Clostridium perfringens, may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro. Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens. Sphingosine, a Fingolimod homolog with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs.There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod, and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium,  Multiple sclerosis (MS) is the most common non-traumatic neurological disease of young adults in Western Europe and North America , causing rapid internalization of S1PR1. In the absence of surface S1PR1, lymphocytes are unable to egress from lymphoid tissues and cannot traffic to target tissues such as the brain; thus the rationale that Fingolimod may reduce the risk of MS relapse and the severity of attacks through immune modulation , ATCC 3626 (type B), ATCC 51880 (type C), ATCC 3631 (type D), ATCC 27324 (type E), and two type A clinical isolates provided by New York Presbyterian Hospital were used for the initial screen while the “type strain,” ATCC 13124, was used for all subsequent experiments. Bacteria were cultured anaerobically at 37°C overnight using the GasPak 100 system (BD). Anaerobiosis was achieved by pre-reducing the culture media using an anaerobic jar containing a GasPak EZ Anaerobe System sachet for a minimum of 6 h before inoculation. After inoculation, the GasPak sachet was replaced for the overnight culture.6 colony-forming units (CFUs) of different C. perfringens strains. Minimal Inhibitory Concentration values  were determined for inhibitory compounds using cation adjusted Mueller Hinton II broth (BD). Inhibitory compounds were diluted serially from 512 μg/ml down to 0.5 μg/ml, inoculated with 5 × 105 CFUs of C. perfringens and then anaerobically cultured at 37°C overnight as previously described. Culture conditions for each compound were performed in triplicate and bacterial growth was determined by measuring OD600-values from 1 ml of re-suspended bacteria.The compounds used for the initial growth inhibition screen were diluted to a final concentration of 500 μg/ml in Mueller Hinton broth (BD) and media was inoculated with 5 × 10SD for three replicates. Statistical analysis was performed using the two-tailed Student t-test .Results are representative of data obtained from repeated independent experiments. Each value represents the mean ± C. perfringens since type B and D strains secrete ETX during log-phase growth. Therefore, we tested if oral DMDs affected the growth of C. perfringens toxinotypes A–E. We compared the growth inhibitory effects of Fingolimod, DMF, and Teriflunomide to that of oral symptom management drugs (SMDs) Baclofen, Bupropion, and Gabapentin; drugs not thought to alter the disease course of MS. We exposed C. perfringens cultures to 500 μg/ml of each compound, allowed for overnight anaerobic growth, and determined the optical density (OD600) the following day. We found that each oral DMD significantly inhibited all C. perfringens toxinotypes and strains tested, while the oral SMDs did not , to Fingolimod, D-sphingosine, and Myriocin and identified that, like Fingolimod, D-sphingosine also displayed inhibitory activity. Myriocin failed to inhibit C. perfringens, but instead, enhanced bacterial growth  donate electrons. The Michael reaction results in covalent alkylation of the sulfhydryl group. This covalent linkage permanently inactivates thiol-containing molecules if the thiol is necessary for the molecule's function, as is the case for glutathione and its antioxidant properties , and fumarate and found that each compound inhibited the growth of type strain, C. perfringens ATCC 13124. However, their saturated succinate counterparts dimethyl succinate (DMS), monomethyl succinate (MMS), and succinate, molecules devoid of Michael acceptor activity due to reduction of the α, β carbon double bond, failed to inhibit C. perfringens , Parthenolide (a sesquiterpenoid), and Curcumin (a curcuminoid) each inhibited C. perfringens .To provide additional evidence that Michael acceptor activity is indeed critical to the antibacterial properties of α, β unsaturated carbonyls, we searched the literature to find compounds for which experimental values of Michael reaction potencies have been determined. Dinkova-Kostova et al. determined the potencies of several plant derived Michael acceptors for their ability to induce cellular quinone reductase activity; a cellular marker for a compound's reactivity with sulfhydryl containing molecules , 87–175 μg/ml for Teriflunomide (MIC95 = 128 μg/ml), and 1500–3000 μg/ml for DMF (MIC95 = 128 μg/ml). Therefore, each compound's MIC95 is within the calculated range of the therapeutic concentration that will enter the small intestine. Furthermore, DMF is a delayed released capsule that dissolves in the more basic pH of the small intestine  and will not grow with strictly inorganic sources .That Michael acceptors such as DMF and its fumarate metabolites inhibit S. aureus and B. anthracis growth, and to suppress S. aureus secretion of toxic shock syndrome toxin 1 , is a synthetic precursor for vitamin K. It is commonly used as a dietary supplement for livestock and as a cost effective vitamin K replacement therapy in developing countries. Of note, Menadione has recently been shown to inhibit In light of the serious side effects associated with current oral DMDs, this study may be of immediate clinical importance. Some of these adverse effects are due to immunosuppression of the CNS, as evidenced by increased risk of JC virus infection and progressive multifocal leukoencephalopathy , the Center for Disorders of the Digestive System (CDDS), and the Weill Cornell Tisch Family Research Fund.Clostridium perfringens epsilon toxin as candidate trigger for multiple sclerosis.All authors are named as inventors on a pending patent entitled, “Methods to protect against and treat multiple sclerosis,” (Publication number CA2899961 A1), which identifies


Dietary recalls over there consecutive days were done in a face-to-face interview. The scores of DBI-07 for each component and three DBI-07 indicators ((Lower Bound Score (LBS), Higher Bound Score (HBS), Diet Quality Distance (DQD)) were calculated according to compliance with the Dietary Guidelines for Chinese residents. Hemoglobin (Hb) concentration was determined using the cyanmethemoglobin method. Univariate and multivariate linear regression models were used to explore the associations between DBI-07 indicators and anemia, as well as scores of DBI-07 components and Hb level. The sample included 336 men and 402 women. Inadequate intakes of vegetables, fruits, dairy, soybean, eggs, fish and excessive intakes of cereals, meat, cooking oil, salt were both common. 91.3% of the participants had moderate or high levels of inadequate food intake, while 37.7% had moderate or high levels of excessive food intake. The mean Hb was 14.2 ± 1.7 g/dL, with a prevalence of anemia of 13.0%. Subjects with high LBS and DQD were more likely to be anemic . After adjustment for potential confounders, there were positive correlations between Hb level and the intakes of vegetables and soybean . In conclusion, dietary imbalance and anemia are common in middle aged and elderly population in southwest China and inadequate intakes of vegetables and soybean may increase the risk of anemia.A balanced diet is essential to achieve and maintain good health. In this study, we assessed diet quality of middle aged and elderly people based on Chinese Diet Balance Index-07 (DBI-07) and explored the associations between DBI-07 and anemia. Data analyzed for this study was from the 2010–2012 National Nutrition and Health Survey in Yunnan province, southwest China ( Anemia remains a major public health concern globally . Anemia Poor nutrition has been identified as the main cause of anemia . People Recent nutritional studies have shifted towards examining diet-diseases associations using overall diet rather than focusing on individual foods or nutrients ,21. BaseIn this study, we evaluated diet quality using DBI-07 among middle aged and elderly people in southwest China, which is a less developed region with a high prevalence of anemia . In addiData used in this study were obtained from the 2010–2012 China National Nutrition and Health Survey in Yunnan Province, southwest China. Participants were recruited from two urban districts and four rural counties using a stratified cluster sampling method. From each of the six counties/districts, which are at different levels of economic development, three townships were randomly selected. Next, two villages were randomly selected from each township and 30 households were randomly selected from each village. All members aged 2 years or over in the households were invited to participate, with a response rate of 96.0%. Altogether, 778 participants aged 50 years or over completed the survey. 40 participants were excluded since they reported to be on a prescribed diet (14 subjects) or having serious illnesses (26 subjects). The final sample analyzed for this paper was 738 participants.The ethical approval was obtained from the Institutional Review Board at China Centers for Disease Control and Prevention (2013–2018). Before administrating the survey, signed informed consent was obtained from all the participants.Diet was assessed with 24 h recalls and a household food inventory over three consecutive days (including two weekdays and one weekend day). Every household member aged 2 or over was asked to report all food consumed, whether at home or away from home. With food picture and model aids, trained interviewers recorded the types and the amounts of food consumed at each meal via face to face interview. Condiments  and cooking oils consumption at the household level were measured by the changes in a household food inventory over the period. Individual consumptions of cooking oil and salt were computed according to per capita consumption in each household and then adjusted by individual energy intake.Data of alcohol consumption was derived from the lifestyle survey. Participants were inquired the drinking frequency and quantity of alcoholic beverages  in the past 12 months. Alcohol in each alcoholic beverage was converted following DBI-07 criteria  and thenDBI-07 has been devised to assess diet quality in the Chinese population. DBI-07 comprises seven components from ‘the Dietary Guidelines for Chinese residents,’ including: (1) cereals; (2) vegetables and fruits; (3) milk and dairy products, soybean and soybean products; (4) Animal food; (5) condiments and alcoholic beverage; (6) Diet variety; (7) drinking water. A score of 0 for each DBI-07 component indicates meeting the recommended intake amounts. Positive scores (range 0–12) were used to assess excessive intake levels of alcoholic beverages and condiments, which the guidelines recommended consuming in a “reduced” or “limited” amount. Similarly, negative scores (range −12–0) were used to assess inadequate intake levels of vegetables and fruits, dairy