We aimed to determine the prevalence of elevated alanine aminotransferase (eALT) in employees of a German chemical company, and analyze its association with sociodemographic, work- and lifestyle-related factors.The cross-sectional study is based on data surveyed from occupational health check-ups between 2013 and 2018 at the site clinic of a chemical company based in Ludwigshafen, Germany. We used logistic regression analyses to assess the association between sociodemographic, work- and lifestyle-related characteristics and eALT. Quantile regression technique was applied to investigate if associations vary across different quantiles of the ALT distribution.pInteraction < 0.001) was found, showing approximately a u-shaped age/ALT relationship in women and an inversely u-shaped relationship in men. Quantile regression showed an increasing positive effect of male gender, overweight/obesity, and for diabetics on ALT level when moving from the lowest (q0.1) to the highest (q0.9) considered quantile. Additionally, from the lowest to the highest quantile an increasing negative effect on ALT for older age was observed.Participants  were predominantly male (78.3%) with a mean age of 42.2 years (SD 10.7). The prevalence of eALT was 18.5% (21.6% in men/7.2% in women) with a geometric mean of 28.9 U/L (32.8 U/L in men/18.5 U/L in women). In the multivariable logistic regression model, odds of eALT were significantly higher for males , manual workers , overweight  or obese respondents , employees who consume any number of alcoholic drinks/week  and diabetics . Additionally, season of participation was significantly associated with eALT, with odds being higher for participation in spring, fall or winter, as compared to summer. A significant interaction between age and gender (Prevalence of eALT in our sample of employees can be considered as high, with almost one in five participants affected. Identification of risk groups allows the implementation of targeted preventive measures in order to avoid transition to severe morbidity. Alanine aminotransferase is a transaminase enzyme present in serum and organ tissue , and a commonly used marker for hepatocellular injury. In the absence of excessive alcohol consumption and viral hepatitis, elevated alanine aminotransferase (eALT) is considered an indicator for non-alcoholic fatty liver disease (NAFLD) which is defined as hepatic steatosis with a fat content of more than 5% of the liver weight or macrosteatosis of hepatocytes of the same degree in the absence of secondary causes of hepatic fat accumulation. NAFLD encompasses a variety of pathological conditions ranging from asymptomatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and the potentially fatal liver cirrhosis.NAFLD is the most common liver disease in Western countries, with approximately one in four persons affected in Europe and the USA according to a recent systematic review and meta-analysis . Thus, NTo the best of our knowledge, comprehensive data on the prevalence of eALT is currently unavailable for Germany. With the current study, our intention is to add to the ever-growing body of literature using a large dataset from occupational health check-ups in a German company. We aimed to determine the prevalence of eALT in a sample of employees and analyze its association with sociodemographic, work- and lifestyle-related factors. Additionally, using quantile regression technique, our aim was to examine if associations are stable across different quantiles of the ALT distribution.2, with approximately 200 industrial plants and about 2000 buildings. The occupational health check-up was introduced in the year 2011 at the Ludwigshafen site of the company and aims at the early detection of chronic diseases. It serves as a supplement to periodic, mandatory occupational health surveillance examinations. Participation in the check-up is entirely voluntary and can be repeated every three years. All employees are personally invited to the check-up by email. Completion of a written questionnaire, a venous blood draw and a comprehensive physical examination including anamnesis and documentation of health behavior by an occupational health physician are the main components of the check-up. Due to a change of laboratory equipment  in December 2012, data from check-ups surveyed between 2011 and 2012 were excluded. If an employee received multiple check-ups during the period of subject recruitment, only first-time visits were considered. Trainees, persons with missing values regarding ALT, pregnant women, participants with a known chronic liver disease other than NAFLD, and employees with known alcohol abuse were excluded from all analyses. Specifically, we excluded participants with alcoholic liver disease , significant alcohol consumption exceeding 21 or 14 standard drinks per week in respectively men and women as defined by the AASLD guideline on the diagnosis and management of NAFLD /height squared [in m2]) and categorized participants according to the classification provided by the World Health Organization . Alcohol consumption was surveyed by self-reported number of drinks per week (without specification of the exact amount or type of drink) and categorized into < 1 drink/week, 1 drink/week, 2 drinks/week, and 3 or more drinks/week. History of diabetes mellitus (DM) was considered present if any of the following was true: HbA1c ≥ 6.5%, diagnosis made by a physician, self-reported diabetes in the check-up questionnaire. In addition, we considered weekday , season , and year of examination (2013–2018) in our analyses.Sociodemographic  and occupational-related variables  were directly extracted from employee records. Participants’ age at the time of examination was categorized into 5-year intervals . Occupational group  roughly represents the socioeconomic status of employees, with manual workers working in production lines and representing physical labor jobs, skilled/supervisory workers having a comparatively better education and performing commercial activities or office work, and managerial staff working in leading positions and usually holding an academic degree. Concerning working time system, we distinguish between day and shift workers. Most shift workers (> 90% in 2013 at the Ludwigshafen site) work 4 × 12 h-rotating shift schedules, with shifts lasting from 6 am to 6 pm followed by a leisure time of 24 h and the following shift beginning at 6 pm and ending at 6am with a subsequent leisure time of 48 h. During medical anamnesis, the responsible occupational health physician ascertained smoking status , alcohol consumption (drinks/week), as well as height and weight of all participants. We calculated body mass index  were considered in the final multivariable logistic regression model. We provided unadjusted and adjusted odds ratios ([a]OR) as measures of effect and corresponding 95%-confidence intervals (95%-CI). Due to reported gender disparities in existing studies on the association of considered factors and elevated ALT ) and the geometric mean (with geometric standard deviation factor [GSD]) as measures of central tendency and dispersion. To calculate the geometric mean and GSD, ALT concentrations below the LOD  were substituted by the LOD divided by the square root of 2, a commonly applied method of imputation . Univariated ALT , 15, 26,echnique  was appln grqreg  STATA-coAltogether, 15,939 employees were first-time participants in the occupational health check-up between 2013 and 2018. Trainees (n = 208), persons with missing values in the outcome (n = 81), pregnant women (n = 61), participants with alcoholic liver disease  or significant alcohol consumption (n = 6), those with drug-induced/toxic liver disease , metabolic liver disease , autoimmune liver disease , infectious liver disease/viral hepatitis  or inflammatory liver disease  were excluded leading to a final sample of 15,348 persons. Participants were predominantly male (78.3%) with a mean age of 42.2 years . Most employees had regular daytime working hours (71.2%) and were employed as manual (35.5%) or skilled/supervisory workers (42.0%). Regarding lifestyle-related factors one in five respondents were smokers (19.7%) and/or obese (19.1%). More than a quarter of participants (28.0%) stated to consume less than one alcoholic drink per week , with an overall prevalence of elevated ALT of 18.5%. The prevalence was considerably higher in men (21.6%) than women (7.2%). Additionally, a comparatively high prevalence was found in manual and shift workers (24.0% and 23.6% respectively), former smokers (23.3%), respondents consuming three or more alcoholic drinks per week (21.7%), obese participants , and respondents with a history of DM  visualized in Fig. In the multivariable model, the odds of eALT were significantly higher for males , manual workers , overweight employees or those with any class of obesity , respondents who consumed ≥ 1 alcoholic drink per week (vs. < 1 drink), participants with a history of DM, if participation took place in spring, fall, or winter (vs. summer), and participation in 2016 (vs. 2013). In contrast, odds of eALT were significantly lower for smokers (vs. non-smokers) and participation in 2017 (vs. 2013). Regarding age, compared to the youngest considered age group (< 35 years), odds of eALT increased by a factor of 1.02 (95%-CI 0.88–1.20) in respondents aged 35–39 years, and continuously decreased thereafter by a factor of 0.88 (95%-CI 0.76–1.03) in persons aged 40–44 years, 0.82 (95%-CI 0.71–0.94) in those aged 45–49 years, 0.67 (95%-CI 0.58–0.77) in those aged 50–54 years, and 0.58 (95%-CI 0.50–0.69) in the oldest age group (≥ 55 years). We found a significant interaction effect between gender and categorical age . For instance, ALT values of males were 7.41 U/L (95%-CI 6.98–7.85) higher compared to females at the lower end of the ALT distribution (at q0.10) and 17.73 U/L (95%-CI 16.11–19.35) at the upper end (at q0.90). In a similar manner, from lowest to highest ALT quantile there was a monotonous and strong increase in the positive effect of being overweight or obese  and in employees with a history of DM (p < 0.05). For participants with a BMI ≥ 40 kg/m2 (obesity class III) for example, ALT values were 8.03 U/L (95%-CI 5.43–10.62) higher compared to normal weight individuals at the lowest considered quantile of the ALT distribution (q0.10) and 43.54 U/L (95%-CI 33.14–53.93) at the highest (q0.90). In contrast, an increasing negative effect was visible for age categories 50–54 years and ≥ 55 years .Correspondingly, the average marginal effect of male gender on the probability of an elevated ALT level  increased from < 35 years to 35–39 years and continuously decreased thereafter . After multivariable adjustment, odds of eALT were comparatively higher for men, manual workers, overweight/obese participants, consumers of one or more alcoholic drinks per week, diabetics, and if participation took place in spring, fall or winter (vs. summer). Contrarily, odds of eALT were comparatively lower with increasing age and for smokers. There was a significant interaction between age and gender, yielding approximately an inverse u-shaped age/ALT relationship in men and a u-shaped relationship in women. Quantile regression showed that associations between considered factors and ALT level vary widely across quantiles. There was e.g. a sharp increase in the positive effect of male gender and overweight/obesity on ALT levels, when moving from the lowest to the highest considered quantile.Considering the prevalence of eALT in our sample of employees 18.5%) in the context of the existing body of literature has to be done with caution since studies may vary widely according to thresholds for elevated ALT, distribution of risk factors within the population studied and diagnosis of NAFLD . A recent systematic review and meta-analysis on the prevalence of NAFLD (diagnosed via imaging techniques) in adults found a global prevalence of 25.2% , and a corresponding estimate of 23.7% for Europe  8.5% in t.The finding of a higher prevalence of eALT in men compared to women aligns with earlier studies on the topic , 11–16. Regarding occupational factors, several studies examined the effect of shift work on liver enzymes –21. In aCompared to abstainers or persons consuming less than one alcoholic drink per week, any frequency of weekly alcohol consumption was significantly associated with ALT levels in multivariable regression models. Findings from the literature are conflicting, with several studies reporting an increase of liver enzymes with increasing alcohol consumption , 38–40, The seemingly protective effect of smoking on ALT-levels found in both multivariable logistic and quantile regression models cannot be explained easily. Earlier studies reported conflicting results on this regard. Whereas some studies did not find effects of smoking on ALT/NAFLD , 46, 47,The prevalence of eALT in participants with a history of diabetes mellitus 35.5%) was amongst the highest besides obese respondents. Significant associations between history of diabetes mellitus and ALT were found in multivariable regression analyses. The association of NAFLD/eALT and diabetes mellitus is well established in the available