Myrceugenia correifolia is a rare arboreal species that grows in deep canyons and hilltops of the Coast Range of north-central Chile between 30° and 35°S. In the northern edge of its distribution M. correifolia grows in small patches of fog-dependent forest surrounding by xeric vegetation. These forest formations are thought to be remnants of an ancient and continuous rainforest that according to some authors became fragmented during aridization of the Neogene (Neogene relict) and to others during warm-dry cycles of the Pleistocene . Here we asked whether the northernmost populations of M. correifolia are Neogene relicts, glacial relicts, or the result of a recent northward colonization. To answer this question we examined genetic diversity and population divergence of M. correifolia using microsatellite markers, tested various competing population history scenarios with an approximate Bayesian computation (ABC) method, and complemented these data with ecological niche modeling (ENM). We detected three genetic clusters with a distinctive latitudinal pattern  and high levels of differentiation (FST = 0.36). Demographic inference supported an admixture event 31 kya between two populations that diverged from an ancient population 139 kya. The admixture time coincides with the beginning of a period of wet conditions in north-central Chile that extended from 33 to 19 kya and was preceded by dry and cold conditions. These results suggest that increased precipitation during glacial periods triggered northward expansion of the range of M. correifolia, with subsequent admixture between populations that remained separated during interglacial periods. Accordingly, ENM models showed that suitable habitats for M. correifolia in north-central Chile were larger and less fragmented during the Last Glacial Maximum than at present, suggesting that northernmost populations of this species are glacial relicts.Rare species frequently occur in areas with microclimatic conditions that are atypical for their regions, but that were more common in the past, and that probably have operated as climatic refugia for a long time.  Species with limited geographic distributions are considered more vulnerable to climatic changes and land use conversion than widespread species, and therefore are of central concern in conservation biology . Rare spThe Mediterranean region of central Chile, one of the world’s plant diversity hotspots, has high levels of richness and endemism . Many trAextoxicon punctatum, distributed from 30° to 43°S  approach to test different historical scenarios. Finally, we used ecological niche modeling (ENM) to identify the climatic niche of this species under current environmental conditions and to predict its potential distribution during the LGM and mid-Holocene. In this way, we explore the “bioclimatic” connectivity of Coast Range summits during glacial and interglacial periods. We combined this information to assess whether the northernmost populations of M. correifolia can be considered: (1) relicts of Neogene that became gradually fragmented by the progressive aridization of north and central Chile; (2) relicts of range expansion during wet conditions of the last glacial period that became fragmented during aridization of Holocene; or (3) the result of a recent northward colonization (this is during Late Holocene). If this species persisted in north-central Chile isolated for a long time, we expect to find strong genetic divergence between northern and southern populations. In contrast, if M. correifolia colonized northern areas more recently, we expect to find low levels of populationdivergence, a south-to-north decline in genetic diversity and isolation by distance.Here we examined genetic diversity and population divergence of the rare Mediterranean species M. correifolia from nine sites located between 30°39′S and 34°41′S in the Coast Range of Chile, covering most of the current distribution range of the species (Table 1). A random sample of 15 individuals was collected from each site (when available).We collected plant material of TM Software version 1.0 (Applied Biosystems).Genomic DNA was extracted from each sample using the DNEasy Plant mini kit . A total of 124 individuals were genotyped for eight microsatellite loci , using the primers and protocol described by N), number of rare alleles (AR), expected heterozygosity (He), observed heterozygosity (Ho), and deviations from Hardy–Weinberg using GenAlEx version 6 (FIS) and its 95% confidence interval (CI) using the R package diveRsity (Table 1). Analyses were performed in R  and assign probabilistically each individual to these clusters without a priori knowledge of putative populations. Ten independent runs were carried out for K ranging from 1 to 9 using a Markov Chain Monte Carlo run length of 500,000, a burn-in of 50,000 and an admixture ancestry model assuming correlated allele frequencies. To determinate the most likely number of clusters (K) we used the rate of change in Ln P(D) (the log probability of data) between successive K values as suggested by FST values for all pair of sites in GenAlEx, and then performed a Mantel test with geographic distances. In order to discount those clusters obtained by STRUCTURE resulted from simple IBD, we conducted a partial Mantel test between genetic distances and clusters using geographic distances as covariate  and when all populations were pooled .Genetic differentiation within and among populations was measured by analysis of molecular variance (AMOVA)  using Geovariate . Given tovariate , we examovariate . We perfM. correifolia we used coalescent-based ABC implemented in DIYABC v2.0 . The third scenario includes an admixture event that resulted from range expansion of two populations that diverged from an ancient population. This scenario is based on the hypothesis that increased precipitation during glacial periods triggered range expansion of M. correifolia, with subsequent admixture between populations that remained separated during the last interglacial period (LIG). We set uniform priors for all demographic parameters. The microsatellite loci were divided into two groups: (1) all dinucleotide repeat loci and (2) the single trinucleotide repeat locus. We ran 4 × 106 simulated datasets for each scenario to estimate posterior probabilities using logistic regression. We selected the scenario with the highest posterior probability and estimated the values of the associated demographic parameters.To reconstruct the history of population divergence of ABC v2.0 . This anM. correifolia from a total of 18 unique-georeferenced species records. We obtained the occurrences from national herbaria: Universidad de Concepción (CONC); Museo Nacional de Historia Natural (SGO), online database Global Biodiversity Information Facility (GBIF); Cooperative Taxonomic Resource for American Myrtaceae (CoTram) and our own field work. We modeled the climatic niche employing the maximum entropy method (2). For the LGM and mid-Holocene model projections we used the pre-industrial calibrated Model for Interdisciplinary Research on Climate . Regression analyses revealed a significant effect of latitude on allelic richness , Shannon genetic diversity index , He , but not on Ho . Three genetic clusters were detected in M. correifolia by Structure analysis with a distinctive latitudinal pattern: (1) north, with the sites Fray Jorge and Talinay; (2) center, with the sites Santa Inés, Zapallar, and Qda. Córdova; and (3) south, with the remaining sites . Most individuals (94%) were assigned to a single genetic cluster (80 of inferred ancestry) and AMOVA indicated high differentiation among regions, with 36% of the variation explained by differences among them and only 8% by differences among populations. Divergence between north and central clusters (FST = 0.22) was weaker than the barrier between central and south clusters (FST = 0.31). The Mantel test between pairwise FST values and geographic distances revealed a strong and significant relationship between geographic and genetic distance , suggesting a significant IBD pattern . A significant association between genetic distances and clusters  was also detected when geographic distance was included as a covariate, indicating the presence of additional barriers to gene flow.Figure 1). This scenario had a probability of 0.53 with a 95% CI ranging between 0.51 and 0.56 that did not overlap with that of scenario 2  and scenario 1 . According to scenario 3, admixture occurred 6,330 generations ago  from two populations that diverged from an ancient population 27,800  generations ago. The rate of admixture was higher between north and central populations (r = 0.64) than between the central and southern groups (1 -r = 0.36). The effective population size was greater in the central group  than in northern  and southern groups .DIYABC analyses indicated that the most likely scenario is scenario 3, which describes an admixture event that resulted from range expansion of two populations that diverged from an ancient population . Paleoclimatic models suggested that areas of high and moderate suitability were larger and less fragmented during the LGM than at present. The potential distribution of M. correifolia during mid-Holocene was similar to the present one, but with a larger area of high suitability, extending between 33° and 34°S .The ENM model for M. correifolia, one of the dominant trees of these fog-dependent forests, suggest that divergence of its northernmost populations located in the emblematic Fray Jorge–Talinay National Park was not as ancient as previously proposed.Fog-dependent forests that today grow isolated on tops of the Coast Range of Central Chile are thought to be remnants of an ancient and continuous rainforest that according to some authors became fragmented during aridization of the Plio-Pleistocene transition , and to M. correifolia, with subsequent admixture between populations that remained separated during interglacial periods. According to DIYABC analysis, admixture occurred 6,330 generations ago between two populations that diverged from an ancient population 27,800 generations ago. The generation time of natural populations of M. correifolia remains unknown, but there are data for individuals grown in the National Botanical Garden of Chile , indicating that they can reach reproductive maturity at 5 years. Assuming a generation time of 5 years, admixture would have occurred 31 kya and ancient divergence at 139 kya. Admixture time coincides with the beginning of a period of wet conditions in north-central Chile that extended from 33 to 19 kya and that was preceded by dry and cold conditions . However, this result is in line with several studies showing that general circulation models tend to underestimate the magnitude of LIG warming respect to proxy reconstructions  is somewhat earlier than the beginning of the LIG. This period, extending between 115 and 130 kya, was characterized by global warming, sea level rise and increased summertime-insolation . The incructions . For theructions .Figure 5). We detected a genetic barrier at 31.5°S separating Fray Jorge and Talinay from the remaining sites of M. correifolia. A similar disjunction has been reported for D. winteri  than the central  and northern groups . Reduction in genetic diversity southward has been documented in species from north-central .Structure analysis also revealed a second barrier at 33.5°S, separating the southern group from the other populations of -central  and sout-central , and hasFP conceived and designed the study, analyzed and interpreted data, and wrote the paper. CI, MC, and GP obtained and analyzed microsatellite data. PM performed niche modeling analysis. LH designed the study and interpreted data.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.


