Possible Role of Docosahexaenoic Acid in Response to Diarrhetic Shellfish Toxins in the Mussel Perna viridis.

Marine bivalves are rich in docosahexaenoic acid (DHA), a polyunsaturated fatty acid known to be beneficial for human health; however, the potential role of DHA in protecting shellfish from the toxicity of diarrhetic shellfish toxins (DSTs) remains poorly understood. Here, we aimed to study the effect of DHA on the response of the bivalve, Perna viridis, to DSTs by using LC-MS/MS, RT-qPCR, and histological examination. In this study, we observed that the DHA content decreased significantly with esterification of DSTs in the digestive gland of the mussel P. viridis after 96 h of exposure to Prorocentrum lima, a DST-producing dinoflagellate. The addition of DHA significantly increased the esterification level of DSTs and increased the expression of Nrf2 signaling pathway-related genes and enzyme activities, alleviating the damage of DSTs to digestive glands. These results suggested that DHA may mediate the esterification of DSTs and activation of the Nrf2 signaling pathway in P. viridis to protect mussels from the toxic effects of DSTs. This study may provide new insights regarding the response of bivalves to DSTs and lay the foundation for uncovering the role of DHA in environmental adaptation of bivalves.

[Anti-tumor effect of Huaier extract supernatant on human gastric cancer HGC-27 and MGC-803 cells].

The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L~(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L~(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.

[Internal circadian clock and liver metabolism].

Circadian clock is an internal autonomous time-keeping system, including central clocks located in the suprachiasmatic nucleus (SCN) and peripheral clocks. The molecular circadian clock consists of a set of interlocking transcriptional-translational feedback loops that take the clock-controlled genes 24 h to oscillate. The core mechanism of molecular circadian clock is that CLOCK/BMAL1 dimer activates the transcription of cryptochromes (CRYs) and Periods (PERs), which act as transcriptional repressors of further CLOCK/BMAL1-mediated transcription. In addition to this basic clock, there is an additional sub-loop of REV-ERBα and RORα regulating the transcription of BMAL1. Approximately 80% protein-coding genes demonstrate significant rhythmicity. The earth rotation is responsible for the generation of the daily circadian rhythms. To coordinate metabolic balance and energy availability, almost all organisms adapt to the rhythm. Studies have shown that circadian clock integrating with metabolic homeostasis increases the efficiency of energy usage and coordinates with different organs in order to adapt to internal physiology and external environment soon. As the central organ of metabolism, the liver performs various physiological activities nearly all controlled by the circadian clock. There are multiple interactive regulation mechanisms between the circadian clock and the regulation of liver metabolism. The misalignment of metabolism with tissue circadian is identified as a high-risk factor of metabolic diseases. This article reviews the recent studies on circadian physiological regulation of liver glucose, lipid and protein metabolism and emphasizes oscillation of mitochondrial function. We also take an outlook for new methods and application of circadian clock research in the future.

[Research progress on anti-tumor effect of Chinese dragon's blood].

As a traditional Chinese medicine, Chinese dragon's blood has multiple effects, such as activating blood to remove blood stasis, softening and dispelling stagnation, astringent and hemostasis, clearing swelling and relieving pain, regulating menstruation and rectifying the blood, so it is called "an effective medicine of promoting blood circulation". It has been widely used clinically to treat a variety of diseases. With the further research on Chinese dragon's blood, its anti-tumor medicinal value is gradually emerging. Modern pharmacological studies have shown that Chinese dragon's blood exerts anti-tumor effects mainly by inhibiting cell proliferation, inducing apoptosis, inducing DNA damage and cell cycle arrest, inducing senescence and autophagy of tumor cells, inhibiting metastasis and angiogenesis, as well as reversing multidrug resistance. This article focuses on the research progress on anti-tumor effects of Chinese dragon's blood extract and its chemical components, with a view to provide new references for the in-depth research and reasonable utilization of Chinese dragon's blood.

