The risk factors of lower dialytic blood pressure and coronary heart disease were identified only in men, whereas peripheral arterial disease only in women. In conclusion, several modifiable and gender specific risk factors were significantly associated with higher prevalence of CKD in Chinese adults with T2DM. The high-risk populations identified in this study shall receive regular screening for renal functions to achieve better patient management in primary care settings.In this study we conducted a cross sectional study to comprehensively evaluated the risk factors of chronic kidney disease (CKD) in a large sample of Chinese adults under primary care for type 2 diabetes mellitus (T2DM). We investigated the risk factors associated with the prevalence of CKD in adults with T2DM, who were enrolled in the Risk Factor Assessment and Management Programme for Patients with Diabetes Mellitus (RAMP-DM) of Hong Kong from July 2014 to June 2017. We collected the individual data of 31,574 subjects, with mean age of 63.0 (±10.8) years and mean DM duration of 7.4 (±6.4) years. Of them 9,386 (29.7%) had CKD and 7,452 (23.6%) had micro- or macro-albuminuria. After adjustment for multiple demographic and lifestyle confounders, we identified several modifiable risk factors associated with higher rate of CKD: obesity (OR = 1.54), current smoking (OR = 1.33), higher systolic blood pressure (OR = 1.01), dyslipidemia (OR = 1.32 and 0.61 for triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C)), hyperglycemia (OR = 1.11 for HbA A clear rising trend was observed in Hong Kong in the past decade, and type 2 (T2DM) accounts for over 90% of all the persons with diabetes2. The International Diabetes Federation estimated that in 2011 the prevalence of diabetes was 9.4% in adults aged 20–79 years of Hong Kong, and this rate could reach 11.9% by 20302.Diabetes Mellitus (DM) is one of the most common chronic diseases worldwide. Globally, in 2014 there were 8.5% of people living with DM which nearly doubled the prevalence rate of 4.7% in 19803. The mortality in DM complicated with CKD was estimated as high as 48 deaths per 1,000 patient-years at risk, and this rate tripled if patients also had cardiovascular conditions3. Previous studies have identified several risk factors for CKD, such as male, older age, obesity, hypercholesterolemia, hypertension and hyperglycemia7. However, most studies used glomerular filtration rate to identify CKD (GFR < 60 ml/min per 1.73 m2) or end-stage kidney disease 8. Few have combined GFR and albuminuria categories, both of which were required for the diagnosis of CKD10. Furthermore, data are relatively limited in Chinese population under primary care.Chronic kidney diseases (CKD) is one of common complications associated with both type 1 and type 2 DM. In the US, 40% of patients with CKD also had DM13. Two recent publications have assessed the risk of developing end-stage renal disease (ESRD) among RAMP-DM participants15. Here we conducted a cross-sectional study using the RAMP-DM data, with the aim to explore the risk factors associated with CKD in Chinese adults. The literature on the risk factors of CKD is less for T2DM than for type 1, and 99% of patients registered to RAMP-DM were diagnosed with T2DM; hence, type 1 and gestational DM were excluded in this study.In Hong Kong, the Risk Factor Assessment and Management Programme for Patients with Diabetes Mellitus (RAMP-DM) was implemented in the General Outpatients Clinics (GOPC) managed by the Hospital Authority (HA) since 2009, to conduct a comprehensive risk assessment and screening for diabetic complications in the primary care setting. Another objective of RAMP-DM was to educate DM patients by involving multi-disciplinary healthcare professionals including doctors, nurses and optometrists. Previous studies in Hong Kong have extensively evaluated the risks of cardiovascular diseases and diabetic retinopathy in patients of RAMP-DMThere were 40,781 people with DM enrolled into RAMP-DM of NTWC from 2 July 2014 to 16 June 2017, and 39,652 diagnosed with T2DM were eligible in this study. Of them, 8,078 (20%) subjects were further excluded from subsequent analyses because of missing data in estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR). The missing data of other variables ranged 0–1.4%, with the only exception of diabetic retinopathy (DR) status, which had 4.4% and 3.3% missing in the subjects with or without CKD. The demographic characteristics of these subjects with missing data were similar to those included in the analysis (data not shown). A final sample size of 31,574 adults with T2DM were eligible for analysis. Table 1c, TG, and lower HDL-C), and higher likelihood of other diabetic complications ; we therefore did a stratified analysis of female and male subjects. The effect estimates after adjustment were similar between female and male, except education, DBP, CHD and BMI categories  in the U.S.17. It has been under debate that eGFR may not accurately reflect the real renal function of T2DM adults18. Our study found that the prevalence of CKD dramatically decreased if only eGFR or albuminuria criteria were applied (29.7% vs 11.6% and 23.6%). This highlights the need of incorporating routine urine albumin tests into the management program of persons with diabetes for early detection of CKD.We conducted a large-scale cross-sectional study in 31,574 participants with individual medical records in primary care setting, to identify the risk factors of CKD and potential gender differences. We found that the prevalence of micro- and macro-albuminuria in Chinese adults with T2DM was 23.6%, which is within the range of prevalence rates reported in a global cross sectional study (9.8–38.8%)4. This could be explained by the fact that female subjects in our study were older and had a longer duration of DM than males. The stratified analysis identified some common risk factors of CKD shared by both female and male, and gender-specific factors such as DBP and CHD that were only significant in men, and PAD only in women. Gender difference has been well documented in the previous studies on ESRD and mortality in populations with diabetes20. Our findings further support that different prediction models shall be separately established for women and men.It is surprising that in our study men had a lower rate of CKD than women, which is contrary to the previous findings21. This association is particularly strong in persons with diabetes, as both are caused by accumulating damages to vascular systems during the disease progress22. We found that the CKD subjects were more likely to have CHD as compared to those without CKD. Similarly, several cross-sectional studies in Chinese adults with T2DM also demonstrated significant association of CHD with CKD24. However, we could not establish the temporal sequence of these adverse events due to the limitation of cross-sectional study design. In a cohort study of T2DM subjects in Hong Kong, CHD was associated with a higher risk of CKD25. It will be of great interest to conduct prospective cohort studies with the aim to explore the possibility of using CHD as an early predictor for CKD events or vice versa.The association of CHD and CKD has been widely demonstrated7. In sensitivity analysis, we adopted the new AHA classification of hypertension and found that compared to subjects with normal BP, those at hypertension stage 1 and 2 had a significantly higher prevalence of CKD, but no significant difference was found between normal and elevated groups. This finding is consistent with one previous study in mainland China that found hypertension could significantly increase the risk of CKD5.The results from the multivariate model showed that SBP was significantly associated with CKD, whereas an inverse association was found between DBP and CKD. This finding echoes a study that were conducted in mainland China with smaller sample size, which also found that SBP, retinopathy, TC, TG and anemia were risk factors of CKD10. Nevertheless, creatinine-based UACR is still widely adopted in current clinical practice. Fourth, this study population were the Chinese adults enrolled in NTWC; hence, the findings might not be applicable to all the Chinese populations. Last but not least, although we have adjusted for multiple variates in regression models, there were potential confounding factors that were not routinely collected in clinical practice still remained unadjusted.There are several limitations in this study. First, we used a cross-sectional study design, therefore could not establish the causal relationship between risk factors and CKD. Nevertheless, a large sample size still allowed us to have statistical power large enough to identify the significant risk factors for CKD. The findings of this study could also shed light on future cohort studies when more follow-up data become available. Second, it was uncertain that CKD in our subjects was diabetic nephropathy, since they might also have abnormal kidney structures or other diseases that deposited them to a higher risk of CKD. Third, in this study we used creatinine-based UACR estimates which have been criticized for lack of accuracy especially within the normal or high range of GFRIn conclusion, we found a high prevalence rate of CKD in Chinese adults with T2DM in this large-scale cross-sectional study. We also identified several modifiable risk factors associated with CKD, including obesity, current smoking, higher SBP, lower DBP (only in men), dyslipidemia, hyperglycemia, the presence of diabetic retinopathy, stroke, PAD (only in women) and CHD (only in men). Given the increasing trend of T2DM prevalence and ageing population, the high-risk populations identified in this study shall receive regular screening for renal functions to achieve better patient management in primary care settings. Future studies on risk or beneficial factors of CKD incidence and disease progress could be conducted in this large patient cohort when more follow-up data become available, in order to achieve better prognosis for Chinese adults with diabetes.26. Briefly, individual data of demographics, education, lifestyle , family history, blood pressure, body mass index (BMI) and waist-hip ratio (WHR) were collected during their first visits to clinics. The participants were assessed for diabetic neurological, macrovascular and microvascular complications, coronary heart disease, stroke, malignancy and diabetic retinopathy. Fasting blood samples were taken during each visit to test for HbA1c, fasting glucose, total cholesterol, LDL-C, HDL-C, triglyceride (TG) and serum creatinine. Urine samples were also taken to test for albumin to calculate urine albumin to creatinine ratio (UACR), urine protein to creatinine ratio (PCR). The estimated glomerular filtration rate (eGFR) was calculated using the following formula27:Individual data of people who were enrolled in the RAMP-DM from 2 July 2014 to 16 June 2017 were obtained from the New Territories West Cluster (NTWC) of the Hospital Authority (HA). The study period was chosen because of the good data completeness. This cluster serves the population of 1.09 million living in the northwest region