The management of post‐stroke epilepsy (PSE) should ideally include prevention of both seizure and adverse effects; however, an optimal antiseizure medications (ASM) regimen has yet been established. The purpose of this study is to assess seizure recurrence, retention, and tolerability of older‐generation and newer‐generation ASM for PSE.This prospective multicenter cohort study (PROgnosis of Post‐Stroke Epilepsy [PROPOSE] study) was conducted from November 2014 to September 2019 at eight hospitals. A total of 372 patients admitted and treated with ASM at discharge were recruited. Due to the non‐interventional nature of the study, ASM regimen was not adjusted and followed standard hospital practices. The primary outcome was seizure recurrence in patients receiving older‐generation and newer‐generation ASM. The secondary outcomes were the retention and tolerability of ASM regimens.n = 322), 98 patients (30.4%) had recurrent seizures and 91 patients (28.3%) switched ASM regimen during the follow‐up (371 [347–420] days). Seizure recurrence was lower in newer‐generation, compared with the older‐generation, ASM . ASM regimen withdrawal and change of dosages were lower in newer‐generation ASM .Of the 372 PSE patients with ASM at discharge , 36 were treated with older‐generation, 286 with newer‐generation, and 50 with mixed‐generation ASM. In older‐ and newer‐generation ASM groups . ASM regimen withdrawal and change of dosages were lower in newer‐generation ASM . These findings suggest potential for newer‐generation ASM as the primary choice in the secondary prophylaxis of PSE.The current study is a real‐world prospective cohort study to evaluate seizure control, retention, and tolerability of older or newer‐generation anti‐seizure medication (ASM) treatment in post‐stroke epilepsy (PSE). Seizure recurrence was lower in newer‐generation, compared with the older‐generation, ASM (hazard ratio [HR], 0.42, 95%CI 0.27–0.70;  Patients hospitalized with PSE between November 2014 and September 2018 were recruited from eight hospitals in Japan. According to the new clinical definition from the International League Against Epilepsy (ILAE) , history of dementia, craniotomy, alcohol drinking, family history of epilepsy, and ASM medication history, were collected during admission. Index stroke were categorized into four types, ischemic stroke, intracranial hemorrhage, subarachnoid hemorrhage, and transient ischemic attack, and assessed for cortical stroke lesions  and size in major axis  by computed tomography (CT) or MRI. Functional disability was evaluated at discharge based on modified Rankin Scale. We also studied the histories of early (within 7 days post stroke) and late (more than 7 days) seizures.PSE seizures were classified as focal aware seizure, focal impaired awareness seizure, focal‐to‐bilateral tonic‐clonic seizure, and others, according to the ILAE classification of seizures , newer‐generation (n = 286), and mixed generation, ASM groups (n = 50). CBZ, VPA, PHT, phenobarbital (PB), clonazepam (CZP), and clobazam (CLB) were defined as “older‐generation ASM,” and LEV, LTG, lacosamide (LCM), zonisamide (ZNS), perampanel (PER), gabapentin (GBP), and topiramate (TPM) as “newer‐generation ASM,” which were launched in Japan after 2006. A “mixed‐generation” ASM group, defined as a combination of older‐ and newer‐generation ASM, was excluded from the main analysis due to various factors on the choice of ASM regimen. These definitions were determined regardless of the number of ASM and selection of regimen was not altered due to the non‐interventional nature of the current study, which followed standard hospital practices. The dosage of each ASM, as well as information on whether the serum concentration of older‐generation ASM reached an adequate therapeutic range at discharge, were collected. After a baseline dataset was collected during admission period, each participant was followed‐up according to usual clinical practice in each hospital over a 1‐year period. The mixed‐generation ASM group was excluded from the main analyses.We evaluated the seizure control, retention, and tolerability of ASM treatment in the secondary prevention of seizure after discharge. We excluded patients without ASM prescription at discharge. For secondary prophylaxis of seizures, PSE patients were categorized into three groups Figure : older‐gThe primary outcome was seizure freedom rate over the complete follow‐up period since discharge. The secondary outcomes were the time to discontinuation or dosage adjustment of initial ASM regimen due to ineffectiveness or intolerable ASM‐related AE (defined as ASM retention) and time to discontinuation of initial ASM regimen due to intolerable ASM‐related AE . The incidence of discontinuation or dosage adjustment due to poor adherence or other reasons were not considered outcomes. Board‐certified epileptologists or neurologists closely assessed episodes of first unprovoked seizure recurrence, ASM regimens, and reasons behind ASM discontinuation or dosage adjustment from hospitalization and/or outpatient visit records and seizure diaries with face‐to‐face consultations. If outpatient visits were not feasible, assessment was made through telephone interview with patients, relatives, or general physicians at 6 and 12 months. Patients who died or were lost to follow‐up were assessed as of the last visit. The maximum period of observation until the last follow‐up was 800 days.2.4n = 69) from the retrospective PSE cohort database of our institution between January 2011 to October 2014, comprising 15 newer‐generation, 18 mixed‐generation, and 69 older‐generation ASM. Retrospective data were obtained from registered PSE patients treated and followed up after discharge completely at our hospitals. During this period, most newer‐generation ASM monotherapies were not covered by Japanese public health insurance.In the current prospective study, the older‐generation ASM group comprised just 36 patients, presumably owing to the trend of using newer‐generation ASM. LEV was predominantly prescribed in preference to older‐generation ASM in Japan  or number (%). Variables were compared using Wilcoxon's test for continuous variables and Pearson's chi‐square test for categorical variables. Time to first seizure recurrence (seizure freedom rates), discontinuation or dosage adjustment (ASM retention rates) and discontinuation (ASM tolerability rates) between older‐ and newer‐generation ASM groups was analyzed by the Kaplan–Meier method and compared by log‐rank test. Follow‐up time was defined as the period from discharge until death, loss to follow‐up or final follow‐up visit. Patients who withdrew due to the above reasons were censored at the time of withdrawal, and those who did not withdraw were censored at their last visit. We also determined cumulative seizure freedom rates by 90 days, 180 days, and 1 year after discharge.p value < .10 between the ASM groups were adjusted in a multivariate model . In addition, we conducted additional analyses by eliminating data on seizure outcomes of PSE patients with inadequate or unknown concentrations of older‐generation ASM (n = 18).Cox proportional hazards modeling was used to calculate hazard ratios (HR) with 95% confidence intervals (CI). Potential confounding factors such as sex . All data were anonymized, stored, and fixed in an electronic data capture system by the data manager prior to analysis.As with verification analyses, prospective newer‐generation ASM were compared against the retrospective older‐generation ASM group, or the combination of prospective and retrospective older‐generation ASM groups, using the same methods. Two‐sided values of 3From November 2014 to September 2018, a total of 392 patients with a diagnosis of PSE in the eight sampled hospitals met the eligibility criteria and were prospectively enrolled into the PROPOSE study , followed by CBZ (n = 26), LCM (n = 18), and VPA (n = 9). The regimen and mean daily dosages of ASM, and proportion of patients with adequate serum concentrations of older‐generation ASM at discharge are described in eTables The demographics and clinical course characteristics of older‐ and newer‐generation ASM groups are shown in Table  LCM n = 8, and VP3.1p = .0003, log‐rank)  days after discharge. The seizure freedom rates were 88.2% at 90 days, 78.3% at 180 days, 69.0% at 1 year, and 65.3% at the end of follow‐up (800 days). Within 1 year of discharge, 23 deaths and 17 difficulties of contact were recorded. The risk of recurrent seizure was significantly lower in newer‐generation, compared with the older‐generation, group  Figure .n = 18) were excluded from analysis, newer‐generation ASM retained a lower risk of seizure recurrence than older‐generation ASM (p < .0001) , while adjusted HRs were 0.40  in model 1 and 0.47  in model 2  . The most common AE leading to discontinuation or dosage adjustment were skin eruptions (n = 11), dizziness (n = 5), and behavioral changes (n = 5)  eTable . No pati3.3n = 69) , the retrospective older‐generation ASM cohort showed significantly lower seizure freedom  and high retention . Of note, the retrospective cases revealed newer‐generation ASM had significantly lower risk in terms of tolerability than older‐generation ASM    for ASM effectiveness, 0.43  for ASM retention, and 0.28  for ASM tolerability.Similar results were obtained if the prospective and retrospective older‐generation ASM cohorts were combined , the ILAE report recommends CBZ, LEV, PHT, and ZNS as initial monotherapy based on level A evidence (Glauser et al., –drug interactions. Moreover, CBZ, a CYP‐inducing ASM, could interfere with the efficacy of statins, consequently increasing lipid levels (Mintzer et al., The current PROPOSE study showed that use of newer‐generation ASM was associated with approximately 50% reduction of seizure recurrence, compared to older‐generation ASM. Similar results were obtained in verification analysis using our retrospective cohort data. Previous meta‐analyses have revealed no significant superiority of seizure control in newly diagnosed focal epilepsy (Lattanzi et al., Our study has several limitations. First, as this was a prospective observational study, ASM regimen could not be controlled, meaning the older‐generation ASM were used less frequently and in smaller dosage than newer‐generation ASM. Second, the PROPOSE study was not a like‐for‐like comparison of each ASM, which may have resulted in insufficient statistical power. However, RCTs may potentially limit generalizability, which cannot be necessarily regarded as general principles valid for all subjects, some of whom may have had difficulties answering more complex questions. In the field of epilepsy, for instance, the various combinations of ASMs are used in a wide variety of clinical, social, and economic conditions. It is impractical to conduct separate RCTs to study the effect of each specific ASM regimen. Observational studies based on routinely collected data have generally a greater external validity than RCTs (Carlson & Morrison, 5The current study is a real‐world prospective cohort study to evaluate seizure control, retention, and tolerability of older or newer‐generation ASM treatment in PSE. These findings suggest potential for newer‐generation ASM as the primary choice in the secondary prophylaxis of PSE. However, further studies are needed to confirm ASM drug interactions and stroke risks and to find the optimal regimen for ASM in PSE.Akio Ikeda belongs to The Department of Epilepsy, Movement Disorders, and Physiology, an Industry‐Academia Collaboration Course, supported by a grant from Eisai Corporation, Nihon Kohden Corporation, Otsuka Pharmaceutical Co., and UCB Japan Co.All other authors report no conflict of interest specifically related to this manuscript.Study concept and design was provided by TT, KF, KK, AS, KN, RM, AI, and MI. Acquisition, analysis, or interpretation of data was performed by TT, KF, and MI. Drafting the manuscript and figures was performed by TT and MI. All authors participated in critical revision of the article for intellectual content. KN and DO were the statisticians. AI and MI provided study supervision.https://publons.com/publon/10.1002/brb3.2330The peer review history for this article is available at Supporting informationClick here for additional data file.


