Dactylellina , Rhizophydium , Clonostachys , Pochonia  and Purpureocillium ) co-occurred in the soybean rhizosphere at a small spatial scale. This study suggests how ‘consortia’ of anti-nematode microbes can derive from indigenous (resident) microbiomes, providing basic information for managing anti-nematode microbial communities in agroecosystems.Cyst and root-knot nematodes are major risk factors of agroecosystem management, often causing devastating impacts on crop production. The use of microbes that parasitize or prey on nematodes has been considered as a promising approach for suppressing phytopathogenic nematode populations. However, effects and persistence of those biological control agents often vary substantially depending on regions, soil characteristics and agricultural practices: more insights into microbial community processes are required to develop reproducible control of nematode populations. By performing high-throughput sequencing profiling of bacteria and fungi, we examined how root and soil microbiomes differ between benign and nematode-infected plant individuals in a soybean field in Japan. Results indicated that various taxonomic groups of bacteria and fungi occurred preferentially on the soybean individuals infected by root-knot nematodes or those uninfected by nematodes. Based on a network analysis of potential microbe–microbe associations, we further found that several fungal taxa potentially preying on nematodes ( Soybearmlands ,6. Howevarmlands , causingTo reduce damage by cyst and root-knot nematodes, a number of studies have evaluated effects of crop varieties/species, crop rotations, fertilizer inputs and tillage intensity on nematode density in farmland soil ,8–10. HoPurpureocillium and Pochonia) were more abundant in the long term than in short-term monoculture fields of soybeans [Based on the technological advances in high-throughput DNA sequencing, more and more studies have examined structures of microbial communities (microbiomes) in order to evaluate biotic environmental conditions in the endosphere and rhizosphere of plants –16. Recesoybeans . Such amsoybeans , there cGlycine max) field and then examined how root and rhizosphere microbiome structures varied among host plant individuals that differed in damage by root-knot nematodes (Meloidogyne sp.). Based on the data of microbiomes at a small spatial scale, we statistically explored microbial species/taxa that occurred preferentially in the roots or rhizosphere soil of nematode-infected soybean individuals. We further investigated the structure of networks depicting co-abundance patterns of microbial species/taxa within the soybean field, thereby examining whether multiple anti-nematode bacteria and fungi form consortia (assemblages) on/around the plant individuals infected by root-knot nematodes. Our results suggest that various taxonomic groups of anti-nematode bacteria and fungi are present within indigenous microbiomes. This study also suggests that microbiome assembly at fine spatial scales is a key to managing populations and communities of such functional microbes.We conducted an Illumina sequencing analysis of bacteria and fungi in a soybean (2.2.1.2O5 : K2O = 3 : 10 : 10 g m−2). In the lines, 69 and 62 individuals , respectively, were sampled at every other position . Among them, ‘green’ individuals exhibited normal growth, while ‘no leaf’ individuals were heavily infected by root-knot nematodes: ‘yellow’ individuals showed intermediate characters. In total, 97 ‘green’, 19 ‘yellow’ and 15 ‘no leaf’ individuals were sampled (figure 1d).Fieldwork was conducted at the soybean field on the Hokubu Campus of Kyoto University, Japan . In the field, the soybean strain ‘Sachiyutaka’ was sown at 15 cm intervals in two lines  on 4 July 2016 .For each individual, two segments of 5-cm terminal roots and rhizosphere soil were collected from around 10 cm below the soil surface. The root and soil samples were transferred into a cool box in the field and then stored at −80°C until DNA extraction in the laboratory. The above-ground bodies of the individuals were placed in drying ovens at 80°C for 72 h to measure dry mass. The dry mass data indicated that ‘green’, ‘yellow’ and ‘no leaf’ soybean individuals differed significantly in their biomass c.2.2.The root segments of each individual were transferred to a 15 ml tube and washed in 70% ethanol by vortexing for 10 s. The samples were then transferred to a new 15 ml tube and then washed again in 70% ethanol by sonication (42 Hz) for 5 min. After an additional sonication wash in a new tube, one of the two root segments was dried and placed in a 1.2 ml tube for each soybean individual. DNA extraction was then performed with a cetyltrimethylammonium bromide method  after puFor DNA extraction from the rhizosphere soil, the ISOIL for Beads Beating kit (Nippon Gene) was used as instructed by the manufacturer. For each sample, 0.5 g of soil was placed into a 2 ml microtube of the ISOIL kit. To increase the yield of DNA, 10 mg of skim milk powder  was added to each sample .−1 [−1) and a final extension at 68°C for 5 min. The PCR amplicons of the 131 soybean individuals were then pooled after a purification/equalization process with the AMPureXP Kit (Beckman Coulter). Primer dimers, which were shorter than 200 bp, were removed from the pooled library by supplemental purification with AMPureXP: the ratio of AMPureXP reagent to the pooled library was set to 0.6 (v/v) in this process.For each of the root and soil samples, the 16S rRNA V4 region of the prokaryotes and the internal transcribed spacer 1 (ITS1) region of fungi were amplified. The PCR of the 16S rRNA region was performed with the forward primer 515f  fused wi−1 . Illumin−1  and a paThe PCR of fungal ITS1 region was performed with the forward primer ITS1F_KYO1  fused wiThe sequencing libraries of the prokaryote 16S and fungal ITS regions were processed in an Illumina MiSeq sequencer . Because the quality of forward sequences is generally higher than that of reverse sequences in Illumina sequencing, we optimized the MiSeq run setting in order to use only forward sequences. Specifically, the run length was set at 271 forward (R1) and 31 reverse (R4) cycles in order to enhance forward sequencing data: the reverse sequences were used only for discriminating between 16S and ITS1 sequences based on the sequences of primer positions.2.3.The raw sequencing data were converted into FASTQ files using the program bcl2fastq 1.8.4 distributed by Illumina. The output FASTQ files were demultiplexed with the program Claident v0.2.2017.05.22 ,32, by wk-nearest neighbour (QCauto) method [For each dataset of 16S and ITS1 regions, filtered reads were clustered with a cut-off sequencing similarity of 97% using the program VSEARCH  as imple) method  and the ) method  as imple) method ,35,36. Ia sample × OTU matrix, in which a cell entry depicted the number of sequencing reads of an OTU in a sample . The cell entries whose read counts represented less than 0.1% of the total read count of each sample were removed to minimize effects of PCR/sequencing errors [For each combination of target region (16S or ITS1) and sample type (root or soil), we obtained g errors . The filg errors  of R 3.4g errors . Samples2.4.Relationship between the number of sequencing reads and that of detected OTUs was examined for each dataset  with the ‘rarecurve’ function of the R vegan package. Likewise, relationship between the number of samples and that of OTUs was examined with the vegan ‘specaccum’ function. For each dataset, difference in OTU compositions among ‘green’, ‘yellow’ and ‘no leaf’ soybean individuals was examined by the permutational analysis of variance  with th2.5.i) for a plant state (j)  as follows:Nobserved  denoted the mean number of the sequencing reads of OTU i among state j soybean samples in the original data, and the Mean ) and s.d. ) were the mean and standard deviation of the number of sequencing reads for the focal OTU–plant state combination across randomized matrices. Regarding this standardized preference index, values larger than three generally represent strong preferences  < 0.05; see results of a previous study [To explore prokaryote/fungal OTUs that preferentially occurred on/around ‘green’, ‘yellow’ or ‘no leaf’ soybean individuals, a randomization test was performed by shuffling the plant state labels in each of the root prokaryote, root fungal, soil prokaryote and soil fungal data matrices (100 000 permutations). We then evaluated preference of a prokaryote/fungal OTU (us study ): hence,2.6.To examine how prokaryote and fungal OTUs co-occurred in root or soil samples, a co-abundance network analysis was performed based on the sparse inverse covariance estimation for ecological association inference (Spiec-Easi) method . In each3.3.1.On average, 107.9 (s.d. = 18.0), 25.4 (s.d. = 8.9), 172.5 (s.d. = 17.3) and 78.3 (s.d. = 10.5) OTUs per sample were observed, respectively, from the root prokaryote, root fungal, soil prokaryote and soil fungal dataset after filtering and rarefaction steps . The total number of OTUs observed was 1387, 346, 1191 and 769 for the root prokaryote, root fungal, soil prokaryote and soil fungal datasets, respectively .a). The fungal community of roots was dominated by the fungal orders Hypocreales, Sordariales, Plesporales, while that of soil consisted mainly of Hypocreales, Agaricales, Eurotiales, Mortierellales and Filobasidiales (b). Regarding the order-level compositions of fungi in the rhizosphere soil, the proportion of Orbiliales reads was much higher in ‘yellow’ (3.62%) and ‘no leaf’ (4.82%) soybean individuals than in ‘green’ ones (0.89%)  . The genIn each dataset , microbial community structure varied among ‘green’, ‘yellow’ or ‘no leaf’ soybean individuals, although the effects of sampling sets on the community structure were much stronger . Even wi3.2.Dyella, Herbaspirillum, Labrys, Phenylobacterium, Gemmata, Chitinophaga, Pedobacter, Niastella and Streptomyces  and Proteobacteria (Kofleriaceae sp.), occurred preferentially on ‘green’ host individuals  and Dactylellina sp.  (Clonostachys (F_0257) and Dactylellina (F_0163) OTUs, respectively, represented 9.5% and 3.5% of the sequencing reads of ‘no leaf’ samples . The indices of preferences for ‘yellow’ soybean individuals are shown in electronic supplementary material, data S5.In the rhizosphere soil microbiome, seven prokaryote OTUs, including those belonging to Chloroflexi  . The rea3.3.c,d) than in the root microbiome data . Within the network representing co-abundance of microbes across root samples, the Clonostachys OTU (F_0257) had a significant link with a Streptomyces OTU, while Dactylellina was absent from the root microbiome network data (figure 4a). Within the positive co-abundance network of the rhizosphere soil microbiome (figure 4c), the Clonostachys (F_0257) and Dactylellina (F_0163) nematophagous fungal OTUs were connected with each other  and fungal OTUs in the genera Calonectria and Purpureocillium , which could capture juveniles of nematodes with hyphal traps [Within the soybean rhizosphere soil microbiome, diverse taxonomic groups of not only bacteria, but also fungi preferentially occurred around ‘no leaf’ soybean individuals . Among tulations ,51 potenulations  or extraulations , but intematodes ,55. An oematodes . The funematodes . Our anaal traps . Speciesal traps –61, ofteal traps –64.Clonostachys and Dactylellina highlighted bacteria and fungi that frequently co-occurred with the nematophagous fungi  have been known to suppress plant parasitic nematodes, insect pests and oomycete phytopathogens [Pochonia  has been known as nematophagous as well and they can kill eggs and females of root-knot (Meloidogyne spp.) and cyst (Globodera spp.) nematodes [Rhizophydium include species that use nematodes as parasites or saprophytes [An additional analysis focusing on us fungi . In the  samples . In the patterns . Moreoveophydium . Among tathogens –68. Anotematodes –72. Specrophytes ,74. Theyrophytes . All therophytes ,37,75. Hrophytes .Figure Calonectria, which causes leaf blight, wilt and root rot of various plant species [Calonectria may have infected host soybeans earlier than root-knot nematodes, followed by the emergence of nematodes and their exploiters (i.e. anti-nematode microbes). Given that fungi can interact with each other both antagonistically and mutualistically in the soil [Calonectria and nematophagous fungi in the genera Clonostachys, Dactylellina, Purpureocillium, Pochonia and Rhizophydium are of particular interest. Studies examining potential interactions involving soybeans, root-knot nematodes, anti-nematode bacteria/fungi and Calonectria will help us understand ecological processes that structure consortia of nematophagous fungi.Along with the consortia of anti-nematode microbes, an OTU in the genus  species ,77, was the soil ,79, direPseudomonas, Purpureocillium and Pochonia), potentially resulting in lowered densities of cyst nematodes [d), tillage can promote the spread of plant damaging nematodes [Although this study did not evaluate potential effects of background environmental conditions (e.g. soil pH and inorganic nitrogen concentration) on microbiome structure, management of edaphic conditions are expected to have great impacts on dynamics of anti-nematode microbiomes. A number of studies have explored ways to suppress nematode populations by optimizing cropping systems . Crop roematodes . Tillageematodes –11 and iematodes –84 have ematodes . Frequenematodes ), but suematodes ,21), morin situ without introducing exogenous ones depending on base compositions and conditions of indigenous microbiomes. In this respect, design of cropping systems  is of particular importance in activating and maximizing ecosystem functions that stem from resident microbial diversity [We herein found that consortia of anti-nematode bacteria and fungi could develop at a small spatial scale within a field of soybeans infected by root-knot nematodes. Given the diversity of those anti-nematode microbes observed in this study, multiple biological control agents are potentially available iversity . Becauseiversity  for revi


P < 0.001) and was an independent risk factor for EOCAD . The early-onset myocardial infarction patients with 3-vessel disease had higher serum UA levels than those with 1- or 2-vessel disease. The serum UA levels of EOCAD patients with acute coronary syndrome were significantly higher than those with chronic coronary artery disease. EOCAD patients with hyperuricemia had higher Gensini scores than those without hyperuricemia. In addition, the serum UA levels were affected by drinking (P < 0.01) and were positively correlated with serum creatinine (r = 0.323) and weight (r = 0.327). Our results show that serum UA was an independent risk factor for EOCAD. The serum UA levels were associated with the presence and severity of EOCAD and suggested that UA may be involved in the progression of EOCAD.Serum uric acid (UA) is the final product of purine metabolism in humans. The present study is aimed at identifying the potential association between serum UA and early-onset coronary artery disease (EOCAD). The study population consisted of 1093 EOCAD patients aged ≤50 years, and 1117 age- and sex-matched apparently healthy people served as controls. The concentrations of UA were measured by uricase method. The severity of CAD was evaluated by Gensini score. The mean serum level of UA was 5.843 ± 1.479 mg/dl in EOCAD patients and 5.433 ± 1.529 mg/dl in controls. Serum UA levels were significantly higher in the EOCAD group than those in the control group ( Uric acid (UA) is the end product of purine nucleotide metabolism, formed from the breakdown of adenosine and guanine . UA is aNitric oxide (NO) plays important roles in the regulation of vascular tone and structure . DecreasMTHFR gene C677T polymorphism was associated with risk of early-onset myocardial infarction (MI) but not in late-onset MI in Caucasians [Compared with late-onset coronary heart disease, early-onset coronary heart disease (EOCAD) has particular components of etiology, including family heredity, lipid metabolism, gender composition, and risk factors –13. For ucasians . Many prn = 1117), free from any signs or symptoms of cardiovascular disease, served as control. They were recruited from a geographic background similar to that of the patients and came from community samples or hospital employees. Written informed consent was obtained from each patient included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was conducted in accordance with the guidelines set by the Ethics Committee of The Affiliated Hospital of Qingdao University.This study was a hospital-based case-control study conducted in patients attending The Affiliated Hospital of Qingdao University from January 2013 to June 2017. The 1093 EOCAD patients were aged ≤50 years in their first onset of symptoms and hospitalization for coronary angiography. All patients fulfilled the criteria of stable chest pain and/or signs of myocardial ischemia on exercise electrocardiography. EOCAD was assessed by review of patients' angiograms by their treating cardiologists. Patients having history of chronic infection diseases, abnormal kidney function, pregnancy, and neoplastic disease and patients on treatment with drugs  were excluded from the study. Age- and sex-matched apparently healthy people , age at onset of CAD symptoms, MI, hypertension, diabetes mellitus, and dyslipidemia were recorded. Height and weight were recorded to the nearest 0.5 cm and 0.1 kg, respectively. The BMI was calculated by using the formula: body weight in kilograms divided by height in meters squared. Systolic and diastolic blood pressure was measured twice at an interval of 30 min using the automated oscillometric device. An average value of the two readings was considered. Diagnosis of hypertension was based on the presence of elevated systolic (≥140 mmHg) and/or diastolic (≥90 mmHg) blood pressure. Diabetes mellitus (DM) was diagnosed when the subject casual fasting glucose level ≥ 7.8 mmol/l, ≥11.1 mmol/l at 2 h after an oral glucose challenge or both.Coronary angiography was used to identify the number of diseased vessels in MI patients. Four major coronary artery branches  were evaluated, and a luminal stenosis degree of 50% or more was defined as a significant lesion. Patients were defined as having single, double, or triple branch involvement if they had one, two, and three or more branches involved, respectively.The severity of CAD was calculated for each patient by the Gensini score system. The Gensini score is a point scale based upon the number of stenotic coronary artery segments, including the degree of luminal narrowing (the score was from 1 to 32) and the localization of the stenosis (the score was from 0.5 to 5). Thus, the Gensini score is calculated as a sum of stenosis score and functional significance score calculated for each segment of the coronary artery tree.Blood samples were drawn from all participants after an overnight fasting of at least eight hours. Serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and serum creatinine (SCr), Lipoprotein (a) (Lp (a)), and uric acid (UA) were determined by using the automatic biochemistry analyzer .t-test or one-way ANOVA for normally distributed parameters. Spearman correlation coefficients were used to discern interrelationships. Logistic regression was used to test the interactive effects of other variables on the observed association between serum UA and EOCAD. All tests were two-sided, and throughout, P < 0.05 was considered statistically significant.All data were analyzed with SPSS statistical software . Values are means ± standard deviation (SD) if not otherwise specified. The distribution of categorical variables was expressed as frequencies and percentages and the comparisons calculated by using chi-square test or Fisher exact test, as appropriate. Comparisons between groups for study variables were done using the unpaired Student's A total of 1093 EOCAD patients and 1117 controls matched for age and gender were enrolled into the study. The demographic and clinical characteristics of the study population are shown in P < 0.01, P > 0.05). The smoking status has no effect on the serum UA levels in male control  and EOCAD groups . In addition, we found that serum UA levels were weakly correlated with SCr , whereas they displayed lower concentrations of HDL-C. Specifically, both groups had similar gender composition, age, serum TC levels, and SCr levels. In addition, the patients had higher smoking and drinking rate compared with controls. 633 patients were diagnosed as MI in the EOCAD group, including 326 patients with 1-vessel disease, 199 patients with 2-vessel disease, and 108 patients with 3-vessel disease. The drinking status could significantly increase the serum UA levels in the male control group . After adjustment for Glu, LDL-C, SCr, TG, TC, HDL-C, and Lp (a), UA remained as a significant factor that correlated with EOCAD . After further adjustment for all these factors, UA remained as an independent risk factor for EOCAD   .n = 101), 5.886 ± 1.505 mg/dl (n = 344), and 5.880 ± 1.479 mg/dl (n = 633), respectively. The serum UA levels in patients with stable angina were significantly lower than those in patients with unstable angina and MI , 5.799 ± 1.503 mg/dl (n = 199), and 6.322 ± 1.554 mgl/dl (n = 108) in MI patients with 1–3-vessel disease. We detected significant difference in the three groups . Levels of serum UA in the group with 3-vessel disease were significantly higher than those in groups with 1-vessel disease and 2-vessel disease  and 48.25 ± 37.23 in male EOCAD patients without hyperuricemia  and only 26.22 ± 22.50 in female EOCAD without hyperuricemia  the serum UA levels were significantly elevated in EOCAD patients, and the serum UA was an independent risk factor for EOCAD. (2) Serum UA levels were associated with disease severity. (3) The serum UA levels were influenced with drinking status and were positively correlated with SCr and weight.UA is a final product of purine metabolism, formed from the breakdown of adenosine and guanine. The transformation of hypoxanthine into xanthine and of the latter into UA is a biochemical chain that leads to UA generation. The processes are catalyzed by the enzyme xanthine oxidase . UA is dThe positive association between serum UA and cardiovascular disease was recognized for 60 years. A number of epidemiologic studies have reported a relation between serum UA levels and a wide variety of cardiovascular conditions, including hypertension , metabolMTHFR gene C677T polymorphism was associated with the risk of early-onset MI in Caucasians  [To compare with late-onset CAD, EOCAD may have different pathogenic mechanisms. Numerous studies have discovered so many biomarkers on EOCAD, including genes, proteins, and other bio-macromolecules , 28. Our7–1.509) , and ser7–1.509) . To our 7–1.509) . Zand et7–1.509) . Dai and7–1.509) . In our The current study should be considered as a preliminary report which has some limitations. First, a control group comprised age- and sex-matched individuals without any signs or symptoms of CAD and having a normal result of routine blood tests. We cannot exclude that since coronary angiography has not been performed in all the controls. Second, the findings are based on cross-sectional data and only measured serum UA at first hospital admission. A temporal association between serum UA and development of EOCAD cannot be inferred with certainty.In conclusion, we observed that increased serum UA levels were associated with EOCAD and first indicated that serum UA is an independent risk factor for the disease. We also demonstrated that the serum UA levels were associated with disease severity, affected with drinking status, and were positively correlated with SCr and weight. The findings may be useful in understanding the mechanism and predicting progression of patients with EOCAD and may contribute to the further prevention of the disease.


