Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance.

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

An epidemiological analysis of Nocardia seriolae isolated from a wide range of aquatic animals in Taiwan, based on their genotype and enzymatic activity.

Chronic disease following Nocardia seriolae infection in a wide range of aquatic animals has been reported in many Asian countries and recently in America and Mexico. This study aimed to investigate the epidemiological relationship among N. seriolae isolates in Taiwan by investigating their genotype and enzymatic activities. A total of 66 strains isolated from 14 known and four unknown host fish from five sites in Taiwan were characterized using five combined methods. High genotypic diversity was recognized among the isolates with 10 pulsotypes being identified from the pulsed-field gel electrophoresis method and 21 reptypes from the repetitive extragenic palindromic amplification method; however, no natural plasmids were detected in this bacterial population. Pulsotypes A8 and RI analysed by PFGE and repPCR, respectively, were found to be predominant within five sites in Taiwan over 17 years of isolation. Enzymatically, the majority of isolates displayed high leucine arylamidase, ß-glucosidase and α-glucosidase activities but were negative for lipase, α-galactosidase, ß-glucuronidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase activities. We identified a strong association between genotype and enzymatic activity since the majority of pulsotypes displayed the same type of enzymatic profile. This study provides comprehensive and potential epidemiological data, which will aid the fish farming activities and prevention method development.

Impact of unintentional coronary angiography on outcomes of emergency surgery in acute type A aortic dissection: a retrospective study.

BACKGROUND: This study investigated the impact of coronary angiography on outcomes of emergency operation in acute type A aortic dissection (ATAAD) patients who were initially misdiagnosed as an acute coronary syndrome. METHODS: From October 2016 to April 2019, 129 patients underwent emergency operation for ATAAD in our institution, including 21 patients (16.3%, coronary angiography group) who received preoperative coronary angiography without knowledge of the ATAAD, and the rest 108 did not (Non-coronary angiography group). Preoperative clinical characteristics, 30-day mortality and postoperative complications were compared. Multivariable logistic regression was performed to confirm the independent prognostic factors for short-term and long-term outcomes. RESULTS: Patients undergoing coronary angiography had higher prevalence of preoperative hypotension or shock (61.9% vs 35.2%, P = 0.022), ischemic changes on electrocardiogram (66.7% vs 37.0%, P = 0.012), platelet inhibition (ADP-induced inhibition 92.0% vs 46.0%, P = 0.001), and coronary involvement (66.7% vs 30.6%, P = 0.002). 30-day mortality was 4.8% versus 9.3% (P = 0.84). Coronary angiography group had more intraoperative bleeding (1900 ml vs 1500 ml, P = 0.013) and chest-tube drainage on the first postoperative day (1040 ml vs 595 ml, P = 0.028). However, preoperative coronary angiography was not independent risk factors for 30-day mortality (OR 0.171, 95%CI 0.013-2.174, P = 0.173) and overall survival (HR 0.407; 95%CI 0.080-2.057; P = 0.277). CONCLUSION: Patients undergoing coronary angiography carried a higher risk of preoperative hemodynamic instability, myocardial ischemia, and perioperative bleeding. However, unintentional coronary angiography did not have a significant impact on short-term and long-term outcomes of emergency surgery in ATAAD.

Factors associated with insomnia and suicidal thoughts among outpatients, healthcare workers, and the general population in Taiwan during COVID-19 pandemic: a cross-sectional study.

