feed the growing world population, expected to reach 9–10 billion people —and nevePrevious investigations have shown that after resistance exercise the skeletal muscle is sensitive to protein supplementation for up to 48 h post-exercise ,6,7. FurAreta et al.  propose The quality of the most commonly consumed isolated protein sources, whey, casein, and soy, differs inter alia (i.a.) with respect to digestibility and amino acid (AA) profile. A high-quality protein contains a large amount of essential amino acids (EAA) and branched-chained amino acids (BCAA), especially leucine, which, besides being a building block for proteins, also seems to have a stimulating effect on skeletal MPS ,14. FurtThe aim of this study was to investigate the potential effect of insect protein isolate as a dietary supplement to increase muscle hypertrophy and strength gains during prolonged resistance training when protein dose and timing meet current recommendations. Primary and secondary outcomes are expressed as changes in fat- and bone- free mass (FBFM) and muscle strength, respectively. We hypothesize that supplementation of insect protein isolate promotes greater muscle hypertrophy and strength gains during prolonged resistance training compared to isocaloric carbohydrate supplementation.Eighteen healthy young males , with no difference between groups, were recruited via posters at Aarhus University and local dorms in Aarhus, and online via posts on Facebook. Inclusion criteria were males from the age of 18 to 30 years who had previous experience performing resistance exercise. Sport and activities up to 10 h per week were accepted, if activities did not include structured resistance training. Exclusion criteria were resistance training in the past year, the use of medication or dietary supplements, smoking and injuries, or health issues conflicting with participation in the training program. At initial contact, subjects were screened by questionnaire against criteria of inclusion and exclusion. Based on the responses to the questionnaire, subjects were deemed suitable. The trial complied with the Declaration of Helsinki and was approved by The Central Denmark Region Committees on Health Research Ethics. Clinical trial registry number: NCT03034239. All subjects gave their informed consent to participate before the experiment.n = 9) or an isocaloric carbohydrate bar  within 1 h after each training and 1 h before night sleep on training days. Before and after the intervention, strength and body composition were measured. Dietary records were obtained (three-day registration periods) before and during the intervention period.The trial was designed as a randomized, controlled, single-blinded trial consisting of eight weeks of resistance training four days/week, performed as a whole-body split routine. The subjects were blinded and randomly assigned to two groups that ingested either an insect-protein bar containing 0.4 g protein/kg  was added for the Pro group supplementation. The manufacturer stated that the total protein content of the insect protein isolate was 80–85%, evaluated on the basis of the Kjeldahl method with a nitrogen-to-protein conversion factor (Kp) of 6.25. A carbohydrate-rich mix of potato flour  and Maxim Sports Drink  ), ratio 1:1, was added for the Con bar to match the Pro bar in terms of energy content. The Con and Pro bars were isocaloric. Thanks to the similar flavors from banana, ginger, and cinnamon, the bars tasted and smelled roughly the same.The dietary supplementations were served as baked bars. All bars contained a mix of banana, ginger, oats, raisins, and cinnamon . A dose of 0.4 g/kg/bar insect protein  from the lesser mealworm  . The system’s software package  was used to determine changes in body composition BW (body weight), FM (fat mass), BMC , and FBFM. In addition to whole-body measurements, the LB and UB regions were analyzed separately. The distinction between LB and UB was made by a line crossing the superior points of the crista iliaca. The head (line crossing chin) was not included in UB. The subjects were scanned at the same time of day at week 0 and 9 and were told to be fasting from 10:00 p.m. the night before the scan, and not to drink 2 h before. Before the scan, subjects had their weight and height determined. All scans were performed at Section for Sports Science, Department for Public Health at Aarhus University.The subjects underwent a series of different strength tests before and after the training period. The protocol consisted of two separate test days with 1 RM test in leg press and bench press, 80% of 1 RM to failure in leg press and bench press (day 2), isometric maximal voluntary contraction (MVC) of right leg knee extensor and flexor, and a counter-movement jump (CMJ) test (day 1). Before 1 RM testing, all subjects performed a familiarization test to avoid any major variation in results affected by the test situation, with a minimum of one day’s rest between the familiarization test and the valid test. Subjects were told to avoid any strenuous exercise the day prior to the test. A standardized warm-up was performed consisting of 5 min of light aerobic exercise followed by 10 repetitions at 50% of estimated 1 RM load with a 2-min rest and 3–4 repetitions at 80% of 1 RM load with a 3-min rest. The test consisted of three to five progressive 1 RM attempts interspersed with a 3-min rest . When 1 MVC was determined seated on a