Glucocorticoids are associated with an increased risk of thrombosis.Healthy volunteers received dexamethasone or placebo in an endotoxin lung instillation model.Dexamethasone suppressed thrombin generation in bronchoalveolar lavage.Glucocorticoids inhibit endotoxin induced pulmonary coagulopathy.Activation of local and systemic coagulation is a common finding in patients with pneumonia. There is evidence that glucocorticoids have procoagulant activity in the circulation, particularly in the context of inflammation. The effects of glucocorticoids on local pulmonary coagulation have not yet been investigated.To use a human model of lung inflammation based on the local instillation of endotoxin in order to investigate whether glucocorticoids alter pulmonary coagulation.BAL) was performed and coagulation parameters were measured.Twenty‐four healthy volunteers were randomized to receive either dexamethasone or placebo in a double‐blind trial. Endotoxin was instilled via bronchoscope into right or left lung segments, followed by saline into the contralateral site. Six hours later, a bilateral bronchoalveolar lavage (1 + 2) was increased three‐fold  and the level of thrombin–antithrombin complex (TATc) was increased by ~ 50%  as compared with saline‐challenged segments. Dexamethasone reduced F1 + 2  and TATc  levels almost to those measured in BAL fluid from the saline‐instilled segments in the placebo group. Dexamethasone even profoundly reduced F1 + 2 levels (80%) in saline‐instilled lung segments . In contrast, dexamethasone had no effect on systemic F1 + 2 levels.Endotoxin induced activation of coagulation in the bronchoalveolar compartment: the level of prothrombin fragment 1 + 2 (FDexamethasone inhibits endotoxin‐induced coagulopathy in lungs. This trial is the first to provide insights into the effects of glucocorticoids on pulmonary coagulation in response to endotoxin. Activation of local and systemic coagulation is a common finding in patients with severe pneumonia http://www.ClinicalTrials.gov (identifier: NCT01714427). Randomization was performed with an open‐access randomization generator (http://www.randomization.com). Two sets of sealed codes/labels with the randomization number containing information about the treatment allocation for the particular subject were prepared for each subject. Randomization was performed by the use of sealed opaque envelopes, which were produced before the start of the study by a staff member not otherwise involved in the study. The trial was conducted in accordance with the Declaration of Helsinki, and informed consent was given by all study participants before trial entry. The major eligibility criteria for inclusion of participants were that they should be healthy male or female volunteers aged 19–40 years and be non‐smokers. Nine women and 15 men were randomly assigned to receive either two infusions of 40 mg of dexamethasone  separated by 12 h  or equivalent placebo  in a double‐blind design , followed by 10 mL of saline and 10 mL of air, was instilled into the contralateral lung in the same way. Instillation of endotoxin through a bronchoscope induces an interleukin‐6‐driven inflammatory and procoagulant response in the bronchoalveolar compartment −1) was administered to all subjects to maintain adequate hydration. After 6 h, BAL was performed at each lung site. Aliquots of 20–40 mL of prewarmed saline  were instilled. The fluid was retrieved by syringe and suction, with avoidance of excessive negative suction pressures. Plasma samples were collected at – 13 h, – 1 h, 6 h and 24 h relative to endotoxin challenge.The trial was performed concurrently with an investigation on the anti‐inflammatory effects of dexamethasone published recently g at 4 °C. The supernatant was aliquoted and stored at − 80 °C until assays were performed. The levels of prothrombin fragment 1 + 2 (F1 + 2), thrombin–antithrombin complex (TATc), soluble TF (sTF) antigen, plasminogen activator inhibitor 1 (PAI‐1) and tissue‐type plasminogen activator (t‐PA) were measured with specific enzyme immunoassays, according to the manufacturers’ instructions . The F1 + 2, TATc and sTF assays are sensitive to detect even low values in plasma and BALF of healthy volunteers as previously described −1 for F1 + 2, 2 μg L−1 for TATc, 0.05 ng mL−1 for sTF, 0.49 IU mL−1 for PAI‐1 activity, 0.313 ng mL−1 for PAI‐1 antigen, 0.05 IU mL−1 for t‐PA activity and 0.1 ng mL−1 for t‐PA antigen). Fibrinogen was measured with the Clauss method in an accredited routine laboratory.After collection, BALFs were immediately put on ice and then centrifuged for 10 min at 900 ×1 + 2 was performed. On the basis of a previous publication, we estimated that the standard deviation of F1 + 2 would be similar to the mean F1 + 2 levels in BALF (0.6 nmol L−1 in patients suffering from pneumonia and 0.15 nmol L−1 in controls) 1 + 2 level in patients with pneumonia than in healthy controls anova was followed by non‐parametric tests because of a non‐normal distribution of data. Statistical comparisons between groups were performed with the Mann–Whitney U‐test, and comparisons between lung sites of subjects were performed with the Wilcoxon test. The median obtained lavage volumes were comparable between lung sites: endotoxin 45 mL (interquartile range [IQR] 35–50 mL) and saline 54 mL (IQR 39–59 mL) in the placebo group; endotoxin 49 mL (IQR 43–64 mL) and saline 53 mL (IQR 45–61 mL) in the dexamethasone group P‐values of the sensitivity analyses or the unadjusted P‐values were used (Table S1). Statistical calculations were performed with commercially available statistical software .The original sample size calculation was based on interleukin‐6 levels in BALF Endotoxin instillation into the lung induced a mild systemic inflammatory response in placebo‐treated individuals, and was generally well tolerated, without the occurrence of any severe adverse events; this has been reported previously, because the trial was performed concurrently with an investigation on the anti‐inflammatory effects of dexamethasone −1 lipopolysaccharide (LPS) increased BALF F1 + 2 levels three‐fold  Glucocorticoid therapy given within 72 h after ARDS onset was associated with a reduction in the duration of mechanical ventilation, intensive care unit (ICU) stay and ICU mortality in a small, double‐blind trial J. Bartko, B. Jilma, J. Wojta, and L. Stiebellehner were responsible for the conception and design. J. Bartko, B. Jilma, C. Schoergenhofer, M. Schwameis, and N. Buchtele were responsible for analysis and/or interpretation. J. Bartko, B. Jilma, G. Schabbauer, and L. Stiebellehner wrote the manuscript. All authors gave final approval of the version to be published.The authors state that they have no conflict of interest.Table S1. Sensitivity analysis for BALF recovery.Click here for additional data file.


A considerable amount of research suggests that the frequent use of caffeinated energy drinks may be associated with undesirable effects, particularly so in children and adolescents. This study aimed to investigate the associations between energy drink intake and mental health problems, in isolation or in combination with junk food consumption, in a nationally representative sample of Korean adolescents.Data from the 2015 Korean Youth Risk Behavior Web-Based Survey, collected from 68,043 adolescents aged 12–18 years (mean age 15.09 ± 1.72 years), were analyzed. Questionnaires were administered to collect information related to dietary behavior including energy drink intake and junk food consumption. Single item measures of sleep dissatisfaction, stress, depression, suicidal ideation, suicide plan, and suicide attempt were also administered. Associations between energy drink intake and sleep dissatisfaction, perceived severe stress, persistent depressive mood, and suicidality were investigated, and a multivariate approach was taken so that additional variance from demographic and lifestyle factors could be controlled for statistically.Energy drink intake was significantly associated with sleep dissatisfaction (adjusted odd ratios [AORs] = 1.64 and 1.25), severe stress (AORs = 2.23 and 1.38), depressive mood (AOR = 2.59 and 1.51), suicidal ideation (AORs = 3.14 and 1.43), suicide plan (AORs = 4.65 and 1.78), and suicide attempt (AORs = 6.79 and 1.91), with a higher risk for more frequent use of energy drinks (≥5 times/wk) than for less frequent use (1–4 times/wk). The detrimental effect of energy drinks on mental health was particularly prominent in frequent junk food consumers.Our data suggest that energy drink intake had detrimental effects related to stress, sleep dissatisfaction, mood, and suicidality, in isolation or in combination with junk food consumption, in Korean adolescents. However, the cross-sectional study design prevents our ability to assess causal relationships. Energy drinks are highly caffeinated soft drinks that are marketed as providing mental and physical stimulation and improving energy, athletic performance, and concentration . ConcernCaffeine and sugar content are the main ingredients in energy drinks , 10. CafThe frequency and amount of consumption of many food and drink products is known to be highly inter-correlated . For insThe current study aimed to examine the associations between energy drink intake and self-reported sleep dissatisfaction, stress, depression, and suicidality in a nationally representative sample of Korean adolescents. The combined effects of energy drink intake and junk food consumption were also investigated. It was hypothesized that 1) energy drink intake is related to mental health problems such as sleep dissatisfaction, depression, and suicidality and 2) frequent energy drink intake in combination with frequent junk food consumption would be the strongest predictor of such undesirable outcomes.Data from the 2015 Korean Youth Risk Behavior Web-Based Survey (KYRBS) were used. The KYRBS is an anonymous self-reported online survey conducted annually since 2005 by the Korea Centers for Disease Control and Prevention to identify teenagers’ health behaviors with the subjects ranging from middle school freshmen to high school seniors. Using stratified cluster sampling, the middle and high school student samples that represent the teenager population were extracted. After given the instructions of the survey, the students who agreed to participate did the survey during class. Personal data including the respondents’ names, schools, phone numbers, addresses, and resident registration numbers were not collected. In 2015, the survey included a total of 800 schools, i.e. 400 middle schools and 400 high schools each, and a total of 70,632 respondents participated. Regarding gender, 35,204 male students and 32,839 female students participated, reaching 96.7 % of participation rate. The average age of the respondents was 15.09 ± 1.72, ranging from 12 to 18. As it can be searched from other sources easily , the desnever, 2 = once or twice a week, 3 = three