and soybeans, dietary diversity and drinking water, which the guidelines recommended that people should consumed in a “sufficient” or “plenty” amount. Moreover, both positive and negative scores were used to evaluate the intake levels of cereals (−12–12) and animal food (−12–8), which the guidelines recommend consuming in an “appropriate” amount. In considering the difference of nutrient requirements in energy consumption, the scoring of cereals, vegetables, fruits, dairy, soybean, animal food and cooking oil is based on seven energy intake levels. The intakes of 12 identified food subgroups are used to assess the diet variety component of DBI-07. These food subgroups were (1) rice and products; (2) wheat and products; (3) corn, coarse grains and products, starchy roots and products; (4) dark-colored vegetables; (5) light-colored vegetables; (6) fruits; (7) soybean and products; (8) milk and dairy products; (9) red meat and products; (10) poultry and game; (11) egg; (12) fish and shellfish. A score of 0 is assigned to a food subgroup if the intake reached or exceeded the lowest recommended intake. Else, a score of −1 is assigned. The lowest recommended intake is 5 g for soybean and products and 25 g for other 11 food subgroups. The score of diet variety component ranges from −12 to 0 ,29,30. SBased on the scores for each DBI-07 component, there indicators of diet quality are calculated. Higher Bound Score (HBS), the indicator for excessive food intake, is calculated by adding all the positive scores. Lower Bound Score (LBS), the indicator for inadequate food intake, is computed by adding all the negative scores. Diet Quality Distance (DQD), the indicator of imbalanced food intake, is calculated by the absolute values of both positive and negative scores. Data on water consumption was not available in the survey and therefore, the component of ‘drinking water’ was not assessed in this study. Therefore, the lower and upper bound of HBS, LBS and DQD were: −72 to 44, 0 to 32, 0 to 60 and 0 to 72, respectively. Each indicator is further divided into five levels to reflect diet quality for simplicity. They are “no problem” (a score of 0), “almost no problem” , “low level” , “moderate level”  and “high level”  ,29,30. CSix milliliter venous blood was collected from each participant using EDTA-containing vacuum tubes. Hemoglobin concentration was determined by the cyanmethemoglobin method. All local laboratory technicians have participated in a national training and passed a qualifying examination. In field examination, one quality control sample was tested in every 30 samples to ensure quality control. The residential elevation above sea level  for each township was obtained from Google Earth Software . Hb concentrations for the diagnosis of anemia was <13 g/L for men and <12 g/dL for women, all Hb values were adjusted for altitude and smoking behaviors (derived from the lifestyle survey) .2), normal weight (BMI 18.5–23.9 kg/m2) and overweight and obesity (BMI ≥ 24.0 kg/m2) [Height and weight were measured without shoes and heavy clothes, by trained local health workers. Body Mass Index (BMI) was computed as measured weight (in kilogram) divided by measured height in meter squared. The BMI cut-off points for Chinese adults were used to determine underweight (BMI < 18.5 kg/m0 kg/m2) . Age grop < 0.05.Numeric variables which normally distributed  were given as mean ± standard deviation (SD). Categorical variables  were given as a percentage (%). Univariate and multivariate linear regression models were used to examine the associations between DBI-07 indicators and socio-demographic predictors, as well as anemia. Multivariate linear regression models were used to evaluate the associations between DBI-07 scores for each component and Hb level. All analyses were performed with SAS 9.4  and statistical significance was set at 2). The overall prevalence of anemia was 13.0% (12.2% in men and 13.7% in women).The contribution rates of DBI-07 components to LBS, HBS and DQD were presented in p < 0.05). People living in rural areas, males and aged 50–59 years had a higher HBS score (p < 0.05). DQD scores were higher among people living in rural areas, of lesser education, of lower income, being underweight and anemic (p < 0.05).vegetables = 1.04, p < 0.01; βsoybean = 0.82, p = 0.04).The findings of this cross-sectional study can be summarized as follows. Firstly, diet imbalance is common among middle aged and elderly people in southwest China, especially for those living in rural, of lower education, of lower income. Secondly, people with anemia were more likely to be inadequate intake of food (LBS) and imbalanced intake of food (DQD). Finally, Hb level was positively correlated with the DBI-07 components of vegetables and soybean intakes.Over the past 20 years, China has experienced an ongoing nutrition transition, which mainly characterized by decreased cereals intake and increased oil and animal food intakes . In contOur results also show that inadequate intakes of vegetables, fruits, dairy, soybean, fish and egg were common in middle aged and elderly population. Over the past decade, consumptions of vegetables and fruits have remained almost unchanged in China, which only meets half of the recommendations . In partPrevious studies have shown that diet quality is generally determined by socio-demographic factors ,40. In tAdhering to evidence-based dietary guidelines is promising for elderly people to achieve and maintain good health. However, the associations between dietary balance and anemia have rarely been investigated, especially in elderly population. A previous study conducted among elderly women in southwest China found that women with anemia usually had higher LBS . In thisWe acknowledge that this study has potential limitations. First, the cross-sectional nature of the study restricts casual inference between dietary intakes and anemia. Second, dietary recalls over three consecutive days are inadequate to assess dietary intakes over a long term. As Zhang et al. reported, seasonal variations in dietary intakes are significant, even in economically well-developed areas of China . Third, In conclusion, imbalanced diet intake and anemia are common in middle aged and elderly population in southwest China and inadequate intake of vegetables and soybeans may increase the risk of anemia. Nevertheless, prospective studies are needed to explore possible mechanisms.


Dry eye disease (DED) is multifactorial, affecting 5–34 % of the global adult population and reducing quality of life. The artificial tears or lubricants are the therapy most used for the treatment of DED, due to their low side effect profile, which attempt to modify the properties of the tear film. The aim of the present study was to evaluate the clinical efficacy of a fixed combination of xanthan gum and chondroitin sulfate preservative free on the ocular surface of patients with dry eye disease during 60 days of intervention.n = 76) or a fixed combination of polyethylene glycol 400 0.4 % and propylene glycol 0.3 % (PEG/PG) (n = 72). Subjects self-dosed four times daily during 60 days. Follow-up was set on days 2, 7, 15, 30 and 60. Assessments of anterior/posterior segment ocular signs were performed. The outcome measures included Schirmer test, tear film break-up time and OSDI score. Security variables included intraocular pressure, lisamine green and fluorescein ocular surface stains.A phase III, double-blind, masked, controlled, multicenter, clinical trial of 148 subjects, randomized to either a fixed combination of xanthan gum 0.09 % and chondroitin sulfate 0.1 % (XG/CS) ophthalmic solution (p = 0.002) and PEG/PG  respectively. Similar results were reported in the tear film break-up time in XG/CS  and PEG/PG  respectively. The OSDI score decreased to normal values in both groups, XG/CS  and PEG/PG  respectively. There was no significant difference between treatments for any parameter. Moreover, both groups decreased the presence of burning sensation, tearing, foreign body sensation, conjunctival hyperemia and photophobia. The adverse events were not related to the interventions.The primary efficacy endpoints were similar between groups at baseline. After intervention time Schirmer test increased in both groups compared to baseline, XG/CS (6.4 ± 2.2 vs 11.0 ± 6.6; Xanthan gum/chondroitin sulfate preservative free showed similar clinical efficacy, evaluated with OSDI score, TBUT and Schirmer test compared to polyethylene glycol/propylene glycol in the treatment of dry eye disease.NCT01657253. Date of registration May 19, 2014.ClinicalTrials.gov:  The tear film is classically schemed as a three-phase emulsion, composed by an aqueous, a mucinous and a lipid layer; together have viscoelastic properties that allows them to adhere to the ocular surface in order to provide moisture, protection, nutrition and effects in optical quality. However, each layer have a particular function; the mucin layer, produced by goblet cells, plays an important role in protecting the ocular surface, stabilizing tears and acting as a gel-like barrier. The quality and quantity from the tear film is highly influenced by the conditions and abnormalities of the ocular surface –3. AlterThe artificial tears or lubricants are the therapy most used for the treatment of DED , due to Xanthan gum is an exopolysaccharide with rheological properties that could provide stability and increase the residence time of the tear film on the ocular surface , 9. ChonThe aim of the study was to evaluate the safety and efficacy of the fixed combination of XG/CS preservative free in subjects diagnosed with mild to moderate dry eye disease in Latin American population compared with PEG/PG.A parallel, randomized, double blind, active-controlled, multicenter, clinical trial was designed to compare the efficacy of two ophthalmic solutions. The study was conducted across 7 investigative sites . An ethics committee in each center reviewed and approved the study. The clinical trial was conducted in accordance with Good Clinical Practice Standards  and Declaration of Helsinki. All patients provided written informed consent. The study was registered at ClinicalTrials.gov with the identifier number NCT01657253.Inclusion criteria were patients (aged >18 years) with best corrected visual acuity < 0.6 logMAR or better in both eyes, mild to moderate dry eye disease based on Ocular Surface Disease Index (OSDI) score between 12–45 and without active ocular disease and no use of topical ocular drops within approximately 24 h before screening. Exclusion criteria were patients with autoimmune disease  Meiboian gland dysfunction, blepharitis, corneal dystrophy, eyelid malformations, history of eye surgery within 3 months before baseline, intolerance or hypersensitivity to any component of study treatments, contact lens users, participation in an investigational drug or device study < 60 days before screening, ocular o systemic infections or conditions  that preclude safe