of Hong Kong Special Administrative Region. Participation into RAMP-DM is voluntary. After 2014, over 90% of people with diabetes have been enrolled to RAMP-DM . The details of RAMP-DM have been described elsewhere1c during hospitalized episodes and outpatient visits were retrieved from the Clinical Data Analysis and Reporting System (CDARS) for the period of 180 days before to 180 days after the first RAMP-DM assessment, by matching the unique patient reference numbers. The laboratory data on the dates closest to the first RAMP-DM assessment were selected for subsequent analyses.Individual data collected at their first RAMP-DM assessment were analyzed in this study. Additional laboratory data of eGFR, UACR and HbA28, which combines eGFR and UACR to define CKD. The eGFR stages include G1 normal and high (≥90 ml/min/1.73 m2), G2 mild reduction (60–89), G3a mild-moderate reduction (45–59), G3b moderate-severe reduction (30–44), G4 severe reduction (15–29) and G5 kidney failure (<15). The UACR stages are A1 normal to miLDL-Cy increased (<3 mg/mmol), A2 moderately increased  (3–30) and A3 severely increased  (>30). CKD is defined as the eGFR stages of G3a through G5 and/or the UACR stages of A2 through A3.The CKD diagnosis followed the 2012 Kidney Disease Improving Global Outcomes (KDIGO) guideline29. Patients are classified into four categories: normal (SBP < 120 mmHg and DBP < 80 mmHg), elevated (120 mmHg ≤ SBP < 130 mmHg and DBP < 80 mmHg), stage 1 (130 mmHg ≤ SBP < 140 mmHg or 80 mmHg ≤ DBP < 90 mmHg), and stage 2 (SBP ≥ 140 mmHg or DBP ≥ 90 mmHg). The diagnosed diabetic complications of coronary heart disease (CHD), stroke, peripheral arterial disease (PAD) were also retrieved for the first RAMP-DM assessment. Diabetic retinopathy was classified into four categories: (1) sight threatening retinopathy were those who had laser, vitrectomy or anti-vascular endothelia growth factor (anti-VEGF) therapy, or were classified as severe non-proliferative DR, proliferative DR or maculopathy); (2) non sight threatening retinopathy were those who are classified as mild/moderate non-proliferative DR, and no maculopathy and no laser for both eyes; (3) No DR are those who were classified as no-DR without any maculopathy, laser, vitrectomy or anti-VEGF therapy; (4) Ungradable were those with at least one eye classified as “Not known” grade of DR, maculopathy and laser, and not classified as severe pre-proliferative DR, proliferative DR, and also without vitrectomy nor anti-VEGF therapy. We also retrieved lipid profiles of TG, HDL-C and LDL-C cholesterol measured at the first RAMP-DM assessment for all the subjects. Individual records of HbA1c, eGFR and UACR were retrieved from the electronic health records of CDARS by matching the unique patient reference numbers.Individual demographic data, such as age, gender, duration of T2DM, occupation, education and whether receiving Comprehensive Social Security Assistance (CSSA), were included in the model as potential risk factors. Smoking status was classified into current, ex- and non-smokers. Alcohol drinking was grouped into current, social, ex- and non-drinkers. Duration of DM was the number of years between the first RAMP-DM assessment date and the first year of DM diagnosis. Obesity status was categorized according to the Asian criteria: underweight as BMI < 18.5, normal as 18.5 ≤ BMI < 23, overweight as 23 ≤ BMI < 25, and obese as BMI ≥ 25. Individual data of blood pressure will be included in the model as continuously variables or alternatively as categorical variable indicating the level of hypertension status. Hypertension status of each subject was classified by systolic blood pressure (SBP) and diastolic blood pressure (DBP) that were measured at their first assessment in RAMP-DM, according to the 2017 Guidelines for High Blood Pressure in Adults from the American College of Cardiology/American Heart Association1c, TG, LDL-C, HDL-C, and both SBP and DBP as continuous variables. Only alcohol drinking was eliminated from the final multivariate model. Adjusted odd ratios (OR) and the corresponding 95% confidence interval (CI) were calculated for the variables in the regression model. The product variables of gender with other significant covariates were respectively added into interaction models to assess the gender difference. If any significance was found in these interaction terms, stratified analysis by gender was also conducted to estimate the age-specific effect estimates. Statistical analysis was conducted by using R version 3.4.2 . A P-value less than 0.05 was considered as statistically significant.The participants with and without CKD were compared for their demographic and clinical characteristics using Chi-square test for categorical variables and t-test for continuous variables  or Mann-Whitney . Each variable was assessed for proportion of missing data, and the subjects with complete and incomplete data were compared for other variables, in order to determine whether data imputation was necessary. Univariate logistic regressions were performed to identify the potential covariates and multivariate logistic regression to estimate the effects after adjustment for covariates. We fit a stepwise multivariate logistic regression model, which adjusted for sex, age, DM duration, education, BMI, smoking, alcohol drinking, receiving CSSA, CHD, stroke, PAD, DR status, HbA29. We did another sensitivity analysis by filling the missing values of LDL-C with multiple imputation on age, sex, DM duration, BMI, HDL-C, TG and comorbidity, and the above multivariate regression was repeated using the new dataset.We replaced the linear covariates of SBP and DBP by hypertension categories defined according to the new AHA classification of hypertensionThe ethical approval has been obtained from the New Territory West Cluster Clinical and Research Ethics Committee. All research was performed in accordance with relevant guidelines. Consent forms were exempted because all the data were extracted from the computerized data system of Hospital Authority, and no personal data were collected for this study.Supplementary file


Candida peritonitis is a crucial disease, however the optimal antifungal therapy regimen has not been clearly defined. Peritoneal fibrosis (PF) can be caused by abdominal surgery, intra-abdominal infection, and malignant diseases, and is also widely recognized as a crucial complication of long-term peritoneal dialysis. However, the influence of PF on Candida peritonitis prognosis remains unknown. Here, we evaluated the severity of Candida peritonitis within the context of PF and the efficacy of micafungin using mice. A PF mouse model was generated by intraperitoneally administering chlorhexidine gluconate. Candida peritonitis, induced by intraperitoneal inoculation of Candida albicans, was treated with a 7-day consecutive subcutaneous administration of micafungin. Candida infection caused a higher mortality rate in the PF mice compared with the control mice on day 7. Proliferative Candida invasion into the peritoneum and intra-abdominal organs was confirmed pathologically only in the PF mice. However, all mice in both groups treated with micafungin survived until day 20. Micafungin treatment tends to suppress inflammatory cytokines in the plasma 12 h after infection in both groups. Our results suggest that PF enhances early mortality in Candida peritonitis. Prompt initiation and sufficient doses of micafungin had good efficacy for Candida peritonitis, irrespective of the underlying PF. Peritoneal fibrosis (PF) is a pathological change occurring in the peritoneal membrane induced by long-term PD, which impairs the efficiency of dialysis and results in withdrawal from PD4. Infective peritonitis is also a common problem occurring in PD patients, which can be crucial. Bacteria, such as staphylococcal species, are common causative organisms of peritonitis6, but fungi, especially Candida species, are also important pathogens, which lead to high mortality rate of up to ≥25%11. Candida albicans is the most common species detected in >50% of cases11. Nevertheless, the optimal antifungal therapy regimen has not been clearly defined.Peritoneal dialysis (PD) is an effective treatment for end-stage renal disease12. Intra-abdominal candidiasis, such as peritonitis or abscesses, is considered much more common than recognized13. The causes of intra-abdominal candidiasis are not only from PD but also from intra-abdominal surgery, anastomotic leakage, and pancreatitis14. The Infectious Disease Society of America (IDSA) guideline on the management of candidiasis, updated in 2016, recommends echinocandin as an initial therapy for intra-abdominal candidiasis irrespective of the presence or absence of PF14. Micafungin is one of the antifungal agents belonging to the echinocandins class; and has been shown to have good antifungal activity even against non-albicans Candida species16. Non-albicans Candida species have recently increased as causative organisms in candidiasis patients19. However, C. albicans remains the predominant species causing Candida peritonitis. Furthermore, echinocandins are easy to use, with minimal adverse effects22. For these reasons, the clinical use of micafungin has recently increased.For fungal peritonitis, the International Society for Peritoneal Dialysis (ISPD) guideline updated in 2016, advocates for immediate catheter removal and antifungal therapy; however, the appropriate antifungal agent, for the initial therapy, has not been clearly definedCandida peritonitis, two complications in PD patients, have been evaluated thus far; and severe or prolonged infective peritonitis is known to induce PF24. Yet, the influence of PF on peritoneal infection has not been adequately investigated. Therefore, evaluation of the efficacy of antifungal therapy for Candida peritonitis developed in the context of PF is essential among PD patients who suffer from Candida peritonitis. Furthermore, it could be useful for treating patients with Candida peritonitis in the context of abdominal surgery, intra-abdominal infection, and malignant diseases who may develop PF27. In this study, we evaluated whether PF has any influence on the prognosis of Candida peritonitis and on the efficacy of micafungin treatment, using PF mouse models.PF and 4  Fig. . In the 4 Fig. .Figure 1We evaluated survival curves of the control and PF mice with or without micafungin treatment Fig. . When unThe fungal burden in the liver, spleen, kidneys, and blood were evaluated at 6 h, 12 h, and 7 days after the infection Fig. . The funCandida cells in most PF mice without micafungin treatment at 12 h after the infection  and PF group (with micafungin treatment), although infiltration inside the organ was not detected in any mice in these groups. No Candida cell was detected in the kidneys in all the groups (photographs not shown).Grocott staining of the liver Fig.  and spleans Fig. . AdhesioCandida peritonitis mouse models. At 12 h after the infection, interferon-gamma, interleukin-10, interleukin-17A, interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha (TNF-α) tended to be suppressed with micafungin treatment in both PF and control mouse models . All