or four times a week, 4 = five or six times a week, 5 = once a day, 6 = twice a day, and 7 = three times or more a day. Participants were classified into three groups: highly frequent energy drink intake (≥5 times/week), moderately frequent energy drink intake (1–4 times/week), and infrequent energy drink intake (<1 time/week).Frequency of energy drink intake during the previous week was assessed on a seven-point scale where 1 = Sociodemographic variables included gender, age, school type , place of residence (name of city), and residential type . Places of residence were dichotomized as rural vs. urban area, and residential type was dichotomized as residence with vs. without family. Perceived academic achievement was assessed using a 5-point Likert scale  and further dichotomized as high achievement vs. others.The frequency of intense-level physical activities was evaluated asking the following. “In recent 7 days, how many days did you engage in physical activities  for more than 20 minutes that made you sweat or pant heavily?” The scales were given from 1(none) to 6(more than 5 days a week). Those who engaged in moderate level physical activities for more than five times a week or those who engaged in intense level physical activities for more than three times a week were categorized as ‘physically active’ group , 27.never, 2 = once or twice a week, 3 = three or four times a week, 4 = five or six times a week, 5 = once a day, 6 = twice a day, and 7 = three times or more a day. Junk food consumption was dichotomized as infrequent consumption (≤2 times/week) vs. frequent consumption (≥3 times/week). Lifetime alcohol use was assessed with the following question: “Have you ever used alcohol?” The response options were “yes” or “no.”Frequency of junk food consumption during a previous week was assessed on a seven-point scale where 1 = The degree of sleep dissatisfaction was measured with the following question: “In the last week, how satisfactory was your sleep in terms of relieving your fatigue?” The response options were very satisfactory (1), satisfactory (2), average (3), unsatisfactory (4), and very unsatisfactory (5). On the basis of the responses, participants were classified into the following two groups for multivariate logistic regression analyses: ≥ average satisfaction with sleep (1–3) and (ii) < average satisfaction with sleep (4–5) .The degree of perceived stress was measured as following: “Usually, to what degree are you stressed? The answers ranged from 1) not at all 2) not so much 3) a little bit 4) quite much 5) very much .The depressed feeling experience in the recent 12 months was measured as following: “Have you experienced sadness or despair to the degree that stopped your daily routine for two weeks?” .Suicidal ideation was evaluated with the following question: “In recent 12 months, have you ever thought of committing a suicide?” Suicide plan was evaluated with the following question: “In recent 12 months, have you planned a suicide in detail?” Suicide attempt was evaluated with the following question: “In recent 12 months, have you ever attempted a suicide?” .Univariate logistic regression tests were performed to compare sociodemographic and lifestyle factors between groups . Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated using energy drink intake (highly or moderately frequent intake) as the predictor and each sociodemographic and lifestyle factor as the main outcome variables.Next, to analyze the associations between the adolescents’ energy drink intake and mental health variables, multivariate logistic regression tests were performed using energy drink intake as the principal predictor and each mental health variable as the main outcome variables, after controlling for age, gender, school type, area of residence, residential type, level of academic achievement, level of physical activity, and junk food and alcohol consumption.Next, in order to further examine the nature of the relationships observed, the variables for frequency of energy drink and junk food consumption were combined so that all four possible groupings of frequent/infrequent intake could be investigated in relation to the dichotomous mental health outcomes, using binary logistic regression analysis. Because consuming energy drinks less than once a week and junk food less than three times a week were considered to be the healthiest dietary practices, the infrequent junk food/energy drink condition was chosen as the comparison group. If the confidence intervals were found not to overlap, then the difference between groups was considered to be significant.All analyses took into account the sampling design parameters, weighting, clustering, and stratification factor. The proportion of general subject characteristics was weighted according to the respondent’s probability of being selected for the sex-, grade-, and school type-specific distributions for the region , 26. SPSThe estimated prevalence of highly frequent energy drink intake (≥5 times a week) was 1.4 %, and prevalence of moderately frequent energy drink intake (1–4 times/week) was 10.5 %. Compared to those who had energy drinks less than once a week, those who had energy drinks 5 times or more per week were more likely to be male (OR = 1.79), attend high school (OR = 1.21), live in a rural area (OR = 1.19), achieve higher academic performance (OR = 1.35), eat junk food (OR = 5.70), drink alcohol (OR = 1.35), be physically active (OR = 1.43), and not to live with their family (OR = 3.73). Those whose energy drink intake frequency was between 1–4 times per week showed similar sociodemographic and lifestyle characteristics as those whose energy drink intake was 5 times or more per week, except that they were less likely to achieve higher academic performance compared to those whose energy drink intake was less than once per week (OR = 0.87) , severe stress (AORs = 2.23 and 1.38), depressive mood (AOR = 2.59 and 1.51), suicidal ideation (AORs = 3.14 and 1.43), suicide plan (AORs = 4.65 and 1.78), and suicide attempt (AORs = 6.79 and 1.91), compared to participants who used energy drinks infrequently or did not use energy drinks, with higher AORs for highly frequent use of energy drinks than for moderately frequent use of energy drinks , severe stress , depressed mood , suicidal ideation , suicide plan , and suicide attempt , with the highest AORs for the frequent energy drinks and frequent junk food group followed by the frequent energy drinks and infrequent junk food group and the infrequent energy drinks and frequent junk food group  deficiency associated with frequent junk food consumption predisposes children to behavioral symptoms, such as hyperactivity, delinquency, and aggressive behaviors –53. In pThis study has several limitations that deserve discussion. First, due to the cross-sectional design of this study, causal relationships between energy drink intake and mental health problems cannot be determined. Given that energy drink consumption did not predict psychiatric problems at 6-month follow up , the assIn conclusion, study findings indicate that adolescents who consume energy drinks regularly are at great risk of mental health problems, particularly when in combination with junk food consumption. Furthermore, the risk of mental health problems was significantly different between highly frequent and moderately frequent consumers. Therefore, the amount of energy drink intake should be carefully monitored, and an educational intervention on the negative effects of energy drinks is needed for adolescents who consume energy drinks regularly.


Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder‐ and xenogeneic‐free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock‐in lines (LMX1A‐eGFP and PITX3‐eGFP) for in‐depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this “next generation” protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno‐free vmDA progenitors from LMX1A‐ and PITX3‐eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC‐derived neurons into the clinic. S The authors report the generation of highly pure midbrain dopamine cultures under feeder‐free, fully defined, and xeno‐free conditions from human pluripotent stem cells. Xeno‐free differentiated cells display gene, protein, and electrophysiological properties of midbrain neurons, as well as improved grafting outcomes in Parkinsonian rodents, observations enhanced by the use of two novel reporter lines of interest to this research field. Furthermore, for the first time, ventral midbrain dopamine neurons were amenable to scalability and cryopreservation, crucial steps for the advancement of cell replacement therapy in Parkinson’s disease.Clinical trials of human fetal tissue grafts in Parkinson’s disease patients provide evidence that donor neural cells can reinnervate appropriate targets, increase dopamine neurotransmission, and reverse phenotypic symptoms for decades after transplantation . Howeverhttps://www.thermofisher.com) or laminin, B27 supplement, and knockout serum replacement (KSR) media ) from D5 to D11. Sonic hedgehog C25II  and the smoothened receptor agonist purmorphamine  were added from D1 to D7 to promote ventralization. GSK3β inhibitor CHIR99021  was added from D3 to D13 to promote caudalization. At D11, media were switched to NBN2B27 , 1% ITS‐A, 0.5% GlutaMAX, and 0.4% penicillin‐streptomycin) with recombinant human brain‐derived neurotrophic factor , recombinant human glial cell line‐derived neurotrophic factor , ascorbic acid , recombinant human transforming growth factor type β3 , dibutyryl cAMP , and the notch inhibitor DAPT , henceforth known as “maturation media.” At D17, cultures were dissociated with Accutase  and seeded onto surfaces coated with human laminin‐521 or poly‐L‐ornithine  and mouse laminin , and fibronectin . Maturation media were maintained until desired end point.Differentiation to vmDA neurons was performed as described previously  with modhttp://www2.grantinstruments.com).vmDA neural progenitor cells (NPCs) were collected after 22 days of differentiation (without passage) using EDTA for 5 minutes at 37°C to generate a cell suspension composed of 10 to 200 cell clusters. Cells were resuspended in maturation media and mixed 1:1 with a xeno‐free cryopreservation solution  and immediately transferred to a slow rate freezer EF600M .Cells were fixed in 4% paraformaldehyde for 7–10 minutes and antibody staining performed as previously described . Images Cells were dissociated with Accutase  and stained with primary antibodies  according to previously described methods . ApproprTotal RNA was extracted at D0, D11, D25, and D40 using Trizol. RNA was converted to cDNA and subsequently analyzed using quantitative real‐time polymerase chain reaction (qPCR) for six genes of interest  using previously described methods . All qPCDopamine and the metabolite homovanillic acid (HVA) levels were measure in xenogeneic and xeno‐free cultures at D40 using reverse phase