Journal of Clinical Medicine — Open Access Journal

Background: Interactions and joint effects of galectin-3 and vascular cell adhesion molecule 1 (VCAM-1) on risks of all-cause and cardiovascular (CV) mortality remain unclear in patients with maintenance hemodialysis (MHD). Methods: Unadjusted and adjusted hazard ratios (aHRs) of mortality risks were analyzed between higher and lower concentration groups of serum galectin-3 and VCAM-1. The modification effect between serum galectin-3 and VCAM-1 on mortality risk was investigated using an interaction product term. Results: During follow-up, galectin-3 and VCAM-1 were associated with incremental risks of all-cause mortality (aHR: 1.038 (95% confidence interval (CI): 1.001–1.077) and 1.002 (95% CI: 1.001–1.003), respectively). Nonetheless, VCAM-1 but not galectin-3 predicted CV mortality (aHR: 1.043 (95% CI: 0.993–1.096) and 1.002 (95% CI: 1.001–1.003), respectively). In the interaction analysis, patients with combined higher galectin-3 (>29.5 ng/mL) and VCAM-1 (>1546.9 ng/mL) were at the greatest risk of all-cause and CV mortality (aHR: 4.6 (95% CI: 1.6–13.4), and 4.2 (95% CI: 1.3–14.4), respectively). The interactions between galectin-3 and VCAM-1 with respect to all-cause and CV mortality were statistically significant (p < 0.01 and < 0.05, respectively). Conclusion: Galectin-3 and VCAM-1 could serve as a promising dual biomarker for prognostic assessment, considering their joint effects on pathogenesis of leukocyte trafficking and atherothrombosis. Full article

This study aimed to identify an association between serum calcium (Ca) and phosphate (P) levels, tested during the pre- and postoperative period, with hospital costs and length of hospital stay (LOS) of patients who underwent major abdominal surgery. This retrospective cohort study analyzed the medical records of patients who underwent major abdominal surgery. A total of 3893 patients were included in the analysis, and multivariable linear regression analysis was performed. For a 1 mg/dL increase in preoperative Ca, total hospital costs decreased by 3997.9 dollars (coefficient: −3997.9, 95% confidence interval (CI): −4900.5, −30,953; p-value < 0.01), and for a 1 mg/dL increase in postoperative P, total hospital costs decreased by 702.5 dollars (coefficient: −702.5, 95% CI: −1274.5, −67.3; p-value = 0.03). Furthermore, for a 1 mg/dL increase in preoperative Ca, LOS decreased by 2.9 days (coefficient: −2.9, 95% CI: −3.7, −2.1; p-value < 0.01). For a 1 mg/dL increase in postoperative P, LOS decreased by 3.4 days (coefficient: −3.4, 95% CI: −4.2, −2.6; p-value < 0.01). This study suggested preoperative Ca and postoperative P could be useful indicators for the reduction of hospital costs and decrease in LOS from the perspective of enhanced recovery after surgery. Full article

Irritable bowel syndrome (IBS) remains a prevalent and difficult-to-manage gastrointestinal condition. There is growing interest in the use of traditional medicine to manage IBS. In particular, curcumin, a biologically active phytochemical, has demonstrated anti-inflammatory and anti-oxidant properties and mucosal protective effects in rat models of colitis. This meta-analysis thus aimed to investigate the hypothesis that curcumin improves IBS symptoms. Using the keywords (curcumin OR turmeric OR Indian saffron OR diferuloylmethane OR curcuminoid) AND (irritable bowel syndrome OR IBS), a preliminary search on the PubMed, Medline, Embase, PsychINFO, Web of Science, and Google Scholar databases yielded 1080 papers published in English between 1 January 1988 and 1 May 2018. Five randomized, controlled trials were systematically reviewed and 3 were included in the final meta-analysis. Random-effects meta-analysis based on three studies and 326 patients found curcumin to have a beneficial albeit not statistically significant effect on IBS symptoms (pooled standardized mean difference from baseline IBS severity rating −0.466, 95% CI: −1.113 to 0.182, p = 0.158). This is the first meta-analysis to examine the use of curcumin in IBS. With its unique anti-oxidant and anti-inflammatory activities and ability to modulate gut microbiota, curcumin is a potentially useful addition to our armamentarium of agents for IBS. It also appears safe and well-tolerated, with no adverse events reported in the available trials. However, current findings are based on a considerably limited evidence base with marked heterogeneity. More robust clinical trials involving a standardized curcumin preparation and larger sample sizes should be encouraged. Full article

