Association of a novel frameshift variant and a known deleterious variant in MMR genes with Lynch syndrome in Chinese families.

BACKGROUND: Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome. This condition is characterized by germline variants in DNA mismatch repair (MMR) genes, including MLH1, MSH2, MSH6, and PMS2. In this study, we analyzed the molecular defects and clinical manifestations of two families affected with CRC and proposed appropriate individual preventive strategies for all carriers of the variant. METHODS: We recruited two families diagnosed with CRC and combined their family history and immunohistochemical results to analyze the variants of probands and those of other family members by using whole exome sequencing. Subsequently, gene variants in each family were screened by comparing them with the variants available in the public database. Sanger sequencing was performed to verify the variant sites. An online platform ( https://www.uniprot.org ) was used to analyze the functional domains of mutant proteins. RESULTS: A novel frameshift variant (NM_001281492, c.1129_1130del, p.R377fs) in MSH6 and a known deleterious variant (NM_000249.4:c.1731G > A, p.S577S) in MLH1 were identified in the two families with CRC. Using bioinformatics tools, we noted that the frameshift variant reduced the number of amino acids in the MSH6 protein from 1230 to 383, thereby leading to no MSH6 protein expression. The silent variant caused splicing defects and was strongly associated with LS. 5-Fluorouracil-based adjuvant chemotherapy is not recommended for patients with LS. CONCLUSIONS: The novel frameshift variant (MSH6, c.1129_1130del, p.R377fs) is likely pathogenic to LS, and the variant (MLH1, c.1731G > A, p.S577S) has been further confirmed to be pathogenic to LS. Our findings underscore the significance of genetic testing for LS and recommend that genetic consultation and regular follow-ups be conducted to guide individualized treatment for cancer-afflicted families, especially those with a deficiency in MMR expression.

AGEs promote atherosclerosis by increasing LDL transcytosis across endothelial cells via RAGE/NF-κB/Caveolin-1 pathway.

OBJECTIVE: To elucidate the mechanism whereby advanced glycation end products (AGEs) accelerate atherosclerosis (AS) and to explore novel therapeutic strategies for atherosclerotic cardiovascular disease. METHODS AND RESULTS: The effect of AGEs on low-density lipoprotein (LDL) transcytosis across endothelial cells (ECs) was assessed using an in vitro model of LDL transcytosis. We observed that AGEs activated the receptor for advanced glycation end products (RAGE) on the surface of ECs and consequently upregulated Caveolin-1, which in turn increased caveolae-mediated LDL transcytosis and accelerated AS progression. Our molecular assessment revealed that AGEs activate the RAGE-NF-κB signaling, which then recruits the NF-κB subunit p65 to the RAGE promoter and consequently enhances RAGE transcription, thereby forming a positive feedback loop between the NF-κB signaling and RAGE expression. Increased NF-κB signaling ultimately upregulated Caveolin-1, promoting LDL transcytosis, and inhibition of RAGE suppressed AGE-induced LDL transcytosis. In ApoE-/- mice on a high-fat diet, atherosclerotic plaque formation was accelerated by AGEs but suppressed by EC-specific knockdown of RAGE. CONCLUSION: AGEs accelerate the development of diabetes-related AS by increasing the LDL transcytosis in ECs through the activation of the RAGE/NF-κB/Caveolin-1 axis, which may be targeted to prevent or treat diabetic macrovascular complications.

Identification of an LDLR variant in a Chinese familial hypercholesterolemia and its relation to ROS/NLRP3-Mediated pyroptosis in hepatic cells.

BACKGROUND: Familial hypercholesterolemia (FH) is a common autosomal dominant hereditary disease. Its early diagnosis and intervention significantly improve the patient's quality of life. However, there are few types of research on the FH pathogenic genes in China. METHODS: In this study, we recruited a family diagnosed with FH and used whole exome sequencing (WES) to analyze the proband variants. Intracellular cholesterol level, reactive oxygen species (ROS) level, and the expression of pyroptosis-related genes were detected after overexpression of wild-type or variant LDLR in L02 cells. RESULTS: A heterozygous missense variant predicted to be deleterious to LDLR (c.1879G > A, p.Ala627Thr) was identified in the proband. Mechanistically, intracellular cholesterol level, ROS level, and the expression of pyroptosis-related genes, nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome and components (caspase 1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NLRP3), gasdermin D (GSDMD), interleukin (IL) -18, IL-1ß was elevated in the variant LDLR group, which was attenuated by inhibition of ROS. CONCLUSIONS: FH is associated with a variant (c.1879G>A, p.Ala627Thr) in the LDLR gene. Regarding the mechanism, the ROS/NLRP3-mediated pyroptosis in hepatic cells may contribute to the pathogenesis of the LDLR variant.