liquid chromatography with electrochemical detection, as previously described , 22. Datn = 21) at D55–D65 using previously described methods . Us. Us‐ andOngoing differentiation promoted the expression of the rate‐limiting enzyme in dopamine synthesis, TH, within both xenogeneic and xeno‐free cultures by D25 –2F. Grea+ populations. To this end we took advantage of our recently generated H9::PITX3‐eGFP knockin reporter line [+ cells were found to coexpress PITX3+ . The increased scale had no obvious effect on the survival, morphology or expression of LMX1A‐eGFP in culture , 4B, com50 flask , with 8050 flask , 4F. vmDentified –4Dii.In addition to the necessity for scalability for clinical translation, we examined the feasibility of cryopreserving vmDA precursors at a stage in the differentiation likely to be amenable to cell transplantation. Such approaches could enable large‐scale standardized differentiation and storage for “on‐demand” needs as reported in other hPSC fields . Scaled + neurons  and cells coexpressing FOXA2+/TH+  resulted in a significant increase in the proportions of TH3 and TH –5Q. ExamCritical for the function of DA neurons is their ability to synthesize and utilize dopamine. To this end, we assessed dopamine metabolism in KCl‐depolarized neurons using high‐performance liquid chromatography (HPLC). Reflective of increases in DA neurons observed at D40, dopamine levels were significantly elevated in both H9 hESC cultures differentiated under xeno‐free conditions compared with xenogeneic cultures  as well as hiPSC RM3.5 cultures  exhibited a resting membrane potential of −62 ± mV, with 38% firing spontaneous action potentials of prolonged duration . Examination of GFP staining however revealed that grafts derived from xeno‐free differentiation cultures contained notably more LMX1A+ progenitors than grafts of xenogeneic‐derived cells  at the transplantation window. The significance of these findings for clinical purposes can be fully realized in the context of preceding clinical trials using fetal tissue where as few as 100,000 surviving DA neurons were sufficient for significant reinnervation of the caudate putamen, F‐DOPA uptake and functional improvements , 37, 38.Our findings address a number of critical stepping‐stones in the advancement of hPSCs for clinical applications. This readily scalable, cyropreservable, and defined xeno‐free protocol, primed for clinical translation, provides a highly efficient and standardized system that has been acutely interrogated with novel LMX1A‐ and PITX3‐eGFP reporter lines. These findings are highly relevant to a wide variety of downstream applications, from in vitro disease modeling and drug screening, to the rapidly approaching aspiration of cell replacement therapy for patients with Parkinson’s disease.J.C.N. and C.W.G.: conception and design, provision of study material and/or participants, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; W.F.A.: collection and/or assembly of data, data analysis and interpretation, final approval of manuscript; S.J.M. and C.R.B.: collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; A.G.E. and E.G.S.: conception and design, provision of study material and/or participants, manuscript writing, final approval of manuscript; J.M.H. and C.W.P.: provision of study material and/or participants, final approval of manuscript; L.H.T.: conception and design, manuscript writing, final approval of manuscript; C.L.P.: conception and design, financial support, provision of study material and/or participants, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.C.W.P. is a consultant for iRiccorgPharm Pty Ltd. and has research funding from Capsugel. The other authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


Bacterial nanocellulose (BNC) has desirable properties for wound healing such as high purity, good shape retention, and high water binding capacity. Bromelain is a protease found in pineapple tissues and has been applied in several fields, it has anti-inflammatory and anticancer properties, promotes cell apoptosis, amongst others. In this work, a BNC based device for the controlled release of bromelain was developed. BNC were submersed in sterilized bromelain solution and incubated at 25 °C under 100 rpm for 24 h. Physical-chemical properties, protein concentration, antioxidant and antimicrobial activities were measured. Results demonstrate that BNC could improve bromelain antimicrobial activity 9 times. Those findings allow concluding that bromelain is a promising molecule to be incorporated into BNC’s. The BNC’s characteristics seem to represent a new promising delivery system of the loaded biomolecule, and protected from external actions. The most abundant biopolymer is cellulose, which is produced by a variety of organisms and constitutes the basic structure of cells walls of almost all plants, many fungi and some algae7.Current advances in biopolymer research demonstrates their potential for a wide range of applications in pharmaceutical and medical fields, including the development of drug delivery systems, wound healing, scaffolds for tissue engineering, novel vascular grafts, and dressing for skin burns and wound healingGluconacetobacter xylinus, among other bacteria of genera Gluconacetobacter, Agrobacterium Rhizobium, Pseudomonas, Sarcina, and Acetobacter10. Unlike vegetable cellulose, BNC is produced in pure form, free of other polymers. In addition, it can be molded into three-dimensional structures, able to retain high water amounts, mechanically resistant and biocompatible11. BNC has a nanofibrilar structure, making it an ideal matrix for use as medical devices, either as an aid in healing skin lesions3 or in tissue engineering, assisting cell regeneration13.Bacterial nanocellulose (BNC) is an extracellular polysaccharide secreted mainly by 14. Nisin-containing BNC films were effective in controlling L. monocytogenes15, while BNC containing sorbic acid was used as a food packaging material with an ability to controlled release the antimicrobial agent16.The scope of applications of BNC can be further expanded through its association with bioactive molecules. For instance, it has been shown that the incorporation of antimicrobial agents into BNC membranes yielded an active packaging systemAnanas comosus) tissues such as stem, fruit and leaves, and has widespread applications, such as in the cosmetic and food industries, as well as in medicine19. In healthcare, bromelain has been used for the treatment of rheumatoid arthritis, thrombophlebitis, hematomas, oral inflammation, diabetic ulcers, rectal and perirectal inflammation, wounds, inhibition of platelet aggregation, angina pectoris, bronchitis, sinusitis, surgical traumas, and pyelonephritis, and for enhancing the absorption of drugs, particularly antibiotics22.Proteolytic enzymes could be used as therapeutics adjuvants, due to their debridement and antimicrobial activities, facilitating tissue renewal process and accelerating ulcerated skin healing. Bromelain is a set of proteolytic enzymes found in pineapple  over a period of 60 minutes at 37 °C. Additionally, the bromelain release rate could be controlled through the selection of the hydrophobicity of the polymers.Bernkop-Schnürch, 19, when incorporating bromelain into alginate and Arabic gum hydrogel. According with suppliers, commercial steam bromelain purity is 35%, and it contains other components with high molecular weight29, which may be not incorporated by BNC. Thus, it is possible to infer that BNC, as well as the alginate and Arabic gum based hydrogel19, selectively absorbed low molecular weight bromelain components, which present higher enzymatic activity.After the first 30 minutes, released bromelain amount was higher than the incorporated amount, for protein concentration and enzymatic activity Fig. . The sam30. However, no relevant changes in BNC network was observed when bromelain was loaded, which was similar to previous results reported by Müller, et al.31 and Moritz, et al.32.SEM analyses, done at 10 kx magnifications, indicated that the BC membranes presented an entangled structure, with void spaces randomly distributed throughout the membrane matrix Fig. . The hig−1, relative to the C grouping=O. The bromelain bacterial cellulose membrane showed a vibration displacement at 1649 cm−1 related to the C=O band. The C=O stretching group corresponds to the amide group from asparagine, glutamine amino acids or residual proteins of BCN membrane. The stretching in 1612 cm−1 can be attribute at N-H from amide I group of bacterial cellulose protein33. The aliphatic amine (1236 cm−1) is observed at 1219 cm−1 on the bromelain-containing bacterial cellulose membrane33. The stretching in the range of 3200–3800 nm−1 for bromelain (a), BCN membrane (b) and BCN with bromelain (c) is corresponding to -OH group. The decrease in OH stretching is related to the formation of hydrogen bonding between immobilisation of bromelain that occurs through the establishment of hydrogen bonding between -OH groups on the surface of -NH2 of BNC membrane19. Bromelain interaction with BCN membranes may also be attributed to hydrophobic interactions or Van der Waals forces, once bromelain contains hydrophobic amino acids exposed on its surface35.Chemical cleavages characteristic of pure bromelain and bacterial cellulose membranes without and with bromelain were determined by Fourier Transform Infrared (FTIR) spectroscopy shown in Fig. For minimal inhibitory concentration (MIC), the following solutions were testes: (i) initial bromelain solution; (ii) residual bromelain solution; and (iii) bromelain solution after being released from BCNs. Table It is possible to note that residual bromelain solution presented a decrease in all parameters when compared with initial solution, indicating that BCN membrane was capable to absorb 7.94 mg/mL of proteins, representing 31% of total protein concentration in the initial bromelain solution. It is also possible to observe, an increase in bromelain specific activity in the release solution , which may be related with BCN membrane selectively absorbing bromelain low molecular weight components with higher enzymatic activity.Those three solutions, were used for the minimal inhibitory concentration assay (MIC), Fig. E. coli, bromelain release solution MIC was 0.99 U/mL, 6 times higher than initial solution MIC. The same behavior was observed for P. aureoginosa, where MIC was achieved at 0.25 U/mL in the release solution, 6 times higher than MIC of initial solution 0.04 U/mL, and for S. aureus bromelain release solution MIC was 0.5 U/mL, 12 times higher than initial solution MIC.Results showed in Fig. et al.36, crude bromelain is more effective in inhibiting Gram-positive bacteria growth than Gram-negative. Our results, however, pointed in a different direction, since bromelain inhibit all tested microorganisms, especially for release solution. Dutta and Bhattacharyya23 reported bromelain antimicrobial