Postmenopausal osteoporosis is the most common form of osteoporosis and one of the major public health problems in developed countries. The prevalence of this condition, associated with the physiological stage of menopause, is continuously increasing. This study evaluated the effectiveness of soy isoflavones as compared to hormone replacement therapy (HRT) in low doses, on the prevention of postmenopausal osteoporosis, by determining bone mineral density (BMD) and urinary deoxypyridinoline (D-pyr) in physiological postmenopausal women. The study was conducted over a period of 12 months, on three parallel groups, which included a total of 325 postmenopausal women (HRT group: n = 95; phytoestrogens group: n = 124; control group: n = 106). At the one-year evaluation, we observed T-score normalization in a small number of cases (5.26%, 2.42% and 0.00%, respectively). The average values of D-Pyr decreased by 11.38% in the group treated with phytoestrogens (p < 0.05) and by 15.32% in the group that followed HRT (p < 0.05); it increased by 4.38% in the control group (p > 0.05). Both therapies have beneficial effects on bone metabolism, leading to a significant decrease in the evolution of bone resorption and there are no major differences between the efficacy of HRT and phytoestrogens in terms of the effects on BMD and bone resorption. Full article

Background: The thyroid plays a key role in development and homeostasis, but it has been difficult to establish causality with diseases and phenotypic traits because of several potential confounders. Methods: To determine the causal effect of euthyroid function, we conducted a two-sample Mendelian randomization study of euthyroid thyrotropin (TSH) and free thyroxine (FT4) levels with respect to 2419 traits assessed in 337,199 individuals from UK Biobank. Additionally, we investigated the molecular differences between hypothyroidism and hyperthyroidism using genome-wide data. Results: After multiple testing correction, sixteen traits appear to be affected by genetically-determined euthyroid TSH, including multiple thyroid-related traits, e.g., hypothyroidism (p = 2.39 × 10⁻¹⁷), height (p = 2.76 × 10⁻¹⁰), body fat distribution (impedance of whole body, p = 4.43 × 10⁻⁸), pulse rate (p = 2.84 × 10⁻⁸), female infertility (p = 4.91 × 10⁻⁶), and hearing aid use (p = 7.10 × 10⁻⁵). Moreover, we found a consistent genetic correlation between hypothyroidism and hyperthyroidism (rg = 0.45, p = 5.45 × 10⁻⁶) with several immune pathways shared between these diseases. Two molecular pathways survived multiple testing correction for specificity to hyperthyroidism, JAK/STAT signaling (p = 1.02 × 10⁻⁶) and Rac guanyl-nucleotide exchange factor activity (p = 4.39 × 10⁻⁶). Conclusion: Our data shed new light on the inter-individual variability of euthyroid function and the molecular mechanisms of the two thyroid disorders investigated. Full article

Individuals at risk (IAR) of familial pancreatic cancer (FPC) are good candidates for screening. Unfortunately, neither reliable imaging modalities nor biomarkers are available to detect high-grade precursor lesions or early cancer. Circulating levels of candidate biomarkers LCN2, TIMP1, Glypican-1, RNU2-1f, and miRNA-196b were analyzed in 218 individuals with sporadic pancreatic ductal adenocarcinoma (PDAC, n = 50), FPC (n = 20), chronic pancreatitis (n = 10), IAR with relevant precursor lesions (n = 11) or non-relevant lesions (n = 5), 20 controls, and IAR with (n = 51) or without (n = 51) lesions on pancreatic imaging. In addition, corresponding duodenal juice samples were analyzed for Glypican-1 (n = 144) enrichment and KRAS mutations (n = 123). The panel miR-196b/LCN2/TIMP1 could distinguish high-grade lesions and stage I PDAC from controls with absolute specificity and sensitivity. In contrast, Glypican-1 enrichment in serum exosomes and duodenal juice was not diagnostic. KRAS mutations in duodenal juice were detected in 9 of 12 patients with PDAC and only 4 of 9 IAR with relevant precursor lesions. IAR with lesions on imaging had elevated miR-196b/LCN2/TIMP1 levels (p = 0.0007) and KRAS mutations in duodenal juice (p = 0.0004) significantly more often than IAR without imaging lesions. The combination miR-196b/LCN2/TIMP1 might be a promising biomarker set for the detection of high-grade PDAC precursor lesions in IAR of FPC families. Full article