literature, although considerable debate remains as to which factor precedes the other. The relationship between these two disease entities has been described as bidirectional with each factor acting as a causative and also exacerbating factor for the other although the detailed underlying pathophysiological mechanism remains to be fully elucidated .5% was a, 49.Surprisingly, we found considerable seasonal variation in the prevalence of eALT, ranging from 15.1% in summer to 20.8% in winter. These findings remained stable in the fully adjusted logistic and quantile regression models. In accordance to that, Miyake and colleagues found ALT values peaking in January and falling to a minimum in July in their time-series analyses on the seasonal variation of liver function tests in Japanese outpatients using a large dataset (> 1 mio. tests) . The autFor employees with eALT/NAFLD, a further work-up is recommended according to existing guidelines and special attention should be paid to those with a high risk of advanced fibrosis. Liver fibrosis is an important prognostic factor which is associated with liver-related morbidity and mortality . As recoThere are limitations to our study which should be acknowledged. Firstly, the cross-sectional design of the current study impedes to draw any causal conclusions. In addition to that, information on lifestyle-related factors such as smoking and alcohol consumption are based solely upon the voluntary self-report of employees and was not objectively validated. It is e.g. possible that isolated cases of alcoholism were not disclosed by the employees and not documented during the medical anamnesis carried out by the responsible occupational physician. We believe this number however to be very low since we used a sample on an active workforce. Furthermore, alcohol consumption was surveyed as “drinks/week”, lacking an exact definition of type and amount of drinks consumed. Thus, inaccuracies in disclosure/misreporting of information cannot be ruled out. Additionally, eALT is an imperfect indicator of NAFLD, since a variety of other factors might be responsible for increased ALT activity and up to 25% of patients with confirmed NAFLD are found to have normal ALT levels according to a recent systematic review and meta-analysis . FurtherIn summary, prevalence of eALT in our sample of employees can be considered as high, with almost one in five participants affected. We found elevated ALT levels to be associated with male gender, overweight and obesity, alcohol consumption, history of diabetes mellitus, lower occupational group, and season of study participation. Identification of risk groups allows the implementation of targeted preventive measures in order to avoid transition to severe morbidity.Additional file 1. Results of the quantile regression model showing coefficients at different quantiles of the ALT-outcome .


Apigenin is a natural flavone with anti-inflammatory and antioxidant properties and antitumor abilities against several types of cancers. Previous studies have found that the antitumor effects of apigenin may be due to its similar chemical structure to 17β-estradiol (E2), a main kind of estrogen in women. However, the precise mechanism underlying the antitumor effects of apigenin in cervical cancer remains unknown. On the other hand, there is increasing evidence that describes a histamine role in cancer cell proliferation. In this study, we examined whether apigenin can attenuate the effects of histamine on tumors by regulating the expression level of estrogen receptors (ERs) to inhibit cervical cancer growth. Our in vitro data indicates that apigenin inhibited cell proliferation in a dose-dependent manner in human cervical cancer cells (HeLa), while histamine shows the opposite effects. After that, the xenograft model was established to explore the antitumor effects of apigenin in vivo, the results show that apigenin inhibited cervical tumor growth by reversing the abnormal ER signal in tumor tissue which was caused by histamine. We also demonstrate that apigenin inhibited cell proliferation via suppressing the PI3K/Akt/mTOR signaling pathway. Collectively, our results suggest that apigenin may inhibit tumor growth through the ER-mediated PI3K/Akt/mTOR pathway and that it can also attenuate the effects of histamine on tumors. Cervical cancer, as a gynecological tumor, remains one of the most common causes of cancer-related deaths worldwide, which consists of 528,000 new cases and 266,000 deaths worldwide each year ,2. Most Apigenin  is a naturally occurring plant flavone, widely present in fruits and vegetables, including onions, oranges, tea and some seasonings ,9. InterTumor development and progression relies on cell proliferation, and high levels of histamine and its synthetizing enzyme are found in growing tumor tissues . SpecifiIn this paper, we establish in vivo and in vitro studies to investigate the mechanism of the effects of histamine and apigenin on cervical cancer. Our results indicate that histamine promoted tumor growth, while apigenin showed the opposite effects. Additionally, we foun that the histamine-induced tumor growth resulted from the expression level change of ERα and ERβ, which is an abnormal estrogen receptor (ER) signal, but apigenin could reverse this effect. We also demonstrated that both apigenin and histamine could influence the autophagy and apoptosis of HeLa cells via regulating the PI3K/Akt/mTOR signaling pathway.To investigate the effects of histamine on tumor growth in vitro, we treated HeLa cells with different concentrations of histamine . After incubation with histamine for 48 h, we found that the proliferation of HeLa cells was stimulated by histamine in a dose-dependent manner between the concentration of 1 ng/mL and 10 ng/mL A. HistamMany gynecologic oncologies, such as breast cancer, are considered ER-positive, indicating the correlation between ER and tumor growth. Therefore, the ERα and ERβ expression levels in normal and cervical cancer tissues were measured by immunohistochemistry. As shown in The PI3K/Akt/mTOR signaling pathway plays an important role in regulating the cell cycle, proliferation and apoptosis. Significant increases in the expression of PI3K, Akt and mTOR, determined by immunohistochemistry analysis, were observed in the cervical tumor tissue A. WesterHeLa cells were incubated with different concentrations of apigenin  for 48 h, and cell proliferation was measured by CCK-8 assays. As shown in Next, we determined whether apigenin can inhibit cervical tumor growth in the xenograft model in female BALB/c nude mice. Compared with control group and histamine treatment group, tumor volume and tumor weight were found to be obviously lower in groups injected with apigenin, AZD9496 and DPN A,B. We aIn this part, we investigated whether the expression change of ER caused by apigenin could inhibit the PI3K/Akt/mTOR signaling pathway, thereby suppressing tumor development. After treatment with apigenin, the activation of PI3K/Akt/mTOR signaling pathway caused by histamine was attenuated, and the expression of Bax was increased A. FurtheApigenin, which has a similar chemical structure to E2, was supposed to influence the endocrine system in nude mice. Therefore, we assessed serum estradiol level in nude mice. Histamine treatment significantly decreased the E2 concentration in serum while the fortification of AZD9496 and apigenin reversed this effect, which is probably due to the competitive binding of apigenin and estradiol to ERα .The development of cervical cancer, as a kind of hormone dependent cancer, was believed to be closely related to the body’s hormone and receptor levels ,18. ReceRegarding the contribution to tumor progression, pro-inflammatory factors, growth factors and immune factors are the best-characterized soluble microenvironmental factors . AccordiPrevious studies have shown that apigenin can inhibit cell proliferation and induce apoptosis: Yang et al. reported that apigenin suppressed proliferation of HeLa cells in a dose-dependent manner and caused apoptosis through PI3K/Akt/mTOR signaling pathway . Chiang In the last decade, several studies have reported that apigenin could act as an estrogen receptor modulator, revealing the potential relation between apigenin and ER ,27,28. AAccording to a previous study, the hormonal treatment enables control of most climacteric symptoms, without any serious side effects . As a kiThe development of cancers is a complicated process involving various factors and pathways. Among the signaling pathways associated with apoptosis and autophagy, the PI3K/Akt/mTOR signaling pathway is well studied. Drugs inhibiting the PI3K/Akt/mTOR pathway are reported to have promoting effects on apoptosis in cancer cells . TherefoApigenin and histamine were from Sigma-Aldrich . The antibodies against Bax (1:1000), PI3K (1:1000), Akt (1:1000), and mTOR (1:1000) were obtained from Abbkine Scientific. . Antibodies for ERα (1:500), ERβ (1:500), p-PI3K (1:1000) and p-Akt (1:5000) were purchased from Abcam . AZD9496 (CAS No.: 1639042-08-2), PHTPP (CAS No.: 805239-56-9), PPT (CAS No.: 263717-53-9) and DPN (CAS No.: 1428-67-7) were obtained from MedChemExpress . FBS  was from Hyclone . Dulbecco’s modified Eagle medium (DMEM) was purchased from Gibco .v/v) penicillin/streptomycin. Cells were maintained at 37 °C in a humidified incubator with 5% CO2. At 80% confluence, the HeLa cells were treated with different concentrations of histamine  and apigenin  for 48 h. Apart from this, AZD9496, PHTPP, PPT and DPN at the concentration of 1 μM were added to other HeLa cells and incubated for 48 h.The human cervical cancer cell line (HeLa) was kindly provided from Cancer Hospital Chinese Academy of Medical Science . The HeLa cells were routinely cultured in DMEM supplemented with 10% FBS and 1%  according to the manufacturer’s instructions. Briefly, 2 × 10The female BALB/c nude mice (6-weeks-old) in our research were purchased from Vital River Laboratories, Inc.  and housed in the specific pathogen-free (SPF) animal laboratory of College of Food Science and Nutritional Engineering, China Agricultural University . The animal laboratory was maintained at a temperature of 22 ± 1 °C, humidity of 55 ± 5%, a 12 h light/dark cycle and air exchanges at 15 times/h. Feed and water were supplied ad libitum. All animal experiments in this study were approved by the Animal Experimental Welfare and Ethical Inspection Committee in China Agricultural University.6) were re-suspended in 100 μL PBS and then injected subcutaneously into the lower right-side flanks of mice to establish tumors. After 6 days of initial implantation, sixty female BALB/c nude mice (6-weeks-old) were randomly divided into six groups with equal body weight (n = 10/group). Groups and treatment are listed in 2. All mice were sacrificed at 30 days and the tumors were dissected and weighed.HeLa cells .Normal tissues and tumor tissues were embedded in paraffin and sectioned at a thickness of 4 μm. Sections of tumor were incubated with respective antibodies at 4 °C overnight after hydration and antigen retrieval. Then, biotin-conjugated secondary anti-mouse IgG was used to incubated with washed tumor sections for 30 min at room temperature. Diaminobenzidine was used to visualize sections according to the manufacturer’s instructions. Results were calculated as follows:After extraction by Trizol reagent (TaKaRa), the total RNA in the HeLa cells was reverse transcribed using a cDNA reverse transcription kit according to the manufacturer’s instructions. Primer sequences of selected genes are shown in g for 15 min at 4 °C. The protein concentrations were quantified by using the Bradford protein assay kit . After that, protein samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes and blocked with fresh 5% nonfat milk for 2 h at room temperature. The membranes were then incubated with specific primary antibody at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies were used to incubated with membranes which were washed with TBST. Proteins were visualized by using chemiluminescence reagents and the signal was detected by a gel documentation system (GelDoc-It 310 Imaging System).Briefly, HeLa cells were homogenized in protein lysate buffer and centrifuged at 12,000× t test using GraphPad Prism 8.01 . A value of p < 0.05 was considered to indicate a statistically significant different.All data are presented as the mean values ± SEM from three independent biological replicates. Statistical significance was determined by one-way analysis of variance (ANOVA) or student’s In conclusion, we demonstrated that histamine induced HeLa cell proliferation in vivo and in vitro, while apigenin showed the opposite effects. In addition, apigenin was found to attenuate the abnormal ER signaling caused by histamine, which resulted in the inhibition of the PI3K/Akt/mTOR pathway. Moreover, this study is the first to show that apigenin can inhibit histamine-induced cervical cancer tumor growth by regulating ER expression. This result may provide a new insight into the anti-tumor activity of apigenin and a series of experimental and theoretical bases which can help us to better utilize phytoestrogens in cancer therapy.