activity against S. aureus. A bromelain solution with 1.95 mg/mL concentration inhibited S. aureus growth by 50%, however bromelain enzymatic activity was not reported. The same study showed the bromelain concentration of 1.86 and 2.11 mg/mL were responsible to inhibit E. coli XL1 blue and E coli DH5α Pet16b (Amp)r growth, respectively. Our results  and standard bromelain (2 mg/mL) created a 22 mm zone of inhibition of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Streptococcus mutans have been predominantly associated with periodontal diseases. Bromelain was investigated as a potential antimicrobial agent to be used as a monotherapy and as an adjunct with mechanical debridement. Bromelain showed antibacterial efficacy against all the isolated strains of both aerobic and anaerobic microorganisms. S. mutans showed sensitivity at the lowest concentration (2 mg/mL), followed by P. gingivalis (4.15 mg/mL). While A. actinomycetemcomitans demonstrated the higher resistance and a MIC of 16.6 mg/mL37.For agar diffusion modified assay, bromelain-loaded BNC membranes showed antimicrobial activity for all tested microorganisms Fig. , differe38. Bromelain antioxidant activity was measured against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, which provides an easy and rapid way to evaluate antiradical activities39.Proteases enzymes, such as bromelain and papain, present antioxidant activity as free radical scavenging and lipid peroxidation inhibitionFigure Both bromelain at 30 and 15 mg/mL were statistically similar to glutathione (GSH), antioxidant standard (0.150 mg/mL), initially and after 15 minutes. However, over time, bromelain at 15 mg/mL showed a statistically lower antioxidant activity than GSH (p < 0.05) and bromelain at 30 mg/mL (p < 0.05). It is important to observe that antioxidant capacity of bromelain increased over time even in the lowest concentrations, a behavior also seen in GSH. This feature is relevant mainly when considering a formulation for topical application for example, in which the antioxidant activity remains and even increases after its application.40, effects on cardiovascular diseases41, modulation of inflammatory system42 and skin debridement properties43. Among them, the latter two are very convenient when associated to the antioxidant and antimicrobial capacity found in this study, once these four properties are essential in skin lesions, as a wound healing.Several studies have been reported about bromelain and its medical use, such as inhibition of platelet aggregation and fibrinolysis44. In this way, mucoadhesion analysis evaluates the interaction between the membrane and the surface of the mucosa at a temperature of 37 °C46.Mucoadhesive property was evaluated by a tensile test, and is a property related to the ability of a material  to adhere to a biological tissue during a certain period of timeet al.44, mucoadhesion may occur by increasing the viscosity of the system or by molecular interactions, such as ionic interactions or formation of hydrogen (OH) bonds. It is worth mentioning that the immobilization of bromelain in the bacterial cellulose membrane occurs by the formation of hydrogen bonds between OH of bacterial cellulose membrane and bromelain NH247. In this way, one can suggest that the decrease of the mucoadhesive property occurred by the decrease of free OH groups in the membrane of bacterial cellulose containing bromelain, results evidenced in the FTIR analysis.Analysis of the results shows that maximum strength values significantly (p < 0.05) reduced from 0.493 ± 3.6 N to 0.112 ± 0.1 N, respectively for bacterial cellulose membrane without and with bromelain. According to Yadav, BCN membranes were able to selectively absorb and release bromelain. Bromelain itself present antimicrobial and antioxidant activities, however, after incorporated into BNC membranes, its antimicrobial activity increased. BNC membranes presented mucoadhesive properties, which decreased after bromelain loading. This can be explained by the presence of H-bonds between bromelain and BNC membrane, leading to a decrease in molecular interactions between BNC and mucin. The broad bromelain solution and BNC membrane antimicrobial spectrum and their tissue adhesion make them an alternative method for wound contamination control, since the membrane may act as platform that delivers antimicrobial gradually avoiding biofilm formation. Thus, bromelain-containing BNC membrane proved to be a promising drug delivery system to minimize healing period, reducing the risk of infection and promoting patient comfort and pain relief.Bromelain, Folin-Ciocateau and bicinchoninic acid assay kit were purchased from Sigma-Aldrich (Brazil). Other reagents were of analytical or food grade. Assays were performed in triplicate.Gluconacetobacter xylinus (ATCC 53582) cultivation in Hestrin & Schramm medium . Microorganism growth in 24-well cell culture plates, containing 1 mL of a 106 CFU/mL inoculum per well.Bacterial nanocellulose membranes (BCN membrane) were produced through Plates were kept for 4 days in static culture at 30 °C, yielding 2 mm thick membranes. After growth, membranes were washed in 2% sodium dodecyl sulfate (SDS) solution overnight, rinsed with distilled water until SDS removal, and immersed in 1 M NaOH solution with stirring at 60 °C for 1 and a half hours. After this period, membranes were washed again until reaching neutral pH. Then they were packaged and autoclaved at 121 °C for 15 minutes in Milli Q water, and stored at 4 °C Fig. .Figure 8®50. Briefly, the method consists in casein cleavage by bromelain for 10 minutes at 37 °C and pH 7.5 and the reaction was stopped by adding trichloroacetic acid (TCA). The resulting solution was centrifuged at 4,000 g for 20 minutes, and a supernatant aliquot placed to react with Folin-Ciocalteau, in the presence of sodium carbonate. The reaction of Folin primarily with tyrosine produces free chromophore with a blue color, which is measured by absorbance analysis at 660 nm. The standard curve was drawn with L-tyrosine. Protein concentration was determined with bicinchoninic acid assay51.Enzymatic activity was determined by adapting the non-specific protease activity assay from SigmaIn 24-well plates, BNC membranes were disposed and 1 mL of 30 mg/mL bromelain solution in citrate-phosphate buffer (pH 5.0) was poured into the plate. In the fourth well only bromelain solution was added. Plates were kept in 100 rpm orbital shaker at 25 °C for various periods ranging for 4, 8, 12 and 24 hours. After each period, supernatants were collected and bromelain activity and protein quantification were evaluated in the isolated residual solutions.This methodology was used to assess bromelain release from BNC membranes. In 24 well plates, membranes loaded with bromelain (after 24 hours loading time) were disposed in triplicate, with a negative control (membrane without bromelain) in the fourth well. In each well 1 mL of citrate-phosphate buffer (pH 5.0) was added. Plates were placed in 100 rpm orbital shaker at 25 °C. At different periods , membranes and supernatant were collected. Bromelain activity and protein quantification were assayed in the isolated released solution.BNC membranes structure was observed using a Leo 440i scanning electron microscopy with 6070 X-ray dispersive energy detector . SEM images were obtained using an accelerating voltage of 15 kV. Before analysis, samples were lyophilized for 24 hours and coated with gold (92 A°) using SC7620 Sputter Coater POLARON .−1 wavelength range after 64 scans, with a resolution of 4 cm−1. The spectra were normalized and the vibration bands were associated with the main chemical groups.The free bromelain and bacterial cellulose membrane with and without bromelain were analysed using Fourier Transform Infrared Spectroscopy (FTIR)  using transmittance modes. Membranes were kept in an oven at 30 °C, after drying they were ground. Approximately 2 mg of the sample was mixed with 300 mg of KBr to form the pellet. Spectra were obtained in the 4000 to 400 cmEscherichia coli (ATCC 25922) and Pseudomonas aeuroginosa (ATCC 9721), representing the class of gram-negative; and Staphylococcus aureus (ATCC 10390), representing the class of gram-positive.In this assay, the microorganisms used were 53, using (i) initial bromelain solution; (ii) residual bromelain solution; and (iii) bromelain solution after being released from BCNs.Minimum inhibitory concentration (MIC) classical method of successive dilution was adapted to 96-well plates, in triplicate55. Membranes were placed in 24-well plates with 1 mL of culture medium and 100 μL of the microorganism suspension (106 CFU.mL−1), and plates incubated for 24 hours at 37 °C. After this time, the resulting membrane and solution were inoculated intro Petri dishes containing agar of the culture medium.The agar diffusion assay was modified to verify qualitatively the antimicrobial activity in BCN membranes itself and after bromelain loadinget al.39.Antioxidant activity was determined based on absorbance decrease at 515 nm after the reaction between radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and initial bromelain solutions, according to Brand-Williams, −1. The force required to separate the surfaces was determined by time versus force ratio (n = 3), and analysis parameters were described on Table Mucoadhesive strength of the bacterial cellulose membrane with and without bromelain was evaluated by the force required to remove the membrane from a mucin disc using the TAXTPlus texture analyzer . Samples were fixed in a water bath with a temperature set at 37 °C. The mucin discs were prepared by compression . The surface of the mucin disc was hydrated and the disk attached to the lower end of the analytical probe. Mucin disc was compressed apical → basal on the surface of the samples, with a compression force of 0.1 N. The contact time of the disc with the surface of the samples was 200 s. Removal of the analytical probe was at the rate of 0.5 mm.sData is presented as mean ± standard deviation. Differences among bromelain concentrations were evaluated by one-way ANOVA, followed by Tukey multiple range tests. P values < 0.05 were considered significant. Data were analyzed using Statistica®8.0 .Supplementary Information