In assisted reproduction procedures, we need to develop and enhance newprotocols to optimize sperm selection. The aim of this study is to evaluatethe ability of the Z potential technique to select sperm with intact DNA innon-normospermic patients and evaluate the impact of this selection onembryonic development.We analyzed a total of 174 human seminal samples with at least one alteredparameter. We measured basal, post density gradients, and post densitygradients + Z potential DNA fragmentation index. To evaluate the impact ofthis technique on embryo development, 54 cases were selected. The embryodevelopment parameters evaluated were fertilization rate, cleavage rate, topquality embryos at the third day and blastocysts rate.We found significant differences in the study groups when we compared thesperm fragmentation index by adding the Z potential technique to densitygradient selection vs. density gradients alone. Furthermore, there was nosignificant difference in the embryo development parameters between the lowsperm fragmentation index group vs. the moderate and high spermfragmentation index groups, when selecting sperms with this newtechnique.The Z potential technique is a very useful tool for sperm selection; itsignificantly reduces the DNA fragmentation index and improves theparameters of embryo development. This technique could be considered routinefor its simplicity and low cost. Primary infertility is mainly defined as the incapacity of a couple to conceivewithin 12 months of periodic sexual intercourse without any contraceptive method;this condition affects approximately 15% of couples in reproductive age and itsprevalence is increasing  and swim-up. These techniques focus theirmethodology on the basics of sedimentation or migration to separate the bestspermatozoa, and they are both efficient in selecting morphologically normal spermswith a high degree of nuclear maturity . HoweverUp to date, different sperm selection methodologies have been developed to improveconventional seminal preparation protocols for assisted reproduction procedures. These min vivo interaction with the oocyte membrane and avoidsnon-specific aggregations in the female tract, as well as among the spermatozoathemselves  combines DGC and a selection based on the sperm membraneelectric potential to obtain sperms with a negative charge (-16mV to -22 mV), thisis evidence for proper epididymal maturation and spermatogenesis at a testicularlevel. , between October 2016 and February 2017, NiuVida's Ethics Committee approved this study.To analyze the Z Potential technique effect on reducing the sperm fragmentationindex in men with low, moderate and high DNA Fragmentation Index (DFI). 174seminal samples were included, with at least one altered parameter according toWorld Health Organization .Samplesp<0.05 as discriminatory value. Data normality wasverified by the Kolmogorov-Smirnov (KS) test, and the equality of variances withthe Levene test. We analyzed the difference of DFI between DGC vs. DGC + PZbetween groups using ANOVA and Games Howell's Post Hoc analysis.We compared the DFI reduction percentage in the post-DGC and post-DGC + ZPpopulations independently by the Student's t-test for related samples with aTo evaluate the ZP technique impact of on embryo development parameters, wechoose 54 randomized high complexity cases with 519 donated oocytes using thealgorithm provided by RANDOM.ORG. Selected cases were assigned two groups, usingDFI as selection criteria, as follows: Group 1, DFI ≤16%, 298 oocytes,and Group 2, DFI ≥17%, 221 oocytes  + Flushing Medium (Origio)). The tubes were centrifuged at 1000rpm for10 minutes. Sperm pellets were recovered and washed in flushing medium (Origio)at 1000rpm for 10 minutes and finally reduced to a volume of 400µl - a25µl aliquot was separated to evaluate the DFI.Sperm selection by Z Potential was carried out modifying the ® commercial kit, followingthe manufacturer's instructions. 500 sperms were counted per sample, and the DFIwas calculated by dividing the number of spermatozoa with small or nil halo bythe total number of analyzed spermatozoa multiplied by 100.Each sample DNA Fragmentation Index (DFI) was measured at 3 different times, using the Spermatic Chromatic Dispersion(SCD) technique with the Halosperm®) on day 3 of the menstrual cycle (MC), rFSH from day 10 to day 12 of MC, cetrorelix acetate(Cetrotide®) from day 10 to day 12 and triptorelin(Gonapeptyl®) at day 14 of MC. Oocyte capture wasperformed by vaginal ultrasound under general anesthesia with Propofol(Diprivan® 1% P/V) in Global®Total® w/Hepes medium.For all cases, a single stimulation protocol was carried out using rFSH among thetotal number of injected oocytes, multiplied by 100, the CR was obtained bydividing the number of cleaved embryos by the number of fertilized embryos,multiplied by 100. Good-quality embryos in day 3 were defined as those that hadbetween 6 and 8 cells, with blastomeres of equal size, and a fragmentation ratebelow 25%. Day 3 TQE was obtained by dividing good quality embryos between thenumber of cleaved embryos multiplied by 100. BR was obtained by dividing thenumber of Blastocysts obtained on day 5 by the total number of fertilizedembryos, multiplied by 100.p<0.05) with the DGC technique.To compare the DFI reduction between DGC and DGC + PZ techniques in Groups A, Band C, the Student's T-test was performed for independent samples in eachpopulation. The results depicted on To determine in which group there is a greater reduction of the DFI by the DGC +PZ technique, we analyzed the difference between the reduction values in eachgroup. p=0.82, CRp=0.97, TQE p=0.29, BRp=0.096).The effects of the Z potential on embryo development was analyzed forming 2groups: Group 1 with basal DFI ≤16 and Group 2 with basal DFI ≥17.The DGC technique results in a sperm homogeneous population with better classicalparameters, and reduces DFI significantly . This trp<0.05) in each comparison betweengroups 1 and 2  for each analyzed parameter as having clinical relevance, we found thatour percentage of fertilization value (78.7%) is comparable to that reported byOur analysis of our CR value (89.4%) in the group of clinical relevance (Group 2)showed that it was in accordance with the one reported by per se is a veryuseful tool for sperm selection in assisted reproduction treatments; itsignificantly reduces the sperm DNA fragmentation index, regardless of the sampleinitial basal value, and improves the parameters of embryo development achieved withconventional techniques for seminal preparation.Our results shown that the Z potential technique, Hence, the Z potential technique is an option that could be considered routine due toseveral advantages in respect to other techniques ,stressing its simplicity, processing velocity and low-cost of implementation andstart-up.This new tool could even replace a previous diagnosis of sperm DNA fragmentationindex when couples have already opted for an IVF treatment, which would reduce costsfor patients by giving them a better chance to success in their treatments,comparable to when more complex and more expensive techniques are used for spermselection.


Desert plants are thought to rely more heavily on nutrient resorption due to the infertile soil. However, little is known regarding the phylogenetic effects on this traits, specifically for halophytes. Here we determined contents of nitrogen (N), phosphorus (P), potassium (K), sodium (Na), calcium (Ca) and magnesium (Mg) in 36 desert plants in a hyper-arid environment. The patterns of resorption or accumulation of the six elements were compared among plant groups with diverse leaf Na regulation strategies: i.e., euhalophytes (Eu), secretohalophytes (Se), pseudohalophytes (Ps) and glycophytes (Gl). Overall, N, P, K presented strict resorption across all groups, but no more efficient than global estimations. Ca and Mg tended to be resorbed less or accumulated during leaf senescence. Significant phylogenetic signal of both leaf Na content and plant group implies the pivotal role of Na regulation in the adaptation of plants to desert environment. Resorption proficiency, rather than resorption efficiency, is more phylogenetically conservative and more relevant to leaf functional traits. Nutrient resorption, a process by which plant withdraws nutrients from senescing structures to developing tissues, is a critical mechanism to reduce the dependence on nutrient uptake and, consequently, increases plant fitness in low-nutrient environments2. Generally, nutrient resorption can be defined as resorption efficiency (RE), i.e., the proportion of green leaf nutrients withdrawn prior to senescence3 and as resorption proficiency (RP), i.e., the terminal nutrient concentration in senesced leaves4. Based on the global estimate, over 50% of leaf N and P , varying among plant groups, are recycled via resorption5. As a critical trait in determining plant fitness, speculation about this process has suggested that plants in desert ecosystem may rely more heavily on the reabsorbed nutrients due to the poor soil fertility7. Comparing data from seven desert shrubs to non-desert shrubs was found the RE of N and P was higher in desert species6. Whereas, another study showed that shrubs in Chihuahuan desert were no more efficient or proficient at resorbing N and P than shrubs growing in other environments8. In part, the mixed results reflect the fact that drought may be an important factor in desert ecosystem affecting nutrient resorption9, as it will advance leaf senescence and, consequently, decrease nutrient resorption10. Although studies have noted lower N and P resorption at drier conditions12, controversial results have also been reported14. Such inconsistencies emphasize that more attention should be paid to resorption patterns of desert plants regarding the influence of drought and nutrient limitation.In most terrestrial ecosystems, growth of wild plant is nutrient-limited15, which induce osmotic stress and ionic toxicity for plant growth16. Osmotic stress can advance leaf senescence, therefore, reduce nutrient resorption9. While ionic toxicity, primarily induced by Na, can reduce N uptake and disrupt whole-plant K homeostasis16, hinder nutrient resorption further. Although drought in conjunction with salinity poses the most severe environmental constraint to plant nutrient uptake and cycling17, it may also be proper conditions for halophytic species18. Desert halophytes are such remarkable plants which can not only tolerate salt concentrations, but also utilize Na as an osmotic solute in coping with water stress16. As they have evolved three tight leaf Na regulation strategies, i.e., compartmentation , secretion  and rejection 19. However, to the best of our knowledge, plants in desert saline environments have received limited attention in nutrient resorption, and the patterns of nutrient resorption in different halophytes remain