BACKGROUND: Insomnia and suicidal thoughts are two of the negative impacts that have been caused by the COVID-19 pandemic. Identifying the factors that contribute to these psychological problems may help develop strategies to sustain the mental health of the public. The present study examined the psychosocial impacts caused by the COVID-19 pandemic among different populations in Taiwan, and investigated the relationships between these psychosocial variables, insomnia, and suicidal thoughts. METHODS: Between September 2020 and May 2021, online questionnaires including psychometrically validated scales were distributed to a convenience sample of outpatients (n = 205), healthcare workers (HCWs) (n = 500), and individuals in the general population (n = 1200) in Taiwan to collect data regarding their insomnia severity, suicidal thoughts, fear of COVID-19, trust of information, and resilience. Multivariate logistic regression methods were used to identify variables associated with suicidal thoughts and insomnia. RESULTS: Greater fear of COVID-19 was significantly associated with suicidal thoughts: odds ratios (ORs) with 95% confidence interval (CI) = 1.155 (1.002-1.330) for outpatients; 1.127 (1.035-1.228) for HCWs; and 1.100 (1.130-1.222) for those in the general population. Higher resilience was significantly associated with lower insomnia: OR (95% CI) = 0.819 (0.725-0.926) for outpatients; 0.803 (0.728-0.887), for HCWs; 0.829 (0.785-0.875), and for those in the general population. In addition, there was a statistically significant association between insomnia diagnosis and greater fear of COVID-19 among HCWs (OR [95% CI] = 1.102 [1.062-1.144]) and those in the general population (OR [95% CI] = 1.079 [1.053-1.106]). Among outpatients, there was a statistically significant association between suicidal thoughts and lower trust of information (OR [95% CI] = 0.794 [0.646-0.976]), while among those in the general population there was a statistically significant association between suicidal thoughts and higher insomnia severity (OR [95% CI] = 1.175 [1.13-1.222]). A statistically significant association was also found between insomnia diagnosis and higher suicidal thoughts among those in the general population (OR [95% CI] = 3.455 [2.338-5.106]). CONCLUSIONS: Trust of information, fear, and resilience were important factors for suppressing suicidal thoughts and insomnia among the three study populations. Health policies that monitor psychological status and build resiliency of the public are recommended to help develop tailored strategies for different populations affected by the COVID-19 pandemic.

Open reduction and locked compression plate fixation, with or without allograft strut, for periprosthetic fractures in patients who had a well-fixed femoral stem: a retrospective study with an average 2-year follow-up.

BACKGROUND: The use of cortical strut allograft has not been determined for Vancouver type B1 or C fracture. This study aimed to evaluate the short-term efficacy of locking compression plating with or without cortical strut allograft in managing these types of fractures. METHODS: We retrospectively assessed 32 patients (17 males, 15 females; 23-88 years, mean: 67.2 years) with Vancouver type B1 or C fractures. Seventeen patients (Group A; B1 fractures in 15 hips, C fractures in 2 hips) were treated with open reduction and internal fixation with locking compression plates (group A). The other 15 patients (Group B; B1 in 14 hips, C in 1 hip) were fixed by locking compression plating combined with cortical strut allografting (group B). The fracture healing rate, healing time, complications and function were compared between these two groups. RESULTS: The mean follow-up time was 32.4 months (12 to 66), and the overall fracture union rate of the 32 patients was 96.9%. Group B had a higher fracture union rate than Group A, but the difference was not statistically significant. Group A had one case of nonunion of type B1 fracture and one case of malunion; the mean time to fracture healing was 5.3 months (3 to 9). In group B, all patients reached bony union without malunion, with a mean time of fracture healing of 5.1 months (3 to 8). CONCLUSION: Treatment of Vancouver type B1 or C fractures by locking compression plating, with or without cortical strut allografting, resulted in similar union rates in these patients. This suggest that, the use of cortical strut allografting should be decided cautiously.

Genotyping and phenotyping of Lactococcus garvieae isolates from fish by pulse-field gel electrophoresis (PFGE) and electron microscopy indicate geographical and capsular variations.

