Anti-ENO1 antibody combined with metformin against tumor resistance: a novel antibody-based platform.

Background: Antibody-based platforms (i.e., ADC) have emerged as one of the most encouraging tools for the cancer resistance caused by cancer stem cells (CSCs) enrichment. Our study might provide a promising therapeutic direction against drug resistance and serve as a potential precursor platform for screening ADC. Methods: The cell migration, invasion, drug resistance, and self-renewal were assessed by the cell invasion and migration assay, wound healing assay, CCK-8 assay, colony formation assay, and sphere formation assay, respectively. The expression profiles of CSCs (ALDH+ and CD44+) subpopulations were screened by flow cytometry. The western blot and cell immunofluorescence assay were used to evaluate pathway-related protein expression in both anti-ENO1 antibody, MET combined with DPP/CTX-treated CSCs. Results: In the present study, western blot and flow cytometry verified that anti-ENO1 antibody target the CD44+ subpopulation by inhibiting the PI3K/AKT pathway, while metformin might target the ALDH+ subpopulation through activation of the AMPK pathway and thus reverse drug resistance to varying degrees. Subsequently, in vitro investigation indicated that anti-ENO1 antibody, metformin combined with cisplatin/cetuximab could simultaneously target ALDH+ and CD44+ subpopulations. The combination also inhibited the CSCs proliferation, migration, invasion, and sphere formation; which may result in overcoming the drug resistance. Then, molecular mechanism exploration verified that the anti-ENO1 antibody, metformin combined with cisplatin/cetuximab inhibited the Wnt/ß-catenin signaling. Conclusions: The study preliminarily revealed anti-ENO1 antibody combined with metformin could overcome drug resistance against CSCs by inhibiting the Wnt//ß-catenin pathway and might serve as a potential precursor platform for screening ADC. More importantly, it is reasonably believed that antibody-based drug combination therapy might function as an encouraging tool for oncotherapy.

Intraoperative electroencephalogram features related to frailty in older patients: an exploratory prospective observational study.

Frailty is an independent risk factor for the increased incidence of postoperative delirium (POD). To date, the effect of frailty on intraoperative electroencephalogram (EEG) changes remains unexplored. The present study, an exploratory analysis of a prospective cohort study, aimed to investigate the differences in EEG characteristics between frail and robust patients. This prospective observational study was conducted between December 2020 and November 2021. The preoperative frailty status was assessed using the FRAIL scale. The patients' baseline (before anesthesia) and intraoperative EEG data were collected using a brain function monitor. Finally, 20 robust and 26 frail older patients scheduled for elective spinal surgery or transurethral prostatectomy under propofol-based general anesthesia were included in the final analysis. Baseline and intraoperative EEG spectrogram and power spectra were compared between the frail and robust groups. No differences were observed in baseline EEG between the frail and robust groups. When the intraoperative EEG spectral parameters were compared, the alpha peak frequency (10.56 ± 0.49 vs. 10.14 ± 0.36 Hz, P = 0.002) and alpha peak, delta, theta, alpha, and beta powers were lower in the frail group. After adjusting for age, Charlson Comorbidity Index (CCI), and mini-mental state examination (MMSE) score, the FRAIL score was still negatively associated with total, delta, theta, alpha, and beta powers. Frail patients had reduced EEG (0-30 Hz) power after the induction of propofol-based general anesthesia. After adjusting for age, CCI, and MMSE score, frail patients still showed evidence of reduced δ, θ, α, and ß power.

A Positive Feedback Loop of E2F4-Mediated Activation of MNX1 Regulates Tumour Progression in Colorectal Cancer.