unknown.Soil salinity is another common environmental problem in desert ecosystem due to the high surface evaporation4. Without recognizing phylogeny, it is not possible to determine the adaptive significance of nutrient resorption14. Several studies have reported that more closely related taxa may have more similar resorption patterns of N and P than more distantly related taxa20. It should be noted that, those studies including phylogenetic effects are simple comparisons with no specific quantization standard, and often have been at broad taxonomic scales 20 or at narrow taxonomic scales 14. While N and P are the main limiting nutrients globally, K, Ca, and Mg also play important roles in plant growth and function22. However, the influences of phylogeny on these essential cations are still unknown.In addition to environmental factors, nutrient resorption is possibly associated with plant evolutionary history, an important intrinsic factor often neglectedHere, by measuring six leaf element content in 36 species from 252 observations, we tested how the leaf resorption or accumulation patterns of five macronutrients  and Na differ in three groups of halophytes, as well as in glycophytes (Gl). Specifically, the two questions were addressed. (1) Are there different nutrient resorption patterns between four plants groups with contrasting Na regulation strategies? (2) Does phylogeny influence these patterns?P = 0.002). The correlation structure for the six elements suggested that Na and K are more important in building the principal components . Na concentration in green leaves was negatively correlated with K/Na ratio  and were positively correlated with leaf dry matter content (LDMC) Table , respectBased on the measurement of six element contents  both in green and senesced leaves of halophytes and glycophytes in an extra-arid environment, the patterns of resorption or accumulation of these elements were explored. The diverse leaf Na regulation strategies are phylogenetically conservative, implying the pivotal role of Na in the survival of plants in desert environment. Over 50% of N and P were resorbed during leaf senescence, indicating nutrient resorption is one of the key component of plant nutrient conservation strategies in water-stressed environment. Significant Na accumulation with leaf senescence is ubiquitous in groups of Eu. Compared with RE, RP is more phylogenetically conservative and more relevant to leaf functional traits .16. Since substantial amount of Na is compartmentalized in the vacuole of Eu, organic osmolytes, such as amino acids, non-protein amino acids, quaternary ammonium compounds and polyamines, are needed in the cytoplasm to prevent adverse effects on metabolism24. The lowest Na concentration in Ps may be attributed to the fact that Ps can prevent Na uptake by roots25. Therefore, Ps is a type of “salt-avoiders” that actively escapes from salinity by locating the active part of the root system into deeper soil26. Interestingly, Na may be a “cheap” osmotic solute for some glycophytes28. For instance, Zygophyllum xanthoxylum, a succulent Gl, accumulates large amount of Na in leaf but only adapts to gravel desert across the study area. In this kind plant, Na should also perform function of osmotic regulation instead of K under low NaCl treatment30. However, the replacement of the non-osmotic functions of K by Na may not be achieved both for glycophytes and halophytes27, hence, the ability to accumulate essential nutrients particularly K is vital for plants adaptation in saline environment16. Across the four plant groups, despite of the large difference in Na concentration, no significant difference was found in K concentration among Eu, Ps and Gl  of terrestrial plants across China31. In contrast, when K/Na ratio <1, it allows plants to use Na as the major osmoregulatory substance28.Across all sampled species in the study are, over 50% of leaf Na variation was explained by plant group Fig . The hig32. It seems reasonable to expect desert plants to rely more heavily on nutrient resorption, because desert environments afford plants less of a chance to recuperate nutrients lost in abscised litter due to the drought-induced slow decomposition6. However, compared to global averages, our results indicate significantly lower RE for N, P and K both in halophytes and glycophytes . These results suggest that more closely related taxa tend to employ similar Na regulation strategies. It also seems likely that the distribution of Na regulation strategies is not random over phylogeny.In the context of plant evolution, adaptive variation has been addressed two complementary perspectives, divergent versus convergent evolutionily Fig. , besidesIn summary, we explored contents of five macroelements and Na both in green and senesced leaves of desert plants with diverse Na regulation strategies in a hyper-arid desert in northwest China. N, P and K showed strict resorption across all plant groups, but no more efficient than global estimations, which suggests that water stress may be the primary factor constraining plant nutrient uptake and resorption in desert environments. Ca and Mg tend to be resorbed less or accumulated during leaf senescence. The significant phylogenetic signals of both leaf Na content and plant group implies the tight Na regulation strategies and its pivotal role in the adaptation of plants to drought and salinity. Compared with RE, RP is more phylogenetically conservative and more relevant to leaf functional traits . In desert ecosystems, it should be more advantageous to understand the resorption process by RE in concert with RP.40. Most of the reserve is typical gravel desert containing large amount of gravel and extra-low moisture content, but with non-saline soils. The Shule River, an interior river runs through the lower areas of the reserve and results in soil salinity due to the high surface evaporation. The saline soils are sandy clay with relatively higher moisture content , west end of Hexi Corridor in Guazhou County, Gansu province, China. The reserve represents a hyper-arid desert ecosystem (aridity index <0.02), with mean annual temperature of 8.7 °C, mean annual precipitation of 45 mm and annul evaporation of 3000 mmnt Table . The vegnt Table .A 20 × 20 m plot was randomly established at each of 18 sites . During the peak growing period (middle of July 2015), sun-exposed and fully expanded green leaves were collected from 5 to eight healthy individual for each woody species and then marked with metal tag. Three 1 × 1 m tagged subplots were applied for sampling of herb species in each plot. At the end of growing season (early October 2015), recently senesced (often yellow), but still attached leaves were collected from the same tagged individual or subplot by gently flicking the branch or leaf. The sampled leaves were rinsed with deionized water to remove surface salts and dust by using a spray bottle in the field. For each species and each sample, at least 60 g of fresh leaves  were collected, of which about 10 g were stored in ice box to keep fresh and the rest were stored in paper envelopes for chemical analyses. Triplicate soil samples were randomly taken by an auger from different layers  within the plot, where each replicate comprised a mixture of three adjacent cores.Flora in Desertis Reipublicae Populorum Sinarum41 and the latest edition of Halophytes in China42.Overall, 252 leaf samples of 36 species belonging to 15 families were collected within the study area. Plants were divided into four groups, including glycophytes (Gl), i.e., non-halophytic plants growth in gravel soils which do not exist in saline environments, and three groups of halophytes, i.e., Eu, Ps and Se. The division of the four plant groups was according to 43. The specific leaf area  was measured by a photographic method43 and analyzed using ImageJ . Leaf succulence was obtained by the ratio of leaf water content/leaf area36, values larger than 500 indicate significant succulence. Leaf samples stored in paper envelopes were oven dried at 60 420 °C to a constant weight, and ground into fine powder using a ball mill  to enable chemical analysis.The leaf dry matter content (LDMC) was obtained by the ratio of leaf saturated weight/leaf dry weight34.Air-dried soil samples were sieved (2 mm), and samples for total N and P were further pulverized through 100-mesh sieve (0.15 mm). Soil pH and electrical conductivity (EC) were measured on 1:2.5 and 1:5 soil: water extracts respectively. Total N in soils (0.15 mm sieved) and leaves (fine powder) were measured by an elemental analyzer  and total P were measured using the ammonium molybdate method after persulfate oxidation3:HClO4:H2SO4, 8:1:1, volume ratio) in 420 °C. Soil samples were measured on 1:10 soil:water extracts (soil soluble cations). The atomic absorption spectroscopy  was used to determine the concentration of Na, K, Ca, Mg in leaf digestive production and soil extracts.Before the analysis of cations, leaf samples (fine powder) were digested in the mixed acid (HNO4. The Resorption efficiency (RE) was quantified as the proportional withdrawal of a nutrient or element during senescence44 and expressed as Eq. (green and Nusenesced are mass based nutrient concentrations in green and senesced leaves.The resorption proficiency (RP) was defined as the nutrient content in senesced leaves, higher resorption proficiency is corresponding to lower final nutrient concentrations in senesced leavesd as Eq. :1\docume45. In this study, the MLCF was calculated as the ratio of dry mass of senesced leaves and the dry mass of green leaves5. The RE for analysis were corrected using Eq.  could be used to compensate the underestimation of REsing Eq. :2\documet-test” was used to test the differences of RE between data obtained in this study and in global scale. Principal component analysis (PCA) was used to evaluate the correlation structure of the six elements in plant leaf, and was performed in the “vegan” package46. Bivariate relationships were examined with standardized major axis regression (SMA) as there are no independent or dependent variables and measurement error exists for both variables, and was performed in “smatr” package47. General linear model (GLM) was used to quantify the contribution of soil (different sites) and plant group/ family to the total variance of leaf chemical traits.To account for the variation in sample size for each species, data were analyzed at two levels i.e., species level, using species means, individual level, using all data. Differences between plant groups were tested using one-way ANOA while the “Independent two-sample 48, the phylogenetic tree of all species . Phylogenetic signal intensity of all leaf traits  were measured by K statistics49 to detect if a given trait of species is phylogenetically conserved. Phylogenetic independent contrasts (PIC) was employed to remove the phylogenetic relatedness of the pairwise correlations among leaf traits50. The phylogenetic analysis was performed in “picante” package51.Based on Angiosperm Phylogeny Group III (APG III) classification of angiospermsies Fig.  was consAll statistical analyses were performed using R software, version 3.2.1 (R Development Core Team 2011).The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary meterial