animal experiments were performed at the Nagasaki University Laboratory Animal Center for Biomedical Research in accordance with the Guide for the Care and Use of Laboratory Animals  and the institutional regulations and guidelines for animal experimentation.The animals used in this study were specific-pathogen-free male ICR mice . They were housed in the Biomedical Research Center, Life Science Support Center, Nagasaki University.32. Mice received injections of 0.1% CG in 15% ethanol, or 15% ethanol alone, at a dose of 0.2 mL/mouse into the peritoneal cavity on alternate days for 3 weeks, 9 times overall.We induced PF by intraperitoneal administration of CG as described previouslyFor the evaluation of PF, the PF and control mice were sacrificed 4 days after the last CG injection (n = 6 for each group). In order to prevent bias, peritoneal membrane was dissected in four locations , and evaluated with Masson’s trichrome staining. Thus, four sections were analyzed for each mouse. The thickness of the submesothelial compact zone for each section was measured at 10 positions and compared between the PF and control groups, using the unpaired t test.Candida peritonitis was induced by intraperitoneal inoculation of the C. albicans wild-type strain SC5314. C. albicans cells grown in yeast extract-peptone-dextrose (YPD) broth overnight were washed and resuspended in sterile saline and adjusted to 5.0 × 107 CFU/mL. Mice were infected intraperitoneally with 1000 µL of the Candida suspension 4 days after the last CG injection. Non-infected groups were intraperitoneally injected with 1000 µL of sterile saline instead of C. albicans cell suspension.C. albicans cell suspension; based on the same administration schedule against candidiasis reported previously38.All the mice in the treatment groups were treated with 5 mg/kg of micafungin (500 µg/mL)  injection subcutaneously in the neck, once a day for seven days, beginning 2 h after the intraperitoneal injection of 38. The dosage of 5 mg/kg corresponds to 125 mg/day in humans39 consistent with serum concentration-time curve data from 0 to 24 h (AUC0-24) between humans and mice41. The mice in the non-treatment groups were administered with sterile saline alone during the same period.The MIC of micafungin was ≤0.015 mg/L, determined by dry plate antifungal susceptibility testing of yeasts; Eiken . We used the subcutaneous route of administration because repeated injection into the tail vein of mice is difficult and it is reported that there is only a marginal difference in the efficacy of this drug between subcutaneous and intravenous administrationsC. albicans until day 20 (n = 8 for each group). Statistical analysis was performed using the Kaplan-Meier method.The survival of the mice was recorded daily after the intraperitoneal injection of Candida in the organs; the liver, spleen, and bilateral kidneys were excised and placed in sterile 0.9% saline at 4 °C immediately after sacrifice, at 6 h, 12 h, and 7 days after the intraperitoneal injection of C. albicans (n = 10 for each group). The homogenate was then serially diluted 1:10, and aliquots were plated on YPD agar. Blood samples were also collected by cardiac puncture and plated on YPD agar without dilution. Viable fungal colony counts were determined after 24–48 h incubation at 30 °C. The lower limit of detection was 10 CFU/organ. The results were expressed as log10 CFU/organ for the three organs and CFU/mL for blood, and analyzed with the Mann-Whitney U test followed by Bonferroni correction.To assess the viable cell count of C. albicans were stained with H & E and Grocott.For the histopathological evaluation, the peritoneum, liver, spleen, and kidney, obtained 12 h after the intraperitoneal injection of TM Mouse Cytokine Th17 Panel A 6-Plex  for the following groups. These were, the control and PF groups just before infection; and infected with or without micafungin administration, in both PF and control groups at 12 h after the infection (four mice each).The plasma cytokine concentrations of interferon-gamma, interleukin-10, interleukin-17A, interleukin-1 beta, interleukin-6, and TNF-α were quantified using a Bio-Plex ProThe plasma TNF-α concentrations in mice exposed to LPS were also quantified as follows: 20 mg/kg of LPS were administered intraperitoneally, after 2 h, 5 mg/kg of micafungin (500 µg/mL) was injected subcutaneously to the treatment group. Sterile saline was subcutaneously injected to the non-treatment groups. Plasma TNF-α concentrations, obtained 6 and 12 h after LPS administration were measured using Mouse TNF alpha Uncoated ELISA . Each of the groups consisted of four mice.The unpaired t test was used to assess differences of the PF thickness. The differences of fungal burden in the target organs and the cytokines in the plasma were evaluated by using the Mann-Whitney U test. Multiple comparisons were adjusted with the Bonferroni method. For all statistical analyses, a value of p < 0.05 was considered significant. All statistical analyses were performed using Prism 5.0 .


Several methods have been used to predict difficult tracheal intubation. Among recently suggested methods, the upper lip bite test (ULBT) could serve as a good predictor. Soft tissue and skeletal hard tissue profiles are affected by many factors including ethnicity. We aimed to assess the clinical utility of the ULBT in Koreans while considering ethnic differences.Three-hundred-forty-four Korean patients undergoing general anesthesia with orotracheal intubation were included. Preoperatively, we recorded the patient’s Modified Mallampati (MMT) classification, ULBT ratings, and the Cormack–Lehane grade.p < 0.0001). The ULBT showed high accuracy (73.83%) and specificity (98.04%). On the other hand, the ULBT showed significantly lower sensitivity (4.49%). Only nine of 344 Korean patients could not bite their upper lip; among them, only three presented a difficult laryngoscopic view.The area under the receiver operating characteristic (ROC) curve (AUC) was lower for the ULBT than the MMT (95% confidence interval: 0.0697–0.191, One factor related to the low sensitivity is the low incidence of a grade III ULBT in Koreans. In Asians, the scarcity of a grade III ULBT is explainable as a result of anteriorly displaced temporomandibular joints and redundant lip soft tissues. Despite its high specificity, the low sensitivity and AUC of the ULBT mean that the test results should be interpreted cautiously in Koreans. Ethnic differences should be considered when evaluating parameters related to soft tissues such as the ULBT.ClinicalTrials.gov Identifier: NCT01908218, Date of registration JUL 2013. Predicting difficult laryngoscopic endotracheal intubation (TI) is an important concern for anesthesiologists. Anticipated difficulties offer opportunities to prepare alternative methods and use proper advanced management techniques. The Mallampati classification and other methods have been used to evaluate anatomical structures during the preoperative period for airway assessment.Khan et al. suggested the upper lip bite test (ULBT) to, evaluates the ability of a patient to cover the mucosa of the upper lip with the lower incisors . This siThis method was based on cephalometric measurements, which differed in skeletal hard tissue and soft tissue profiles of Asian and Caucasian populations –10. The This study aimed to assess the differences in the ULBT in Koreans while considering ethnic differences. We tried to figure out the influence of the cephalometric differences to ULBT in Koreans.After obtaining approval from the Severance Hospital Institutional Review Board (IRB number: 4–2008-0583), the trial was performed at Yonsei University Severance Hospital.The written informed consent was obtained from all subjects participating in the trial.ClinicalTrials.gov identifier: NCT01908218, Principal investigator: So Woon Ahn, Date of registration JUL 2013). This manuscript adheres to the applicable EQUATOR Network guidelines. Patients were excluded if they had facial anomalies, had temporomandibular(TM) joint disorder, were edentulous or required a rapid sequence induction. In the pre-anesthetic care unit, we recorded each patient’s age, sex, weight, height, American Society of Anesthesiologists (ASA) classification, Modified Mallampati (MMT) class [Three hundred forty-four Korean adult patients undergoing general anesthesia with orotracheal intubation were included in our registered prospective observational study. (T) class , ULBT raT) class  In the cBased on an institutional pilot study, the area under the receiver operating characteristic (ROC) curve (AUC)s of the MMT and ULBT were 0.61 and 0.52, respectively. We determined that 344 patients would be required to demonstrate a difference between two predicting tools with a type 1 error (α) of 5% and power (1-β) of 90%(two-sided) using the PASS program .For continuous variables, a Student’s t-test was used to assess differences in means between the groups. For categorical data, a chi-square test was used to assess differences in proportions across the categories. The cut-off points of thyromental, sternomental and mouth opening distances were obtained from the analysis on the ROC which was calculated maximize the sensitivity and specificity . Each teData were collected from four hundred forty-four elective surgical patients. Seventy-five patients were excluded because they were edentulous or the patients did not undergo TI because of changes in their anesthetic plans. A total of 344 patients’ data were analyzed Fig. , Table 1During direct laryngoscopy, 89 patients presented with a difficult laryngoscopic view (Cormack–Lehane grade of 3 or 4) without external manipulation Table . Among tAn MMT class > II, IID ≤ 4.5 cm, TMD ≤ 8.3 cm, and SMD ≤ 17.9 cm were defined as cut-off points for difficult intubation  was higher than that of the MMT 62.80%), and the specificity of the ULBT (98.04%) was higher than that of the MMT (61.18%). Particularly, the ULBT showed significantly lower sensitivity (4.49%) compared with that of the MMT in our trial (67.42%). The prevalence of a difficult laryngoscopy (DL) was 25.87% (89 of 344), and the percentage of patients with an MMT class > II was 67.4% (60 of 344), while only nine patients (2.6%) showed a grade III ULBT  could easily be classified as a grade II or better with the application of external pressure to the larynx (the BURP maneuver) to move the epiglottis. These patients would have been described as having an easy TI. Therefore, it is difficult to predict a difficult TI (DTI) only by mandibular movement, and a DL does not predict a DTI.Moreover, in this trial, the prevalence of a DL was 25.87% 89 of 344), and the DTI/DL ratio was 3.4% (3/89). These are similar to the results shown in previous studies (4.3–77.8%) , 33, 34.4, and thIn Koreans, the ULBT shows very high specificity. The ULBT presented low false-positive rates and high accuracy. Cephalometric ethnical differences may present in Far East Asians are one reason. Therefore, especially in Asians, soft tissue redundancy and skeletal variance should be considered based on ethnic differences when evaluating parameters related to soft tissue such as the ULBT.