Lactococcus garvieae is the etiological agent of Lactococcosis, an evolving disease affecting many fish species and causing significant economic losses worldwide. Assessing pathogen relatedness and bacterial population structure is critical for determining the epidemiology of L. garvieae infections and in establishing effective pathogen management methods. The previously published morphological and genetic studies point to a clonal population structure, as seen in other fish bacteria. In the present study, the pulsed-field gel electrophoresis (PFGE) method was utilized to define a population of 41 Taiwanese isolates from outbreaks with comparisons to four well-characterized non-Taiwanese isolates previously published. Two restriction enzymes (ApaI and SmaI) were utilized individually for PFGE analysis (cut-off value = 90.0%), revealing genetic heterogeneity across L. garvieae isolates, with ApaI and SmaI yielding 12 and seven distinct PFGE band patterns, respectively. The phylogenic analysis using internal transcribed spacer region clustered all L. garvieae isolates in the same clad. Furthermore, the electron microscopic results confirmed the absence of capsular gene cluster (CGC) in previously characterized Taiwanese vaccine strain (S3) from grey mullet. Overall, our findings emphasize the importance of analysing the morphological and genetic diversity in L. garvieae being correlated for proper taxonomic classification in vaccine strain selection and epidemiological studies.

Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum.

Edwardsiella spp. is a gram-negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty-nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed-field gel electrophoresis [PFGE], and virulence-related gene detection). SpeI digestion revealed 10 pulsotypes and I-CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

The Role of Autophagy and Apoptosis in Neuropathic Pain Formation.

Neuropathic pain indicates pain caused by damage to the somatosensory system and is difficult to manage and treat. A new treatment strategy urgently needs to be developed. Both autophagy and apoptosis are critical adaptive mechanisms when neurons encounter stress or damage. Recent studies have shown that, after nerve damage, both autophagic and apoptotic activities in the injured nerve, dorsal root ganglia, and spinal dorsal horn change over time. Many studies have shown that upregulated autophagic activities may help myelin clearance, promote nerve regeneration, and attenuate pain behavior. On the other hand, there is no direct evidence that the inhibition of apoptotic activities in the injured neurons can attenuate pain behavior. Most studies have only shown that agents can simultaneously attenuate pain behavior and inhibit apoptotic activities in the injured dorsal root ganglia. Autophagy and apoptosis can crosstalk with each other through various proteins and proinflammatory cytokine expressions. Proinflammatory cytokines can promote both autophagic/apoptotic activities and neuropathic pain formation, whereas autophagy can inhibit proinflammatory cytokine activities and further attenuate pain behaviors. Thus, agents that can enhance autophagic activities but suppress apoptotic activities on the injured nerve and dorsal root ganglia can treat neuropathic pain. Here, we summarized the evolving changes in apoptotic and autophagic activities in the injured nerve, dorsal root ganglia, spinal cord, and brain after nerve damage. This review may help in further understanding the treatment strategy for neuropathic pain during nerve injury by modulating apoptotic/autophagic activities and proinflammatory cytokines in the nervous system.

[Changes in the disease spectrum in the pediatric intensive care units within 2 years before and after the outbreak of coronavirus disease 2019]. / æ–°åž‹å† çŠ¶ç— æ¯’è‚ºç‚Žç–«æƒ å‰åŽ2å¹´å„¿ç«¥é‡ç—‡ç›‘æŠ¤ç— æˆ¿ç–¾ç— è°±çš„å˜åŒ–.

OBJECTIVES: To investigate the changes in the disease spectrum among hospitalized children in the pediatric intensive care units (PICU) within 2 years before and after the outbreak of coronavirus disease 2019 (COVID-19). METHODS: The related data on disease diagnosis were collected from all children who were hospitalized in the PICU of Affiliated Hospital of Jining Medical College from January 2018 to December 2019 (pre-COVID-19 group) and from January 2020 to December 2021 (post-COVID-19 group). A statistical analysis was performed for the disease spectrum of the two groups. RESULTS: There were 2 368 children in the pre-COVID-19 group and 1 653 children in the post-COVID-19 group. The number of children in the post-COVID-19 group was reduced by 30.19% compared with that in the pre-COVID-19 group. There was a significant difference in age composition between the two groups (P<0.05). The top 10 diseases in the pre-COVID-19 group by number of cases were respiratory diseases, neurological diseases, sepsis, critical illness, circulatory system diseases, severe neurosurgical diseases, digestive system diseases, unintentional injuries, endocrine system diseases, and tumors. The top 10 diseases in the post-COVID-19 group by number of cases were respiratory diseases, neurological diseases, sepsis, circulatory system diseases, unintentional injuries, endocrine system diseases, severe neurosurgical diseases, acute abdomen, trauma surgical diseases, and digestive system diseases. The proportions of respiratory diseases, critical illness and severe neurosurgical diseases in the post-COVID-19 group were lower than those in the pre-COVID-19 group (P<0.05), while the proportions of unintentional injuries, acute abdomen, endocrine system diseases, trauma surgical diseases and sepsis were higher than those in the pre-COVID-19 group (P<0.05). CONCLUSIONS: COVID-19 epidemic has led to a significant reduction in the number of children admitted to the PICU, and there are significant changes in the disease spectrum within 2 years before and after the outbreak of COVID-19. Relevant prevention and control measures taken during the COVID-19 epidemic can reduce the incidence of respiratory diseases, neurological diseases, and other critical illness in children, but it is necessary to strengthen the prevention of unintentional injuries and chronic disease management during the epidemic.