Being Present single arm pilot study, we designed and tested an 8-week audio-based mindfulness meditation program for patients with metastatic colorectal cancer receiving chemotherapy with or without a participating caregiver. The study accrued 33 of 74 (45%) eligible patients consenting together with 20 family caregivers  within nine months. Forty-one participants were evaluable (77%); 10 of 12 cases of attrition were attributable to hospitalization or death. Median participant age was 51 (range 21–78 years); 38% were men. Baseline levels of distress were similar in patients and caregivers. The top reasons for participation cited in pre-intervention interviews were to increase relaxation/calm, improve mood/emotions, and reduce stress/anxiety. In measures of adherence, 59% of responses to weekly texts asking: “Have you practiced today?” were “Yes” and 59% of interviewees reported practicing >50% of the time. Compared to baseline, post-intervention surveys demonstrated significantly reduced distress (p = 0.01) and anxiety (p = 0.03); as well as increased non-reactivity (p<0.01), and feeling at peace (p<0.01). Post-intervention qualitative interviews, where 71% of participants reported benefit, were consistent with quantitative findings. In the interviews, participants spontaneously described reduced stress/anxiety and increased relaxation/calm. Benefits appeared to be accentuated in patient-caregiver pairs as compared to unpaired patients. Seventy-nine percent of participants reported plans for continued practice after study completion. We conclude that the Being Present audio-based mindfulness meditation program is of interest to, feasible, and acceptable for patients with metastatic colorectal cancer and caregivers, with initial evidence of efficacy. These results will guide plans for a follow-up study.A metastatic cancer diagnosis is associated with high levels of distress in patients and caregivers. Mindfulness interventions can reduce distress and improve quality of life in cancer patients. However, standard mindfulness training relies on in-person instruction, which is often not practical for either patients receiving chemotherapy or their caregivers. In the Trial registration: ClinicalTrials.gov NCT02423720 A cancer diagnosis is associated with high levels of distress, leading to frequent anxiety, depression, fear of recurrence/progression, sleep disturbance, and fatigue in both patients and caregivers –8. The NWhile an estimated 35–45% of cancer patients report psychological distress , 12, disTo help cope with the distress associated with the diagnosis, disease symptoms, and treatment, individuals affected by cancer are increasingly turning to complementary and integrative medicine –30, inclTo overcome the challenges of physically attending mindfulness classes, recent studies have begun to explore the use of technology to replace face-to-face approaches to deliver mindfulness interventions –42. We pBeing Present study on patients with metastatic colorectal cancer receiving chemotherapy and their caregivers. Although an audio-based mindfulness meditation intervention may be useful to patients with other cancer types, we chose to focus the current study on metastatic colorectal cancer because it is the second leading cause of cancer mortality in both US men and women, accounting for an estimated 50,000 deaths each year. Also, patients with metastatic colorectal cancer and related intestinal malignancies tend to receive similar treatments and live for many months with relatively stable health   pain or suffering and go to another level and be able to see from outside. Mindfulness, the word that comes to me is empathy.” This discussion led to recognition of the need to provide clear definitions of meditation and mindfulness. Definitions were provided in an audio track introducing the Being Present intervention and in the printed study booklet.Both focus groups opened by asking participants to share any prior experience with mindfulness practices. In addition to meditation and yoga, participants cited walking, dancing, and prayer. Mention of these other modalities was incorporated into the intervention text messages and emails. Next, participants were asked what came to mind when they thought of meditation or mindfulness. Here, patients and caregivers provided a wide range of responses. Words associated with meditation included: calm/tranquility/relaxation; health; concentration/focus; consciousness; and blank mode/no thought. A participant who defined meditation as “putting your mind in blank mode” postulated:”I would think mind-Being Present includes a Progressive Muscle Relaxation exercise, which is not part of MBSR. Perceived barriers to practice for both patients and caregivers related to having busy lives.Next focus group participants were asked how they thought mindfulness meditation practice could be helpful. Stress relief and relaxation were answers common to both focus groups. A caregiver shared: “it provides a moment of time for yourself… I think it’s a nice little window to have a break and have a calm space to go to.” Patients cited potential utility for management of symptoms including: intrusive thoughts and emotions (e.g. fear), pain, nausea, insomnia, and muscle tension. Based on these responses, The intervention was then demonstrated, and feedback on the schedule, delivery, and content was requested. There was consensus that an expectation of 20 minutes of practice, 5 days per week without in-person visits was feasible. Both emails and daily text messages were acceptable, with a preference to provide short responses via text. Refinements to the intervention content resulting from the focus groups included the incorporation of quotes from the focus group participants into weekly emails to intervention participants . AdditioRecruitment occurred over nine months (August 2015-May 2016). Eighty-one patients were invited by letter or in clinic . ReasonsEight patients and four caregivers who consented to participate, and in some cases completed baseline assessments, were not evaluable because they completed no on-study assessments . Compared to evaluable patients, non-evaluable patients had poorer health status, with statistically significantly worse performance status, more hospitalizations and deaths . Six nonDemographics of the focus group and intervention participants were comparable, including widespread technology use . Both paIn the pre-intervention semi-structured interviews, prior exposure to meditation ranged from none (N = 17) to having a regular meditation practice in the past (N = 2). Participants shared a wide variety of spontaneously reported reasons for participation or perceived benefits of meditation . Many ofIn post-intervention interviews, 71% of participants reported some form of benefit in response to open-ended questions about effects of study participation . The mosd effect size (d) for change in distress was 0.51 , anxiety (p = 0.03), and fatigue (p = 0.03); as well as significant improvement in the mindfulness facet, non-reactivity to inner experience (p<0.01); and feeling at peace (p<0.01) . The Cohwas 0.51 . There wwas 0.51 . The hiswas 0.51 . No statwas 0.51  Tables. was 0.51 . The effwas 0.51 .d = 1.1), anxiety , non-reacting , feeling at peace , as well as sleep disturbance   . There wIn post-intervention interviews, participants reported practicing at a variety of times and in different locations. One 40-year-old female patient explained:I was able to do it usually in the morning right after everybody left for school…. I was hoping I could fit it in in the evenings after everybody went to bed, but I just found I was too tired then and…it kind of felt like work at that point…I’m able to do it in the acupuncture chair, and then I used it a couple times when I had [a chemotherapy] treatment at the infusion center.d = 0.77), global mental health  and feeling at peace  were observed among participants who reported practicing at least “most of the time” (>50%) and who completed both baseline and week 8 surveys (N = 12), whereas changes did not reach statistical significance in survey respondents who reported ≤50% adherence or who did not complete the exit interview , with another 29% reporting about 50% adherence to the instruction to practice 5 times per week for 8 weeks . Signifinterview  Tables.Text messaging was another method used to estimate adherence. There were 36 unique responders out of 41 evaluable participants (89%). We received a median of 6 responses per participant to the 13 questions sent via text (46% response rate). Congruent with the qualitative interview data, 59% of respondents answered “Y” (yes) to the question “Have you practiced today?”, which was asked 9 times over the 8 weeks. Response rates and content were consistent over the course of the study. In week 6, we texted “How many days did you practice mindfulness meditation this week?”–the median answer was 4 (range 2–6) . QuestioThe most frequently reported barrier to full participation was allocating time, and this was more common among caregivers vs. patients (71% vs. 41% of interviewees). Thirty-five percent of patients reported that illness or chemotherapy side-effects got in the way of practice. A third of participants cited issues associated with the MP3 player, including track numbering confusion; automatic continuation onto the next track; and battery charging, as hindrances . A 23-yeBeing Present intervention made a lasting impact on their daily life; and 10 of 14 (71%) reported that since the conclusion of the study they had made a conscious effort to practice forms of mindfulness such as meditation and yoga.Seventy-nine percent of interviewees reported plans for continued practice and 14 participants submitted 3-month follow-up surveys. On the 3-month follow-up survey, 9 of 14 (64%) respondents indicated that participating in the Being Present, specifically stress around not feeling “like I was doing it right,” guilt about not practicing when “you know you should be,” and, in a couple of instances, not understanding the material  and sleep disturbance (paired participants). However improved sleep was not mentioned in post-intervention interviews. Of note, the NCCN Distress Thermometer is intended for patients and “Are you at peace” was designed to probe spiritual concerns at end of life. These instruments were not intended for caregivers. Yet we found comparable distress levels in patients and caregivers, and emotional distress in particular may be even higher in caregivers, corroborating previous findings –6.Being Present. As patients are living longer with metastatic cancer, more attention should be paid to distress in both patients and caregivers  could be one of the first opportunities where you’re both going through the same thing… It’s a positive shared experience … It’s a different focus, a healthy focus.  with a Patient and Caregiver Advisory Council. Recruitment and diversity may be augmented by expanding the eligibility criteria to include other cancer types; conducting a multi-center trial; translating study materials; and use of social media. A waitlist control, which has been used in studies of cancer survivors [Based on what we learned from urvivors , 40, wouBeing Present participants offered valuable insights that will guide the design of follow-up studies. One goal of the Being Present pilot was to gather information on technology use in our target population. In order to remove possible barriers to participation posed by access to/discomfort with technology, each participant was given an MP3 player. While some participants liked having a separate player for meditation, issues related to the MP3 player predominated. We learned that smartphone use, including mobile applications, was almost universal—irrespective of participant age Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOC)Click here for additional data file.S1 FigDesigned by Kameron Allen.(TIFF)Click here for additional data file.S2 FigN = 16. A linear regression line was created from the 3 points for each patient (blue lines) and overall (red dashed line). A) National Comprehensive Cancer Network (NCCN) Distress Thermometer ratings; B) National Institutes of Health Patient Reported Outcomes Measurement Information System (NIH PROMIS) Anxiety Short Form 4A scores; C) Five Facet Mindfulness Questionnaire Short Form (FFMQ-SF) “Non-React” scores; D) “Are You at Peace?" one-item spiritual probe ratings.(DOC)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 FileWith Institutional Review Board approval notification.