dynamometer  with 90° hip flexion and the hands were placed on the lap. All subjects performed a warm-up of 5 min light aerobic exercise (self-chosen load) on a stationary bicycle  before the test. Their isometric knee extensor and flexor MVC were then measured in the dynamometer by five attempts at 70° knee flexion  . Jumping height, subjects’ weight, and flight time were used to assess the maximal power output during the jump. Subjects were given five attempts with a 1-min recovery between them. The best of the five attempts were used for further analysis.n = 3).Before the intervention, the participants completed a 3-day weighed dietary record on three consecutive days, including one weekend day. The data were analyzed for habitual intake of energy and macronutrients. During the intervention, a similar dietary record was performed. All dietary data (except the Con and Pro supplements) were registered in the software program . Records not meeting the criteria of Goldberg cut-off limits  < 1.00 or >2.4)  were excz-test. A two-way repeated measure analysis of variance (ANOVA) with time as a within factor and group as a between factor was used to determine any time-by-group interaction in all body composition variables, dietary variables, and strength changes. Results are presented as mean ± standard deviation (SD) with delta values shown as mean ). Alpha level was set to ≤0.05.Statistical analyses were performed using the EpiBasic v3.0  . All datAll 18 subjects included in the study completed the intervention period with a training compliance of 97.6 ± 2.7%. The dietary supplementations were consumed with no major complications in both groups. Two subjects (Pro) suffered an injury in the shoulder complex from activity not related to the training protocol . Upper-bOver eight weeks of resistance training with dietary supplementation, total body weight increased significantly in both groups . FM incrn = 3, Con).p < 0.05) from pre- to mid- intervention, while Pro increased protein intake by 48% (p < 0.001). No significant difference in total energy and fat intake were observed between groups. However, there was a tendency towards a higher energy intake in Con compared to Pro (p = 0.10) from pre- to mid-intervention. Furthermore, a significant time-by-treatment interaction for protein intake in Pro (p < 0.05) and carbohydrate intake in Con (p < 0.05) was evident.Con increased total energy, protein and carbohydrate intake by 30%, 26% and 43% (p < 0.05), except bench press volume Pro (p = 0.54) Con (p = 0.97) and leg press volume Pro (p = 0.08). However, a tendency towards a time effect for leg press volume for Pro was observed. No difference in group-by-time interaction was detected at any point.Changes in 1 RM, leg- and bench- press volume, MVC knee extension (Ext.), knee flexion (Flex.) and CMJ between pre- and post- intervention are shown in p = 0.57) or UB .Total training volume was calculated for all subjects. As training intensity of load was defined as 80% of 1 RM , total training volume was calculated as sets × repetitions (reps). Statistical tests revealed no significant difference between groups in LB  and 2.7 ± 1.5 kg (4.2%) in FBFM for Con and Pro, respectively, is notably high compared to similar studies with protein supplementation ,32,33. SComparable to studies examining strength gains following resistance training for 7–16 weeks ,35,36,37p < 0.05). Recommendations on optimal daily protein intake vary between 0.8 and 1.6 g/kg/day depending on physical activity level, gender, and age [p = 0.10). On non-training days, Con increased total energy intake from 2764 ± 312 kcal (Pre) to 3069 ± 621 kcal (Mid) (p = 0.28) and Pro decreased from 3142 ± 747 kcal (Pre) to 2785 ± 441 kcal (Mid) (p = 0.2218), which may reflect different changes in FM between groups   . MoreoveThe present study had no dietary restrictions before and after training, which may contribute to the fact that no differences in body composition changes were observed between groups. In addition, the time of training was not controlled and we observed that most subjects trained in the late afternoon. This may interfere with optimal timing strategies for protein supplementation, as suggested by Areta et al. , as bothAn intervention period of eight weeks with resistance training may seem short compared to similar studies of 10–14 weeks ,41,49. AAlphitobius diaperinus) equally match the profile of soybean in regards to EAA, although no AA analysis was conducted on the specific protein isolate  used in this study. A review by Devries and Phillips [Although we did not detect any benefits of timed protein supplementation during exercise intervention in healthy, well-nourished young men, insect protein may still have value as an alternative protein source. AA profiles by Yi et al.  showed tPhillips  examinedPhillips  suggesteWith a growing world population and increased need for dietary protein, insect protein should be considered a valuable alternative to other dietary animal protein sources given its environmental profile . Future In conclusion, we found that eight weeks of resistance training effectively promote gains in FBFM and muscle strength in healthy young men with a habitual intake of protein corresponding to the general population of young Danish men. However, no difference in morphological adaptations such as hypertrophy or muscle strength was observed between supplementation groups, as hypothesized.