Microbial origin polysaccharides have gained popularity due to lesser toxicity, better degradability and selectivity as compared to their synthetic counterparts and can be used as emulsifier, stabilizer, thickener, texturizer, flocculating and gelling agent. Here main emphasis on exopolysaccharide production from potential lactic acid bacteria that has GRAS status.Lactobacillus paraplantarum KM1. L. paraplantarum KM1 was isolated from human milk and identified by conventional and molecular techniques. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KX671558. L. paraplantarum KM1 was found to produce EPSs in lactose containing MRS medium, and the maximum yield (47.4 mg/ml) was achieved after 32-h incubation. As evident from TLC and HPLC analyses, the polysaccharide was found to be a heteropolymer-containing glucose, galactose and mannose as main sugars. Different oligosaccharides namely hexoses were obtained after partial hydrolysis of the polymer using MALDI-ToF-MS. The total molecular weight of all polysaccharides present was 348.7 kDa with 100 °C thermal stability as well as water soluble in nature. Cell cytotoxicity revealed that the purified EPS was safe for consumption; thus, it can be used in various food industries as emulsifying and texture agent.This work was aimed at isolating, purifying and characterizing an extracellular polysaccharide (EPS) produced by a foodgrade lactic acid bacteria The present study highlighted that exopolysaccharides could be harnessed to improve food products in terms of texture, emulsifying agents, pharmaceutical industry  and as safety purposes. Microbial origin polysaccharides based emulsifiers have gained popularity during recent times due to lesser toxicity, better degradability and selectivity as compared to their synthetic counterparts . Apart fInvestigations on the chemical compositions and molecular structures of EPSs are important to establish their structure–function relationship. Recurring sugar units joined together by glycosidic linkages form EPSs and numerous inter- and intra-molecular hydrogen bonds exists because of hydroxyl groups in the structure , 13. CroLactobacillus paraplantarum KM1 probiotic microorganisms isolated from human milk origin as well as its GRAS status.To investigate the possible applications of EPS in food and pharmaceutical industry, this research aims to focus on the production, purification and physico-chemical characterization of an EPS produced by All of the chemicals used were of analytical reagent grade. Sephadex G75 was obtained from Bangalore Genie Private Ltd . The remaining chemicals were procured from Merck Specialities Private Ltd .http://www.ncbi.nlm.nih.gov/) and Basic Alignment Search Tool (BLAST). The registered accession number of Lactobacillus paraplantarum KM1 is KX671558.KM1 bacterial isolate showing high EPS content was isolated from pooled human milk and was maintained on MRS agar and used throughout the study Fig. a. The cu8 CFU/ml) was obtained in MRS medium after inoculating under static condition at 37 °C. EPS production  was used to filter supernatant. Two volumes of 10% (w/v) trichloroacetic acid were added to the filtrate to obtain protein precipitates, and after keeping it overnight at 4 °C under static conditions, it was recentrifuged at 25,000g for 20 min. Four volumes of prechilled 95% (v/v) ethanol were added to the supernatant, further keeping it for 24 h at 4 °C and centrifuging at 25,000g for 20 min at 4 °C to obtain polysaccharide precipitates. The pellet was further lyophilized to make the EPS dry powder. The EPS powder was dissolved in 5-ml distilled water and dialysed using a 14-kDa cut-off dialysis membrane . The EPS which was dialysed was relyophilized and assessed for carbohydrate content. Carbohydrate content was spectrophotometrically analysed at 490 nm  following the phenol–sulphuric acid method  was helpful in removing acid resistant material from the digest by diluting it. The ethanol soluble oligosaccharides were then lyophilized. The 1-ml deionized water was used to dissolve 1 mg of oligosaccharide. The sample was mixed with equal volume of 2,5-dihydroxybenzoic acid (10 mg/ml) as matrix, prior to MALDI analysis. MALDI analysis was done on an Applied Biosystem Voyager-DE PRO MALDI ToF mass spectrometer with a nitrogen laser (337 nm) operated in an accelerating voltage (20 kV). Every spectrum was obtained in the positive ion reflector mode as an average of 100 laser shots. External calibration of the data was done using angiotensin and ACTH . The reproducibility of each spectrum was checked five times by comparing it with the prepared sample.Surface analysis of purified EPS was studied by scanning electron microscope (SEM) at different magnifications. EPS was fixed on aluminium stub and gold sputtered. The sample was examined under VEGA TESCAN SEM  using services provided NIPER, Mohali, Punjab, India.l-glutamine , 100 μg/ml streptomycin  and 100 IU/ml penicillin. By seeding cells in 96-well culture plates , MTT assay [The extracted purified EPS was evaluated for cytotoxicity using mammalian epithelial cell line (HEp-2C) by MTT assay for their safety usage pattern. HEp-2C cells were maintained in Earle’s Minimum Essential Medium , supplemented with 10% foetal calf serum , 1% non-essential amino acids , 2 M TT assay  was repeStatistical tool applied for the analysis of data was central composite design (CRD).L. paraplantarum KM1 was fractionated on sephadex G75 chromatography column  than others, followed by L. monocytogenes (14 mm) and S. aureus (13 mm) the last one being the most resistant.The agar well diffusion method was used to study antimicrobial activity of purified EPS of L. paraplantarum KM1 EPS; the masses range found it between 102.67 and 372.97 m/z which corresponded to the mass of one hexose sugar residue (m/z 180).The EPS fractions of the one peak containing high concentration of total sugar were collected for further investigation. The intense peaks of MALDI-ToF mass spectrum of partially acid-digested EPS Fig.  could beThe MALDI-ToF MS of the EPS fraction revealed six peaks with molecular weight of ~32, ~54, ~72, ~32, ~80 and ~76 kDa, respectively with an average molecular weight of 348.7 kDa. The lower molecular weight fractions could mainly be attributed the presence of oligomers present in the polysaccharide.L. paraplantarum KM1 showed the presence of 3 spots in total. When the computed Rf (Retention factor) value of the spots were compared with the literature Rf value, the compounds were identified as glucose (Rf = 0.18), galactose (Rf = 0.16) and mannose (Rf = 0.21). As TLC results had shown the presence of glucose, galactose and mannose in L. paraplantarum KM1, further quantification of these sugars was done by using high-performance liquid chromatography (HPLC). After hydrolysis and anthranilic acid derivatization, EPS I was analysed for its sugar composition by HPLC. The hydrolysed EPS was fractioned by high-performance liquid chromatography (HPLC). The sugar moieties identified were glucose, galactose and mannose with additional sugars which were not traced in TLC analysis. HPLC analysis depicted that EPS contains 615 mg/ml, 22.1 mg/ml and 4.1 mg/ml of glucose, mannose and galactose respectively in purified EPS . TLC analysis of EPS in  Rf = 0.1 and mannL. paraplantarum KM1 revealed a compact structure composed of similar sizes of cubes .The KM1 Fig. . Only smLactobacillus kefiranofaciens ZW3 isolated from Tibet kefir. Due to good holding capacity of KM1 EPS, it can be used in various fermented food products for their texture enhancement.KM1 EPS is water soluble with good water-holding capacity. These properties are attributed to the permeable structure of polymer chains which can hold large amounts of water through hydrogen bonds . Our resAntimicrobial activity was determined by measuring the diameter of zone of inhibition, indicating that the purified EPS was inhibitory against the test organisms. The zones of inhibition of indicator organisms tested ranged from 13 to 16 mm in diameter. The antimicrobial activity of purified EPS was thought to be a significant incident for competitively excluding or inhibiting the activities of pathogenic intestinal microflora making the host safer against the harmful microorganisms .x axis representing m/z (mass divided by charge) and a y axis representing absolute intensity. Similar results of EPS were confirmed by Chowdhury et al. [m/z in purified EPS from Bacillus megaterium RB-05. Similarly, Liang et al. [Paenibacillus mucilaginosus TKU032 using MALDI-ToF MS and revealed two mass peaks with molecular weight of 105 and 135 kDa. The difference in molecular weights of the EPSs could be attributed to the different producing strains, the culture media and the growth conditions used [MALDI-TOF analysis of the EPS fraction was completed. The resultant mass spectrum contained an y et al.  using hag et al.  studied ons used . The molons used .L. plantarum C88 was composed of galactose and glucose [L. plantarum KF5 produced two EPS, both containing glucose mannose and galactose in varied ratios [L. paraplantarum KM1 was found to contain high concentration of glucose in its EPS.In TLC, the retention times (RT) of monosaccharide compositions of EPS were determined comparing with the chromatograms of each respective standard monosaccharide. The presence of sugars in EPS has been confirmed in several studies. The EPS from  glucose . L. pland ratios . In our L. paraplantarum KM1 had a smooth cube surface, and additionally, the SEM scan showed that the EPS was made of homogeneous matrix. Similar structure was observed where the structure of their EPS was smooth and cubic [.e. butylated hydroxyanisole and butylated hydroxytoluene) have been demonstrated to be cytotoxic [L. paraplantarum KM1 was free from the cytotoxic effects and could be of importance in food industry. Similarly, Challouf et al. [Arthrospira platensis on colon cancer cell lines ‘Caco-2’ and kidney cell lines ‘Vero’ and reported that their EPS have no cytotoxic effect. El-Deeb et al. [SEM is considered to be a powerful tool to study the morphological features of polysaccharides and could be used to elucidate their physical properties . The EPSnd cubic  and was nd cubic . Since mytotoxic , the curf et al.  also stub et al.  performeL. paraplantarum KM1 was isolated and identified from human milk. L. paraplantarum KM1 was found to produce an efficient amount of EPS and was purified using gel chromatography. Acidic hydrolysis, HPLC and MALDI-TOF MS studied reviled glucose, galactose and mannose sugar moieties in purified EPS. Microstructural characterization of the purified EPS indicated that the EPS had a highly compact structure with smooth cube surface, facilitating formation of films. Future studies will be focused on further characterization of the EPS structure, its biological functions and possible roles in determining the potential probiotic properties of the strain in question.


