Vibrio coralliilyticus, a known temperature-dependent pathogen of some corals, and Halobacteriovorax, a unique bacterial predator of Vibrio and other gram-negative bacteria. We challenged reef-building coral with V. coralliilyticus in the presence or absence of Halobacteriovorax predators, and monitored microbial community dynamics with 16S rRNA gene profiling time-series. Vibrio coralliilyticus inoculation increased the mean relative abundance of Vibrios by greater than 35% from the 4 to 8 hour time point, but not in the 24 & 32 hour time points. However, strong secondary effects of the Vibrio challenge were also observed for the rest of the microbiome such as increased richness (observed species), and reduced stability (increased beta-diversity). Moreover, after the transient increase in Vibrios, two lineages of bacteria  increased in coral tissues, suggesting that V. coralliilyticus challenge opens niche space for these known opportunists. Rhodobacterales increased from 6.99% (±0.05 SEM) to a maximum mean relative abundance of 48.75% (±0.14 SEM) in the final time point and Cytophagales from <0.001% to 3.656%. Halobacteriovorax predators are commonly present at low-abundance on coral surfaces. Based on the keystone role of predators in many ecosystems, we hypothesized that Halobacteriovorax predators might help protect corals by consuming foreign or “alien” gram negative bacteria. Halobacteriovorax inoculation also altered the microbiome but to a lesser degree than V. coralliilyticus, and Halobacteriovorax were never detected after inoculation. Simultaneous challenge with both V. coralliilyticus and predatory Halobacteriovorax eliminated the increase in V. coralliilyticus, ameliorated changes to the rest of the coral microbiome, and prevented the secondary blooms of opportunistic Rhodobacterales and Cytophagales seen in the V. coralliilyticus challenge. These data suggest that, under certain circumstances, host-associated bacterial predators may mitigate the ability of other bacteria to destabilize the microbiome.Coral microbiomes are known to play important roles in organismal health, response to environmental stress, and resistance to disease. The coral microbiome contains diverse assemblages of resident bacteria, ranging from defensive and metabolic symbionts to opportunistic bacteria that may turn harmful in compromised hosts. However, little is known about how these bacterial interactions influence the mechanism and controls of overall structure, stability, and function of the microbiome. We sought to test how coral microbiome dynamics were affected by interactions between two bacteria:  Vibrio coralliilyticus is a well described model bacterium for the study of interactions between corals, the environment, and pathogenic bacteria , to consortia of archaea, fungi, and bacteria. Although the role of Symbiodinium in the coral holobiont is well studied, the exact roles of each member of the bacterial portion of the holobiont remains far from clear. Experiments and metagenomic analyses have provided some insights into the roles of individual members of the coral microbiome . In free-living attack phase, BALOs actively seek out prey in order to attach, burrow inside, and restructure their host cell into a rounded bdelloplast. This kills their prey and provides BALOs with an osmotically stable structure free from competition to utilize prey resources for growth and replication. A new generation of attack-phase predators then bursts forth from the bdelloplast to seek new hosts.We have recently described how the predatory bacteria ial prey . Halobacathogens . HalobacHalobacteriovorax and other members of the coral holobiont using co-occurrence network analysis of an in-field experimental time series of three coral genera, across three years, several treatments, and range of temperature conditions. These networks showed that Halobacteriovorax are core members of the coral microbiome, present in >78% of samples from three coral genera Porites, Agarica, and Siderastrea , a foreign bacterium , and a coral host (M. cavernosa) interact to affect the complex system of the coral microbiome in a laboratory-based system. While V. coralliilyticus is not associated with causing disease in M. cavernosa, our study still provides baseline experiments for predator and prey interactions on a coral host.Bacterial predators in the coral microbiome could be a type of top-down control, that directly alters the structure and function of the coral microbiome as demonstrated in other aquatic systems by bacterivorous predators , were grown on Marine 2216 Agar (MA) overnight. A single colony was re-suspended in 50 mL Marine 2216 Broth (MB) in a 250 mL flask at 30 °C and 250 rpm overnight. Cultures were diluted 1:100 in fresh media and incubated until late exponential growth before use in any experiment.We first evaluated if Halobacteriovorax sp. PA1 (Accession # KR493097), was grown as previously reported  and one milliliter of the appropriate predator dilution was mixed with 3 ml of molten agar (PP20 medium containing 1.1% Difco agar) held at 42 °C, for a final top layer agar concentration of 0.66%. The mixture was immediately spread over the surface of 1.8% agar PP20 Petri dish plates, and three replicates were plated for each predator–prey combination. Plaques were measured after 3–5 days of incubation . Tecan OD values were reported without conversion to a 1-cm path length. Halobacteriovorax in attack phase do not significantly alter the absorbance reading of the prey at 600 nm due to their small cell size. Predation rates in the liquid co-culture assay were measured by the host cell density reduction compared to the predator-free controls. Based on our observed predation rates and the biological relevance of the strain, we chose to conduct our predator–prey addition experiment using V. coralliilyticus BAA 450 (accession # AJ440005).Triplicate biological replicates for each vibrio prey species were grown overnight in marine broth at 28 °C and shaking at 250 rpm, transferred using a 1:100 ratio into fresh MB, and monitored until late exponential phase. Prey were washed three times in 0.2 µm filtered and autoclaved seawater (FSW) and resuspended to a concentration of 10Montastraea cavernosa was selected as a model for this work as it is both a common reef-building Caribbean coral and is susceptible to a variety of coral diseases  from the Key West , and was maintained for 10 weeks in a shaded flow through raceway tank at the University of Miami Experimental Hatchery. This single M. cavernosa colony was split into 3.5 cm diameter cores with skeleton trimmed to ∼2 cm. Coral cores were transferred back into a common garden experimental aquaria that only contained the M. cavernosa cores and were allowed to acclimate for an additional four weeks where they demonstrated signs of growth including lateral tissue extension over exposed skeleton and feeding behavior during recovery. Just prior to being subjected to the various experimental treatments as described below, the M. cavernosa cores were then transferred to a second common garden at FIU which was composed of a recirculating seawater tank that only contained the M. cavernosa cores. All coral cores were distributed randomly into new 40L recirculating treatment tanks and only used once in the experiment. Seawater for the experiment was obtained from the University of Miami Experimental Hatchery (sand and UV-filtered seawater pumped in from Biscayne Bay).diseases . The 33 Vibrio coralliilyticus virulence as previously described  were taken from common garden tank at time zero, scored with a file to mimic tissue damage, and inoculated in the beakers by transferring the sterile media (control), V. coralliilyticus or Halobacteriovorax cells using a sterile q-tip .Bacterial challenges were conducted in sterile beakers with water temperatures held at 31 °C to induce escribed . To furt9Vibrio coralliilyticus, (3) a total of 106Halobacteriovorax, and (4) a total of 109V. coralliilyticus and 106Halobacteriovorax. Three replicate cores )  sterile media as a control, (2) a total of 10 total)) . Once chHalobacteriovorax culture was passed through a 0.45 µm filter to remove prey and washed three times under the same condition as the V. coralliilyticus cells. The final cell pellet was resuspended in 100 µl of ASW and transferred using a dual q-tip approach  to apply cells to freshly abraded corals in sterile beakers  in 2 mL tubes by centrifugation at 10,000 × g for 10 min and gently re-suspension with sterile media. The late exponential  beakers . Q-tips T = 4, 8, 24, and 32 h) one coral fragment from each replicate tank of each treatment (n = 3 per time per treatment) was removed, photographed, placed in a Whirlpak , flash frozen in liquid nitrogen, and transferred to −80 °C freezer for microbial DNA analysis.At each time point (−1), followed by incubation at 37 °C for 30 min. A 200 µl aliquot of homogenized sample was used for DNA extraction with the Power Soil DNA extraction kit ; the remainder was stored at −20 °C. Microbial amplicon libraries were generated using 515F and 806R primers to the V4 region of the 16S rRNA gene with Schloss sequencing adapters  containing 500 µl of TES Buffer . A 1.5 mL microtube pestle was used to homogenize the tissue before adding 400 µl of TES buffer with lysozyme  was performed using QIIME (v.1.8) . Sequenct-tests with 999 Monte Carlo permutations. For beta diversity analysis, weighted UniFrac distances . To account for multiple comparisons between treatments, Bonferroni-corrected p-values are reported for both alpha- and beta-diversity analyses in the text and figures.To avoid artifacts due to uneven sampling depth during comparisons of alpha and beta diversity, all samples were rarified (randomly subsampled) to equal sequencing depth. After quality control steps, the least sequenced sample had 11,716 reads; this value was thus chosen as the rarefaction depth. For alpha diversity (richness), total observed OTUs and Chao1 diversity statistics  were calistances  were calq-values less than 0.05.To analyze how order-level taxa responded to bacterial challenge, a generalized linear model (GLM) was fitted with the R package DESeq2 . The GLMV. coralliilyticus strains, BAA 450 was most susceptible to predation in the liquid assay while strains RE22 and RE98 were less susceptible  ranged from 8.96 h in V. cholerae N1696 to 24.16 h in V. coralliilyticus RE98. The prey rate for V. coralliilyticus BAA 450, a model coral pathogen, was 19.00 h.Strain and species level differences in susceptibility to predation were detected among some of the Vibrio species . For exaceptible . In the TCC19106 ; Fig. 1CHalobacteriovorax sp. PA1 was capable of killing V. coralliilyticus BAA 450 in co-culture, we conducted an in situ challenge experiment that directly inoculated M. cavernosa corals with this foreign bacterium in the presence and absence of the predator (8 (±6.63 ×107) total V. coralliilyticus