Melatonin is considered a potential plant growth regulator to enhance the growth of plants and increase tolerance to various abiotic stresses. Nevertheless, melatonin’s role in mediating stress response in different plant species and growth cycles still needs to be explored. This study was conducted to understand the impact of different melatonin concentrations  applied as a soil drench to maize seedling under drought stress conditions. A decreased irrigation approach based on watering was exposed to maize seedling after drought stress was applied at 40–45% of field capacity.The results showed that drought stress negatively affected the growth behavior of maize seedlings, such as reduced biomass accumulation, decreased photosynthetic pigments, and enhanced the malondialdehyde and reactive oxygen species (ROS). However, melatonin application enhanced plant growth; alleviated ROS-induced oxidative damages by increasing the photosynthetic pigments, antioxidant enzyme activities, relative water content, and osmo-protectants of maize seedlings.Melatonin treatment also enhanced the stomatal traits, such as stomatal length, width, area, and the number of pores under drought stress conditions. Our data suggested that 100 μM melatonin application as soil drenching could provide a valuable foundation for improving plant tolerance to drought stress conditions. Furthermore, by altering the primary and secondary metabolites, plants can also accumulate soluble sugar and proline in cells, thus encouraging water retention to be adopted in the dry environment [Drought is one of the abiotic stresses that limit plant growth and production, directly affecting the yield of crops . China hironment –12. TherThe use of phytohormones to increase adaptability and protect plants from adverse environmental conditions is one of the innovative strategies . VariousAlthough the function of melatonin in crop plant stress tolerance has been previously reported in various studies. However, the impact of melatonin as a soil drenching application and its effect on root growth and leaf ultrastructure of maize seedlings under drought stress is still unclear. Therefore, melatonin-mediated physiological, biochemical, and growth changes in maize seedlings exposed to drought stress were investigated in our current research. We also examined the effect of plant growth, photosynthetic capacity, oxidative system, osmolytes accumulation, and the leaf stomatal ultrastructure traits of maize seedlings under drought stress conditions. This research would lead to elucidating the mitigating effects of melatonin on oxidative damage caused by drought stress.Drought stress had a significant inhibitory effect on the overall growth of maize plants, but the application of melatonin significantly increased the growth characteristics of maize seedlings. Under control drought stress (CKD), fresh weight (FW) and dry weight (DW) root biomass of maize seedlings were significantly lower compared to well water conditions (CKW). Table Similarly, average root diameter showed a steady increase in melatonin-treated plants compared to CKD. We observed that melatonin-treated seedling had a thicker root diameter and was greater by 22.18% in MT2 than CKD (Table Relative water content (RWC) is the key indicator that shows the plant’s survival capability and leaf water status. The CKD significantly reduced the RWC compared to CKW. The leaf RWC was markedly greater in all melatonin treatments than in CKD. Under drought stress conditions, the most significant increase in RWC was observed in melatonin treatment MT2 by 21.30% compared to CKD  showed a substantial increase with an increasing the melatonin concentrations for an instant. The results showed that melatonin MT2 treatment increased the SOD activity by 31.64% compared with CKD under drought stress conditions Fig. . The res2 due to stomatal closure, and finally inhibited cell division under drought stress [Drought stress is one of the most destructive abiotic stresses to plants, limiting their physiological, biochemical functions, growth, and biomass production . Melatont stress . Melatont stress . While mt stress . The higt stress , 26, 27.Melatonin regulates plant physiology and improves photosynthetic pigments , 29. Chl2 and water vapor. However, certain abiotic variables such as atmospheric CO2 concentration, water state, temperature, and light can affect these functions [2 from entering mesophyll cells [2 in plant cells directly decreases water stress by reducing diffusion through stomata and reducing CO2 in mesophyll by improving leaf carbon metabolism and photochemistry [Leaf stomata are recognized as the primary pores for controlling the transport of COunctions . The elill cells . CO2 in hemistry , 38. Phohemistry , 40.• radicals to protect turgor cells membrane, DNA and protein, from ROS-induced damage under abiotic stress conditions [citrus aurantium L. and B. napus L.) seedling has sustainably increased solutes concentration under abiotic stress [Plants also adopt a potential strategy to adjust stress conditions by preserving osmotic adaptation by developing substances known as osmolytes with low molecular weight, including soluble sugar, proline, soluble protein, etc. , 41. Pronditions , 43. Our2O2, O2, and MDA increased under water deficit conditions; however, the exogenous melatonin treatments significantly reduced the MDA content compared to CKD. Higher antioxidant enzyme activity has been linked to lower ROS accumulation in melatonin-treated plants [Oxidative damage due to excessive accumulation of ROS in plant cells is the primary consequence of environmental stresses . Despited plants . Plants d plants .2 status and is involved in the main cell defense process [2O2 accumulation was controlled by CAT and APX and reduced to H2O [In contrast to the regulation, all enzymatic antioxidants enzyme activities in our experiment increased in melatonin-treated plants under drought stress compared to well water control. Exogenous melatonin application enhanced the antioxidant enzyme activities including SOD, POD, CAT, and APX that protect plants from ROS induce oxidative damage mediated by melatonin allowed to survive under drought stress conditions. SOD is a central enzyme that controls the O process . In addid to H2O . Severald to H2O , 48. OurThe present research work concludes that the exogenous application of melatonin could be an effective approach to improving maize seedling’s resistance to drought stress. The application of melatonin as a soil drenching may enhance enzymatic and non-enzymatic antioxidant activities. This has mainly contributed to improving the antioxidant defense mechanism of maize seedlings, minimizing the oxidative damage caused by drought, as shown by significantly decreasing MDA and ROS accumulation. Also, by osmotic modification, exogenous application of melatonin simulated drought stress by enhancing the levels of osmo-protectants. Melatonin controls root growth to absorb moisture from greater depth in the soil and thus promoted stomatal behavior resulting in an increased net photosynthesis rate in maize seedlings under drought stress conditions. Our research results show the beneficial effects of the melatonin-soil drenching at an appropriate concentration (100 μM) on maize seedling under drought stress conditions. Keeping in view, the morphological, physiological, biochemical, stomatal features from the present study could be employed as an effective approach in many crops and will be useful in elevating drought stress.Seeds of hybrid Maize (Wanchuan-1306), the most commonly grown variety in the southern areas of China was used in the current experiment. The seed were obtained from Guangxi Wanchuan seed Industry Co. Ltd., China. The selected seeds permission was granted from the respective authority. The pot experiment was conducted in the glass-shed (net house) of Guangxi University, Nanning, Guangxi, China. The healthy hybrid maize seeds were selected, and five seeds were planted in each pot. The pots size was (32.5 cm diameter and 29 cm height), with 20 kg of soil mixture from arable topsoil was added to each pot. The pots were arranged in a randomized complete block design (RCBD) under natural light in the glass shed. In the early growth stage of maize seedlings, the soil moisture contents were maintained at normal 80–85% field capacity (F.C). A progressive drought was subsequently enforced during the experiment by withholding watering (40–45% F.C), based on daily measurements of pot weight from