Correlations among disability, anti-AQP4 antibody status and prognosis in the spinal cord involved patients with NMOSD.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare neuroinflammatory disorder of the central nervous system that typically involves the optic nerve, the spinal cord and other specific brain regions. In relapse of the disease, factors associated with clinical features and lesion severity are important for clinicians to predict disease-related disability. METHODS: We retrospectively analyzed 22 female patients with NMOSD who had spinal cord lesions. Detailed clinical features, onset symptoms, motor disability, relapse episodes, serum aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) autoantibodies and MRI characteristics were documented to correlate their associations with the nadir and three-month Expanded Disability Status Scale (EDSS) scores. Patients with three-month EDSS scores below four (< 4) were categorized as the good outcome group, while those with scores of four or more (> 4) were categorized as the poor outcome group. RESULTS: In patients with NMOSD, the mean age was 44.5 ± 12.8 years, and the mean three-month EDSS score was 4.3 ± 1.9. A significantly higher all-limb muscle power score was found in the good EDSS group than in the poor EDSS group (p = 0.01). A tendency toward longer follow-up periods and lower anti-AQP4 antibody levels was found in the good outcome group. Serum anti-AQP4 antibodies were present in 86% of patients with NMOSD, and MOG autoantibodies were found in one anti-AQP4 antibody-negative patient (33.3%). In patients with NMOSD, more than 40% of spinal cord lesions were distributed at the middle cervical and upper thoracic levels. CONCLUSIONS: Our findings suggest that EDSS scores and MRC scores at the nadir had significant associations with three-month EDSS scores. The topographic distributions of the spinal cord lesions might relate to different serum anti-AQP4 antibody status. However, further studies will be needed to corroborate this finding.

Association between metabolic parameters and risks of anemia and electrolyte disturbances among stages 3-5 chronic kidney disease patients in Taiwan.

BACKGROUND: Anemia and electrolyte disturbances are adverse outcomes of chronic kidney disease (CKD). This study explored the association between metabolic parameters with anemia and electrolyte and mineral disorders among CKD patients in Taiwan. METHODS: This cross-sectional study with a total of 2176 CKD stages 3-5 patients were collected from the Department of Nephrology at Shuang Ho Hospital, Taipei Medical University through the "Chronic Kidney Disease Common Care Network" database from December 2008 to April 2019. A multivariable-adjusted logistic regression expressed as odd ratios (OR) was performed to assess the association of metabolic parameters with anemia and electrolyte and mineral disorders. RESULTS: Elevated diastolic blood pressure, fasting blood glucose, and glycated hemoglobin A1c (HbA1c) were associated with presence of anemia. Similarly, elevated fasting blood glucose and HbA1c were associated with hyponatremia (OR = 1.59 and 1.58, P for both < 0.01) and hypercalcemia (OR = 1.38 and 1.33, P for both < 0.05). There was no significant association in serum lipid levels with presence of anemia. However, total triglycerides, total cholesterol and low-density lipoprotein-cholesterol were only associated with presence of hypercalcemia (OR = 1.43, 1.95 and 3.08, respectively, P for all < 0.05). CONCLUSIONS: Elevated diastolic blood pressure, fasting blood glucose, HbA1c and blood lipids are associated with anemia or electrolyte and mineral disorders in CKD patients.