cells were transferred using the swab transfer method  as measured by Chao1 and observed species diversity metrics  that were similar to control conditions, but distinct from the V. coralliilyticus treatment  were also significantly different between the control treatment and the V. coralliilyticus treatment (p = 0.012)  . In a sieatments . The HalVibrionales, Rhodobacterales, Alteromonadales, Cytophagales, and Burkholderiales  taxa. To identify specific pair-wise differences in the relative abundance of these taxa across all the individual treatments  we used Wald post hoc tests  in white syndrome affected corals have recently been reported  relative abundance to a maximum mean value of 48.75% (± 0.14 SEM) in the final time point. However, during joint inoculation of Halobacteriovorax and V. coralliilyticus, Rhodobacterales showed no significant differences vs. controls. Similarly, there were no differences in the abundance of Rhodobacterales vs. controls in the treatment where Halobacteriovorax was added alone  in the V.  coralliilyticus treatment (p = 0.015) in the V. coralliilyticus versus the combined Halobacteriovorax and V. coralliilyticus treatment that had a relative abundance of 21.86%   alter the microbiome of a compromised host in a laboratory setting (2) Can predators of this taxa prevent or ameliorate the downstream effects of the alien challenges, and (3) Does the predator itself affect the coral microbiome?High-throughput sequencing allows researchers to more easily document membership dynamics and community topology, yet we often lack the ability to confirm causal relationships among them. Manipulative studies are necessary to link cause and effect. While some host-microbe models can be more readily manipulated , there remain considerable methodological barriers for many systems, especially those for which gnotobiotic (germ-free) host animals are not available. Here we used individual and combinatorial bacterial challenges to a coral host in order to ask three specific questions: (1) How can inoculation of a foreign bacteria  but also alters the microbiome in various ways, including increases in alpha and beta diversity  will likely elicit variable downstream responses in the microbiome, unlike taxa that core members.Here we show that a iversity . HoweverV. coralliilyticus led to an increase in relative abundance in a known group of opportunists of corals, the Rhodobacterales  positively co-occurred with both Vibrionales and Cytophagales in the field (Halobacteriovorax directly alters the abundance of both of these taxa. We show here that a V. coralliilyticus challenge is associated with significant increases in Cytophagales relative abundance in vivo as well, suggesting there is a more direct interaction between these two taxa (A small but growing body of research suggests crobiome . Furtherher taxa . Here inhe field . Here wetwo taxa . This woVibrio coralliilyticus alters the microbiome of coral tissue, whether these downstream responses of the microbiome are the result of wounding and challenge with any putative pathogen remains untested. At the same time, both the absolute numbers of V. coralliilyticus added in this experiment were not environmentally relevant and whether V. coralliilyticus is a pathogen of M. cavernosa is still unknown, shedding doubt on whether these same effects would be seen in true infection scenarios. Yet the patterns of change in the community after addition of V. coralliilyticus remarkably mirror the changes we found in corals exposed to stressors in the environment. For example, we found that overall beta-diversity and the relative abundance of opportunists such as Rhodobacterales increase in corals under threat (While this limited laboratory study suggests that r threat . This stV. coralliilyticus resulted in a destabilized host microbiome in which opportunists bloomed, potentially further exacerbating the negative effects of the initial inoculation. Recent evidence also supports the hypothesis that commensal or mutualistic host-associated microbes offer protection against invasive pathogens either by depriving these alien bacteria of essential nutrients or acting as a physical barrier to host attachment (Corals and other marine organisms are in constant contact with an array of distinct microbes. In the face of this constant microbial challenge healthy host microbiomes are robust to change unless challenged with foreign agents or poor environmental conditions. In this laboratory system, a challenge by tachment . Here wetachment . As our 10.7717/peerj.3315/supp-1Table S1Estimation of the actual number of cells transferred using the swab transfer method. Direct cell counts using epifluorescent microscopy to quantify the number of cells transferred using the swab pellet transfer method.Click here for additional data file.10.7717/peerj.3315/supp-2Table S2Alpha diversity at a rarefaction sequence depth of 11716 reads from coral samples in microbiome manipulation experiment.Click here for additional data file.10.7717/peerj.3315/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.3315/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.3315/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.3315/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.3315/supp-7Supplemental Information 7Click here for additional data file.


Since the 1990s, urban agriculture (UA) has contributed to improving food security in low- and middle- income countries. Now, it is implemented as a multifunctional intervention that can influence various determinants of health . Studies of interest stem from several research disciplines, use a wide range of methods, and show results that are sometimes inconsistent. Current studies have not summarized the overall effects of UA on health and its determinants.The objective of this protocol is to develop a research strategy for a scoping review that characterizes studies of beneficial and adverse impacts of UA on health and its determinants in a wide range of disciplines.Initially, with the help of a library specialist, a list of publications will be obtained through a systematic search of seven electronic bibliographic databases: PubMed, Embase, MEDLINE (Embase), CINAHL Plus with full text, Academic Search Premier (EBSCO host), CAB Abstract (Ovid), and Web of Science. Secondly, a three-step screening by two independent reviewers will lead to a list of relevant publications that meet eligibility and inclusion criteria. Finally, data on the bibliography, type of participants, type of study, results of study, and countries will be extracted from included articles and analyzed to be presented in a peer-reviewed article.The findings are expected to identify research gaps that will inform needs for UA research in specific fields , among certain population groups  or within different economic contexts . Furthermore, the findings are expected to identify knowledge gaps and direct future research needs.This is an original study that seeks to integrate beneficial and adverse effects of UA on health at different level of influence  in order to facilitate a better understanding of UA impacts. This protocol is a first of its kind and is expected to lead to a characterization of UA impacts based on sociodemographic profiles of participants and income levels of the studied countries. This will be relevant for policy makers and UA practitioners. Since the 1990s, urban agriculture (UA) has been a strategy contributing to improving income and food security for individuals and households in low- and middle- income countries, particularly in Africa . In citiUA as an intervention can have social and economic impacts on individuals, households, and/or an entire community by directly influencing health or its determinants. It can influence food security, mental or physical health, or social relationships at different population levels. A significant number of studies have already attempted to demonstrate the contribution of UA to food security ,23 by asOther studies have focused on the negative effects of UA. For example, several studies highlight the potential public health risks associated with UA  by addreAlthough some systematic reviews have been conducted on UA and health, specifically food security and wellbeing , there aThe consideration of high-income countries in literature reviews of interventions similar to UA is not new. Other systematic studies have already evaluated gardening or school gardening, which to some extent are similar interventions to UA. These studies do not allow to draw conclusions about the impacts of UA on health. For example, Ohly et al  used a mAt this time, current studies have not been able to draw definite conclusions on the effects of UA on specific determinants of health or health in general. Given that UA is a multidisciplinary topic , it may be better to address it first in a more general systematic process such a scoping review and consider a broader impact outcome like health prior to engaging future systematic reviews.With this scoping review we seek to identify evidence from peer reviewed literature that demonstrates beneficial and adverse impacts of UA on the determinants of health according to countries’ income level as defined by the World Bank . The detWhat are the impacts of UA on health and its determinants?How do these impacts differ according to countries’ income level or sociodemographic characteristics of studied participants?Conceptually, by answering these questions, we will have a better understanding of how UA as an intervention can affect different health outcomes such as food security, nutrition, social relationships, physical or mental health. Furthermore, we are interested in categorizing these outcomes according to level of influence  and countries’ income level . The findings will allow us to draw a global picture of the potential impacts of UA on health present in the existing literature. Identifying research gaps will also allow researchers and policy makers to make informed decisions about future UA research needs and implications for public policy.The specific objectives of this study are:To identify UA impacts on health and its main determinantsTo characterize the results according to population and country income levelsThis scoping review will follow the five steps described by Arksey and O'Malley  for simiIdentification of the research questions (listed above)Identification of relevant studiesSelection of relevant and reliable studiesData extraction from included studiesCollating, summarizing, and reporting the findingsThis scoping review will use the method suggested by Aromataris and Riitano  to constTypes of participants: This study considers all human participant groups  at different level of influence  who have been implicated by UA.Intervention or concept: For the purpose of this review, UA is defined as food growing initiatives that include the production of edible plants and livestock in