To examine the association between sleep duration trajectories over 28 years and measures of cognition, gray matter volume, and white matter microstructure. We hypothesize that consistently meeting sleep guidelines that recommend at least 7 hours of sleep per night will be associated with better cognition, greater gray matter volumes, higher fractional anisotropy, and lower radial diffusivity values.We studied 613 participants (age 42.3 ± 5.03 years at baseline) who self-reported sleep duration at five time points between 1985 and 2013, and who had cognitive testing and magnetic resonance imaging administered at a single timepoint between 2012 and 2016. We applied latent class growth analysis to estimate membership into trajectory groups based on self-reported sleep duration over time. Analysis of gray matter volumes was carried out using FSL Voxel-Based-Morphometry and white matter microstructure using Tract Based Spatial Statistics. We assessed group differences in cognitive and MRI outcomes using nonparametric permutation testing.N = 29), 6.2 ± 0.3 hours , 7.0 ± 0.2 hours , and 7.9 ± 0.3 hours . No differences in cognition, gray matter, and white matter measures were detected between groups.Latent class growth analysis identified four trajectory groups, with an average sleep duration of 5.4 ± 0.2 hours (5%, Our null findings suggest that current sleep guidelines that recommend at least 7 hours of sleep per night may not be supported in relation to an association between sleep patterns and cognitive function or brain structure. Up to a third of adults report between 6 and 7 hours of sleep per night, and thus fail to meet sleep guidelines which recommend at least 7 hours of sleep per night. Although extreme short sleep (e.g. ≤5 hours per night) has repeatedly been associated with poor cognitive health, it is currently unclear if such relationships subsist for more moderate short-sleep durations. We found no differences in cognitive or structural MRI measures between groups that reported, on average, 5.4 hours, 6.2 hours, 7.0 hours, and 7.9 hours sleep per night over 5 timepoints spanning 28 years. If replicated with longitudinal markers of cognitive health, such null results could challenge the suitability of current sleep guidelines on cognitive outcomes.Both short- and long-sleep durations are consistently associated with increased mortality and unfavorable health outcomes, including elevated risk of stroke, diabetes, heart disease, and psychiatric disorders , 2. As aWith regard to cognitive health, meta-analyses have associated extremes in sleep duration (most frequently defined as ≤5 hours or ≥9 hours) with reduced scores for overall cognition, executive function, verbal memory, and working memory .In order to understand the mechanisms that underlie associations between sleep and cognition, a number of studies have used magnetic resonance imaging (MRI) techniques to examine the relationship between sleep duration and brain structure. Although both short- and long-sleep durations have been linked with increased rates of brain atrophy , 10 and Here, we examine the relationship between patterns of sleep duration over 28 years and measures of cognition, gray matter volume and white matter microstructure in members of the Whitehall II Imaging Sub-Study. We hypothesize that consistently meeting sleep guidelines (i.e. sleeping at least 7 hours per night) will be associated with higher cognitive scores, greater gray matter volumes, increased fractional anisotropy (FA) and reduced radial diffusivity (RD) values compared with consistently missing the guidelines, or transitioning in and out of the guidelines over time.All participants were members of the prospective occupational cohort study, Whitehall II  and the We excluded from analyses participants who were selected based upon depressive symptoms in 2010, reported a history of dementia or neurological illness, displayed significant abnormalities on structural MRI scans, reported current sleep apnea or prescribed medication for a sleep disorder at the time of the MRI scan, had any missing data for cognitive or MRI outcomes, or had missing data at more than one time point for sleep duration. Thus, the analytic sample included 613 participants . Participants selected their answers from the following options: “5 hours or less,” “6 hours,” “7 hours,” “8 hours,” or “9 hours or more.” For the purposes of this study, “5 hours or less” was coded as 5 hours and “9 hours or more” was coded as 9 hours.Cognition was assessed using a previously described battery of cognitive tests, at the same timepoint as the MRI scan 2012–2016) . With th . With thttp://www.fmrib.ox.ac.uk/fsl) [MRI data were acquired at the Oxford Centre for Functional MRI of the Brain (FMRIB) using a 3-Tesla Siemens Magnetom Verio  scanner (April 2012–December 2015) with a 32-channel head coil or a 3-Tesla Siemens Magnetom Prisma (June 2015–December 2016), with a 64-channel head-neck coil. Data processing and analysis were carried out using tools from the FMRIB Software Library (.uk/fsl) , 23. Gra.uk/fsl)  and Trac.uk/fsl) . Full dep-value < 0.05 for the Lo-Mendel-Rubin likelihood ratio test (LMR-LRT) and Bootstrap likelihood ratio test (BLRT) [To identify how sleep patterns changed over time, a set of unconditional latent growth curve models were performed. Several patterns of change were assessed, including: (1) no change, (2) linear change, and (3) quadratic change over time. Variances of the observed sleep variables were constrained to be equal over time. Adequate model fit  was detet (BLRT) , 30. An t (BLRT) . We alsoYears since the baseline assessment was used as the time scale for both the latent growth curve and latent class growth analyses and these values were divided by either 10 (former ) or 100 F tests, and t-tests performed when an F test was significant (p < 0.05).We employed permutation-based methods for nonparametric testing to examine differences between sleep trajectory groups . Differep values for each cognitive domain [The FSL tool Permutation Analysis of Linear Models (PALM) was used for the statistical analysis of demographic, cognitive, and global MRI measures. To reduce multiple comparisons for cognitive outcomes, Fisher nonparametric combination (NPC) testing was used to assess the overall e domain . The FSLA total of 613 participants were included in analyses  based on their self-reported sleep duration, measured over five time points . Twenty-Demographics for each group are presented in p = 0.158), executive function (Fisher NPC testing p = 0.266), memory (p = 0.983), or processing speed (p = 0.571).Descriptive values for all cognitive tests are provided in Supplementary Material F test minimum p = 0.358) and TBSS .MRI outcomes are presented in The aim of this study was to examine sleep duration trajectories over a 28-year period and their relationship with measures of cognition, gray matter volume, and white matter microstructure. We hypothesized that consistently meeting recommendations for sleep duration (i.e. self-reporting a minimum of 7 hours sleep per night) would be favorably associated with cognition, gray matter volume and white matter microstructure, compared with consistently not meeting the guidelines or transitioning in and out of the guidelines over time. In contrast to our hypotheses, our results did not show any differences in cognitive measures, gray matter volume, or white matter microstructure between different sleep trajectory groups.To the best of our knowledge, only one study has previously applied latent class growth modeling to examine trajectories of self-reported sleep duration over time within an adult population. In a study of 8,673 Canadian adults, Gilmour et al. identified four sleep trajectory groups with intercepts of 5.57 hours (11% of participants), 6.68 hours (49%), 7.65 hours (37%), and 8.34 hours (2%) . We alsoOf particular note within our study is that 37% of participants were included in a group with an average sleep duration of 6.2 hours. Guidelines published by the American Society for Sleep Medicine and the Sleep Research Society state that “adults should sleep 7 or more hours per night on a regular basis to promote optimal health” . In addiextremes in sleep duration within our sample. At each phase, between 92% and 96% of participants reported 6, 7, or 8 hours sleep per night . Sleep duration may be more sensitively measured if sleep was measured in hours and minutes and if there were no lower or upper thresholds for sleep duration. A further limitation is that participants were asked to report their sleep duration only on an “average week night.” The discrepancy between weeknight and weekend sleep duration is common in working-age populations and there is debate regarding whether long weekend sleep can compensate for short weeknight sleep for health outcomes such as mortality , weight,measures . Self-remeasures . Importameasures . As selfmeasures , are theDue to the results from previous observational studies, we hypothesized that consistently meeting sleep guidelines (i.e. sleeping at least 7 hours per night) would be associated with improved cognition, increased gray matter volumes and improved white matter microstructure (increased FA and reduced RD) compared with consistently missing the guidelines, or transitioning in and out of the guidelines over time. However, such hypotheses were not supported by our results, as we found that sleep duration remained relatively stable over 28 years and that there was no evidence of differences in cognition or MRI findings between sleep trajectory groups. If replicated, such null results could challenge the suitability of current sleep guidelines on cognitive outcomes and open doors to new directions in research regarding the appropriateness of considering sleep duration and quality together.This research was supported by the UK Medical Research Council  and the NIHR Oxford Health Biomedical Research Centre. The Wellcome Centre for Integrative Neuroimaging is supported by core funding from the Wellcome Trust (203139/Z/16/Z). Whitehall II was supported by the MRC ; the British Heart Foundation (PG/11/63/29011 and RG/13/2/30098); the National Heart, Lung, and Blood Institute (R01HL036310); the National Institute on Aging, National Institute of Health ; and the Economic and Social Research Council (ES/J023299/1). JZ received funding from the Global Brain Health Institute (GBHI), Alzheimer’s Association, and Alzheimer’s Society (GBHI ALZ UK-19-585532). MK is supported by the MRC , NIA (R01AG056477), and the Academy of Finland (311492). NF was funded by the NIHR Oxford Health Biomedical Research Centre. YL is supported by the National Institute on Aging (NIA) (1K99AG056598), and received funding from GBHI, Alzheimer’s Association, and Alzheimer’s Society (GBHI ALZ UK-19-591141).zsz290_suppl_Supplementary_MaterialClick here for additional data file.