the five to six-leaf stage. After starting drought stress, the melatonin having different concentrations was applied as soil drenching at 250 mL solution to each pot for three consecutive days at the V-8 stage of maize seedling. The treatments designated for this study include 1. Melatonin 0 μM + Control well water condition (CKW), 2. Melatonin 0 μM + Control drought stress (CKD), 3. Melatonin 50 μM + drought stress (MT1), 4. Melatonin 100 μM + drought stress (MT2), 5. Melatonin 150 μM + drought stress (MT3). All sampling and measurements for different tests, such as leaf ultrastructure, morphological, physiological, and biochemical characteristics, were performed after one week of treatment at the V-9 stage. Three plants  were immediately processed or stored at − 80 °C for further analysis from each treatment.Plants from each group treatment were sampled and divided into roots, shoots, and leaves to assess fresh and dry biomass. Root length and diameter were calculated by root image analysis using WinRHIZO 2003a software . A tape meter was used to measure the plant height and leaf area to detect growth changes. The plant samples were then dried in an oven at 75 °C until constant weight and its dry weight (DW) was determined.The technique of Su et al.  was usedThe following formula was used to determine the RWC (%);2, stomatal conductance, and transpiration rate on a sunny day between the 10:00 and 12:00 pm. Each treatment was repeated three times. The method of Arnon [A portable infrared gas analyzer photosynthetic system LI-6800XT  was used to evaluate the net photosynthetic rate, intercellular COof Arnon  was used2), and count the number of stomata.The imaging of leaf stomata was done with the help of SEM. Five youngest fully open middle parts of leaves (1 mm) were collected from each treatment. The samples were immediately fixed with a 4% glutaraldehyde solution in 0.1 M phosphate-buffered saline . Samples were dehydrated in a graded ethanol series, vacuum dried, and gilded after rinsing five times with PBS for 5, 10, 15, 20, and 30 min . After t2SO4). The mixture was incubated in a boiling water bath for 10 min, after which the absorbance was recorded at 630 nm using a UV-spectrophotometer. The leaf proline content was determined by the method to describe by Tiwari et al. [Soluble sugar content was determined by using the Anthrone method, according to Shi et al. . 0.2 g fi et al. . The 0.5− 1 FW. MDA as an end product of lipid peroxidation as measured by Heath et al. [− 1 FW.Soluble protein content was estimated by using the Coomassie Brilliant Blue G-250 solution method according to Zhao et al. . 0.5 g fh et al. . A fresh2O2, the method described by Zhang et al. [2O2 with potassium iodide (500 mM). Incubate the reaction mixture at 25 °C for 1 h, and H2O2 is used as a standard, and the absorbance was measured at 390 nm by using a UV-spectrophotometer. Following the protocol with a slight modification of Zhang et al. [2 generation was calculated. A fresh leaf sample of 0.2 g was extracted in 1 mL  and then centrifuged at 10,000×g. A reaction mixture of sulfanilamide (17 mM) and naphthalene diamine hydrochloride was then applied to 1 mL of supernatant (7 mM). The resulting mixture was incubated at 37 °C for 10 min, and 3 mL of ether was added to each tube. Samples were centrifuged at 24 °C for 5 min (5000×g), and absorbance was determined using a UV-spectrophotometer at 540 nm.For the determination of Hg et al.  was usedg et al. , the rat− 1 min− 1 FW.Fresh leaf sample (0.2 g) was harvested by removing midrib, weighed, and washed with distilled water and then homogenized with a 5 ml chilled sodium phosphate buffer mortar and pestle . The homogenates were centrifuged at 15,000×g for 15 min at 4 °C. The supernatant was kept at 4 °C and used for assays of SOD, CAT, POD, and APX activities and expressed as U mgThe activity of SOD was determined according to the protocol of Giannopolitis et al.  after ph2O2 was added to the enzyme extract (20 μL). The reaction was started after the addition of H2O2, and a spectrophotometer observed the decomposition rate of H2O2 at 240 nm.The CAT activity procedure was described by Wang et al. . The CAT2O2 (200 mM), and 0.5 mL water. Using a spectrophotometer to measure POD enzyme activity, increases in absorbance due to guaiacol oxidation at 470 nm were observed.POD activity was calculated according to Ekemkci et al.  with lit2O2. The absorbance was measured immediately at 240 nm with a UV-spectrophotometer.APX activity was tested according to the method of Nakano et al. . To 3 mLp ≤ 0.05. For each treatment, means were expressed as ± standard deviation (SD) of triplicates (n = 3). Image-Pro Plus 6.0 software was used to measure the clear stomata from each slice, measure the stomatal width (μM), stomatal length (μM), stomatal area (μM2), and count the number of stomata.Statistical analysis was done using Statistic 8.1 software to evaluate the obtained data; the Least Significance Difference (LSD) test was used to equate the means at the significance level of


Alcohol use disorders (AUD) cause 7.2% of UK hospital admissions/year. Most are not managed by hepatologists and liver disease may be missed. We used the Enhanced Liver Fibrosis (ELF) test to investigate prevalence and associations of occult advanced liver fibrosis in AUD patients not known to have liver fibrosis.Liver fibrosis was assessed using ELF in prospective patients referred to the Royal Free Hospital Alcohol Specialist Nurse (November 2018–December 2019). Known cases of liver disease were excluded. Patient demographics, blood tests, imaging data and alcohol histories recorded. Advanced fibrosis was categorised as ELF ≥ 10.5.p = 0.081), or inflammation (p = 0.574).The study included 99 patients  with median alcohol intake 140 units/week (IQR 80.9–280), and a mean duration of harmful drinking of 15 years (IQR 10–27.5). The commonest reason for admission was symptomatic alcohol withdrawal (36%). The median ELF score was 9.62, range 6.87–13.78. An ELF score ≥ 10.5 was recorded in 28/99 (29%) patients, of whom 28.6% had normal liver tests. Within previous 5-years, 76% had attended A&E without assessment of liver disease. The ELF score was not associated with recent alcohol intake (Over a quarter of patients with AUD had previously undetected advanced liver fibrosis assessed by ELF testing. ELF was not associated with liver inflammation or recent alcohol intake. The majority had recent missed opportunities for investigating liver disease. We recommend clinicians use non-invasive tests to assess liver fibrosis in patients admitted to hospital with AUD.The online version contains supplementary material available at 10.1186/s12876-021-01728-2. While aHospital admissions related to alcohol are rising annually, with 350,000 alcohol related admissions per year in 2019, (an increase of 20% in a decade) . This isWhile a proportion of people admitted to hospital with harms arising from their drinking behaviour are recognised to have liver fibrosis and are managed by liver specialists, many are managed by a wide range of doctors and their liver disease may be missed, even if their alcohol use disorder (AUD) is recognised.Moreover, it is estimated that up to 75% of people with Chronic Liver Disease (CLD) first present to healthcare when their liver disease is advanced often with decompensated cirrhosis, when it is too late for behaviour change or interventions to avert poor outcomes , 5, 6.Part of the reason for this is because cirrhosis is often asymptomatic, and it is not reliably detected using routine liver function blood tests (LFTs) or ultrasound . The lasCurrently non-invasive testing for liver fibrosis with the Enhanced Liver Fibrosis test (ELF) is widely used for Non-Alcoholic-Fatty Liver Disease (NAFLD) to determine which patients with fatty liver have advanced fibrosis and warrant referral to hepatology, versus those with low ELF scores who can remain in primary care . HoweverWe aimed to investigate the prevalence of advanced liver fibrosis using the ELF test employing the literature-based ELF cut-off of 10.5 , in patiThis was a prospective service evaluation conducted at the Royal Free Hospital from November 2018 to December 2019.Consecutive referrals to the Royal Free Hampstead alcohol specialist nurse (ASN) were included if aged ≥ 18 and excluded if already under the care of a hepatologist, if they had a known chronic liver condition, a diagnosis of alcoholic hepatitis, or acute liver injury secondary to a cause other than alcohol. Out of 100 consecutive referrals to the ASN 98 were in-patients or emergency department attendees. The vast majority of out-patient referrals were ineligible for inclusion because they had known ArLD, leaving 2/100 eligible out-patient referrals. One patient was excluded from the analysis as they were found to have an ALT of 1,023 following a pregabalin overdose, reducing the sample size from 100 to 99.Data extracted from patients’ electronic medical records included patient demographics, reason for presentation to hospital, BMI, alcohol intake (detailed in next section), postcode to enable deprivation score calculation, results of any imaging or fibrosis tests performed within 6 months of referral to ASN, blood test results to enable calculation of FIB4, AST:ALT ratio and APRI , and numData were anonymised and entered into a password protected spreadsheet held on a secure NHS computer.Current alcohol consumption was recorded in units per week (U/w), and duration of ‘excess alcohol consumption’ in years. This was obtained from patients’ self-reported consumption extracted from free text in clinical notes. AUDIT scores were not available. It was noted if the patient had been actively drinking up to the point of presentation to hospital.