urban areas. The review will seek studies that assess UA in all its forms when it is used as an intervention consisting to grow food or raise animals for domestic consumption, local sales, or as a leisure activity.Outcomes: The targeted outcomes are a set of determinants of health inspired from Dahlgren and Whitehead . For exaContext: To be included, studies must have been conducted in urban settings of a high-, middle-, or low-income country according to the World Bank's income-based country classification .Type of study: Peer reviewed quantitative or qualitative studies demonstrating one or more effects of UA on health or its determinants will be included. Narratives, essays, gray literature and theses will be excluded. Other systematic studies will not be included in the analysis but the list of their references will be examined to identify relevant studies.food security does not appear as a MeSH, so in the search for MesH, we will use food supply as an alternative but the keyword food security will also be searched as it is written in the titles and abstracts. Boolean operators OR will also be used to combine individual keywords while the Boolean operator AND will be used to combine sets of keywords  AND (agriculture OR farm). An example of the complete search strategy used on PubMed is described in The search strategy has been designed with the help of a library specialist and searches will be performed in the following seven electronic bibliographic databases: PubMed, Embase, MEDLINE (Embase), CINAHL Plus with full text, Academic Search Premier (EBSCO host), CAB Abstract (Ovid), and Web of Science. The outlined keywords in ReferenceAuthorYearStudy locationCity, countryCountry income levelPopulationType of participants Characteristics of participants Type of studyStudy purposeStudy designOutcomes measuredResultsType of impacts Results of studyDue to a limited accessibility of UA scientific papers prior the 1980s, the search will be restricted to articles published between 1980 and 2017. Titles in languages other than English, French and Spanish will be excluded in the selection phase. All identified publications will be transferred to EndNote  and articles whose publication dates and languages do not meet our requirements will be removed. All remaining publications will be transferred to an online systematic review software , to remove duplicates and for title and abstract screening by two independent reviewers. The full text of eligible articles will be screened by two independent reviewers according to the following inclusion criteria:Relevance: The study must be relevant to the question and objectives of our research. It will be considered relevant if it demonstrates one or more beneficial or adverse impacts of UA on human health or its determinants.Study design: To be included into the scoping review, the study must also present data collected from human participants. Furthermore, the design of the study must be appropriate to answer the studied research questions. Studies that report environmental impacts will be considered only if they report effects on humans .A list of all excluded articles at this stage will be provided with the reasons for exclusion. The reference lists of included studies will also be reviewed to identify relevant studies. The identified studies will be assessed with the same eligibility criteria to validate their inclusion or exclusion. Final inclusion of the publications will be discussed by the two reviewers and any disagreement on the inclusion or exclusion will be resolved by consensus.The quality of the included studies will be evaluated using the criteria of the Effective Public Health Practice Project (EPHPP) guide for quantitative studies, and the qualitative study evaluation criteria of Wallace et al  used by Data as described in Once this information is extracted, the results will then be presented in two forms to make a narrative account of the literature . As a fiThe findings are expected to identify research gaps that will inform needs for UA research in specific fields , among certain population groups  or within different economic contexts . Furthermore, the findings are expected to identify knowledge gaps and direct future research needs.To our knowledge, this scoping study is the first of its kind to explore both beneficial and adverse impacts of UA on health determinants. Other systematic studies have already provided valuable information on specific benefits of UA. However, in the current context of urbanization and climate change where health and environmental challenges are related to food production in cities, it is obvious that the adverse impacts of UA are a concern . TherefoThis study will highlight the state of research on the association between UA and health. A holistic approach that considers beneficial and adverse effects of UA, may inform better public policies and target intervention populations. The scoping review will allow for a better understanding of the contributions or consequences of UA on specific determinants of health. It may also be used by policy makers to target indicators that can help better evaluate UA as an intervention that directly impacts individuals, households, or communities. Such approach will also serve to inform urban planning decisions where the role of agricultural production has not always been evident .


The pathogenic mechanism of ALS remains unclear. However, increasing evidence has indicated that uric acid (UA) may play a protective role in the pathogenesis of ALS. The aim of this study was to evaluate the association between serum UA levels and ALS. A comprehensive literature search in PubMed, Embase, Web of Science, and Cochrane Library was conducted up to 31st August, 2017, using keywords. A random-effects model or fixed-effects model was used to calculate the pooled estimate according to the inter-group heterogeneity. Finally, we indentified 8 case-control and 3 cohort studies. The results indicated that patients with ALS had significant decreased levels of serum UA compared to healthy controls (standardized mean difference (SMD) = −0.72, 95% CI , P < 0.001). Increased serum UA levels were associated with lower all-cause mortality risk among ALS patients (risk ratio (RR) = 0.70, 95% CI , P = 0.001). To summarize, there is an inverse association between serum UA levels and risk of death among ALS patients. Randomized controlled trials with high quality are required to elucidate the role of UA on ALS. Its incidence rate is nearly 2 and 0.8 per 100,000 people-years in Western countries and East Asia, respectively3. The average survival time of ALS patients is approximately 2 to 5 years after symptom onset because of respiratory failure4. The pathogenic mechanism of ALS is still elusive. However, central nervous system seems to be especially vulnerable to oxidative stress and increasing evidence supports the theory that oxidative stress plays a crucial role in the pathogenesis of ALS6.Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal neurodegenerative disease that is characterized by the degeneration of upper and lower motor neurons8. While there is growing evidence to support a possible protective role of UA, a cause-effect relationship between UA and disease outcomes has not been firmly established yet11. Thus, we conducted this study to comprehensively assess the association between UA and ALS by integrating all available data.Uric acid (UA), which is one of the metabolic products of purine, is a natural antioxidant. UA has been hypothesized to reduce oxidative stress by scavenging reactive oxygen radicals and other reactive species21. The flow chat is shown in Fig. 21. Among the included studies, 7 were performed in Asians, 3 in Causations, 1 in both Asians and Causations. Most case-control studies controlled for some conventional risk factors, including age (n = 8), and gender (n = 8). However, only 3 case-control studies controlled for BMI. Those cohort studies adjusted for several variables, including age (n = 3), gender (n = 3), and BMI (n = 3). In addition, two cohort studies adjusted for disease duration. The follow-up time ranged from 13 to 21.3 months. The detailed characteristics of the included studies are presented in Tables The literature research identified 355 papers from computerized databases. Among these articles, 84 were removed because they were duplicate publications. After reviewing the titles and abstracts, 251 citations were excluded for various reasons, and 20 articles were evaluated through a full-text review. Ten articles were further excluded for not meeting the inclusion criteria. Therefore, 8 case-control studies and 3 cohort studies were identified in this studyThere was substantial heterogeneity between those case-control studies . Thus, a random-effects model was used. The mean concentration of serum uric acid was lower in ALS patients compared to control subjects . The forest plot was shown in Fig. Heterogeneity analysis suggested that there was no obvious heterogeneity between those cohort studies, and a fixed-effects model was used to pool the risk estimate. The results suggested that higher serum UA levels were associated with decreased all-cause mortality among ALS patients . The forest plot was shown in Fig. Publication bias was assessed by funnel plot and Egger’s test. The funnel plot showed that there was no obvious asymmetry Fig. . In addiIt is important to explore the association between UA and ALS. The present meta-analysis included both case-control studies comparing the serum UA levels in ALS patients and control subjects, and cohort studies assessing the risk of death among ALS patients with different categories of serum UA levels. Our results identified that the level of serum UA in ALS patients was significantly lower than that in control subjects. We also demonstrated that elevated serum UA levels were associated with lower all-cause mortality risk in patients with ALS. The top tertile of UA levels was associated with 30% lower risk of death compared to the bottom tertile among ALS patients. Therefore, there is an inverse association between serum UA levels and risk of death among ALS patients, and UA plays a protective role in ALS. Considering the protective effect of UA, elevated UA levels could be a potential therapeutic method for ALS, and randomized controlled trails are needed.22. There were some differences when compared these two meta-analyses. First, we included cohort studies assessing the all-cause mortality risk among ALS patients with different serum UA levels, which could help us assess the effect of UA levels on the risk of death among ALS patients. Second, we conducted a comprehensive search and included more recent studies (8 case-control studies and 3 cohort studies). Third, study quality was strictly evaluated, and robustness of the results was examined in this meta-analysis.A previous meta-analysis, which included 3 studies, found that ALS