Purpose: Colorectal cancer (CRC) is the 3rd most prevalent malignant tumour globally. Although significant strides have been made in diagnosis and treatment, its prognosis at the moment remains unpromising. Therefore, there is an urgent and desperate need to identify novel biomarkers of CRC and evaluate its mechanism of tumourigenesis and development. Methods: JASPAR and RNAinter databases are used to analyze target genes associated with colorectal cancer. Western blotting, q-PCR and immunohistochemistry et, al. were used to detect the level of MNX1 in patients with colorectal cancer, and Chip-PCR was used to detect the targeted binding ability of E2F4 and MNX1. The cells and animal models overexpressed MNX1 and E2F4 were constructed by shRNA transfection. Results: Herein, MNX1 was highly expressed and linked to favourable overall survival curves in colorectal cancer. The functional assay revealed that MNX1 overexpression could promote proliferation, migration, and invasion of CRC cells. Based on the prediction of the JASPAR and RNAinter databases, the transcription factor, E2F4, was bound to the MNX1 promoter region. The Chromatin Immunoprecipitation (ChIP) assay verified the interactions between MNX1 and E2F4 in CRC. Additionally, we found that sh-E2F4 markedly downregulated the MNX1 levels and reduced CRC progression in vivo and in vitro, which reversed MNX1 overexpression. Conclusion: Therefore, our research discovered that E2F4-mediated abnormal MNX1 expression promotes CRC progression and could become a novel diagnostic or therapeutic target of CRC.

Posterior Atlantoaxial Fusion With C1-2 Pedicle Screw Fixation for Atlantoaxial Dislocation in Pediatric Patients With Mucopolysaccharidosis IVA (Morquio a Syndrome): A Case Series.

OBJECTIVE: To investigate the efficacy and safety of posterior atlantoaxial fusion (AAF) with C1-2 pedicle screw fixation for atlantoaxial dislocation (AAD) in pediatric patients with mucopolysaccharidosis IVA (MPS IVA). METHODS: This study included 21 pediatric patients with MPS IVA who underwent posterior AAF with C1-2 pedicle screw fixation. Anatomical parameters of the C1 and C2 pedicle were measured on preoperative computed tomography (CT). The American Spinal Injury Association (ASIA) scale was used to evaluate the neurological status. The fusion and accuracy of pedicle screw was assessed on postoperative CT. Demographic, radiation dose, bone density, surgical, and clinical data were recorded. RESULTS: Patients reviewed included 21 patients younger than 16 years with an average age of 7.4 ± 4.2 years and an average of 20.9 ± 7.7 months follow-up. Fixation of 83 C1 and C2 pedicle screws was performed successfully and 96.3% of them were identified as being safe. One patient developed postoperative transient disturbance of consciousness and one developed fetal airway obstruction and died about 1 month after the surgery. Out of the remaining20 patients, fusion was achieved, symptoms were improved, and no other serious surgical complications were observed at the latest follow-up. CONCLUSIONS: Posterior AAF with C1-2 pedicle screw fixation is effective and safe for AAD in pediatric patients with MPS IVA. However, the procedure is technically demanding and should be performed by experienced surgeons with strict multidisciplinary consultations.

Salidroside ameliorates orthopedic surgery-induced cognitive dysfunction by activating adenosine 5'-monophosphate-activated protein kinase signaling in mice.

Perioperative neurocognitive disorders (PND) are the most common postoperative complications with few therapeutic options. Salidroside, a plant-derived compound, has gained increased attention as a treatment for various neurological diseases and particularly as a modifier of microglia-mediated neuroinflammation. However, the effect of salidroside on orthopedic surgery-induced cognitive dysfunction and the underlying mechanisms are largely unknown. Here, we found that salidroside greatly attenuated cognitive impairment in mice after orthopedic surgery. Neuroinflammation in the mouse hippocampus was also attenuated by salidroside. Meanwhile, salidroside treatment induced a switch in microglial polarization to the anti-inflammatory phenotype. In vitro, salidroside suppressed the expression of proinflammatory cytokines and induced a switch in microglial phenotype to the anti-inflammatory phenotype. Mechanistically, molecular docking studies revealed the potential AMPK activation activity of salidroside. And salidroside did up-regulated the AMPK pathway proteins. Moreover, AMPK antagonist abolished the effects of salidroside in vivo and in vitro. Taken together, our results demonstrated that salidroside effectively suppressed PND by suppressing microglia-mediated neuroinflammation through activating AMPK pathway, and it might be a novel therapeutic approach for PND.