Remnant cholesterol is independently asssociated with an increased risk of peripheral artery disease in type 2 diabetic patients.

Background: Remnant cholesterol (RC) has been correlated with a higher risk of atherosclerosis. It has been confirmed that in the general population, an elevated RC level is related to a 5-fold higher risk of peripheral arterial disease (PAD). Diabetes is one of the strongest risk factors for PAD development. However, the association between RC and PAD in the specific population of type 2 diabetes mellitus (T2DM) has not been investigated. Herein, the correlation was investigated between RC and PAD in T2DM patients. Methods: In the retrospective study, the hematological parameter data of 246 T2DM patients without PAD (T2DM - WPAD) and 270 T2DM patients with PAD (T2DM - PAD) was collected. Differences in RC levels between the two groups were compared, and the association between RC and PAD severity was examined. Multifactorial regression was used to determine whether RC was a significant contributor to the development of T2DM - PAD. The diagnostic potential of RC was tested using receiver operating characteristic (ROC) curve. Results: The RC levels in T2DM - PAD individuals were considerably greater than in T2DM - WPAD individuals (P < 0.001). RC had a positive correlation with disease severity. Further, multifactorial logistic regression analyses found that elevated RC levels were a major contributor to T2DM - PAD (P < 0.001). The area under the curve (AUC) of the RC for T2DM - PAD patients was 0.727. The cut-off value of RC was 0.64 mmol/L. Conclusion: The RC levels were higher in T2DM - PAD patients, and were independently linked with its severity. Diabetic patients with RC levels > 0.64 mmol/L had an elevated risk of developing PAD.

Combination model of neutrophil to high-density lipoprotein ratio and system inflammation response index is more valuable for predicting peripheral arterial disease in type 2 diabetic patients: A cross-sectional study.

Background: Neutrophil/high-density lipoprotein (HDL) ratio (NHR), monocyte/HDL ratio (MHR), lymphocyte/HDL ratio (LHR), platelet/HDL ratio (PHR), systemic immune-inflammation index (SII), system inflammation response index (SIRI), and aggregate index of systemic inflammation (AISI) have been recently investigated as novel inflammatory markers. Herein, the correlation was investigated between these inflammatory biomarkers and peripheral arterial disease (PAD) in type 2 diabetes mellitus (T2DM) patients. Methods: In this retrospective observational study, the hematological parameter data of 216 T2DM patients without PAD (T2DM-WPAD) and 218 T2DM patients with PAD (T2DM-PAD) at Fontaine stages II, III or IV stage had been collected. Differences in NHR, MHR, LHR, PHR, SII, SIRI, and AISI were analyzed, and receiver operating characteristic (ROC) curves were used to analyze the diagnostic potential of these parameters. Results: The levels of NHR, MHR, PHR, SII, SIRI and AISI in T2DM-PAD patients were significantly higher than in T2DM-WPAD patients (P < 0.001). They were correlated with disease severity. Further, multifactorial logistic regression analyses showed that higher NHR, MHR, PHR, SII, SIRI, and AISI might be independent risk factors for T2DM-PAD (P < 0.001). The areas under the curve (AUCs) of the NHR, MHR, PHR, SII, SIRI, and AISI for T2DM-PAD patients was 0.703, 0.685, 0.606, 0.648, 0.711, and 0.670, respectively. The AUC of the NHR and SIRI combined model was 0.733. Conclusion: The levels of NHR, MHR, PHR, SII, SIRI, and AISI were higher in T2DM-PAD patients, and they were independently linked with its clinical severity. The combination model of NHR and SIRI was most valuable for predicting T2DM - PAD.

Scavenger receptor a mediates glycated LDL transcytosis across endothelial cells to promote atherosclerosis.