(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Resistance training is often recommended for combined increases in traditional and alternative hamstrings-to-quadriceps (H:Q) ratios in order to reduce knee strength imbalance and associated hamstrings and knee ligament injury risk. The aim of this study was to investigate the effect of different concentric and eccentric resistance training programs on traditional and alternative H:Q ratios. Forty male volunteers were assigned to one of 4 groups: concentric quadriceps and concentric hamstrings , eccentric quadriceps and eccentric hamstrings , concentric quadriceps and eccentric hamstrings , or no training (control (CNTRL), n = 10). Traditional conventional (CR) and functional (FR), alternative rate of torque development (RTD), muscle size (MS), and muscle activation (MA) H:Q ratios were measured before and after six weeks of unilateral nondominant knee extension–flexion resistance training performed on an isokinetic dynamometer. The ECC/ECC training significantly increased FR , whereas the lack of training (CNTRL) decreased the RTD H:Q ratio . There were no differences between groups for the other traditional and alternative ratios following resistance training protocols. These findings suggest eccentric exercise for quadriceps and hamstrings as the most beneficial training program for inducing increases in the traditional FR. However, different resistance training strategies may be needed to also elicit increases in the alternative RTD, MS, and MA H:Q ratios for fully restoring muscle balance and reducing potential hamstrings and knee ligament injury risk. Anterior cruciate ligament (ACL) and hamstring strain injuries are common lower extremity noncontact injuries in sports. These may occur when the hamstrings fail to produce enough strength to decelerate high anterior tibial shear and rotation movements induced by maximal quadriceps strength actions ,2,3,4. TTraditional H:Q strength ratios can be classified as conventional or functional based upon how they are calculated. The conventional ratio is calculated by the balance between hamstring and quadriceps concentric (CON) PT, whereas the functional ratio calculates balance between hamstring eccentric (ECC) and quadriceps CON PT. The CR is commonly used to describe the absolute muscle strength relationship between hamstrings and quadriceps ,3. In coAdditionally, since these traditional methods of calculating H:Q ratios only account for knee balance based on the maximum strength capacity , recent investigations have proposed alternative methods of determining H:Q ratios that consider balance between other neuromuscular variables that can influence the antagonist to agonist muscle relationship. This is the case of H:Q ratios calculated by rate of torque development (RTD) ,12,13,14Previous studies have suggested that future longitudinal investigations should be performed to provide greater insight about the most appropriated resistance training program for reducing potential injury risk as indicated by increased traditional and alternative H:Q ratios ,14,16. HForty healthy men  volunteered for this study. Participants reported participating in sport and recreational activities, but they did not perform any endurance or resistance training programs for 3 months prior to initiation of the study. This ensured participants had similar low levels of strength before participating in the study. Therefore, based on the classification used in previous studies ,21, partParticipants were randomly allocated to one of four groups (n = 10 participants per group): CON quadriceps and CON hamstring (CON/CON), ECC quadriceps and ECC hamstring (ECC/ECC), CON quadriceps and ECC hamstring (CON/ECC), or no training (control (CNTRL)). The three training groups performed six weeks of unilateral nondominant knee extension–flexion exercise on a Biodex isokinetic dynamometer . In thisAll pretesting sessions were performed on two separate occasions (separated by 48 h). On day one, participants were tested for quadriceps and hamstrings muscle thickness (MT), followed by isometric maximal testing, which included RTD and MA measurements. On day two, participants performed isokinetic quadriceps and hamstrings CON and ECC maximal testing for PT measurements. The training sessions started 72 h after the last pretesting.lh), semitendinosus (ST), and semimembranosus (SM) muscles of the nondominant leg using a real-time portable B-mode ultrasound device . For this, a linear array probe  was placed perpendicular to quadriceps and hamstrings muscles at 50% of the distance between the greater trochanter and the lateral condyle of the femur, except for the VM, which was at 30% of this same distance [lh), 0.69% and 0.48% (ST), and 0.52% and 0.44% (SM). Previous studies have determined muscle thickness (MT) as a measure of muscle size (MS) [lh + ST + SM MT, and quadriceps MS was considered as the sum of RF + VI + VL + VM MT [Participants laid supine on a table with legs relaxed for 10 min in order to allow body fluids to stabilize. An experienced researcher measured MT images of quadriceps rectus femoris (RF), vastus intermedius (VI), vastus lateralis (VL), and vastus medialis (VM) and hamstrings biceps femoris long head  . Therefo + VM MT . These vMaximal quadriceps and hamstrings isometric strength for the nondominant leg was measured on a Biodex System 2 isokinetic dynamometer . Participants sat comfortably on the seat of the machine, having straps across chest, thighs, and hips. The lateral femoral condyle of the test leg was aligned to the machine’s axis of rotation. The ankle was attached to the lever arm by an ankle cuff positioned slightly above the medial malleolus ,30,31. Plh muscles for MA measurements. The same anatomical sites used for ultrasound measurements were used for placement of electrodes. The areas surrounding these sites were shaved, abraded, and cleaned, and electrodes were replaced if high levels of noise were identified, which may indicate high skin impedance. Raw EMG signals were filtered  and amplified (bandwidth = 1–500 Hz amplifier) using a Myopac EMG device  with a sampling rate of 1 kHz. The calculation of the root mean square (RMS) was performed over a 1 s plateau  of each knee flexion and extension maximal isometric torque by using a custom LabVIEW . The average of 3 EMG values for each muscle action was considered for further analysis. The three pre- and post-testing EMG measurements had maximal CVs of 6.02% and 1.07% (quadriceps) and 1.69% and 6.26% (hamstrings).During the test, participants were fitted with 2 bipolar (3.5 cm center-to-center) disposable electromyography (EMG) surface electrodes  on their quadriceps VL and hamstrings BFParticipants were tested for maximal quadriceps and hamstrings CON and ECC muscle actions for the nondominant leg on the same Biodex isokinetic dynamometer and using the same procedures as the isometric maximal test. Before testing, participants performed a specific isokinetic warm-up consisting of 10 CON knee extension–flexion repetitions at 180°/s, followed by three repetitions at 60°/s through 90° of range of motion (0° = full extension). Testing consisted of 5 maximal CON and 5 ECC knee extension–flexion repetitions at 60°/s, separated by 2 min rest. Isokinetic torque data were collected using LabVIEW software (sampling rate: 1 kHz). The highest quadriceps and hamstrings CON and ECC PT values across all repetitions were considered for further analysis. lh) EMG by quadriceps (VL) EMG values.Traditional H:Q ratios were represented by CR and FR. CR was calculated by dividing hamstrings CON PT by quadriceps CON PT, whereas FR was calculated by dividing hamstrings ECC PT by quadriceps CON PT. Alternative ratios were represented by RTD, MS, and MA H:Q ratios. RTD H:Q ratios were calculated by dividing hamstrings RTD by quadriceps RTD values, MS H:Q ratios were calculated by dividing hamstrings MS by quadriceps MS values, and MA H:Q ratios were calculated by dividing hamstrings (BFThe training sessions were performed for 6 weeks (2 sessions a week separated by at least 48 h). The first training session began 72 h after the second day of pretesting. Participants sat on the same Biodex used for isokinetic testing, using the same set-up and warm-up procedures. The CON/CON group started the first week of training by performing 1 set of 10 maximal repetitions at 210°/s for quadriceps and hamstrings. The ECC/ECC group started the first week of training by performing 1 set of 10 maximal repetitions at 60°/s for quadriceps and hamstrings. The CON/ECC group started the first week of training by performing 1 set of 10 maximal CON repetitions at 210°/s for quadriceps and 1 set of 10 maximal ECC repetitions at 60°/s for hamstrings. The training intensity was increased every week by increasing the isokinetic angular velocity for eccentric and decreasing it for concentric in 30°/s increments ,31,32. APost-testing sessions were performed on two days (separated by 48 h) with the same order and procedures as the pretesting sessions. All tests were performed 72 h after the last training session.t tests and posthoc analysis (Fisher’s least significant difference), respectively. Data are expressed as mean and SD. An a priori alpha level of 0.05 determined statistical significance. Analyses were performed with SPSS 21.0 . Effect sizes were calculated by Cohens d. According to Rhea [Normality of data was verified by the Shapiro–Wilk test. A 2 × 2 × 2 × 4 (muscle × action × time × group) mixed factor ANOVA was used to compare PT. Three 2 × 2 × 4 (muscle × time × group) mixed factor ANOVAs were used to compare RTD, MS, and MA. A 5 × 2 × 4 (ratio × time × group) mixed factor ANOVA was used to compare all traditional (CR and FR) and alternative  H:Q ratios. Significant interactions and main effects were followed up by paired  to Rhea , values Results and effect sizes of PT, RTD, MT, and MA are presented in t tests per group. For groups CON/CON and CNTRL, hamstrings PT increased from pre- to post-testing (p < 0.05), but quadriceps PT was not different from pre- to post-testing (p > 0.05). For groups ECC/ECC and CON/ECC, hamstrings and quadriceps PT increased from pre- to post-testing (p < 0.05).For PT, there was a significant three-way interaction (muscle × time × group). This was followed up with four two-way ANOVAs (muscle × time), one for each group. All groups demonstrated significant interactions, which were followed up with two paired For RTD, there were no interactions. However, there was a main effect for muscle (p < 0.001), where quadriceps was greater than hamstrings.t test per group. For groups CON/CON, ECC/ECC, and CON/ECC, MS increased from pre- to post-testing (p < 0.05). For group CNTRL, MS decreased from pre to post-testing (p < 0.05).For MS, there was a significant two-way interaction (time × group). This was followed up with one paired For MA, there was a significant two-way interaction (time × group). This was followed up with one paired t-test per group. For group CON/CON, MA increased from pre to post-testing (p < 0.05). For groups ECC/ECC, CON/ECC, and CNTRL, MA did not differ from pre- to post-testing (p > 0.05).t tests per group. For group ECC/ECC, FR increased from pre- to post-testing (p < 0.05), but there were no differences for the other ratios (p > 0.05). For CNTRL, RTD H:Q ratios decreased from pre- to post-testing (p < 0.05), but there were no differences for the other ratios (p > 0.05) . This was followed up with four two-way ANOVAs (ratio × time), one for each group. For groups CON/CON and CON/ECC, there were no interactions or main effects. Groups ECC/ECC and CNTRL demonstrated interactions, which were followed up with five paired  > 0.05) .The aim of the present study was to investigate the effects of different resistance training protocols involving CON and ECC muscle actions on traditional and alternative H:Q ratios. Results revealed that ECC/ECC training led to increases in FR, but no other resistance training protocols involving CON and ECC muscle actions were effective at increasing traditional or alternative H:Q ratios. These findings suggest ECC exercise may be the most beneficial training intervention impacting the quadriceps and hamstrings and inducing increases in the traditional FR. However, given the importance of increases in both traditional and alternative H:Q ratios for fully restoring muscle balance and reducing potential hamstrings and knee ligament injury risk, different strategies of resistance training may be needed in order to induce increases in the alternative RTD, MS, and MA H:Q ratios.Previous studies have recommended that resistance training interventions should focus on increasing hamstrings ECC strength