administration of study treatment, and patients that were pregnant, at risk for pregnancy without birth control treatment, or breastfeeding. Written informed consent was received from each subject prior to any study related procedure. Patients were randomly allocated 1:1 to received XG/CS  or PEG/PG  using random numbers software. Baseline data including demographics, relevant medical and ocular history, and concomitant medications were noted at visit 1 (Day 0). Subjects instilled one drop of study drug topically in the inferior conjunctival sac of both eyes four times daily. The compliance was evaluated according this formula: weight of the bottle after intervention × 100 / weight of the bottle before intervention was started. An adequate compliance was considered >80 %. The compliance was evaluated in 1 bottle each 30 days. The final compliance was determined using the mean of both bottles. Moreover, the pharmacist verified the register of eyedrop instillation from patient diary. Investigators were masked to the study medication. Because the active control bottle (Systane) was visibly different than the investigational bottle, a designee at each study site, other than the investigator, was responsible for the dispensing study treatment. Attempts were made to mask the subjects by removing commercial labeling, replacing it with identical investigational labels and packaging in identical kit boxes and were separated during the evaluations. Patients were evaluated during six study visits: Visit 2 (Day 2 ± 1), Visit 3 (Day 7 ± 1), Visit 4 (Day 15 ± 1), Visit 5 (Day 30 ± 1) and, Visit 6 (Day 60 ± 1) after randomization. Clinical assessments during baseline and the final visit consisted of intraocular pressure (IOP), using a calibrated Goldman applanation tonometer, tear break-up time (TBUT), Schirmer I test with anesthesia, and indirect ophthalmoscopy. Slit lamp assessment (biomicroscopy) and fluorescein and green lisamine stain were performed. Safety assessments included adverse events (AEs) and ocular tolerability .The primary efficacy endpoints were the increase from baseline in mean Schirmer test, TBUT and, a reduction of OSDI score at visit 6 (Day 60). The safety endpoint was the incidence of ocular and systemic AEs and their severity and relationship to the study drug.p- value of < 0.05 (two-tailed) was considered statistically significant. Adverse reactions were evaluated using the collection method. All statistical analyses were conducted using SPSS software  version 19.The results are presented in mean and standard deviation. Kolmogorov-Smirnov test was made to know the normal distribution of data. We considered both eyes for statistical analysis. An intent-to-treat analysis was performed. Inter-eye correlation was made within the same subject. The mean of IOP, Schirmer test, TBUT and OSDI score were compared using paired two-sided t tests. Ocular signs and symptoms were summarized using proportions and were analyzed with the chi-square method. In all analyses, a n = 93, XG/CS; n = 90 PEG/PG) and 148 subjects completed the study  without statistical difference. According to Kolmogorov-Smirnov test the data have a normal distribution. The primary efficacy endpoints were similar between groups at baseline. After intervention time, Schirmer test and TBUT increased in both groups compared with baseline , PEG/PG ; tearing, XG/CS , PEG/PG ; foreign body sensation, XG/CS , PEG/PG ; conjunctival hyperemia XG/CS , PEG/PG ; and photophobia, XG/CS , PEG/PG . There were no alterations in corneal stains. The adverse events were not related to the interventions.The treatment groups were comparable regarding to demographics and baseline characteristics Table . Of the The ideal lubricant should be able to restore the affected component of tear film regardless etiology, with less frequency of instillation, and above all non adverse effects on the ocular surface –16. A maXanthan gum is a polymer with mucoadhesive properties that creates synergic interactions with mucin molecules playing an important role in the formation of the mucus layer of the tear film . In addiIn other hand, PEG/PG works by binding to the hydrophobic exposed areas of the epithelial cells, attaching a protective HP-Guar tear-gel matrix that helps restore the ocular surface. In a review, PEG/PG has showed in several clinical trials its efficacy and safety studies compared with different lubricants.Clinical symptoms, TBUT, Schirmer test, and OSDI score are the most sensitive and specific tests performed to evaluate the severity of DES , 21. ChaThese results could be due to mucoadhesivity properties of xanthan gum, which increased the corneal residence time of the tear film. On the other hand, CS has moisturizing properties delaying evaporation of aqueous layer due to its effect like-coat on the ocular surface –24.In addition to rheological properties above described, both, XG and CS act on different pathophysiological points involved in DED development. Xanthan gum is rich in OH-groups allowing it to react with reactive oxygen species and preventing oxidative stress damage implicated as a possible pathogenic cause of DED . MeanwhiOne limitation of this study is the lack of measurement of some inflammatory and oxidative markers described in the pathogenesis of DED , 27.Xanthan gum/chondroitin sulfate preservative free showed similar clinical efficacy, evaluated with OSDI score, TBUT and Schirmer test compared to polyethylene glycol/propylene glycol in the treatment of dry eye disease.


Results are in accordance with transrectal ultrasound prostatic biopsy (TRUSPB) results, with an area under curve (AUC) value of 0.83±0.03. Therefore urinary Spm shows potential to serve as a novel PCa diagnostic biomarker, which in turn can help to address the limited sensitivity and specificity problem of serum PSA test.Current screening methods towards prostate cancer (PCa) are not without limitations. Research work has been on-going to assess if there are other better tests suitable for primary or secondary screening of PCa to supplement the serum prostate specific antigen (PSA) test, which fails to work accurately in a grey zone of 4-10ng/ml. In this pilot study, the potential roles of urinary polyamines as prostate cancer biomarkers were evaluated. PCa, benign prostatic hyperplasia (BPH) patients and healthy controls (HC) showing PSA>4.0ng/ml were enrolled in the study. Their urine samples were obtained, and the urinary levels of putrescine (Put), spermidine (Spd) and spermine (Spm) were determined by ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-MS/MS). Receiver operating characteristics (ROC) curve and Student’s t-test were used to evaluate their diagnostic accuracies. Among the three biogenic polyamines, Spm had demonstrated a good diagnostic performance when comparing their levels in PCa patients with BPH patients (1.47 in PCa vs 5.87 in BPH;  Given the latency of early, treatable PCa and the lethality in its late and discernible stage, there is an urgent demand for more sensitive and accurate diagnostic methods to detect early PCa so that the treatment outcome can be significantly improved and more lives being saved.PCa is one of the most common non-skin male-related cancers in the world, and it is one of the leading causes of mortality and momentous public health impact in many developed countries, like most western European nations and the United States –2. No exCurrent diagnosis of PCa relied mainly on clinical suspiciousness raised by digital rectal examination (DRE) and serum PSA test, followed by TRUSPB confirmation. Although DRE is a simple procedure, it brings discomfort to patients. It is also a strongly-investigator-dependent technique, showing poor accuracy for PCa diagnosis.  In partiUrine-based assays present an important area for biomarker research. Urine collection is non-invasive and readily available; more importantly, it affords detection samples for either malignancy-related metabolites excreted in urine, directly exfoliated cancer cells or prostatic products secreted into the genitourinary tract.  This is One example of such cancer biomarkers are natural polyamines. Interests on these analytes have been starting in 1971 when Russell reported a considerable increase of urinary polyamines such as Put, Spd and Spm in patients with various types of solid tumors and leukaemias.  AfterwarIn this pilot study, we attempted to evaluate the potential of three main urinary polyamines  as biomarkers for PCa detection by comparing the cases between diagnosed PCa, BPH patients and HC. Through a well validated chromatographic method, urinary Spm had been shown to possess usefulness in differentiating PCa from non-cancerous cases including BPH, and it could help to act as a secondary screening tool to serum PSA test to address its high false-positive rate when using 4.0ng/ml as a cut-off point.This study was reviewed and approved by the Clinical Research Ethical Committee of the Chinese University of Hong Kong, and it was performed strictly according to the guidelines developed by that committee. Written consent was acquired from all of the patients. Urine samples were obtained at noon time after lunch prior to prostatic biopsy from 165 male patients (age > 50) having serum PSA level greater than 4.0ng/ml between Oct-2014 and Mar-2016. These patients’ urine samples were accepted only when they didn’t have clinically active urinary tract infection which might pose a biasing effect. When patients did not consent to take part in the study, or they showed clinical evidence of other types of cancers, they were excluded from our sampling scheme.Amongst these 165 patients, there were three subsets for the clinical samples enrolled in the current study. 66 were diagnosed to have PCa and the remaining 99 were evaluated as having no evidence of malignancy (NEM) by using TRUSPB as the reference standard. To further categorize these 99 NEM patients, using the criteria of prostate volume >30ml, 88 were found to have BPH while others were considered as HC. All pathological examinations were conducted at Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong under supervision of experienced uro-pathologists. 8) dihydrochloride (98 atom % D), spermidine–(butane-d8) trihydrochloride , spermine–(butane-d8) tetrahydrochloride  and heptafluorobutyric acid  were purchased from Sigma-Aldrich  and used without further purification. Strong Anion Exchange solid phase extraction (SPE) cartridges were obtained from Phenomenex . Centrifugation was performed using a Refrigerated centrifuge obtained from Eppendorf .Methanol was obtained from TEDIA . Acetonitrile was obtained from ACS . Water was purified in a MilliQ Direct Water Purification System . All standard compounds, including 1,4-Diaminobutane , spermidine , spermine , 1,4-Diamino. Briefly, urine samples and standards were thawed, deproteinized and centrifuged. The supernatant was separated and reacted with picric acid in alkaline solution to produce tangerine condensate through Jaffe reaction. [The creatinine concentration inside urine samples were determined by LabAssayeaction.  