All obese pregnant women are considered at equal high risk with respect to complications in pregnancy and birth, and are commonly managed through resource-intensive care pathways. However, the identification of maternal characteristics associated with normal pregnancy outcomes could assist in the management of these pregnancies. The present study aims to identify the factors associated with uncomplicated pregnancy and birth in obese women, and to assess their predictive performance.2) with singleton pregnancies included in the UPBEAT trial were used in this analysis. Multivariable logistic regression was used to identify sociodemographic, clinical and biochemical factors at 15+0 to 18+6 weeks’ gestation associated with uncomplicated pregnancy and birth, defined as delivery of a term live-born infant without antenatal or labour complications. Predictive performance was assessed using area under the receiver operating characteristic curve (AUROC). Internal validation and calibration were also performed. Women were divided into fifths of risk and pregnancy outcomes were compared between groups. Sensitivity, specificity, and positive and negative predictive values were calculated using the upper fifth as the positive screening group.Data form obese women (BMI ≥ 30 kg/m2), the prevalence of uncomplicated pregnancy and birth was 36% (505/1409). Multiparity and increased plasma adiponectin, maternal age, systolic blood pressure and HbA1c were independently associated with uncomplicated pregnancy and birth. These factors achieved an AUROC of 0.72 (0.68–0.76) and the model was well calibrated. Prevalence of gestational diabetes, preeclampsia and other hypertensive disorders, preterm birth, and postpartum haemorrhage decreased whereas spontaneous vaginal delivery increased across the fifths of increasing predicted risk of uncomplicated pregnancy and birth. Sensitivity, specificity, and positive and negative predictive values were 38%, 89%, 63% and 74%, respectively. A simpler model including clinical factors only (no biomarkers) achieved an AUROC of 0.68 (0.65–0.71), with sensitivity, specificity, and positive and negative predictive values of 31%, 86%, 56% and 69%, respectively.Amongst 1409 participants  contains supplementary material, which is available to authorized users. Maternal obesity is associated with increased risk of maternal and perinatal mortality and morbidity, including gestational diabetes mellitus (GDM), preeclampsia and birth complications . In commA key strategy of the UK 2016 Maternity Transformation Programme is the evidence-based provision of antenatal care that recognises different levels of risk of adverse outcomes between different groups of women . The UK The aim of the present study is to identify factors in the early second trimester associated with subsequent uncomplicated pregnancy and birth in obese women and to assess their predictive performance.The UK Pregnancies Better Eating and Activity Trial  was a multicentre, randomised controlled trial of a complex behavioural intervention of diet and physical activity advice versus standard antenatal care in obese pregnant women to prevent GDM and delivery of large for gestational age infants. The study involved eight centres located in London (three centres), Bradford, Glasgow, Manchester, Newcastle, and Sunderland. Regulatory approvals were obtained from the UK research ethics committee  and local research and development departments in participating centres. All women provided written informed consent prior to entering the study.2), aged 16 years or older, and with a singleton pregnancy between 15+0 and 18+6 weeks’ gestation. Women were recruited from March 2009 to June 2014. Exclusion criteria were multiple pregnancy, current use of metformin, unwilling or unable to provide written informed consent, or underlying disorders . Extensive data were collected on sociodemographic and clinical characteristics, and anthropometric measures and blood samples were also obtained. Women were followed up at 27+0 to 28+6 and 34+0 to 35+6 weeks’ gestation, at delivery and at 6 months postpartum [UPBEAT recruited 1555 obese women , GDM, preeclampsia, hypertensive disorders of pregnancy, antepartum haemorrhage, placental abruption, venous thromboembolism, delivery of a small for gestational age infant  were selected on the basis of known associations with one or more pregnancy complications. The sociodemographic characteristics and clinical factors explored were maternal age, ethnicity , adjusted index of multiple deprivation, BMI, parity, previous history of GDM or preeclampsia, smoking, history of threatened miscarriage in the index pregnancy, systolic blood pressure (SBP) and maternal anthropometry. Maternal anthropometric measures included were mid-arm circumference and sum of skinfold thickness . Blood pressure was recorded using the pregnancy validated Microlife BP3BT0-A blood pressure monitor  and maternal skinfold thicknesses  were measured in triplicate, using Harpenden skinfold Calipers  [Factors from early pregnancy  .+0 to 18+6 weeks’ gestation were assessed. The biomarkers measured in blood samples were markers of glucose homeostasis (haemoglobin A1c (HbA1c), fructosamine, insulin and C-peptide), adipokines (adiponectin and leptin), inflammatory and endothelial markers , lipids , liver-associated markers , sex hormone binding globulin and ferritin) and vitamin D. Analytical methods for these biomarkers are shown in Additional file A total of 19 selected biomarkers measured in blood samples obtained at 152 , so that odds ratios (ORs) showed the effect associated with doubling the concentration. Ethnicity was transformed into a binary (white and non-white) variable for analysis to reduce the degrees of freedom. The index of multiple deprivation was categorised into fifths for a clearer interpretation. Continuous data were summarised by mean (standard deviation) and categorical data reported as number (percentage). Comparisons between groups were performed using a t-test or χ2 test, as appropriate. Univariable logistic regression was used to estimate the effect size for each factor. Clinical factors with a P value less than 0.05 in group comparison were included in multivariable analyses. Biomarkers with a statistically significant association following Bonferroni correction for 19 tests (P < 0.0026) were included in multivariable analyses.All participants with complete pregnancy outcome data were included in the analysis. To ensure that the study population was representative of the overall UPBEAT participants we compared demographic characteristics between women included and excluded from this analysis. Missing data on potential predictors were minimal (< 2%), except for biomarkers, as blood samples were not available for 27% of women (286/1409). Of those with blood samples taken, biomarker missing data were minimal (< 2%), except for HbA1c . Biomarkers were assessed for normality and variation with gestational age. None of the biomarkers showed variation with gestational age at measurement and biomarkers with highly skewed distributions were transformed into logBackward stepwise logistic regression was then used to identify factors that were independently associated with uncomplicated pregnancies and birth . One mul+0 and 18+6 weeks’ gestation. In this analysis, the study population comprised 1409 women with complete outcome data  had antenatal complications alone, 32% (287/904) had labour complications alone and 30% (275/904) experienced both. Women who experienced both antenatal and labour complications accounted for 20% 275/1409) of the whole study population .In univariable analyses, multiparity was associated with increased odds of an uncomplicated pregnancy and birth, and factors associated with a reduced odds were higher maternal age, SBP, BMI, mid-arm circumference and sum of skinfolds Table . A previWomen were then divided in fifths of predicted risk and the prevalence of each adverse outcome in the fifths is described in Table The model including only clinical factors  was also used to explore the prevalence of each adverse outcome in each fifth of predicted risk  that were associated with an uncomplicated pregnancy and birth, namely multiparity, lower maternal age, SBP and HbA1c levels, and higher adiponectin levels. In combination, these achieved an AUROC of 0.72 (0.68–0.76) for the prediction of an uncomplicated pregnancy and birth. The use of the upper fifth of predicted risk of uncomplicated pregnancy and birth as the screen positive group provided a sensitivity of 38% and a specificity of 89%.Over one-third of this contemporary, multi-ethnic, inner city population of obese pregnant women had an uncomplicated pregnancy and birth. Nevertheless, this also highlights the extent to which obesity is an important risk factor for pregnancy complications. Combining clinical factors and biomarkers, we identified five independent predictors in early pregnancy  in this group might be desirable. This also applies to the prediction tool based on clinical factors . We therefore recommend that the effectiveness of any alternative pathway of care based on these prediction tools should be fully evaluated prior to implementation in clinical practice. Inclusion of other relevant variables, for example, previous obstetric history, weight change from pre-pregnancy to early second trimester or additional biomarkers, may further improve the accuracy of these models. Further studies should also focus on identifying possible causal mechanisms (i.e. biomarkers of good glycaemic control and lower insulin resistance) contributing to uncomplicated pregnancy and birth, which could be amenable to targeted interventions.2 should receive recommendation to deliver in an obstetric unit [2) [2 was 62% (57–67%), similar to that in women with a BMI of 30–35 kg/m2 . Importantly, women identified as most likely to have an uncomplicated pregnancy and birth (upper fifth) by our prediction model had a higher rate of spontaneous vaginal delivery of 92% (87–97%) following onset of labour. This figure was markedly similar  in the simpler model including clinical factors alone. In common with our findings, the Birthplace study also identified a subgroup of lower risk women with a BMI ≥ 35 kg/m2 in which the risk for obstetric interventions or maternal adverse outcomes was lower than in nulliparous women of normal BMI (21% vs. 53%) [Our results have immediate relevance to the choice of place of birth. According to NICE, all women with a BMI ≥ 35 