Glycated low-density lipoprotein (G-LDL) is an established proatherosclerotic factor, but the mechanism is not completely understood. In vitro, we evaluated the uptake and transcytosis rates of N-LDL and G-LDL in endothelial cells and the uptake and transcytosis rates of G-LDL were much higher than those of N-LDL. Then, using small interfering RNAs, the receptor mediating G-LDL uptake and transcytosis was screened among eight candidate receptors, and the mechanism of the receptor regulation was thoroughly examined. We discovered that scavenger receptor A (SR-A) knockdown dramatically decreased the uptake and transcytosis rates of G-LDL. Additionally, endothelial cells with overexpressed SR-A had enhanced G-LDL uptake and transcytosis. In vivo, G-LDL was injected in the tail vein of ApoE-/- mice to investigate whether G-LDL affects atherosclerotic plaque formation. Compared with the injection of N-LDL, the injection of G-LDL accelerated atherosclerotic plaque formation in ApoE-/- mice, which was ameliorated by endothelial cells specific SR-A knockdown. Together, our results provide the first demonstration that the transcytosis of G-LDL across endothelial cells is much faster than that of N-LDL and SR-A is the major type of receptor responsible for G-LDL binding and transcytosis across endothelial cells.

C-band 100-GBaud/λ PAM-4 transmission enabled by hardware-efficient nonlinear equalizer with weight-sharing pruning.

A Volterra nonlinear equalizer (VNLE) can effectively mitigate both linear and nonlinear impairments arising in intensity-modulation direct-detection (IM-DD) transmission systems. However, the high computational complexity of the VNLE hinders its applications. Here, we propose a hardware-efficient Volterra equalizer with weight-sharing pruning (VE-WSP). Such an equalizer first uses the k-means++ clustering algorithm for the weight sharing within the same cluster, and then ranks the cluster centroid weight for pruning, leading to a significant computational complexity reduction without sacrificing any equalization performance. We experimentally verified that the use of VE-WSP can enable the C-band 100-GBaud/λ PAM-4 transmission over a 1-km standard single-mode fiber (SSMF), to reach the 20% soft-decision forward error correction (SD-FEC) threshold. Meanwhile, the proposed VE-WSP can reduce the computational complexity by 51% and 21%, in terms of multiplication and addition, respectively, in comparison with the VNLE.

VCAM-1-binding peptide targeted cationic liposomes containing NLRP3 siRNA to modulate LDL transcytosis as a novel therapy for experimental atherosclerosis.

BACKGROUND: Activation of NLRP3 inflammasome accelerates the formation of atherosclerotic plaques. Here, we evaluated the effects of inflammation on the expression of the NLRP3 inflammasome in endothelial cells (ECs). METHODS: The effect of TNF-α on transcytosis of LDL was measured. VCAM-1 binding peptide targeting cationic liposomes (PCLs) were prepared as siRNA vectors. Methylated NLRP3 siRNA was encapsulated into the PCLs to knock down NLRP3 in vitro and in vivo. In rats with partial carotid ligation, TNF-α-induced LDL retention in the carotid artery endothelium was observed. In ApoE-/- mice, NLRP3 siRNA-PCLs were injected intravenously to observe their effect on the formation of atherosclerosis. RESULTS: Our results showed that TNF-α upregulated NLRP3 in ECs, promoting the assembly of the NLRP3 inflammasome and processing of pro-IL-1ß into IL-1ß. Moreover, TNF-α accelerated LDL transcytosis in ECs. Knockdown of NLRP3 prevented TNF-α-induced NLPR3 inflammasome/IL-1ß signaling and LDL transcytosis. Using optimized cationic liposomes to encapsulate methylated NLRP3 siRNA, resulting in targeting of VCAM-1-expressing ECs, to knockdown NLRP3, TNF-α-induced NLRP3 inflammasome activation and LDL transcytosis were prevented. Using the partial carotid ligation as an atherosclerosis rat model, we found that local administration of NLRP3 siRNA-PCLs efficiently knocked down NLPR3 expression in the carotid endothelium and dramatically attenuated the deposition of atherogenic LDL in carotid ECs in TNF-α-challenged rats. Furthermore, NLRP3 siRNA-PCLs were injected intravenously in ApoE-/- mice, resulting in reduced plaque formation. CONCLUSION: These findings established a novel strategy for targeting the NLRP3 inflammasome using NLRP3 siRNA-PCLs to interrupt LDL transcytosis, representing a potential novel therapy for atherosclerosis.

Deacetylation of Caveolin-1 by Sirt6 induces autophagy and retards high glucose-stimulated LDL transcytosis and atherosclerosis formation.