Quantita8, Spm-d8, Spd-d8) were prepared in water individually. The three stock solutions were mixed and diluted to give an IS working solution (1μg/ml) in water.Stock solutions (5000μg/ml) of each polyamine  were prepared in water separately. The three stock solutions were mixed and diluted to give an intermediate standard (50μg/ml), which was then used to prepare a series of working standards with polyamine concentrations of 10, 25, 50, 100, 250, 500, 1000ng/ml in water. For internal standards, the stock solutions (5000μg/ml) of each polyamine  working solutions were analyzed to verify the accuracy of calibration curves and ensure comparability among batches. The solutions were prepared using analyzed control urine samples from our research group. The polyamines concentrations of controls’ urine samples were determined and then mixed equally to give a pooled urine sample. Afterwards, three QC working solutions with different polyamine concentration ranges  were prepared by mixing this pooled urine sample with standard solutions. For low QC working solution, the SPE-treated pooled urine sample was mixed with SPE-treated 10ng/ml standard in a 1:7 ratio. For medium QC working solution, the SPE-treated pooled urine samples were mixed with SPE-treated 100 ng/ml standard in a 1:1 ratio. For high QC working solution, the SPE-treated pooled urine sample was mixed with SPE-treated 1000ng/ml standard in a 1:1 ratio.et al. had previously demonstrated that both the standard mixtures and QC samples were stable after storing at six hours at room temperature (short-term stability), after storage at -20°C and -80°C for two months (long-term stability) and after going three cycles of freezing and thawing before sample preparation (freeze thaw stability). [For stability studies, Häkkinen bility).  For furtThe quantitation of polyamines was performed by Ultra-high Performance Liquid Chromatography coupled with a triple quadrupole mass spectrometer (UPLC-MS/MS). LC separation was done by an Agilent 1290 Infinity Quaternary LC System while mass analysis was done by an Agilent 6460 Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream technology electrospray ionization source. The column used was an Agilent EclipsePlus C18 RRHD  protected with an Agilent SB-C18 guard column .The LC elution profiles were optimized as follows: Eluent A was water with 0.1% HFBA while eluent B was acetonitrile with 0.1% HFBA. Eluent A was decreased from 95% to 60% in 10 minutes, and from 60% to 10% in 1 minute. Afterwards the gradient was held constant for 5 minutes. The gradient was then increased from 10% to 95% in 1 minute, and held constant for 8 additional minutes. The autosampler and column temperatures were set at 4 and 35°C respectively. Injection was achieved by 5-second needle wash in Flush Port mode for 3 times with eluent B. 10 μL was injected each time.For the source parameter, drying gas (nitrogen) temperature was set as 300°C with 5 L/min flow rate. Nebulizer pressure was 45 psi. Sheath gas temperature was set as 250°C with 11 L/min flow rate. Capillary voltage was set as 3500V. For mass detection, scheduled multiple reaction monitoring (MRM) was performed. The information of MRM transitions can be found in The result was calculated using Agilent MassHunter Workstation software. Calibration curves were fitted linearly without any weighing. The correlation coefficients should not be smaller than 0.995. Acceptance values for each calibration points and quality control working solutions were ±30% to ensure accuracy. For precision verification, after every 10-sample injection, a 250ng/ml standard was injected and checked if it can be reproduced (±15%).p value smaller than 0.05 (two-tailed) was considered as statistically significant during comparison based on Student’s t-test.For statistical analysis, the ROC curve and the AUC were obtained by using GraphPad Prism 6 . A 2 not less than 0.995 Among the three polyamines monitored, normalized Spm showed a significant decrease in PCa patients compared to non-cancerous cases including BPH patients and HC in terms of statistical means (Unpaired student’s t-test). In detail, the mean value was 1.47 in PCa vs 5.87 in BPH vs 5.43 in HC. Correlations between urinary Spm had also been performed with patients’ clinicopathologic characteristics, like age, serum PSA, creatinine content, and prostate volume. However, all of them showed weak correlation with correlation coefficients < 0.1. (Data not shown)et al. reported an increase in N1, N12–Diacetylspermine in patients with early and late stage colorectal and breast cancers and established its role to be a novel marker for these cancers. [et al. had shown a significant elevation in polyamines level in Put, Spd and Spm. [et al. had monitored the level differences between polyamines, polyamine precursors and catabolites in both patients’ plasma and urines. [The relationship between polyamines and cancer has long been investigated by scientists. It is generally believed that increase of polyamine levels in blood or urine reflect the enhanced levels of polyamine synthesis in rapid-growing cancer tissues/cells, since they are associated with increased cell proliferation, decreased apoptosis and increased expression of genes affecting tumor invasion and metastasis. , 26, 27 cancers.  In casesand Spm.  For hepa urines.  By analyet al. had reported a significant elevation of urinary Spd content in PCa patients by electrophoresis, but not Put and Spm. [et al. analyzed urinary Spd and Put contents in patients with tumors in either breast, stomach, prostate, female genital tract, or metastatic carcinomas of unknown origins by LC with fluorometric detector in 1984 yielding an indeterminant conclusion. [Nevertheless, very few reports focused on detecting the effects of PCa on urinary polyamines levels, which in turn might provide a potential diagnostic tool for this increasingly common cancer. In 1975 Fair and Spm.  Horn et clusion.  With theet al. reported a reduced Spm content in tumor prostatic tissues compared to normal and benign hyperplastic prostatic tissues by high performance liquid chromatography with fluorometric detector. [et al. also reported a decreased Spm level in prostate tissue samples by Proton high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and quantitative histopathology. [et al. [et al. reported that in human expressed prostatic secretions, citrate, myo-inositol and Spm are potentially important markers of PCa, and all of them showed a decreased level in PCa patients compared to control samples. The AUC value reported in their study for Spm is 0.87 in EPS, which is close to the value discovered in this research, despite we only investigated patients with serum PSA value over 4.0 ng/ml. [The observation of such a declined level in urinary Spm actually was reasonable from results of previous literature about PCa studies. Although only a limited number of tissue specimens had been examined, van der Graaf etector.  Swanson thology.  High gra [et al.  Apart fr0 ng/ml.  With res0 ng/ml.  In essenet al. suggested a possible explanation that changes of cell organization caused by cancer cell proliferation finally result in a decreased luminal volume, which in turn reduces the amount of secreted compounds in prostate tissue, prostatic fluid or even urine. [et al. reported that Spm was a proposed endogeneous inhibitor to prostate cancer growth, and a linear correlation was found between Spm content and the volume percentage of normal prostatic epithelial cells as quantified by histopathology. [N1-acetyltransferase (SSAT) expression were observed in both precursor prostatic inflammatory atrophy lesions and early prostatic intraepithelial neoplastic lesions, which resulted in a depletion of Spm content.  Instead, it acts like a diagnostic biomarker working in accordance with TRUSPB for PCa diagnosis.Nevertheless, unlike what GF Giskeødegård siveness , we coulet al. reported an even poorer sensitivity and specificity for it in their study . [et al. showed that total PSA only gave AUC value of 0.52±0.07. [Compare to the currently adopted serum PSA test, it is generally accepted that it leaves much to be desired as a primary screening test. It had been shown to cause over-diagnosis especially to patients showing value in the grey zone.  For exam 0.684).  Another 52±0.07.  Our dataThe current research is not without limitations. Firstly, all patients sample are obtained from one hospital in Hong Kong, while collecting samples from multi-center and multi-country may strengthen the outcome of our study. Secondly, diet control had not been performed to completely exclude the effect from food habit, despite Vargas et al. reported before no significant association was found between dietary polyamine intake and urinary polyamines measured.  Finally,To conclude on the basis of this pilot study, the potential roles of the three main urinary polyamines as PCa biomarkers were evaluated. Among Put, Spd and Spm, Spm demonstrated an outstanding diagnostic performance for PCa, in particular for patients with elevated serum PSA level, upon comparison of their levels in PCa and BPH patients. Its AUC value is 0.83±0.03. This work takes a step towards tackling the current medical challenge of poor specificity of the serum PSA test.S1 Fig2 = 0.9996) (B) Spd (r2 = 0.9993) (C) Spm (r2 = 0.9995).(A) Put (r(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(* Denoted the quantifier transitions)(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