Liraglutide Ameliorates Lipotoxicity-Induced Oxidative Stress by Activating the NRF2 Pathway in HepG2 Cells.

Although glucagon-like peptide-1 (GLP-1) analogue has been reported to suppress oxidative stress in non-alcoholic fatty liver disease (NAFLD), an effective therapeutic agent for NAFLD is currently unavailable. Therefore, in this study, we aimed to investigate the protective effects of the GLP-1 analogue liraglutide against lipotoxicity-induced oxidative stress in HepG2 cells and to elucidate the underlying mechanisms. HepG2 cells were cultured for 48 hours and treated with a free fatty acid (FFA) mixture: FFA mixture and liraglutide or FFA mixture, liraglutide, and exendin (9-39). Lipid accumulation was examined by oil red O staining. Oxidative stress was assessed by measuring the levels of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate and thiobarbituric acid-reactive substances, whereas antioxidant capacity was assessed by measuring the activity of superoxide dismutase and catalase. Expression of the nuclear factor erythroid-2-related factor 2 (NRF2) gene and the genes encoding antioxidant enzymes was analyzed using quantitative RT-PCR. Cellular and nuclear NRF2 expression levels were assessed using immunofluorescence cell staining and western blotting. Liraglutide treatment reduced high fat-induced lipid formation and the levels of oxidative stress markers and increased antioxidant enzyme activity in HepG2 cells. Liraglutide treatment increased the mRNA expression of NRF2 target genes, induced NRF2 nuclear translocation, and increased nuclear NRF2 levels without altering NRF2 mRNA expression. Collectively, these results indicate that liraglutide exhibits a protective effect against lipotoxicity-induced oxidative stress, possibly via modulation of NRF2 and expression of antioxidant enzymes in liver cells.

Premature Stroke Secondary to Severe Hypertension Results from Liddle Syndrome Caused by a Novel SCNN1B Mutation.

INTRODUCTION: Liddle syndrome (LS), an autosomal dominant and inherited monogenic hypertension syndrome caused by pathogenic mutations in the epithelial sodium channel (ENaC) genes SCNN1A, SCNN1B, and SCNN1G. OBJECTIVE: This study was designed to identify a novel SCNN1B missense mutation in a Chinese family with a history of stroke, and to confirm that the identified mutation is responsible for LS in this family. METHODS: DNA samples were collected from the proband and 11 additional relatives. Next-generation sequencing was performed in the proband to find candidate variants. In order to exclude genetic polymorphism, the candidate variantin SCNN1B was verified in other family members, 100 hypertensives, and 100 healthy controls by Sanger sequencing. RESULTS: Genetic testing revealeda novel and rare heterozygous variant in SCNN1B in the proband. This variant resulted in a substitution of threonine instead of proline at codon 617, altering the PY motif of ß-ENaC. The identified mutation was only verified in 5 relatives. In silico analyses indicated that this variant was highly pathogenic. In this family, phenotypic heterogeneity was present among 6 LS patients. Tailored medicine with amiloride was effective in controlling hypertension and improving the serum potassium concentration in patients with LS. CONCLUSIONS: We identified a novel SCNN1B mutation (c.1849C>A) in a family affected by LS. Patients with LS, especially those with severe hypertension, should be alert for the occurrence of premature stroke. Timely diagnosis using genetic testing and tailored treatment with amiloride can help LS patients to avoid severe complications.

[Effect of Huaier aqueous extract on growth and metastasis of human non-small cell lung cancer NCI-H1299 cells and its underlying mechanisms].