This study assessed the significance of the neutrophil-to-lymphocyte ratio (NLR) in head and neck squamous cell carcinoma (HNSCC), and the relationships of the NLR with the aldehyde dehydrogenase 1 (ALDH1) level in tumors and the proportion of myeloid-derived suppressor cells (MDSCs) in the peripheral circulation. In total, 227 HNSCC patients who had received curative treatment at our hospital were enrolled into the present study. The NLR of each HNSCC patient before treatment was calculated. The associations of NLR with various clinicopathological parameters and prognoses were then examined. In addition, correlations between the proportion of MDSCs and level of ALDH1 with the NLR were assessed. Our data revealed that an elevated NLR was significantly correlated with the risk of developing locoregional recurrence and with a reduced overall survival in HNSCC patients. Multivariate analyses revealed that the NLR pretreatment and surgical resection were significantly correlated with the rate of treatment failure and the overall survival rate in HNSCC patients. Furthermore, the levels of ALDH1 in tumors and MDSCs in the peripheral circulation were significantly correlated with the prognosis of HNSCC, and the NLR was positively correlated with MDSC levels in the circulation and ALDH1 staining intensity in tumor specimens. In conclusion, the NLR has power in predicting the expression of ALDH1 in tumors, the circulating level of MDSCs, and the prognosis in HNSCC. We suggest that the NLR is an important biomarker that can assist the clinician and patient in making informed decisions regarding treatment options for HNSCC patients. Full article

Mitochondrial dysfunction is related to reproductive decline in humans, with consequences for in vitro fertilization (IVF). We assessed whether dehydroepiandrosterone (DHEA) could regulate mitochondrial homeostasis and mitophagy of cumulus cells (CCs) in poor ovarian responders (PORs). A total of 66 women who underwent IVF treatment at the Reproductive Medicine Center of Kaohsiung Veterans General Hospital were included in this study. Twenty-eight normal ovarian responders (NOR) and 38 PORs were enrolled. PORs were assigned to receive DHEA supplementation (n = 19) or not (n = 19) before IVF cycles. DHEA prevents mitochondrial dysfunction by decreasing the activation of DNM1L and MFF, and increasing MFN1 expression. Downregulation of PINK1 and PRKN occurred after DHEA treatment, along with increased lysosome formation. DHEA not only promoted mitochondrial mass but also improved mitochondrial homeostasis and dynamics in the CCs of POR. We also observed effects of alterations in mRNAs known to regulate mitochondrial dynamics and mitophagy in the CCs of POR. DHEA may prevent mitochondrial dysfunction through regulating mitochondrial homeostasis and mitophagy. Full article

We evaluated the efficacy of progressive resistance training of the pelvic floor muscle for post-prostatectomy incontinence. In this prospective study, 59 patients who underwent radical prostatectomy were evaluated preoperatively. Continence was sequentially assessed within 2 weeks postoperatively, and an exercise regimen was initiated at 6- and 12-weeks. The primary outcome was continent status and the secondary outcome was changes in muscle strength and endurance after the exercise intervention. Continence was defined as no urine loss in a 1h pad test. A total of 59 patients participated in this study. Six patients dropped out of the study because of non-compliance and orthopedic problems. Of the remaining 53 patients, 31 (58.5%) achieved pad-free continence at 12 weeks postoperatively. The patients were divided into two groups based on their continence status, and no statistically significant difference was observed in age, body mass index, prostate volume, prostate-specific antigen, pathological Gleason score sum, and pathological T stage. Meanwhile, preoperative maximal urethral closure pressure and change in hip extensor muscle strength and endurance during the 12-week exercise program were significantly higher in the continent group. In multivariate analysis, change in hip extensor muscle strength was the only significant parameter predicting achievement of continence status (Odds ratio, 1.039; p = 0.045). The changes in hip extensor muscle strength in the current exercise program was an independent predictor of continence status after radical prostatectomy. A large-scale prospective study on the relationship between extensor muscle strength and urinary incontinence should be explored in future. Full article