® immunoassay system. ELF Scores were calculated from test results using the manufacturer’s published algorithm. An ELF threshold of 10.5 was pre-selected for detection of advanced fibrosis based on recommendations by Thiele et al. [An ELF test was performed on consecutive eligible patients referred to the ASN. Serum was extracted from 5 mL blood per patient which was analysed at the Central ELF laboratory . The samples were analysed for HA, PIIINP and TIMP1 levels using the proprietary assays developed by Siemens Healthineers Inc  for the ELF test, on a Siemens ADVIA centaure et al. .The primary outcome was the proportion of patients referred to the ASN at the Royal Free Hospital who had previously undetected advanced fibrosis as determined by an ELF score of ≥ 10.5 . SecondaPatients with ELF scores ≥ 10.5 were sent a letter inviting them to attend a hepatology outpatient clinic to see a hepatologist with an interest in ArLD, and to have a FibroScan. Blood samples were also taken to screen for viral, immunological and metabolic causes of liver disease in accordance with current protocols if these tests had been omitted during their hospital admission.As this is an exploratory investigation, following statistical advice we accepted a precision of estimate at 0.1 which would generate a sample size of between 62 and 89 using literature-based estimates of prevalence of advanced fibrosis. A post-hoc sample size calculation for 0.29 prevalence of advanced fibrosis, using a precision of estimate at 0.1 and 0.95% CI results in a minimum required sample size of n = 80.Demographic information was described using frequencies and percentages for categorical variables. Continuous data were described using means and SD or medians and IQR, depending on the normality of the data.t test for normally distributed data.For the comparison of categorical variables, Chi-Squared or, if sample size was less than 5, Fisher’s exact test was used as a conventional test, and for the continuous data the Mann Whitney U (for non-parametric data) or Student’s p values less than 0.25 in univariate analyses (either against ELF < / ≥ 10.5 or as a continuous variable). All p values were 2-sided and they were considered significant if p < 0.05. All data were analysed using SPSS software .Alcohol ‘units per week’ was analysed both as continuous data, and in quartiles. After univariate analyses, to determine the variables associated with the presence of advanced fibrosis with the most significance, a multiple binary logistic regression analysis model was used, using the literature-based ELF threshold of 10.5  and mult2 (SD 5.94) and 84% were current or past smokers. Alcohol intake was high with a median consumption of 140 U/w (80.9–280), and in this cohort men and women drank similar amounts (p = 0.73). The two patients seen in the ASN outpatient clinic had not been drinking alcohol within the past 3 months, and one inpatient had stopped drinking three weeks prior to hospital admission. All of the other 96/99 patients were drinking alcohol up to the point of presentation to hospital. The median duration of alcohol consumption was 15 years (IQR 10–27.5). This cohort of patients were from deprived areas, with 69% of them positioned within the lowest 4 deprivation deciles.The analysis included 99 patients  with a mean age of 53.1 years (SD 14.4)  of patients were seen as inpatients or in the emergency department. The most common reason for presentation to hospital was symptomatic alcohol-withdrawal (36.4%) including seizures, followed by injuries from falling over 13.1%) and mental health presentations (11.1%) including overdose. The vast majority (73.7%) were under the care of a general medical team .LFTs were performed in 95/99 patients of which 66.3% of these patients had abnormal LFTs (raised transaminases or ALP + GGT), with median ALT of 39 21–73) and AST of 43 (24–86.5) (see Table –73 and ATwenty-eight participants 28.3%) had an ELF score of ≥ 10.5, indicating advanced fibrosis . In addition, correlations were investigated between these same characteristics and ELF as a continuous variable.p = 0.031, Fig. p = 0.357).When comparing clinical characteristics with ELF score < 10.5 versus ≥ 10.5 there was no significant difference in age, sex, or BMI between the two groups Table d. There p = 0.074). On univariate analysis the AST:ALT ratio did significantly predict advanced fibrosis based on ELF (OR 2.081 (95% CI 1.145–3.779), p = 0.016 , AST:ALT ratios trended towards being higher in the advanced fibrosis group , than in the group with lower ELF scores   and ELF score 81) Fig. a and thi81) Fig.  and bina81) Fig. . Alcohol Fig. a ar = 0.303, p = 0.002), and this was confirmed in multivariable analysis, when adjusted for alcohol intake, AST:ALT ratio, ALP, MCV and bilirubin   on univariate analysis Fig. c, (r = 0p = 0.27) . The mean FibroScan value was 10.9 kPa (± 7.1 kpa), . Using a literature-derived threshold of 9.5 kPa for advanced fibrosis , 10/17 (Over a quarter (28.3%) of patients with AUD presenting to hospital for a variety of reasons had an ELF score of ≥ 10.5, indicating the presence of advanced liver fibrosis. None of these patients had been assessed previously for liver fibrosis or referred to a liver specialist. All of them were at high risk of liver damage, with a current median alcohol consumption of 140 U/w, and history of excess alcohol consumption lasting more than 15 years, and yet none had been investigated for liver disease during their index presentation or at any time previously. Moreover, 76% of the cohort had presented to hospital on a median of four times per person over the preceding five years without a diagnosis of ArLD, indicating missed opportunities for detection and treatment of liver fibrosis in a high-risk population. Missed opportunities for recognising and assessing liver damage in primary care were not investigated in this study, but none of the patients in this study had been referred to hepatology services for assessment of liver disease prior to diagnosis in this study.We found that LFTs were not a reliable predictor of advanced fibrosis, with 28% of patients who had ELF ≥ 10.5 having normal LFTs, in concordance with previous reports .r = 0.303, p = 0.002), there was no difference in the median ages of those with or without advanced fibrosis, as determined by ELF scores ≥ 10.5 or < 10.5 respectively. ELF score has been found to correlate with age in some [Whilst ELF scores were positively correlated with increasing age . Thiele et al. [Increasing AST:ALT ratio was the only other marker significantly associated with advanced fibrosis (ELF ≥ 10.5) in this study   with eitAdditional file 1. Table S1: Multiple logistic regression analysis to investigate for effect of alcohol unit quartiles on presence or absence of advanced fibrosis (as per ELF ≥ 10.5). Table S2: Multiple linear regression analysis to investigate for effect of alcohol unit quartiles on continuous ELF score. Fig. S1: Influence of AST:ALT ratio on binary and continuous ELF scores. a Scatter plot of ELF by AST:ALT ratio . b Boxplot of AST:ALT ratio by presence or absence of advanced fibrosis (ELF ≥ 10.5). Statistical test: Mann Whitney U, p value significance set at 0.05, AST:ALT ratio displayed with IQR (interquartile range).