Tridacna maxima were characterized over a two-year-period in the lagoon of the high island of Tubuai  and in the semi-closed lagoon of Tatakoto (Tuamotu Archipelago) in French Polynesia. We also recorded temperature, water level, tidal slope, tidal range, and mean wave height in both lagoons. Lower lagoon aperture and exposure to oceanic swells at Tatakoto than at Tubuai was responsible for lower lagoon water renewal, as well as higher variability in temperature and water level at Tatakoto across the studied period. These different environmental conditions had an impact on giant clams. Firstly, spawning events in the lagoon of Tatakoto, detected by gonad maturity indices in June and July 2014, were timed with high oceanic water inflow and a decrease in lagoon water temperature. Secondly, temperature explained differences in shell growth rates between seasons and lagoons, generating different growth curves for the two sites. Thirdly, local mortality rates were also found to likely be related to water renewal patterns. In conclusion, our study suggests that reef aperture and lagoon water renewal rates play an integral role in giant clam life history, with significant differences in rates of shell growth, mortality and fertility found between open versus semi-closed atoll lagoons in coral reef ecosystems.Shell growth, reproduction, and natural mortality of the giant clam  Tridacna maxima and T. squamosa, are widespread throughout the Indo-Pacific and can be found from the Red Sea and southeast Africa in the west to the Central Pacific in the east [T. maxima), where light provides them most of the energy necessary for survival, growth, and reproduction, owing to the ability of their symbiotic zooxanthellae to photosynthesize [Among the 12 species of giant clams , nthesize .T. maxima [Giant clams play various ecological roles in coral reef ecosystems . Their t. maxima .Most species of giant clams were traditionally exploited by local fishers for meat consumption or shell use as common household items . Over thT. maxima , is still found in relatively high densities at various locations throughout the Central Pacific [Tridacna maxima is currently classified as lower risk/conservation dependent  under the IUCN Red List for threatened species. Densities and stocks can vary locally by several orders of magnitude, depending on past and present fishing pressure and fluctuations in environmental drivers [T. maxima’s meat and the related depletion of their stocks throughout their range has highlighted the need for conservation measures designed to address the state of the resource at local levels. Thus, quantifying stocks and their spatial distribution are the first steps necessary to relevant management planning.The small giant clam  Pacific . Tridacn drivers . The incT. maxima stock and individual density estimates of T. maxima were derived for a number of locations and reef types . Densit Tuamotu , 5. At iubstrate , 10. Theubstrate ) and supubstrate , 11).T. maxima densities has also been reported for the enclosed lagoon of Millennium atoll  and their interaction with the environment. When estimated at a specific point in time, stock distribution alone cannot provide an accurate view of population dynamics. Any conclusions drawn from such an assessment may therefore affect the success of long term management. For example, a re-evaluation of stocks at Tatakoto in 2012 highlighted a tenfold decrease in observed densities . Howeveriribati; ). This rT. maxima’s life traits  based on a sampling scheme spatially-structured at a number of scales. At lagoon and regional scale, our study sampled two sites in French Polynesia differing in their location and reef type. At intra-lagoon scale, the sampling scheme was designed according to the abiotic factors previously identified to structure the spatial distribution of densities at both locations [T. maxima life traits at different scales.Besides the monitoring of stocks at regular time intervals, sustainable management also requires a clear understanding of population dynamics and biological processes, as well as their interaction with environmental factors at regional scale , reef scale , and intra-lagoon scale  . Howeverocations . We alsoT. maxima collections for this study were authorized by French Polynesia Ministerial Order no. 2850/MRM.All Our study focused on two sites in French Polynesia: the semi-closed atoll of Tatakoto  located in the Tuamotu Archipelago, 1,200 km East of Tahiti; and the island of Tubuai  located in the Austral Archipelago, 600 km South of Tahiti . Offshor-1), bringing cool, dry weather that may last for several days. Such wind patterns are less common in the Tuamotu. These differences in wind patterns result in stark differences in annual rainfall and sunshine hours at both locations: around 2,000 mm.yr-1 and 2,200 h.yr-1 at Tubuai compared to 1,000 mm.yr-1 and 2,800 h.yr-1 at Tatakoto respectively [The two sites are also exposed to different wind regimes : northeaectively , 19.2) is open as it is permanently connected to oceanic waters through a large reef pass in the northern part of the lagoon, and a small reef pass at the southwest end of the lagoon (T. maxima in the context of this study). The atoll rim of Tatakoto is closed in its northern part, but the lagoon (11.46 km2) is connected to the ocean by way of several shallow channels that bissect the southern part of its rim (T. maxima were recorded in 2004 at the eastern extremity of the lagoon (up to 544 ind.m-2) leading to this area’s classification as a no take area (NTA) that same year [The two study sites are also representative of two contrasting reef aperture configurations . The lae lagoon . All ree its rim . Tatakotame year . Our stuTo characterize the physical environment of Tatakoto’s and Tubuai’s lagoons, we deployed temperature and pressure sensors at a number of locations at both sites. The exact positions, measurement types, and depths at which the sensors were deployed are summarized in At Tatakoto, one sensor (RBRduo n°3) at station 15 recorded temperature every 30 minutes, and pressure in bursts of 512 measurements every 30 minutes from November 2012 to October 2014. Pressure measurements provided (i) a proxy for water levels inside the lagoon, (ii) tidal slope and tidal range, and (iii) mean wave