Since enzyme replacement therapy for Gaucher disease (MIM#230800) has become available, both awareness of and the natural history of the disease have changed. However, there remain unmet needs such as the identification of patients at risk of developing bone crisis during therapy and late complications such as cancer or parkinsonism. The Spanish Gaucher Disease Registry has worked since 1993 to compile demographic, clinical, genetic, analytical, imaging and follow-up data from more than 400 patients. The aims of this study were to discover correlations between patients’ characteristics at diagnosis and to identify risk features for the development of late complications; for this a machine learning approach involving correlation networks and decision trees analyses was applied.p = 0.005); serum levels of IgA, delayed age at start therapy (> 9.5 y.o. since diagnosis) also correlates with severe bone disease at diagnosis and with the incidence of bone crisis during therapy. High IgG (> 1750 mg/dL) levels and age over 60 y.o. at diagnosis were found to be related with the development of cancer. When modelling the decision tree, patients with a delayed diagnosis and therapy were the most severe and with higher risk of complications.A total of 358 patients, 340 type 1 Gaucher disease and 18 type 3 cases were selected. 18% were splenectomyzed and 39% had advanced bone disease. 81% of cases carried heterozygous genotype. 47% of them were diagnosed before the year 2000. Mean age at diagnosis and therapy were 28 and 31.5 years old (y.o.) respectively. 4% developed monoclonal gammopathy undetermined significance or Parkinson Disease, 6% cancer, and 10% died before this study. Previous splenectomy correlates with the development of skeletal complications and severe bone disease (Our work confirms previous observations, highlights the importance of early diagnosis and therapy and identifies new risk features such as high IgA and IgG levels for long-term complications. Gaucher Disease (GD) is the most common lysosomal storage disorder (LSD) , 5. ClinGD has a pan-ethnic distribution with cases described worldwide. Outside the Ashkenazi Jewish population, incidence ranges from 1 in 40,000 to 1 in 100,000 inhabitants; however, in the Ashkenazi population a higher incidence has been found (1 in 2500) and GD is not considered a rare disease . Three tThe application of Enzymatic Replacement Therapy (ERT), which began in 1991, has significantly improved awareness of the disease, and has changed the characteristics and expectations of patients as well as the experience of everyone involved in GD management. Nowadays, ERT offers a secure therapy for GD patients with 3 different available enzymes worldwide, two of them in Europe , taliglucerase alfa obtained from plant cell-expressed is until now non approval in EU . Since 2GBA (MIM*606463) variants distribution in the population [The Spanish Gaucher Disease Registry (SGDR) has worked since 1993 to compile demographic, clinical, genetic, analytical and imaging data of Spanish GD patients . The Registry has allowed us to calculate GD prevalence in Spain  and to identify the pulation , 18.In the last decades, the explosion of all kind of data has driven to the use of different big data and machine learning techniques for many applications in the healthcare and bioinformatics fields , a total of 418 GD patients have been reported in Spain. All patients included in the SGDR provided informed consent for the collection and use of the information and biological samples for research projects, all according to the Helsinki declaration of 1963 revised in October 2013, and in accordance with European Regulation 2016/679 on the protection of personal data and the free movement of such data. For this study, ethics and scientific FEETEG boards gave their approval.All the registered patients were included except those diagnosed with GD2 and those who had less than 70% of baseline data available Table . Of 418 In collaboration with Kampal Data Solutions demographic, clinical, analytical, imagining data at diagnosis and comorbidities during the follow-up were evaluated Table .GBA genotype (NM_000157), presence of absence of the variant NM_0003465:c.1049_1072dup24 on CHIT1, age to start therapy, type of therapy (enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) or no therapy, new bone crisis or joint replacement, development of malignancies or PD, collected over a follow-up period of 5 to 25 years.Variables: Birthdate, age at diagnosis, gender, concomitant diseases, family history of Parkinson disease (PD), death date, severity category of disease according to Gaucher Disease Severity Score System category (GD-DS3) , liver size, spleen size, spleen removal, previous bone crisis and bone disease degree according to the Spanish magnetic resonance image score (S-MRI) , bone mineral density (DEXA), GD biomarkers (chitotriosidase activity (ChT), CCL18/PARC and Glucosilsphyngosine (GluSph) concentrations), B12 vitamin level, iron concentration, serum ferritin, cholesterol, triglycerides, high density lipoprotein cholesterol (HDL), Low density lipoprotein cholesterol (LDL), aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), acid phosphatase, bilirubin, hemoglobin concentration, white blood cells (WBC) count, platelets count, serum gammaglobulin fraction, immunoglobulins  -serum concentrations, glucocerebrosidase (GCase) activity, The aimed conditions for which the analysis sought correlations were the presence of severe bone disease at diagnosis, development of bone crisis during follow-up, and the development of neoplasia or PD.The statistical analysis of the data was made in two parts.A descriptive analysis was performed by splitting the variables between numerical and categorical. To establish correlation, Pearson, Chi-Square, Mann-Whitney and Mann-Whitney normalized tests were used.Based on the results of the first step and the correlation between the different variables, we proceeded to the development of a predictive model using decision trees.To implement the models, a training and validation cohort  were useGBA genotype was complex heterozygosity  with the most common variant being NM_000157:c.1226A > G . Forty-seven GD1 patients (13. 9%) were homozygous for c.1226A > G, and 9 GD3 patients (42.8%) were homozygous for c.1448 T > C. Diagnosis was made before the year 2000 for 168 (46. 9%) and 36 (10.1%) died before this study. Most of patients  were treated with ERT. At diagnosis, 65 patients (18.2%) were splenectomized, and 139 (38.89%) had advanced bone disease with bone complications. Regarding comorbidities, 14 (4.1%) GD1 patients developed monoclonal gammopathy of undetermined significance (MGUS), another 14 (4.1%) suffered PD, and 20 (5.6%) malignant neoplasia  and the rest were GD3 21, 5.6%). The most frequent 1, 5.6%. p ≤ 0.05). The weight of the link is equal to the correlation between the two variables. The position algorithm used for its creation tries to place more closely those nodes that are joined by stronger links, while those that are unrelated are further away. The highest correlation was established between the age of diagnosis and the age of onset of treatment. The statistical analysis was performed in order to stablish correlation among all variables, however, there were some correlations that need to be taken carefully in an individualized manner, in special the one involving baseline characteristics and variables such as the age at diagnosis, time since diagnosis to therapy, time on therapy. At this respect, for example, some patients did not start therapy because the ERT was not available, and as consequence their age at therapy correlates with the delay of therapy.A detailed correlation between the numerical variables Table S and cate2n = 10.87, p < 0.01) and repeat bone crises . Almost all of the patients who suffered new bone crises had previous bone lesions .Table SGBA genotypes no NM_000157. 4:c.1226A > G in homozygosity were the variables related to the development of PD .Family history of PD and 2n = 3.80, p = 0.05)  and spleen removal  and IgA levels , Table Sp-values, such as high levels of ferritin , triglycerides , delayed age at diagnosis (> 9.5y.o.) (p < 0.001), time in years between diagnosis and the start of treatment  or delayed age of initiation of ERT  , high IgA levels , and delayed age of initiation of ERT   Fig. B1.n = 0.62, p = 0.00001)  Table SA2.Fig. n = 0.91, p = 0.01) and time delays before the start of therapy (mean age at diagnosis: 28.1 y.o. (0. 5-87); mean age at start therapy 31.5 y.o. (1-83); mean delay time: 7.8 years (0–46)  were related to the development of neoplasia  and age at diagnosis ; in this last correlation the age of PD onset probably has more weight . Almost all of the patients who suffered new bone crises had previous bone lesions (p = 0.0005) in spite of long-term ERT exposure.High correlation were found between spleen removal and the severe bone disease and repeated bone crises .The family history of PD and The last correlation between categorial variables with statistical significance was cancerp = 0.05) Fig. development  and spleen removal  and also with the existence of relatives with PD (p = 0.08), although this was not statistically significant. In the supplemental material, Tables SIn the case of PD, it was not possible to design a tree that improves the prediction of the risk of disease development, because the percentage of patients was very small. However, it has been observed that there is an important correlation with the Large-scale data (big data) in our case when referring to a rare disease have been adapted to the number of cases available; this kind of analysis is a new tool that has recently been incorporated into biomedical activity; machine learning is the study of computer algorithms that improve automatically through experience and it involves a wide series of algorithms, classification and regression models such as decision trees being some of them –29. ThisRegistries have an important role in this kind of analysis, because they include complete information about patients, which is especially important for rare disease research. This collected information helps in diagnosis, patient management, treatment strategy planning, health care planning and follow-up. It enables the acceleration of research and paves new pathways for personalized medicine , 40.This study is the first attempt to establish a correlation network among different biochemical and clinical characteristics in a national-base cohort. We have aimed to analyse diagnostic data and to relate them with long-term complications as bone crises, development of neoplasia or PD, which are the most common and disabling complications –45.p = 0.05). This last observation is in line with the observation that c.1226A > G variant provides a mild phenotype [Two observations, already accepted in Gaucher research, were also confirmed in this machine-learning study: first, the fact that spleen-removal patients have a higher risk of presenting more serious and extensive bone disease; second, our observation that almost all patients with new bone crisis – despite having received long-term ERT – had previous bone lesions, which remind us that the most feared complication in GD1 are not solved merely by starting ERT. These two facts confirm previous reports and provide validity of our analysis , 45–47. henotype , 49.It is a priority to identify accurate risk factors of bone crisis to improve treatment dosage and to avoid this complication. The standard biomarkers related to GD  have been discarded as risk factors for bone complication , 51 evenp = 0.001). The age of onset of treatment (mean 30.6 y.o.) (p = 0.01) also shows a clearer relevance for the occurrence of bone crises (p = 0.01).Surprisingly, the high serum IgA concentration correlates with the degree of bone involvement and with the development of bone crisis (p < 0.01) and the increased concentration of IgG (p = 0.01). Many aspects remain to be unraveled in the complexity of the immune system, but aging is an important factor clearly related to humoral immune dysfunction and the appearance of malignancies [In this study, the development of malignancies appears strongly correlated with the delayed age at the start of ERT . Correlation between numerical variables. Table S2. . Correlations between categorical variables. Table S3. : Correlations of the conditions with numerical variables. Table S4.  Correlations of the conditions with categorical variables Bone disease.