Clostridioides difficile infection represents a growing clinical challenge. The new compound omadacycline is a potential treatment alternative, as many antibiotics have limited activity or are rarely used due to costs and side effects. The activity of omadacycline and five comparators was assessed with agar dilution on a 2015-to-2018 collection of 65 C. difficile isolates from Sweden. Omadacycline demonstrated in vitro activity against the contemporary ribotypes of C. difficile, and further clinical investigation is needed.IMPORTANCE Evaluating the activity of novel antimicrobials like omadacycline is of great interest, as a reliable and efficient antimicrobial treatment for Clostridioides difficile infections is in demand. Clostridioides difficile is an important nosocomial pathogen worldwide, causing C. difficile infection (CDI). Historically, metronidazole, given orally, has been the mainstay therapeutic agent for the treatment of CDI. A low efficacy of metronidazole among patients with a severe form of the disease and a high recurrence rate among patients despite receiving adequate treatment has prompted the need for treatment alternatives. Even though recurrence rates remain similar, comparative studies proved that vancomycin was superior, and therefore, it is now preferred as an initial treatment C. difficile isolates obtained from patients diagnosed with CDI in the Stockholm County region between 2015 and 2018 was used in the present study. Data regarding the contemporary and prevalent ribotypes (RTs) in Sweden during 2015 to 2018 were extracted from the Swedish Public Health Agency database. C. difficile isolates belonging to nine different ribotypes were obtained from the Clinical Microbiology Laboratory, Karolinska University Hospital, Huddinge, Stockholm, Sweden, and one isolate belonging to ribotype 027 was obtained from the Department of Clinical Microbiology, Jönköping Hospital, Jönköping, Sweden. A reference strain of C. difficile, ATCC 43594 (ribotype 005), was used for quality control. All isolates were freshly subcultured before testing and confirmed as C. difficile using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Diagnostics). The antibiotics tested alongside omadacycline were metronidazole, vancomycin, fidaxomicin, tigecycline, and moxifloxacin; these were incorporated within blood agar at various concentrations . The values for all the study isolates were determined in triplicate, and the geometric mean MICs, MIC50s, and MIC90s are reported.A collection of clinical trations  to perfoC. difficile isolates were tested. The ribotypes included were 014 (n = 20 isolates), 002 (n = 11), 023 (n = 8), 001 (n = 7), 005 (n = 5), 078 (n = 5), 020 (n = 4), 078/126 (n = 3), 027 (n = 1), and 012 (n = 1). The distribution of the study isolates based on their MICs for the antibiotics tested is depicted in  from a previous study that used the agar dilution method for MIC determination among 21 clinical isolates of C. difficile (50 (0.031 μg/ml) and MIC90 (0.031 μg/ml) reported in a more recent study from Texas, USA  definitions for characterizing the study isolates against the individual antibiotics tested. The geometric mean MIC of omadacycline (0.67 mg/liter) was comparable with the geometric mean MIC of metronidazole (0.55 mg/liter) and higher than the values for fidaxomicin (0.17 mg/liter) and tigecycline (0.07 mg/liter) among our strain collection. Despite the higher geometric mean MICs for omadacycline in comparison with those of fidaxomicin and tigecycline, the new agent can be a potential candidate for treatment of CDI due to its favorable pharmacokinetic and pharmacodynamic properties and its limited impact on the normal gut microbiota , 13, 14.m region , and hen


Orthostatic hypotension (OH) is a common nonmotor symptom in patients with Parkinson's disease (PD), with an incidence ranging from 14% to 54%.This study explored changes in cognition and transcranial sonography (TCS) findings in patients with PD and OH.We enrolled PD patients who visited the outpatient or inpatient department from 2017 to 2020. Blood pressure was measured in different positions, and demographic data were collected. Motor and nonmotor symptoms were evaluated using standard scales. A subset of 107 patients underwent TCS.p < .05). Among PD‐OH patients, there was a statistically significant difference in UPDRS II and III scores between patients with or without clinical symptoms (p < .05). The substantia nigra (SN) area was significantly larger in PD‐NOH patients (0.45 ± 0.18 cm2) than PD‐OH patients (0.34 ± 0.16 cm2) (p < .05).We enrolled 66 PD‐OH patients and 92 PD‐no orthostatic hypotension (NOH) patients. There were no significant differences in gender, age, disease duration, or Hoehn and Yahr stage between groups. Binary logistic regression revealed age as an independent risk factor for OH in PD patients. There were statistically significant group differences in visuospatial and executive function and Unified Parkinson's Disease Rating Scale (UPDRS) I and II scores (PD‐OH patients had poorer visuospatial and executive function and lower UPDRS I and II scores compared with PD‐NOH patients. Within the PD‐OH group, there was no significant difference in cognition between patients with or without clinical symptoms. The difference in the SN area may indicate different subtypes of PD or a tendency to develop parkinsonism syndrome. PD patients with OH have poorer visuospatial and executive function and life quality. What's more, symptomatic OH have higher scores in motor sympotoms and daily life. Finally, PDOH patients have smaller substantia nigra area in transcranial sonography. OH reflects autonomic nervous system dysfunction, and the prevalence in PD is about 53%  is a noninvasive method for PD diagnosis. Patients with PD show significant lager substantia nigra (SN) area in TCS compared to normal controls  and/or 10 mm Hg in diastolic BP (DBP) within 3 min from shifting from the supine to upright position. Upright mean blood pressure (MBP) < 75 mm Hg is highly specific and sensitive for identifying symptomatic OH  and Montreal Cognitive Assessment (MoCA) including visuospatial/executive functions, naming, attention, learning, abstraction, delayed verbal memory, and orientation. Motor symptoms were assessed with the Unified Parkinson's Disease Rating Scale (UPDRS III). Nonmotor symptoms were evaluated by nonmotor symptom questionnaire (NMSQ). Emotional problems were measured with the Hamilton Depression Scale (HAMD)−24 items and the Hamilton Anxiety Scale (HAMA)−14 items. Daily life was assessed with the UPDRS II and Parkinson's Disease Quality of Life Questionnaire (PDQL)−39. Fatigue was measured with the Fatigue severity scale (FSS).TCS was performed as previously described  for all statistical analyses. If continuous variables were normally distributed, independent sample 3p < .05). The PD‐OH and PD‐NOH groups did not differ in gender, age, duration, or disease stage  had OH and 92 (58.2%) patients did not. The PD‐OH group had significantly higher supine systolic pressure (SSP), orthostatic systolic pressure (OSP), orthostatic diastolic pressure (ODP), and orthostatic mean arterial pressure (OMAP) . There were no significant differences in other evaluations of motor and nonmotor symptoms including MMSE, MoCA , UPDRS I, UPDRS III, NMSQ, FSS, HAMA, Hamilton Rating Scale for Depression (HRSD), and PDQ‐39 . However, patients with symptomatic OH tended to have higher lower SDP, OSP, ODP, and MBP measurements. Symptomatic OH was also associated with higher scores for UPDRS II (p = .003) and UPDRS III (p = .016). The groups did not differ in the other assessments of motor and nonmotor symptoms  patients had symptomatic OH. There were no differences in age, gender, duration, or H‐Y stage between subjects with asymptomatic and symptomatic OH . There were no significant differences in middle cerebral velocity or resistance or third ventricle width  patients completed TCS. Those who could not schedule the examination or had poor temporal windows were excluded. The side with larger SN area and the lower velocity middle cerebral artery were used for comparisons between groups. The level of SN was adjusted as previously described  than those with PD‐NOH . PD‐OH patients showed smaller SN hyperechogenic areas. It may indicate that different clinical subtypes of PD are associated with different SN patterns ; Jiangsu Provincial Medical Key Discipline Project, Grant Number: ZDXKB2016022; Suzhou Science and Technology Development Program, Grant Number: SYS2020131; Discipline Construction Program of the Second Affiliated Hospital Soochow University, Grant Number: XKTJ‐XK202001.


GBA gene. As to PD, eight subjects were either homozygous or compound heterozygous for GAA mutations, including three novel mutations: (a) c.472 A > G; p.T158A, (b) c.503G > T; p.R168L, (c) c.1985del. Regarding FD, two subjects had pathogenic GLA mutations, and four had single nucleotide polymorphisms in the 5'UTR, previously implicated in modulating gene expression. The findings highlight a higher incidence of abnormal enzyme levels and pathogenic mutations in the target population reflecting ancestry‐based specific genotype and phenotype variations.Population studies point to regional and ethnicity‐specific differences in genetic predisposition for some lysosomal storage disorders (LSDs). The aim of the study was to determine the prevalence of the three treatable forms of lysosomal storage disorders  in a cohort of mostly urban‐dwelling individuals of African ancestry, a previously unknown genetic landscape for LSDs. Large‐scale selective multistep biochemical and genetic screening was performed in patients seeking healthcare for various health concerns. Fluorimetric enzyme assays for GD, PD, and FD were performed on dried blood spots. Targeted gene sequencing was performed on samples that showed significantly lower enzyme activities (<10% of control mean) after two tiers of enzymatic screening. A total of 5287 unique samples representing a cross section of patients who visited Howard University Hospital and College of Medicine from 2015 to 2017 were included in the study. Study samples were obtained from a population where ~90% reported as African‐American, ~5% Hispanic, and <5% Caucasian or other. Regarding GD, three subjects had either homozygous or heterozygous mutations in the  Selective screening for lysosomal diseases in minority groups in the United States shows higher prevalence rates and novel variants.1Lysosomal storage disorders (LSDs) are a group of about 50 inherited metabolic disorders resulting usually from mutations in the genes encoding lysosomal enzymes or enzymatic cofactors in the substrate degradation pathways. The advent of enzyme replacement therapy followed by the development of substrate reduction and pharmacological chaperone therapies enabled different treatment alternatives for some of the common LSDs, including Gaucher (GD), Pompe (PD), and Fabry (FD) diseases . As a result of treatment access, the natural history of these disorders has significantly changed, with a marked decrease in morbidity and mortality.All three of these LSDs have juvenile and/or adult presentations with attenuated phenotypes.GBA gene.Whereas individual LSDs are categorized