Cardiotoxicity monitoring of pyrotinib in combination with nab-paclitaxel, doxorubicin, and cyclophosphamide in HER2-positive breast cancer: a single-armed clinical trial.

Background: Human epidermal growth factor receptor 2 (HER2) inhibitors play a vital role in the treatment of HER2-positive breast cancer. Numerous studies have shown that traditional HER2 inhibitors and chemotherapeutics such as albumin-paclitaxel, liposomal doxorubicin, and cyclophosphamide (TAC regimen) have different degrees of cardiotoxicity. Pyrotinib is a novel small-molecule HER2 inhibitor and has no cardiotoxicity. Here, the purpose of this study was to investigate the cardiac safety of pyrotinib with TAC regimen for HER2-positive breast cancer. Methods: In this study, 22 patients with stage I-IIIA HER2-positive breast cancer were screened, enrolled, and assigned to receive either neoadjuvant or postoperative adjuvant treatment with pyrotinib (320-400 mg, once daily) combined with TAC (albumin-paclitaxel 260 mg/m2, liposomal doxorubicin 20 mg/m2, cyclophosphamide 600 mg/m2) from December 2019 to May 2021. Patients' heart function was monitored using electrocardiogram, echocardiogram, and serological indicators. ST segment and T wave change, left ventricular ejection fraction (LVEF, %), N-terminal pro-B-type natriuretic peptide (NT-pro-BNP), creatine kinase (CK), creatine kinase myoglobin band (CK-MB), together with patients' weight, white blood cells (WBC), red blood cells (RBC), platelets, plasma lipid, and glucose were recorded. Results: Before and after the 2nd, 4th, and 6th cycles of treatment, the incidence of abnormal electrocardiogram of patients enrolled in the neoadjuvant treatment group was 36.4%, 27.3%, 27.3%, and 27.3%, respectively, while in the postoperative adjuvant treatment, the incidence was 45.5%, 36.4%, 36.4%, and 36.4%, respectively. LVEF before and after treatment in the neoadjuvant chemotherapy group was 65.36%±2.25% and 65.00%±2.15% (t=1.305, P=0.221), while in the postoperative adjuvant treatment group, LVEF was 66.27%±2.69% and 65.18%±1.89% (t=1.359, P=0.204). Pyrotinib combined with a TAC regimen may have induced a decrease in RBC. No obvious abnormality was found in the level of NT-pro-BNP, CK, CK-MB, patients' weight, WBC, platelets, plasma lipid, or glucose in all enrolled patients during the entire treatment process. Conclusions: Our findings indicated that neither neoadjuvant nor postoperative adjuvant treatment using pyrotinib combined with a TAC regimen to treat patients with HER2-positive breast cancer increased cardiotoxicity. However, the treatment may have induced a decrease in RBC and further research is needed.

Neoadjuvant pyrotinib plus nab-paclitaxel, doxorubicin, and cyclophosphamide for HER2-positive locally advanced breast cancer: a retrospective case-series study.

BACKGROUND: The anti-tumor activity of pyrotinib has been confirmed in human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This study investigated the effect of pyrotinib plus nab-paclitaxel, doxorubicin, and cyclophosphamide as neoadjuvant therapy in patients with HER2-positive locally advanced breast cancer. METHODS: In this single-center retrospective study, female patients with HER2-positive locally advanced breast cancer received pyrotinib 320 mg orally once a day and the TAC regimen (nab-paclitaxel 260 mg/m2, liposomal doxorubicin 20 mg/m2, and cyclophosphamide 600 mg/m2) on day 1 of each 21-day cycle. Surgery was performed after 4-6 cycles of neoadjuvant therapy. The outcomes included total pathological complete response (tpCR, ypT0/Tis ypN0) rate, objective response rate (ORR) after neoadjuvant therapy, progression-free survival, overall survival, and the incidence of adverse events (AEs). RESULTS: Between March 2019 and January 2020, a total of 22 patients were included. The median age was 48 years (range, 32-60). The ORR was 100% after the completion of neoadjuvant therapy. Ten (45.5%) patients achieved tpCR, including four of ten (40.0%) patients with positive hormone receptor, and six of 12 (50.0%) patients with negative hormone receptor. As at December 2020, no disease recurrence, progression, or death occurred. All patients suffered AEs after neoadjuvant therapy, most of which were grade 1-2. Grade ≥3 AEs included diarrhea [4 (18.2%)], rash [2 (9.1%)], and hand-foot syndrome [1 (4.5%)]. CONCLUSIONS: Neoadjuvant pyrotinib combined with the TAC regimen showed promising clinical benefit in patients with HER2-positive locally advanced breast cancer, with an acceptable safety profile.