in order to increase H:Q muscle strength balance ,4,5,31. Our results are in agreement with most of these studies Although all training groups had significant hamstrings and/or quadriceps torque increases, the achieved ES revealed that the ECC/ECC training was the only intervention that exhibited a moderate magnitude of change for hamstrings ECC PT. The other groups presented only small, trivial magnitudes for quadriceps and hamstrings CON and ECC PT. Therefore, the moderate hamstrings ECC PT to small quadriceps CON PT magnitude increases may explain the significant increases in FR found for the ECC/ECC group. This may also help explain the lack of changes in the traditional but less functionally relevant CR. Additionally, it is also possible that quadriceps ECC training reduces hamstrings co-contraction . This maMS increases have been thought to be a primary determinant of strength gains following resistance training . BecauseHamstrings strains and ACL injuries have been thought to occur in the early phases of sporting movements. Therefore, the RTD H:Q ratios have been proposed as an alternative method to calculate the knee strength balance necessary during explosive sporting movements ,12,13. Ilh muscles during isometric muscle actions before and after different resistance training protocols, which may only describe an absolute relationship between hamstrings and quadriceps MA. Interestingly, agonist resistance training has been suggested to be effective at inducing neural changes, such as reductions in antagonist MA, to optimize force production [The MA H:Q ratio has been proposed as a method to quantify the hamstrings relative to quadriceps MA, which functionally occurs to decelerate the high anterior tibial shear and rotation movements induced by maximal knee extension movements ,18,19. Aoduction . Howeveroduction ,45. Addioduction  reportedIn this study, participants had preliminary repetitions before isometric (including RTD) and isokinetic maximal testing and received proper instruction and verbal encouragement to perform each test. However, it is possible that additional familiarization sessions performed before the intervention could have minimized the greater variability we found in some functional pretesting compared to post-testing measures between groups.Our findings suggest that ECC training for quadriceps and hamstrings is an advantageous resistance training program for inducing increases in the traditional (FR) H:Q ratio, which could be effective for rehabilitation and injury prevention purposes. However, given the importance of increases in both traditional and alternative H:Q ratios for fully restoring muscle balance and reducing potential hamstrings and knee ligament injury risk, different strategies of resistance training may be needed for also inducing increases in the alternative RTD, MS, and MA H:Q ratios.


Cyclophosphamide may induce autoimmune diabetes through a decrease in suppressor T cells and increase of proinflammatory T helper type 1 response in animal models. In humans, this association is not as clear due to the presence of other risk factors for hyperglycemia, but it could be a precipitant for acute complications.A 31-year-old Mestizo-Mexican woman with a history of systemic lupus erythematosus presented with severe diabetic ketoacidosis, shortly after initiating a multi-drug immunosuppressive therapy. She did not meet the diagnostic criteria for type 1 or type 2 diabetes and had no family history of hyperglycemic states. She persisted with hyperglycemia and high insulin requirements until the discontinuation of cyclophosphamide. After this episode, she recovered her endogenous insulin production and the antidiabetic agents were successfully withdrawn. After 1 year of follow up she is still normoglycemic.Cyclophosphamide may be an additional risk factor for acute hyperglycemic crisis. Glucose monitoring could be recommended during and after this treatment. Most patients with diabetes mellitus (DM) are classified into the commonly accepted groups: type 1 DM (T1DM), type 2 DM, gestational DM, latent autoimmune diabetes of the adult (LADA), or maturity-onset diabetes of the young (MODY). However, almost 10% of patients may prove difficult to classify, especially in the younger groups [T1DM is usually associated with other autoimmune diseases. In this type of diabetes the CD4+ and CD8+ T cells play an important role in the initiation and progression of the disease. The recruitment and activation of these lymphocytes stimulates further secretion of inflammatory cytokines that accelerate beta cell destruction, resulting in insulin depletion . Due to Cyclophosphamide (CY) is a cytotoxic chemotherapeutic agent used in the treatment of hematological diseases. A possible relation between CY and T1DM has been suggested in experimental animal models due to its immunomodulatory properties. It has been shown to promote susceptibility to T1DM in young prediabetic non-obese diabetic (NOD) mice , 4–7. AlWe present the case of a patient with systemic lupus erythematosus (SLE), not previously diagnosed as having diabetes and with no evidence of autoimmune DM, who developed DKA after six cycles of CY therapy.A 31-year-old Mestizo-Mexican woman presented to our emergency department (ED) with DKA. She did not have a family history of diabetes or previous autoimmune diseases. She mentioned multiple hospitalizations due chronic idiopathic pancreatitis between 12 and 16 years of age. At that time, laboratory tests ruled out the presence of gallstones, tumors, autoimmune diseases, or metabolic diseases. Her pancreatic exocrine and endocrine functions were completely normal after each episode. Fifteen years after the last episode of pancreatitis, she presented with alopecia, malar rash, and oral ulcers. She was initially diagnosed as having discoid lupus with the skin biopsy results. However, a week later, she developed severe neuropsychiatric manifestations of SLE, including focal motor seizures, with a Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score of 18 points. Rheumatologists prescribed methylprednisolone (three doses of 1 g daily by intravenous injection) with improvement of neurological symptoms. Her treatment at hospital discharge was: chloroquine (225 mg/day), levetiracetam (1.5 g twice a day), phenytoin (100 mg twice a day), lorazepam (1 mg/day), and a monthly bolus of CY. In addition, she was treated with weekly down-titrating doses of prednisone .Seven days after administration of the sixth dose of CY (cumulative dose of 6.25 g), she presented with asthenia, adynamia, polydipsia, nausea and vomiting, food intolerance, impaired visual acuity, and abdominal pain. The physical examination at our ED revealed tachycardia (125 beats per minute), tachypnea (27 breaths per minute), drowsiness, and dehydration. She presented with a normal body mass index and no stigmata associated with insulin resistance. Laboratory tests reported: serum glucose of 1240 mg/dl, sodium of 127 mEq/L, potassium of 5.56 mEq/L, chlorine of 86 mEq/L, arterial pH of 7.07, bicarbonate of 3.00 mmol/L, and ketonuria (80 mg/dl) with effective serum osmolarity of 322.8 mOsm/kg and total osmolarity of 334.1 mOsm/kg, with an anion gap of 43.56, urea 63 mg/dl, and creatinine of 1.58 mg/dl. Liver tests, amylase, and lipase were normal. After the diagnosis of a mixed hyperglycemic state with predominance of severe DKA, she was started on an aggressive intravenously administered fluid schedule with saline solution 0.9% and intravenous continuous insulin infusion (CII). During the first day she required a total of 165 units of insulin per day (2.75 U/kg per day). Table During the first hours, traditional precipitant factors for DKA, such as infections or cardiac ischemia were ruled out. With an aliquot from the first serum sample in the ED, we measured anti-insulin, anti-GAD65, and anti-IA2 antibodies that were reported as negative; C-peptide was determined to be 0.5 ng/ml and the glycated hemoglobin (HbA1c) was 12.0%. The diagnostic consideration was of T1DM, so once DKA remission was achieved, she was prescribed an intensive insulin scheme at a dose of 1.09 IU/kg per day with 70% of basal insulin glargine daily and 30% of lispro boluses divided in three doses.With the multi-drug immunosuppressive treatment her symptoms improved and 3 months after discharge the CY was switched to azathioprine and then to chloroquine. Three weeks after the switch from CY to other immunosuppressive therapies, she presented with symptomatic hypoglycemia and insulin doses were tapered down. Five months after the switch, insulin was completely withdrawn. At last follow up, 1 year after the hyperglycemic crisis, she had no symptoms related to hyperglycemia and her blood glucose remains under 100 mg/dl. She continues to have a well-balanced, low-carbohydrate diet and her last HbA1c was 5.2% and C-peptide was 1.8 ng/ml without any antidiabetic drugs.Atypical diabetes accounts for up to 10% of cases of DM. A differential diagnosis is important in order to provide the best initial and long-term management, especially in patients with systemic comorbidities in which glucose elevations may jeopardize their quality of life and overall wellbeing. Initially, in this case, we had four diagnostic possibilities: drug-induced DM (due to prednisone that affects the metabolism of carbohydrates); ketosis-prone diabetes; type 1b DM, and finally a possible association with CY.The use of glucocorticoids is widely associated with hyperglycemia. They stimulate the activity of phosphoenolpyruvate carboxykinase (PEPCK) in the liver and decrease its activity in the adipose tissue. This enzyme participates in liver gluconeogenesis and increases the level of glycerol 3-phosphate, increasing triacylglycerol (TAG) levels. In the adipose tissue, the inhibition of PEPCK decreases glyceroneogenesis, also decreasing TAG formation. Both, the increase in liver TAG and decrease in adipose tissue TAG formation induce serum elevation of free fatty acids, which are involved in the decrease of sensitivity to insulin in peripheral tissues with a consecutive rise in blood sugar . HoweverAnother entertained diagnosis was ketosis-prone diabetes. This type of diabetes was described in the 1960s in patients who maintain glycemic control without insulin therapy after an episode of DKA. These patients were obese and had a family history of diabetes, preserved insulin secretion, low prevalence of beta cell autoimmunity, and the ability to discontinue insulin therapy . At the et al. subsequently confirmed that CY accelerates T1DM development in NOD mice and that this phenomenon could be prevented through an increase in the amount of mononuclear cells [+CD25+FoxP3+ Treg cells from peripheral lymphoid tissues and the pancreas. Those Treg cells are involved in the suppression of the accumulation and function of effector T cells that attack pancreatic B cells [After ruling out all common causes of diabetes, we considered that the hyperglycemic crisis may have been related to the CY treatment or precipitated by it. Previous reports of insulin-dependent DM in non-obese mice  also notar cells . The priar cells –8. Speci B cells . Further B cells  and indi B cells . These met al. [In our patient, traditional islet autoantibodies were negative, and she discontinued the use of insulin after 5 months. However, tests for other antibodies, such as anti-insulin receptor antibodies or Zn8, could not be performed. We suspect that CY could have precipitated hyperglycemia by a cytotoxicity mechanism, but other non-identified mechanisms may also have played a role. We also hypothesize that the effect may have been cytotoxic but not cytocidal, or that the surviving beta cells are currently enough to maintain a long-term normoglycemia and C-peptide production. The association of other factors predisposing to hyperglycemia cannot be completely ruled out in most humans, including our patient. After an exhaustive review of the literature, we only found one clinical case reported by Sharma et al.  of a 51-et al. . We consWe present the case of a patient with a difficult to classify diabetes who lacked the traditional clinical or biochemical markers for the common types of diabetes. She developed insulin deficiency, with high insulin requirements and decreased serum C-peptide. After ruling out the most common causes for diabetes, we considered that her hyperglycemic crisis might be at least partially related to the CY treatment. Until further evidence is published, glucose monitoring of patients being treated with this drug may be useful, especially if other risk factors for hyperglycemia are present.