as rare orphan disorders, LSDs as a group are relatively common and affect a considerable number in the general population with a combined incidence of 1:5000 to 1:8000.In the current study, the incidence of three LSDs was investigated in a cohort of mostly urban‐dwelling African‐Americans, compared to previous work primarily in Caucasians. These LSDs were chosen for two reasons, (a) several treatments are available, with more novel therapies in the pipeline, and (b) these LSDs have adult onset forms of the disease. In this prospective screening study, leftover blood samples were screened by enzymatic assays and targeted gene sequencing to determine the prevalence of GD, PD, and FD in individuals of African ancestry. Our results show a higher than expected incidence of these rare LSDs in the study cohort of 5287 samples; the incidence for GD was 1:1800, 1:700 for PD, and 1:900 for FD when assayed for enzyme activity followed by exon sequencing. Furthermore, at least three novel variants were identified for PD.22.1The study was reviewed and approved by Howard University Institutional Review Board (IRB‐14‐MED‐09) and the Western Institutional Review Board (NCT02120235) and was conducted in accordance with the ethical principles described in Declaration of Helsinki of 1975. Samples (n = 5287) consisted of the randomized collection of left‐over blood samples during a period spanning 2015 to 2017, at Howard University Hospital, Washington, DC. Blood samples used as positive controls for GD (n = 25), obligate carriers of GD (n = 25), PD (n = 12), FD (n = 25), and healthy controls (n = 60) were obtained at the Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA.2.2Leftover blood samples that were collected in K2 EDTA blood collection tubes were used to prepare dried blood spots (DBS) within 24 hours of blood draw. The positive and negative control samples collected at LDRTC in a similar manner were also used within 24 hours of blood draw. About 60 μL and 30 μL of blood sample from each tube was spotted onto Whatman 903 protein saver cards and FTA cards  respectively. The cards were air‐dried overnight at room temperature and protein saver cards were stored at −20° C, while FTA cards were stored at room temperature until further use. The samples were stripped of personal identifiers and given unique identification codes to match only with the age, sex, and race of the blood donor.2.3Fluorimetric enzyme assays for lysosomal enzymes β‐glucocerebrosidase , acid α‐glucosidase  and α‐galactosidase  were performed following the original method developed by Chamoles et al,2.4Statistical analysis was performed using GraphPad Prism software . Enzyme values were plotted, and graphs were generated as Box & Whisker (Tukey) plots. Data are expressed as average values ± the SE of the mean (SEM).2.5GBA (gene ID: 2629), GLA (gene ID: 2717) and GAA (gene ID: 2548) were performed in seven, four, and five reactions, respectively, using the primers listed in Table GBA were designed to selectively amplify the GBA gene and not the pseudogene sequence. PCR amplification was optimized by using Takara PrimeSTAR GXL Polymerase . The DNA was denatured for 30 seconds at 94°C followed by 40 cycles of denaturation at 98°C for 10 seconds, annealing at 59°C for 50 seconds, and elongation at 68°C for 4 minutes. Amplified DNA was purified with DNA Clean and Concentrator kit , followed by fragmentation and preparation of libraries using NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent . Concentration of libraries was measured with quantitative PCR using the universal library quantification kit for Ion Torrent . Samples were sequenced on the Ion Torrent Personal Genome Machine. In‐house scripts written on C++ and publicly available sequence aligner programs  were used to analyze the generated DNA‐sequencing data. For the analysis of DNA‐sequencing, sequence reads were aligned against GBA, GAA or GLA genes sequence and single nucleotide profile was further analyzed against NCBI mutation database and Human Genome Mutation Database (HGMD) for GD, PD, and FD. Data were searched for pathogenic mutations.The samples were genotyped using 1 mm punches from FTA cards that were added to the PCR mix in a 50 μL PCR reaction. Amplifications of 3The blood samples analyzed reflected 5287 urban‐dwelling, mostly African ancestry patients seeking clinical care for various health concerns and of these, 90% reported as African‐American, 5% Hispanic and 5% Caucasian or other. The age range of patients was 15 to over 100 years, with almost equal distribution of males to females. The experimental flow is represented in Figure For all three LSDs, the average and range of enzymatic values for this mostly African‐American population are different from the LSD‐negative control reference group Figure . β‐Glu aGBA gene  samples presented with activity below the cutoff and the β‐glu assay on these samples was repeated in 96‐well plates for second tier testing. Of these, 53 (1.0%) samples showed enzyme activity lower than 1 nmol of substrate released/mL of blood/hour. FTA cards corresponding to the 53 samples were used to sequence the GAA and GLA genes. Results from GAA sequencing showed a total of eight samples with two or more mutations. Two subjects had premature stop codon mutations (c.2560C > T/p.R854*) while one showed a deletion mutation (c.1985 del). Two subjects with undetectable or low α‐Gal activity showed hemizygous pathogenic mutations, c.335 G > A/p.R112H and c.427 G > A/p.A143T, respectively. Four samples showed variants in 5'UTR of GLA gene  and 22 (0.42%) samples with low enzyme activities were identified. The corresponding FTA cards were sequenced for 4GAA gene impacted in PD.There are extensive studies accounting for epidemiological, clinical, biochemical, genetic, and therapeutic inquiries of LSDs in Caucasian populations in the United States and internationally. A recent search of Pubmed in December 2020 for “African‐American” and “Gaucher,” or “Pompe,” or “Fabry” yielded 13, 6, and 5 publications, respectively. Many of these articles were single case reports. Therefore, the findings in this study are opportune and much needed for more inclusive healthcare delivery regarding these rare disorders. Here, we report a higher than expected incidence of GD, PD, and FD in a mostly Black patient population. We further report three novel polymorphisms in the GD and PD are collectively, two of the most common LSDs inherited in an autosomal recessive mannerIn addition to specific mutations in the various genes associated with GD, PD, and FD in this unique population, our findings suggest a never before reported increased biochemical activity for β‐glu, α‐gal, and α‐glu that bypasses by up to 10‐fold the highest expected range in “control” samples. At present, the significance of increased enzymatic activity in this or other populations is not known. In addition, even when enzymatic activity was zero or very low, this did not always correspond to a mutation following gene sequencing. Therefore, it is possible that some low enzymatic values correspond to other as of yet unidentified metabolic problems.Recognizing the importance of screening for common forms of LSDs, they are included in many pilot newborn screening (NBS) programs.GBA gene (GD), 8:5256 in the GAA gene (PD), and 3:5287 in the GLA gene (FD). These high rates could be because this cohort includes sick individuals seeking inpatient and outpatient healthcare. Nonetheless, this was not an LSD clinic, and such findings have not been previously reported for other groups, including Caucasians at other healthcare settings.Since GD, PD, and FD can be managed therapeutically,It is highly relevant to US health care system to be aware of any racial influences in the prevalence rates in order to properly focus on clinical care needs. According to the 2017 Census Bureau data, the population of the District of Columbia (DC), was 47.1% Black or African‐American, 45.1% White (36.8% non‐Hispanic White), 4.3% Asian, 0.6% American Indian or Alaska Native, and 0.1% Native Hawaiian or Other Pacific Islander. Individuals from two or more races made up 2.7% of the population. Hispanics of any race made up 11.0% of the District's population.The results from the large‐scale undertaking in the current study provide much needed population data of GD, PD, and FD prevalence in African‐Americans. One of the limitations of the present study is that the study population was already under clinical care for various reasons and thus the results may not represent the true prevalence rates in the general non‐hospital African‐American population. Another limitation is the inability to assign clinical symptoms and phenotypic correlation with the enzymatic levels and genetic variants. Nonetheless, given the fact that the diagnostic assays for these LSDs can be performed with relatively low cost and results can be confirmed using targeted single‐gene sequencing, it would be advisable for the physicians responsible for the clinical care of these patients to be aware that these LSDs, which are considered rare diseases in the general population, may be more common in people of African ancestry and other minority groups. Moreover, a higher incidence of LSDs observed in African‐American predominant minority groups provides another example for current existing disparities in health care. Underrepresentation of minority groups for both participation in clinical research trials and seeking specific medical care further hamper the clinical practice specifically addressing the medical needs of patients of African descent.Ozlem Goker‐Alpan acts as a consultant and has received speaker honorarium from Shire  and Pfizer. Renuka Pudi Limgala, Vyacheslav Furtak, Margarita M. Ivanova, Erk Changsila, Floyd Wilks, Marie N. Fidelia‐Lambert, and Marjorie C. Gondré‐Lewis have no conflicts of interests to disclose. The reporting of results of the study and decision to publish this manuscript are independent of the above conflicts.Ozlem Goker‐Alpan and Marjorie C. Gondré‐Lewis: Study concept and design. Renuka Pudi Limgala, Vyacheslav Furtak, Margarita M. Ivanova, Erk Changsila, Floyd Wilks, and Marie N. Fidelia‐Lambert: Acquisition of data. Renuka Pudi Limgala and Marjorie C. Gondré‐Lewis: Data curation. Renuka Pudi Limgala, Vyacheslav Furtak, Margarita M. Ivanova, Ozlem Goker‐Alpan, and Marjorie C. Gondré‐Lewis: Analysis and interpretation of data. Renuka Pudi Limgala, Ozlem Goker‐Alpan, and Marjorie C. Gondré‐Lewis: Drafting of the manuscript. Vyacheslav Furtak, Margarita M. Ivanova, Erk Changsila, Floyd Wilks, and Marie N. Fidelia‐Lambert: Critical revision of the manuscript for important intellectual content. Ozlem Goker‐Alpan and Marjorie C. Gondré‐Lewis: Funding acquisition. Ozlem Goker‐Alpan andMarjorie C. Gondré‐Lewis: Study supervision.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was not obtained from the patients for being included in the study. The study was reviewed and exempted by Howard University Institutional Review Board (IRB‐14‐MED‐09) and the Western Institutional Review Board (NCT02120235) based on federal regulation 45 CFR 46 and associated guidance.Supplemental table 1 Primers for GBA, GAA, and GLA genes PCR amplification.Click here for additional data file.