was associated with a lower concentration of serum uric acid24. UA has the ability to reduce oxidative stress, to inhibit glutamate toxicity, and to protect central nervous system25. ALS could benefit from these properties. First, superoxide, like reactive oxygen radicals, could increase oxidative neurotoxicity to neurons26.UA can play a protective role by removing it27. Second, UA could also chelate copper and iron, which meant that they cannot catalyze free-radical reactions28. Third, glutamate toxicity involved in the pathogenesis of ALS30. UA could reduce damage to neurons elicited by reducing glutamate toxicity31.Previous epidemiological studies proved that decreased uric acid levels were associated with an increased risk of PD and ADOf course, there were several limitations in this meta-analysis, so the results should be treated cautiously. First, this meta-analysis only included studies published in English and Chinese, which could inevitably lead to language bias. Second, diet and lifestyle differences might influence the metabolism of UA, while these differences could not be considered in this study. Third, selection bias might have been introduced because we fail to obtain complete data from some studies. Fourth, it has been proved that UA levels were correlated with BMI. But several included studies did not adjusted BMI between ALS patients and healthy controls, which might reduce the reliability of the study results. Fifth, we failed to conduct the dose-response analysis between serum UA levels and all-cause mortality risk among ALS patients because of limited data.In conclusion, there is an inverse association between serum UA levels and risk of death among ALS patients. UA plays a protective role in ALS. Well-designed randomized controlled trials are required to assess the therapeutic effect of UA on ALS.32.This study was conducted according to the “Preferred Reporting Items for Systematic Reviews and Meta-analyses” (PRISMA) guidelinesFour main computerized databases, including PubMed, Web of Science, Embase and Cochrane Library were searched to collect studies regarding the association between UA levels and ALS before 31st August, 2017. The research language was restricted to English and Chinese. The following search terms were used: “uric acid” or “urate”, or “urine acid”, or “UA”, and “amyotrophic lateral sclerosis” or “ALS”. Furthermore, reference lists of relevant articles were evaluated to collect all eligible articles.The inclusion criteria were as follows: (1) having definite diagnostic criteria for ALS; (2) case-control studies or cohort studies evaluating the association between serum UA levels and ALS; (3) comparing the serum UA levels in ALS patients and control subjects or assessing the all-cause mortality risk among ALS patients with different categories of serum UA levels; (4) odds risk(OR), risk ratio (RR), or Hazard risk (HR) with 95% confidence interval (CI) were reported in cohort studies, or mean value of serum UA levels with standard derivation (SD) was reported in case-control studies. The exclusion criteria were as follows: (1) necessary data were not available; (2) duplicate reports; and (3) animal studies.33. The maximum point was 9. A score of 7 or above was considered high quality. The literature search and data extraction were completed by two investigators separately. The disagreements between investigators were resolved by discussion.The following data were extracted from each included studies: author name, publication year, country of origin, gender, controlled factors, mean value and SD of serum UA levels, sample size of ALS cases and healthy controls, risk estimates of all-cause mortality in ALS cohorts, and adjusted confounders. Study quality was assessed by the Newcastle-Ottawa Scale (NOS)35. Briefly, log risk estimates were transformed with the comparison of top and bottom tertiles being equivalent to 1.37 times the log risk ratio for comparison between the top and bottom halves, and to 2.18 times the log risk ratio for 1-standard deviation (SD) increase.To compare serum UA levels in ALS patients and control subjects, standardized mean difference (SMD) with 95% CI was used. To evaluate the association between serum UA levels and all-cause mortality risk in ALS patients, the RR with 95% CI was used. The UA concentration was converted from μmol/L to mg/dl using a ratio of 16.81 (1μmol/L = 0.01681 mg/dl). HRs and ORs were assumed equally to RR. Risk estimates for UA were differently reported by individual studies , and thus transformed as previously described36. If I² < 50% and P > 0.1 in the Q test, the studies were not obviously heterogeneous, and the fixed-effects model was used to calculate the pooled estimate. Otherwise, the random-effects model was used when there was substantial heterogeneity. Publication bias was assessed by funnel plot and Egger’s test37. To evaluate the influence of each individual study on the pooled estimate, sensitivity analysis was conducted by omitting each study by turns. All data were analyzed using the STATA12.0 software. P < 0.05 was considered statistically significant.Heterogeneity analysis was assessed using the Cochrane Q test and I² statisticAll data generated or analysed during this study are included in this article and its supplementary materials.Supplementary Information


Baeolophus atricristatus), a species that experiences substantial changes in population density and competition for food over the annual cycle. We compared the size of the prominent head-crest to foraging success at community-used feeding stations; we tested this relationship when competition was seasonally high, and when competition was seasonally low. We then experimentally decreased the number of feeders to increase competition (during the season of low-competition), and again tested whether male crest size predicted access to feeders. When competition was seasonally high, males with longer crests had greater access to feeders, but this pattern was not apparent when competition was seasonally low. When competition was experimentally increased, males with longer crests were again more successful at maintaining access to feeders. These findings provide evidence of a context-dependent status signaling system, where the status signal only mediates access to resources during periods of high competition. We discuss possible hypotheses for why the signaling system may not be functional, or detectable, during periods of low competition, including that competitors may interact less frequently and so have reduced opportunity for signaling, or that status signals are disregarded by receivers during periods of low competition because signalers are unlikely to escalate a contest into a fight. In any case, these results indicate that resource availability affects a status signaling system, and that the potential for status signaling persists in this system between seasons, even though such signaling may not be overtly present or detectable during periods of low competition.Status signals allow competitors to assess each other’s resource holding potential and reduce the occurrence of physical fights. Because status signals function to mediate competition over resources, a change in the strength of competition may affect the utility of a status signaling system. Status signals alter competitor behavior during periods of high competition, and thus determine access to resources; however, when competition is reduced, we expect these signals to become disassociated from access to resources. We investigated seasonal changes in status signaling of the male black-crested titmouse ( Competition for limited resources is ubiquitous among social organisms, and highly competitive individuals often enjoy fitness advantages because they are able to garner priority access to resources . HoweverThe degree to which individuals invest in resource defense is determined by optimizing the benefits associated with the resource against the costs of maintaining access against competitors . SeasonaIn addition to resource value, the cost of aggression can also dramatically impact an individual's decision to engage in a fight. Models predict that the risk of injury and the expenditure of energy during a fight must be outweighed by the value of the resource ,25. Due When individuals strategically allocate investment into aggression, we expect the utility of a status signaling system, which mediates such aggressive interactions, to vary over the same time period. When resources are of high value, we expect that a status signaling system will function as expected, and that a status signal will determine access to resources. However, when competition is reduced, and the motivation to fight will be lost by both dominant and subordinate signalers, we expect status signals to become disassociated from access to resources. Indeed, a context-dependent relationship between ornamentation and signaler or receiver behavior has been described in other systems –34, as wBaeolophus atricristatus), a species that experiences substantial changes in population density and competition for food over the annual cycle. We carried out two observational studies during different times of the annual cycle, and then experimentally reduced the amount available food by reducing the number of supplemental feeders. We compared the size of the prominent head crest  is a socially monogamous passerine with a conspicuous black crest . The black-crested titmouse breeds between March and June and is a year-round resident in this region. Although core-territories are defended throughout the year, the species forages within larger home-ranges that overlap with multiple neighboring territories , and aggOur study is divided into two periods that vary in population density. We conducted our first observational study between June 27-July 15 2013, which corresponds to the high-density period, when large nuclear family groups of 3–8 birds maintain territories (breeding pairs plus young produced that year). These groups can remain together for up to six-months . Both thTo measure access to supplemental feeders, we created an array of 11 feeders outfitted with RFID antennae and processors ,43 spreaBetween May-June 2013, we captured birds using funnel traps placed around seed feeders. Upon capture, we measured size (tarsus) and body mass. Birds were banded with USGS metal band, individualized color bands, and a 12 mm PIT tag was attached to the color bands on one leg. Within our study site, we captured and tagged 15 adult males for the purpose of these studies.1,12 = 0.14, p = 0.72).Upon capture, crest length was measured digitally using standardized photos in which the crest was flattened and held parallel to the camera's sensor. A size scale was included in each picture and Image J  was usedFor study 1, which occurred during the high-density period in the summer, we compared crest length to our measures of access to resources. We monitored