The aim of this study was to investigate the occurrence of postoperative bleeding following dentoalveolar surgery in patients with either continued vitamin K antagonist medication or perioperative bridging using heparin.A retrospective study was performed analyzing patients who underwent tooth extraction between 2012 and 2017. Patients were retrospectively allocated into two comparative groups: un-paused vitamin K antagonist medication versus bridging using heparin. A healthy, non-anticoagulated cohort with equivalent surgery served as a control group. Main outcome measures were: the occurrence and frequency of postoperative bleeding, the number of removed teeth, the surgical technique of tooth removal (extraction/osteotomy/combined extraction and osteotomy) and the prothrombin time.p = 0.004; CG versus BG p < 0.001, BG versus VG p < 0.001. A significant correlation of number of the extracted teeth in the BG (p = 0.014) and no significance in VG (p = 0.298) and CG (p = 0.210) and in the BG versus VG and CG with p < 0.001 in terms of surgical intervention extraction. No difference observed in terms of prothrombin time.In total, 475 patients were included in the study with 170 patients in the group of un-paused vitamin K antagonist medication VG, 135 patients in the Bridging group BG and 170 patients in the control group CG. Postoperative bleeding was significant: CG versus VG Bridging with heparin increases the risk for bleeding compared to un-paused vitamin K antagonist medication. The perioperative management of anticoagulated patients requires a well-coordinated interdisciplinary teamwork to minimize or at best avoid both: postoperative bleeding and thromboembolic incidences. Due to the increase in life expectancy and the rising number of patients with cardiovascular diseases, the number of anticoagulated patients continues to increase worldwide . AlthougAnticoagulation therapy can be carried out using various classes of drugs , which all intervene differently in the coagulation cascade and have their assets and drawbacks. Despite the availability of substances, which are easier to handle , the coumarin derivative and vitamin K antagonist Phenprocomoun (Marcumar®), is still widely used for the prevention of thromboembolic events in atrial fibrillation or following heart valve replacement or pulmonary embolism. There are widely discussed approaches to the management of vitamin K antagonists prior to oral surgery: suspension for several days , 5, tempIn general, available literature on this topic is heterogeneous. Whilst there is a relative consensus pro Bridging regarding major surgery, especially for small-to-medium-sized surgical interventions including oral surgery the recommendations differ considerably even in official guidelines , 17. It A monocentric retrospective patient cohort analysis was performed in a German university dental clinic, including all patients with a permanent vitamin K antagonist medication, who had oral surgery done between 2012 and 2017 in the clinic or were transferred to the clinic for treatment following oral surgery. As a first step, patient search was conducted by screening the digital clinic documentation system  and the digital patient file  using the following key words: tooth extraktion, tooth osteotomy, surgical intervention, tooth, bleeding event, Marcumar®, Bridging, heparin, anticoagulation and thromboembolic event. As a next step, further selection of patients was carried out, by including only patients, who had an oral surgical intervention . Both, in- and outpatients were considered. Furthermore, all patients with hemorrhagic diatheses or blood-thinning medication other than vitamin K antagonists  were excluded.Depending on whether the vitamin K antagonist medication was temporarily paused and substituted by heparin perioperatively (= Bridging) or continued without interruption, we retrospectively allocated the selected patients into two groups: a Bridging group named BG and a vitamin K antagonist group named VG. Additionally, a control group of healthy patients without any anticoagulants, who had equivalent oral surgery, was added as a control group (named CG).For each patient the following data was acquired from the digital patient file:Number of postoperative bleeding events .Surgical intervention and postoperative bleeding events .Anticoagulation monitoring.Correlation of postoperative bleeding events and basic diseases..Patients with incomplete documentation of the above listed information were not considered for the study. The primary outcome of the study was the frequency of postoperative bleeding in each group. Secondary outcomes were the type of surgery (tooth extraction with or without osteotomy), the number of extracted teeth and the INR. Ethical approval was obtained from the local medical faculty ethics committee (registration No.192_19Bc).p < 0.05 in all tests performed.Statistical analysis was performed using the statistical programming language R V3.6.1 (R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). The non-parametric Kruskal–Wallis and Mann–Whitney U tests were used, as well as the Chi-square test, Fisher’s exact test and Cochran–Mantel–Haenszel test. The level of significance was p < 0.001).As a result of the data analysis, a total of 475 patients were included in the study, distributed to the three groups as follows: Bridging group (BG: n = 135), vitamin K antagonist group (VG: n = 170) and control group (CG: n = 170). The overall mean age was 71.76 years. Mean age in the groups was as follows: 79.67 years (BG), 78.76 years (VG) and 58.49 years (CG) with a statistical significance between CG and BG/VG (p < 0.001) and the control group versus the vitamin K antagonist group (p = 0.004). Furthermore, bleeding occurred significantly more often in the Bridging group than in the vitamin K antagonist group (p < 0.001), as shown in Fig. p < 0.001; CG vs. VG: p = 0.002). The comparison of the Bridging group and the vitamin K group revealed a higher number of bleeding events in the Bridging group (p < 0.001).Postoperative bleeding occurred in 22 out of 170 patients (12.9%) in the control group, in 44 out of 170 patients (25.9%) in the vitamin K antagonist group and in 65 out of 135 patients (48.1%) in the Bridging group. Comparing the groups statistically, significant differences were found for the control group versus the Bridging group (p = 0.210) and in the vitamin K antagonist group (p = 0.298). The number of teeth had a significant impact on postoperative bleeding events in the bridging group (p = 0.014) as shown in Fig. p < 0.001) and the vitamin K antagonist group (p < 0.001) as shown in Fig. A total of 584 teeth were removed in the control group, 520 teeth in the Bridging group and 443 teeth in the vitamin K antagonist group. The procedures varied from single tooth extractions to row extractions of up to 24 teeth as a maximum. On average, 3.44 teeth were removed per procedure in the control group, 3.85 teeth in the Bridging group and 2.61 teeth in the vitamin K antagonist group. No significant difference was determined between the number of removed teeth and the number of postoperative bleeding events per procedure in the control group (p < 0.001), but it did not significantly differ between the groups of bleeding/non-bleeding patients (p > 0.05).The Prothrombin time (PT) and International normalized ratio (INR) had been routinely determined before surgery and additionally when postoperative bleeding occurred. Bridging group patients without postoperative bleeding had an average INR of 1.4, whereas the average INR of those with bleeding was 1.45. In the vitamin K antagonist group the average INR was 2.1 for patients without bleeding and 2.7 for those with bleeding. The INR differed significantly between the groups of Bridging and vitamin K antagonist medication . Single tooth extraction within the vitamin K antagonist group resulted in a rate of postoperative bleeding events of 10.5%, a rate of serial extraction of 16.7% and a rate of osteotomy of 10%. In the control group with 603 procedures, they found 0% postoperative bleeding events in single tooth and serial extractions and 1.3% in osteotomies. These results correspond to those of another study where 214 patients, who underwent tooth extraction of one to five teeth per procedure without a significant correlation . In our In a review, Wahl et al. examined more than 2775 patients with dental procedures under bridging with heparin conditions. Postoperative bleeding occurred in 161 patients (6%), which needed intervention in four patients 0.14%) with more than local hemostatic measures % with mo, 25–27. However, also the continuation of vitamin K antagonists still poses a challenge. This is because even in this case, inconsiderable secondary bleeding may occur, although it can be easily treated by local hemostatic measures , 5, 22. There are shortcomings of this study that need to be discussed. First, the retrospective study design led to discrepancies between the groups in terms of group size and composition. The extent and type of the surgical procedure varied between the groups and since operations were performed by different surgeons, the surgical techniques varied to a certain extent. Furthermore, a possible confounding factor of different platelet counts within the groups could not be included in the evaluation.Within the limitations of the current study, it can be concluded that postoperative bleeding events occur significantly more frequently in bridged patients than in patients with un-paused vitamin K antagonist medication. It therefore appears reasonable to continue vitamin K antagonist medication perioperatively for the investigated class of small-to-medium sized oral surgery cases. A close interdisciplinary collaboration between oral surgeons and other medicine specialists is essential to minimize perioperative risks for the patients.