Severe combined immunodeficiency (SCID) is fatal unless treated with hematopoietic stem cell transplant. Delay in diagnosis is common without newborn screening. Family history of infant death due to infection or known SCID (FH) has been associated with earlier diagnosis.The aim of this study was to identify the clinical features that affect age at diagnosis (AD) and time to the diagnosis of SCID.From 2005 to 2016, 147 SCID patients were referred to the Asian Primary Immunodeficiency Network. Patients with genetic diagnosis, age at presentation (AP), and AD were selected for study.IL2RG mutations, 12 RAG1 mutations, 8 RAG2 mutations, 7 JAK3 mutations, 4 DCLRE1C mutations, 4 IL7R mutations, 2 RFXANK mutations, and 2 ADA mutations. A total of 29 mutations were previously unreported. Eighty-three of the 94 patients fulfilled the selection criteria. Their median AD was 4 months, and the time to diagnosis was 2 months. The commonest SCID was X-linked (n = 57). A total of 29 patients had a positive FH. Candidiasis (n = 27) and bacillus Calmette–Guérin (BCG) vaccine infection (n = 19) were the commonest infections. The median age for candidiasis and BCG infection documented were 3 months and 4 months, respectively. The median absolute lymphocyte count (ALC) was 1.05 × 109/L with over 88% patients below 3 × 109/L. Positive FH was associated with earlier AP by 1 month (p = 0.002) and diagnosis by 2 months (p = 0.008), but not shorter time to diagnosis (p = 0.494). Candidiasis was associated with later AD by 2 months (p = 0.008) and longer time to diagnosis by 0.55 months (p = 0.003). BCG infections were not associated with age or time to diagnosis.A total of 88 different SCID gene mutations were identified in 94 patients, including 49 9/L.FH was useful to aid earlier diagnosis but was overlooked by clinicians and not by parents. Similarly, typical clinical features of SCID were not recognized by clinicians to shorten the time to diagnosis. We suggest that lymphocyte subset should be performed for any infant with one or more of the following four clinical features: FH, candidiasis, BCG infections, and ALC below 3 × 10 IL2RG), which accounted for 45 and 19% of SCID cases before and after the T-cell receptor excision circle (TREC) newborn screening, and was introduced in USA (Severe combined immunodeficiency (SCID) is a group of genetic diseases causing profound developmental and functional impairment of T cells, affecting cellular and humoral immunities. Currently, at least 49 genes are identified to be responsible for SCID and its variants –3. Of ald in USA , chronicd in USA . PatientThe definitive treatment for SCID is hematopoietic stem cell transplant (HSCT). In addition, gene therapy serves as an alternative for X-linked and adenosine deaminase (ADA)-deficient SCID if suitable HSC donors are not available . SCID paThe Asian Primary Immunodeficiency Network (APIN) is a primary immunodeficiency (PID) referral network established in 2009 by The University of Hong Kong as a platform for consultation and offering free genetic testing for suspected PID in over 70 centers in Asia and Africa. Its database stores clinical information provided by the referring doctors, laboratory results, and genetic test reports –14. FromCandida, Pseudomonas aeruginosa, Acinetobacter baumannii, Pneumocystis jiroveci (PCP), Aspergillus, cytomegalovirus (CMV), and herpes zoster virus. We defined opportunistic infections by Candida if patients were documented to have invasive candidiasis, candidemia or persistent oral candidiasis  and those with B− genotypes   as the age when clinical diagnosis of SCID was made, and time to diagnosis as the duration between AP and AD. We only considered patients to have certain clinical feature if that feature was stated in the referral summary. We defined recurrent infections as more than one episode of infections affecting similar systems. We considered the infection to be severe if at least one of the following was present: life-threatening complications (such as acute respiratory distress syndrome and sepsis), intensive care unit (ICU) admission, and life support being used . We defined opportunistic infection as an infection with at least one of the following pathogens was involved: BCG, didiasis . We preseviously . Since tGenetic analysis was performed in the Department of Pediatrics and Adolescent Medicine of the University of Hong Kong using PCR and direct sequencing  . GeneticU test; multivariate linear regression was performed for all factors that were significant (p < 0.05) in univariate analysis. We defined statistical significance as p < 0.05, and 95% confidence interval did not contain 0 in multivariate analysis. We did not include opportunistic infection group in the multivariate linear regression to avoid multicollinearity.For descriptive statistics, all data were expressed in median and range (month). Univariate analysis was performed using Mann–Whitney Patients with missing categorical data such as clinical features were considered to be without the features. Patients with missing numerical data such as ALC were not analyzed when analyzing median and range.n = 4), the lack of AD (n = 9), and being diagnosed by screening (n = 3). Among the 16 patients excluded from the study, 11 of them had genetic diagnosis. Altogether, molecular diagnosis of SCID was identified in 94 patients in our cohort.From 147 SCID patients referred to the APIN, 131 of them had documented AP and diagnosis. Among these patients, 83 of them had genetic diagnosis Figure . SixteenIL2RG (n = 65), followed by RAG1 (n = 7), RAG2 (n = 7), JAK3 (n = 5), DCLRE1C (n = 4), IL7R (n = 3), RFXANK (n = 2), and ADA (n = 1). Eighty-eight different mutations were identified in this study . There was no difference in clinical features between X-linked and autosomal recessive SCID patients .The genetic mutations of the 83 SCID patients included in our study and 11 SCID patients excluded from our study are shown in Table IL2RG mutation. c.104G>T mutation was observed in three unrelated patients with RAG2 mutation.Genetic mutations in all SCID genes were not evenly distributed, and two mutations were seen three or more times in unrelated patients. c.854G>A mutation was seen in six unrelated patients with IL2RG mutations, 5 RAG1 mutations, 4 RAG2 mutations, 3 JAK3 mutations, 1 IL7R mutation, and 1 ADA mutation). These mutations accounted for 25% of all mutations and were involved in 31 patients .Twenty-two C>T or G>A mutations within CpG dinucleotides were documented . Effects of these unreported mutations on protein functions were predicted by multiple tools and are shown in Table SE3 in Supplementary Material.There were 29 unreported mutations identified in our patients, including 23 n = 131) are shown in Tables n = 83), 88.0% were male (n = 73) and 75.9% were Chinese (n = 63). The median AP was 2 months (0.1–6 months), AD 4 months (0.5–18 months), and time to diagnosis 2 months (0–14 months). Twenty-nine patients (34.9%) had a family history of early infant death (FH), among them one patient had a family history of SCID and one patient had a family history of PID. Parental consanguinity was present in four kindreds. The median ALC was 1.05 × 109/L  with 88.6% below 3 × 109/L (n = 62). The major immunophenotype was B+ SCID (n = 51) (Tables Characteristics of patients included in our study n = 11 are shon = 42) and 60.2% of patients recurrent infections (n = 50). The commonest site of infection was the respiratory system (n = 61), followed by gastrointestinal system (n = 42). 47.0% of infections were severe (n = 39).50.6% of patients presented with chronic diarrhea (n = 27), followed by BCG infection (n = 19) and viral infection (n = 9). The median age for candidiasis documented was 3 months, the median age for BCG infection was 4 months, and the median age for CMV infection was 2.25 months.Fifty patients developed opportunistic infection (60.2%). The commonest opportunistic infection was candidiasis (n = 83) and without genetic diagnosis (n = 48). Patients without genetic diagnosis had higher frequency of FTT  and CMV infections  , 107 of them (93.9%) had at least one of the following four clinical features: FH, candidiasis, BCG infection, and ALC below 3 × 109/L. 65 of them (57.0%) had at least two of the four clinical features mentioned.For patients with documented ALC . Upon multivariate analysis, only FH and pneumonia were associated with earlier AP .In univariate analysis, FH, pneumonia, and recurrent infections were associated with earlier AP . Upon multivariate analysis, only FH was associated with earlier AD .In univariate analysis, FH and CMV infections were associated with an earlier AD . Upon multivariate analysis, only candidiasis was associated with later AD .In univariate analysis, candidiasis, FTT, opportunistic infections, and BCG infection were associated with a later AD . Upon multivariate analysis, both candidiasis and recurrent infections were associated with longer time to diagnosis .Candidiasis, opportunistic infections, and recurrent infections were shown to be associated with longer time to diagnosis  identified in our study, which was at a lower frequency when compared to that of 45–57% reported previously , 29, 30.The median age for BCG infection documented in our patients was 4 months, which was beyond the optimal time for HSCT. Our findings were in line with a previous report in which 74% of 349 BCG-vaccinated SCID patients developed BCG infection at or after 4 months of age . TherefoWe found that opportunistic infections were associated with later AD, while recurrent infections and opportunistic infections were associated with longer time to diagnosis. Therefore, such clinical features were likely the consequences of delay in the diagnosis of SCID, reflecting that clinicians in Asia were unable to recognize these as SCID features.We found that pneumonia was associated with an earlier AP but did not affect AD and time to diagnosis. Therefore, parents may perceive pneumonia as a severe medical condition and then brought their children to seek medical care earlier. However, pneumonia also commonly affects children without SCID in Asia –42, and 9/L were not associated with AD and time to diagnosis, likely due to the distributions of the AD and the time to diagnosis that were quite wide in patients with these features . The low rate of documented CMV infections may be due to the lack of diagnostic capacity , which was comparable with previous study  reported previously in Chinese literature (RFXANK mutations reported in Asia. RFXANK mutation causes bare lymphocyte syndrome type 2B (We reported two patients (P091 and P092) with terature . These p type 2B , commonl type 2B  and some type 2B . MHC cla type 2B –62, whil type 2B  compared type 2B , as well type 2B , 64. The type 2B , 65. How type 2B .The care of patients with SCID in Asia is still at an early phase of development, as reflected by delay in diagnosis and suboptimal management with no easy access to HSCT , 14. Eig9/L, and reaffirming lymphopenia is a feature commonly seen in SCID patients .ADL, PL, and YL designed the study; ADL and YL wrote the manuscript with extensive appraisal from PL, K-WC, and HM; YL, K-WC, WW, and ADL analyzed the data; K-WC, WY, and WT performed the genetic and immunological studies; PL, HM, YL, XC, T-XC, JH, NK, DS, YT, YX, LJ, WL, OJ, TD, AG, SS, AR, AHL, ACL, LS, TN, TC, YC, ZL, TML, NL, BL, QL, DR, M-RB, M-KT, MA, XW, CX, HY, H-HY, TLL, FY, PC, and MH contributed clinical data and took care of patients in this study. PL and YL established the Asian Primary Immunodeficiency Network (APIN).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.