Enolase 1 regulates stem cell-like properties in gastric cancer cells by stimulating glycolysis.

Recent studies have demonstrated that gastric cancer stem cells (CSCs) are a rare sub-group of gastric cancer (GC) cells and have an important role in promoting the tumor growth and progression of GC. In the present study, we demonstrated that the glycolytic enzyme Enolase 1 (ENO1) was involved in the regulation of the stem cell-like characteristics of GC cells, as compared to the parental cell lines PAMC-82 and SNU16, the expression of ENO1 in spheroids markedly increased. We then observed that ENO1 could enhance stem cell-like characteristics, including self-renewal capacity, cell invasion and migration, chemoresistance, and even the tumorigenicity of GC cells. ENO1 is known as an enzyme that is involved in glycolysis, but our results showed that ENO1 could markedly promote the glycolytic activity of cells. Furthermore, inhibiting glycolysis activity using 2-deoxy-D-glucose treatment significantly reduced the stemness of GC cells. Therefore, ENO1 could improve the stemness of CSCs by enhancing the cells' glycolysis. Subsequently, to further confirm our results, we found that the inhibition of ENO1 using AP-III-a4 (ENOblock) could reduce the stemness of GC cells to a similar extent as the knockdown of ENO1 by shRNA. Finally, increased expression of ENO1 was related to poor prognosis in GC patients. Taken together, our results demonstrated that ENO1 is a significant biomarker associated with the stemness of GC cells.

Long-term prognostic utility of low-density lipoprotein (LDL) triglyceride in real-world patients with coronary artery disease and diabetes or prediabetes.

BACKGROUND: Recent guidelines highlighted the association between atherosclerosis and triglyceride-enriched lipoproteins in patients with impaired glucose metabolism. However, evidence from prospective studies for long-term prognostic utility of low-density lipoprotein triglyceride (LDL-TG) in real-world patients with prediabetes (Pre-DM) or diabetes mellitus (DM) and coronary artery disease (CAD) is currently not available. The aim of the present study was to evaluate the impact of LDL-TG on major adverse cardiovascular events (MACEs) in patients with stable CAD under different glucose metabolism status. METHODS: A total of 4381 patients with CAD were consecutively enrolled and plasma LDL-TG level was measured by an automated homogeneous assay. They were categorized according to both status of glucose metabolism [DM, Pre-DM, normal glycaemia regulation (NGR)] and tertiles of LDL-TG. All subjects were followed up for the occurrence of MACEs. RESULTS: During a median of 5.1 (interquartile range 3.9 to 5.9) years' follow-up, 507 (11.6%) MACEs occurred. Cubic spline models showed a significant association between LDL-TG and MACEs in DM and Pre-DM but not in NGR. When the combined effect of elevated LDL-TG and glucose disorders was considered for risk stratification, the medium tertile of LDL-TG plus DM, and the highest tertile of LDL-TG plus Pre-DM or plus DM subgroups were associated with significantly higher risk of MACEs after adjustment of confounders including triglyceride [hazard ratios (95% confidence intervals): 1.843 (1.149-2.955), 1.828 (1.165-2.867), 2.212 (1.396-3.507), all p < 0.05]. Moreover, adding LDL-TG into the original model increased the C-statistic from 0.687 to 0.704 (∆C-statistic = 0.016, p = 0.028) and from 0.734 to 0.749 (∆C-statistic = 0.014, p = 0.002) in Pre-DM and DM, respectively. CONCLUSIONS: In this longitudinal cohort study on real-world practice, higher LDL-TG was associated with worse outcomes among Pre-DM and DM patients with stable CAD.