height (H). Two further temperature sensors (SBE56 n°1 and n°2) and one pressure sensor (DR1050 n°4) were placed at opposite ends of the lagoon to characterize the wave exposure and thermal variations of these shallow areas. A greater sampling effort was allocated to temperature measurements at Tatakoto, as previous research has suggested this factor as critical to structuring the giant clam population in the atoll’s lagoon [T. maxima densities in this lagoon [At Tubuai, one sensor TWR2050 n°3) at station 7 recorded temperature every 30 minutes and pressure in bursts of 512 measurements every 30 minutes from April 2013 to December 2014. Four further pressure sensors  were also deployed at various locations to compare wave exposure between the western reef flat (sensors n°1 and n°5), the southern reef flat (sensor n°4), and patch reefs located in the eastern part of the lagoon (sensor n°2). The absolute values of the difference between two pressure measurements taken at one-minute interval (δP) were used as a proxy of wave exposure. Sampling at Tubuai focused on pressure (instead of temperature) as wave exposure has previously been identified as the main factor structuring 0 n°3 at We estimated growth and natural mortality of giant clams through regular monitoring of tagged individuals.At Tatakoto, where individuals occur in high density aggregations, we used a tagging technique commonly referred to in the literature , 26, 18:At Tubuai, the lower densities of giant clams allowed us to implement a method that is not stressful for the animals. Individuals were identified by their colours and their position along permanently established transects. Overall, 424 individuals were virtually tagged in April 2013. We checked whether individuals were still alive and estimated their shell length (anterior-posterior measurements) to the nearest millimetre using callipers in November 2013, May 2014, and December 2014. No supplementary tagging was needed at Tubuai as the majority of initially tagged clams were relocated in resurveys.Analyses of natural mortality and shell growth were first performed at lagoon-scale  for the entire study period. For considerations at intra-lagoon-scale, we structured the analyses by the same factors that structured sampling . The monitoring field visits allowed us to define four time periods at Tatakoto and three at Tubuai, each including either the coldest time of the year  or the warmest .G) between field visits were calculated using the following equation:Lt is the shell length  at time t and Δt denotes the time interval between two measurements (in days). The instantaneous natural mortality rate (M) was calculated using Nt and Nt+1 are the number of survivors between the start and the end of the time period considered. We assumed for giant clams that “disappeared” between two sampling events to be dead in our analyses , from the western part of the lagoon. The sampling location was chosen for practical and logistical reasons. At Tubuai, the monitoring of gonad maturation could not be conducted in as systematic a fashion as at Tatakoto because of operational constraints. Thus, samples were collected opportunistically 5 times over a two year period: August 2013, November 2013, September 2014, December 2014, and August 2015.Wbiomass , and the gonad weight Wgonad, to calculate the “Gonado-Somatic Index” (GSI)  .versus male tissues  and the proportion of male tissues harbouring spermatozoids (SPZ) were also calculated using histological treatments and used as proxy of sexual maturity, following the procedure recommended by a recent study [The proportion of female nt study .Tv) from the recorded values of temperature (T) using the following equation:We calculated the mean temperature over the study period  for parameters G) and instantaneous rate of natural mortality (M)—both calculated over the entire study period—were expressed as a linear model of mean shell length (function lm of stats package in R.3.1.0). To allow for comparison of shell growth rates published elsewhere, the estimates of intercept and slope were used to calculate the growth parameter k and the asymptotic length at which growth is zero (L∞) of the Von Bertalanffy growth equation:At the scale of the lagoon, shell growth rate (M) were square-root transformed and expressed as a function of seasons, stations, depth and interactions between these factors on the basis of a linear model. Shell growth rates (G) calculated over each monitoring period were expressed as a function of shell length and sampling-structuring factors  using a linear mixed model (function lme of nlme package in R. 3.1.0.). We could not test for interactions between factors, but a random effect considered the existence of dissimilarities between individuals . For all models, we checked the assumption of homogeneity of variance and normality of residuals graphically using normal quantile-quantile plots and scale-location plots of sqrt. The significance of each parameter was tested using an analysis of variance (anova function in R.3.1.0.).At intra-lagoon scale, instantaneous mortality rates (Tv) estimated at 2.02 ± 0.007 for Tatakoto and 2.01 ± 0.004 for Tubuai. Even though the temperature sensor n°3 at Tatakoto was located slightly deeper than the temperature sensor at Tubuai  at Tatakoto than Tubuai . Seasonat Tubuai , the shot Tubuai ; with a -1 and 0.13 m.h-1 respectively for Tubuai compared to -0.03 m.h-1 and 0.04 m.h-1 for Tatakoto. Analyses highlighted that a 10-day period of low mean wave height (< 1×10−3 m) at Tatakoto (−3 m), and the water level of the lagoon was permanently influenced by tide .The tidal slope was an order of magnitude higher at Tubuai than Tatakoto, with 5% and 95% quantile values at -0.12 m.hTatakoto  was assoTatakoto , a decreTatakoto , and a mTatakoto . At TubuAt intra-lagoon-scale, the three temperature sensors deployed at Tatakoto yielded similar results despite being placed at different locations and depths  showed high variability. However, on average, growth rates (± SD) at lagoon-scale were higher and more variable during the warm season (1.84×10−3 ± 3.21×10−3 cm.d-1 and 2.29×10−3 ± 2.68×10−3 cm.d-1 for Tatakoto and Tubuai respectively) than the cold season (0.83×10−3 ± 2.20×10−3 cm.d-1 and 1.94×10−3 ± 2.15×10−3 cm.d-1 for Tatakoto and