kg/mric unit . Howeverunit [2) . In our vs. 53%) . TogetheIt is generally accepted that obese women should be offered a 75 g OGTT between 24 and 28 weeks’ gestation. Lower risk obese women in this study had a GDM prevalence of 16%, similar to the prevalence in an unselected population with the same IADPSG diagnostic criteria . Therefo2 higher compared to women who declined participation [A key strength of this study lies in the prospective collection of detailed clinical, anthropometric and biomarker data related to obesity with contemporaneous data monitoring across all sites. Another strength lies in the assessment of uncomplicated pregnancy and birth, and in the principle of a prediction tool applicable to all pregnancies in obese women rather than one for each outcome . This novel concept simplifies stratification in obese women who are at increased risk of multiple adverse outcomes. A limitation was the number of women without biomarker data . To explore potential selection bias, we assessed the effect size of each clinical factor in the whole study population and in the restricted study cohort (with available biomarkers); similar effect sizes were observed in both samples. A ‘healthy cohort’ effect cannot be excluded; obese women who took part in UPBEAT may have been healthier than the general obese pregnant population, and our prediction model performance would not be generalisable. However, we consider this unlikely as UPBEAT participants had maternal demographic characteristics usually associated with the poorest pregnancy outcomes, being on average 10 months older and with a BMI 0.7 kg/mApproximately one-third of the obese women studied had an uncomplicated pregnancy and birth. We have shown that risk stratification could be achieved by a combination of clinical factors and biomarkers. Stratification of risk for an uncomplicated pregnancy is an innovative approach to the management of obese pregnant women, with the potential to improve clinical management and the choices for women as well as to ensure efficient resource allocation. Further studies are needed to explore alternative pathways of care for the sub-groups of obese women who differ according to risk profile for uncomplicated pregnancy and birth.Additional file 1:Analytical methodologies for the biomarkers measured. (DOCX 13 kb)Additional file 2:Sociodemographic characteristics of study population and UPBEAT participants excluded from this analysis. (DOCX 13 kb)Additional file 3:Distribution of antenatal and labour complications. (DOCX 38 kb)Additional file 4:Factors assessed in relation to uncomplicated pregnancy and birth. (DOCX 15 kb)Additional file 5:Pregnancy outcomes according to predicted chance in fifths of uncomplicated pregnancy and birth using the model with clinical factors and HbA1c. (DOCX 15 kb)Additional file 6:Sensitivity analysis to assess consistency of association of clinical factors in a restricted sample (women with biomarker data). (DOCX 12 kb)


Therefore, natural convection solar dryer can be used to dry pepper with acceptable microbial and sensory qualities, as an alternative to open-sun drying. The objective of this work was to determine the effects of blanching and two drying methods, open-sun drying and natural convection solar drying, on the quality characteristics of red pepper. A 2 × 3 factorial design with experimental factors as 2 drying methods (open-sun drying and use of solar dryer) and 3 levels of pepper blanching  was conducted. Dried pepper samples were analysed for chemical composition, microbial load, and consumer sensory acceptability. Blanching of pepper in 2% NaCl solution followed by drying in a natural convection solar dryer reduced drying time by 15 hours. Similarly, a combination of blanching and drying in the solar dryer improved microbial quality of dried pepper. However, blanching and drying processes resulted in reduction in nutrients such as vitamin C and minerals content of pepper. Blanching followed by drying in natural convection solar dryer had the highest consumer acceptability scores for colour and overall acceptability, while texture and aroma were not significantly ( Capsicum annuum L.) is used as a spice or major ingredient in dishes around worldwide and perhaps considered the first spice to have been used by man with archaeological evidence of pepper and other fossil foods dating back to 6000 years old [Red pepper  and 3 levels of pepper blanching  were conducted. The samples were analysed for their chemical composition including moisture, crude protein, ash, and fibre contents . Additio Capsicum annuum L. (red pepper) were purchased from the local open market in Navrongo. The pepper was first washed thoroughly under running tap water and then blanched at 93°C in plain or in 2% NaCl solution for 4 min. Prior to drying experiments, the initial moisture content of fresh untreated pepper was determined by drying in an oven set at 120°C until constant mass was observed. The initial and final masses of the red pepper samples were recorded with an electronic balance. Initial moisture content of red pepper was found to be about 75% on wet basis.Fresh2 and dried by suspending them on weighing balance in the drying chamber of the direct solar dryer and in the open sun simultaneously. The samples were spread evenly in a single layer on the stainless-steel wire-mesh trays and kept inside the drying chamber of the natural convection solar dryer or in the open sun. The drying experiments were carried out in the month of May, 2016, at University for Development Studies, Navrongo Campus, Ghana. Each experiment started at 09:00 a.m. and continued until 5:00 p.m. daily. Relative humidity during the experimental period ranged between 55% and 65%. During the experimental period, ambient temperature, temperatures within the dryer, and the absorber plate temperatures were measured at 1 h intervals. Additionally, the mass of pepper was measured at 1 h intervals throughout the experiments until a final moisture content of 5%  was achieved [Differently treated pepper samples were loaded onto wire-mesh trays at 5 kg/machieved .Crude protein, fibre, and ash were determined following the procedures by AOAC methods 970.22, 985.29, and 972.15, respectively . Mineral E. coli) colonies on CHROMagar ECC plates were enumerated. Microbial counts were reported as CFU/g of pepper sample.Serial 10-fold dilutions were made by weighing 10 g of dried pepper samples into 90 ml of Butterfield's phosphate buffer . Appropriate dilutions were surface-plated onto Tryptic Soy Agar  supplemented with cycloheximide (50 mg/l) to inhibit the growth of mold and onto CHROMagar ECC plates and incubated at 37°C for 24 h. Following incubation, all visible colonies on TSA were enumerated as Aerobic Mesophilic count (AMC), and all pink (coliforms) and blue .All experiments were carried out in triplicate and values are presented as means with standard deviations (SD) where applicable. Data obtained were subjected to analysis of variance (ANOVA) and Least Significant Difference (LSD) was used to separate means at Variations in ambient temperature and temperatures in the drying chamber of solar dryer and absorber plate during the experimental period are shown in p < 0.05) than pepper samples dried in the open sun. Blanching either in 2% NaCl solution or in water significantly (p < 0.05) improved drying rate of pepper  after blanching treatments and drying  counts in dried pepper are shown in Mean aerobic mesophilic counts (AMC) andp < 0.05) lower microbial load when compared to samples that were dried in the open sun with or without blanching (aw) in pepper, microbial load can be controlled although spores may not be killed. These may bring microbial load of dried pepper to allowable safe limits while supporting long shelf life of these products.Generally, pepper samples that were blanched and dried in solar collector had significantly  in aroma and texture between the different pepper samples. However, significant differences (p < 0.05) were observed in colour and overall acceptability between samples. For colour, pepper samples that were blanched either in plain water or in 2% NaCl solution and subsequently dried using the solar dryer scored significantly higher (7.0), whereas pepper samples that were not blanched and subsequently dried in the open sun scored significantly (p < 0.05) lower (3.8). For overall acceptability, the highest score of 7.1 was attained for pepper samples that were blanched in 2% NaCl solution and subsequently dried in the solar dryer, indicating the panelist likeness for the product.Consumer sensory evaluation of the differently treated and dried pepper samples assessed using a 9-point hedonic scale is shown in It has been suggested that inactivating the enzymes responsible for browning , during blanching improves both colour and flavour of vegetables . Howeverp > 0.05) affected by the various treatments. Therefore, natural convection solar dryer has the potential for commercial small-medium scale industrial application for drying pepper with acceptable microbial and sensory qualities, especially in developing rural communities where equipment cost as well as recurring cost of fuel or energy to operate mechanical dryers can prove to be high. This in turn would ensure reduction in postharvest losses of pepper and thus better economic returns for farmers and processors.Blanching of pepper in 2% NaCl solution followed by drying in a natural convection solar dryer reduced drying time by 15 hours when compared to drying in the open sun which takes at least 28 hours. Similarly, a combination of blanching and drying in the solar dryer improved microbial quality of the dried pepper. However, the blanching and drying processes resulted in reduction of nutrients such as vitamin C and minerals contents. Consumer sensory analysis showed that blanching, followed by drying in natural convection solar dryer, had the highest scores for colour and overall acceptability, while texture and aroma were not significantly (