Methylation status of ORMDL3 regulates cytokine production and p-ERK/MMP9 pathway expression.

Orosomucoid like-3 (ORMDL3) has been identified to be associated with the development of asthma according to previous studies. However, the definite role of ORMDL3 in the pathogenesis of asthma remains unclear. In this study, we found ORMDL3 was highly expressed in PBMC specimens from childhood asthma patients. Cytokines production and p-ERK/MMP-9 pathway expression was also increased in childhood asthma patients compared with controls. In addition, ORMDL3 overexpression induced IL-6 and IL-8 release and activated p-ERK/MMP-9 pathway in vitro. Increased ORMDL3 expression was observed after treated with 5-Aza-CdR. 5-Aza-CdR decreased the percentage of the CpG island in the ORMDL3 promoter region and increased its promoter activity. In addition, 5-Aza-CdR significantly increased IL-6 and IL-8 levels in NHBE cells while there was no obvious alteration after knocking down ORMDL3. Knockdown of ORMDL3 also significantly decreased the expression of p-ERK/MMP-9 pathway in the presence or absence of 5-Aza-CdR. In conclusion, our study provided novel evidence for the association between ORMDL3 and asthma-associated cytokines. Moreover, DNA methylation plays an important role in ORMDL3-mediated increased IL-6 and IL-8 levels and p-ERK/MMP-9 pathway expression.

Familial hypercholesterolemia in very young myocardial infarction.

Familial hypercholesterolemia (FH) is one of the most common causes of premature myocardial infarction (MI). However, The patterns of FH remained unrecognized in clinical care, especially in very young patients (VYPs, ≤35 years) with MI. The present study enrolled a total of 1,093 VYPs (≤35 years) presenting a first MI. Clinical diagnosis of FH was made using Dutch Lipid Clinic Network criteria. Coronary severity was assessed by Gensini score (GS). Patients were followed for a median of 40-months with cardiac death, stroke, MI, post-discharge revascularization or unstable angina as primary endpoints. The detected rates of definite/probable FH were 6.5%. The prevalence reached up to 10.3% in patients ≤25 years. The FH had similar levels of comorbidities but was younger, more likely to be very high risk (VHR) and had higher GS (p < 0.05) than unlikely FH. Notably, the FH on prior lipid-lowering medication presented a lower GS compared to those untreated. Differences in event rates were similar in FH as unlikely FH (11.8% vs. 8.1%, adjusted hazard ratio 1.35 [0.64-2.86], p = 0.434) but patients on treatment improved outcome (6.5% vs. 10.5%, adjusted hazard ratio 0.35[0.13-0.95], p = 0.039). The early identification and treatment might be critical to reduce cardiovascular risk in VYPs with MI.

Comparison of Antioxidant Constituents of Agriophyllum squarrosum Seed with Conventional Crop Seeds.

Twelve chemical constituents were identified from the Agriophyllum squarrosum seed (ASS). ASS contained large amounts of flavonoids, which were more concentrated in the seed coat. ASS-coat (1 g) contained 335.7 µg flavonoids of rutin equivalent, which was similar to the flavonoid content in soybean (351.2 µg/g), and greater than that in millet, wheat, rice, peanut, and corn. By LC-MS analysis, the major constituents in ASS were 3-O-[α-L-rhamnopyranosyl-(1→6)-ß-D- glucopyranosyl]-7- O-(ß-D-glucopyranosyl)-quercetin (1), rutin (4), quercetin-3-O-ß-D- apiosyl(1→2)-[α-L-rhamnosyl(l→6)]-ß-D-glucoside (2), isorhamnetin-3-O-rutinoside (5), and allantoin (3), compared with isoflavonoids-genistin (16), daidzin (14), and glycitin (18) in soybean. Among constituents in ASS, compounds 1, 2, 4, protocatechuic acid (8), isoquercitrin (11), and luteolin-6-C-glucoside (12) potently scavenged DPPH radicals and intracellular ROS; strongly protected against peroxyl radical-induced DNA scission; and upregulated Nrf2, phosphorylated p38, phosphorylated JNK, and Bcl-2 in HepG2 cells. These results indicate that ASS is rich in antioxidant constituents that can enrich the varieties of food flavonoids, with significant beneficial implications for those who suffer from oxidative stress-related conditions. PRACTICAL APPLICATION: This study found that A. squarrosum seed contains large amounts of antioxidative flavonoids and compared its chemical constituents with those of conventional foods. These results should increase the interest in planting the sand-fixing A. squarrosum on a large scale, thus preventing desertification and providing valuable foods.