Tubuai respectively). The seasonal difference in mean growth was more pronounced at Tatakoto, but remained significant at both sites (p < 0.001 and p < 0.01 for Tatakoto and Tubuai respectively).Results for shell growth rates (2 = 0.44 for Tubuai and R2 = 0.13 for Tatakoto). Means (± SE) for the Von Bertalanffy parameter k was higher at Tubuai (2.7×10−4 ± 1.6×10−5 d-1) than Tatakoto (1.5×10−4 ± 3.9×10−5 d-1), yielding contrasting growth curves (L∞ were comparable at both sites (19.7 and 20.0 cm for Tubuai and Tatakoto respectively).Shell growth rate was found to decline significantly with increasing shell length  was estimated at 0.47 ± 0.07 in December 2013, declining to 0.12 ± 0.02 in June 2014 . This deM) per shell length class and over the entire study period differed markedly between Tatakoto and Tubuai , whereas it decreased significantly with shell length at Tatakoto (p < 0.001). Natural mortality did not show any seasonal trend (warm season versus cold season)  were significantly lower (p < 0.01) at station 15 and station 21 . Both stations are located in close proximity to the narrow and shallow channel in the fringing reef connecting the lagoon with the ocean. At Tubuai, there were no significant differences between stations (p = 0.458), despite lower values in M for patch reefs in the eastern part of the lagoon  because of high individual variability and measurement error. However, findings did suggest slower growth at Tatakoto compared to Tubuai and similar maximum sizes for the two lagoons  and lower theoretical maximum sizes (L∞ = 21.85 for Tatakoto and Fangatau against 24.7 for Tubuai) [The shell growth rate of bivalves is often reported as a function of their shell length or age. For giant clams, the acquisition of energy may influence the relationship between shell growth rate and shell length since the number of zooxanthellae per unit of flesh decreases with age . Our res lagoons . These r Tubuai) . The difGrowth curves reported in the literature usually suggest that old individuals (> 10 years) are characterized by smaller shell length in atoll lagoons compared to other reef systems . The groAt intra-lagoon scale, higher growth rates and higher variability in growth were recorded at Tatakoto for giant clams located in shallow areas, corroborating other studies’ findings . ConsideTo date, very few studies have looked into the reproductive activity of giant clams , 44. In T. maxima’s reproductive activity in enclosed reef systems [In this study, we found a decline in maturity indices in May and June 2014 at Tatakoto, indicative of a mass spawning event over that time period. This spawning event followed a decline in temperature, associated with high mean wave heights and a greater connection between the lagoon and the ocean . We cann systems , 46.Estimates of instantaneous rates of natural mortality were higher at Tatakoto than at Tubuai, particularly for the small shell length classes . It is uT. gigas; [Perkinsus sp. was detected in 2012. This parasite is known to affect juveniles [Factors that may cause elevated natural mortality rates among juvenile invertebrates include: disease, predation, competition, loss of energy, and unfavourable physical and chemical properties of seawater  . At Tatuveniles , but infOctopus cyanea) were regularly observed during field trips, as well as high predation rates on small giant clams by numerous fish (Labridae and Tetraodontidae) and gastropods (Pyramidellidae). The presence of predators and parasites in atoll lagoons may follow cyclical predator-prey relationships, with high abundances possibly the result of previously high giant clam densities [The thin and delicate shells of small giant clams make them more vulnerable to predators . Some auensities . HoweverIntra-specific competition related to density-dependent processes can also negatively influence the natural mortality and shell growth of bivalves . In the versus island). The reef pass and submerged reef flat at Tubuai allow for increased exchange between lagoon and oceanic waters. In this highly open lagoon, water levels are permanently driven by the tide (hoa) that allow for oceanic and lagoon waters to mix during periods of high southerly swell (more common between March and September) and east/southeasterly winds. We found that a 10-day period of low waves inside the lagoon  was associated with a decrease in lagoon water level and an increase in water temperature. At intra-lagoon scale, the lowest mortality rates were recorded at stations 15 and 21, located in close proximity to where the lagoon and ocean connect, while the highest mortality rates were recorded in areas subject to greater variations in temperature and more intense warming. This suggests that high water renewal rates and the associated physico-chemical water characteristics  in these areas increase survival. These results strengthen the idea that a prolonged period of “lagoon closure”  may decrease giant clam survival throughout the lagoon.Finally, the higher mortality rates recorded at Tatakoto compared to those recorded at Tubuai may be the result of contrasting physical characteristics of lagoon waters between the two sites. In this study, we highlighted differences in temperature, wave exposure, tidal slope, and water level variability between both locations, partly due to their distinct reef aperture configuration Click here for additional data file.S1 FigA-2013: April 2013; N-2013: November 2013; S-2014: September 2014; D-2014: December 2014; A-2015: April 2015. SPZ (grey): male maturity index. GSR (red): Gonadal Sex-Ratio. GSI (black): Gonado-Somatic Index.(EPS)Click here for additional data file.S2 FigDashed vertical lines indicate field trip during which the survival of giant clams was checked. The lower panel display values for the instantaneous rate of mortality (M) obtained at the end of each period. The water level line is greyed out before 15/10/2013 as the sensor was placed slightly deeper than usual on its pedestal (20 to 40 cm).(EPS)Click here for additional data file.S3 FigDashed vertical lines indicate field trip during which the survival of giant clams was checked. The lower panel display values for the instantaneous rate of mortality (M) obtained at the end of each period.(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.