Duchenne’s muscular dystrophy is an X-linked neuromuscular disease that manifests as muscle atrophy and cardiomyopathy in young boys. However, a considerable percentage of carrier females are often diagnosed with cardiomyopathy at an advanced stage. Existing therapy is not disease-specific and has limited effect, thus many patients and symptomatic carrier females prematurely die due to heart failure. Early detection is one of the major challenges that muscular dystrophy patients, carrier females, family members and, research and medical teams face in the complex course of dystrophic cardiomyopathy management. Despite the widespread adoption of advanced imaging modalities such as cardiac magnetic resonance, there is much scope for refining the diagnosis and treatment of dystrophic cardiomyopathy. This comprehensive review will focus on the pertinent clinical aspects of cardiac disease in muscular dystrophy while also providing a detailed consideration of the known and developing concepts in the pathophysiology of muscular dystrophy and forthcoming therapeutic options. Full article

Arterial blood gas (ABG) analysis is the traditional method for measuring the partial pressure of carbon dioxide. In mechanically ventilated patients a continuous noninvasive monitoring of carbon dioxide would obviously be attractive. In the current study, we present a novel formula for noninvasive estimation of arterial carbon dioxide. Eighty-one datasets were collected from 19 anesthetized and mechanically ventilated pigs. Eleven animals were mechanically ventilated without interventions. In the remaining eight pigs the partial pressure of carbon dioxide was manipulated. The new formula (Formula 1) is PaCO₂ = PETCO₂ + k(PETO₂ − PaO₂) where PaO₂ was calculated from the oxygen saturation. We tested the agreements of this novel formula and compared it to a traditional method using the baseline PaCO₂ − ETCO₂ gap added to subsequently measured, end-tidal carbon dioxide levels (Formula 2). The mean difference between PaCO₂ and calculated carbon dioxide (Formula 1) was 0.16 kPa (±SE 1.17). The mean difference between PaCO₂ and carbon dioxide with Formula 2 was 0.66 kPa (±SE 0.18). With a mixed linear model excluding cases with cardiorespiratory collapse, there was a significant difference between formulae (p < 0.001), as well as significant interaction between formulae and time (p < 0.001). In this preliminary animal study, this novel formula appears to have a reasonable agreement with PaCO₂ values measured with ABG analysis, but needs further validation in human patients. Full article

Increased physical activity (PA) affects outcomes in patients with anorexia nervosa (AN). To objectively assess PA patterns of hospitalized AN patients in comparison with healthy, outpatient controls (HC), and to analyze the effect of PA on Body Mass Index (BMI) change in patients with AN, we measured PA in 50 female patients with AN (median age = 25 years, range = 18–52 years; mean BMI = 14.4 ± 2.0 kg/m²) at the initiation of inpatient treatment and in 30 female healthy controls (median age = 26 years, range = 19–53 years; mean BMI = 21.3 ± 1.7 kg/m²) using the SenseWear™ armband. Duration of inpatient stay and weight at discharge were abstracted from medical records. Compared with controls, AN patients spent more time in very light-intensity physical activity (VLPA) (median VLPA = 647 vs. 566 min/day, p = 0.004) and light-intensity physical activity (LPA) (median LPA = 126 vs. 84 min/day, p < 0.001) and less time in moderate-intensity physical activity (MPA) (median MPA = 82 vs. 114 min/day, p = 0.022) and vigorous physical activity (VPA) (median VPA = 0 vs. 16 min/day, p < 0.001). PA and BMI increase were not associated in a linear model, and BMI increase was mostly explained by lower admission BMI and longer inpatient stay. In a non-linear model, an influence of PA on BMI increase seemed probable (jack knife validation, r² = 0.203; p < 0.001). No direct association was observed between physical inactivity and BMI increase in AN. An altered PA pattern exists in AN patients compared to controls, yet the origin and consequences thereof deserve further investigation. Full article

The clearance of malignant clonal cells significantly correlates with clinical outcomes in many hematologic malignancies. Accurate and high throughput tools for minimal residual disease (MRD) detection are needed to overcome some drawbacks of standard molecular techniques; such novel tools have allowed for higher sensitivity analyses and more precise stratification of patients, based on molecular response to therapy. In this review, we depict the recently introduced digital PCR and next-generation sequencing technologies, describing their current application for MRD monitoring in lymphoproliferative disorders. Moreover, we illustrate the feasibility of these new technologies to test less invasive and more patient-friendly tissues sources, such as “liquid biopsy”. Full article