Association between plasma proprotein convertase subtisilin/kexin type 9 concentration and coronary artery calcification.

Background Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) has been reported to be related to several risk factors and diseases such as inflammatory markers and coronary artery disease. The aim of present study was to investigate whether plasma PCSK9 concentration was associated with coronary artery calcification. Methods A total of 403 consecutive untreated patients with angina-like chest pain, who received electron beam computed tomography, were enrolled and a coronary artery calcification score (CACS) was also measured. The baseline clinical characteristics were collected and blood sample was taken after 12-h fasting. The plasma PCSK9 concentrations were determined by ELISA in all patients, and the relationship between plasma PCSK9 concentrations and CACS was investigated. Results Patients with coronary artery calcification (CACS > 0) had significant higher plasma PCSK9 concentrations compared with those (CACS = 0) without coronary artery calcification (258.58 ± 69.53 ng/mL vs. 202.53 ± 52.17 ng/mL, P < 0.001). Patients with highest PCSK9 concentrations had the highest CACS. Multivariable linear regression analysis suggested that PCSK9 was independently associated with coronary artery calcification ( P = 0.002) after adjusting for traditional cardiovascular risk factors. Furthermore, the area under the curve for the plasma PCSK9 concentration in predicting coronary artery calcification was 0.736 (95% CI: 0.687-0.785, P < 0.001), with a sensitivity of 66% and specificity of 70%. Conclusion A positive association between plasma PCSK9 concentration and coronary artery calcification in untreated patients with angina-like chest pain was observed in our study, suggesting that further investigation may be needed in order to confirm our primary findings and explore the clinical implications.

Up-regulation of IRF-3 expression through GATA-1 acetylation by histone deacetylase inhibitor in lung adenocarcinoma A549 cells.

Interferon regulatory factor 3 (IRF-3) is an important transcription factor for interferon genes. Although its functional activation by viral infection has been widely explicated, the regulatory mechanism of IRF-3 gene expression in cancer cells is poorly understood. In this study, we demonstrated treatment of lung adenocarcinoma A549 cells with trichostatin A (TSA) and valproic acid (VPA), two different classes of histone deacetylase inhibitors, strongly stimulated IRF-3 gene expression. Truncated and mutated IRF-3 promoter indicated that a specific GATA-1 element was responsible for TSA-induced activation of IRF-3 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TSA treatment increased the binding affinity of GATA-1 to IRF-3 promoter. Using immunoprecipitation assay and immunoblotting, we demonstrated that TSA increased the level of acetylated GATA-1 in A549 cells. In summary, our study implied that TSA enhanced IRF-3 gene expression through increased GATA-1 recruitment to IRF-3 promoter and the acetylation level of GATA-1 in lung adenocarcinoma A549 cells.

Impact of Smoking Status on Lipoprotein Subfractions: Data from an Untreated Chinese Cohort.