This study aims to investigate the effect of Huaier aqueous extract on the growth and metastasis of human non-small cell lung cancer NCI-H1299 cells and its underlying mechanisms. MTT assay was used to detect the effect of Huaier aqueous extract on the proliferation of NCI-H1299 cells. Flow cytometry was used to examine the effect of Huaier aqueous extract on the apoptosis, cell cycle, and ROS level of NCI-H1299 cells. Wound healing assay was used to evaluate the effect of Huaier aqueous extract on the migration ability of NCI-H1299 cells. Western blot was used to detect the levels of proteins involving apoptosis, epithelial-mesenchymal transition(EMT), and MAPK signaling pathway in NCI-H1299 cells exposed to Huaier aqueous extract. The results showed that Huaier aqueous extract inhibited the proliferation of NCI-H1299 cells, and induced cell-cycle arrest at the phase S. Huaier aqueous extract promoted the apoptosis of NCI-H1299 cells by down-regulating the expression of anti-apoptotic protein Bcl-2. Moreover, Huaier aqueous extract increased ROS level and induced ferroptosis in NCI-H1299 cells. EMT played a critical role in cancer metastasis. Huaier aqueous extract reduced the migration ability of NCI-H1299 cells by inhibiting EMT of NCI-H1299 cells. In addition, this study revealed that Huaier aqueous extract inhibited MAPK signaling pathway in human non-small cell lung cancer NCI-H1299 cells, which may be one of Huaier's mechanisms in inhibiting growth and metastasis of NCI-H1299 cells. This study provides a new theoretical basis for the clinical treatment of lung cancer with Huaier, and important reference significance for further studies on the anti-tumor mechanisms of Huaier.

A Human Health Risk Assessment of Trace Elements Present in Chinese Wine.

The concentrations of trace elements in wines and health risk assessment via wine consumption were investigated in 315 wines. Samples were collected from eight major wine-producing regions in China. The concentrations of twelve trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS) and Duncan's multiple range test was applied to analyze significant variations (p < 0.05) of trace elements in different regions. Based on a 60 kg adult drinker consuming 200 mL of wine per day, the estimated daily intake (EDI) of each element from wines was far below the provisional tolerable daily intake (PTDI). Health risk assessment indicated the ingestion influence of individual elements and combined elements through this Chinese wine daily intake did not constitute a health hazard to people. However, Cr and Mn were the potential contaminants of higher health risk in Chinese wines. The cumulative impact of wine consumption on trace elements intake in the daily diet of drinkers should not be ignored due to the presence of other intake pathways.

Effect of Nano-Carbon Black Surface Modification on Toxicity to Earthworm (Eisenia fetida) Using Filter Paper Contact and Avoidance Test.

Engineered nanomaterials (NMs) may enter the soil through various channels and pose potential harm to soil animals, especially those proactively applied for soil heavy metal remediation. Effects of nano-carbon black (CB) and surface modified carbon black (MCB) on catalase (CAT) activity and malondialdehyde (MDA) content in earthworms exposed on filter paper for 48 h were tested. Avoidance test was used to determine hazard of soil treated with 0.015% and 1.5% CB and MCB. Surface properties of NMs were also characterized. MCB has a significant effect on CAT activity at 70 and 1000 mg/L (1.1 and 15.7 µg/cm2), but has no impact on MDA content in earthworm. Strongly avoidance behavior of worms was also found in soil added 1.5% MCB. Negative charges and oxygen functional groups increased for MCB and its adverse effect on earthworm was higher than CB. The application of MCB in soil remediation warrants more attention.

Genetic screening of SCNN1B and SCNN1G genes in early-onset hypertensive patients helps to identify Liddle syndrome.

BACKGROUND: Liddle syndrome is an autosomal dominant form of monogenic hypertension. Phenotypic variability makes it difficult to identify patients with Liddle syndrome, resulting in misdiagnosis and severe complications at early age. OBJECTIVES: To identify mutation in SCNN1B and SCNN1G genes in an adolescent with suspicious Liddle syndrome and his family members and to explore the screening target subjects of Liddle syndrome. METHODS: Genetic analysis of the C-terminus of SCNN1B and SCNN1G genes was conducted in an adolescent, with treatment-resistant hypertension and hypokalemia, who was suspected of having Liddle syndrome, and his family members. A Medline research of the reported cases with Liddle syndrome was also performed. RESULTS: A recurrent SCNN1B mutation, c.1853C>A (p.P618H), was detected in the 19-year-old male patient, and family screening identified five additional members who were heterozygous for the mutation. The diagnosis of Liddle syndrome was made in all affected individuals. Despite the phenotypic variability, a systematic review of 54 reported index cases revealed the early-onset hypertension, aged no more than 30 years, as a common feature. CONCLUSIONS: Genetic screening for Liddle syndrome should be considered in hypertensive subjects with early penetrance, maybe no more than 30 years, after exclusion of common secondary causes of hypertension.