Our data showed significantly higher serum and sputum PlGF levels in asthma patients, especially asthmatic smokers, than in healthy controls. Serum and sputum PlGF levels correlated negatively with post-bronchodilator forced expiratory volume in 1 s (FEV1) and the FEV1/forced vital capacity, but positively with airway-wall thickening. Stimulation of HLMECs with rhPlGF promoted all of the steps of airway-microvascular remodelling. These findings provide insights into the influence of cigarette smoking on the structural changes in the airways of asthmatics and the important pathogenic role played by PlGF.The increased expression of placental growth factor (PlGF) in chronic obstructive pulmonary disease and allergy-related asthma suggests its role in the pathogenesis of these diseases. In asthmatic smokers, airway remodelling is accompanied by an accelerated decline in lung function. However, whether PlGF contributes to the persistent airflow obstruction and vascular remodelling typically seen in asthmatic smokers is unknown. In this study we measured lung function, airway-wall thickening, and PlGF levels in serum and induced sputum in 74 asthmatic and 42 healthy smokers and never-smokers. Using human lung microvascular endothelial cells (HLMECs), we evaluated the  In China, the prevalence of smoking in the asthmatic population is as high as 34.51%25The airways of smokers with asthma are characterized by an increase in the number and size of vascular structures. These alterations are mediated by angiogenic factors such as vascular endothelial growth factor (VEGF), osteopontin, and angiopoietin-1 and angiopoietin-2, all of which contribute to airway remodelling and airflow obstruction56712PlGF is a member of the VEGF family, which includes VEGF-A, B, C, D, and E18in vitro study, we evaluated the effects of recombinant human PlGF (rhPlGF) on the proliferation, migration, stress-fibre expression, and tubule-formation of primary HLMECs.The work described herein was a part of a study on biomarkers of COPD and asthma2122The 74 asthmatics (30 smokers and 44 never-smokers) and 42 healthy individuals (20 smokers and 22 never-smokers) who participated in the study were matched for age, sex, and smoking history. Their demographic characteristics are summarized in Serum PlGF levels were significantly higher in patients with asthma than in the healthy controls. Smoking significantly increased the serum PlGF levels in smokers with asthma  vs. never-smokers with asthma [15.8 (9.6–19.9)] and in healthy smokers [16.3 (10.3–24.8)] vs. healthy never-smokers [10.8 (6.3–14.9)] . SimilarDLCO  and a higher FeNO50 . As expected, healthy never-smokers had better lung function parameters than healthy smokers. Among smoking asthmatics, serum and induced sputum PlGF levels were negatively associated with post-bronchodilator FEV1% pred  ] ] . None of−0.138)] .p = 0.016)] and the airway lumen area was reduced , but with no significant differences in RB10% wall area  vs. 1.52 mm (1.21–1.70 mm) ], which indicates that the associated production of PlGF contributes to the compromised lung diffusion characteristic of these patients. However, the exact mechanism remains to be elucidated.A significant negative association between PlGF levels and post-bronchodilator FEVHigh-resolution CT has been used to quantify airway abnormalities arising from airway remodelling, and a correlation between the HRCT score and the severity of asthma as well as airflow obstruction has been reportedex-vivo cell studies can only suggest a potential mechanism and further studies are needed to corroborate the findings and extend them.Airway-wall neovascularization, manifested as increases in the number and size of bronchial blood vessels and angiogenic sprouts, is a prominent feature seen in asthma patients who are smokers and it correlates with airway-wall thickness, airway obstruction, and the degree of airway hyper-responsiveness1226There were several limitations to this study, including the absence of bronchial biopsies to confirm the serum, sputum, and CT findings. The cross-sectional design of the study design precluded the collection of data on longitudinal changes in PlGF levels in serum and sputum. Patients with asthma were not further assigned to sub-groups, as it would have reduced the statistical validity of the study. Also, HLMECs used in our experiments were not further purified to obtain bronchial arterial endothelial cells. Nonetheless, our results provide useful pilot data for larger studies of the effects of PlGF on asthmatic smokers.In summary, our study revealed increases in the PlGF levels of the serum and induced sputum of asthmatic patients who are cigarette smokers. In this group, PlGF levels correlated positively with segmental airway-wall thickening and inversely with lung function. Our findings suggest that, in asthmatic smokers, PlGF contributes airway remodelling might through promoting angiogenesis. As such, they provide new insights into the pathophysiologic role of PlGF.1 of >12% and 200 ml after salbutamol administration or hyper-responsiveness to inhaled methacholineThe 74 asthmatic patients (current smokers and never-smokers) were recruited between October 2014 and February 2016 from respiratory clinics in the Affiliated Hospital of Guangdong Medical University, China. As controls, 42 healthy volunteers  were recruited from the surrounding communities. All participants were on stable medication. Patients with a respiratory tract infection or an exacerbation of their asthma in the past 8 weeks were excluded. Smokers were defined as individuals with a smoking habit of at least 10 pack-years who currently smoke five or more cigarettes per day. Asthma was diagnosed according to the Global Initiative for Asthma (GINA) guidelines, based on a patient history of recurrent episodes of wheezing and chest tightness, with or without cough, impaired spirometry with reversibility, and a FEVThis open cohort cross-sectional study of patients with asthma and healthy controls (smokers or never-smokers) was performed as part of a study on biomarkers of chronic inflammatory airway disease2122Sputum was induced as previously describedSerum samples collected from patients with asthma and from healthy controls were kept at −70 °C. PlGF levels in serum and induced sputum were assessed using ELISA kits for human PlGF  according to the manufacturer’s protocol.50) in concordance with standardized guidelinesDLCO) were determined according to ATS guidelines Spirometry was measured using standard spirometric techniques  at least 6 h after the patient’s most recent treatment with albuterol, according to the guidelines of the American Thoracic Society (ATS)2). The measurements were performed once. Interobserver reproducibility was good (k = 0.7).CT of the chest was performed at full inspiration using a spiral CT scanner with 16-slice Brightspeed and 64-slice Lightspeed  imaging and the following parameters: 125 kV, 100 mA·s, matrix size of 512 × 512, and a slice thickness of 1 mm. The window level was −600 Hounsfield units (HU), with a width of 1600 HU, as generally recommended for the evaluation of the bronchi and lung parenchyma2 incubator. The culture medium was replaced every 2 days until the cells were confluent. All experiments were carried out using HLMECs between passages 5 and 8.Primary human HLMECs were isolated from lung tissue obtained from donors undergoing resection for localized lung carcinoma, as described previously4/well and treated for 48 h with different doses of recombinant human PlGF . A CCK-8 assay  to determine cell proliferation was performed according to the manufacturer’s instructions. HLMEC migration was assessed in a ‘scratch-wound’ assayThe cell proliferation assay was carried out in a 96-well plate using cells seeded at a density of 1 × 104. The cells were cultured overnight and then stimulated for 1 h with different concentrations of rhPlGF . They were then washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized for 5 min with 0.5% Triton X-100 . Staining with TRITC-phalloidin (100 nM) for 30 min in the dark was followed by counterstaining for DNA with 4′, 6-diamidino-2-phenylindole . F-actin was detected with a Leica-TCS-SP confocal microscope and the accompanying software (Leica v2.6.1).The structure of fibrillary actin (F-actin) was determined by TRITC-phalloidin  staining using HLMECs seeded on LabTek-II chamber slides  at a density of 5 × 10The formation of vascular-like structures by HLMECs on growth-factor-reduced Matrigel (BD Biosciences) was induced as previously described2 tests. Categorical variables were summarized as the frequencies and percentages per category and compared using Fisher’s exact test or Pearson’s χ2 test. Associations between serum, induced sputum PlGF levels, and sputum cells were examined in a multivariate linear regression analysis in which either serum or the induced sputum PlGF level was the dependent variable. Linear regression analysis was performed with the covariates smoking, age, sex, body surface area, disease duration, and treatment regimen. A similar regression analysis was performed in two subgroups identified on the basis of smoking habit. The data were interpreted as standardized coefficients with 95% confidence intervals. Spearman’s or Pearson’s correlations were performed, according to data normality, between serum or induced sputum PlGF levels and lung function or chest CT parameters. SPSS 16.0  and GraphPad Prism 5.0 software  was used for the analyses and graphs. Statistical significance was set at p < 0.05.Normally distributed data are presented as the mean ± standard deviation (SD), and skewed data as the median (interquartile ranges). The Kolmogorov–Smirnov test was performed to examine the normality of the distribution. Statistical comparisons between groups were performed using a one-way analysis of variance (ANOVA) for normally distributed data and a Kruskal–Wallis test for skewed data, with the appropriate post-hoc tests performed for multiple comparisons . Differences in numerical variables between two groups were evaluated using unpaired t-tests or Mann–Whitney U-tests for normal and skewed data, respectively. Proportions were compared using χHow to cite this article: Wu, D. et al. Elevated expression of placental growth factor is associated with airway-wall vascular remodelling and thickening in smokers with asthma. Sci. Rep.7, 43017; doi: 10.1038/srep43017 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Objectives. To determine the relationship between fecal calprotectin (FCAL) and imaging studies and other biochemical inflammatory markers and the impact of FCAL measurements on decision-making in IBD patient management in usual clinical practice. Methods. 240 persons with IBD were enrolled. The correlation between FCAL values and other markers for disease activity such as serum albumin , hemoglobin (Hg), and C-reactive protein (CRP) and diagnostic imaging or colonoscopy was examined. FCAL ≥ 250 mcg/g of stool was considered a positive result indicating active IBD. Results. 