The development of the rumen is an important physiological challenge for young ruminants. Previous studies have shown that starter feeding can effectively facilitate the growth and development of the rumen in ruminants. However, the mechanism through which starter feeding stimulates the development of the rumen is not clear. Here, we performed an integrated analysis in ruminal microbiota and a host transcriptomic profile in a lamb model with the intervention of starter feed to understand the ruminal microbiome-host crosstalk in stimulating the development of the ruminal epithelium.Mitsuokella spp., lactate-producing Sharpea spp., lactate-utilizing Megasphaera spp., and Entodinium spp. was enriched in rumen microbial communities in the starter-feed group. The abundances of genes involved in sugar degradation were decreased in starter-feed lambs, but the GH13 encoding α-amylase was obviously increased. Rumen epithelial transcriptome analysis revealed that seven differentially expressed genes, including MAPK1, PIK3CB, TNFSF10, ITGA6, SNAI2, SAV1, and DLG, related to the cell growth module were upregulated, and BAD’s promotion of cell death was downregulated. Correlation analysis revealed that the increase in the concentrations of acetate and butyrate significantly correlated with the expression of these genes, which indicates acetate and butyrate likely acted as important drivers in the ruminal microbiome-host crosstalk.Decreased ruminal pH and increased acetate and butyrate concentrations in the rumen, followed by increasing rumen organ index, were observed in lambs supplemented with starter. Using metagenome sequencing in combination with 16S rRNA and 18S rRNA gene amplicon sequencing, the results showed the abundance of acetate-producing The present study comprehensively describes the symbiotic relationship between the rumen microbiota and the host in lambs after starter feeding. Our data demonstrates that the microbiome-driven generation of acetate and butyrate mediated the growth-related genes’ regulation of the growth-associated signalling pathway in the ruminal epithelium. These co-development networks regulated many physiological processes in the epithelium, including papillae morphology and rumen epithelial growth.The online version of this article (10.1186/s40168-019-0701-y) contains supplementary material, which is available to authorized users. For ruminants, the rumen is important for the host’s metabolism, immunity, and health. Vast microbes colonize in the rumen including bacteria, archaea, fungi, and protozoa, which play important roles in diet fermentation and the host’s energy supply. The rumen is also a natural bioreactor, in which microbial extracellular enzymes catalyse the hydrolysis of refractory dietary plant fibre that is otherwise resistant to the animal’s endogenous digestive enzymes . This unMost interestingly, the rumen epithelium is a unique place of interaction between host and microbial metabolism in that the rumen epithelium affects the net use of nutrients of the whole body, which physically serves as a barrier to the rumen epithelium’s contents, by providing VFA absorption capacity , 7. EmerTo date, there have been few studies that explore the underlying mechanism of microbiome-host crosstalk in stimulating the development of ruminal papillae by diverse dietary niches. Here, to develop a deeper understanding of ruminal microbiome-host crosstalk in the development of the rumen epithelium, we used 16S rRNA gene, 18S rRNA gene, shotgun metagenome, and transcriptome sequencing techniques to explore the interactions between the ruminal microbiota and host. This study brings new insights into the interactions between the ruminal microbial population and the host and highlights the importance of the co-development of the host and its microbiota in terms of host fitness.p < 0.001). The total VFA (p = 0.034), acetate (p = 0.028), and butyrate (p = 0.007) were increased after starter feeding, while propionate (p = 0.650) and other VFA (p = 0.496) remained unchanged. In the molar proportion, butyrate was higher in the group supplemented with starter feed (ST) (p = 0.019), whereas acetate (p = 1.000), propionate (p = 0.151), other VFA proportions (p = 0.326), and the acetate to propionate ratio (p = 0.131) were not significantly different between the two groups. As for the physiological indicators of the rumen, starter feeding increased the emptied weight of rumen (p = 0.034), as well as, increased the length (p < 0.001), width (p < 0.001), and surface (p = 0.001) of papillae in the ventral sac of the rumen .Compared with the control (CON) group . There was a total of 875,402 high-quality reads and an average of 43,770 ± 1737 reads per sample via 16S rRNA sequencing. Rarefaction curves approximately trended to a plateau at 27,942 reads revealed that the sequencing coverage was saturated  and Firmicutes (34.05–33.50%). Significant shifts were detected (p < 0.05) in three phyla, including Proteobacteria, Tenericutes, and Actinobacteria, during the period of starter feeding. Among these affected phyla, the relative abundance of Proteobacteria and Actinobacteria increased (p < 0.05), whereas the relative abundance of Tenericutes decreased (p < 0.05) in the ST group. At the genus level  in the ST group. Moreover, the relationship between Megasphaera and Sharpea in terms of relative abundance was significantly strong . In addition, starter feeding significantly reduced the proportion of RC9_gut_group, unclassified Christensenellaceae, unclassified Lachnospiraceae, Butyrivibrio, Oribacterium, and Quinella compared to the CON group .Based on the above results, we identified all bacterial OTUs Fig. a. In thep = 0.005). After a quality filter was applied, 928,193 high quality reads and an average of 46,410 ± 2339 reads per sample were observed. Rarefaction curves approximately trended to a plateau at 22,952 reads, which revealed that the sequencing coverage was saturated . Across the 18S rRNA gene samples, we found that almost all ciliate protozoal sequences were assigned to six genus-equivalent protozoal groups all belonging to Ciliophora with an identical threshold that means relative abundance in one group was more than 0.5% , was an only significantly increased genus (p = 0.010). However, Diplodinium, Ophryoscolex, Polyplastron, and unclassified Trichostomatia were comparatively enriched in the CON group ; the others had no significant differences  and the top 10 identified phyla or genera were linked based on the sequences , the regulations of three modules, namely protein activity processes (modification and degradation), substance transport, and cell growth (apoptosis and proliferation), were observed , whose ruminal DNA samples were used for the metagenomic analysis. We generated 54.82 Gb of clean data, with an average of 6.85 Gb (± 0.34 SEM) of clean data per subject. Among the encoded genes, 604 DEGs were identified from the comparison of the two groups . Among these DEGs, there were 358 upregulated genes and 246 downregulated genes. Using DAVID, the DEGs were then used to conduct GO enrichment analysis of the biological processes. We found that, among 73 significantly altered GO terms  consisted of 10 lambs randomly kept with the dam, and the lambs were fed breast milk without receiving the starter feed. The starter treatment group (ST) consisted of the other 10 lambs that were fed breast milk for 1 h at 0630, 1030 and 1530 h, and also daily fed the starter feed ad libitum from 0400 to 1900 h in individual pens. The amount of DMI of the starter feed was targeted to 200 g·animal−1·d−1 throughout the experimental period, and the amounts of feed offered and residue were recorded daily for calculation of the DMI. To meet the nutrient requirement of Hu sheep lambs, we designed the formula of starter feed . The ingredients and nutrient composition of the starter feed is provided in Additional file This study was conducted in accordance with the guidance of the Animal Care and Use Committee of Nanjing Agricultural University (SYXK (Su) 2015-0656) in China. We used 20 healthy 10-day-old w/v) metaphosphoric acid  and mixed and stored at − 20 °C for analysis of VFA concentrations in gas chromatography  [On the last day of the experimental period, all lambs were slaughtered 3 h after the morning feeding. The lambs were stunned and exsanguination immediately followed. The internal organs were immediately dissected and the rumen was separated; the content within the rumen was collected for sampling. Five milliliters of homogenized ruminal content samples were collected in triplicates and stored at − 20 °C for microbial DNA extraction. The other aliquot of the ruminal content sample was screened through a four-layer cheesecloth. Immediately, we used a portable pH meter  to measure the pH of the rumen fluid. Then, the fluid was kept in 25%  . SubsequThe DNA of the rumen microbiota was extracted from 0.3 g ruminal content per sample using a DNA Kit . The specific operation was strictly in accordance with the standard process specification of the DNA Kit. A bead-beating procedure was used to break down the cell wall of the microbes and separate the DNA. The purity and concentration of the obtained DNA was determined by a NanoDrop 1000 spectrophotometer . All the DNA samples were stored at − 80 °C until subsequent processing.Through sequencing the region of bacterial 16S rRNA gene and ciliate protozoal 18S rRNA gene, we obtained the structure of the rumen microbial communities. For bacteria, we used universal primers to amplify the V3 and V4 regions of 16S rRNA gene and with a 6 bp barcode unique to each sample. The primers were . For protozoa, we used special primers to amplify the ciliate protozoal 18S rRNA gene and with a 6 bp barcode unique to each sample. The primers were . After PCR amplification, all amplicon libraries were sequenced using an Illumina MiSeq PE 300 platform. We removed the barcodes and sequencing primers before data processing.Eight samples (four per group) were selected to conduct shotgun metagenome sequencing. For each sample, 1 mg of genomic DNA was used with Illumina’s TruSeq for library preparation. Libraries were sequenced using an Illumina HiSeq PE 150 platform.http://drive5.com/uparse/) [We used FLASH (version 1.2.7)  to mergeuparse/) . Then, tuparse/) .After sequencing, low-quality reads and contaminated adaptor and sheep host reads were removed from the raw sequenced reads by the FastQC software (version 0.11.8)  and BWA The total RNA of the rumen epithelium across all samples was extracted using TRIzol  . Then, ahttps://ccb.jhu.edu/software/hisat2/index.shtml) [https://david.ncifcrf.gov/) [We first used an inhouse perl script to remove low-quality reads. HISAT2 (x.shtml)  was usedx.shtml) . Gene exx.shtml) ). The GOrf.gov/) . KOBAS (rf.gov/) . After sU test in SPSS software . A value of p < 0.05 was regarded as statistically significant and p values were adjusted into FDR using the