Active form of vitamin D ameliorates non-alcoholic fatty liver disease by alleviating oxidative stress in a high-fat diet rat model.

The purpose of this study was to determine whether treatment using the active form of vitamin D (1,25(OH)2D3) could protect against high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats and ameliorate oxidative stress. Male Sprague-Dawley rats were divided into three groups and treated with standard chow, HFD, or HFD plus intraperitoneal injection of 1,25(OH)2D3 (5 µg/kg body weight, twice per week), respectively, for 16 weeks. Serum lipid profiles, hepatic function, intrahepatic lipid, and calcium levels were determined. Hepatic histology was examined using hematoxylin/eosin, Masson's trichrome, and Oil Red O staining. Oxidative stress was assessed by measuring hepatic malondialdehyde (MDA) and F2α-isoprostane content. Expression of nuclear factor-erythroid-2-related factor 2 (Nrf2) and downstream target genes was analyzed using quantitative RT-PCR. 1,25(OH)2D3 treatment improved the serum lipid profile, reduced intrahepatic lipid levels, and attenuated hepatic steatosis and inflammation in HFD rats. Furthermore, MDA and F2α-isoprostane levels in liver tissue were reduced by 1,25(OH)2D3 administration. Although 1,25(OH)2D3 did not regulate the expression of Nrf2 mRNA, it did induce Nrf2 nuclear translocation. The expression of Nrf2 target genes, including Gclc, Nqo1, Sod2, and Cat, was up-regulated by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 protects against HFD-induced NAFLD by attenuating oxidative stress, inducing NRF2 nuclear translocation, and up-regulating the expression of genes encoding antioxidant enzymes.

[Clinical application features of Fufang Kushen injection in treating malignant esophageal tumor： real world study based on hospital information system].

To analyze the clinical application features of Fufang Kushen injection in treating malignant esophageal tumors in the real world by using hospital information system database, and provide reference for clinical application of Fufang Kushen injection. The electronic medical data from 2 550 patients with malignant esophageal tumors using Fufang Kushen injection from 22 large-scale hospitals nationwide were extracted based on the hospital information system (HIS) established by Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences. Then the descriptive analysis based on frequency and rate was conducted for general characteristics, diagnostic characteristics, characteristics of dosage and medication information, characteristics of drug combination, and discharge outcome characteristics. The results showed that the average age of patients treated with Fufang Kushen injection for malignant esophageal tumors was 62.3 years old; more males than females; admitted to hospital mainly in department of cardiology, oncology and digestology first. The total efficiency was 47.15% based on the discharge outcome characteristics; the most common dosage was 10-20 mL for single use; the course of treatment was mainly 4-7 d; and the common drugs in drug combinations included dexamethasone, tropisetron injection, thymosin injection, compound amino acid injection, pantoprazole sodium injection, fluorouracil, et al. The characteristics of the crowd using Fufang Kushen injection to treat the malignant esophageal tumors were clear and in line with the general rule of malignant esophageal tumors; its clinical dosage and scope of treatment for malignant esophageal tumors in the real world were basically consistent with the specification; and the types of clinical drug use combinations were more extensive.

A novel frameshift mutation of epithelial sodium channel ß-subunit leads to Liddle syndrome in an isolated case.