Development and validation of a prediction model (AHC) for early identification of refractory thrombotic thrombocytopenic purpura using nationally representative data.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and life-threatening haematological emergency. Although therapeutic plasma exchange together with corticosteroids achieve successful outcomes, a considerable number of patients remain refractory to this treatment and require early initiation of intensive therapy. However, a method for the early identification of refractory iTTP is not available. To develop and validate a model for predicting the probability of refractory iTTP, a cohort of 265 consecutive iTTP patients from 17 large medical centres was retrospectively identified. The derivation cohort included 94 patients from 11 medical centres. For the validation cohort, we included 40 patients from the other six medical centres using geographical validation. An easy-to-use risk score system was generated, and its performance was assessed using internal and external validation cohorts. In the multivariable logistic analysis of the derivation cohort, three candidate predictors were entered into the final prediction model: age, haemoglobin and creatinine. The prediction model had an area under the curve of 0.886 (95% CI: 0.679-0.974) in the internal validation cohort and 0.862 (95% CI: 0.625-0.999) in the external validation cohort. The calibration plots showed a high agreement between the predicted and observed outcomes. In conclusion, we developed and validated a highly accurate prediction model for the early identification of refractory iTTP. It has the potential to guide tailored therapy and is a step towards more personalized medicine.

Comparative Study of Amide Proton Transfer-Weighted Imaging and Intravoxel Incoherent Motion Imaging in Breast Cancer Diagnosis and Evaluation.

BACKGROUND: Amide proton transfer-weighted imaging (APTWI) and intravoxel incoherent motion imaging (IVIM) are valuable MRI techniques applied to cancer. PURPOSE: To compare APTWI and IVIM in the diagnosis of benign and malignant breast lesions and to evaluate the correlations between different parameters (MTRasym [3.5 ppm], D, D*, and f) and prognostic factors for breast cancer. STUDY TYPE: Retrospective. POPULATION: In all, 123 breast lesions were studied before treatment, including 58 benign lesions and 65 malignant lesions. FIELD STRENGTH/SEQUENCE: Conventional MRI (T1 WI, T2 WI, and diffusion-weighted imaging [DWI]), APTWI, and IVIM MRI at 3T. ASSESSMENT: The magnetization transfer ratio asymmetry at 3.5 ppm (MTRasym [3.5 ppm]), diffusion coefficient (D), pseudo diffusion coefficient (D*), and perfusion fraction (f) values were compared between the benign and malignant groups and between groups with different expression levels of prognostic factors. STATISTICAL TESTS: Individual sample t-test, χ2 test, Spearman correlation, logistic regression, and the Delong test. RESULTS: The D and MTRasym (3.5 ppm) values of the malignant group were lower than those of the benign group; however, D* and f values were higher than those of the benign group (all P < 0.05). The areas under the curve (AUCs) of D, MTRasym (3.5 ppm), D*, and f were 0.809, 0.778, 0.670, and 0.766, respectively; however, only the difference between AUC (D) and AUC (D*) was significant (Z = 2.374, P < 0.05). The D value showed a low correlation with the pathological grade and Ki-67 expression (| r | = 0.294, 0.367); the f value showed a low correlation with estrogen receptor (ER) expression (| r | = 0.382); and the MTRasym (3.5 ppm) value showed a low correlation with pathological grade (| r | = 0.371). DATA CONCLUSION: This analysis revealed that both IVIM and APTWI could be used for the differential diagnosis of benign and malignant breast lesions, and APTWI-derived MTRasym (3.5 ppm), IVIM-derived D, D*, and f values showed correlations with some prognostic factors for breast cancer. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2 J. Magn. Reson. Imaging 2020;52:1175-1186.

A prospective, multicenter, noninterventional study in Taiwan to evaluate the safety and tolerability of lacosamide as adjunctive therapy for epilepsy in clinical practice.