Benjamini-Hochberg method. The “Hmisc” package in the R software was used to complete the Spearman rank correlation coefficient. In addition, the “circlize” package was used to calculate the correlation between the dominant phylum or genus and carbohydrate-active enzymes classes. Cytoscape (version 3.5.1) [All the significant differences in the present paper were tested using the Mann−Whiney n 3.5.1)  was perfAdditional file 1:Table S1. Compositions of the starter diet (DM basis). (DOCX 15 kb)Additional file 2:Table S2. Effects of starter feeding on rumen fermentation in lambs. (DOCX 16 kb)Additional file 3:Table S3. Effects of starter feeding on rumen epithelium papillae parameters in lambs. (DOCX 14 kb)Additional file 4:Figure S1. The rarefaction of the rumen bacteria and ciliate protozoa based on the 16S rRNA and 18S rRNA genes in lambs. (PDF 295 kb)Additional file 5:Table S4. The alpha diversity of rumen bacterial community based on 16S rRNA genes at 3% dissimilarity level. (DOCX 14 kb)Additional file 6:Table S5. Effects of starter feeding on the relative abundance (%) of rumen bacteria at the phylum level. (DOCX 15 kb)Additional file 7:Table S6. Effects of starter feeding on the relative abundance (%) of rumen bacteria at the genus level. (DOCX 17 kb)Additional file 8:Effects of starter feeding on the relative abundance (%) of rumen ciliate protozoa at 3% dissimilarity level. (DOCX 15 kb)Additional file 9:Table S8. Effects of starter feeding on the relative abundance (%) of rumen ciliate protozoa the at genus level. (DOCX 15 kb)Additional file 10:Table S9. Effects of starter feeding on the relative abundance (TPM) of carbohydrate-active enzymes genes. (DOCX 15 kb)Additional file 11:Table S10. Effects of starter feeding on the relative abundance (TPM) of GH family genes coded amylolytic enzymes. (DOCX 15 kb)Additional file 12:Figure S2. Phylogenetic distribution of sequences of carbohydrate-active enzyme classes assigned to the top 10 phyla or genera. (PDF 1801 kb)Additional file 13:Table S11. GO enrichment analysis of differentially expressed genes in biological processes. (XLSX 18 kb)Additional file 14:Table S12. The expression profile (FPKM) of differentially expressed genes related to the cell growth module in the rumen epithelium of lambs. (DOCX 15 kb)Additional file 15:Table S13. The primer sequences of differentially expressed genes related to the cell growth module in the rumen epithelium of lambs for qRT-PCR. (DOCX 16 kb)Additional file 16.The parameters of the software and packages mentioned were presented. (DOCX 17 kb)


A majority of current microbiome research relies heavily on DNA analysis. However, as the field moves toward understanding the microbial functions related to healthy and disease states, it is critical to evaluate how changes in DNA relate to changes in proteins, which are functional units of the genome. This study tracked the abundance of genes and proteins as they fluctuated during various inflammatory states in a 4.5-year study of a patient with colonic Crohn’s disease. Our results indicate that despite a low level of correlation, taxonomic associations were consistent in the two data types. While there was overlap of the data types, several associations were uniquely discovered by analyzing the metaproteome component. This case study provides unique and important insights into the fundamental relationship between the genes and proteins of a single individual’s fecal microbiome associated with clinical consequences. Faecalibacterium and a positive association of Escherichia with calprotectin. Despite concordant associations of genera, the specific genes correlated with these metrics were drastically different between metagenomic and metaproteomic data sets. This resulted in the generation of unique functional interpretations dependent on the data type, with metaproteome evidence for previously investigated mechanisms of dysbiosis. An example of one such mechanism was a connection between urease enzymes, amino acid metabolism, and the local inflammation state within the patient. This proof-of-concept approach prompts further investigation of the metaproteome and its relationship with the metagenome in biologically complex systems such as the microbiome.Although genetic approaches are the standard in microbiome analysis, proteome-level information is largely absent. This discrepancy warrants a better understanding of the relationship between gene copy number and protein abundance, as this is crucial information for inferring protein-level changes from metagenomic data. As it remains unknown how metaproteomic systems evolve during dynamic disease states, we leveraged a 4.5-year fecal time series using samples from a single patient with colonic Crohn’s disease. Utilizing multiplexed quantitative proteomics and shotgun metagenomic sequencing of eight time points in technical triplicate, we quantified over 29,000 protein groups and 110,000 genes and compared them to five protein biomarkers of disease activity. Broad-scale observations were consistent between data types, including overall clustering by principal-coordinate analysis and fluctuations in Gene Ontology terms related to Crohn’s disease. Through linear regression, we determined genes and proteins fluctuating in conjunction with inflammatory metrics. We discovered conserved taxonomic differences relevant to Crohn’s disease, including a negative association of IMPORTANCE A majority of current microbiome research relies heavily on DNA analysis. However, as the field moves toward understanding the microbial functions related to healthy and disease states, it is critical to evaluate how changes in DNA relate to changes in proteins, which are functional units of the genome. This study tracked the abundance of genes and proteins as they fluctuated during various inflammatory states in a 4.5-year study of a patient with colonic Crohn’s disease. Our results indicate that despite a low level of correlation, taxonomic associations were consistent in the two data types. While there was overlap of the data types, several associations were uniquely discovered by analyzing the metaproteome component. This case study provides unique and important insights into the fundamental relationship between the genes and proteins of a single individual’s fecal microbiome associated with clinical consequences. Due to the growing evidence for a connection between microbial communities and human health, exploration of the microbiome has rapidly expanded in the past decade. To date, the primary avenue for studying the microbiome has been through genomic technologies –3. These–2/MS3)-based quantitation workflow to increase accuracy and reduce the sparsity associated with label-free proteomics , a subtype of inflammatory bowel disease (IBD), represents a chronic autoimmune condition associated with large fluctuations in the microbiome –22. A st–A distinguishable aspect of our study is a shift from contrasting IBD cohorts and healthy subjects to exploring a time series perspective from a single patient. Previous studies investigated metaproteome stability in the context of healthy subjects , 28; how29–n = 1 patient was a nonsmoker male. He was diagnosed in 2011, at age 63, with CCD by William J. Sandborn at the University of California Health System. The inflamed region of the colon was determined, via colonoscopy and abdominal magnetic resonance imaging (MRI) analysis, to be confined to 6” to 8” of the sigmoid colon. Specifically, a 2012 colonoscopy revealed that this region had extensive diverticulosis and inflammatory focal ulceration, inflammatory pseudopolyps, and patchy friability not associated with the diverticular orifices. During the time interval covered in this work (28 December 2011 to 22 May 2016), the patient had one period of antibiotic therapy, which consisted of ciprofloxacin 500 mg administered twice daily and metronidazole 250 mg administered three times daily for 1 month starting 31 January 2012. During that period, the patient was also taking 40 mg prednisone daily. In another 4-month period from August through November 2013, the patient had simultaneous courses of mesalamine  and budesonide (Uceris) administered at 9 mg daily. During the reported period, the patient had episodic symptoms of rectal bleeding, abdominal cramps, bloating, and malaise. Lastly, there was no surgery performed on the patient during the time period covered by this work.The r = 0.96 and 0.50, respectively), which led to overlapping results in downstream analysis. Because calprotectin is more widely used for the assessment of IBD , lysozyme, S-IgA, calprotectin, and lactoferrin were selected for their unique properties and clinical applications in IBD. We observed similar expression patterns over time for calprotectin, lactoferrin, and S-IgA . Lactofet of IBD , we focu3)  .3 data. Our first approach utilized the shotgun metagenomic reads generated within the study to create a personalized database (pDB) containing 1.3 million protein-coding regions  Protein group identifications using two database methods for proteomic data and gene-level identifications against the pDB. (b) Comparisons of peptide sequence identification results from two search database methods. (c) KEGG orthologous annotation comparisons between the two search database methods for proteomics data (d) Distance comparisons of samples from cases of high and low inflammation  from BraCopyright © 2019 Mills et al.2019Mills et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Notably, a lack of sequences shared between samples is a known trait of microbiome studies . We obse10–P < 0.001). We also observed clustering based on the presence of a state of high or low inflammation  using the Bray-Curtis distance metric . To overammation . Group dammation .To investigate the relationship between gene-level and protein-level fluctuations, the data were subsetted to the 3,598 ORFs with quantitation in both the metagenome and metaproteome. Spearman correlations between the protein and gene abundances in each of the samples were assessed. Overall, the Spearman correlations were normally distributed around ρ = 0.317 . This liMethanobrevibacter smithii and Methanosphaera stadtmanae    and b. Dlistipes . For easlistipes . Metaprojustment .10.1128/mSystems.00337-18.2FIG S2FIG S2, PDF file, 1.2 MB.Taxonomic relationships to disease severity. (a) Lowest common ancestor-adjusted phylum-level composition of metaproteome data. (b) Lowest common ancestor-adjusted genus-level composition of metaproteome data. (c) Correlations between the microbial dysbiosis disease index and clinical markers of severity. Best-fit lines are displayed surrounded by 95% confidence intervals. Download Copyright © 2019 Mills et al.2019Mills et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Escherichia coli and Fusobacterium nucleatum, which are increased in abundance in CD, and Faecalibacterium prausnitzii, Eubacterium rectale, and Bacteroides