Nephrops norvegicus is a commercially valuable fishery in the EU but management of stocks is challenging due to difficulties in aging individuals and calculating growth and biomass production. Growth of N. norvegicus was estimated by releasing 1177 tagged individuals in western Ireland in Summer 2013 and recapturing these in 2014  and 2015 . Moulting occurred twice per year in approximately half of the males and only once in females. Mean growth increments after approximately one year were 5.1 mm Carapace Length (CL) in males and 1.4 mm CL in females. After two years, males had grown by 12.0 mm CL and females by 4.6 mm CL, on average, across size classes. Low variation in growth increments was seen across female size classes, but significantly lower growth was observed in larger males, meeting an important assumption of the Von Bertalanffy Growth Function. Asymptotic carapace lengths were 70.8 mm  and 55.2 mm  with respective growth constants (k) of 0.161 yr−1 and 0.077 yr−1. The results suggest that this is a very productive fishery and that survivability of returns from creel fishing is high. Nephrops norvegicus, is one of the most commercially important demersal species in the Northeast Atlantic, with increased landings over the past 50 years1The Norway lobster, N. norvegicus, is unresolved due to a lack of verified aging criteria and the difficulties in measuring growth in moulting species. Previous growth studies on N. norvegicus have mainly employed indirect methods for estimating growth; for example, identifying cohorts within length frequency distributions and determining the rate of their progression over time  as well as frequency of moulting, which is important since smaller individuals may moult more frequently than larger ones per unit timeN. norvegicus on the Irish west coast via tagging with substantially increased recapture rates compared with most previous studies. The study population was located inshore, away from the trawling grounds and was generally lightly-exploited. To maximise tagged returns, the experimental area was left undisturbed by local creel fishermen (apart from scientific fishing) for >24 months of this study. Tagged returns provided size-specific growth averages and this information was used to generate growth descriptors for wild N. norvegicus, as well as to evaluate assumptions of a standard growth model . The frequency distribution of moult increments was used to estimate moult frequency in wild individuals and this parameter was also monitored in captivity. The importance of resolving uncertainties about growth is all the more critical since several functional management units  are overexploited; for example, N. norvegicus in FU15 (Western Irish Sea), FU17 (Aran Grounds), and FU19 (SE and SW Ireland), leading to separate management measures being proposed in these areas6N. norvegicus in certain circumstances6815The present study investigated the growth of N. norvegicus were recaptured in year one at Clew Bay in the west of Ireland  (captive = 1.00 (1.00–2.00) mm CL, medianwild = 1.40 (0.20–4.20); Mann-Whitney U = 2732.5, nwild = 100, ncaptive = 63, P = 0.154)  or November and December  . Growth < 0.001) . Growth = 0.154) .Analysis of growth increments in the wild was broken down by year. In year one, the mean number of days that males were at liberty was 344 ± 3.4 standard error mean (S.E.), during which time mean growth was 5.1 ± 0.2 mm CL. Females were at liberty for an average of 344 ± 2.5 days, with mean growth of 1.4 ± 0.1 mm CL. Mean growth within size classes  showed tSubstantial growth increments were seen in individuals which were at liberty for two years of growth i.e. 654 ± 6 days in males and 665 ± 6 days in females . LargestN. norvegicus were captured between April and September 2014 by creel fishing . The overall modal size for the total catch was identical for males and females, whereas mean size was larger in males (40.2 ± 0.2 mm CL) than females (37.6 ± 0.1)  . Monthlyther sex . There wther sex .∞ was estimated separately in males and females using the Powell-Wetherall method for N. norvegicus captured between April and September 2014 . Based on the mean size distribution of individuals in Clew Bay, males were considered to be fully recruited to the fishery at a length of 38 mm CL and females also recruited at a length of 38 mm CL. The Powell–Wetherall analysis estimated asymptotic carapace lengths, L∞, in N. norvegicus to be 70.8 mm CL for males and 55.2 mm CL for females  or unreadable tags (n = 1) were omitted from these plots, which resulted in an overall n = 99 males and n = 100 females. For males, k was estimated to be 0.157. For females this growth rate was considerably lower at 0.067. The Gulland and Holt plot calculations were repeated for year two growth . Higher values of k were seen in 2015, i.e. 0.192  and 0.117 . Pooling both years, k was estimated to be 0.161 in males and 0.077 in females at Clew Bay  progression1718rvegicus  and therrvegicus . Thus itN. norvegicus (unpublished data). In addition, the present study was carried out in inshore areas, where agreements were made with local fishermen to allow the release area to remain ‘fallow’ over the experimental period. Local fishermen were eventually compensated as they were chartered to recover tagged N. norvegicus in their creels the following year. As a result, the recapture rate of tagged N. norvegicus was relatively high in the current study . Most N. norvegicus were recaptured after approximately one year of growth, with a good balance of returns across males (n = 100) and females (n = 107) to provide robust growth estimates for both sexes.On the other hand, tag-recapture is difficult for more practical reasons because tagging can result in issues arising from repeat handling of animals, interference with the normal range of movements or moulting, and this type of study has traditionally shown a low return rate of tagged individuals . Although the relatively low numbers of returns in this case means that these data are likely to suffer from small sample artefacts, again we observed that the smallest size classes of males and females showed the largest growth increments after two years of growth. Overall, the growth of Nephrops appeared to conform to assumptions that permit use of the VBGFN. norvegicus are somewhat contradictory. Although, like us, others have found decreased growth increment with increased starting size25et al.19It was apparent that mean annual growth increments were much larger in males (5.1 ± 0.2 mm CL) than in females (1.4 ± 0.1 mm CL). In females, growth increment was reasonably uniform within and across size classes apart from the smallest size class (<28 mm CL), which had a much bigger increment . The traN. norvegicus at this latitude1320293032The second major element of growth is the question of moulting frequency. The observed changes in size demonstrated that annual moulting occurred in almost all wild individuals over the size range examined (26–40 mm CL). Zero or negative growth was rarely observed in this group. In fact, almost half of the males moulted twice , which hN. norvegicus females (April and May). Moulting in wild females also occurs in April and May, since this is the period immediately following spawning of their embryo masses at Clew Bay . Females not bearing eggs are free to moult outside of this period, however. Moulting in captive males mainly occurred in November and December, but was not confined to this period. The majority of male moulting in the wild appears to occur over an extended period from Spring-AutumnBoth moulting frequency and increment at moult were much reduced in captive individuals, particularly in males. Reduced growth in captivity has been shown in numerous studies9∞ or growth rate k) lead to drastic changes in the biological reference points for fisheries. Depending on which growth parameters are used, the management advice could either advocate increased or reduced fishing effort. It is recommended to calculate growth rates (k) independently of L∞, as in the present study∞ for males in Clew Bay, Co. Mayo in the west of Ireland was 70.8 mm CL. Since the largest male encountered during an independent fishing study at Clew Bay in 2015 was 68.0 mm CL, i.e. less than theoretical maximum L∞, this provides confidence in our L∞ estimation. This value was similar to an estimate of 72.9 mm CL from the SkagerrakEven minor changes in values of VBGF parameters (asymptotic length LN. norvegicus stocks on the east coast (Irish Sea) which occur at high-densities and lower-density stocks on the west coast∞ for east coast males was 18% lower (60 mm CL) compared to the west  in inshore grounds. The average weight of a single individual tagged in 2013 was 0.029 kg, so that 35 individuals produced ~1 kg of whole product. In 2014, the average weight of a single tagged N. norvegicus increased to 0.039 kg, with only 25 individuals required to produce ~1 kg of product. Therefore the average increase in weight per individual was equivalent to progression into a new size-grading categoryN. norvegicus in inshore areas and potentially seeding with stock from trawling grounds. Growth in the wild after two years was at a slightly higher rate than after one year . Although emergence behaviours along diel, tidal and other rhythms strongly influence catchability of N. norvegicus in trawling gear3637N. norvegicus during fishing operations. Once arrived at the aquaculture facility, animals were housed in individual plastic tubes, which were stacked by attaching the tubes to a vertical support within large tanks (5 m3) in a flow-through aquaculture system (flow rate of 50–65 m3/hour and an average monthly temperature 10–17 °C). N. norvegicus in the system were fed once a week with frozen mussel until the time came to use them in the tag-recapture growth experiment.All experimental animals were captured in Clew Bay (-see below) during April 2014 using static gear (creels or ‘pots’). Creels were baited with salted herring and soaked for 48 hours before being lifted and examined for rd abdominal segment of N. norvegicus . At recapture, it is necessary to dissect out these tags in order to read the unique sequence under the microscope, thereby identifying individuals. Animals were allowed several days to recover from the tagging procedure prior to release onto fishing grounds.Tagging was carried out using sequential CWTs (Northwest Marine Technology Inc.), which are passive tags that have been successfully employed to tag a range of crustaceans3940N. norvegicus at the release site.Tagged animals were released back to their original capture location in Clew Bay, which is a broad bay on the west coast of Ireland that faces the Atlantic Ocean . The bayN. norvegicus were tagged and released at Clew Bay on three dates in 2013: 5 June, 19 June and 17 July. The releases were conducted at 18–20 m depth, depending on the state of the tide, which varies ~5 m in vertical extent. Tagged individuals were released at three closely-located sites, approximately 30 m apart. The size of individuals varied between 26.9–40.2 mm CL in males and 22.0–44.6 mm CL in females. Tagged individuals were released to the seafloor in a fisherman’s cassette that was placed in a weighted device that sat on the seabed for ~72 hours, allowing individuals to escape at will. Creel fishing for N. norvegicus at Clew Bay is seasonally restricted to Spring and Summer months, but in the present case, the release location was left ‘fallow’ to fishing activity post-release. This ‘closed’ experimental period handled the problem of