Aim: To determine the levels of Wnt inhibitors in patients treated with aromatase inhibitors (AIs) prior to therapy and to investigate their association with bone mineral density (BMD) and lifestyle parameters. Methods: 137 breast cancer patients were divided into a group treated with 1 mg of anastrozole and a group w/o anastrozole therapy. Serum concentrations of sclerostin and dickkopf1 (DKK1) were measured by ELISA. BMD was measured by dual-energy X-ray absorptiometry (DXA). Lifestyle factors were investigated by a self-reported questionnaire. Results: Sclerostin was significantly higher in the AI-treated group (31.8 pmol/L vs. 24.1 pmol/L; p < 0.001), whereas DKK1 was significantly lower in the AI-treated group (24.3 pmol/L vs. 26.02 pmol/L; p < 0.001). Total hip and femoral neck BMD were significantly lower in the AI-treated group. Conclusion: AI treatment was associated with increased levels of sclerostin and decreased levels of DKK1. Full article

Carotid artery stenting (CAS) is a minimal invasive procedure used to resolve carotid occlusion that can be affected by peri-procedural complications. Statin use before CAS has shown to reduce peri-procedural risk and improve survival, though time-dependent cofactors that influence mortality has not been considered. The aim of this study was to evaluate long-term survival of patients who undergo CAS considering new occurred major adverse cardiovascular event (MACE) as time-dependent cofactor. In this study, 171 high cardiovascular risk patients (age 72 ± 8 years, 125 males) were enrolled after CAS procedure and were followed for a median of 8.4 years. Death occurred in 44% of patients with a mean time to death of 69 ± 39 months and MACE in 34% with a mean time of 35 ± 42 months. In patients who used or not statins at baseline, death occurred in 33% and 65%, respectively (p < 0.001). Survival analysis showed that statin use reduced risk of death (hazard ratio HR 0.36, 95% confidence interval CI 0.23–0.58, p < 0.0001). Including MACE as time-dependent variable did not change beneficial effects of statins. Additionally, statin use was associated with a protective effect on MACE (HR 0.48, 95% CI 0.27–0.85, p = 0.012); particularly, the prevalence of stroke was reduced by 59% (p = 0.018). In multivariate analysis, effects of statins were independent of demographic and anthropometric variables, prevalence of cardiovascular risk factors, renal function, antiplatelet use, and MACE occurrence. In conclusion, use of statins before CAS procedure is associated with increased long-term survival and reduced MACE occurrence. This evidence supports the hypothesis that statin use before CAS might be beneficial in high risk patients. Full article

Although statin treatment is recommended for patients with chronic kidney disease (CKD) stages I–IV, its potential benefits have not been reported in advanced CKD patients. Non-diabetic patients with advanced CKD (pre-dialysis patients, estimated glomerular filtration rate <15 mL/min/1.73 m²) were enrolled from a National Health Insurance Research Database with a population of 23 million. Statin users and non-users were matched using propensity scoring and analyzed using Cox proportional hazards models, taking mortality as a competing risk with subsequent end-stage renal disease (ESRD) and statin doses as time-dependent variables. A total of 2551 statin users and 7653 matched statin non-users were identified from a total 14,452 patients with advanced CKD. Taking mortality as a competing risk, statin use did not increase the risk of new-onset diabetes mellitus (NODM) or decrease the risk of de novo major adverse cardiovascular events (MACE), but reduced all-cause mortality (hazard ratio (HR) = 0.59 [95% CI 0.42–0.84], p = 0.004) and sepsis-related mortality (HR = 0.53 [95% CI 0.32–0.87], p = 0.012). For advanced CKD patients, statin was neither associated with increased risks of developing NODM, nor with decreased risk of de novo MACE occurrence, but with a reduced risk of all-cause mortality, mainly septic deaths. Full article