Onychophorans, commonly known as velvet worms, display a remarkable diversity of reproductive strategies including oviparity, and placentotrophic, lecithotrophic, matrotrophic or combined lecithotrophic/matrotrophic viviparity. In the placentotrophic species, the embryos of consecutive developmental stages are attached to the uterus via a placental stalk, suggesting they might be transported passively towards the vagina due to proximal growth and distal degeneration of tissue. However, this assumption has never been tested using specific markers. We therefore analyzed the patterns of cell proliferation and apoptosis in the genital tracts of two placentotrophic peripatids from Colombia and a non-placentotrophic peripatopsid from Australia.All three species show a high number of apoptotic cells in the distal portion of the genital tract near the genital opening. In the two placentotrophic species, additional apoptotic cells appear in ring-like vestigial placentation zones of late embryonic chambers. While moderate cell proliferation occurs along the entire uterus in all three species, only the two placentotrophic species show a distinct proliferation zone near the ovary as well as in the ring-like implantation zone of the first embryonic chamber. In contrast to the two placentotrophic species, the non-placentotrophic species clearly does not show such regions of high proliferation in the uterus but exhibits proliferating and apoptotic cells in the ovarian stalks. While cell proliferation mainly occurs in stalks carrying maturating oocytes, apoptosis is restricted to stalks whose oocytes have been released into the ovarian lumen.Our results confirm the hypothesis that the uterus of placentotrophic onychophorans grows proximally but is resorbed distally. This is supported by the detection of a proximal proliferation zone and a distal degenerative zone in the two placentotrophic species. Hence, cell turnover might be responsible for the transport of their embryos towards the vagina, analogous to a conveyor belt. Surprisingly, the distal degenerative zone is also found in the non-placentotrophic species, in which cell turnover was unexpected. These findings suggest that the distal degenerative zone is an ancestral feature of Onychophora, whereas the proximal proliferation zone might have evolved in the last common ancestor of the placentotrophic Peripatidae.The online version of this article (10.1186/s12983-019-0317-x) contains supplementary material, which is available to authorized users. Onychophorans, also called “velvet worms” Fig. a–c, are sensu ref. [Mesoperipatus tholloni from tropical Africa, as the reproductive mode and embryonic development are unknown from the latter [Even though the anatomy of onychophorans has changed little since the Early Cambrian , they hansu ref. ), which nsu ref. , 14–23. nsu ref. . The remnsu ref. , 24–30. nsu ref. , 31–33, nsu ref. –38, and nsu ref. , 31. By Among these types of nourishment supply, placentotrophic viviparity is unique, as a similar developmental mode has not been described from any other invertebrate taxon so far , 20. ThiEuperipatoides rowelli from Australia. To detect cell proliferation, we used an antibody directed against phospho-histone H3 as a mitosis marker. Apoptotic cells were detected using the terminal deoxinucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique, which labels free 3′-OH ends of the degrading DNA of apoptotic cells [E. rowelli and the placentotrophic viviparous peripatid Principapillatus hitoyensis [However, this hypothesis has never been tested by using specific markers for cell proliferation and controlled cell death –43. In tic cells . Additioic cells . The comtoyensis .E. rowelli and the placentotrophic Peripatidae might provide insights into the evolution of reproductive strategies in onychophorans.The results of this study might help to clarify whether the complementary growth and resorption of uterine tissue is an ancestral feature of all onychophorans or a derived feature of the placentotrophic Peripatidae, which has evolved along with the placental attachment of embryos to the uterine wall. Hence, the comparison of cell proliferation and apoptosis patterns in the genital tracts of sensu ref. [Drosophila melanogaster [E. rowelli, this succession might occasionally be interrupted due to the occurrence of embryos that have not developed beyond stage I . Each hsensu ref. [sensu ref. [The paired tubular ovaries of the placentotrophic Peripatidae have a smooth external surface without externally visible oocytes sensu ref. [E. rowelli bears externally visible oocytes of different sizes, the largest of which are raised above the ovarian surface via stalks  of E. rlks Fig. c. Additinsu ref. ) are absrus Fig. c. Even tE. rowelli  of largIn addition to cell proliferation, we detected apoptosis using TUNEL and anti-caspase-3 labeling Table . In contE. rowelli, except for individual apoptotic cells appearing in the empty stalks of the ovaries , while the tissue is resorbed distally  , 20, thuE. rowelli, whereas a distinct proliferation zone is missing. This indicates that the uterus of E. rowelli is not growing in a defined, proximal region but rather along its entire length, similar to the distal half of the uteri of the placentotrophic species in which the embryos have lost their connection to the uterus and lie freely within its lumen  indicates the presence of a distinct distal degeneration zone.Interestingly, our results further revealed cell proliferation along the entire proximo-distal extent of the paired uteri in the lecithotrophic/matrotrophic peripatopsid men Fig. b [14, 17In summary, our data revealed that cell proliferation occurs along the entire genital tract of all species investigated whereas apoptosis is restricted to a distal degeneration zone. In contrast to this, a distinct proximal proliferation zone is only present in the placentotrophic Peripatidae. Thus, our results indicate that the distal degenerative zone of the uterus is an ancestral feature of Onychophora, while the proximal proliferation zone might have evolved in the last common ancestor of the placentotrophic Peripatidae . SimilarIn addition to the distinct patterns in the uterus, the cell proliferation marker used yielded signal in the ovaries of all investigated species. This signal might originate from oogonia situated within the germinal epithelium, which undergo mitosis and eventually give rise to cells that begin meiosis and become early maturing oocytes . Since tE. rowelli  of the Based on an outgroup comparison with arthropods, some of which display exogenous ovaries –71, it hE. rowelli, in which the embryos are not attached to the uterine wall and thus cell turnover was unexpected. These findings suggest that the distal degenerative zone might be an ancestral feature of onychophorans, whereas the proximal proliferation zone might be a derived feature of the placentotrophic Peripatidae. To test this hypothesis, future studies should focus on the existence of the distal degenerative zone in additional representatives of Peripatopsidae and South-East Asian Peripatidae as well as the proximal proliferation zone in the peripatid Mesoperipatus tholloni from tropical Africa. The occurrence of a distinct pattern of cell proliferation and apoptosis in the stalks of the exogenous ovary of E. rowelli indicates that these processes are involved in the formation and degeneration of stalks. The absence of a corresponding pattern in the endogenous ovary of the placentotrophic Peripatidae confirms the proposed [Our results confirm a previous assumption that the genital tracts of the placentotrophic viviparous Peripatidae is constantly growing at the proximal end while tissue is resorbed at the distal end near the genital opening, thus retaining a constant length of the uterus while the embryos are transported towards the genital opening in a conveyor-like fashion. Surprisingly, we also detected a similar distal degenerative zone  in the genital tract of the lecithotrophic/matrotrophic viviparous species proposed  loss of Euperipatoides rowelli Reid, 1996 (Peripatopsidae) from Australia and two undescribed placentotrophic viviparous species of Peripatidae (Neopatida) from Colombia, gen. sp. 1 and gen. sp. 2, were studied , those stored in PBS with sodium azide were rinsed several times in 1 mL PBS (5 × 5 min). Afterwards, they were incubated in 1 mL of 0.1 M sodium citrate (pH 6.0) for 30 min at 70 °C and rinsed in 1 mL PBS containing 0.3% Triton X-100 (PBS-Tx) at room temperature. Apoptotic cells were detected using the In Situ Cell Death Detection Kit  according to the manufacturer’s protocol. The genital tracts were incubated in 450 μL labeling solution containing 50 μL enzyme solution for 2 h at 37 °C in a hybridization oven with gentle shaking. Positive controls were performed by treating the tissue with 0.25 mg/ml DNAse I (Roche) for 30 min at room temperature prior to the TUNEL labeling procedure. In these controls, all nuclei were TUNEL positive  and pre-incubated in 1 mL 5% normal goat serum  in PBS-Tx for 3 h at room temperature. They were then incubated with the primary rabbit polyclonal anti-phospho-histone H3 (Ser10) mitosis marker  overnight at room temperature with gentle shaking. After several washes with 1 mL PBS-Tx , a secondary antiserum  was applied overnight at room temperature in the dark with gentle shaking. The samples were washed several times with 1 mL PBS-Tx , counterstained with the DNA-selective fluorescent dyes SYBR®Green  or 4′,6-Diamidin-2-phenylindol  for 1 h and mounted between two cover slips in Vectashield® Mounting Medium .For anti-caspase-3 immunolabeling, genital tracts stored in methanol were rehydrated stepwise in PBS at 4 °C , washed several times with PBS-Tx (3 × 5 min) and incubated in 5% NGS for 10 min at room temperature. After replacing the blocking solution with fresh 5% NGS, the genital tracts were incubated in the blocking solution for 3 h at room temperature. Incubation with the primary antiserum  was carried out for three nights at 4 °C with gentle shaking. After several washing steps with 1 mL PBS-Tx , the secondary antiserum  was applied for three nights at 4 °C in the dark with gentle shaking. The samples were washed several times with 1 mL PBS-Tx , counterstained with DAPI (diluted 1:1000 in PBS) for 1 h at room temperature and mounted in Vectashield® mounting medium. The resulting signal was similar to the patterns detected with the TUNEL technique, indicating that the detected signals are specific to apoptotic cells.The samples were analyzed with the confocal laser scanning microscopes Leica TCS 5 STED  and LSM 880 , and the stereomicroscope Axio Zoom V16 equipped with an Axiocam 503 color digital camera (Carl Zeiss MicroImaging GmbH). Confocal optical sections were taken at intervals ranging from 1 to 5 μm and the resulting image stacks were merged digitally into maximum projection micrographs. The colors were adjusted using Leica AS AF, v2.3.5 (Leica Microsystems), and ZEN 2012 SP1 software packages . The stereomicrographs were taken at different focal planes and merged to single projections using the ZEN 2012 blue edition software version 1.1.2.0 (Carl Zeiss MicroImaging GmbH) or the Auto-Blend Layers function in Photoshop version CS 5.1 . All micrographs were adjusted for brightness and contrast using Photoshop CS 5.1. Final panels and diagrams were designed using Illustrator CS 5.1 (Adobe Systems Inc.).Additional file 1:A) or SYBR®Green  are represented in gray. Proximal is up in all images. A Proximal