short recapture times and growth over-estimation, which can occur in animals with periodic growth (moulting) when growth is measured over too short a time period. Creel fishing resumed from April to September 2014 (i.e. approximately one year post-release) and again in June–July 2015 (approximately two years later). Scientific observers were on board during all fishing operations to measure the entire catch (mm CL) and to recapture as many of the tagged N. norvegicus as possible. Tagged individuals were identified due to the steel tag emitting a ‘beep’ after passing animals over a T-wand detector (Northwest Marine Technology Inc.). All tagged individuals were returned to the laboratory for measurement (mm CL) and dissection of CWTs for reading under the microscope.A total of 1177 N. norvegicus. Experimental animals were caught and held in an aquaculture system, as outlined above, and moulting frequency was observed over 12 months in April 2014–March 2015. A total of 232 N. norvegicus were held over size ranges of 26.2–53.0 mm CL for males and 23.5–41.2 mm CL for females. CL measurement was carried out approximately once every four weeks, however no observations were made in two of the months (March and October). Regular checks determined whether N. norvegicus within the holding system had moulted. Moulting was detected either by the presence of shell fragments from the moulted exoskeleton, by a change in the appearance of the individual e.g. a pink/soft shell, or by a sudden change in CL (defined as an increase of >0.5 mm which was sustained over subsequent months).A frequency distribution of the increment (i.e. change in size in mm CL) while individuals were at liberty was generated separately in males and females to investigate the presence of modes in the distribution, and hence, the number of moults. As the moulting frequency cannot be measured directly in the wild, moulting frequency was also observed in captive N. norvegicus. Spearman rank correlation was used to examine a statistical relationship between CL before growth and growth increment. Comparison of growth increment after year one was carried out in wild and captive N. norvegicus using a Mann-Whitney U test. These statistical analyses were carried out using MINITAB 17 Statistical Software Package.Growth increment expressed in both CL and weight (g) was tabulated according to starting size (i.e. size at tagging) in wild ∞, which is the asymptotic length i.e. the length that an individual would reach if it was allowed to grow indefinitely; and k, which is the rate at which L∞ is approached. These quantities are commonly used in growth models including the VBGF∞ and k because of their strong inter-dependence (one quantity implies the other). L∞ was calculated from analysis of length-at-catch distributions obtained during 2014 fishing activity while k was calculated from growth increments obtained from the tag-recapture experiment. The Powell–Wetherall method can be used to obtain an estimate of L∞ using catch data by estimating length classes equal to and above the fully recruited or ‘cut-off’ length. This method uses linear regression of terms in equation , which corresponds to L∞ and ‘b’ the regression coefficient (slope).A mean length sample LThe assumptions of this method are that growth follows the VBGF, i.e. there is a linear decline in growth with increased length. The sample population is also assumed to be in equilibrium (at a steady-state) with constant exponential mortality, no changes in selection pattern of the fishery and constant recruitmentN. norvegicus. A Gulland and Holt plot calculates k based on the length of individuals when they are first measured, the length of individuals when they are measured again, and the difference between these two measurements per unit time. The regression was “forced” using L∞ calculated in the previous analysis, allowing k to be estimated separate from L∞ –see ref. ‘Forced’ Gulland and Holt plots were used to estimate the growth rates k, with data from recaptured tagged All animal sampling was carried out in accordance with relevant guidelines and regulations from the relevant authority . Animals were held in a licensed aquaculture facility. Animal handling and experimental procedures were carried out under supervision of certified personnel , which is in keeping with University Policy.How to cite this article: Haynes, P. S. et al. Growth in Nephrops norvegicus from a tag-recapture experiment. Sci. Rep.6, 35143; doi: 10.1038/srep35143 (2016).


There was no significant difference between δ13CEW and δ13CLW in P. kesiya, while δ13CEW was significantly higher than δ13CLW in P. armandii. For both P. kesiya and P. armandii, δ13CEW was highly correlated with previous year’s δ13CLW, indicating a strong carbon carry-over effect for both pines. The intrinsic water use efficiency (iWUE) in the earlywood of P. armandii was slightly higher than that of P. kesiya, and iWUE of both pine species showed an increasing trend, but at a considerably higher rate in P. kesiya. Respective δ13CEW and δ13CLW series were not correlated between the two pine species and could be influenced by local environmental factors. δ13CEW of P. kesiya was positively correlated with July to September monthly mean temperature (MMT), whereas δ13CEW of P. armandii was positively correlated with February to May MMT. Respective δ18OEW and δ18OLW in P. kesiya were positively correlated with those in P. armandii, indicating a strong common climatic forcing in δ18O for both pine species. δ18OEW of both pine species was negatively correlated with May relative humidity and δ18OEW in P. armandii was negatively correlated with May precipitation, whereas δ18OLW in both pine species was negatively correlated with precipitation during autumn months, showing a high potential for climate reconstruction. Our results reveal slightly higher iWUE in natural forest pine species than in secondary forest pine species, and separating earlywood and latewood of for δ18O analyses could provide seasonally distinct climate signals in southwestern China.Stable isotopes in wood cellulose of tree rings provide a high-resolution record of environmental conditions, yet intra-annual analysis of carbon and oxygen isotopes and their associations with physiological responses to seasonal environmental changes are still lacking. We analyzed tree-ring stable carbon (δ Due to the intense human impact, including large scale forest harvesting and resultant land use changes, the area of natural forests in tropical and subtropical China is remarkably decreasing in favor of secondary forest or plantation . The tra13C, δ18O) are frequently used in environmental research, as they provide a continuous, annually resolved record of environmental conditions and show stronger correlations between tree individuals and environmental variables than radial tree growth  and to estimate differences in water use among different plants  is positively correlated with annual precipitation in western and central India, but a negative correlations with annual precipitation was found in southern India , whereas latewood δ18O was correlated with late monsoon precipitation and RH. Therefore, separate analyses in tree ring earlywood and latewood stable isotopes may shed light on the physiological responses of tree species to seasonal environmental change.The earlywood in tree rings is produced during spring and early summer, while the latewood is produced in late summer and autumn, thus earlywood and latewood may potentially record climatic signals in different seasons . In addiighlands , whereasPinus kesiya Royle ex Gord var. langbianensis is an important type of natural forest replacement and have a wide distribution across southern Yunnan, southwestern China  and oxygen (δ18O) isotope variations in the earlywood and latewood of the two pine species. Our aims were (1) to study whether the earlywood and latewood of the two pine species differed in their δ13C and δ18O and if δ13C in earlywood are influenced by a carry-over effect of carbohydrates assimilated in the previous growing season; (2) to investigate if there exist differences in iWUE of pine species from the secondary forest and the natural forest and their response to elevated CO2; (3) to detect the seasonal climatic signals that control tree ring δ13C and δ18O in earlywood and latewood of the studied species. We hypothesized that (1) natural forest pine would have higher iWUE than the secondary forest pine; (2) tree ring δ18O in earlywood and latewood of both pine species might capture seasonal moisture signals , that earlywood recorded early growing season moisture signal, while latewood recorded autumn moisture signal.The secondary forest formed by dominant stands of rn China . These sin 1970s . Pinus aFigure 1).This study was carried out in a subtropical forest ecosystem of the Ailao Mountains, Yunnan Province, southwestern China. The Ailao Mountains form the major climatic border between regions influenced by the southwestern Asian summer monsoon and East Asian summer monsoon , and harFigure 2A). In January, temperature may drop below 0°C. The investigated forest is a subtropical cloud forest with heavy fog during June to August and November to December. Annual mean precipitation at Jingdong County amounts 1118 mm and mean annual temperature is 18.6°C . Although differing in absolute values, the temporal variations of temperature and precipitation between Jingdong County and ALS are synchronous, therefore the data of both stations are highly correlated . However, RH during the late summer and autumn months shows poor correlation between the two stations , and 36 cores (18 trees) of P. armandii from a natural forest close to ALS . The stem density in the tree layer of the P. kesiya is lower  than those of the natural Lithocarpus forest   exceeds 20 cm  of han 95%) . The samds 20 cm . There aTM 6, Rinntech, Germany). Tree-ring measurements were cross-dated to the calendar year of their formation by growth pattern matching, and statistical tests using the software package TSAP-Win  indicate the reliable quality of the ring-width chronologies.Tree-ring widths were measured with traditional technique under the stereomicroscope linked to a LINTAB digital positioning table with a resolution of 0.001 mm  and latewood (LW) of the annuals rings were cut separately with a razor blade, according to their bright and dark color.We selected cores from each of six 2 solution at 60°C, an 8 h × 5 times process was applied in order to achieve an adequate chemical treatment. The remaining hemicelluloses were extracted with 17% NaOH for 2 h at room temperature. A washing procedure with boiled water was interposed for three times across different steps. Finally, samples were treated once with 1% HCl and rinsed with boiling de-ionized water for three times and transferred from the filter funnels into Eppendorf tubes with 1 ml de-ionized water. The samples were then homogenized by using an ultrasound unit  while laboratory standards were periodically interposed to test analytical replication. The δ13C and δ18O values were referred to International Standards  and their analytical error lie within typically reported analytical precisions δ13C = ±0.15′, δ18O = ±0.3′Alpha-cellulose was extracted by using the Multiple Samples Isolation Systems according to 2 (13C and CO2 concentration (ca) of ambient air during 1956–2004 were obtained from The measured carbon isotope values were corrected for anthropogenically induced trends of a rising atmospheric CO2 . The δ13According to 13Cplant and δ13Cair refer to