183 stool samples (76.3%) were returned. The return rate in the pediatric and adult cohorts was 91% (n = 82) and 67.3% (n = 101), respectively (P < 0.0001). Positive FCAL was associated with colonoscopy findings of active IBD (P < 0.05), low albumin (P < 0.05), anemia (P < 0.01), and elevated CRP (P < 0.01). There was no significant difference for FCAL results by outcomes on small bowel evaluation among the 21 persons with small bowel CD. Most persons (87.5%) with normal FCAL and no change in therapy remained in remission during subsequent 3 months. Conclusions. FCAL is a useful marker of disease activity and a valuable tool in managing persons with IBD in clinical practice. Clinicians have to be cautious in interpreting FCAL results in small bowel CD. Inflammatory bowel disease (IBD) is a group of closely related diseases of immune dysregulation including ulcerative colitis (UC), Crohn's disease (CD), and IBD type unclassified (IBD-U). IBD is a chronic disease with remissions and relapses that requires long-term monitoring and runs a variable course. Poor control of ongoing inflammation has been associated with poor outcomes . In clinCalprotectin is a calcium and zinc binding protein found in the cytosol of human neutrophils and macrophages. It is released extracellularly in times of cell stress or damage and can be detected within feces and thus can be used as a sensitive marker of intestinal inflammation. Fecal calprotectin (FCAL) has emerged as a potential noninvasive, inexpensive, and more accurate tool than serological measures in monitoring IBD disease activity and diagnosing a flare. It has also been found to be useful in differentiating irritable bowel syndrome (IBS) from IBD , 5. AlthIn this single-centre cross-sectional study, 240 persons with IBD seen in the Gastroenterology Outpatient Clinics of three university hospital-based gastroenterologists (two adult and one pediatric) for either routine follow-up or new symptoms, between August 2013 and February 2016, were asked to bring in a stool sample for FCAL measurements. The samples were collected at home and processed at the laboratory of the University of Manitoba IBD Clinical and Research Centre. Persons with IBD aged four or above and with confirmed diagnoses of IBD by endoscopy and/or radiography were included. FCAL was done using the Quantum Blue® Lateral Flow Reader and within 24 hours of stool collection. FCAL ≥ 250 mcg/g was considered a positive test indicative of active IBD.Participating clinicians were given a questionnaire to collect patients' demographics, diagnoses, physician global assessment (PGA), and the impact of FCAL testing on the management of patients in terms of plans for investigation(s) or changes in therapy. The clinicians determined at the time of ordering FCAL whether they would undertake other investigations, but they also could determine to pursue other investigations once the FCAL result became available. Once the FCAL result was available the clinicians recorded whether and how it changed their management. The ordering of diagnostic imaging and blood work was left to the discretion of the clinicians. Clinicians had the FCAL test results within one week.Medical records of all included persons were reviewed and data were collected on serum levels of C-reactive protein (CRP), albumin, and haemoglobin (Hg) done within two weeks of collecting stool samples. In our local laboratory an abnormal CRP was equal to or greater than 8 mg/L. A low level of serum albumin was equal to or less than 33 g/L. We used World Health Organization (WHO) definition of anemia, which included the following values: male age ≥ 15 yr Hg < 140 g/L, female age ≥ 15 yr Hg < 120 g/L, children 12–14 yr < 120 g/L, and children 4–11 yr Hb < 115 g/L. Abdominal diagnostic imaging, colonoscopies, and flexible sigmoidoscopies were performed within one month of collecting stool samples. This time frame was chosen because MRE, CTE, or endoscopy can be generally arranged within one month in our centre. We reviewed reports of all abdominal diagnostic imaging (computed tomographic enterography (CTE) and magnetic resonance enterography (MRE)) and used the radiologic report from the gastrointestinal radiologist to determine disease activity. The definition of active disease on CTE included findings such as stricturing with mucosal hyperenhancement; bowel wall thickening; or thumb printing and findings on MRE included abnormal enhancement, wall thickening, or ulcerations. The presence of erythema, loss of vascularity, friability, or ulcerations were included in the definition of active disease on endoscopy.In order to determine the value of normal FCAL in real-world practice, patients who had normal FCAL measurements and no investigation or change in therapy were followed up prospectively for 3 months to assess relapse rate which was defined by hospitalization, change in therapy, or endoscopy or imaging showing active disease.P value <0.05 was considered significant. Odds ratios (ORs) and 95% confidence interval (CI) of the multiple variables of interest in relation to FCAL measurement results were calculated. Kruskal-Wallis test was used to compare medians of ages of returners of stool samples versus nonreturners.Nominal variable comparisons were performed using Fisher's Exact test. A The study protocol was approved by the University of Manitoba Health Research Ethics Committee.n = 150 and children n = 90)  compared to the adult cohort . The adult subjects (age ≥ 18) who did not return stool samples were younger than those who returned the stool samples ; however a similar proportion of sample returnees and non returnees were female (63.2% versus 64.4%)  . The oves 64.4%) . Ninety-P = 0.31).Twenty-three subjects underwent CTE or MRE. Of 9 persons with active disease on CTE or MRI, only 6 had a positive FCAL. Of 11 subjects with a positive FCAL who underwent imaging, only 6 had active disease on imaging. A positive FCAL was not significantly associated with radiologic evidence of active disease , 8.79; 95% CI 1.54–65.55). Hence positive FCAL had 80.9% sensitivity and 69.2% specificity in comparison to the findings of active disease on endoscopy.Clinicians chose to undertake colonoscopy or flexible sigmoidoscopy in 33, either based on their assessment or because of elevated FCAL. Twenty-one (67%) with evidence of active disease on endoscopy had positive FCAL . 147 subjects had serum Hg measured. Of 56 persons with anemia 40 had positive FCAL . 132 had serum CRP measured. Of 36 with high CRP, 28 had positive FCAL . Of persons with positive FCAL (n = 99), 78 were clinically diagnosed to have active disease including mild (n = 52), moderate (n = 23), and severely active disease (n = 3) as assessed by the participating clinicians using the PGA scale before the FCAL testing was performed   . The mean = 10) or endoscopy , there was no significant difference in outcome by FCAL results (P = 0.73 for imaging and P = 0.39 for endoscopy).Of the twenty-one persons with small bowel CD and either diagnostic imaging (n = 86) 87% (n = 84), clinicians made no change in therapy or further investigations in (n = 74) 88% (For the 99 persons with a positive FCAL test clinicians made a change in therapy or investigations in ( 86) 87% . On the  74) 88% .Of the 74 persons who had normal FCAL (<250 mcg/g) and had no change in therapy or investigations, 8 (12.5%) had a flare within 3 months of the date FCAL test. Three of them required hospitalization. Five of these 8 persons had FCAL test results between 150 and 249 mcg/g. Six additional persons had FCAL results between 150 and 249 mcg/g and they did not have a flare of symptoms within 3 months. Hence, a total of eleven persons had a FCAL result of 150–249 mcg/g and five of eleven had a flare within 3 months.FCAL is a surrogate marker of intestinal mucosal inflammation. Several studies and meta-analyses have shown that FCAL is useful for discriminating IBD from other diseases , 10 and Only 12.5% of persons who had no change in therapy or investigations based on negative FCAL results had a flare within 3 months. Allowing for the lack of controls and short follow-up time, this may indicate that the FCAL test can be used reliably in clinical practice. Given the noninvasive nature, quick turnaround of FCAL test, and good correlation with endoscopy in colonic inflammatory bowel diseases as we have shown in this study, FCAL can help in monitoring disease activity and may be used as a tool along with other serologic and clinical measures to guide further investigation and therapy. While adherence rates in children were excellent they could be improved in adults.We used a cut-off of less than 250 mcg/g as normal for an IBD population. In a meta-analysis including 13 studies (744 patients with UC and 727 with CD) using a cut-off value of 250 mcg/g the areas under the curve values for diagnostic accuracy of disease activity were 0.89 , 0.93 (0.89–0.97), and 0.88 (0.83–0.93) in IBD, UC, and CD groups, respectively. The pooled sensitivity was 0.80 (0.76–0.84) and specificity was 0.82 (0.77–0.86). It was concluded that FCAL was a reliable marker for assessing IBD disease activity and may have greater ability to evaluate disease activity in UC than CD . While wn = 30). The bowel wall T2 hyperintensity  and total length of disease  were found as the best independent predictors of active CD. CDAI, C-reactive protein, and FCAL were not found to be independent predictors of active CD [P = 0.01), with a modest overall correlation . FCAL reflected MRE inflammatory activity with an area under the receiver operating characteristic curve of 0.91 . A cut-off value of 166.5 mg/g had 90% sensitivity, 74% specificity, 89% positive predictive value, and 76% negative predictive value for diagnosis of inflammation [r = 0.46, P < 0.001) and , respectively [n = 21), our study did not show a signification association between FCAL levels and disease activity on MRE/CTE. To our knowledge no systematic review or meta-analysis has addressed the question of small bowel CD activity and FCAL levels. Clinicians have to be cautious in interpreting FCAL results in this subtype of CD patients.Data on assessing disease activity in CD comparing FCAL with MRE or CRE results are conflicting. Quaia et al. studied 91 patients with CD who underwent MRE. They compared MRE findings to the Crohn's Disease Endoscopic Index of Severity (CDEIS) and with histologic analysis of those who underwent elective small bowel resection (ctive CD . Cerrillammation . Elsewheectively . AllowinThe limitations to our study were that the same testing was not undertaken in all subjects to fully determine the correlations between the FCAL and various outcomes, especially cross-sectional imaging. However, at study outset, we were particularly interested in determining how clinicians would use FCAL results. A positive FCAL often triggered a clinical response, either more investigations or a change in therapy. Unfortunately, not having done imaging in all subjects, we cannot discern how many false negative FCAL tests are represented within our sample. Nonetheless, our results do show how much faith clinicians have placed in FCAL based on the medical literature since all participating clinicians were utilizing FCAL for the first time in their practices.Our study represents a snapshot of how FCAL testing is used in monitoring disease activity in IBD in clinical practice outside the context of clinical trials. We found that in a referral population of persons with IBD, positive FCAL was significantly associated with abnormal endoscopy, elevated serum CRP, low serum Hg, and low serum albumin. While it was a small cohort that had imaging, FCAL results were not significantly associated with cross-sectional imaging evidence of disease activity, likely reflecting a weaker association with small bowel disease. Our study confirms that FCAL can be used as a surrogate marker for disease activity in IBD. However, clinicians are placing considerable faith in the test even though its role in small bowel CD requires further research. Finally, optimizing adherence to stool testing in adults is needed. If persons with IBD do not actually have the test done because they will not submit a stool sample, FCAL will not function as a useful predictor of disease activity.