The use of gamification in healthcare has been gaining popularity. This prospective, randomized, clinical trial was designed to evaluate whether gamification of the preoperative process—via virtual reality (VR) gaming that provides a vivid, immersive and realistic experience—could reduce preoperative anxiety in children. Seventy children scheduled for elective surgery under general anesthesia were randomly divided into either the control or gamification group. Children in the control group received conventional education regarding the preoperative process, whereas those in the gamification group played a 5 min VR game experiencing the preoperative experience. Preoperative anxiety, induction compliance checklist (ICC), and procedural behavior rating scale (PBRS) were measured. Sixty-nine children were included in the final analysis (control group = 35, gamification = 34). Preoperative anxiety (28.3 [23.3–36.7] vs. 46.7 [31.7–51.7]; p < 0.001) and intraoperative compliance measured using ICC (p = 0.038) were lower in the gamification group than in the control group. However, PBRS (p = 0.092) and parent/guardian satisfaction (p = 0.268) were comparable between the two groups. VR experience of the preoperative process could reduce preoperative anxiety and improve compliance during anesthetic induction in children undergoing elective surgery and general anesthesia. Full article

Thymus bovei Benth. (TB) is an important plant in the traditional medicine of the Mediterranean region. This study investigates the health-promoting properties of TB essential oil (TB-EO) for its possible use in clinical practice with regards to its cytotoxic, anti-herpes simplex virus type 2 (HSV-2), and antihypertensive (through inhibition of human angiotensin-converting enzyme; ACE) properties. The phytochemical profile of EO (99.9%) was analyzed by Gas Chromatography with Flame-Ionization Detection (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS). In this study, all biological methods were performed at the level of in vitro studies. The results showed that TB-EO exerted remarked cytotoxic properties against human cervical carcinoma cells, colon cancer cells, and lung adenocarcinoma cells with the half-maximal inhibitory concentration (IC₅₀) values of 7.22, 9.30, and 8.62 µg/mL, respectively, in comparison with that of standard anticancer drug cisplatin with IC₅₀ values of 4.24, 5.21, and 5.43 µg/mL, respectively. Fascinatingly, TB-EO showed very weak cytotoxicity on the healthy human fetal lung fibroblast cells with an IC₅₀ value of 118.34 µg/mL compared with that of cisplatin (IC₅₀ = 10.08 µg/mL). TB-EO, its main component geraniol, TB-EO combined with acyclovir (ACV) along with standard ACV, have displayed pronounced inhibitory properties against the replication of HSV-2 with the half-maximal effective concentration (EC₅₀) values of 2.13, 1.92, 0.81 and 1.94 µg/mL, respectively, with corresponding selectivity indices (SI) 98.59, 109.38, 259.26 and 108.25, respectively. TB-EO and geraniol at a concentration of 15 µg/mL showed prominent inhibitory activities against ACE with % of inhibition 95.4% and 92.2%, respectively, compared with that of standard inhibitor captopril (99.8%; 15 µg/mL). Molecular docking studies were performed to unveil the mechanism of action of geraniol as well as structural parameters necessary for anti-HSV-2 activity (through the inhibition of HSV-2 protease) and ACE inhibition. This is the first report on the chemical composition of Egyptian TB-EO along with the above-mentioned biological activities. Our results may be considered as novel findings in the course of a search for new and active anticancer, anti-HSV-2 and antihypertensive agents, and expand the medicinal value of this plant and its phytochemicals in clinical practice. Full article

There have been few large-scale studies on the relationship between smoking and gut microbiota. We investigated the relationship between smoking status and the composition of gut microbiota. This was a population-based cross-sectional study using Healthcare Screening Center cohort data. A total of 758 men were selected and divided into three groups: never (n = 288), former (n = 267), and current smokers (n = 203). Among the three groups, there was no difference in alpha diversity, however, Jaccard-based beta diversity showed significant difference (p = 0.015). Pairwise permutational multivariate analysis of variance (PERMANOVA) tests between never and former smokers did not show a difference; however, there was significant difference between never and current smokers (p = 0.017) and between former and current smokers (p = 0.011). Weighted UniFrac-based beta diversity also showed significant difference among the three groups (p = 0.038), and pairwise PERMANOVA analysis of never and current smokers showed significant difference (p = 0.01). In the analysis of bacterial composition, current smokers had an increased proportion of the phylum Bacteroidetes with decreased Firmicutes and Proteobacteria compared with never smokers, whereas there were no differences between former and never smokers. In conclusion, gut microbiota composition of current smokers was significantly different from that of never smokers. Additionally, there was no difference in gut microbiota composition between never and former smokers. Full article