This study aimed to propose a new surgical strategy, i.e., the transcervical video-assisted mediastinoscopic lymphadenectomy (VAMLA) with esophagectomy via the left transthoracic approach for patients with esophageal cancer (EC), and to compare the outcomes with those of esophagectomy via the right thoracic approach.From December 2014 to March 2016, 49 cases were enrolled in this non-randomized concurrent control study. Twenty-eight patients with EC who underwent transcervical VAMLA with esophagectomy via the left transthoracic approach were assigned into the study group, while 21 EC patients undergoing esophagectomy via the right transthoracic approach during the same period were enrolled into the control group. Operative outcomes including operative time, the numbers of removed lymph nodes, intraoperative blood loss, the length of hospital stay, and postoperative complications in both groups were evaluated and compared.p < 0.05], dissected superior mediastinal lymph nodes , and dissected in the recurrent laryngeal nerve lymph nodes . No significant differences were observed in the operative time, intraoperative blood loss, length of postoperative hospital stay, number of dissected abdominal lymph nodes, postoperative pulmonary complications (pneumonia and atelectasis), anastomotic fistula, chylothorax, and vocal cord paralysis (p > 0.05).There were no significant differences in the baseline profiles between the two groups, and all patients in the two groups successfully underwent the surgery. There was a significant difference between transcervical VAMLA with esophagectomy via the left thoracic approach and esophagectomy via the right thoracic approach with regard to the number of all dissected lymph nodes [(29.0 ± 8.7) vs. (17.8 ± 8.1), Transcervical VAMLA combined with esophagectomy via the left thoracic approach appears technically feasible and safe and shows advantages in the number of dissected superior mediastinal lymph nodes, suggesting that it may serve as a new treatment option for patients with esophageal carcinoma. Esophageal cancer (EC) is one of the worst malignant digestive neoplasms and the sixth leading cause of cancer-related deaths around the world , produciFor the radical resection of EC, the most common open surgical protocols include transthoracic and transhiatal esophageal resections , 6. The Recently, the widely used minimally invasive esophagectomy (MIE) for the patients with EC has reduced the surgical trauma and the incidence of complications; however, this operation still requires abdominal incisions that increase trauma and may cause complications such as abdominal incision infection and hernia . FurtherThe application of surgical resection with the left or right transthoracic approach in patients with EC is always controversial, and the core dispute focuses on which approach can achieve better mediastinal lymph node dissection . The lefIn this non-randomized concurrent control trial, we identified consecutive 49 patients with EC between December 2014 and March 2016. All patients were divided into either study group or control group by the method that was not random. The chief surgeon defined and managed the alternatives based on the wills of patients and their family members. In order to reduce the deviation, only the EC patients who met the criteria of VAMLA combined with esophagectomy via the left transthoracic approach were enrolled in the control group. Patients in the study group underwent VAMLA combined with esophagectomy via the left transthoracic approach, while others in the control group received esophagectomy via the right transthoracic approach. The surgery was performed by the same group of surgeons, who was well trained and had performed more than 200 cases of esophagectomy.The inclusion criteria were as follows: (1) tumor located in the middle-lower thoracic region, a clear pathological diagnosis confirmed using gastroscopy and endoscopic ultrasound, clinical stage under cT3N1M0 by preoperative evaluation according to UICC 7th edition TNM staging , and no VAMLA combined with the left transthoracic esophagectomy was divided into two steps:First, the patients were placed in the right lateral decubitus position, and a routine left posterolateral thoracotomy was performed. The esophagus was then freed, and a gastric tube was created. Lymph nodes in the thoracic cavity and abdominal cavity including the nodes along the esophagus, the right and left cardiac node, the nodes along the left gastric artery, and greater and lesser gastric curvatures were resected. For three cases with lower thoracic tumors in this group, the esophagogastric anastomosis was constructed below the aortic arch. The remaining 25 cases underwent left neck anastomosis as follows: esophageal tumor was resected about 2 cm above the edge of the esophageal tumor, and the stump of the remnant esophagus was embedded using sterile finger cot, fixed with silk suture, and then hung with highest point of the gastric conduit and ligated with silk suture to prepare the neck incision, gastric tube traction, and esophagogastric anastomosis. Additionally, the gastric conduit should be placed carefully in the esophageal bed with a particular direction. Finally, the placement of the chest drainage tube and the suture of thoracic incision were performed after the diaphragm was closed.Second, the patients were repositioned in a supine position with shoulder padded high and head hypsokinesis as far as possible. We made an approximate 5-cm transverse incision 2 cm above the sternal notch. The skin, subcutaneous tissue, anterior muscles, and tracheal fascia were incised and separated with the finger along both sides of the trachea. Subsequently, the mediastinoscope was inserted along the right side of the trachea, and the lymph nodes in the space between the trachea and the superior vena cava were removed up to the bottom of the innominate artery and down to the bottom of the azygos vein Fig. a, b. TheRadical esophageal surgery via right transthoracic approach was performed with the similar steps reported in the previous literature , 14. IntAfter surgery, all cases would be nursed and treated in the same ICU.After the complete surgical preparation, all patients underwent general anesthesia and double-lumen tracheal tube intubation.The primary endpoint of the study was the incidence of major complications in both groups. Major complications included anastomotic fistula, chylothorax, vocal cord paralysis, and respiratory complications (pneumonia and atelectasis). Four weeks after the operation was considered as the cut-off point for postoperative complication evaluation, while perioperative mortality was defined as death within 30 days. Secondary endpoints included the number of dissected regional lymph nodes, perioperative mortality, operative time, intraoperative blood loss, and the length of postoperative hospital stay. The mean follow-up duration in both groups was 12 months (range 6–20 months).U test [χ2 test, and the difference with p < 0.05  was considered statistically significant.The SPSS18.0 statistical software was used for the data analysis. The continuous data were expressed as mean ± standard deviations, and the Mann-Whitney U test  was usedAccording to the abovementioned criteria, 28 patients  with EC in the study group received VAMLA combined with esophagectomy via the left transthoracic approach, while the 21 patients  in the control group received esophagectomy via the right transthoracic approach , anastomotic fistula, chylothorax, and vocal cord paralysis between the two groups Table . No patiIn the study group, one patient occurred to have multiple pulmonary and bone metastases 8 months after the operation, and two cases confronted anastomotic recurrence . In the control group, one case had multiple pulmonary metastases 9 months after the operation, and three cases had anastomotic recurrences , including two cases of mediastinal and abdominal lymph nodes recurrence.p < 0.05, Table p > 0.05).In the study group, the number of total dissected lymph nodes, superior mediastinal lymph nodes, and RLN lymph nodes was 29.0 ± 8.7, 11.2 ± 5.0, and 5.6 ± 3.5, respectively; in the control group, the number was 17.8 ± 8.1, 3.7 ± 2.9, and 2.3 ± 2.1, respectively. The total number of dissected lymph nodes, the harvested number of superior mediastinal lymph nodes, and the number of RLN lymph nodes were significantly different between the two groups  [Lymph node dissection plays an essential role in EC radical resection , 27. Lym < 0.05) . Thus, t < 0.05) , 34, 35. < 0.05) . AdditioIn the study of Matsuda et al., the total number of dissected LNs was 20.02 ± 8.16 and 27.93 ± 11.75, respectively, in the thoracic duct (TD)-preserved and TD-resected groups via right transthoracic approach , which sTranscervical mediastinoscopy is a relatively mature surgical procedure in thoracic surgery, which is mainly used for the diagnosis of a mediastinal mass, lymph node biopsy, and preoperative staging of lung cancer. In 1990, Buess and Becker  first reVAMLA combined with radical operation via left transthoracic approach for EC has the following potential advantages: (1) not only retaining the benefits of the traditional esophagectomy via the left transthoracic approach but also achieving the superior mediastinal lymph nodes and bilateral RLN lymph node dissection without increasing surgical incisions; (2) satisfactory exposure of the upper mediastinal anatomical structure, in particular, the exposure of bilateral RLN ,and better lymph node dissection; (3) achieving the cervical and upper thoracic esophageal dissociation simultaneously; and (4) less trauma and bleeding, as well as less postoperative pain and complications.Our results indicated that the total number of dissected lymph nodes and the numbers of upper mediastinal lymph nodes and RLN lymph nodes were significantly higher in the study group than that in the control group, while the number of dissected abdominal lymph nodes was similar in both groups. It suggested that VAMLA combined with radical operation via left transthoracic approach for EC was not inferior to esophagectomy via the left transthoracic approach in the dissection of abdominal lymph nodes. Furthermore, it has distinct advantages in dissecting the upper mediastinal lymph nodes, especially for the RLN lymph node. In this study, the RLN lymph node metastatic rate was 25% (7/28), namely four cases of right RLN lymph node metastasis and three cases of left RLN lymph node metastasis, which was similar with that reported in domestic and foreign relevant literature , 34.There were no significant differences in postoperative complications including respiratory system complications, arrhythmia, chylothorax, anastomotic fistula, postoperative hospital stay, and vocal cord paralysis between the two groups. Four patients  in the study group were subjected to RLN paralysis, and the symptom in three of them significantly alleviated after 3 months of operation. The incidence of nerve palsy, however, has been reported to be 3.1–22.5% in other studies . The posIn the present study, VAMLA was used to make up the deficiency of the dissection of recurrent laryngeal nerve lymph nodes (RLN LNs) in esophagectomy via left transthoracic approach. Although the Ivor Lewis approach is the most routinely performed approach by most surgeons , we alsoIn summary, VAMLA combined with esophagectomy via left transthoracic approach is technically feasible and safe. It shows advantages in the number of the upper mediastinal lymph node dissection, especially the RLN lymph node dissection, which suggests that it may offer a new treatment option and a complementary therapy for patients with EC. However, more cases and long-term follow-up should be included in the further study.