OBJECTIVE: Liddle syndrome, an autosomal dominant form of monogenic hypertension, is attributed to mutations in the genes encoding ß and γ subunits (SCNN1B and SCNN1G) of the epithelial sodium channel (ENaC). The aim of this study was to search for pathogenic mutations of SCNN1B and SCNN1G in an adolescent under the impression of Liddle syndrome and no family history of hypertension. DESIGN AND PATIENTS: We screened the C-terminus of SCNN1B and SCNN1G in an adolescent with poorly controlled hypertension who was clinically diagnosed as having Liddle syndrome. We also screened for the mutation in his parents, 100 hypertensive patients and 100 controls. RESULTS: Genetic analysis of SCNN1B revealed a frameshift mutation induced by insertion of an additional cytosine into a string of six located between codons 617 and 618, which is predicted to introduce a new termination codon at position 621 and produce a protein truncated by 20 amino acids. This frameshift mutation was not detected in the patient's parents, the 100 hypertensive patients or the 100 controls, indicating that this is a de novo mutation and not a common genetic polymorphism. There was no mutation of SCNN1G in any of the individuals examined. CONCLUSION: Based on direct DNA sequencing, we identified a novel frameshift mutation in the ßENaC gene in an isolated case of Liddle syndrome. Confirmation of the diagnosis and effective tailored treatment in the patient were achieved, implying that genetic testing is a useful tool to diagnose Liddle syndrome.

Mutation of Disulfide Bond Sites Reduces the Immunoreactivity of Cra a 4 by Changing the Structural Characteristics.

Sarcoplasmic calcium-binding protein (Cra a 4) from Crassostrea angulata belongs to the EF-hand superfamily, and understanding of its structure-allergenicity relationship is still insufficient. In this study, chemical denaturants were used to destroy the structure of Cra a 4, showing that disruption of the structure reduced its IgG-/IgE-binding activity. To explore which critical amino acid site affects the allergenicity of Cra a 4, the mutants were obtained by site-directed mutations in the disulfide bonds site (C97), conformational epitopes (I105, D114), or Ca2+-binding region (D106, D110) and their IgG-/IgE-binding activity was reduced significantly using serological tests. Notably, C97A had the lowest immunoreactivity. In addition, two conformational epitopes of Cra 4 were verified. Meanwhile, the increase of the α-helical content, surface hydrophobicity, and surface electrostatic potential of C97A affected its allergenicity. Overall, the understanding of the structure-allergenicity relationship of Cra a 4 allowed the development of a hypoallergenic mutant.

Enhancement of ionospheric heating effect by chemical release.

The ionosphere can be artificially modified by employing ground-based high-power high-frequency electromagnetic waves to irradiate the ionosphere. This modification is achieved through the nonlinear interaction between the electromagnetic waves and the ionospheric plasma, leading to changes in the physical properties and structure of the ionosphere. The degree of artificial modification of the ionosphere is closely related to the heating energy density of high-frequency pump waves. Due to the high density of neutral constituents in the lower ionosphere and the high frequency of electron-neutral collisions, the energy of heating pump waves will be absorbed and attenuated during the penetration of the low ionosphere, seriously affecting the heating effect. This paper proposes a method to reduce the absorption of ionospheric heating pump waves by releasing electron attachment chemicals into low ionosphere to form a large-scale electron density hole. A model for mitigating pump waves absorption based on SF6 release is established, and the absorption at different frequencies is quantitatively calculated. The propagation characteristics of high-frequency signals in ionospheric holes are studied using a three-dimensional ray tracing method, and the results demonstrate that the chemical release method not only reduces the absorption attenuation of heating pump waves but also forms spherical electron density holes, which exhibit a focusing effect on the heating beam and enhance the heating effect. The results are of great significance for understanding the nonlinear interaction between electromagnetic wave and ionospheric plasma and improving the ionospheric heating efficiency.

Damage Effect of Amorphous Carbon Black Nanoparticle Aggregates on Model Phospholipid Membranes: Surface Charge, Exposure Concentration and Time Dependence.