CD44 has been proposed as a prognostic marker and a stem cell marker but studies in patients with prostate cancer have yielded inconsistent results. Patients submitted to radical prostatectomy between 2008 and 2013 at a university hospital were followed with biannual serum PSA tests to determine the biochemical recurrence (BR). Archived paraffin blocks with neoplastic and nonneoplastic tissue were evaluated immunohistochemically for a panCD44 and MYC.p = 0.042) and higher preoperative serum PSA levels when evaluated continuously (p = 0.029). CD44 expression was not associated with tumor stage (p = 0.668), surgical margin status (p = 0.471), or BR (p = 0.346), nor was there any association between CD44 and MYC expression in neoplastic tissue (p = 1.0). Sixty-nine patients completed follow-up and were included. CD44 positivity was observed in inflammatory cells (42%), nonneoplastic epithelium (39.7%), and neoplastic tissue (12.3%). In nonneoplastic tissues staining was observed in basal and luminal cells with the morphology of terminally differentiated cells. In neoplastic tissues, CD44 negativity was correlated with higher Gleason scores (Rho = −0.204;  In the bulk of cells, the minority of cancer stem cells would not be detected by immunohistochemistry using panCD44. As a prognostic marker, its expression was weakly correlated with Gleason score and preoperative PSA level, but not with surgical margin status, tumor stage, or BR. The cell-surface glycoprotein CD44 plays a central role in cell-cell and cell-matrix adhesion –5. AlongOn the other hand, it has been hypothesized that stem cells in the basal epithelium are capable of self-renewal and differentiation into terminal cells , 14. CanMYC is one of the proteins regulated by CD44 . MYC-encThe purpose of the present study was to evaluate the immunohistochemical expression of panCD44 in neoplastic and nonneoplastic prostate epithelium, determining its association with tumor aggressiveness  and C-MYC expression.The sample consisted of all patients with localized prostate cancer   submitteThe immunohistochemical evaluation used archived paraffin blocks. Fragments were selected which contained adenocarcinoma, benign gland tissue, and specimens with highly variable Gleason scores . Three-micrometer sections were placed on silanized slides according to standard laboratory procedures , followep ≤ 0.05).The statistical analysis was made with the software IBM SPSS v.22. The normality of the data was verified with the Shapiro-Wilk test, while the Mann–Whitney test was used to verify potential associations between CD44 expression and serum PSA levels. The frequency analysis of the categorical variables was done with Fisher's exact test and odds ratios . The Spearman test was employed to verify correlations between CD44s expression and Gleason patterns. Kaplan-Meier curves were generated and compared with the log rank test. Univariate Cox regression was used to detect associations between BR and CD44s expression. The level of two-tailed statistical significance was set at 5% , with a mean follow-up time of 41 months (range: 2–89), during which 20 (29%) were diagnosed with BR. In four paraffin blocks, the neoplasia was exhausted . The same occurred with one sample of benign epithelium.The membrane and cytoplasm of inflammatory cells stained positive for CD44 (intensely and/or extensively) in 42% of the samples . As for p = 0.862), but significantly weaker in neoplasia (p ≤ 0.001).The immunohistochemical findings for CD44 are summarized in p = 0.029). The CD44 staining score was inversely but weakly correlated with Gleason pattern , but the association was nonsignificant when the PSA values were categorized and the Gleason scores were stratified  in positive cases and 8.42 ng/mL (range: 3.06–94.3) in negative cases (ratified .p = 0.836) for the relation between CD44 expression and BR.The presence or absence of immunoexpression for CD44 was not associated with tumor stage, surgical margins, or BR . The uniNuclear staining for MYC was observed in rare endothelial cells (40.6% of cases), nonneoplastic basal epithelial cells (25%), and neoplastic cells (27.3%). MYC expression and CD44 expression were significantly associated in nonneoplastic epithelium, but not in neoplastic tissue.The ability of CD44 expression in biopsies and transurethral resections to predict tumor aggressiveness in prostatectomies had been evaluated , 38, as The detection of CD44 on immunohistochemistry may be approached in many different ways . CD44 goCut-off values for CD44 generally indicate “superexpression” or “intense expression,” as in Aaltomaa et al. (2001) , in whicIn our study, preoperative serum PSA levels were lower in CD44-positive patients than in CD44-negative patients, but the association was nonsignificant when PSA levels were stratified using 10 ng/mL as cut-off. An association between CD44 status and PSA level was also reported by Aaltoma et al. (2002) , but notp value. However, when the Gleason scores were summed up and stratified, the association was nonsignificant. De Marzo et al. [CD44 positivity and Gleason score were correlated, showing a decline in CD44 expression from Gleason pattern 1 to Gleason pattern 5 , in our o et al.  tested ao et al. .p = 0.001) [The lack of association between CD44 expression and tumor stage observed in this study matches the findings of several authors , 46, 47,= 0.001)  as evideMatching the results of other authors , 46, in Finally, CD44 was not associated with BR. Only one of the reviewed studies found an association with BR in all analyses  . In the α2β1 integrin. While the number of commercially available antibodies against CD44v is still limited, ongoing research is likely to expand the range of options in the near future [A large proportion of neoplastic and nonneoplastic cells express CD44, as shown in the literature in general, with some authors reporting positivity rates approaching 100% , a rate r future .Our study was limited by the small sample size, by the use of archived specimens, and by the short time of follow-up. However, our results are supported by other studies showing high rates of CD44 positivity in prostate tissues, contrary to what would be expected for a stem cells marker. Our method of CD44 analysis was similar to that of Tei et al. (2014) , as wereCD44 expression was stronger in inflammatory and nonneoplastic cells than in tumor cells. Within the latter, expression was stronger in well-differentiated than poorly differentiated tumors. The loss of CD44 expression on immunohistochemistry was weakly associated with greater tumor aggressiveness (lower preoperative PSA levels and Gleason scores), but no correlation with biochemical recurrence was observed. The wide range of panCD44 expression in prostate tissues would not detect the minute minority of cancer stem cells by immunohistochemistry.