RFID data to assess foraging over 19 consecutive days . We began collecting RFID data after birds already had approximately 2 months experience with the feeders , and so all birds in the area already had an opportunity to utilize the feeders. The RFID readers acquired data on foraging visits during all daylight hours (ca. 14 hrs per day). There were two occasions in which data from a single feeder was lost due to hardware damage by mammals, and so data were removed during this time-period from all 11 feeders so that all feeders recorded identical periods.For study 2, we again tested for a relationship between male crest size and access to resources by re-deploying RFID-equipped feeders during the low-density period before the breeding season . Before we began collecting RFID data, we replaced the 11 feeders for approximately one-month to allow all birds in the area to rediscover the resource. We conducted this second study as described above, and collected 10 days of baseline foraging data using the same 11 feeders outfitted with RFID readers.After we established baseline usage of feeders during the low-density period in study 2, we conducted study 3: we experimentally increased competition by reducing the number of available feeders by approximately half (from 11 to 6 feeders). Immediately following study 2, we conducted Study 3 for 10 days, between February 11–20 2014. The removed feeders were evenly distributed spatially throughout the study site, and the removed feeders were balanced so that a similar number of feeders were removed from areas where dominant and subordinate males typically foraged. In other words, the number of short-crested individuals that lost their primary feeder was similar to the number of long-crested individuals that lost their primary feeder . We removed the feeders at night so as not to interfere with foraging behavior. After feeders were removed, we collected RFID data in the same manner as listed above . There was one instance where a feeder was emptied of seed for 7 hours during the baseline period (study 2), and so data from all other feeders during these hours were removed from this dataset, and a corresponding period of data of equal duration, from the same time of day, and from the same day within the 10-day cycle, was removed from all feeders from the study 3 dataset.To test whether crest length predicted maintenance to access to food when competition was increased, we calculated the percent reduction in foraging success for each individual upon the reduction of feeders , divided by total visits during the baseline low-density period. This measure represents an individual’s success at maintaining its own baseline level of access to feeders after competition was increased.This study was carried out in strict accordance local and federal laws; vertebrate work was approved by an Institutional Animal Care and Use Committee (IACUC) (Permit Number: 090512_TM2). Handling of titmice was kept to a minimum, following recommendations of the Ornithological Council's Guidelines to the use of wild birds in research. All sampling procedures and experimental manipulations were approved as part of obtaining IACUC certification. The research on private property was conducted with permission of the owner.General linear models were used to analyze foraging success data. Separate analyses we run for studies 1–3 to compare crest length to foraging success. Body size (tarsus) and body mass were included in all models to control for their possible effect on dominance. Factors were removed using backwards stepwise simplification if P > 0.10. All statistics were performed in JMP 13. Three tagged males were not observed after initial capture, and one male was removed from analyses because it was missing half of the distal feathers on the crest at the time of capture. Sample size was thus reduced from 15 to 11 males in study 1. For studies 2 and 3, sample size was further reduced from 11 to 7 males because 4 individuals disappeared after the first study. The males that disappeared had crest-lengths that were evenly distributed from the distribution of crest length , so their absence did not add a directional bias to our results. All research was performed following strict animal care guidelines and protocols were approved by IACUC (Trinity University 090512_TM2).1,9 = 5. 82, p = 0.039, R2 = 0.39, We recorded a total of 9,412 foraging events from 11 male titmice over the 19-day high-density period. Each male foraged 855.6 ± 132.9 (mean ± SE) times throughout the study period, with a mean of 45.0 (range: 5–76) visits per bird per day. During this high-density period, crest length was positively correlated with total number of visits across all feeders  visits per bird per day. During this low-density period, there was not a significant relationship between crest length and total visits  visits per bird per day. Use of feeder resources decreased with the reduction in the number of feeders, with a significant decrease in total feeding events between baseline and the experimental removal of feeding stations . Individuals suffered a loss, on average, of 52.4% ± 9.8% of their baseline foraging success.1,5 = 12.25, p = 0.017, R2 = 0.71, n = 7, After the experimental reduction in the availability of food, crest length was significantly positively related to an individual's ability to maintain competitive status: individuals with larger crests maintained a larger percentage of their baseline feeding rate . The birds that disappeared before experimental study 3 were equally distributed between large- and short-crested individuals, so their absence did not directionally drive differences in the relationship between crest length and foraging success across studies. Furthermore, our failure to detect an effect of crest on foraging success in study 2 was unlikely due to low statistical power: studies 2 and 3 had the same sample size, yet there was sufficient statistical power in experimental study 3 to detect a moderate effect size. As such, we conclude that the relationship between crest length and access to food was reduced or absent during the period when the titmouse population density was low, when feeders were likely to represent a low-value resource.The observation that male crest length is related to foraging success only during periods of high competition is consistent with our expectation that status signal systems will have an increased function when there is greater risk of fighting. Multiple avian species have been found to use status signals only during discrete times of the year, although in these examples, the trait is absent, or is noticeably diminished, when the signaling system is not fully functional. For example, the black throat badge of male house sparrows is partially obscured by white feather tips during the non-breeding season when signaling is reduced  and thenThere are a number of hypotheses that could explain why a status signaling system would lose functionality during periods of low competition. The first possibility is that competitors may be simply less likely to interact during periods of reduced competition , and so have reduced opportunity to signal to one another. As a result, access to resources would not be mediated by a signal. A second possibility is that signalers may modulate the way their signal is displayed depending on the level of competition –54. If aOne limitation of our study is that we did not conduct behavioral observations to confirm that long-crested males actively exclude short-crested competitors. As such, it is possible that the pattern we detected  may not relate directly to signaling. For instance, males with longer crests may win fights with short crested birds over access to supplemental feeders without the use of a signal. However, theory predicts that fights will be relatively rare if they can be avoided through signaling , and we Our results indicate that resource availability affects a status signaling system, and that the potential for status signaling persists in this system between seasons, even though such signaling may not be overtly present or detectable during periods of low competition. Because our results provide evidence that a status signal is actively used during only part of the year, we urge future research to consider the possibility that a signaling system may not be functional during the time-period under investigation (also see , as this


Calpain-10 belongs to a commonly expressed member of the Calpain-like cysteine protease family, which acts as risk marker for some diseases. The purpose of this study is to elucidate correlation between Calpain-10 single-nucleotide polymorphisms (SNPs) and the incidence of OSAHS followed by ischemic stroke (IS).Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common chronic disorder which is followed by various complications. Calpain-10 protein expression, polymerase chain reaction (PCR)-restriction fragment length polymorphism for detection of gene polymorphisms of SNP 43 and SNP 19, and PCR-allele specific amplification for SNP 44. Polysomnography was conducted to check the nocturnal polysomnography indicators, and also Montreal Cognitive Assessment (MoCA), Scientific Data System scores cognition and anxiety of patients, respectively. Logistic analysis was used for the risky factors for OSAHS.OSAHS patients were divided as OSAHS + IS, OSAHS, and control groups, respectively. Immunohistochemistry was performed for Calpain-10 protein expression was significantly increased in the OSAHS + IS and OSAHS groups compared with the control group. Significant differences in SNP 43 and SNP 44 genotype, and also allele frequency were observed in 3 groups, among which the OSAHS + IS group had higher SNP 43 and SNP 44 allele frequency than the control and OSAHS groups. There were differences regarding apnea-hypopnea index, minimum fingertip blood oxygen saturation (LSaO2 [%]), oxygen reduction index (ODI) between patients with different genotypes of SNP 43 and SNP 44 in OSAHS patients, and also GC and AT frequency in the OSAHS + IS and OSAHS groups. As compared with the OSAHS group, the MoCA scores and MoCA subitems in the OSAHS + IS group were declined, whereas the Scientific Data System scores were elevated. Additionally, GG 43 genotype, high apnea-hypopnea index, and body mass index were detected as the risk factors of OSAHS.Calpain-10 SNP 43 may be related to OSAHS with IS, with SNP 43 GG genotype as a risk factor for OSAHS with IS.These findings indicate that the  The prevalence of OSAHS in the United States is estimated up to 24% for men and 9% for women. The pathogenesis of OSAHS involves an interaction between ventilatory control instability and poor pharyngeal anatomy. It is also believed that OSAHS may be triggered by anatomic factors that promote pharyngeal narrowing, such as large neck circumference, cervical soft tissue, vessels, and bony structures. OSAHS exhibits the highest incidence rate among