vulgatus, which are decreased in abundance in CD. After summing gene and protein abundances and determining the relationship between log ratios and each biomarker, fecal calprotectin was found to have the strongest association with the microbial dysbiosis index in both the metagenome and metaproteome. This result was not statistically significant, which was likely a result either of the small sample size or of the extrapolation of methods developed from hundreds of patients for use with a single subject . Interestingly, most of the individual genes and proteins associated with each of the inflammatory markers were unique, with only 0.5%  of associations shared between data types  after calculations of linear regressions with respect to the abundance of each clinical marker were performed . Red, metaproteome; black, metagenome. Download FIG S3, PDF file, 0.3 MB.Comparison of highly correlated proteins and genes from shared identifications. The Venn diagrams represent the overlapping genes and proteins from the 3,598 ORFs representing both gene counts and protein abundances. Genes and proteins were deemed highly correlated by effect size , we contrasted the taxonomic compositions of the positive and negative correlations. Several genera had >30-fold differences between compositions  were compared, and the log ratio per phylum was plotted at the metagenomic and metaproteomic levels. (b) Proteins and genes used as described for panel a were analyzed at the genus level. Genera were grouped by Gram staining results. Download FIG S4, PDF file, 0.4 MB.Taxonomic relationships to lysozyme. (a) The compositions of highly correlated proteins and genes related to lysozyme levels  had differences of 3-fold or greater . CompariBiologically relevant relationships were observed in the metaproteome that were not detectable in the metagenome. Free amino acids and urease enzymes have previously been associated with gut dysbiosis and Crohn’s disease . InteresAnother observation that was exclusively related to the metaproteome data was the relationship of chaperone proteins to several of the inflammatory metrics. There were 15 chaperone proteins with similar trends in expression with respect to CRP . This coFaecalibacterium and Butyrivibrio were identified  proteins or genes derived from each functional group were compared. The log ratios of the compositions of positive and negative proteins are plotted on the x axis for each clinical marker. Metaproteome values are plotted in red, and metagenome values are plotted in black. (c) The function-level associations determined for lactotransferrin were similar to those determined for calprotectin. The methods used for the determinations were described to those described for panel b, but eggNOG categories were compared. (d) Time series plots of selected proteins of interest. Proteins with GO terms that included iron are plotted with associated 68% confidence intervals. A legend describing the protein names and associated genera is shown to the right of the plot. Download FIG S5, PDF file, 1.7 MB.Analysis of other immune biomarkers. (a) Other immune markers (lactotransferrin and secretory IgA) showed trends similar to those seen with calprotectin. The proteome relative abundances of IGHA1 and lactotransferrin and calprotectin are displayed for each time point. (b) The genus-level associations with lactotransferrin were similar to those seen with calprotectin. Calculations of linear regressions with respect to clinical markers were performed, and the numbers of highly correlated  had a larger portion of GO terms related to iron (15.5% of 470 positive associations and 10% of 233 negative associations). Many of these proteins were pyruvate oxidoreductases, which are used in anaerobic bacteria for forming acetyl-coenzyme A (acetyl-CoA) from pyruvate . In the single-species context, bacterial systems have generally shown correlations between mRNA and proteins to range from ρ = 0.5 to 0.6 . Our exp formate . Nitrateammation . Tables Faecalibacterium is a genus depleted in IBD  and HRPP 150275 . Both protocols were approved by the Human Research Protection Program (HRPP) of the University of California, San Diego. Written informed consent obtained from the patient concerning dissemination and scientific publication of the results is also included in the approved protocols.Naturally passed fecal samples were collected and immediately stored without buffer at −80°C. Eight samples were selected. A personal symptom log entry was generated at the time that each fecal sample was passed. Additionally, the weight and body mass index (BMI) of the patient were determined on the day associated with each sample.Samples were extracted according to the Earth Microbiome Project  protocolhttps://github.com/knightlab-analyses/Crohns-MG-MP-Comparisons).Because typical metagenomics and metaproteomics workflows require a reference database, it was necessary to use a minimal approach to create from scratch a single reference database that could be used for both metagenomics and metaproteomics from the individualized data. All reads from the technical triplicates of each sample were concatenated. Next, the MEGAHIT alignment program  was utilThe program Salmon  was applhttps://www.uniprot.org/; accessed 28 November 2016) using Proteome Discoverer 2.1 (Thermo Fisher Scientific). Further data processing is described below.The generation of mass spectra data is described below. Spectral data were searched against the pDB with a concatenated human reference library (18 Sep-Paks (Waters) and eluted with 40% and 80% acetonitrile solutions containing 0.5% acetic acid . Samples were suspended through vortex mixing and homogenized through the use of a blender apparatus. A Steriflip (Millipore) filter (20 μM vacuum) was used to remove particulate from the samples. Cells were pelleted through centrifugation at 4,000 rpm for 10 min. Next, cells were lysed in 2 ml of buffer containing 75 mM NaCl (Sigma), 3% sodium dodecyl sulfate , 1 mM NaF (Sigma), 1 mM beta-glycerophosphate (Sigma), 1 mM sodium orthovanadate (Sigma), 10 mM sodium pyrophosphate (Sigma), 1 mM phenylmethylsulfonyl fluoride , 1× Complete Mini EDTA free protease inhibitors (Roche), and 50 mM HEPES (Sigma), pH 8.5 . An equatic acid . Concenttic acid . These btic acid . Each satic acid . Reagent18 column (Thermo Scientific) (4.6 mm by 250 mm) on an Ultimate 3000 high-performance liquid chromatography (HPLC) system fitted with a fraction collector, degasser, and variable-wavelength detector. The separation was performed over a 22% to 35%, 60-min linear gradient of acetonitrile–10 mM ammonium bicarbonate (Fisher) at 0.5 ml/min. The resulting 96 fractions were combined as previously described (2/MS3 for identification and quantitation.Sample fractionation was performed by basic pH reverse-phase liquid chromatography with concatenated fractions as previously described . Brieflyescribed . Fractio2/MS3 experiments were carried out on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) with an in-line EASY-nLC 1000 instrument (Thermo Fisher Scientific) and a chilled autosampler. Separation and acquisition settings were as previously defined . The Sequest searching algorithm (2 fragments. Static modification of TMT 10-plex tags on lysine and peptide N termini (+229.162932 Da), carbamidomethylation of cysteines (+57.02146 Da), and variable oxidation of methionine (+15.99492 Da) were included in the search parameters. Raw data were searched at a peptide and protein false-discovery rate (FDR) of 1% using a reverse-database-search strategy (–Data were processed using Proteome Discoverer 2.1 (Thermo Fisher Scientific). MSlgorithm  was usedlgorithm , 79 and strategy –82.3 spectra for quantitative analysis, and signal-to-noise values were used for quantitation. Additional stringent filtering was used, removing any moderate-confidence peptide spectral matches (PSMs) or ambiguous PSM assignments. Additionally, any peptides with a spectral interference level above 25% were removed, as well as any peptides with an average signal-to-noise ratio of less than 10. In accordance with false discovery rate benchmarking . A two-step database search method was utilized , and records of the code are available in corresponding Jupyter Notebooks for this project . All displayed metaproteomic data were generated using the pDB metaproteomic data unless otherwise specified. Qiime was used for principal-coordinate analysis (http://pandas.pydata.org/). Linear regressions were performed on metagenome sums and metaproteome averages against the metaproteome abundances of each of the biomarker abundances. Protein and gene associations were ranked by the associated coefficient of correlation, and taxonomic and functional annotations of the top associated genes and proteins (|r| < 0.7) were compared. Linear regressions were performed using the python package scipy (https://www.scipy.org). Friedman tests were also performed through scipy, comparing genus compositions within the metagenome and metaproteome between samples.Data analysis was performed in python version 3.5 . Metagenomic data are available through the European Bioinformatics Institute (EBI) (https://www.ebi.ac.uk/ena) under the study identifier PRJEB28712 (ERP110957).Proteomic data and supplementary files are available online at


Obstetric dfficult airway is a leading cause of maternal morbidity and mortality. The laryngeal mask airway (LMA) is often used as a rescue airway device after failed intubation, however, little is known about predictors of difficult LMA insertion, particularly in obstetrics. Since Mallampati scores of III/IV has been associated with difficult tracheal intubation, our present study aims to investigate if Mallampati score (MP) could predict airway outcomes for LMA use in obstetrics.This prospective cohort study was performed at a single-center: Quanzhou Women’s and Children’s Hospital, Fujian Province, China. Five hundred and eighty-four parturients undergoing elective cesarean section under general anesthesia were recruited. The primary outcome was time to effective ventilation, and secondary outcomes included first attempt insertion success, seal pressure, ventilation and hemodynamic parameters, occurrence of clinical aspiration, and maternal and fetal outcomes.p <  0.0001). There was no difference in maternal age, ASA status and gestational age. There was similar time to effective ventilation (mean (SD) High MP: 14.9 (4.5) vs Low MP: 15.7 (4.4) seconds, p = 0.2172), and first attempt success rate, seal pressure, and peak airway pressure. No clinical aspiration was noted. The incidence of blood on SLMA was higher in the High MP group than in Low MP (4 (6.6%) vs 4 (0.8%), p = 0.001). There was no difference in sore throat, voice hoarseness, maternal satisfaction and fetal outcomes.The parturients were classified into two groups based on MP of III/IV (High MP: 61) versus I/II (Low MP: 523). BMI was higher in the High MP group than