Oncorhynchus Spp.) that has provided rich genetic baseline data of allozymes, microsatellites, and single‐nucleotide polymorphisms (SNPs) across the Pacific Rim. Here, we analyzed published data sets for adult chum salmon (Oncorhynchus keta), namely 10 microsatellites, 53 SNPs, and a mitochondrial DNA locus  in samples from 495 locations in the same distribution range . TreeMix analysis of the microsatellite loci identified the greatest convergence toward Japanese/Korean populations and suggested two admixture events from Japan/Korea to Russia and the Alaskan Peninsula. The SNPs had been purposively collected from rapidly evolving genes to increase the power of GSI. The largest expected heterozygosity was observed in Japanese/Korean populations for microsatellites, whereas it was largest in Western Alaskan populations for SNPs, reflecting the SNP discovery process. A regression of SNP population structures on those of microsatellites indicated the selection of the SNP loci according to deviations from the predicted structures. Specifically, we matched the sampling locations of the SNPs with those of the microsatellites and performed regression analyses of SNP allele frequencies on a 2‐dimensional scaling (MDS) of matched locations obtained from microsatellite pairwise FST values. The MDS first axis indicated a latitudinal cline in American and Russian populations, whereas the second axis showed differentiation of Japanese/Korean populations. The top five outlier SNPs included mtDNA3, U502241 (unknown), GnRH373, ras1362, and TCP178, which were identified by principal component analysis. We summarized the functions of 53 nuclear genes surrounding SNPs and the mtDNA3 locus by referring to a gene database system and propose how they may influence the fitness of chum salmon.Genetic stock identification (GSI) is a major management tool of Pacific salmon , U502241 (unknown), GnRH373, ras1362, and TCP178, which significantly differentiated in Japanese/Korean populations. The functions of the outlier SNPs related to fitness, namely endurance exercise training, oxygen consumption, reproduction, and growth. GSI has been a major management tool of Pacific salmon  . The data consisted of the allele frequencies at 14 loci from chum salmon populations at 381 localities in a distribution range   Table  (Beacham5) Table . These dOke_U401‐220, Oke_GHII‐2943, Oke_IL8r‐272, and Oke_U507‐87, to avoid pseudo‐replication following the original study. A total of 53 nuclear SNPs and a combined mtDNA locus (mtDNA3) were included in our analysis . The mtDNA3 locus included three gene loci, Oke‐Cr30 and Oke‐Cr386 (mtDNA control region), and Oke‐ND3‐69 (mtDNA NADH‐3).SNP genotypes were retrieved from the Dryad data repository  from a 14‐microsatellite locus panel were found in Japanese, Russian, and representative North American populations  to estimate pairwise FST between populations. Because only allele frequency data could be accessed, we used the “read.frequency” function and computed pairwise FST values averaged over loci using the “GstNC” function in the R package FinePop v1.5.1 on CRAN.We used microsatellite allele frequencies  as the explained variation measure, which was computed in the R function as Ck=∑j=1kλj/∑j=1Kλj, where λj is the eigenvalue and jλ = 0 if jλ < 0. Following the original study  analysis was performed on the pairwise L^ + 2 × s, where L^ is the maximum composite likelihood and s is the number of parameters to be estimated, which, in this study, was three per migration event, namely, migration edge, branch length, and migration weight. For example, for the model with two migration events, the number of parameters was six. TreeMix maximizes composite likelihood and the 2 × log likelihood ratio is expected to follow an approximate χ2 distribution with three degrees of freedom. Because TreeMix calculates composite likelihoods, the penalty in AIC needs to account for the correlation in the likelihoods. To avoid unconscious bias toward selection of parameter‐rich models, we kept all the candidate models except those where the AIC values were decisively different.To infer demographic history with admixture, we applied TreeMix  values for the data sets. For microsatellites, we computed He for each population at each locus based on the allele frequencies. We then averaged He values over loci. For the 53 nuclear SNPs, we converted the original genotype data to genepop format , where He0,i = /(max He−min He). This conversion represents the standardized magnitude of an He value at the sampling point, with a continuous color gradient ranging from blue to red for the lowest and highest He values, respectively. Population‐specific He values of microsatellites and SNPs were grouped by seven geographical areas: Japan/Korea, Russia, Alaskan Peninsula, Western Alaska, Yukon/Tanana/Kuskokwim, SEA/BC, and WA.Sampling points were visualized by a color gradient of population‐specific He values between all pairs of the groups were tested by one‐way ANOVA, where p‐values were adjusted for the multiple comparisons using the “TukeyHSD” function in R.Differences in average 2.6https://www.genecards.org/) and published literature.To compare the genetic differentiation of SNP allele frequencies with the population structure inferred using the microsatellite markers, we analyzed 10 microsatellite loci and 53 nuclear SNPs loci plus the combined mtDNA3 locus. The mtDNA3 locus had five alleles, and the major allele (second one) was used for the meta‐analysis. The functions of all 54 analyzed gene loci were confirmed by referring to the GeneCards database system  of the MDS of the pairwise FST estimated using the microsatellite allele frequencies as:pj(i) is the allele frequency of SNP i  in population j . The explanatory variables mds1j and mds2j are the 1st and 2nd MDS coordinates of pairwise FST values estimated from the microsatellite allele frequencies for the matched populations j . These explanatory variables are population‐specific (J = 114). The regression analysis was performed for each marker , and 54 sets of regression coefficients  were obtained. The p‐values for the coefficients were adjusted for the multiple comparison  by the method of Benjamini and Hochberg , where jp0 = (jp−min p)/(max p−min p). This conversion represents the standardized allele frequency values at the sampling point, with a continuous color gradient ranging from blue to red for the lowest and highest allele frequencies, respectively.To identify genes that best characterize geographical areas, we conducted a principal components analysis (PCA) for the allele frequencies of the 53 nuclear SNPs and mtDNA3 locus using the “prcomp” function in R. Finally, we visualized the geographical distributions of the identified highly differentiated genes using boxplots and geographical maps of the allele frequencies and 33.1FST values based on the microsatellite allele frequencies averaged 0.019 ± 0.010 . The MDS of pairwise FST values , substantial gene flow between American and Asian populations was identified for the microsatellites . Large He values were found in Japanese, Russian, and Alaskan populations, whereas He values were slightly smaller in SEA/BC, WA, and Canadian Yukon populations . Japan and WA populations had the smallest He values. The He values were similar in populations from Western Alaska, Yukon, Alaskan Peninsula, and Russia , locus names, and raw data locus names used in our analysis Table . Five ofq‐values, showing that most of SNPs were differentiated beyond the neutral population structure. The top five outlier loci  was significantly larger in Japanese/Korean populations than it was in Western Alaska for mtDNA3  and TCP178 loci , whereas it was similar in Western Alaska for U502241 , and ras1362 . GnRH373 had smaller He values in Japanese/Korean populations (0.33) than in Western Alaskan populations (0.40) .The allele frequencies of the top five outlier loci were distinct in Japanese/Korean populations in the distribution range Figure . The maj4FST identified the north–south cline in American and Russian populations, and mds2 clearly showed divergence of Japanese/Korean populations, in agreement with the original study  in the early 1980s.mtDNA3  and may indicate a common effect. The original study  is a key regulator of vertebrate reproduction, including that of salmonids ; Data curation (lead); Formal analysis ; Funding acquisition ; Investigation (lead); Methodology ; Project administration (lead); Resources ; Software ; Supervision (lead); Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing . Hirohisa Kishino: Conceptualization ; Data curation (supporting); Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration (supporting); Resources ; Software ; Supervision (supporting); Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing .Fig S1‐S10Click here for additional data file.Table S1‐S3Click here for additional data file.