Moreover, the combined predictive value of ηc and patient age provided for sensitivity and specificity of 100% and 95.5%, respectively. This simple and rapid diagnostic tool can be immediately implemented after EUS-FNA sampling.Differentiation between mucinous and non-mucinous pancreatic cysts is exceedingly important and challenging, particularly as the former bears malignant transformation potential. Pancreatic cyst fluid (PCF)-based diagnostics, including analyses of biochemical markers, as well as cytology, has shown inadequate accuracy. Herein, a preliminary single-center study of 22 PCF samples, collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), assessed the rheological behavior of PCF and its correlation with lesion type. The dependence of PCF shear viscosity on shear rate was found to follow a power law and could be fitted using Ostwald–de Waele model. Three types of flow curves were identified, where two types correlated with non-mucinous cysts, differing by their power law exponent, and the third type corresponding to mucinous cysts. Viscosity measured at a high shear rate was shown to serve as an accurate and independent marker distinguishing between mucinous and non-mucinous cysts, with an optimal cutoff value of  Pancreatic cancer is the fourth leading cause of cancer deaths among men and women, accounting for 6% of all cancer-related deaths, with a collective median survival time of 4–6 months. Pancreatic cancer is difficult to diagnose in its early stages; at the time of diagnosis, 52% of patients have distant metastases and 26% present regional spread23Imaging plays a vital role in both detection and characterization of pancreatic cyst lesions. An example of representative CT, MRI, and endoscopic ultrasound (EUS) images of pancreatic body cysts, are presented in 5PCF analysis for cytology and markers has been widely used for the differential diagnosis of pancreatic cysts891011131421A total of 22 patients   , resulte and η∞) . More defindings , we can findings  demonstrηc values were significantly different between type I and II lesions versus type III lesions (p < 0.0001).For the purpose of comparing between predictive factors in their diagnostic capacity to differentiate between mucinous and non-mucinous PCFs, the statistical significance of mean values measured for flow curve types I + II versus III are presented in ηc, established that the optimal cutoff value for differentiating between MC (flow curve III) versus NMC (flow curve I and II) cysts was 1.3 cP  was greater than that of CEA (72.7%), amylase (50%), cytology (72.7%) and string-sign (59.1%). When considering cyst fluid viscosity, ηc jointly with patient age, the sensitivity and the accuracy increased to 100% and 95.5%, respectively, but the specificity remained 91.7%.Receiver operator curve analysis of the cyst fluid viscosity, s 1.3 cP , with ars 1.3 cP . The senη as a function of shear rate, n∼0, where the absolute values of the viscosity were close to viscosity of water at room temperature. This behavior is typical of low molecular weight fluids as well as for diluted polymer solutions or dispersions. Types II and III PCF samples demonstrated yield behavior n < 0, i.e., strong shear thinning at low shear rates. This kind of behavior is typically associated with the presence of a structure in the fluid , which is destroyed upon application of the shear field, resulting in a sharp drop in viscosity. The viscosity tends to level off, approaching a constant value  as the shear rate increases. The lowest constant viscosity reflects the properties of the fluids when its structure has been completely destroyed (disentangled macromolecules). Such behavior is expected of a multicomponent fluid, such as PCF, whose bulk is comprised of mucins, characterized by high molecular weight, heavily glycosylated proteinsWhen focusing on the change in viscosity, nguished . For typηc, measurements to that of the CEA and amylase. For instance, viscosity evaluations, as well as pathology, categorized PCF samples nos 5, 15 and 21 as MC, while CEA and amylase levels of samples nos 15 and 21 differed from sample no. 5 by four orders of magnitude  and were carried out in accordance with the approved guidelines. All patients signed an informed consent form prior to the EUS-FNA or blood sampling.All experimental protocols were approved by the Rambam Medical Center Ethics Committee  patients with a suspicious pancreatic cystic lesion, detected by cross-sectional imaging (CT and/or MRI scan) and consequentially referred for an EUS-FNA. Pancreatic cyst fluid samples were collected for cytological and biochemical analysis and patients were followed-up. In addition to routine cyst fluid analysis , rheological measurement were performed (see below).Pancreatic cysts were classified as mucinous  or non-mucinous , based on surgical and/or clinical findings .A curvilinear array echoendoscope  was used to insert a 22-gauge needle , under ultrasound guidance, to aspirate the cyst fluid. Cyst fluid characteristics were recorded. The levels of biochemical markers of cyst fluid CEA and amylase were measured using commercial solid-phase double- antibody .Specimens were cytopathologically analyzed for the presence of mucinous epithelium, extent of cytologic atypia and presence of malignant cells. A cellblock was prepared for further immunohistological staining, as described previouslyThe string-sign was determined at aspiration, by the endosonographer, as described previouslyPCF samples were stored at 4 °C, incubated at room temperature for one hour prior to measurements, and vortexed for 30 sec just before testing, to ensure homogenization.Use of a rotational viscometer supports simulation of true rheological conditions (the stepping change of either the shear stress or the shear rate is programmed but the parameter remains constant during each step). The viscosity of the PCF samples was measured with a DHR-2 Rheometer  at 25 °C. The preferred geometry was cone-and-plate, with a cone diameter of 40 mm and a surface-plate angle of 1°. The rheometer was operated in shear rate control mode. Several time sweep tests at different constant shear rates (5–2000 1/s) were performed. The measured steady-state shear viscosity values (when the viscosity was constant in time) were used to construct flow curves of the fluids. Measurements were repeated 1–4 times for each sample, depending on the amount of fluid aspirated. The flow curves (viscosity versus shear stress or shear rate), characterizing the viscous response of the fluid under the shear field over a wide range of conditions, were then graphed.Statistical analysis was performed using the nonparametric Mann-Whitney U-test, Pearson’s chi-squared test and receiver operating characteristic (ROC) curve. Cutoff points of age, CEA and viscosity were evaluated by ROC curve analysis and Youden’s statistics. Sensitivity, specificity and accuracy were calculated. A p value < 0.05 was considered significant. All analyses were performed using SPSS 21.0 .How to cite this article: Khamaysi, I. et al. Differentiation of Pancreatic Cyst Types by Analysis of Rheological Behavior of Pancreatic Cyst Fluid. Sci. Rep.7, 45589; doi: 10.1038/srep45589 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A trade-off between fertility and longevity possibly exists. The association of the male hypothalamic–pituitary–gonadal (HPG) axis with familial longevity has not yet been investigated.To study 24-h hormone concentration profiles of the HPG axis in men enriched for familial longevity and controls.We frequently sampled blood over 24 h in 10 healthy middle-aged male offspring of nonagenarian participants from the Leiden Longevity Study together with 10 male age-matched controls. Individual 24-h luteinizing hormone (LH) and testosterone concentration profiles were analyzed by deconvolution analyses to estimate secretion parameters. Furthermore, the temporal relationship between LH and testosterone was assessed by cross-correlation analysis. We used (cross-)approximate entropy to quantify the strength of feedback and/or feedforward control of LH and testosterone secretion.p = 0.51) from the total LH secretion of controls [186 (130–242) U/L/24 h]. Likewise, mean (95% CI) total testosterone secretion of the offspring [806 (671–941) nmol/L/24 h] and controls [811 (676–947) nmol/L/24 h] were similar (p = 0.95). Other parameters of LH and testosterone secretion were also not significantly different between offspring and controls. The temporal relationship between LH and testosterone and the strength of feedforward/feedback regulation within the HPG axis were similar between offspring of long-lived families and controls.Mean [95% confidence interval (CI)] total LH secretion of the offspring was 212 (156–268) U/L/24 h, which did not differ significantly (This relatively small study suggests that in healthy male middle-aged participants, familial longevity is not associated with major differences in the HPG axis. Selection on both fertility and health may in part explain the results. These latter comprise the hypothalamic–pituitary–thyroid (HPT) axis, the hypothalamic–pituitary–growth hormone (GH) axis, the hypothalamic–pituitary–adrenal (HPA) axis, the hypothalamic–pituitary–prolactin (PRL) axis, and the hypothalamic–pituitary–gonadal (HPG) axis. To maintain homeostasis, these centrally regulated hormonal axes respond to stressors, such as inflammation and starvation. As a response to systemic inflammatory disease, the HPA axis is activated and available resources are reallocated by suppressing the GH, HPG, and HPT axes among others  included offspring of long-lived families that are enriched for exceptional longevity and partners thereof, serving as a control group . To datevia telephone questioning, controls with a nonagenarian parent who had one or more nonagenarian siblings were also excluded. Participants were middle-aged (52–76 years) and had a stable body mass index (BMI) between 18 and 34 kg/m2. The Switchbox Leiden Study was approved by the Medical Ethical Committee of the Leiden University Medical Centre and was performed according to the Helsinki declaration. All participants gave written informed consent for participation.The LLS comprises 421 families with at least two long-lived Caucasian siblings fulfilling the age criteria (men ≥89 years and women ≥91 