Conventional meta-analyses and randomized controlled trials have shown inconsistent results regarding the efficacy of immunosuppressants for pediatric steroid-resistant nephrotic syndrome (SRNS).To conduct a network meta-analysis aimed at evaluating the efficacy and safety of available immunosuppressive agents in pediatric patients with SRNS.MEDLINE, the Cochrane Central Register of Controlled Trials, and EMBASE were searched on January 2017. Data from randomized controlled trials (RCTs) were included. The main outcomes analyzed were efficacy  and safety [adverse secondary event (ASE) rates].A meta-analysis of 18 RCTs showed that tacrolimus was more efficacious for achieving CR than intravenous (i.v.) cyclophosphamide, mycophenolate mofetil (MMF), oral cyclophosphamide, leflunomide, chlorambucil, azathioprine, and plaebo/nontreatment (P/NT), and more efficacious than i.v. cyclophosphamide, oral cyclophosphamide, and P/NT in terms of TR outcomes. Cyclosporin was associated with a greater CR rate than i.v. cyclophosphamide, MMF, oral cyclophosphamide, chlorambucil, azathioprine, or P/NT, and associated with a greater TR rate than i.v. cyclophosphamide, oral cyclophosphamide, or P/NT. MMF was found to be more efficacious than i.v. cyclophosphamide and oral cyclophosphamide in terms of TR.Tacrolimus and cyclosporine may be preferred initial treatments for children with SRNS. MMF may be another option for this patient population. Further studies of the efficacy and safety of these three drugs in children with SRNS should be pursued. The incidence of pediatric primary nephrotic syndrome (NS) is about 1–3/100,000 children 16 years old or younger . The appTreating SRNS, which should be done under the care of a pediatric nephrologist, can be challenging because there is a paucity of strong evidence to inform SRNS treatment decisions due to the lack of large-scale randomized controlled trials (RCTs). Children with SRNS may be treated with immunosuppressive agents, such as cyclosporin, cyclophosphamide, or tacrolimus . RemissiIn recent decades, several new lower-toxicity immunosuppressive medications have been introduced for the treatment of SRNS in children . HoweverHere, we report a network meta-analysis in which nine nonsteroidal immunosuppressive agents were compared with respect to efficacy and safety in children being treated for SRNS. The aim of this work was to identify a preferable SRNS therapy drug in children.A total of 7,681 potentially relevant studies were retrieved, which included 6,146 non-repetitive potentially eligible articles. On the basis of our eligibility criteria , 6,102 articles were excluded during the title/abstract review process and, subsequently, 26 articles were excluded consequent to a full-text review. Finally, 18 articles published from 1970 to 2015 were included in our network meta-analysis. The 18 selected studies involved a total of 790 individuals who were assigned randomly to an immunosuppressive medication or placebo/nontreatment (P/NT) group. The trial selection process is summarized in Figure The characteristics of the 18 included trials are summarized in Table Our efficacy analyses included 730 patients with a CR in 17 trials examining a total of ten treatments Figure , 569 pat2 = 0%). Cyclosporin also showed better efficacy than i.v. cyclophosphamide for both PR and TR outcomes (both I2 = 0%), as well as greater efficacy than MMF for TR (I2 = 0%), though in the latter case the benefits were less clear (95% CI for OR slightly more than 1). Additionally, tacrolimus was more efficacious than i.v. cyclophosphamide for TR (I2 = 0%) and i.v. cyclophosphamide was more efficacious than oral cyclophosphamide for CR (I2 = 37.8%).The efficacy and safety analysis results obtained for individual immunosuppressive medications as determined by direct pairwise meta-analyses are shown in Figure 2 = 15.1%). There were no significant differences in safety revealed by other direct comparisons between immunosuppressants. However, it should be noted that the 95% CIs obtained for most of the comparisons were reflective of either high or no heterogeneity due to the small number of studies included in the pairwise comparisons; overall, heterogeneity was moderate.In terms of ASEs, oral cyclophosphamide was found to be safer than i.v. cyclophosphamide (95% CI for OR slightly more than 1) and tacrolimus was found to be safer than both cyclosporin and i.v. cyclophosphamide . The guidelines suggested that tacrolimus be considered as a treatment option for patients with SRNS when cyclosporine is counter-indicated due to cosmetic side effects (recommendation grade C1), and that cyclophosphamide not be considered for induction therapy in children with SRNS (recommendation grade C2) .The most problematic ASE of long-term cyclosporine use is chronic nephrotoxicity, an increased risk for which has been associated with cyclosporine treatment for ≥ 2 years –35. The The results of this analysis apply to treatment periods of 2 years or less. Longer term clinical efficacy and safety beyond 2 years may differ substantially from outcomes recorded within 2 years . AdditioThis study focused on examining RCTs of available immunosuppressive agents in pediatric SRNS patients. Recently, ongoing or completed small sample case series of new biologics—including anti-CD20 ofatumumab, abatacept, adalimumab, fresolimumab, and saquinavir which target immune cell subsets or activation pathways selectively—have spurred a new direction of hypothesis-driven therapies that may improve outcomes of children with kidney disease , 38. FurIn conclusion, on the basis of all available direct and indirect evidence, our results suggest that tacrolimus and cyclosporine are preferable first-line medications for initial SRNS in children. MMF may be an acceptable option for patients with SRNS. Further studies are needed to evaluate the relative benefits and harms of tacrolimus versus cyclosporin for pediatric SRNS treatment. Additional information about the safety and efficacy of MMF in children with SRNS is also needed.ClinicalTrials.gov was searched for trials in progress.In preparation for this network meta-analysis, we drafted a study protocol and published it on the PROSPERO website (CRD42017062564). Clinical trials comparing at least two different treatments were searched in MEDLINE (1950 to January 2017), the Cochrane Central Register of Controlled Trials , and EMBASE (1974 to January 2017) with the following search terms: “immunosuppressive agents” or “alkylating agents” or “azathioprine” or “cyclosporine” or “cyclophosphamide” or “mycophenolic acid” or “rituximab” or “chlorambucil” or “levamisole” or “tacrolimus”; and “nephrotic syndrome” or “minimal change nephrotic syndrome” or “glomerulonephritis membranoproliferative” or “focal segment glomerulosclerosis” or “membranoproliferative glomerulonephritis”. The search results were restricted to articles reporting studies involving children. Additionally, the reference lists of all included publications and relevant reviews were screened and 2/h after four weeks or more of daily corticosteroid use. Trials involving patients experiencing steroid-sensitive nephrotic syndrome, congenital nephrotic syndrome, and other kidney or systemic forms were excluded. Trials for which only abstracts were published  were also excluded. No language restrictions were applied.Parallel RCTs in which children with initial SRNS and children with delayed SRNS were the subjects and comprehensive comparisons of any of the following agents were included: cyclophosphamide, cyclosporine, tacrolimus, mycophenolate mofetil (MMF), azathioprine, chlorambucil, rituximab, or levamisole. The experimental interventions could be compared to a placebo/nontreatment (P/NT) and/or another immunosuppressive medication. SRNS was defined as persistence of proteinuria > 3+ on a dipstick test, urinary protein-creatinine ratio (UP/C) > 0.2 g/mmol or 40 mg/m2/h for three or more consecutive days. We defined PR as proteinuria < 2+, urinary UP/C < 0.2 g/mmol or 40 mg/m2/h. TR was defined as the total number in remission, including patients in CR or PR. We defined ASEs as sequelae occurring within the initial posttreatment period (determined by each study's authors).The primary efficacy outcomes were number in CR, number in PR, and total number in remission (TR). The primary safety outcome was the incidence of adverse secondary effects (ASEs). We defined CR as edema-free and urine protein was < 1+ on dipstick tests, urinary UP/C < 0.02 g/mmol or 4 mg/mTwo investigators (SL and LT) reviewed all of the abstracts and references, evaluated the integrity of data abstraction, and rated the quality of included studies independently. We sent emails to the authors of articles with incomplete information and asked them to provide supplemental data. Methodological quality and risk of bias were assessed with the Cochrane risk of bias tool.2 statistic was obtained as an index of heterogeneity.All data analyses were conducted in Stata, version 14 software . First, we performed pairwise random-effects meta-analyses using the Knapp-Hartung method with the metan command, from which we report odds ratios (ORs) and 95% confidence intervals (CIs) . When a P < 0.05 was considered significant. We looked at a plausible range for population difference magnitude. We used the network rank option to evaluate the probability that each drug could be the most  efficacious treatment. The surface under the cumulative ranking curve (SUCRA) was determined as an estimation of the ranking probability for each medication, and the resultant SUCRA estimates were used to rank the treatments in a hierarchy [Second, we conducted a network meta-analyses within a frequentist framework using the Stata network suite . A multiierarchy . We rankierarchy . A loop-ierarchy . Inconsi