Background and Objectives: The aim of this study is to identify risk factors for the development of delayed neurocognitive recovery (dNCR). Materials and Methods: 140 patients underwent neurocognitive evaluations  and middle cerebral artery (MCA) blood flow velocity (BFV) measurements, one day before cardiac surgery. BFV was re-evaluated after anesthesia induction, before the beginning, middle, end, and after cardiopulmonary bypass (CPB) and postsurgery. To measure glial fibrillary acidic protein (GFAP) and neurofilament heavy chain (Nf-H), blood samples were collected after anesthesia induction, 24 and 48 h after the surgery. Neurocognitive evaluation was repeated 7–10 days after surgery. According to the results, patients were divided into two groups: with dNCR (dNCR group) and without dNCR (non-dNCR group). Results: 101 patients completed participation in this research. GFAP increased in both the non-dNCR group (p < 0.01) and in the dNCR group (p < 0.01), but there was no difference between the groups . Nf-H increased in both groups (p < 0.01), but there was no difference between them . MCA BFV was significantly lower in the dNCR group during the bypass  and after surgery . Results of neurocognitive tests correlated with CO2 concentration , hematocrit , MCA BFV during bypass , and age . The probability of developing dNCR increases 1.21 times with every one year of increased age (p < 0.01). The probability of developing dNCR increases 1.07 times with a decrease of BFV within 1 cm/s during bypass (p = 0.02). Conclusion: Risk factors contributing to dNCR among the tested patients were older age and middle cerebral artery blood flow velocity decrease during bypass. Delayed neurocognitive recovery (dNCR) remains a frequent condition after surgery and is characterized as impairment of memory, attention, and concentration . The freThe etiology of dNCR is associated with genetics, inflammatory neurotransmitters, stress, anesthesia, and other factors ,2. ThereMany researchers have identified that neuropsychological complications emerge from insufficient cerebral perfusion ,5,6,7. TEarly detection of blood-based brain biomarkers could correlate with neurological injury . NeuroinThe aim of this study is to determine the most important factors in dNCR development based on neurocognitive evaluation and brain biomarkers neurofilament heavy chain (Nf-H) and GFAP. This prospective observational study was conducted for 140 patients undergoing elective heart surgery at the Department of Cardiothoracic and Vascular Surgery in the Hospital of Lithuanian University of Health Sciences Kauno Klinikos from April 2018 to April 2019. Permission was obtained from, and the research was approved by the Kaunas Regional Bioethics Committee (Nr BE-2-3) and the Institutional Review Board (NCT03641833). All patients signed a “participation in research” form. Inclusion criteria were elective heart surgery with CPB and age > 18 years. Exclusion criteria were patients suffering from diseases causing cognitive dysfunction  or using agents affecting the central nervous system, multiple organ dysfunction syndrome, deficiency, emergency or re-surgery, carotid artery atherosclerosis with a reduction in artery diameter of 50% or more, and patient’s disagreement. The study design is shown in The Adenbrooke test (ACE-III) —validated to detect cognitive impairment and is often used instead of the Mini mental state examination (MMSE) test .Confusion Assessment Method (CAM)—used to identify delirium.Neurocognitive evaluation tests were performed one day before surgery and 7–10 days postsurgery before leaving the hospital. They were chosen to determine many different domains and included:All patients received premedication of 1–2 mg oral clonazepam and the half dose of the used dose of metoprolol. Patients received general anesthesia. Induction was performed with fentanyl 1–2 μg/kg i.v.; propofol 2–2.5 mg/kg i.v.; rocuronium 0.5–0.7 mg/kg. Lungs were ventilated with a supply of oxygen/air 50–60% after intubation. Anesthesia was maintained with sevoflurane (MAC 1–2%) and fentanyl (10–12 μg/kg) for analgesia. Middle sternotomy section and standard technique for anastomosis formation and valve surgery were performed for all the patients. After systemic heparinization and cannulation, CPB was established using a roller pump, “Stockert III”, and hollow-fiber membranous oxygenator “Compactflo D703” . The CPB circuit was primed with 1200–1500 mL of Ringer’s acetate crystalloid solution and 10,000 IU of heparin. After full CPM support, pump flow was maintained at a cardiac index of 2.4 L/min/m [Standard monitoring of vital signs consisted of an electrocardiogram, heart rate, invasive arterial blood pressure, pulse oximetry, esophageal and body core temperature, central venous pressure, and urine output. Bispectral index (BIS) was maintained 40–60 for adequate depth of anesthesia. Mean arterial pressure was maintained between 50 and 75 mmHg by infusion of intravenous fluids and administration of sympathomimetics if needed. Normothermia was maintained during surgery. All patients were transferred to the intensive care unit (ICU) after surgery and treated according to ICU protocols.The TCD technique was performed with a Sonara transcranial Doppler device with 2 MHz ultrasound transducer through the temporal window. Mean flow velocity was evaluated 1 day before surgery. Patients were excluded from the study if the window for MCA blood flow evaluation was not found or the signal was not clear enough. According to the literature, about 5–37% of the patients had inadequate Doppler signals because of an insufficient acoustic temporal window or window failure during transcranial Doppler examination ,16,17,18Blood samples for GFAP and neurofilaments heavy chain (Nf-H) were collected after anesthesia induction, 24 and 48 h after surgery. Both samples were analyzed by an enzyme-linked immunosorbent assay kit and designed for the quantitative measurements of GFAP and Nf-H in tissue and cell extracts. Measurements were made according to the manufacturer recommendations: GFAP—abcam, United Kingdom, Nf-H—antibodies-online, Germany. A signal was generated to the amount of bound analyte, and intensity was measured at 450 nm wavelength for both biomarkers. The lowest detection limits are defined as a mean-value of an analite-free sample plus three times the standard deviation and are the smallest clearly detectable concentration. According to the manufacturer’s recommendation, GFAP values in blood were 0.781 ng/mL and Nf-H 78.13–5000 pg/mL.t-test. Non-normal distribution data were expressed as median . For nonparametric statistics, a Mann–Whitney U-test was performed for comparison between groups. The difference in frequency distribution between groups was determined by the chi-square test. Pearson correlation analysis was performed to determine the association between two variables . The normality of data was assessed with Kolmogorov–Smirnov or Shapiro–Wilks tests. The study power (β) was calculated according to the incidence of dNCR. The dNCR was determined for 40% of patients. The estimated study power was 0.984, with a sample size of 101 patients and a 0.05 rate of type I error. The estimated size effect was 0.87, with a rate of type I error 0.05, type II error 0.2, the number of patients in the group I—41, in groups 0—60, and proportion 0.6. Normal distribution data were expressed as mean ± standard deviation (SD). Groups were compared by independent samples 2, hematocrit) determine the probability of developing dNCR (dependent variable). dNCR was coded 0/1. The backward conditional method was chosen.The independent factors selection process was made with a univariate analysis of each variable. Any variable having a significant univariate test was selected as a candidate for logistic regression. Logistic regression was used to analyze how independent variables . Statistical tests were two-sided, with In total, 101 of 140 patients completed ACE-III, MoCa, and CAM tests. They consisted of 33 females (32.70%) and 67 males (67.30%), mean age 69 (SD 9.10). dNCR was diagnosed if at least one test determined cognitive impairment, and it was identified for 41 (40.60%) patients. Delirium was diagnosed for 11 (10.90%) patients according to the CAM scale. According to the neurocognitive test results, patients were enrolled in two groups: patients without cognitive dysfunction after surgery were included in the first (non-dNCR) group and patients with delayed neurocognitive recovery were included in the second (dNCR) group. Groups were not differentiated according to sex, surgery type, comorbidities, duration of bypass and cross-clamping, ejection fraction, or bypass pump flow.Demographic, preoperative, intraoperative, and postoperative characteristics are shown in GFAP changes are shown in Nf-H concentration changes in both groups are shown in MCA BFV changes are shown in p = 0.006).BFV changed during surgery in the non-dNCR group. It was lowest during bypass, but this difference was not statistically significant. BFV notably decreased during bypass on the dNCR group  in the non-dNCR group and 1–36% (12.87% SD 9.58) in the dNCR group.2 concentration, hematocrit, and MCA BFV during bypass and moderate negative correlation with age (Results of neurocognitive tests had a moderate positive correlation with COwith age . p < 0.01). The probability of developing dNCR decreases 1.07 times with an increase of blood flow velocity within 1 cm/s during bypass (p = 0.02)  .2, hematocrit) determine the probability of developing dNCR (dependent variable). Method backward conditional was chosen. Only age and blood flow velocity during bypass have an influence on dNCR.Logistic regression was used to analyze how independent variables  are a group of proteins integrated into the neuronal and axonal cytoskeleton . They arNf-H increased in both groups, but we did not find a correlation with dNCR. There is not much research with this biomarker in cardiac surgery, and the results are contradictory. Szwed K. compared several biomarkers suitable for cardiac surgery and did not find Nf-H elevation in patients with cognitive dysfunction . Saller Nfs are new biomarkers with controversial results. One of the hypotheses is that preinterventional ischemia could mask any further alteration of pNf-H levels. Additionally, most of the investigations are of small sample size, and the ideal point for measurement is unknown . Saller GFAP is a specific biomarker for astrocytes and found only in the central nervous system. For this reason, it is widely used as a biomarker for brain damage and in research. GFAP has been shown to be involved in astrocyte functions, which are important during regeneration, synaptic plasticity, and reactive gliosis . In our Research has confirmed that pre-existing brain vulnerability and neuronal damage are associated with dNCR ,39,40. Gp < 0.05). It is interesting that a significant difference between the mean of MCA BFV during CPB (41.3 vs. 37.1 cm/s) was not found in delirium and no-delirium groups. Baseline MCA BFV was lower in patients who developed postoperative delirium . Benvenuti M. suggested that even preoperative BFV hypoperfusion may cause long term dNCR [Another important factor for dNCR is cerebral autoregulation and blood flow velocity. It is considered that cerebral hypoperfusion during surgery may lead to cognitive impairment. Our results and the results of many other investigations support this statement. Kosir G. determined hypoperfusion episodes in 9 of 15 