Commercial nano-scale carbon blacks (CB) are being harnessed widely and may impose potentially hazardous effects because of their unique properties, especially if they have been modified to grow reactive functional groups on their surface. Cytotoxicity of CB has been well studied but the membrane damage mechanisms and role of surface modification are still open to debate. Negatively and positively charged giant unilamellar vesicles (GUVs) were prepared using three lipids as model cell membranes to examine the mechanistic damage of CB and MCB (modified by acidic potassium permanganate) aggregates. Optical images showed that both anionic CB and MCB disrupted the positively charged but not the negatively charged GUVs. This disruption deteriorated with the rise and extension of exposure concentration and time. Lipids extraction caused by CBNs (CB and MCB together are called CBNs) was found. MCB caused more severe disruption than CB. MCB was enveloped into vesicles through an endocytosis-like process at 120 mg/L. MCB mediated the gelation of GUVs, perhaps through C-O-P bonding bridges. The lower hydrodynamic diameter and more negative charges may have been responsible for the distinction effect of MCB over CB. The adhesion and bonding of CBNs to the membrane were favored by electrostatic interaction and the practical application of CBNs warrants more attention.

Clinical characteristics and management of coexistent cardiomyopathy in patients with bicuspid aortic valve.

BACKGROUND: Bicuspid aortic valve (BAV) is the most common congenital heart disease. However, the prevalence, clinical characteristics, and current management of BAV associated with inherited cardiomyopathy, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC) have not been well described. METHODS: Consecutive patients diagnosed with BAV at a large tertiary cardiovascular referral center between 2009 and 2018 were retrospectively assessed for HCM, DCM, and LVNC based on clinical and echocardiographic criteria. Patients with coexistent conditions were investigated further. RESULTS: Of 3533 patients with BAV screened, 57 (1.6%) had concomitant cardiomyopathy. BAV was combined with HCM in 30 of these patients, with DCM in 19, and with LVNC in eight. Forty-six patients (80.7%) were male, and the mean age at first diagnosis was 47 years for BAV with HCM, 49 years for BAV with DCM, and 35 years for BAV with LVNC. Heart failure and aortic valve dysfunction were common in these patients, and the prevalence of coexisting aortopathy was 43.3%, 26.3% and 25.0%, respectively, for BAV with HCM, DCM and LVNC. During the index hospitalization, 24 of the 57 patients (42.1%) underwent surgery, 16 (28%) underwent aortic valve and/or aortic surgery, and 16 of the 30 patients with HCM had a Morrow procedure. There were no deaths or other major adverse cardiovascular events. CONCLUSIONS: The prevalence of inherited cardiomyopathy was higher in our patients with BAV than in the general population. Aortopathy and heart failure were common, with almost half of patients requiring surgery at diagnosis.

Implication of a novel truncating mutation in titin as a cause of autosomal dominant left ventricular noncompaction.

BACKGROUND: Mutation in the titin gene (TTN) in left ventricular noncompaction (LVNC) has been reported with a highly heterogeneous prevalence, and the molecular mechanisms underlying the pathogenesis of TTN gene mutation are uncharacterized. In the present study, we identified a novel TTN mutation in a pedigree with LVNC and investigated the potential pathogenic mechanism by functional studies. METHODS: The whole-genome sequencing with linkage analysis was performed in a 3-generation family affected by autosomal dominant LVNC cardiomyopathy. The clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) technology was used to establish novel truncating mutation in TTN in a rat cardiomyoblast H9C2 cell line in vitro, in which functional studies were carried out and characterized in comparison to its wild-type counterpart. RESULTS: A novel truncating mutation TTN p. R2021X was identified as the only plausible disease-causing variant that segregated with disease among the five surviving affected individuals, with an interrogation of the entire genome excluding other potential causes. Quantitative reverse transcription-polymerase chain reaction and cellular immunofluorescence supported a haploinsufficient disease mechanism in titin truncation mutation cardiomyocytes. Further functional studies suggested mitochondrial abnormities in the presence of mutation, including decreased oxygen consumption rate, reduced adenosine triphosphate production, impaired activity of electron translation chain, and abnormal mitochondrial structure on electron microscopy. Impaired autophagy under electron microscopy accompanied with activation of the Akt-mTORC1 signaling pathway was observed in TTN p. R2021X truncation mutation cardiomyocytes. CONCLUSIONS: The TTN p. R2021X mutation has a function in the cause of a highly penetrant familial LVNC. These findings expand the spectrum of titin's roles in cardiomyopathies and provide novel insight into the molecular basis of titin-truncating variants-associated LVNC.