the δ13C values of the α-cellulose and of atmospheric CO2, respectively. Δ was also showed to be closely correlated with ci/ca by Where δa represents isotopic discrimination occurring during diffusion of CO2 from the atmosphere into the intercellular spaces of leaves with a = 4.4‰; b represents the isotopic fractionation through discrimination that occurs during enzymatic carboxylation with b = 27‰. By combining Equations (1) and (2), the intercellular CO2 (ci) can be calculated. iWUE was calculated according to Where 13C and δ18O chronology for the six individuals of P. kesiya and five individuals of P. armandii were used to investigate the correlations between mean δ13C and δ18O chronologies in earlywood and latewood and climate data. The mean Pearson’s correlation coefficients among carbon and oxygen isotope series of tree individuals within each species (Rbar) were calculated to examine the common climate signal carried by individual series. The expressed population signal  station, in Kunming .The carbon and oxygen isotope series of different individuals were analyzed using the numerical mix method to produce a mean isotope chronology via arithmetic average . To remonal EPS;  was also13CEW and δ13CLW of secondary forest pine P. kesiya showed similar mean values , while δ13CEW was higher than δ13CLW in natural forest pine P. armandii . There was no significant difference between δ18OEW and δ18OLW in P. kesiya , while δ18OEW of P. armandii was higher than that of δ18OLW . For δ13C, the inter-series correlations are higher in earlywood than latewood of both pine species. In contrast, δ18O showed higher inter-series correlations in latewood than earlywood for both pine species. There were significant correlations between δ13CEW and previous year’s δ13CLW in both pine species (Table 1). δ18OEW was positively correlated with previous year’s δ18OLW in P. armandii, while there were no significant correlations between δ18OEW and previous year’s δ18OLW in P. kesiya (Table 1). δ13CEW of P. kesiya vs. δ13CEW of P. armandii, and δ13CLW of P. kesiya vs. δ13CLW of P. armandii were not correlated between two pine species, whereas both δ18OEW and δ18OLW in P. kesiya were positively and significantly correlated with those of P. armandii, respectively (Table 2).Both δ13C on the tree ring δ13C, both δ13CEW and δ13CLW of P. kesiya showed an increasing trend . In contrast, both δ13CEW and δ13CLW of P. armandii showed an increasing trend before 1980 and then remain stable after 1980 with reduced inter-annual variability . The δ18OEW and δ18OLW in P. kesiya showed no significant trends , however, δ18OEW in P. armandii displayed slightly decreasing trends .Despite of removing the impact of atmospheric δFigures 4A,B). There was no significant difference in iWUE between earlywood and latewood in P. kesiya , while the iWUE in earlywood was consistently higher than that of latewood in P. armandii . The earlywood of P. armandii had higher iWUE than that of P. kesiya (P < 0.001). The mean intercellular CO2 concentration (ci) tended to increase with time in both pine species , and ci in earlywood was consistently lower than that of latewood in P. armandii .iWUE in earlywood and latewood of both pine species showed increasing trends from the 1960s to 2010s (13CEW of P. kesiya was positively correlated with monthly mean temperatures (MMT) of the previous year’s July and August, and current July to September (1975–2012), while δ13CLW of P. kesiya was positively correlated with MMT of previous July and December, and current January and July . We found no significant correlations between δ13CEW and δ13CLW of P. kesiya and precipitation. The δ13CEW of P. armandii was positively correlated with MMT from February to May (1958–2011), and negatively correlated with monthly precipitation of current February .The δ18OEW of P. kesiya showed significantly negative correlation with previous October MMT (1975–2012), while δ18OLW of P. kesiya correlated negatively with current September, October, and November precipitation . The δ18OEW of P. armandii was negatively correlated with MMT of previous year’s July to December, and current August and September, and was negatively correlated with previous year’s July and current May precipitation (1958–2011). The δ18OLW of P. armandii was negatively correlated with current September and October precipitation .The δ13CEW of P. kesiya was negatively correlated with November relative humidity (RH) , while while δ13CEW of P. armandii was negatively correlated with previous year’s October and current June RH, and δ13CLW of P. armandii was negatively correlated with previous year’s September and current April RH . The δ18OEW of both pine species were negatively correlated with May relative humidity (RH), while δ18OEW of P. armandii was positively with July RH . δ18OLW of P. kesiya was negatively correlated with previous year’s July and November RH, and δ18OLW of P. armandii was negatively correlated with previous year’s July and August RH .The δ13CEW are closely related with previous year’s δ13CLW in both secondary forest pine P. kesiya and natural forest pine P. armandii, indicating that there was a significant carbon carry-over effect, which is consistent with 13C) in both earlywood and latewood showed low correlations between P. kesiya and P. armandii (Table 2), indicating that the carbon isotope chronologies reflect mainly species-specific environmental signal.We found that δP. kesiya at our site is 30 years., so the carbon isotope series of P. kesiya might potentially be affected by age effects. However, the canopy at our study site had a crown density of ca. 20% only, so air may ventilate freely between stems. Hence, there is no dense canopy that may possibly cause CO2 enrichment of 13C depleted air from soil respiration inside the stand, which is the most responsible factor for age effects in tree-ring 13C  than P. kesiya (0.56‰) , but these amplitudes are lower than those found from high-altitude fir (Abies forrestii) in the nearby Yulong Snow Mountains, southwestern China , indicating that oxygen isotope variations in the earlywood and latewood represent different climate signals. Moreover, respective δ18OEW and δ18OLW of natural forest pine are positively correlated with those of secondary forest pine (Table 2), pointing to a strong common seasonal climatic forcing imprinted on oxygen isotope. Our results suggest that separating EW and LW δ18O may provide seasonally distinct climate information.The seasonal amplitude of δependent . IntraspP. armandii) had a slightly higher iWUE compared to pine species from a secondary forest (P. kesiya) , indicating that the former could have a more conservative water use than the secondary forest pine species, which is consistent with our first hypothesis. Conservative water use strategy associated with higher iWUE and lower growth rates were also reported from tree species in East Africa , which could be due to the lower water status and reduced stomatal conductance in the early growing season  and iWUE in both species have been increasing, which is consistent with previous studies (-1 per ppm) is twice as high as that of forest pine (0.38 μmol mol-1 per ppm), and the increase rate of iWUE of P. kesiya is also considerably higher than those found from other coniferous species . Our results indicated that both δ13C in earlywood and δ18O in latewood of the two pine species carried a stronger common signal that most likely related to climatic factors. Earlywood δ13C of both pine species mainly stored a temperature signal , while latewood δ18O recorded precipitation signal .We found that inter-series correlations of earlywood δ13CEW of P. kesiya was positively correlated with MMT of previous year’s July and current July to September (1975–2012), however, due to the impact of resin collection on P. kesiya, correlations between δ13C and climate should be interpreted with care. In contrast, δ13CEW of P. armandii was positively correlated with MMT from February to May (1958–2011). The spring season is characterized by increasing temperature as well as relatively dry conditions before the onset of the rainy summer monsoon season . Stomatal conductance and intercellular CO2 concentration would be reduced due to lower soil water status in the spring season, and there would be a weaker carboxylation discrimination against δ13C and resulting in positive correlations between δ13CEW of P. armandii and MMT in the early growing season. In contrast to earlywood, δ13CLW of P. armandii was not significantly correlated to any climate parameter, which was consistent with the fact that the inter-series correlations of latewood δ13C were lower than those from δ13CEW.δ18O in both P. armandii and P. kesiya shared common moisture signals, that δ18OEW of both pine species was negatively correlated with May RH, and δ18OLW of both pine species was negatively correlated with the autumn (September to November) precipitation, which is consistent with our second hypothesis. As can be derived from dendrometer measurements nearby from our study site, cambium growth of P. kesiya last until October . Moreover, we found that the δ18O in the earlywood and latewood of both P. kesiya and P. armandii were negatively with the pre-monsoon (May) RH and autumn RH, respectively , which were also convinced by other studies  is characterized by sunny conditions with little rainfall and low humidity, while ample monsoon precipitation and high humidity occurs during summer .The present study showed that δ studies . This su18O and the weighted mean δ18O of precipitation in Kunming were higher in earlywood growth period (EWG) than that of latewood period (LWG) for both pine species  than that of P. kesiya (r = 0.25). However, the correlations between reconstructed δ18O and weighted mean of precipitation were much lower than other studies (The correlations coefficients between reconstructed source water δ studies , which c13C in both secondary forest pine species P. kesiya and natural forest pine species P. armandii. In P. kesiya, there was no significant difference between earlywood and latewood for either δ13C or δ18O, however, both δ13C and δ18O in the earywood were significantly higher than those of latewood in P. armandii. Our results showed that P. armandii had slightly higher iWUE in than that of P. kesiya. Water use efficiency is increasing in both pine species, however, with a higher rate in P. kesiya. δ13C variations in earlywood and latewood differed among the studied species and represented a site-specific or species-specific climate signals. Due to the impact of resin collection on P. kesiya, care should be taken to interpret the correlations relationships between tree-ring δ13C and climate factors. In contrast to δ13C, δ18O variations showed strong coherence between the study species. δ18OEW of both pine species was negatively correlated with May RH, whereas δ18OLW of both species had a strong significant autumn precipitation signal and thus has the potential to reconstruct autumn precipitation in the study area. The contrasting climatic signals in δ18OEW and δ18OLW imply that separate analyses of earlywood and latewood are recommendable to derive clear climatic signals in δ18O series from conifer species growing in the mid-mountain zone of the summer monsoon region of southwestern China.We found a strong carry-over effect for δAB, AG, Z-XF, and P-LF designed the experiment. P-LF, JG, and AG performed the experiment. P-LF and Z-XF analyzed the data. All authors contributed to the writing 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.