High blood glucose and type 2 diabetes are associated with a range of adverse health and cognitive outcomes. One factor that contributes to high blood glucose and type 2 diabetes is dietary intake. This study investigated the relationship between dietary patterns, fasting blood glucose and diabetes status in a sample of 209 participants aged 60–65. Blood plasma glucose was measured from venous blood samples. Individual Prudent and Western dietary patterns were estimated from a self-completed food frequency questionnaire. The relationship between dietary patterns, diabetes, and blood glucose was assessed via general linear model analyses controlling for age, sex, height, and total caloric intake. Results indicated that there was no association between Prudent diet and fasting blood glucose levels, or type 2 diabetes. In contrast, an individual in the upper tertile for Western dietary score had a significantly higher risk of having diabetes than an individual in the lower tertile for Western dietary score. However, there was no significant association between Western diet and fasting blood glucose. Western diet may be associated with type 2 diabetes through mechanisms beyond impacting blood plasma glucose directly. The fact that the association between Western diet and type 2 diabetes remained even when total caloric intake was controlled for highlights the need for policy and population health interventions targeting the reduction of unhealthy food consumption. Dietary management can lower blood glucose levels in those with Rather than focussing on a particular food group, dietary pattern analysis is conceptually attractive as it takes into account the whole diet, and so accounts for the synergistic effects of nutrients on health. As described in Hu A number of cross-sectional and longitudinal studies have used dietary pattern analysis and found that the Prudent dietary pattern is associated with lower fasting blood glucose and a lower incidence of type 2 diabetes, while the Western dietary pattern has been linked to higher fasting blood glucose and higher incidence of type 2 diabetes , 22. StuThe aim of the present study was to investigate the relationship between Prudent and Western dietary patterns and fasting blood glucose, considering participants across the full range of blood glucose levels (those without diabetes as well as those with impaired fasting glucose or type 2 diabetes), and to examine whether the association is changed when stratifying analyses on the basis of impaired fasting glucose and type 2 diabetes diagnosis. Further investigation of the relationship between diet and impaired fasting glucose or type 2 diabetes incidence will help clarify if there are categorical differences in dietary patterns between individuals with blood glucose in the normal range, with impaired fasting glucose or with type 2 diabetes which may underlie differences in blood plasma glucose.22.1The sample for this study included participants from the PATH Through Life project, a longitudinal study of ageing described elsewhere 2.2The study was approved by the Australian National University Ethics Committee and all participants provided written informed consent.2.3The Commonwealth Scientific and Industrial Research Organisation Food Frequency Questionnaire was used to measure dietary intake. Validated for use in an Australian population, this measure includes food types, serving sizes, cooking methods and general eating habits 2.4Venous blood was collected after a skipped breakfast. Plasma glucose was measured on an LX20 analyser by an oxygen rate method . Individuals had either normal blood glucose (blood glucose <5.6 mmol/L), impaired fasting glucose (blood glucose 5.6–<7 mmol/L), or type 2 diabetes (blood glucose> = 7 mmol/L or self-reported as having diabetes).2.5Multinomial logistic regression was then used to examine the relationship between dietary score tertile and blood glucose as a continuous variable, as well as blood glucose as a continuous variable. Where appropriate, the lowest tertile group and individuals with normal blood glucose were used as the base group for comparisons. Linear models were then used to investigate the association between dietary tertiles and continuous blood glucose levels. Adjusted models controlled for age, gender, height, and total energy intake. Height was controlled for to take into account body size while not over-correcting by using related measures such as body mass index . Total energy intake was centred on the sample mean. One individual with very high fasting blood glucose (16.1 mmol/L) was removed from analysis as their data comprised an influential outlier (Cook’s distance >1), and destabilised coefficients in sensitivity analyses. Alpha was set at 0.05.33.1Participant demographic characteristics can be found in 3.2A prudent score tertile was not significantly associated with either diabetes or IFG . Individ3.3When all participants were included in the model, prudent diet was not significantly associated with fasting glucose . In the 3.4p = 0.68, RR = 0.83, 95%CI; high estimate = −0.32, p = 0.50, RR = 0.72, 95%CI), and a pattern similar to the previously reported association between Western dietary tertiles and IFG . When adjusting for BMI rather than height, coefficient directionality and significance for prudent dietary tertiles were unaffected. However, the high Western tertile gained significance for IFG . In linear analyses, no dietary tertile was significantly associated with blood glucose when analyses were limited to individuals with diabetes only, though results are likely unstable due to small cell sizes. Analyses considering dietary pattern scores as continuous variables . This indicated no significant association between IFG and diabetes and Prudent dietary tertiles  5In conclusion, this study found no association between a Prudent dietary pattern and blood glucose levels, or type 2 diabetes. The pattern of results suggests that a Western dietary pattern is associated with type 2 diabetes and that this effect may either be due to sustained dietary intake in the past or through mechanisms that do not directly affect blood plasma glucose. Because of the well-documented health impacts of type 2 diabetes diagnosis, the nature of these mechanisms requires further investigation. The fact that the Prudent diet did not have a protective effect for blood glucose levels or type 2 diabetes, and that the association between Western dietary pattern and type 2 diabetes remained significant even after total caloric intake was controlled for, highlights the need for policy and population health interventions to initiate behaviour change specifically targeted at reducing unhealthy food consumption in addition to those aimed at reducing excessive caloric intake.Erin I. Walsh: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Felice N. Jacka, Peter Butterworth: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.Kaarin J. Anstey: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.Nicolas Cherbuin: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data.National Health and Medical Research Council . Nicolas Cherbuin and Kaarin Anstey’s Research Fellowship are funded by the Australian Research Council (grant number 120100227) and the National Health and Medical Research Council (grant number 1002560). Felice Jacka has received Grant/Research support from the Brain and Behaviour Research Institute, the National Health and Medical Research Council (NHMRC), Australian Rotary Health, the Geelong Medical Research Foundation, the Ian Potter Foundation, Eli Lilly, the Meat and Livestock Board, Woolworths Limited, Fernwood Gyms and The University of Melbourne and has received speakers honoraria from Sanofi-Synthelabo, Janssen Cilag, Servier, Pfizer, Health Ed, Network Nutrition, Angelini Farmaceutica, Eli Lilly and Metagenics. She is supported by an NHMRC Career Development Fellowship (2) (#1108125).This work was supported by the The authors declare no conflict of interest.No additional information is available for this paper.


Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities  in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2%  DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers , protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement. In aquatic ecosystems, heterotrophic prokaryotes play important roles in organic matter cycling, including the transformation and remineralization of organic molecules and in its transfer to other organisms via trophic interactions. In order for heterotrophic bacteria that are osmotrophs to obtain nutrients from polymeric biomolecules such as proteins, these high molecular weight organic molecules must be hydrolyzed extracellularly to smaller sizes ” activities by taking measurements separately on seawater that passes through filters of a specified pore size, as well as unfiltered samples. These fractionations may provide insights into the natural forms of hydrolytic enzymes in seawater and the ecological roles that each play Arnosti, . Smith eProteins and peptides should be good nutrition for heterotrophic prokaryotes after suitable hydrolysis by extracellular enzymes, and thus extracellular proteases are one of the most important hydrolytic enzymes in aquatic microbial ecosystems. Measurements of extracellular proteolytic enzyme activity in aquatic samples using fluorogenic model substrates have been popular since their introduction  into bottles to remove large particles. Polycarbonate 500 mL bottles were used as a sample container, except for the experiment to compare several types of containers. Samples were immediately placed on ice for transport to the laboratory where the samples were refrigerated at 4°C until proceeding with the assays within several hours. In general, to assess the extracellular enzyme activities in natural aquatic samples the activities should be measured as soon as possible after sampling, to minimize the possible alteration after sampling such as degradation of dissolved enzymes and changing of microbial community and their activity. Even though the main purpose of each experiment in this study was to compare the data for the same water sample using different methodological conditions, we conducted each experiment as soon as possible after seawater sampling , except for the preservation test in different sample containers. When we kept water samples for a short time before measurement, samples were kept cool and in the dark for the least degradation of enzymes possible, although there were still unavoidable possibilities of microbial cell lysis at 4°C.For some experiments, an aliquot of sample was filtered through a 0.2 μm pore size polycarbonate Nuclepore filter (Whatman), then both unfiltered and filtered (<0.2 μm) seawater samples were used.The potential activities of extracellular proteolytic enzymes in seawater samples were measured using 17 MCA substrates (Table MCA substrates were dissolved in solvents dimethyl sulfoxide (DMSO) or 2-methoxyethanol (MTXE) to prepare stock solutions (10 or 20 mM). For assay, 10× substrate working solutions were prepared from the stock solutions in autoclaved artificial seawater and solvent to control for solvent concentration in the solution, with substrate and solvent concentrations 10 times higher than the target final concentrations in assay. Seawater samples and 10× substrate solutions were mixed in disposable cuvettes or 96-well microplates and incubated at 25°C in the dark to measure potential enzyme activities. The fluorescence of the hydrolytic product, 7-amino-4-methylcoumarin (AMC), was measured several times at intervals  during the incubation. The excitation/emission wavelengths for fluorescence measurements were 380/460 nm on a spectrofluorometer (Hitachi F-2500) or 380/440 nm on a microplate reader (Corona SH8100Lab). A solvent blank (seawater sample with solvent but without substrate) was also prepared and subjected to fluorescence measurements along with samples. After subtracting the solvent fluorescence blank, the concentration of AMC generated during the incubation was calculated using a calibration curve prepared by measuring fluorescence intensity of AMC solutions at seven concentrations (0–1 μM) under the same conditions as the sample measurements. To measure the non-enzymatic produced AMC during incubation, collected seawater was autoclaved and prepared and assayed as an inactivated control under the same conditions as the intact sample. The hydrolysis rate of the substrate in the seawater sample, namely, extracellular enzyme activity in seawater, was calculated by determining the increase in AMC concentration with time after subtracting the concentration of non-enzymatic produced AMC estimated in autoclaved seawater.Every assay was performed in triplicate using three cuvettes or