Pathogenicity and Long-Term Outcomes of Liddle Syndrome Caused by a Nonsense Mutation of SCNN1G in a Chinese Family.

Objective: Liddle syndrome (LS) is a monogenic hypertension consistent with autosomal dominant inheritance, often with early onset high blood pressure in childhood or adolescence. This study aimed to identify the pathogenicity of a nonsense mutation in SCNN1G in a Chinese family with LS and the long-term outcomes of tailored treatment with amiloride. Methods: To explore the pathogenicity of candidate variant reported in 2015 by our team, we constructed mutant and wild-type models in vitro and measured amiloride-sensitive current in Chinese Hamster Ovary (CHO) cells using patch clamp technique. Participants were followed up for 7 years after tailored treatment with amiloride. Results: A nonsense variant was detected in six members, two of whom were pediatric patients. This mutation resulted in a termination codon at codon 572, truncating the Pro-Pro-Pro-X-Tyr motif. The mutant epithelial sodium channels displayed higher amiloride-sensitive currents than the wild-type channels (P < 0.05). Tailored treatment with amiloride achieved ideal blood pressure control in all patients with normal cardiorenal function, and no adverse events occurred during follow-up. Conclusion: We found the pathogenicity of a nonsense SCNN1G mutation (p.Glu571*) with enhanced amiloride-sensitive currents in a LS family with young patients. Tailored treatment with amiloride may be an effective strategy for the long-term control of blood pressure and protection from target organ damage or cardiovascular events, including children and youth patients with LS.

A Novel Frame-Shift Mutation in SCNN1B Identified in a Chinese Family Characterized by Early-Onset Hypertension.

Background: Liddle syndrome is a form of monogenic hypertension caused by mutations in the three homologous subunits of the epithelial sodium channels (ENaCs), α, ß, and γ. It is characterized by early-onset refractory hypertension, hypokalemia, low renin activity, and hypoaldosteronism. In this study, we report a novel frame-shift mutation in SCNN1B responsible for Liddle syndrome in a Chinese family. Methods: DNA samples were collected from all participants. Whole-exome sequencing was performed in the proband to detect possible causative variants. Sanger sequencing was then conducted in the other family members to verify the candidate variant, and in 100 patients with hypertension and 100 normotensive controls to exclude population genetic polymorphism. Results: We identified a novel frame-shift mutation (c.1691_1693delinsG) in SCNN1B that was responsible for Liddle syndrome in this family. This mutation leads to the substitution of Arg in place of Gln at codon site 564 and generates a new stop codon at 592, influencing the crucial PY motif and resulting in reduced inactivation of the ENaCs. Aside from the proband, eight family members carried the mutation. Intra-familial phenotypic heterogeneity was observed in the blood pressure and serum potassium levels. Amiloride therapy combined with a low sodium diet is effective to alleviate the symptoms of patients with Liddle syndrome. Conclusion: c.1691_1693delinsG, a novel frame-shift mutation in the ß subunit of ENaC, was identified in a Chinese family with Liddle syndrome by whole-exome sequencing. Phenotypic heterogeneity can make diagnosis of Liddle syndrome difficult on the basis of clinical or biochemical characteristics alone. Genetic analysis is a useful tool allowing timely and accurate diagnosis of Liddle syndrome and playing a guiding role in precise treatment of the disease.