RATIONALE: Lacosamide (LCM) was initially approved in Taiwan in March 2014 for use as adjunctive therapy for focal impaired awareness seizures and secondarily generalized seizures (SGS) in patients with epilepsy ≥16 years of age. The efficacy and tolerability of adjunctive LCM for the treatment of patients with focal seizures have been demonstrated in randomized, placebo-controlled trials. However, the trials do not reflect a flexible dose setting. This study (EP0063) was conducted to assess the safety and tolerability of LCM in real-world clinical practice in Taiwan. Effectiveness of LCM was also assessed as an exploratory objective. METHODS: EP0063 was a multicenter, prospective, noninterventional study with an expected observation period of 12 months ±â€¯60 days. Eligible patients were ≥16 years of age, had focal impaired awareness seizures and/or SGS (in line with approved indication in Taiwan at the time of the study), were taking at least one concomitant antiseizure medication (ASM), and had at least one seizure in the 3 months before baseline. Patients were prescribed LCM by their treating physician in the course of routine clinical practice. The primary safety variable was treatment-emergent adverse events (TEAEs) spontaneously reported to, or observed by, the treating physician. Based on safety data from previous studies of LCM and known side effects of other ASMs, certain TEAEs (including but not limited to cardiac and electrocardiogram, suicidality, and rash related terms) were analyzed separately. Effectiveness variables included Clinical Global Impression of Change (CGIC) and change in 28-day seizure frequency from baseline to 12 months (or final visit), and freedom from focal seizures. RESULTS: A total of 171 patients were treated with LCM, of whom 139 (81.3%) completed the study. The Kaplan-Meier estimated 12-month retention was 82.9%. Patients had a mean (standard deviation [SD], range) age of 38.5 (14.0, 16-77) years, and 96 (56.1%) were male. Patients were taking a mean (SD, range) of 2.8 (1.1, 1-6) ASMs at baseline. Mean (SD, range) duration of LCM treatment was 288.7 (111.9, 2-414) days, and the mean (SD, range) daily dosage of LCM was 205.0 (82.7, 50.0-505.2) mg/day. Overall, 95 (55.6%) patients reported at least one TEAE, most commonly dizziness (33 [19.3%] patients). Drug-related TEAEs were reported in 74 (43.3%) patients, and drug-related TEAEs leading to discontinuation of LCM were reported in 14 (8.2%) patients. Two (1.2%) patients died during LCM treatment, which were considered not related to LCM. Two (1.2%) patients had suicidality-related TEAEs; these TEAEs were considered either not related to LCM or the relationship was not recorded. Rash-related TEAEs were reported in five (2.9%) patients (considered LCM-related in two patients). Based on the CGIC, at 12 months (or final visit), 109 (63.7%) patients were considered to have improved, 54 (31.6%) had no change, and the remaining eight (4.7%) were minimally worse. At 12 months (or final visit), the median percentage change in focal seizure frequency was -50.0. During the first 6 months of the study, 21 (12.3%) patients were free from focal seizures; 37 (21.6%) patients were free from focal seizures in the last 6 months of the study; and 14 (8.2%) were free from focal seizures for the full 12 months of the study. CONCLUSIONS: Results of this prospective, noninterventional study suggest that adjunctive LCM was generally safe and well tolerated in this patient group in real-world practice in Taiwan. Effectiveness was also favorable, with more than 60% of patients considered to be improved by their physician at 12 months (or final visit).

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

BACKGROUND: The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees. METHODS: We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions. RESULTS: Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees. CONCLUSIONS: We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies.

Urine cell-free DNA as a promising biomarker for early detection of non-small cell lung cancer.

BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.

Electrical cortical stimulation for treatment of intractable epilepsy originating from eloquent cortex: surgical accuracy and clinical efficacy.