three wells in a microplate for each sample and substrate pair.Extracellular proteolytic enzyme activities in natural seawater samples were measured using different assay methods  simultaneously with the same working solutions. In Method A, we used a spectrofluorometer with disposable cuvettes, while Methods B and C were conducted on a fluorometric microplate reader with regular and low protein binding microplates, respectively. The excitation/emission wavelengths recommended by the supplier of MCA substrates (Peptide Institute) for the assay were 380/460 nm; however, to obtain a higher intensity fluorescence signal from smaller sample volumes used on microplates (Methods B and C), we set the emission wavelength at 440 nm, the wavelength of maximum fluorescence intensity for AMC. We confirmed that readings taken at 440 and 460 nm provided equivalent results when the corresponding calibration curve was applied. Following are brief overviews of Methods A, B, and C:Method A (cuvette) was conducted with a reaction volume of 1 mL in a disposable cuvette made from polymethylmethacrylate (PMMA). Fluorescence was measured by a spectrofluorometer at excitation/emission wavelengths of 380/460 nm.Method B (regular microplate) was conducted with a reaction volume of 300 μL in regular 96-well black microplates made from polystyrene (Nunc #237107). Fluorescence was measured by a microplate reader in fluorescence mode at an excitation/emission wavelength of 380/440 nm.Method C (low protein binding microplate) was conducted as for Method B except with a low protein binding, black, 96-well microplate made from polystyrene coated with methacryloyloxyethyl phosphorylcholine (MPC) (Nunc #245393).Pearson's correlation coefficient and simple regression were used to compare data obtained from different methods in pairwise comparisons . Regression analyses of pairs of methods were performed with combined dataset from unfiltered and filtered seawater samples for “all estimated activities” and for the “aminopeptidase activities,” “trypsin-type activities,” and “chymotrypsin-type activities.”Using selected MCA substrates for proteases Table , kineticTo determine whether differences in results between Methods B and C are proteolytic enzyme-specific, the same experiment was conducted using MUF substrate for phosphatase  at a final concentration of 0, 10, 30, 60, 100, 160, and 200 μM. Measurement of the hydrolysis rate of the MUF substrates was conducted by the same method as for the MCA substrates, except that the excitation/emission wavelengths of 365/445 nm were used to detect the 4-methylumbelliferon product.V, and the substrate concentrations, [S], the theoretical maximum activity, Vmax, and the Michaelis constant, Km, were estimated by curve fitting using software OriginPro 9.1 to the Michaelis–Menten equation:Based on the measured hydrolytic activities, Low protein binding 96-well black microplates from different suppliers were tested using seawater collected by bucket and strained through 50 μm nylon mesh into polycarbonate bottles. An aliquot of seawater 180 μL) was mixed with 20 μL of each of 15 MCA substrate solutions (Table 80 μL wasThe manufacturer of the MCA substrates (Peptide Institute) recommends using DMSO to prepare stock solutions. Considering the possibility of solvent bias in activities measurement, a lower concentration of solvent in assay is better. However, lower solvent concentration may result in reduced solubility of the substrate in seawater. To examine the effect of DMSO, two experiments were conducted with different DMSO concentrations: (1) kinetic experiments comparing 10% (v/v) and 2% (v/v) DMSO, and (2) comparison of the activities measurement with 2 and 1% DMSO with 200 μM substrate final concentration.Substrate working solution 10×) were prepared for each substrate concentration with 100% DMSO for final 10% DMSO in the assay, while those were prepared with autoclaved artificial seawater containing 20% DMSO for final 2% in the assay. Using low protein binding, 96-well microplates (Nunc), natural seawater sample 270 μL and 10× substrate working solution 30 μL were mixed in each well of the microplate. For this experiment, five MCA substrates  were used  as a solvent of substrates to measure potential activities of extracellular hydrolytic enzymes in seawater  were tested by Student's To test the effect of sampling container material, the following five types of containers were used: 500 mL polycarbonate bottles (PC500) , 50 mL polypropylene tubes supplied by Corning (PPC) and Eppendorf (PPE), 50 mL polyethylene terephthalate tubes (PET) (Corning), and low protein binding ProteosaveSS 50 mL tubes (SS) . The four 50 mL tubes had a similar shape of ordinary plastic centrifugal tubes. Natural seawater samples were collected by a bucket and immediately transferred to these containers through 50 μm nylon mesh. All seawater samples were kept cool until measurements of proteases activities. Proteases activities were measured using five MCA substrates  at several hours after sampling, and at 1 day (26 h) and 2 days (47 h) after sampling. Reaction volume for the assay was 200 μL (180 μL seawater sample + 20 μL substrate solution) in low protein binding microplate (Nunc), and the final concentrations in the mixture were 200 μM substrate with 2% DMSO. Other procedures for fluorescence measurement and enzyme activities estimation were the same as described above.Figure n = 64 (unfiltered and filtered samples were combined), r = 0.953, p < 0.0001] with a regression coefficient of 0.394 ± 0.015  and trypsin-type activity . Regression line was not shown for chymotrypsin-type activity in Figure t = 4.12, p < 0.0005) than that for aminopeptidase activity (0.643 ± 0.023). Although, there is not absolute evidence that higher estimation is more accurate, these results seem to suggest that Method B (microplate reader with regular polystyrene microplates) resulted in an underestimation of extracellular enzyme activity in natural seawater, and this effect was greater for trypsin-type activity than for aminopeptidase activity. Similarly, for the comparison of activities measurements by Method B and Method C  were systematically lower than those by Method A  and the regression coefficient was near 1 (1.015 ± 0.031), indicating that proteases activities measurements by these two methods are equivalent. Separate analyses for each of the enzyme types also were significant with regression coefficients for aminopeptidase and trypsin-type activity of 0.805 ± 0.039 and 1.038 ± 0.044, respectively . These results indicated that the systematic underestimation of enzyme activities using Method B can be avoided by using low protein binding microplates.The linear relationship between the enzyme activities measurements obtained by Methods A and C was significant  should be the same for a sample by both assay methods. However, the Vmax of hydrolysis of MUF-phosphate, was 5.5 ± 0.4 nmol L−1 h−1 by Method B, which is 45% of that measured by Method C (12.3 ± 0.4 nmol L−1 h−1) for the same seawater sample  are attributable to the adsorption of both enzymes and substrates to the microplate surface. Although, the relationship between enzyme adsorption (binding) onto the polystyrene surface and deactivation of the adsorbed enzymes was not determined, Calliou et al.  reportedFigure Most MCA substrates need to be dissolved in organic solvent prior to mixing with the seawater sample. DMSO is a good solvent for dissolving MCA substrates; however, toxic effects to microbial cells in seawater and other unexpected effects could occur during the incubation and affect the measurement of extracellular enzyme activities in seawater samples. To minimize these types of artifacts, a lower concentration of organic solvent in the assay mixture is preferred. However, too low of a solvent concentration with a high concentration of substrate might result in solubility difficulties during the assay.Michaelis plots for assays conducted with 10 and 2% DMSO with various substrate concentrations of five selected substrates  are shown in Figure Among the 17 tested MCA substrates listed in Table p = 0.295 for all pairs, n = 39; p = 0.274 for aminopeptidase, n = 12; p = 0.256 for trypsin-type, n = 24; p = 0.965 for chymotrypsin-type, n = 3). Steen et al. .In previous studies, MTXE was used to prepare stock solutions of hydrolytic substrates. Here, we compared the enzyme activities measurements in assays with the only difference being the use of solvents 2% DMSO and 2% MTXE. Among the MCA substrates tested, two could not be used with MTXE: Suc-Leu-Leu-Val-Tyr-MCA (for chymotrypsin) did not dissolve in MTXE for preparation of the stock solution, and Arg-MCA (for aminopeptidase) appeared as a suspension in the 10× working solution . Figure To assess the potential extracellular protease activities in natural seawater, the use of both 2% DMSO and 2% MTXE in assay give similar results for the hydrolysis of most MCA substrates with a final substrate concentration of 200 μM.Underestimation of hydrolytic enzyme activity in seawater using regular polystyrene microplates in the assay implies that similar concern is needed for the water sample container used to store seawater samples until assay. In general, adsorption effects are less for larger volume containers. While keeping seawater samples in smaller volume containers is convenient, the sample can be easily affected by differences among materials of the containers. For samples stored five types of container materials Figure , the estThese results show that protease activity in seawater samples stored in small polypropylene tubes decreased rapidly, causing the underestimation of natural activities, especially for trypsin-type and chymotrypsin-type endopeptidases. The choice of containers for water samples is an important consideration, even when assays are conducted as soon after sampling as possible, for assessing the level of enzyme activity in natural environmental seawater samples. In general, adsorption of organic molecules onto glassware is thought to be less than that on plastics, especially if the glass surfaces are silanized. We did not test glassware in this study, however, glass vials could be thought as a good sample container, depending on the research purposes.It is thought that various enzymes in nature have a range of characteristics and behaviors in natural aquatic environments. Some enzymes in seawater are easily adsorbed onto the surfaces of some materials, and that may cause artificial effects or biases in the enzyme activities measurement. To assess the actual natural activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems, materials used for both sampling and measurement assays should be carefully selected.Water sample containers must maintain enzyme activity in the natural seawater samples for the short-term; protease activities in seawater decreased rapidly in small volume polypropylene tubes. Using fluorogenic substrates and a fluorometric microplate reader with low protein binding, black, 96-well microplates was effective for obtaining high-resolution and reliable measurements of hydrolytic enzyme activities in small volume (180 μL) of seawater samples, while regular polystyrene microplates showed significant underestimation of activities, especially for trypsin-type proteases.For measuring the potential activities of extracellular proteases in seawater, a final substrate concentration at 200 μM in the assay (seawater sample with substrate solution) appeared to be a good saturation level for most of the tested oligopeptide analog MCA substrates for aminopeptidase, trypsin, and chymotrypsin. Stock solutions of MCA substrate are usually dissolved in a solvent, and both 1 or 2% DMSO and 2% MTXE in assay provided similar and reliable activities measurements, except for some substrates which were not sufficiently soluble. Calibration curves of AMC (product of substrate hydrolysis) should be generated under the same conditions as are used for the sample measurement. A solvent blank (sample with solvent without substrate) and an inactivated control (autoclaved seawater) should be also prepared and assayed with the samples for reliable calculation of hydrolytic enzyme activity in the sample.Substrate concentrations at saturation level for assay of potential activity and substrate solubility may depend on sample type, targeted enzyme, and its substrate. It is important to optimize these factors for the sample types and target enzymes to obtain data that are as reliable as possible.YO designed and conducted all experiments, analyzed data, constructed discussion, and prepared the manuscript; CWB conducted portions of the experiments with YO; and SS supported all stages of the research and contributed to preparation 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.