part of the genital tract including the first five uterine chambers containing embryos of subsequent developmental stages. B Distal part of one uterus horn. C Detail of the common duct/vagina. Abbreviations: cd, common duct/vagina; ch1–ch5 and ch7, uterine chambers containing embryos of subsequent developmental stages; d, distal; p, proximal; pz, proximal proliferation zone; ut, uterus. Scale bars: A, B: 500 μm; C: 200 μm. (TIF 7280 kb)Apoptosis in the genital tract of the placentotrophic peripatid gen. sp. 1. Nuclei of TUNEL-positive cells are marked in orange, nuclei stained with DAPI  are marked in cyan. Proximal is up in all images. A Overview of a genital tract of E. rowelli. B Proximal portion of the genital tracts of gen. sp. 1 (left and middle) and distal portion of the genital tract of gen. sp. 2 (right). Abbreviations: cd, common duct/vagina; ch1–ch5 and ch7, uterine chambers containing embryos of subsequent developmental stages; d, distal; dz, distal degeneration zone; oc, oocytes; ov, ovary; p, proximal; pz, proximal proliferation zone; sr, seminal receptacle; ut, uterus. Scale bars: 500 μm. (TIF 5269 kb)Apoptosis and cell proliferation in the genital tracts of Additional file 3:E. rowelli detected with an antibody directed against cleaved caspase-3 (Asp175). Caspase-positive cells are illustrated in orange, cell nuclei stained with DAPI are represented in gray. Proximal is up in all images. A Ovary with stalked oocytes. B Common duct/vagina. Abbreviations: cd, common duct/vagina; oc, oocyte; ov, ovary. Scale bars: A: 500 μm; B: 200 μm. (TIF 2090 kb)Apoptosis in the genital tract of Additional file 4:TUNEL control in the proximal uterus near the implantation zone of the placentotrophic peripatid gen. sp. 1. Note that after the DNAse treatment, all cell nuclei are TUNEL-positive (orange). Abbreviations: ut, uterus. Scale bars: 200 μm. (TIF 1682 kb)


Background: Early prediction of cerebral palsy (CP) using the General Movement Assessment (GMA) during the fidgety movements (FM) period has been recommended as standard of care in high-risk infants. The aim of this study was to determine the accuracy of GMA, alone or in combination with neonatal imaging, in predicting cerebral palsy (CP). Methods: Infants with increased risk of perinatal brain injury were prospectively enrolled from 2009–2014 in this multi-center, observational study. FM were classified by two certified GMA observers blinded to the clinical history. Abnormal GMA was defined as absent or sporadic FM. CP-status was determined by clinicians unaware of GMA results. Results: Of 450 infants enrolled, 405 had scorable video and follow-up data until at least 18–24 months. CP was confirmed in 42 (10.4%) children at mean age 3 years 1 month. Sensitivity, specificity, positive and negative predictive values, and accuracy of absent/sporadic FM for CP were 76.2, 82.4, 33.3, 96.8, and 81.7%, respectively. Only three (8.1%) of 37 infants with sporadic FM developed CP. The highest accuracy (95.3%) was achieved by a combination of absent FM and abnormal neonatal imaging. Conclusion: In infants with a broad range of neonatal risk factors, accuracy of early CP prediction was lower for GMA than previously reported but increased when combined with neonatal imaging. Sporadic FM did not predict CP in this study. Cerebral palsy (CP) is the major cause of motor impairment in young children ,2, and tThe General Movement Assessment (GMA) during the fidgety movements (FM) period (2–4 months post-term age (PTA)) is currently considered the best clinical test for early prediction of CP ,10, and Fidgety movements can be classified according to their presence and length of interspersed pauses . IntermiIn this study, the predictive accuracy of GMA assessed once during the FM period for later CP was explored in a large population of neonatal intensive care unit (NICU) graduates referred to neurodevelopmental follow-up at discharge. The effect of the temporal organization of FM on CP-risk and gross motor function was also explored, as well as how neonatal imaging contributed to the prediction of CP.https://clinicaltrials.gov/ct2/show/NCT00515281). Although one infant could have several risk factors for adverse neurodevelopment, all infants were classified into one risk group according to their main reason for referral. Extremely preterm infants (ELBW/ELGAN) were classified as such irrespective of other risk factors, whereas preterm infants with GA 280–306 weeks and BW > 1000 g were classified as such only if they had no other risks of perinatal brain injury .High-risk infants were consecutively recruited before discharge from the NICU at five sites from 2009–2014. Sites 1–3 recruited infants in three Norwegian university hospitals based on at least one of the following: (a) Birth weight (BW) ≤1000 g and/or gestational age (GA) <28 , (b) neonatal arterial ischemic stroke, (c) neonatal encephalopathy, (d) other significant risk factors for perinatal brain injury  or MRI) was done for clinical purposes and according to each unit’s guideline. A central classification of the imaging results into normal or abnormal was based on local written reports, by an experienced neonatologist (RS) who was unaware of the infant’s medical history, GMA result, and neurodevelopmental outcome. Cerebral imaging was classified as abnormal if there was intraventricular hemorrhage (IVH) over grade II according to Papile , other sInfants were video recorded during active wakefulness at 10 to 15 weeks PTA using a standardized set-up with a commercially available digital video camera . Video recordings and gestalt assessment of GMs were done according to Prechtl and co-workers . Two traChildren were followed clinically to establish the diagnosis of CP or no CP. Some children with CP were followed to the age of 6 years but follow-up to exclude CP continued until a minimum of 18–24 months at all sites.CP and CP subtype were diagnosed as part of regular follow-up by trained pediatricians who were unaware of the GMA classification and in accordance with the decision-tree published by the Surveillance of cerebral palsy in Europe (SCPE) . The usep values less than 0.05.Statistics: Data were analyzed using SPSS Statistics version 23.0 . The data are presented as numbers with proportion (%) or mean with standard deviation (SD). Group differences were examined using the chi-square test or Fisher exact test for dichotomous variables. Sensitivity, specificity, positive and negative predictive values, and test accuracy were estimated. Logistic regression was used to assess the association between the explanatory variables GMA , neonatal imaging , GA and sex, and the dichotomous outcome variable CP. Results from logistic regression are presented as odds ratios (OR) with 95% CI. Results were considered statistically significant for Ethical Approval: The study was approved by the regional committee for medical and health research ethics  in Norway. Each of the two study sites in Chicago, USA also had approvals from their local Institutional Review Boards. Written parental consent was obtained before inclusion.n = 5), infant state of crying/fussing (n = 8) or had external distractions during recording (n =2), and infants with restricted movements due traumatic injury of an extremity (n = 1) were excluded. In addition, infants who had significant medical incidences occurring after GMA assessment (n = 3), or who were categorized as having exaggerated FM (n = 7) or were lost to follow-up (n = 19) were excluded. These exclusions left 405 infants available for analysis. Demographic characteristics and the clinical condition considered the most relevant for increased risk of perinatal brain injury are shown in A total of 450 infants were included. Infants with video recordings which could not be assessed due to inadequate video set-up (Mean age at video recording for GMA was 12.3 weeks (SD 1.3). Ninety-six (23.7%) of the infants were categorized with abnormal GMA (60 (14.8%) with absent and 36 (8.9%) with sporadic FM), and 309 (76.3%) infants were categorized as normal (258 (63.7%) with intermittent, 51 (12.6%) with continual FM).Neonatal cUS and/or MRI results were available in all but two infants. In preterm infants, cUS was the most common modality with results available for all those born before GA 32 weeks. MRI results were available in 104 (68.4%) of those born at GA ≥ 32 weeks.p < 0.001).Eighty-seven (21.5%) infants had abnormal cerebral imaging. The proportion of infants with abnormal GMA during the FM period was significantly higher among those with abnormal compared to normal imaging  children received a diagnosis of CP, and mean age at the last available confirmation of the diagnosis was 3 years 1 months . Of the 42 children with CP, eight (19%) had unilateral spastic, 26 (62%) had bilateral spastic, five (12%) had dyskinetic, and one (2%) child had ataxic CP. In two children data on CP subtype was not available.Predictive accuracy of GMA and neonatal imaging is presented in The odds of developing CP were 14 times higher for infants with absent/sporadic FM in a model which included GA, sex, and neonatal imaging .p < 0.001).Gross motor function classification (GMFC) was available for all children with a diagnosis of CP. Twenty-one (50%) had a GMFCS level of IV–V, corresponding to non-ambulatory function. There was a significant association between absence of FM and being non-ambulatory among those who developed CP. Nineteen (66%) of the 29 infants with absent FM who developed CP had a GMFCS level of IV–V compared to one (8%) of 13 with any presence  of FM , which was supported by the high accuracy of abnormal imaging for later CP. Furthermore, the categorization of imaging results was based on local reports of imaging done for clinical purposes, making the results generalizable to clinical settings.Among children with CP, those who presented with sporadic or intermittent FM, had significantly better gross motor function than those who presented with absent FM. A retrospective study of 61 children with CP concluded that sporadic FM did not indicate a milder type of CP . Howeverhttp://yourcpf.org/early-recognition). Byrne and co-workers [Successful implementation of “Early diagnosis and intervention guidelines for CP” has already been reported , and The-workers  reportedLimitations: A few children with CP in our study were assessed before two years of age for gross motor function. The GMFCS is more reliable after two years of age , and theThe present study only focused on CP outcome of participants. Absent FM may be associated with genetic disorders and disabilities such as autism , which aAll study sites did not provide GMA results to parents, and the present study did not include any evaluation of parental reactions to early CP prediction. Although a delay in providing a diagnosis of CP is harmful to parents’ well-being , little Presence of FM was a strong marker for a non-CP outcome in high-risk infants but did not exclude milder CP phenotypes. The predictive accuracy of GMA increased when sporadic FM were not categorized as a marker for later CP, and absence of FM correctly predicted CP in approximately 50% of cases. Neonatal cerebral imaging in combination with GMA increased the predictive accuracy. Future research should focus on how neurodevelopmental follow-up could be individualized and targeted based on an early developmental profile. In infants with present FM, a redirection of resources to closer assessment of other areas of neurodevelopment, such as cognition, communication, and behavior, may be considered.