in the Low MP group (mean (SD) 29.3 (7.0) vs 26.8 (3.1), High MP was not associated with reduced SLMA airway outcomes in cesarean section under general anesthesia, but may increase the risk of blood found on SLMA upon removal.http://www.clinicaltrials.gov, identifier: NCT02026882, retrospectively registered. Date of registration: December 31, 2013.This study was registered at  Airway complication is one of the main causes of anesthesia-related obstetric adverse events. There are several complex clinical challenges faced in managing the parturient including the risk of pulmonary aspiration, physiological changes of pregnancy, urgent nature of cesarean section, and balancing the potentially conflicting needs of the mother and fetus. Endotracheal intubation has long been considered standard of care for obstetric airway management , 2, but With the additional risk of failed intubation posed by the obstetric airway (estimated at 0.4%), the laryngeal mask airway (LMA) has emerged as a second-line airway device . AdditioNotwithstanding its utility as a rescue airway device, we should be cognizant that the incidence of first-attempt LMA failure in obstetrics is approximately 2% , 14. AddTherefore, it is vital to predict when LMA failure is likely to occur, and formulate an alternate airway plan. Additionally, to be an effective rescue airway the factors associated with LMA failure could be different from those predicting failure to intubate. This prospective cohort study aims to investigate the association between the modified Mallampati score and airway outcomes of Supreme™ LMA insertion for cesarean section under general anesthesia.This is a secondary analysis of a previously-published prospective cohort study , which w2, upper respiratory tract or neck pathology, or self-reported reflux disease. After fasting for a minimum of 4 h, the modified Mallampati score was determined by an independent assessor and the patients divided into two groups: Mallampati III/IV (High MP) and Mallampati I/II (Low MP).Parturients who were healthy or with well-controlled medical conditions (American Society of Anesthesiologists (ASA) physical status classification II) undergoing elective cesarean section under general anesthesia were recruited. We excluded patients with BMI > 35 kg/m2O via a manometer, assessment of successful SLMA placement was made by auscultation of breath sounds and the capnographic evidence of end-tidal carbon dioxide, and cricoid pressure was released. The use of additional maneuvers such as chin lift, jaw thrust, or head tilt were permitted. Rocuronium was used to maintain intraoperative muscle relaxation, and fentanyl was administered for analgesia after delivery of the fetus.The induction of anesthesia and SLMA insertion reflects the clinical practice at the study center, as well as the cohort study that forms the basis for this secondary analysis . As per 2O, positioning of the SLMA bite block between the incisors, successful orogastric tube insertion, and leak pressure > 20cmH2O. If successful SLMA insertion was not accomplished (1) after two attempts, (2) within one minute, or (3) before desaturation ensued, endotracheal intubation would be performed.We recorded (1) the number of insertion attempts required, with each attempt defined as insertion and complete removal of the SLMA; and (2) the time to effective airway placement, from when the SLMA was picked up until the first end-tidal carbon dioxide capnogram appeared. Subsequently, a pre-mounted #14 orogastric tube was inserted through the gastric drainage port, and placement confirmed by aspiration of gastric contents and auscultation of a “swoosh” over the epigastrium with air injection, followed by suctioning of the orogastric tube before surgery commenced. Finally, oropharyngeal leak pressure was measured by shutting the adjustable pressure-limiting valve and maintaining 3 L/min fresh gas flow into the closed circuit. Cesarean section was allowed to commence if the following were met: presence of square-wave capnogram, cuff pressure of 60 cmHPerioperative anesthesia management during the original cohort study  reflectsOur primary outcome was time to effective ventilation, as defined above. Secondary outcomes included the rate of successful first-attempt SLMA insertion, oropharyngeal leak pressure, ventilation parameters such as tidal volume and respiratory rate, hemodynamic parameters including heart rate and blood pressure at 2 and 5 min after induction, amount and pH of gastric aspirate, the pH of the SLMA laryngeal surface, and the presence of clinical aspiration. Obstetric and fetal outcomes included neonatal weight, Apgar scores, and umbilical venous pH.p <  0.05 and all tests were two-sided. Data analysis was generated using SAS 9.3 software .All demographic, anesthetic, and clinical categorical data were summarized as frequency with corresponding proportion, and continuous variables were expressed as mean (standard deviation (SD)) or median [interquartile range (IQR)], whichever appropriate. The difference between categorical data was tested using the Chi-Square test, while Student’s t-test and Mann-Whitney U test were used for parametric and non-parametric continuous data, respectively. Simple linear and multivariable linear regression methods were utilized to determine associated risk factors for our primary outcome of time to effective ventilation. Associations identified from linear regression were expressed as estimate (β) with 95% confidence interval (95%CI). Significance level was set at We performed a sample size calculation based on the following assumptions: a meaningful clinical difference of at least 10 s in time to effective ventilation between “High MP” and “Low MP” groups, pooled SD of 4.5, allocation ratio of 1:8, use of two-sample independent t-test, level of significance of 5% and > 95% power. The study was adequately powered (> 95%) with 584 parturients.p = 0.0001), but there was no significant difference in maternal age, fetal weight, or gestational age. The duration of surgery was similar, with mean (SD) 31.0 (11.1) minutes in the High MP group vs 29.3 (9.2) minutes in the Low MP group (p = 0.4159).We recruited 584 parturients. Of these, 61 were found to have Mallampati III/IV (High MP) and 523 with Mallampati I/II (Low MP) (Table p = 0.2172). Similarly, there was no difference in first attempt insertion success rate of 98.4% for the High MP group versus 98.3% for the Low MP group. The lowest SpO2, seal pressure and peak airway pressures were similar between the groups. No episodes of hypoxemia, laryngospasm, or bronchospasm were observed intra-operatively.Anesthetic outcomes are presented in Table p = 0.0191). The presence of blood on the SLMA was more frequently noted in the High MP group with 4 (6.6%) versus 4 (0.8%) in the Low MP group (p = 0.0010), but there were no significant differences in sore throat (p = 0.6071), voice hoarseness (p = 0.1585), or maternal satisfaction (p = 0.5131). Fetal outcomes, including Apgar scores at 1 and 5 min, and venous cord pH were similar between the groups.Maternal and fetal outcomes are summarized in Table Univariate linear regression method showed that age and baseline systolic blood pressure were significantly associated with time to effective ventilation Table ; while mThis prospective cohort study of 584 parturients undergoing cesarean section under general anesthesia compared the airway outcomes of the SLMA in patients with lower Mallampati scores (I or II) versus higher scores (III or IV). Out of 584 patients, 61 (10.4%) had Mallampati scores of III or IV. We found no association between Mallampati scores and time to effective ventilation, first attempt success rate, or SLMA seal pressure. There was no clinical aspiration detected, and the pH of the SLMA laryngeal surface was not indicative of gastric regurgitation. However, higher Mallampati scores was associated with increased risk of blood found on the SLMA, which may indicate increased risk of oropharyngeal trauma on insertion, but without corresponding change in sore throat, voice hoarseness, or maternal satisfaction.Our results suggest that LMA outcomes in obstetric general anesthesia was not significantly affected by higher Mallampati scores. The Mallampati score is postulated to be related to the size of the tongue base during difficult laryngoscopy due to occlusion of the view of pharyngeal structures . This isAlthough previous studies associated higher Mallampati scores with increased LMA failure rates , our finA common concern of LMA use in obstetrics relates to the inherently higher risk of gastric regurgitation and pulmonary aspiration, exacerbated by concomitant obesity, labor pain, or opioid analgesia. In this study, we did not detect any clinical aspiration or regurgitation, likely due to (1) the ability of second-generation LMAs to isolate the alimentary tract and facilitate gastric decompression, (2) better pharyngeal seal that prevents stomach insufflation and regurgitated gastric contents from entering the airway, (3) use of rapid-sequence induction and cricoid pressure, and (4) careful patient selection with exclusion of patients at high risk for regurgitation. However, this study was not powered to investigate the risk of pulmonary aspiration.There are several limitations in this study. First, we are cognizant that bedside tests, including the Mallampati score, are considered by some to be poor predictors of difficult intubation especially if used in isolation , 21. How2O greater than the average peak airway pressure regardless of Mallampati score, as well as gastric decompression via an orogastric tube.Second, we utilized clinical markers of SLMA airway outcomes, such as time to ventilation, first attempt insertion success rate, and seal pressure, in contrast to other studies that assessed LMA position via a fiberoptic grading scale . Several2 or higher. The retrospective study by Ramachandran et al. reported that elevated BMI of 29.3 versus 26.9 was associated with LMA Unique failure, but Mallampati scores of III to IV were not [2 in the “Low MP” group, we did not find any corresponding increased SLMA failure, although we were not specifically assessing the effects of increasing BMI on SLMA outcomes. Finally, we did not directly correlate failed intubation with LMA failure, and therefore cannot comment on the use of SLMA as a rescue airway.Third, we deliberately excluded patients at high risk of gastric regurgitation, including parturients with BMI of 35 kg/mwere not . DespiteOur prospective cohort study suggests that higher Mallampati score is not associated with longer time to effective ventilation when using the Supreme LMA in parturients undergoing elective cesarean delivery under general anesthesia, with similar time to effective ventilation, first attempt SLMA success rate, and seal pressure. More work is required to evaluate the accuracy of other bedside tests on predicting LMA airway outcomes in obstetrics.