sleep-related diseases and is also followed by various complications among which cardiovascular diseases are the most common and severe. To the best of our knowledge, recent years have witnessed an intensification of the study with aims to establish the genetic contribution to the occurrence of OSAHS, and also its sequelae.Obstructive sleep apnea-hypopnea syndrome (OSAHS) is clinically defined as a disordered breathing event that may cause a blockage or narrowing of the upper airway, which is in occurrence with symptoms such as sleepiness, and also snoring.Calpain-10 is identified as the first type 2 diabetes mellitus gene in a genome-wide scan followed by positional cloning, and is located on chromosome 2q37, encoding Calpain-10, which is a commonly expressed member of the Calpain-like cysteine protease family.,7 Functional genetic data reveals that Calpain-10 plays an instrumental part in insulin resistance and intermediate phenotypes, including those related to adipocytes.,9Calpain-10 may promote the translocation of GLUT4 through reorganization of the cytoskeleton. Furthermore, the Calpain-10 gene has been implicated in several aspects of metabolism syndrome including plasma cholesterol concentrations, elevated body mass index (BMI), hypertension, and hypertriglyceridemia. Four Calpain-10 single-nucleotide polymorphisms (SNPs) 19 (rs3842570), 43 (rs3792267), 44 (rs2975760), and 63 (rs5030952) have been commonly investigated for their potential roles, acting as risk markers for type 2 diabetes that is a complex metabolic disorder with increased risk of cardiovascular disease. In accord with previous studies, this study aims to perform an association study between Calpain-10 SNPs including SNP 43, SNP 44, and SNP 19, and also the occurrence of OSAHS and OSAHS with ischemic stroke (IS).22.1 and the diagnosis of OSAHS patients with IS was done according to the criteria of both OSAHS and IS. The included criteria were as follows: OSAHS patients confirmed by computed tomography (CT) and magnetic resonance imaging (MRI); OSAHS patients confirmed by polysomnogram; OSAHS patients with symptoms like sleeping in the daytime and snoring at night; and OSAHS patients with good compliance. Exclusion criteria were as follows: patients with coma, much oral secretion, and no ability to expectorate; patients with pulmonary infection; patients with mass bunamiodyl in sternum, severe aerothorax, or mediastinal emphysema; patients with acute myocardial infarction and acute left ventricular failure; patients with uncontrollable acute ear, nasitis, and nasal sinusitis infection; patients with large area of cerebral infarction and brain stem infarction; patients with glaucoma; and patients with hypertension or diabetes, or undergoing antihypertension and antidiabetic treatment. All subjects included in this study were not blood-related. Additionally, this study also recruited 240 healthy individuals as the control group, who were inquired for medical history before undergoing screening for exclusion of OSAHS using a Stardust portable sleep monitor, followed by a confirmation of normal blood pressure, heart rate, electrocardiogram, breathing state, liver and kidney function, and hemogram. The clinical trial regime was approved by the Ethics Committee of Xianyang Hospital of Yan’an University and strictly complied with the Ethical Guidelines for Biomedical Research on Human Subjects in the Declaration of Helsinki. All subjects or their legal representatives signed informed contents and well understood the trail contents, processes, and possible side-effects.In all, 186 OSAHS patients (OSAHS group) and 198 OSAHS patients with IS (OSAHS + IS group) were admitted into the Xianyang Hospital of Yan’an University between October 2013 and November 2015. The OSAHS was diagnosed by the American Academy of Sleep Medicine (AASM) criteria,2.2Baseline data of all subjects were collected, including sex, age, BMI, diabetes history, hypertension, and smoking. Each fasting subject underwent the extraction of peripheral venous blood (10 mL) in the morning, and 5 mL of the extracted blood was put into heparinized tubes. After 20 minutes of centrifugation (3000 revolutions/min), the blood sample was packed in Eppendorf tubes. Enzyme method was used to detect triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) by Hitachi 7600 automatic biochemical analyzer . The remaining 5 mL of peripheral venous blood was put in ethylene diamine tetra acetic acid (EDTA) tubes and reserved at −80°C in a refrigerator for genomic DNA extraction and further experiments.2.3Enzyme-linked immunosorbent assay (ELISA) was conducted for the Calpain-10 protein expression in the peripheral blood both in the case and control groups. The enzyme-labeled plate coated by antibodies were taken out, and in its wells added with standard samples and samples to be tested of various concentrations, respectively, which were then incubated at 37°C for 2 hours. After the solution elimination, 0.1 mL of biotinylated antibodies were added in each well at 37°C for 30 minutes, followed by phosphate buffer saline (PBS) washing for 5 times. Then 0.1 mL of tetramethylbenzidine (TMB) for each well was used for developing at 37°C for 20 minutes and TMB stopping solution in the same dosage for stopping reaction. The optical density (OD) in the enzyme-labeled plate was detected at the wavelength of 450 nm. The ELISA kit was purchased from the Shanghai Ximei Biotechnology Co. Ltd .2.42 saturation (SaO2) <90%, oxygen reduction index (ODI), lowest SaO2 (LSaO2%), mean SaO2 (MSaO2%), the proportion (%) of stage I sleep and stage II sleep (S1 + S2 sleep) time in the total sleep time, the proportion (%) of stage III sleep and stage IV sleep (S3 + S4 sleep) time in the total sleep time, and the proportion (%) of rapid eye movement (REM) sleep time in the total sleep time.All subjects were prohibited against alcohol, coffee, tranquilizer, and hypnotic within 24 hours before monitoring, and then they continuously received sleep apnea monitoring with no less than 7 hours by a polysomnography  at night in a sleep laboratory. Monitor index of sleep apnea included: apnea-hypopnea index (AHI), figure arterial O2.5 were extracted using EPPendorf UV spectrophotometry to measure DNA concentration and normalize it to 50 ng/mL. The 3 SNP primers of Calpain-10 gene were synthesized by Shanghai Boya biological Co. Ltd  as shown in Table  included 2.5 μL of 10× PCR buffer solution, 11.3 μL of ddH2O, 2 μL of dNTP, 0.2 μL (5 U/μL) of Taq polymerase, 2 μL of template DNA, 1 μL of the upstream and downstream primers, and 0.75 μL of R2 and 0.25 μL of L primer in the SNP44 locus R1 primer. The PCR conditions are as follows: predenaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and amplification at 72°C for 30 seconds, and final extension at 72°C for 10 minutes. The products of SNP 43 were identified by 2% gel electrophoresis after digested by NsiI incision enzyme in water bath at 37°C for 16 hours. However, the products of SNP 19 and SNP 44 are directly identified by 2% gel electrophoresis.A total of 2 mL of EDTA anticoagulant, genomic DNA from peripheral blood according to the conventional phenol/chloroform/isopropanol method2.6The products of PCR SNP43 were identified by NsiI enzyme. After restriction enzyme treatment, 3 kinds of genotypes were produced: wild-type GG for 144 bp, heterozygous mutation type for 144, 121, and 23 bp, and pure mutant type AA for 121 and 23 bp. SNP19 was the insertion deletion polymorphism, and its PCR products after electrophoresis were directly divided into 3 types: wild-type 11 for 142 bp, heterozygous mutant 12 for 174 and 142 bp, and pure mutant type 22 for 174 bp. PCR SNP44 products was directly assigned into 3 kinds of genotypes after electrophoresis: wild-type TT for 60 bp, heterozygous mutant TC for 60 and 75 bp, and pure mutant CC for 75 bp  was used to evaluate the anxiety of the study objects, with SDS <50 meaning no anxiety, 50 to 59 meaning mild anxiety, 60 to 69 meaning severe anxiety, and over 70 meaning severe anxiety.All patients were assessed using the Montreal cognitive function rating scale (MoCA)2.8t test was used in the 2 groups for comparison and 1-way analysis of variance (ANOVA) for single-factor analysis of variance for groups, and tested by SNK-q test. If not, the nonparametric Kruskal-Wallis h rank-sum test was conducted, and Mann-Whitney U test was conducted for group comparisons. The count data were expressed by constituent ratio or rate using chi-square test. Relative risk of genotype was expressed by odds ratio (OR) and 95% confidence interval (CI). Hardy-Weinberg equilibrium method was used to detect group representation of samples. SHEsis software was utilized for haploid analysis and logistic regression method for the risk factors for OSAHS with IS, with P < 0.05 meaning statistically difference.SPSS 21 statistical software  was used to statistically analyze the data. The measurement data were expressed by mean ± standard deviation (SD), and before data analysis, normality and homogeneity of variance tests were conducted. If the distribution and variance were normally presented, the 33.1P < 0.05), whereas no significant difference of age, sex, smoking history, history of diabetes, history of hypertension, and 3 acyl glycerin (TG), HDL-c, LDL-c content ratio was found among the OSAHS, OSAHS + IS, and control groups .As shown in Table 3.2P < 0.05).The Calpain-10 protein expression in serum among 3 groups was detected by ELISA, as shown in Fig. 3.3Calpain-10 genotypes was assessed by the goodness-of-fit chi-square test. This study included a total of 208 subjects. After inspection, the results showed that there were no significant differences between observed value and estimated value of the genotype distribution frequency of Calpain-10 SNP 43, SNP 44, and SNP 19 (P > 0.05), which indicated that the samples were from a large group with randomly assigned equilibrium , whereas no significant difference was shown between the control and OSAHS groups (P > 0.05). SNP 43 G and SNP 44 C allele frequencies in OSAHS + IS group were significantly higher than those in the control and OSAHS groups . However, there was no remarkable difference regarding SNP 19 genotype and allele frequency among the OSAHS, OSAHS + IS, and control groups (P > 0.05) , but no differences in MSaO2, S1 + S2 (%), S3 + S4 (%), and REM (%) among patients with different SNP 43 and SNP 44 genotypes . The differences of sleep respiratory monitoring indexes were not significant (P > 0.05) among the patients with various SNP 19 genotypes  . The MoCA sub items in OSAHS + IS group such as attention and calculation, abstract, visual space, and executive function and delayed recall significantly decreased (P < 0.05). No significant difference was found in other subitems (P > 0.05), as shown in Table Patients underwent SDS and MoCA scale evaluation after admission to the hospital. Compared with the OSAHS group, the