NBAS variants, confirmed by either trio analysis or cDNA studies. Functional characterization was undertaken, measuring NBAS and p31 levels by Western blotting, demonstrating reduced NBAS levels in two of three families, and reduced p31 levels in all three families. These results provided functional characterization of the molecular impact of a missense VUS, allowing reclassification of the variant and molecular confirmation of NBAS‐associated RALF. Importantly, p31 was decreased in all individuals, including an individual with two missense variants where NBAS protein levels were preserved. These results highlight the importance of access to timely functional studies after identification of putative variants, and the importance of considering a range of assays to validate variants whose pathogenicity is uncertain. We suggest that funding models for genomic sequencing should consider incorporating capabilities for adjunct RNA, protein, biochemical, and other specialized tests to increase the diagnostic yield which will lead to improved medical care, increased equity, and access to molecular diagnoses for all patients.Variants of uncertain significance (VUS) are commonly found following genomic sequencing, particularly in ethnically diverse populations that are underrepresented in large population databases. Functional characterization of VUS may assist in variant reclassification, however these studies are not readily available and often rely on research funding and good will. We present four individuals from three families at different stages of their diagnostic trajectory with recurrent acute liver failure (RALF) and biallelic  Access to rapid genomic and adjunct functional studies will improve clinical care of patients with NBAS‐associated recurrent acute liver failure and other monogenic conditions.1Rapid genomic sequencing is revolutionizing management of seriously ill infants and children, with the potential to confirm a molecular diagnosis within a few days with a high degree of clinician‐reported clinical utility.NBAS variants were first associated with short stature, optic nerve atrophy, and Pelger‐Huet anomaly  in 2010,NBAS variants and recurrent acute liver failure (RALF) or Infantile liver failure syndrome 2 in 2015 .NBAS variants and RALF have been reported, with a mutational spectrum including loss of function, missense, and deep intronic variants.NBAS variants may result in decreased NBAS protein levels, however some variants, particularly biallelic missense variants, may be associated with normal NBAS protein levels despite altered NBAS function.NBAS variants result in decreased p31 protein levels.Biallelic NBAS. Our functional data assisted reclassification of one VUS to likely pathogenic, and two likely pathogenic variants to pathogenic, thus confidently securing molecular diagnoses in these families.We present clinical, genomic and functional data from three families with biallelic variants in 22.12.1.1An 18‐month‐old female presented with RALF. At 8 months, she was admitted to hospital with vomiting and fever and was found to have alanine aminotransferase (ALT) activity of 739 IU/L , which resolved with supportive treatment. Coagulation studies were not performed. At age 11 months, she developed symptoms suspicious for sepsis and was found to have hypoglycemia, hypernatremia, lactic acidosis, coagulopathy, and liver derangement  9.9 ). She was treated with intravenous fluids, antibiotics, antivirals, fresh frozen plasma, vitamin K and corticosteroids, and her symptoms resolved over a few days. Adenovirus was isolated from a stool sample. At 18 months, she developed symptoms of a viral respiratory infection and was found to have an ALT of 11 524 IU/L and lactate dehydrogenase (LDH) of 20 823 U/L  accompanied by a coagulopathy with an INR of 9.1 and unexplained hypoglycemia. There was no history of an ischemic insult or exposure to toxins. A liver biopsy was performed for diagnostic purposes, as investigations for metabolic and infective causes of liver failure were non diagnostic. Results were nonspecific with severe diffuse acute pauci‐inflammatory hepatocellular injury, with zonal hepatocyte necrosis. Extensive metabolic investigations were nondiagnostic. There was no history of an ischemic insult or exposure to toxins.She has subsequently experienced three further episodes requiring hospital admission , all in the context of viral precipitants. The peak INR at these presentations was 2.0, 1.0, and 2.7, respectively. All episodes resolved with supportive therapy. Apart from these six episodes, she remains otherwise well with normal intervening blood test results. She has normal growth and neurodevelopment with no craniofacial dysmorphism. There is no family history of liver disease.2.1.2l‐3‐hydroxyacyl‐CoA dehydrogenase were normal. She died aged 2 years 9 months during an acute episode. Respiratory chain enzymology in liver was borderline low for complex III and normal for all other enzymes.Individual B1 had an antenatal history of intrauterine growth restriction from 32 weeks of gestation. Physical examination showed rhizomelia and large fontanelles. Although reported to be irritable, his neurodevelopment was normal prior to the onset of viral illness with elevated liver enzymes at age 7 months. Hepatitis A and B serology and blood lactate and pyruvate at this time were normal. Subsequently, he had recurrent episodes characterized by initial signs of a viral illness and then deterioration with prostration, drowsiness, and markedly elevated liver enzymes. However, coagulopathy was not documented. Fasting studies showed a blood glucose level of 2.5 mmol/L with B‐hydroxybutyrate increased markedly, reaching 9.1 mmol/L. Succinyl‐CoA transferase and acetoacetyl‐CoA thiolase activities in cultured skin fibroblasts were normal. The episodes would resolve in a few days with liver enzyme levels returning to baseline. He had several admissions to hospital until he died at age of 14 months. Respiratory chain enzymology in skin fibroblasts and skeletal muscle were normal, while liver homogenate results were deficient for complex II in both a biopsy and perimortem sample . His female sibling, individual B2, had a similar presentation with episodic severe RALF with intercurrent viral infections. Unlike her brother, she had no documented hypoglycemia, ketosis or metabolic acidosis. The episodes would resolve in a few days with the liver function enzymes returning to baseline. Fibroblast fatty acid oxidation screen, and specific assays for Long‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase deficiency and short chain 2.1.3Individual C has been previously described.2.2Genomic sequencing was performed by clinically accredited laboratories at the Victorian Clinical Genetics Services  , The Center for Applied Genomics  , and Kinghorn Centre for Clinical Genomics  , using genome sequencing (GS) or exome sequencing (ES) techniques. For individual A, GS was performed using massively parallel sequencing (MPS)  with a mean target coverage of 30×, and a minimum of 90% of bases sequenced to at least 10×. Data were processed, including read alignment to the reference genome (GRCh38) and variant calling, using CpipeCuration of variants was phenotype‐driven with pre‐curated or custom gene lists used for variant prioritization. Variant classification was based on modified ACMG/AMP guidelines.2.3Mycoplasma. Skin fibroblasts were cultured at 37°C in 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) 3.7 g/L NaHCO3 (HyClone) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Protein was extracted from cultured fibroblasts by resuspending in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail (Roche), then sonicated and incubated on ice for 30 min before centrifugation at 18000g for 20 min at 4°C. Total protein concentration was determined with the Pierce Bicinchoninic acid (BCA) kit (ThermoFisher) following the manufacturer's protocol. Protein lysates containing 20 μg of protein samples were analyzed by SDS‐PAGE Western blot (BioRad system) using primary antibodies against NBAS , USE1 (p31)  and GAPDH , and detected by anti‐rabbit IgG secondary‐horseradish peroxidase (HRP) conjugated antibody  using Enhanced chemiluminescence reagents (ECL) . Protein band intensities were quantified using ImageJ software and normalized to GAPDH.All fibroblast cell lines were tested for 2.4NBAS exons 12–15  and 25–30 .To inhibit nonsense‐mediated decay, Case B1 and control fibroblasts at ~70% confluency were treated with 100 ng/μL cycloheximide  for 24 h before harvesting.33.13.1.1NBAS with results available in 71 h. The paternally inherited variant, NM_015909.3(NBAS):c.2951T>G; p.(Ile984Ser), was classified as likely pathogenic according to modified ACMG/AMP criteria.https://gnomad.broadinstitute.org/ [accessed 15/11/2019]). It has been previously reported in individuals with RALF.https://genome.ucsc.edu/ [accessed 15/11/2019]). There is a large physicochemical difference between isoleucine and serine (Grantham Distance of 142).https://gnomad.broadinstitute.org/ [accessed 15/11/2019]). It has not been previously reported in association with disease. Computational tools predict a deleterious effect of the variant on protein function, and the variant is highly conserved . There is a large physicochemical difference between arginine and glycine (Grantham Distance of 125).NBAS).Ultra‐rapid trio GS, including analysis of the mitochondrial genome, was performed during an acute episode of RALF, identifying compound heterozygous missense variants in 3.1.2NBAS: the NM_015909.3(NBAS):c.2951T>G; p.(Ile984Ser) likely pathogenic variant described in individual A, and a novel variant, NM_015909.3(NBAS):c.1213C>T; p.(Arg405*), classified as pathogenic according to modified ACMG/AMP criteria. The p.(Arg405*) variant was absent from a large population database :c.2617C>T p.(Arg873Trp) was classified as likely pathogenic according to modified ACMG/AMP criteria. The variant was present in a large population database at a frequency of <0.01%  :c.2423+404G>C, is a deep intronic variant and was classified as likely pathogenic. The variant was present in a large population database at a frequency of <0.01%  . However, the NBAS protein levels in individual A who carried biallelic missense variants, were similar to controls. There was an overall reduction in p31 levels in all cases compared to controls Figure .3.3NBAS):c.2423+404G>C variants identified in individual C could be reclassified to Pathogenic.The missense VUS (p.(Arg136Gly)) identified in individual A was reviewed by the diagnostic genomic laboratory following completion of the adjunct functional studies, and reclassified as Likely Pathogenic. The missense variant identified in individual A and B, previously classified as Likely Pathogenic (p.(Ile984Ser)), was upgraded to Pathogenic, based on the functional data. Likewise, the p.(Arg873Trp) and NM_015909.4 receiving ultra‐rapid genomic sequencing results which required functional characterization to confirm pathogenicity of a VUS, the fourth patient  receiving stepwise genetic results until a molecular diagnosis was achieved, and the second family  receiving molecular confirmation of a diagnosis over 30 years after death. Ideally, infants and children presenting with RALF and other conditions suspicious for a monogenic disorder should be able to access rapid genomic testing followed by timely adjunct studies (if required) to allow reclassification of VUS in a clinically relevant timeframe. A timely diagnosis of NBAS‐related RALF. Based on published case reports and series', NBAS‐related acute liver failure is usually self‐limiting and resolves with conservative and supportive management in most,NBAS‐related RALF may provide reassurance to treating clinicians and avoid invasive or expensive investigations.There are no published management guidelines for In summary, adjunct functional studies have an important role in assisting with reclassification of VUS identified by genomic sequencing. Funding models for genomic sequencing services should consider incorporating capabilities for adjunct functional studies as part of their service.All authors declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000 Parents provided written informed consent for participation in the study. Human research ethics committee approvals were obtained from the Royal Children's Hospital Human Research Ethics Committee  and the Children's Hospital at Westmead . Parents provided written informed consent for participation in the study.