patients in a pump–on group. MCA BFV was 15–25 cm/s in 5 patients and 5–15 cm/s in 4 patients. This study stated that blood flow velocity changes and pulsatility play an important role in postoperative neurologic outcomes . Thudiumerm dNCR . Nuttal erm dNCR . These rBFV during bypass becomes nonpulsatile, which may be one of the risk factors of various complications ,52. EvenA validation study on the reproducibility of transcranial Doppler velocimetry was made by Maeda. CoV was determined at 6.7–19.5%, good enough to warrant the applicability of this method for the repeated measurements of the intracranial arterial blood flow velocity in future studies . These mp = 0.45). This may have led to a better response of these patients’ blood vessels to various factors, and therefore, the blood flow velocity may have changed minimally. Increased variability (>20%) was determined for 4 patients (3.96%); all these patients suffered from dNCR and were associated with a less favorable outcome. Reduced variability (less than 6%) was determined for 24 patients (23.7%). These patients were statistically younger than the rest of the group  vs. 69 y ; A combined technique (blood biomarkers and BFV changes) was used to identify risk factors for dNCR. There are currently no studies combining these two methods in cardiac surgery. Our investigation determined that the most important factors for dNCR are age and blood flow velocity changes. Most patients undergoing cardiac surgery are elderly, so anesthesiologists should be especially careful. Monitoring anesthesia depth, brain oximetry, and blood flow velocity during surgery provide useful information and help to determine target doses of medicines, identify hypoperfusion or desaturation periods, and allow faster adjustment in treatment. Brain biomarkers GFAP and Nf-H are not helpful in identifying dNCR.This study has several limitations. TCD was performed and BFV measured at the selected points of surgery. It would be more useful to monitor blood flow velocity continuously during all operations. This is a small sample of a one-center study. A multicentric study with a bigger sample size will provide more precise results. Additionally, there was no assessment of long-term neurocognitive disorders. Patients were discharged for rehabilitation or home, and we did not succeed in assessing mild neurocognitive disorders for most patients.p < 0.01). The probability of developing dNCR decreases 1.07 times with an increase of blood flow velocity within 1cm/s during bypass (p 0.02) Neurofilaments heavy chain (Nf-H) and GFAP were not helpful in diagnosing dNCR, and we do not recommend their routine use.The most important factors in developing dNCR are older age and middle cerebral artery blood flow velocity decrease during bypass. The probability of developing dNCR increases 1.22 times with every one year of increased age (


Asp680Ser) (rs6166) and c. −29 G/A (rs1394205) One-hundred twenty-seven healthy men underwent evaluation of the anthropometric parameters, assessment of metabolic and lipid profile, measurement FSH serum levels, and genotyping of both the aforementioned FSHR SNPs. Data grouped according to the FSHR rs6166 or rs1394205 genotypes underwent to statistical analysis.FSHR rs6166 SNP, glucose levels were significantly lower in men with the GG genotype compared with those with the AA genotype. Men with AG had lower insulin levels and HOMA index values compared with those carrying the genotype AA (p < 0.05). The GG group showed a negative correlation between serum FSH levels and insulin and between serum FSH levels and HOMA index (p < 0.05). In contrast, men grouped according to the FSHR rs1394205 genotype showed no significant difference in blood glucose, serum insulin levels, and HOMA index. The AG group showed a negative correlation between FSH insulin and between serum FSH levels and HOMA index (p < 0.05).The three groups of men for each FSHR SNP did not differ statistically significantly for body mass index and serum FSH levels. As for FSHR rs6166 SNP have lower blood glucose levels than those with the AA genotype. Their FSH levels inversely correlated with insulin and HOMA index. In contrast, the genotype FSHR rs6166 A/G did not reveal any role of FSH on glucose metabolism in healthy men. The inverse relationship between FSH and insulin or HOMA index in the group with the genotype GG of the FSHR rs6166 SNP suggests a possible cross-talk between FSH and insulin.Men with the genotype GG of the  The follicle-stimulating hormone (FSH) is a glycoprotein made of an α and a β subunit. It exerts its peripheral effects by binding to its receptor (FSHR), a 678 amino acid protein belonging to the G-coupled receptor family, expressed in granulosa and Sertoli cells, where FSH stimulates follicle genesis and spermatogenesis, respectively .follicle-stimulating hormone receptor (FSHR) gene, mapping in the 2p16.3 chromosome, contains ten exons and nine introns. The first nine exons encode for the majority of the extracellular domain. The exon 10 encodes for the C-terminal region of the extracellular domain, the transmembrane and the intracellular domains . A total of 146 men were evaluated for inclusion in this study. The following exclusion criteria were used and this resulted in the exclusion of a number of men that is indicated in parentheses: azoospermia . Reference values were as follows: FSH 0.95–11.95 IU/l, glycemia 74–100 mg/dl, insulin 1.9–23 µIU/ml, total cholesterol 0–200 mg/dl, HDL cholesterol 35–55 mg/dl, LDL cholesterol <160 mg/dl, and triglycerides 35–200 mg/dl. The HOMA index was evaluated using the formula: [glycemia (mg/dl) × insulin (µIU/ml)]/405. The upper normal value of HOMA index was 2.5.FSHR polymorphism. Probes and primers for rs6166 FSHR and rs1394205 FSHR polymorphisms were chosen on https://www.thermofisher.com/it/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/snp-genotyping-taqman-assays.html?SID=fr-taqman-2. The reaction was carried out according to manufacturer’s instructions . Each DNA sample was analyzed in triplicate [Genomic DNA was extracted from blood cells using the PureLink® Genomic DNA Kits  for purification of genomic DNA according to the manufacturer’s instructions. The concentration and the quality of the DNA was determined using a ND-1000 spectrophotometer . Allelic Discrimination was performed with TaqMan assay in order to show a different genotyping distribution of p value < 0.05 was accepted as statistically significant.The normality of the variables was evaluated with the Shapiro–Wilk test. Descriptive statistical results have been reported as mean ± standard deviation (SD) for not-skewed variables. Statistical analysis was performed by one-way analysis of variance, followed by the Duncan’s Multiple Range Test, using SPSS 22.0 for Windows . For correlation analysis, the Pearson or the Kendall tests were used, according to the data distribution. A FSHR rs6166 genotypes were: 33 (26.0%), 63 (49.6%), and 31 (24.4%) for AA, AG, and GG, respectively. For the genotype FSHR rs1394205, they were: 14 (11.0%), 38 (29.9%), and 75 (59.1%) for AA, AG, and GG genotypes, respectively. The FSHR allelic frequencies and the estimated FSHR haplotype frequencies found in this cohort were consistent with previously published data from unrelated Caucasian populations [The number and the prevalence (in parentheses) of 0%), 63 4.6%, and were: 14 .0%, 38 , with the exception of total cholesterol, which was higher in men with FSHR rs1394205 AA genotype compared with those with the AG genotype (p < 0.05). According to their BMI, 42.2% of patients were overweight, 20% obese; 8.3% had hyperglicemia, 20.5% insulin resistance, and 26.3% hypercholesterolemia. The prevalence of these features did not differ after classification by the FSHR genotype.Data were normally distributed. Anthropometric data and biochemical parameters of men grouped according to the FSHR rs6166 GG vs. men with the AA genotype. Men with AG SNP had lower insulin levels and HOMA index values (Table p < 0.05). Men grouped for the FSHR rs1394205 genotype did not show any difference in glycaemia, insulin, and HOMA index , insulin (r = −0.26) or HOMA index (r = −0.19) in the entire cohort. Correlation analysis in men grouped by FSHR rs6166 or rs1394205 genotypes are shown in Table FSHR rs6166 GG and for the genotype FSHR rs1394205 AG. All the other correlations were not significant.No significant correlation was found between FSH and glycaemia  do not develop obesity as wild-type ovariectomized mice do. These effects were also reported in male Fshr+/− mice. Furthermore, the administration of a blocking antibody against FSH was found to efficiently induce thermogenic adipose tissue and to reduce body fat in both sexes [Scanty evidence has been published on the impact of FSH on glucose metabolism so far. A preclinical study reported that female mice FSHR gene deficient  with hypergonadotropic hypogonadism, including patients with Klinefelter syndrome. Remarkably, the authors reported that FSH could enhance soluble-RANKL (sRANKL) secretion from human adipocytes. sRANKL levels, in turn, are negatively associated with fat percentage, fasting insulin, and glucose . sRANKL FSHR rs6166 SNP suggests the hypothesis that FSH and insulin may share a common molecular signaling. Generally, tyrosine kinase receptors  are able to influence the FSHR signaling [The inverse relationship between FSH and insulin or HOMA index observed in men with the GG genotype of the ignaling –21. In tignaling , 23. Intignaling , 25; thuignaling . In lineignaling . Unfortuignaling . FSH admignaling . These fignaling . In addiFSHR SNPs assessment. A possible FSHR SNPs usefulness has been classically searched on fertility so far. However, the research promoted after the identification of FSH effects on bone tissue has led to studies aimed at exploring its protective role against the development of osteoporosis in women with the FSHR rs6166 GG genotype; thus supporting extra-gonadal effects that FSH may likely have [FSHR SNPs in the metabolic field needs to be further addressed.Finally, the results of the present study expand the view of the possible clinical applications of ely have . SimilarOur results need to be taken with care due to the relatively small sample size examined. Hence, further studies on greater cohorts are warranted.FSHR rs6166 A/G genotypization could reveal the impact of FSH on glucose metabolism in healthy men. The inverse relationship of FSH with insulin and the HOMA index in the FSHR rs6166 GG group may support the existence of a cross-talk between FSH and insulin. Accordingly, the higher is insulin, the lower is FSH, thus reflecting the importance of treating insulin resistance prior to FSH administration in oligozoospermic patients [FSHR to signal transduction) could confirm the occurrence of a molecular interaction between insulin and the FSHR. Clearly, this topic represents a black hole and deserves further investigation.In conclusion, we found significantly lower blood glucose levels in men with the GG genotype of the FSHR rs6166 SNP, and significantly lower insulin levels and HOMA index in AG compared with AA men. Furthermore, FSH serum levels correlated inversely with insulin levels and the HOMA index in men with GG, no effects were observed on these parameters when men were classified according to the rs1394205 genotype, although a significant indirect correlation between FSH and insulin and a negative trend between FSH and HOMA index was found in the AG group. patients . Moleculpatients  may some