BACKGROUND: Atherosclerosis (AS) is the basis of diabetic macrovascular complications. The plasma low-density lipoprotein (LDL) particles transcytosis across endothelial cells (ECs) and deposition under the endothelium is the initiation step of AS. We previously reported that high glucose inhibits the autophagic degradation of Caveolin-1 and promote LDL transcytosis across ECs, which in turn accelerates atherosclerotic progression. Since Sirt6 is a chromatin-associated protein with deacetylation activity, whether it can regulate Caveolin-1 acetylation and regulating the autophagic degradation of Caveolin-1 remains elusive. METHODS: Autophagy and histone acetylation were assessed in the umbilical cords of patients with gestational diabetes mellitus (GDM) by immunohistochemistry. An in vitro model of LDL transcytosis was established, and the role of Sirt6 in LDL transcytosis across endothelial cells was clarified. The effect of Sirt6 on the autophagic degradation of Caveolin-1 under hyperglycemic conditions was explored in a streptozotocin (STZ)-induced diabetic AS model established using the ApoE-/- mice. RESULTS: Caveolin-1 and acetylated histone H3 levels were significantly increased, while LC3B and Sirt6 were downregulated in the monolayer of the vascular wall from GDM and type 2 diabetes mellitus (T2DM) patients. Immunoprecipitation assays showed that Sirt6 interacts with Caveolin-1 and specifically mediated its acetylation levels. Immuno-electron microscopy (EM) further indicated that Sirt6 overexpression triggered the autophagic lysosomal degradation of Caveolin-1. ECs-specific overexpression of Sirt6 by adeno-associated viral vector serotype 9 (AAV9) induced autophagy, reduced Caveolin-1 expression, and ameliorated atherosclerotic plaque formation in STZ-induced diabetic ApoE-/- mice. CONCLUSION: Sirt6-mediated acetylation of Caveolin-1 activates its autophagic degradation and inhibits high glucose-stimulated LDL transcytosis. Thus, the Sirt6/Caveolin-1 autophagic pathway plays a crucial role in diabetic AS, and the overexpression or activation of Sirt6 is a novel therapeutic strategy.

Age-related changes of human serum Sirtuin6 in adults.

BACKGROUND: Aging is a natural life process and with an aging population, age-related diseases (e.g. type 2 diabetes mellitus (T2DM), atherosclerosis-based cardiovascular diseases) are the primary mortality cause in older adults. Telomerase is often used as an aging biomarker. Detection and characterization of novel biomarkers can help in a more specific and sensitive identification of a person's aging status. Also, this could help in age-related diseases early prevent, ultimately prolonging the population's life span. Sirtuin 6 (Sirt6) - a member of the Sirtuins NAD+-dependent histone deacetylases family - is mainly intracellularly expressed, and is reported to be involved in the regulation of aging and aging-related diseases. Whether serum Sirt6 is correlated with aging and could be used as an aging biomarker is unknown. In the present study, we aimed to investigate the age-related Sirt6 changes in the serum of human adults. METHODS: Participants were divided into three groups according to age: 20-30 years (Young); 45-55 years (Middle-aged); and ≥ 70 years (Old). The Sirt6 and telomerase serum concentrations were determined by ELISA. The Sirt6 and human telomerase reverse transcriptase (hTERT) expression in vessels from amputated human lower limbs were analyzed using real-time quantitative PCR (RT-qPCR) and immunohistochemical staining. The relationships between variables were evaluated by Pearson correlation analysis. RESULTS: The Sirt6 and telomerase serum levels reduced with an increase in age. A similar tendency was observed for Sirt6 and hTERT in the vessel. Serum levels of Sirt6 were higher in females compared with males. Pearson's regression analysis revealed that the Sirt6 serum level positively correlated with telomerase (r = 0.5743) and both were significantly negatively correlated with age (r = - 0.5830 and r = - 0.5993, respectively). CONCLUSIONS: We reported a negative correlation between serum Sirt6 concentration and aging in human beings. Therefore, the Sirt6 serum level is a potential sex-specific aging marker.

Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system.

Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.

Salidroside-Mediated Autophagic Targeting of Active Src and Caveolin-1 Suppresses Low-Density Lipoprotein Transcytosis across Endothelial Cells.