Although Mitochondrial DNA depletion syndrome (MDS) can be classified into three forms: myopathic, encephalomyopathic and hepatocerebral form, it is difficult to identify its form due to its clinical heterogeneity. Therefore, it is very important to conduct molecular genetic analysis on suspected patients. This study presented a male 38 weeks and 5 days infant with liver cytolysis and leukodystrophy.TWNK: c.1186 C > T / p.Pro396Ser and c.1844 G > C / p.Gly615Ala inherited by an autosomal recessive form from his parents, of which protein conservative analysis and structural modeling supported the pathogenicity of the two mutations. Unfortunately, the conditions described above were not improved until he was discharged from the hospital on the 23rd day and died at 4 months of age.A male infant proband was admitted to the department of NICU for feeding intolerance, irregular rhythm of respiration, hypoglycemia, lactic acidosis, liver cytolysis and neurological abnormalities. He was onset of mild jaundice with leukodystrophy and high lactate and phenylderivatives for urine organic acids on the 7th day. Whole exome sequencing (WES) and Sanger sequencing were performed to screen and confirm the suspicious pathogenic mutations. The results revealed this proband carried two compound heterozygous mutations in TWNK c.1186 C > T/ c.1844 G > C compound heterozygous mutations which were probably the disease-causing mutations of hepatocerebral form of MDS and described the clinical manifestations of the proband, which expanded the phenotypic spectrum of MDS caused by variants in TWNK. This study also emphasized WES technology can provide the genetic diagnosis of Mendelian genetic disease.In this study, we investigated a Chinese family with the hepatocerebral form of MDS and conducted WES and Sanger sequencing to explore the causative mutations for this proband born from non-consanguineous and healthy parents. We identified two novel  TK2) [RRM2B) [SUCLA2 (succinate-CoA ligase ADP-forming beta subunit) [SUCLG1 [TWNK, OMIM # 606075), DNA polymerase gamma, catalytic subunit (POLG) [DGUOK) [MPV17) [The mitochondrial DNA (mtDNA) encodes 13 essential subunits of the respiratory chain complexes, 2 ribosomal RNA and 22 transfer RNA, whereas all the other components required for mitochondrial structure and function are encoded by the nuclear genome. Mutations in nuclear genes involved in the replication and maintenance of the mtDNA can result in the reduction of the mtDNA copy number, the accumulation of mtDNA multiple deletions, or both alterations in critical tissues [TK2)  and ribo [RRM2B)  are respsubunit)  and SUCLsubunit)  are relat (POLG) , 9, the  [DGUOK)  and the  [MPV17)  are the TWNK with 5 exons mapped to 10q24.31 can bring about hepatocerebral form of MDS with characteristic enlargement of liver and spleen, damage of liver function, retardation of psychomotor development, nystagmus and muscle hypotonia [TWNK and located in the mitochondrial matrix and mitochondrial nucleoids, plays an important role in mtDNA replication [TWNK was also associated with autosomal dominant progressive external ophthalmoplegia  which can result in ophthalmoparesis with exercise intolerance, ataxia, peripheral neuropathy and multiple mtDNA deletions and at least 40 causative mutations for adPEO have been identified up to now [TWNK mutations in an autosomal recessive inherited trait can cause infantile onset spinocerebellar ataxia (IOSCA) (OMIM #271245) with the main clinical features including muscle hypotonia, athetosis, ataxiaophthalmoplegia, hearing deficit, sensoryaxonal neuropathy, female hypogonadism and epilepticencephalopathy [Hepatocerebral form of MDS identified in children between birth and six months old presents multisystem phenotypes including liver failure, hypoglycemia, hypotonia and neurological symptoms, and patients usually die within one year of age  . Indeedypotonia –15. Twinlication . TWNK wap to now . Besidesalopathy  and Perralopathy .TWNK.Here, we described an infant proband born in a Chinese non-consanguineous family with clinical features of hepatocerebral form of MDS and identified novel compound heterozygous variants in This study investigated an infant proband with hepatocerebral form of MDS from a Chinese family. However, his parent didn’t exhibit similar symptoms of the disease. Written informed consent was obtained from the patient according to the protocol approved by the Institutional Review Board of The Affiliated Hospital of Qingdao University.http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.bii.a-star.edu.sg) whereas evolutionary conservation of the missense mutation sites were analyzed by aligning Twinkle amino-acid sequences of multiple species obtained from the UniProt website (http://www.uniprot.org).WES technology was performed to screen the mutational sites. Genomic DNA of the proband and his parent were extracted from the peripheral blood using the QIAamp DNA Blood Mini Kit . The DNA samples were prepared as Illumina sequencing libraries. The sequencing libraries were constructed with SureSelect Human All Exon V5 , according to the manufacturer’s protocols and were sequenced using Hiseq 4000 next-generation sequencing platform . Paired-end sequence reads were aligned to the human reference genome hg19 with Burrows-Wheeler Aligner (BWA), and then the variant calling process was run using the Genome Analysis Tool Kit (GATK). Variants were submitted to ANNOVAR for functional annotation and genetic filtering. Common variants were excluded by comparison with > 1000 exomes sequenced in our laboratory for other conditions and subsequently filtered with dbSNP v137, 1000 Genomes Project, National Heart, Lung, and Blood Institute (NHLBI), Exome Sequencing Project (ESP) and ExAC databases. Finally, functional impacts of the variants were predicted in silico by corrsponding softwares. Missense mutations were analyzed by PolyPhen-2 .The variants detected by WES were validated using Sanger sequencing. Amplifications were performed with primers targeting the variant loci as follows: forward, 5′-CAGTTCCGGCGGATTGTATT-3’and reverse, 5′-GTCCTACCCTTGAAACCAAGACA-3’for c.1186 C > T in The structure of T7 bacteriophage primase-helicase gp4 (PDB 1e0kA), a Twinkle ortholog, was used to predict the structural roles of Twinkle residues . Compara2 18 mmHg, Normal = 35~48 mmHg, buffuer excess − 18.6 mmol/L, Normal = − 2~ − 3 mmHg), liver cytolysis  and neurological abnormalities including: hyperspasmia, peripheral hypertonia and no primitive reflex. Biochemical investigations showed high plasma creatine kinase: 1360.6 UI/L , high isoenzyme of creatine kinase concentration:177 UI/L, high lactate dehydrogenase , high plasma ammonia: 90 μmol/L, high uric acid: 510 μmol/L and high blood urine nitrogen:10.31 mmol/L, while blood and body fluids showed no evidence of infection. In addition, there was elevated level of plasma tyrosine:1054 μmol/L  without associated evaluations in urinary succinylacetone. Urine organic acids revealed high lactate and phenylderivatives. Brain MRI showed leukodystrophy. The mild jaundice was noted  on the 7th day. The clinical manifestations were not get better until he left the hospital on the 23rd day and died at 4 months old.The proband was a male infant, born in 38 weeks and 5 days with a weight of 2950 g (10th centile). Apgar scores were 10 at 1, 5 and 10 min. He presented feeding intolerance, irregular rhythm of respiration, hypoglycemia , lactic acidosis  , Exome Sequencing Project (ESP) and ExAC databases, we identified potentially shared homozygous or compound heterozygous potentially damaging variants. We found the proband carried two compound heterozygous mutations in er) Fig. a, b. TheTWNK mutations in the proband. The contributed structure was energy minimized by Amber and shown as PyMOL Viewer representation, demonstrating that the two structures were similar , gp4 primase/helicase from phage T7 (PDB code 1e0kA) was used to model the possible structure of the TWNK: c.1186 C > T / p.Pro396Ser in exon 1 and c.1844 G > C / p.Gly615Ala in exon 5, which were transmitted in a autosomal recessive trait. The former was inherited from the father while the later was from the mother, both of which were not described associated with mitochondrial disorders before.MDS may affect either a specific tissue  or multisystem including heart, brain and kidney. Clinically, MDS were classified into three forms: myopathic, encephalomyopathic and hepatocerebral form . Due to TWNK, plays an important role in the integrity and maintenance of the mitochondrial DNA and displays 5′ → 3′ DNA helicase activity in unwinding the mitochondrial DNA at replication fork [TWNK have been summarized as Perrault syndrome 5(OMIM #616138) and Mitochondrial DNA depletion syndrome 7 . The main clinical features of Perrault syndrome 5 are hearing loss, female hypergonadotropic hypogonadism and ataxia [Twinkle, encoded by ion fork . Autosomd ataxia , howeverd ataxia , 24 and d ataxia , 25, 26.TWNK mutations leading to MTDPS7 have been identified, with four of these various causing the severe hepatocerebral form of MDS. Compound heterozygous TWNK mutations (p.A318T and p.Y508C) were found in two siblings with severe encephalopathy and signs of liver involvement at 6 months old. The clinical manifestations included hypotonia, athetosis, sensory neuropathy, ataxia, hearing deficit, ophthalmoplegia, intractable epilepsy and elevation of serum transaminases [TWNK in two patients presented neurological abnormalities, liver enlargement and failure, lactic acidosis at birth and abnormal eye movements [TWNK as the cause of multisystem failure including renal tubulopathy in three siblings [TWNK.Up to now, a total of 13 aminases . p.T457Iovements . In 2013siblings . In our Three functional domains forms the Twinkle: an N-terminal primase, a linker region involved in oligomerization and required for helicase activity and a C-terminal helicase . In thisTWNK mutations which are probably pathogenic mutations of hepatocerebral form of MDS. Our study emphasizes the utility of WES for the genetic diagnosis of complex multisystem disorders. Due to the diverse clinical manifestations of mitochondrial disorders, identification of causive allele by WES is now the gold standard for evaluation.Further involving multiple systems makes MDS exhibit a variety of clinical phenotypes, however the heterogeneity of clinical features of this disease makes it challenging to reach a definitive diagnosis in this case. By performing WES and Sanger sequencing, we identified two novel