MoCA scale scores were significantly lower, whereas the SDS scores were significantly higher in the OSAHS + IS group . As shown in Table P < 0.05).Using 4Calpain-10 gene polymorphisms and the occurrence of OSAHS and OSAHS with IS. SNP 63 was identified as minor allele with the lowest allele frequency, always selected for some rare polymorphism studies; thus it was excluded in this study.,21 Therefore, we demonstrated the positive association between Calpain-10 SNP 43 and SNP 44 polymorphisms, and the risk of OSAHS and OSAHS with IS.The study was designed to elucidate association between  It was reported that Calpains played a pivotal role in neurodegeneration, cell motility, and synaptic plasticity, among which mu-calpain  and m-calpain  were 2 major calpain isoforms shown in brain. Calpain-10 protein, as intracellular Ca (2+)-dependent cysteine protease, exerts effects on regulation of thermogenesis, pancreatic β-cell function, and glucose metabolism, and several sites of Calpain-10 polymorphism are researched for their potential markers for the metabolic syndrome and type 2 diabetes. It is sited in mitochondria, which is of importance in mitochondrial homeostasis, implicated in diabetes-induced renal dysfunction, indicating that loss of Calpain-10 induced by glucose in vivo leads to organ failure and renal cell apoptosis via collections of mitochondrial dysfunction and mitochondrial calpain-10 substrates. To our knowledge, there is a connection between the severity of OSAHS and both damaged glucose tolerance, and also insulin resistance, suggesting that obstructive sleep apnea-related diseases are risk factors for the occurrence of cardiovascular disease, such as type 2 diabetes. Also, the occurrence rate of obstructive sleep apnea-related diseases was found significantly higher in the patients with type 2 diabetes. These data have proposed the presumption of type 2 diabetes as a risk factor for OSAHS, arising with a bidirectional fashion, and Calpain-10 was defined as the first gene influencing the risk of type 2 diabetes by positioning cloning and the possibility of association between SNP 43 and SNP 44 polymorphisms, and transcriptional regulation Calpain-10 expression has been raised. A study performed by Ling et al has reported that the Calpain-10 mRNA level was increased by 64% in pancreatic islets from patients with type 2 diabetes in comparison with nondiabetic individuals. Horikawa et al also discovered that mutation in Calpain-10 gene has been linked to a 3-fold increased risk of type 2 diabetes in Mexican-Americans, and also in Northern European populations. It was also believed that variation of the Calpain-10 gene (SNP 43 GG and SNP 44 TT) had connections with elevated levels of total cholesterol.,29 Taken together, our study assumed that SNP 43 and SNP 44 polymorphisms of Calpain-10 gene might affect the variance in Calpain-10 mRNA level so as to increase the risk of cardiovascular diseases, ultimately contributing to the development of OSAHS. In our study, however, there was no significant difference in SNP 19 genotype and allele frequency between the OSAHS, OSAHS + IS, and control groups. In the study by Zaharna et al, they uncovered that, in the donors with type 2 diabetes, the total cholesterol levels were higher in the SNP 19 22 genotype than those in 12 or 11 genotype, which was significantly different from our results. Nevertheless, the above positive data were also changed by the studies conducted by Carlsson et al and Daimon et al. This difference in results could be due to the limited studies that investigated total cholesterol, and also triglyceride levels and their associations with Calpain-10, which needs further investigation.The key findings from this study indicated that the frequency of SNP 43 GG genotype and SNP 44 TC genotype in the OSAHS and OSAHS + IS groups were remarkably higher than that in the case group. Calpains are a very common family of the calcium-dependent cysteine proteases which are always involved in a large range of differentiation processes and cell regulatory. In our study, we found that the OSAHS patients with IS show increased AHI compared with the OSAHS patients, possibly suggesting that OSAHS with IS would deteriorate OSAHS. The ODI refers to the number of events per hour in which oxygen saturation declines by 4% or more. Interestingly, hypopnea implicates a reduction of airflow by 50% to 80% for at least 10 seconds related to either oxygen desaturation of at least 4% or arousals. Our results also demonstrated that the OSAHS patients with IS had increased ODI compared with the OSAHS patients. Consistent with our study, Park et al supported the possibility of the severe OSAHS patients with greater oxygen desaturation. Also, our study revealed that the OSAHS patients with IS exhibit decreased LSaO2 and MSaO2. As reported in the study performed by Ursavas et al, the patients with OSAHS had remarkable lower MSaO2, but longer time of SaO2 <90% than the healthy controls. Also, there were significant differences in AHI, LSaO2, MSaO2, ODI, time of SaO2 <90%, and REM/TST among the SNP 43 GG, GA, and AA, and between the SNP 44 TT and TC, suggesting that SNP 43 GG and SNP 44 TG is linked to OSAHS.Additionally, there were significant differences in these monitor indexes of sleep apnea between the OSAHS patients and OSAHS patients with IS. The severity of OSAHS is commonly measured by the AHI that reflects the number of AHI per hour of sleep, with 5/h regarded as normal, AHI 5 to 15/h as mild, AHI 15 to 29/h as moderate, and AHI 30/h as severe.Calpain-10 SNP 43 and SNP 44 polymorphisms were correlated with the risk of OSAHS and OSAHS with IS. Disease association with certain variants of Calpain-10 found in certain populations may not be applicable for other populations due to detecting the expected variants in only part of all populations. Increased or decreased frequency of particular genotypes depends on genetic and environmental factors, such as linkage disequilibrium, founder effect together with selection. Apart from this, our study consisted of a small sample size, since it is better to recruit a large sample size in genetic association studies. Thus, further in-depth researches will be performed to explore the mechanism of Calpain-10 SNPs on the influencing factors, such as total cholesterol and triglycerides in cardiovascular diseases and metabolism syndromes.In conclusion, the logistic regression analysis indicated that SNP 43 AA, SNP 44 TC, and larger neck circumference were the risk factors of OSAHS and OSAHS with IS. Collectively, We thank the reviewers for critical comments.


Post-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays.This study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2. Immunohistochemistry studies revealed the presence of Quiescin Q6-Sulfydryl Oxidase 2 in a broad range of porcine tissues; the pronounced vesicle-contained Quiescin Q6-Sulfydryl Oxidase 2 at the apical region of epididymis and seminal vesicles epithelium suggested its involvement in sperm physiology and its participation in semen formation. The majority of porcine Quiescin Q6-Sulfydryl Oxidase 2 could be purified via either antibody affinity column or be salted out using 10%–40% ammonium sulfate. Higher amount of low molecular weight Quiescin Q6-Sulfydryl Oxidase 2 observed in the seminal vesicle likely represents the secretory form of Quiescin Q6-Sulfydryl Oxidase 2 and reflects an exuberant secretory activity in this organ.We demonstrated for the first time, the presence of Quiescin Q6-Sulfydryl Oxidase 2 in porcine species; moreover, two forms of Quiescin Q6-Sulfydryl Oxidase 2 were identified and exhibited distinct molecular weights and properties during protein purification processes. This study also provided feasible Quiescin Q6-Sulfydryl Oxidase 2 purification methods from slaughterhouse materials that could potentially allow obtaining sufficient amount of Quiescin Q6-Sulfydryl Oxidase 2 for future functional investigations. The formation of a functional male gamete is an outcome of complex events involving biochemical, physiological and morphological modifications of the spermatocyte. Spermatogenesis starts in the testis with multiple mitotic divisions, followed by two subsequent cycles of meiotic division, resulting in the formation of a haploid spermatocyte . Althoug2→ R-S = S-R + H2O2 [Upon ejaculation, spermatozoa are mixed with secretions from the accessory glands . Amongst constituent components that form seminal plasma, proteins are of most important ones that directly or indirectly regulate sperm functions and activities. One of the protein families that have been identified in the seminal plasma is the Quiescin Q6-Sulfydryl Oxidase (QSOX) family. QSOX protein was first discovered by Otrowoski and co-workers from rat seminal vesicles back in 1979 [The substrates of QSOXs range from small thiol compounds such as dithiothreitol (DTT) to thiol groups in proteins. The formation of the covalent linkages between thiol groups is considered as a reversible post-translational modification process commonly observed in secreted proteins. Intra molecular disulfide bonds are required for proper protein folding, function and its stability . MoreoveInterestingly, despite an early identification (from human neuroblastoma cells) of QSOX2 protein and its associated gene , up to dChemicals and reagents were obtained from Sigma-Aldrich  unless otherwise stated. Rabbit polyclonal anti-QSOX1 and anti-QSOX2antibodies were purchased from Abcam . Magnetic Dynabeads®Protein A Immunoprecipitation Kit was obtained from Thermo Fisher Scientific .Tissues used in this study were obtained from slaughterhouse materials upon routine visits for health and meat production inspections under the guidance of Council of Agriculture, Taiwan. Tissue sampling for pigs was carried out under the supervision of a certified slaughterhouse veterinarian and under IACUC protocol (NTU-103-EL-86). For porcine analyses, materials from eight male Landrace pigs, aged between 2 to 2.5 years were collected in late March. Samples were collected immediately after slaughter. For murine analyses, 8-week-old male ICR mice were purchased from National Laboratory Animal Center, all experiments were approved by IACUC committee of National Taiwan University .n = 4) were collected. After collection, both porcine and murine samples were divided for immunohistochemistry, immunoblotting analysis, and protein purification.For porcine analyses, reproductive  and non-reproductive organs  were collected from slaughterhouse. Tissue samples were immediately transferred onto dry ice before further storage at −80 °C. For murine analyses, seminal vesicles and caput epididymis from 8-week-old male ICR mice (v/v) hydrogen peroxide (H2O2) for 30 min at room temperature (RT). To minimize non-specific signals, sections were incubated with 2.5% filtered normal goat serum (NGS) diluted in Tris-buffered saline  for 30 min, RT. Anti-QSOX2 