To investigate the prevalence of poppers use and its relationship with sexual risk behaviors among men who have sex with men (MSM) in southwestern China.This cross-sectional study was conducted in three cities of southwestern China between July and September 2016. Anonymous questionnaire survey was administered to collect data on demographics, drug use, sexual behaviors, history of STIs and HIV infection. Logistic regression analysis was performed to explore factors correlated with sexual risk behaviors including group sex and unprotected anal intercourse (UAI).Of the 1122 participants included in the study, 24.1% reported a history of poppers use. 11.6% MSM reported ever engaging in group sex and 36.2% participants reported UAI with non-regular male partners in the past 12 months. Multivariate logistic analysis showed that age > 25 , seeking sex partners through the internet , preferring receptive anal intercourse  and ever using poppers  were positively associated with engaging in group sex. Lower levels of education  and ever using poppers  were significantly correlated with UAI with non-regular partners.The study suggested poppers was prevalent among MSM and its use was significantly associated with sexual risk behaviors. Given high prevalence of HIV among this subpopulation, comprehensive measures are needed to decrease poppers use and its potential risk for HIV transmission in southwestern China. Recreational drug use among men who have sex with men (MSM) has been a worldwide public health concern in the past decades –4. DiffePrior studies from western countries reported that methamphetamine or ecstasy were the most commonly used drugs among MSM , 9, whicFindings from specific countries have demonstrated the associations between poppers use and sexual risk behaviors among samples of MSM , 16. HowThis cross-sectional study was conducted in three cities  of Sichuan province during the period of July and September, 2016. Convenience sampling method was used to recruit subjects and MSM participants were invited by local community-based organizations (CBOs). Each CBO is operated by 2~ 3 full-time MSM staff who are trained and guided by the local Centers for Disease Control and Prevention (CDCs). CBO staff introduced the study to potential participants when providing voluntary counseling & testing (VCT) service for MSM and regularly reaching out in community  to carry out health education activities. Recruited participants were encouraged to refer their MSM peers.The inclusion criteria for participants were: male, ≥18 years, self-reported sexual experience with men and willing to provide informed consent. After confirming the eligibility of recruited participants, trained staff from CBOs fully explained the purpose, contents and procedures of the study to them. Verbal informed consent was obtained from MSM participants and then self-administered paper-and-pencil questionnaire survey was conducted anonymously at private rooms of local CBOs. Each participant was paid 25 CNY (~4 USD) for transportation fees.The questionnaire survey took 10~ 20 min to complete and was used to collect socio-demographic characteristics , drug use behaviors, sexual behaviors, self-reported history of STIs and HIV infection from MSM participants.Participants were asked to report their lifetime use of rush poppers, capsule zero , crystal meth, ecstasy, magu, ketamine, happy water , GHB, cannabis, bath salt (or cathinone), red crystal meth (extracts from methamphetamine) and heroine. Participants were asked to specify the drug’s name in case it was not listed.Participants were asked about their age at sexual debut, sexual orientation , preferred sexual role with males (insertive/receptive/versatile), routes to seek sex partners  and number of sex partners in the past 12 months. They were also asked whether they had regular  or non-regular  sex partners and their status of condom use (never/sometimes/every time) in the past 12 months. Condom use variables were re-coded as unprotected (never or sometimes) versus protected (every time). Furthermore, MSM participants were asked whether they had engaged in group sex. Other variables included history of STI/HIV test (yes vs. no) and results of STI/HIV test (positive vs. negative).P < 0.2 level in the univariate analysis were selected for multivariate logistic regression analysis and variables with P < 0.05 in the multivariate logistic regression analysis were retained in the final model.Data were entered by Epi Data software 3.1  and analyzed by SPSS 16.0 . Continuous variables and category variables were expressed as means and proportions, respectively. Chi-square tests were conducted to compare the differences in socio-demographics, sexual behaviors and HIV/STI infection by poppers use status. Stepwise logistic regression analysis was used to evaluate associated factors for UAI with non-regular partners and engaging in group sex. Variables with A total of 1167 MSM were invited to participate in this study. After excluding MSM who were < 18 years (1.1%) and those who were not willing to report their drug use status (2.7%), 1122 participants were included in the final analysis. The mean age of enrolled MSM was 30.0 years (SD = 9.0). Of the 1122 eligible participants, almost all (98.2%) officially resided in Sichuan province and over two-fifths (44.1%) had obtained at least a college education level. Most (70.5%) of MSM participants were single. More than half (52.6%) reported to be solo business owners or service providers and three-fifths (60.0%) reported a monthly individual income of 3000 Yuan or more. Overall, 128 (11.6%) MSM reported ever engaging in group sex and more than one-third (36.2%) reported having UAI with non-regular partners in the past 12 months. 142 (12.7%) MSM reported being HIV-positive and 149 (13.3%) MSM reported a history of STIs.Among the participants, 311 (27.7%) participants reported using at least one type of recreational drug in their lifetime, of whom 36.0% were polydrug users. Poppers (24.1%) were the most commonly used recreational drug, followed by methamphetamine (7.1%), zero capsule (6.1%), magu (2.0%), ecstasy (0.4%), ketamine (0.4%) and happy water (0.2%). Comparisons of socio-demographic characteristics, sexual behaviors, history of STIs and HIV infection between poppers users and non-users were presented in Table P < 0.2): age > 25 years old, being bisexual, seeking sex partners through the internet, preferring receptive anal intercourse and ever using poppers. These factors were further included in multivariate analysis and the following variables were retained in the final multivariate model (P < 0.05): age > 25  (versus age ≤ 25), seeking sex partners through the internet , preferring receptive anal intercourse   and ever using poppers .Results exploring factors associated with engaging in group sex are summarized in Table P < 0.2). In the final multivariate logistic regression model, UAI with non-regular partners were significantly correlated with the following factors (P < 0.05): having less than senior high school education  and ever using poppers .Table Consistent with results from the coastal metropolises –14, the In the present study, 11.6% MSM reported having ever participated in group sex, which was comparable with related reports in China , 21, butMoreover, older MSM were more likely to report participating in group sex, and similar result was indicated among MSM in Australia . MSM witIn addition, poppers use was found to be significantly correlated with UAI with non-regular sex partners in the past 12 months. Like other recreational drugs, poppers may also have the neurological effects on sexual behaviors such as heightening libido and reducing sexual inhibition . Thus, MP-values. This could cause a problem that some predictor variables may not meet the criterion of biological plausibility. For example, we are unable to explain why men who preferred receptive anal intercourse was more likely to be engaged in group sex and further studies are needed to figure it out.There are a few potential limitations to our study. First, although MSM participants were recruited from different venues, still selection bias may exist, as there was no sampling frame and non-probabilistic sampling method was used. Second, the results were based on self-administered questionnaire survey, which may result in recall bias. Third, data on drug use and sexual behaviors are still sensitive in China, and thus social desirability bias and concealment of information are possible. However, the purpose and contents of the study were fully explained to participants and confidentiality of their information was guaranteed by self-administered anonymous questionnaire survey in private rooms, which could possibly minimize the bias. Fourth, the causal relationship between associated factors and risky sexual behaviors was weak, as data were collected from a cross-sectional study. Finally, this is an exploratory study and the variable selections in the multivariable models were solely based on Our study indicated recreational drugs, especially poppers, were prevalent among MSM and use of poppers was significantly associated with sexual risk behaviors including group sex and UAI. Given high prevalence of HIV among MSM, popularity of poppers use may further fuel the epidemic within this subpopulation. Thus, Close attention should be given to the dynamics of poppers use and interventions targeting to both poppers use and sexual risk behaviors are needed to prevent the spread of HIV transmission in China.