Subendothelial retention of apolipoprotein B100-containing lipoprotein, such as low-density lipoprotein (LDL), is the initial step of atherogenesis. Activation of autophagy exhibits beneficial effects for the treatment of atherosclerosis. In our previous study, we demonstrated that hyperglycemia suppressed autophagic degradation of caveolin-1, which in turn resulted in acceleration of caveolae-mediated LDL transcytosis across endothelial cells and lipid retention. Therefore, targeting the crossed pathway in autophagy activation and LDL transcytosis interruption may be a promising antiatherosclerotic strategy. In metabolic diseases, including atherosclerosis, salidroside, a phenylpropanoid glycoside compound (3,5-dimethoxyphenyl) methyl-ß-glucopyranoside), is the most important compound responsible for the therapeutic activities of Rhodiola. However, whether salidroside suppresses LDL transcytosis to alleviate atherosclerosis has not yet been elucidated. In the present study, we demonstrated that salidroside significantly decreased LDL transcytosis across endothelial cells. Salidroside-induced effects were dramatically blocked by AMPK (adenosine monophosphate-activated protein kinase) inhibitor (compound c, AMPKα siRNA) and by overexpression of exogenous tyrosine-phosphorylated caveolin-1 using transfected cells with phosphomimicking caveolin-1 on tyrosine 14 mutant plasmids (Y14D). Furthermore, we observed that salidroside promoted autophagosome formation via activating AMPK. Meanwhile, the interaction between caveolin-1 and LC3B-II, as well as the interaction between active Src (indicated by the phosphorylation of Src on tyrosine 416) and LC3B-II, was significantly increased, upon stimulation with salidroside. In addition, both bafilomycin A1 (a lysosome inhibitor) and an AMPK inhibitor (compound c) markedly prevented salidroside-induced autophagic degradation of p-Src and caveolin-1. Moreover, the phosphorylation of caveolin-1 on tyrosine 14 was disrupted due to the downregulation of p-Src and caveolin-1, thereby directly decreasing LDL transcytosis by attenuating the number of caveolae on the cell membrane and by preventing caveolae-mediated LDL endocytosis released from the cell membrane. In ApoE-/- mice, salidroside significantly delayed the formation of atherosclerotic lesions. Meanwhile, a significant increase in LC3B, accompanied by attenuated accumulation of the autophagy substrate SQSTM1, was observed in aortic endothelium of ApoE-/- mice. Taken together, our findings demonstrated that salidroside protected against atherosclerosis by inhibiting LDL transcytosis through enhancing the autophagic degradation of active Src and caveolin-1.

Wilson Disease With Novel Compound Heterozygote Mutations in the ATP7B Gene Presenting With Severe Diabetes.

OBJECTIVE: To determine the relationship between ATP7B mutations and diabetes in Wilson disease (WD). RESEARCH DESIGN AND METHODS: A total of 21 exons and exon-intron boundaries of ATP7B were identified by Sanger sequencing. RESULTS: Two novel compound heterozygous mutations (c.525 dupA/ Val176Serfs*28 and c.2930 C>T/ p.Thr977Met) were detected in ATP7B. After d-penicillamine (D-PCA) therapy, serum aminotransferase and ceruloplasmin levels in this patient were normalized and levels of HbA1c decreased. However, when the patient ceased to use D-PCA due to an itchy skin, serum levels of fasting blood glucose increased. Dimercaptosuccinic acid capsules were prescribed and memory recovered to some extent, which was accompanied by decreased insulin dosage for glucose control by 5 units. CONCLUSIONS: This is the first report of diabetes caused by WD.

Low density lipoprotein mimics insulin action on autophagy and glucose uptake in endothelial cells.

Elevated plasma low density lipoprotein (LDL) is an established risk factor for cardiovascular disease. In addition to being able to cross the endothelial barrier to become accumulated in subendothelial space and thereby initiate atherosclerosis, LDL may exert a direct effect on vascular endothelial cells through activation of LDL receptor and its downstream signaling. Whether LDL can modulate the signaling for autophagy in endothelial cells is not clear. The present study firstly demonstrated that LDL can suppress endothelial autophagy through activation of the PI3K/Akt/mTOR signaling pathway and can promote glucose uptake by translocating glucose transporter 1 (GLUT1) from cytoplasm to cell membrane, actions similar to those of insulin. A co-immunoprecipitation assay found that LDL receptor (LDLR) and insulin receptor (IR) formed a complex in HUVECs. Knock down of the insulin receptor by small interfering RNA blocked the suppression of autophagy by LDL, as well as the signaling pathway involved. We conclude that LDL may mimic the action of insulin in endothelial cells, which might partly explain the increased incidence of diabetes in patients receiving some LDL-lowering therapy.