antibody was used at a dilution of 1:100 and anti-QSOX1 antibody was in 1:200 dilution as recommended by the company datasheet. Polymer-HRP reagent (BioGenex HRP kit) was used as secondary antibody after intensive washes of sections with TBS. Reactions were developed with 2% diaminobenzidine  for 10 min and slides were counter stained with hematoxylin  for 30 s.For histo-chemical analysis, representative tissue samples were collected, fixed in 10% neutral buffered formalin, processed routinely for paraffin blocks and sectioned at 10 μm. Hematoxylin and eosin (H&E) stain was used for general histological and morphological examinations. For IHC staining, tissue sections were deparaffinized with 100% xylene and rehydrated with 100%–80% ethanol. Antigen retrieval was carried out by heating tissue sections in 10 mM citrate buffer (pH 6.0) using regular microwave at 95 °C for 2 cycles. Endogenous peroxidase was subsequently removed by incubating tissue sections with 3%  at room temperature, blots were incubated with primary antibody (1:250 diluted in TBST) for overnight at 4 °C. After three times washing with TBST, secondary antibody was subsequently added and blots were further incubated at RT for 1 h. After rinsing with TBST, protein was visualized by using chemiluminescence . When necessary, blots were stripped (Thermo Scientific Restore Western Blot Stripping Buffer) and re-probed for other proteins of interests.Immunoblotting procedures were followed as previously described . For pro4)2SO4) was used for total protein precipitation from tissue homogenates of epididymis and seminal vesicle mixture, procedures were followed by company’s instructions (BioVision Inc.). Cells or tissue debris were span down by centrifugation at 3000 g for 10 min prior to precipitation processes. Supernatant was mixed in 1:1 ratio with phosphate buffered saline (PBS) in the presence of 1 M Tris-HCl. Proteins were salted out using 10%, 25%, 40%, 60%, 80% and saturated (100%) ammonium sulfate in this study. Sample homogenates were mixed and stirred with different percentages of ammonium sulfate for 15 min and centrifuged at 15,000 g (20 min at 4 °C). Protein pellets obtained from each ammonium sulfate concentration were re-dissolved in PBS and further quantified for total protein concentration as described earlier.Ammonium sulfate , mixed thoroughly with 50 μl Magnetic Protein A Dynabeads® on an end-to-end rotor for 30 min at RT to form beads-Ab complex. After washing with washing buffer (provided in the kit), tissue homogenate mixtures  were incubated with beads-Ab complex for 1 h at RT for allowing QSOX2 to bind to the preformed beads-Ab complex. Beads-Ab-QSOX2 complexes were subsequently separated from unbound proteins and supernatant on the magnet holder. Elution of the target antigen (QSOX2) was followed by adding 30 μl of elution buffer (provided in the kit) for 10 min at RT. Elute was quantified for protein concentration and stored at −80 °C in the presence of protease inhibitor as described above.Sus scrofa).To investigate the presence and tissue distribution of QSOX2 in porcine, IHC studies were carried out. Our results demonstrated that QSOX2 was present in most of the tissues tested including in heart, lung, liver, spleen, small intestine and kidney. In line with previous reports from rodents or human, no signal can be detected in skeletal muscle; however, unlike in humans, we observed a positive signal in porcine heart Fig. , even thmarked with arrows). This pronounced and specific epithelium staining pattern is similar to our observations in mouse reproductive tracts . In contrast to a single band detected in other tissue homogenates from non-reproductive organs, two distinct bands at 50kD and 75kD were detected in both epididymis and seminal vesicles 2SO4). From Fig. upper panel), proteins were mostly precipitated when 10%–80% ammonium sulfate were used. Western-Blotting analysis of the precipitates revealed that the long form QSOX2 (at 75 kDa) could be precipitated using 10%–25% (NH4)2SO4 while the short form QSOX2 (at 50 kDa) could be precipitated using 40% (NH4)2SO4. We further performed immunoprecipitation using commercially available anti-QSOX2 antibody. From SDS page of immunoprecipitated samples, clear bands were visible at 75 kDa, 68 kDa and 50 kDa . As skeletal muscle was considered negative for QSOX2 in porcine species based on our data presented above , we detected a 2.4 and 2.2-fold increase in 50 kDa signal in seminal vesicle (yellow bar) and epididymis (grey bar), respectively as compared to the signal detected in skeletal muscle. For long form porcine QSOX2 detected at 75 kDa, a 1.3 and 1.6 fold increase was measured in seminal vesicle (yellow bar) and epididymis (grey bar), respectively. This was further confirmed by Western-Blotting analysis from the seminal vesicle and epididymis tissue mixture immuno-precipitates, which showed two major bands at 50 kDa and 75 kDa in the eluted fraction. Interestingly, under the same elution condition, a large portion of long form QSOX2 remained on the bead while most of the short form QSOX2 was eluted suggesting biochemical differences between two porcine QSOX2 isoforms  and biochemical properties (contained both long and short form), which are not the same as the rest of tissues examined Fig. . To incrkDa Fig. . In our ove Fig. , b, we crms Fig. .Fig. 4TwAfter the spermatozoa are liberated from testis, membrane surface modifications continue with constant re- and de-coating events induced by male accessory fluid , 29. TheCebuscapucinus imitator, white headed capuchin, chimpanzee and gray short-tailed opossum (NCBI data base). It is reasonable to predict that in boars, tissues or organs with less or no secretion function may consist mainly of transmembrane domain containing QSOX2, exhibiting a higher protein molecular weight at ~75 kDa; whereas organs with exuberant secretory activities like seminal vesicles and epididymis contain besides transmembrane QSOX2, an additional secretory form of QSOX2 with a smaller molecular weight due to the lack of transmembrane domain as seen from our Western-blotting analysis  in porcine species and further showed its ubiquitous tissue distributions in heart, lung, liver, spleen, small intestine, kidney, testis, seminal vesicle and epididymis, except in skeletal muscle Fig. . This wiis Figs. . TherefoFunctional analyses of QSOX2 rely on the purity of the protein. However, compared with QSOX1 expression, QSOX2 expresses a much lower level in most of the human tissues . To overAs protein composition of porcine seminal fluid is complex and contains many proteins other than QSOX2 , 40, to In conclusion, in this study, we showed for the first time, the presence of QSOX2 in porcine species, and porcine QSOX2 exhibits two isoforms which may represent transmembrane  and secretory  forms of QSOX2; moreover, both QSOX2 can further be purified via immunoprecipitation using specific antibody. Our data not only provides evidence on the identification of QSOX2 in porcine species, but also indicates a new source and approach for QSOX2 purification for future functional analyses.QSOX2 is enriched in porcine epididymis and seminal vesicles and obtaining sufficient amount of QSOX2 is critical for in-depth functional analyses of QSOX2. Porcine materials from the slaughterhouse serve as ideal sources for protein purification without additional sacrifice of individuals. Large-scale QSOX2 protein purification and accompanied functional studies will shed lights on functional relevance of QSOX2 on sperm physiology.


However, the presence of a sustained chronically active cytotoxic T-cell response to HTLV-1 antigens in virtually all HTLV-1-infected individuals, regardless of their proviral load, argues against complete latency of the virus in vivo. There is an immediate burst of plus-strand transcription when blood from infected individuals is cultured ex vivo. How is the HTLV-1 plus strand silenced in PBMCs? Is it silenced in other anatomical compartments within the host? What reactivates the latent provirus in fresh PBMCs? While plus-strand transcription of the provirus appears to be intermittent, the minus-strand hbz transcripts are present in a majority of cells, albeit at low levels. What regulates the difference between the 5′- and 3′-LTR promoter activities and thereby the tax-hbz interplay? Finally, T lymphocytes are a migratory population of cells that encounter variable environments in different compartments of the body. Could these micro-environment changes influence the reactivation kinetics of the provirus? In this review we discuss the questions raised above, focusing on the early events leading to HTLV-1 reactivation from latency, and suggest future research directions.HTLV-1 plus-strand transcription begins with the production of doubly-spliced  In ∼5–10% of the infected individuals, HTLV-1 infection leads to either an aggressive T-cell malignancy, adult T-cell leukemia/lymphoma (ATL), or a chronic progressive neuro-inflammatory condition called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Both these conditions have limited treatment options, and ATL in particular carries a very poor prognosis.Human T-lymphotropic virus type 1 (HTLV-1), also known as Human T-cell leukemia virus type 1, is a retrovirus that mainly infects CD4+ T-cells 4 to 105 different T-cell clones, each with a unique proviral integration site  to devise effective preventive and therapeutic approaches against HTLV-1-associated diseases such as HAM/TSP and ATL.A peculiar characteristic of HTLV-1 is the absence of detectable cell-free virions in infected individuals . HTLV-1 ividuals . This obcis-perturbation of these genes, perhaps contributing to leukemogenesis  situated at each end of the genome.in vivo: p30 and p13 promote infectivity in a rabbit model ], DNA methylation inhibitors  and histone methyltransferase inhibitors  . Similarex vivo. The importance of glucose in the HTLV-1 life cycle is emphasized by the fact that the glucose receptor GLUT-1 is a cellular receptor for HTLV-1 , PBMCs in peripheral venous blood are exposed to significantly higher levels of oxygen (∼6–10%)  . We rece(∼6–10%) . Again, The transcriptional regulation of the plus- and minus-strands of HTLV-1 is affected by a number of virus-specific and host-specific factors. Advances in single-cell genomics and transcriptomics have made it possible to study HTLV-1 infection at the single-cell level. We anticipate that these approaches will shed light on the effect of cellular heterogeneity on viral replication and help identify novel mechanisms that were missed by earlier studies which depended on population-level analyses. Precise identification of the molecular mechanisms involved in HTLV-1 reactivation from latency would help us design and test novel therapeutic strategies to reduce the disease burden associated with this infection.Both authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.