years) without selection on health or demographics, as described previously in more detail . In the ® 1500 Ltd., Isle of Man, British Isles) (Full details on the 24-h blood sampling procedure have been described previously . In shorh Isles) . Waist cAll hormonal assays were performed with fully automated equipment and diagnostics from Roche Diagnostics  at the Department of Clinical Chemistry and Laboratory Medicine of the Leiden University Medical Centre in The Netherlands, which is accredited according to the National Coordination Committee for Quality Assurance for Health Care Laboratories in The Netherlands. LH  and testosterone  were measured in EDTA plasma samples collected every 10 min using ECLIA (Electrochemoluminiscentie immunoassay) on a Roche Modular E170 immunoanalyser. The measuring range of LH is 0.100–200 U/L, and the interassay coefficients of variation (CV) were 4.47% at 4.10 U/L and 2.83% at 56.43 U/L. The measuring range of testosterone is 0.087–52.0 nmol/L, and the interassay CV were 4.12% at 1.77 nmol/L and 3.78% at 34.84 nmol/L. Testosterone is mostly bound to binding proteins including sex hormone-binding globulin (SHBG) and albumin. Albumin  and SHBG  were measured on Roche Modular analyzers in six EDTA plasma samples with 4-h intervals for each participant. Free testosterone was calculated based on albumin and SHBG levels as described by Vermeulen et al.  with catalog number L2KDS2 was measured using a solid-phase competitive chemiluminescent enzyme immunoassay with an Immulite 2000 XPi system from Siemens Healthcare diagnostics . Total 25-hydroxyvitamin D  was measured using ECLIA with an E170 module of Modular Analytics from Roche Diagnostics. Interleukin 6  and tumor necrosis factor-α (TNF-α)  were measured by ELISA from R&D Systems. High-sensitivity C reactive protein (hsCRP) with catalog number 04628918190 was determined by a particle-enhanced turbidimetric assay using Cobas Integra 800 from Roche Diagnostics.For each participant, 3 μL of all (144) samples taken during the 24-h blood sampling were pooled. In this mixture, the levels of testosterone , estradiol , and prolactin  were determined using ECLIA and E170 module of Modular Analytics from Roche Diagnostics.To determine underlying components of LH and testosterone secretion, 24-h LH and testosterone concentration profiles were analyzed by validated deconvolution analysis . By decom (window length) = 1 and r (criterion of similarity) = 20% of the SD of the individual subject time series were used (Jack-knifed approximate entropy (JkApEn) is a measure for the strength of feedforward and feedback control signals in a hormone system. First, approximate entropy (ApEn), which is a scale- and model-independent statistic that quantifies the regularity of consecutive time-series data, was calculated using the Matlab software program  . ApEn haere used . Subsequere used .Bivariate cross-ApEn is a scale- and model-independent regularity statistic, which quantifies the relative pattern synchrony of two time series . ChangesBesides ApEn, other proxies for the strength of the feedforward and feedback signaling in the HPG axis are the ratios of LH and testosterone concentrations. LH/testosterone ratio reflects the feedback of testosterone on LH secretion, with a higher ratio signifying lower feedback. Testosterone/LH ratio reflects the strength of the feedforward drive of LH on the testosterone secretion, with a higher ratio denoting higher feedforward .The temporal relationship between LH and testosterone concentrations was determined by cross-correlation. Cross-correlation assesses the relative strength between a hormone pair for all possible time shifts. For the offspring and control groups, the strongest correlation coefficient with corresponding lag time was compared.U-test and independent-samples T-test were used to assess differences between offspring and controls in, respectively, variables that were not normally distributed and normally distributed variables. LH and testosterone secretion parameters were compared between offspring and controls using linear regression adjusted for age. Variables were examined for normality. Variables that were not normally distributed were logarithmically transformed prior to analysis and are presented as geometric means with 95% confidence intervals (CIs), except for two variables, LH slow half-life and testosterone total secretion, because logarithmic transformation did not improve normality. p ≤ 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS for Windows, version 23 . Graphs were made using the GraphPad Prism version 6 .Descriptive statistics was used to summarize the characteristics of study groups. The non-parametric Mann–Whitney p = 0.02) than those of the controls. Offspring and controls were similar in age, body composition, available immunological markers, DHEAS, and 25-hydroxyvitamin D. Group characteristics of female offspring of long-lived families and controls are presented in Table S1 in Supplementary Material. Similar to men, female offspring and controls did not differ significantly in age, body composition, available immunological markers, DHEAS, and 25-hydroxyvitamin D, except for mean age of the parents.Group characteristics of male offspring of long-lived families and controls are presented in Table p = 0.51). Likewise, basal LH secretion, pulsatile LH secretion, and characteristics of pulsatile LH secretion were similar between the groups. Mean (95% CI) total testosterone secretion of the offspring was 806 (671–941) nmol/L/24 h, which was similar (p = 0.95) to the mean (95% CI) total testosterone secretion of controls [811 (676–947) nmol/L/24 h]. Other parameters of testosterone secretion were also not significantly different between the offspring and controls.To illustrate the wide variation between participants in LH and testosterone concentration profiles, Figures S1 and S2 in Supplementary Material present the individual 24-h LH and testosterone concentration profiles of all participants, respectively. Results of the deconvolution analyses on 24-h concentration profiles of plasma LH and testosterone are shown in Table Approximate entropy of LH and of testosterone, representing the strength of feedback and feedforward regulation, were similar between offspring and controls, as shown in Table r) of 0.16 for offspring and r = 0.29 for controls, which was not significant different (p = 0.21) when adjusted for age.The maximal correlation between LH and testosterone concentrations was found at a lag time between 60 and 80 min with a correlation coefficient (Because all women were postmenopausal, determination of sex hormones was limited to a single measurement in the 24-h pool. No differences were observed in testosterone, estradiol, and prolactin levels between offspring and controls, neither in men nor in women (Table In this study, we did not observe significant differences in 24-h secretion of HPG axis parameters and their regulation between male subjects enriched for familial longevity compared to controls. LH and testosterone secretion parameters, and the strength of feedforward and feedback regulation within the HPG axis, were similar between offspring of long-lived families and controls. Also, testosterone, estradiol, and prolactin levels measured in a 24-h pool were similar between the offspring and controls in men and in women.According to the disposable soma theory, which suggests a trade-off between fertility and longevity, longevity would be associated with reduced reproductive capacity, i.e., reduced HPG axis action . HoweverPossibly, this observation is related to the selection criteria of the study population. First, due to the recruitment of the study population based on the presence of at least two long-lived siblings, we may have selected on fertility, which is strongly associated with the HPG axis. Second, we applied strict exclusion criteria based on health. Health is highly associated with the HPG axis; health status can influence the HPG axis, and conversely, the HPG axis can influence health. Inflammation is a major factor influencing the HPG axis, which was also shown recently by Veldhuis et al. reporting on healthy male subjects receiving an IL-2 injection which led to an immediate decrease in testosterone secretion . Even thSupporting our findings, no mutations in the HPG axis have been associated with changes in lifespan in model organisms, indicating that a constitutively lower activity of the HPG axis is likely not associated with longevity. Long-living IGF-1 and GH-mutant mice do show suppression in HPG hormones and reproduction, but mainly in female mice . HoweverOne of the limitations of the current study was the relatively small sample size. This study is underpowered to detect many small differences but adequately sampled to identify major differences in hormonal axes. Indeed, in the same study population, comprising men and women, offspring had a 59.9% higher total TSH secretion and a higher temporal correlation between TSH and fT3 compared to controls , 14. FurInherent to the study design of the LLS, in which families are included based on two proband nonagenarian siblings, the age range of the offspring was 52–76 years. Therefore, LH and testosterone profiles could not be obtained at young age. Another limitation of this study was that there could have been dilution of potential differences between offspring and controls, since possibly not all offspring may have inherited the favorable predisposition for longevity of their long-lived parent. Strength of our study is that we sampled blood every 10 min during 24 h, creating the opportunity to study the HPG axis in detail. Moreover, we performed our study in a special cohort, in which we were able to study human familial longevity.To conclude, this relatively small study suggests that in healthy male middle-aged participants, human familial longevity is not associated with major differences in the HPG axis.Conceptualization and methodology: DH, FR, RW, and HP; formal analysis: ES, FR, SJ, and DH; investigation: SJ, AA, and ES; resources: BB and CC; writing – original draft: ES and DH; writing – review and editing: ES, SJ, AA, BB, CC, PS, GB, RW, HP, FR, and DH; visualization: ES and DH; supervision: DH, HP, and FR; funding acquisition: RW, DH, and HP.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.