BACKGROUND: Electrical cortical stimulation is shown effective in treating patients with drug-resistant epilepsy. We demonstrated how detailed procedures of pre- and intra-operative planning of cortical stimulation implantation may influence the results of seizure reduction rate. METHODS: To confirm the precision of subdural grids covering the epileptogenic foci in the eloquent regions, pre- and intra-operative video-electroencephalography (VEEG) were performed in patients with drug-resistant epilepsy during a 4-day 24-h monitoring. The localization of the grid was determined via 3D reconstruction imaging of subdural electrodes co-registered onto the patient's cortex. A final quadripolar lead in cyclic stimulation mode was then placed and secured on the target cortex area. Post-operative 3D CT ensured the accurate location of stimulation lead without any misplacement. Bipolar cyclic stimulation and post-implantation VEEG were performed for 7 days. Patients were discharged and followed up regularly for parameters adjustment and recording of seizure outcomes. RESULTS: Eight patients received chronic cortical stimulation implantations between February 2003 and December 2017. The mean age of these patients was 21.1 years old and the average post-operative follow-up was 77.3 months. Comparisons of their seizure frequency at baseline and during the postoperative period revealed a mean reduction in seizures of 60.4% at the first year and 65.6% at the second year. CONCLUSIONS: Pre-surgical planning enhanced the accuracy of electrode placement and led to a favorable seizure reduction rate. Our report confirms that electrical cortical stimulation with detailed implantation procedures is safe and effective for patients with drug-resistant epilepsy originating from eloquent cortex.

The incidence and interpretation of large differences in EIT-based measures for PEEP titration in ARDS patients.

Positive end-expiratory pressure (PEEP) can be titrated by electrical impedance tomography (EIT). The aim of the present study was to examine the performance of different EIT measures during PEEP trials with the aim of identifying "optimum" PEEP and to provide possible interpretations of largely diverging results. After recruitment (maximum plateau pressure 35 cmH2O), decremental PEEP trial with steps of 2 cmH2O and duration of 2 min per step was performed. Ventilation gain and loss, the global inhomogeneity (GI) index, trend of end-expiratory lung impedance (EELI) and regional compliance (Creg) for estimation of overdistension and collapse were calculated. Largely diverging results of PEEP selection among the measures were defined as differences ≥ 4 PEEP steps (i.e. ≥ 8 cmH2O). In 30 ARDS patients we examined so far, 3 patients showed significant differences in PEEP selections. Overdistension and collapse estimation based on Creg tended to select lower PEEP while the GI index and EELI trend suggested higher PEEP settings. Regional inspiration times were heterogeneous indicating that the assumption of a uniform driving pressure in the calculation of Creg may not be valid. Judging by the predominant ventilation distribution in the most dependent regions, these patients were non-recruitable with the applied recruitment method or pressure levels. The existence of differences in the recommended PEEP among the analyzed EIT measures might be an indicator of non-recruitable lungs and heterogeneous airway resistances. In these extreme cases, the largely diverging results may prompt the attending clinician to develop individual ventilation strategies.Clinical Trial Registration Registration number NCT03112512, https://clinicaltrials.gov/ Registered 13 April 2017.

GPCR structure and function relationship: identification of a biased apelin receptor mutant.

Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained full capabilities in ligand binding and G protein activation, but was defective in GRK recruitment, ß-arrestin recruitment, and downstream receptor-mediated ERK activation. Based on molecular dynamics simulations, we proposed a molecular mechanism for biased signaling of I109A mutant receptor. We postulate that due to the extra space created by I109A mutation, the phenyl group of the last residue (Phe-13) of apelin rotates down and initiates a cascade of conformational changes in TM3. Phe-13 formed a new cluster of hydrophobic interactions with the sidechains of residues in TM3, including F1103.33 and M1133.36, which stabilizes the mutant receptor in a conformation favoring biased signaling. Interruption of these stabilizing interactions by double mutation F110A/I109A or M113A/I109A largely restored the ß-arrestin-mediated signaling. Taken together, we describe herein the discovery of a biased APJ mutant receptor and provide detailed molecular insights into APJ signaling selectivity, facilitating the discovery of novel therapeutics targeting APJ.