OBJECTIVE: Cigarette smoking is one of the established risk factors of atherosclerotic cardiovascular disease, however, its impact on lipids is not completely understood, especially in the Chinese population. Therefore, this study evaluated the impact of smoking status (non, former, and current smoking) on the distribution of lipoprotein subfractions in untreated patients with angina-like chest pain. METHODS: A total of 877 patients were consecutively enrolled and divided into nonsmoking (n = 518), former smoking (n = 103), and current smoking (n = 256) groups. Both low- and high-density lipoprotein cholesterol (LDL-C and HDL-C) subfractions were measured using the Quantimetrix Lipoprint System. The distributions of lipoprotein subfractions were evaluated among the groups. RESULTS: Compared with nonsmoking subjects, the current smoking group had significantly lower large/medium HDL-C (both P < 0.001) concentration and large HDL subfraction percentage but higher small HDL-C and medium LDL-C concentrations as well as medium LDL subfraction percentage. Importantly, former smoking subjects showed elevated levels of large HDL-C concentration, large HDL particle percentage, and mean LDL particle size and attenuation in small HDL/LDL percentages and small LDL-C concentration, but these levels did not reach the optimal status compared with those of the non-smoking group (data not shown). CONCLUSION: Smoking has an adverse impact on the lipoprotein subfractions, presented as lower large HDL particles besides higher small HDL and medium LDL particles, whereas smoking cessation could reverse these change to a certain degree.

Eight novel mutations in the ABCD1 gene and clinical characteristics of 25 Chinese patients with X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disease caused by mutations in the adenosine triphosphate-binding cassette D1 (ABCD1) gene. This study aimed to retrospectively investigate the clinical characteristics of 25 patients with X-ALD including members of large pedigrees, to analyze ABCD1 gene mutations, the effect of gene novel variants on ALD protein (ALDP) structure and function, and to expand gene mutation spectrum of Chinese patients. METHODS: Twenty-five male patients diagnosed with X-ALD were enrolled in this study. The clinical characteristics of the patients were retrospectively summarized by reviewing medical records or telephone consultation. ABCD1 gene mutations were analyzed. The pathogenicity of novel missense variants was analyzed using cobalt constraint-based multiple protein alignment tool, polymorphism phenotyping, sorting intolerant from tolerant, Align-Grantham variation and Grantham deviation, and Swiss-Program Database Viewer 4.04 software, respectively. RESULTS: Childhood cerebral form ALD (CCALD) is the most common phenotype (64%) in the 25 patients with X-ALD. The progressive deterioration of neurological and cognitive functions is the main clinical feature. The demyelination of the brain white matter and elevated plasma very long chain fatty acids (VLCFAs) were found in all patients. Different phenotypes were also presented within family members of the patients. Twenty-two different mutations including 8 novel mutations in the ABCD1 gene were identified in the 25 patients. Of the mutations, 63.6% were missense mutations and 34.8% located in exon 1. The amino acid residues of three novel missense mutations in eight species were highly conserved, and were predicted to be "probably" damaging to ALDP function. The other five novel mutations were splice, nonsense, deletion or duplication mutations. CONCLUSIONS: CCALD is the most common phenotype (64%) in our patients with X-ALD. Eight novel mutations in the ABCD1 gene identified are disease-causing mutations. Brain magnetic resonance imaging and plasma VLCFA determination should be performed for the patients who present with progressive deterioration of neurological development.

New cytotoxic triterpene glycoside from the East China Sea cucumber Holothuria nobilis.

A new cytotoxic triterpene glycoside named nobiliside E (1) has been obtained from the East China Sea cucumber Holothuria nobilis Selenka. Its structure was determined on the basis of NMR spectroscopic and MS analyses, together with chemical evidence. The new compound showed significant cytotoxicity to eight human tumor cell lines with IC50 values in the range of 0.53-4.06 µg/mL.

QDPR gene mutation and clinical follow-up in Chinese patients with dihydropteridine reductase deficiency.

BACKGROUND: This study aimed to investigate the mutation spectrum of the QDPR gene, to determine the effect of mutations on dihydropteridine reductase (DHPR) structure/function, to discuss the potential genotypephenotype correlation, and to evaluate the clinical outcome of Chinese patients after treatment. METHODS: Nine DHPR-deficient patients were enrolled in this study and seven of them underwent neonatal screening. QDPR gene mutations were analyzed and confirmed by routine methods. The potential pathogenicity of missense variants was analyzed using Clustal X, PolyPhen program and Swiss-PDB Viewer 4.04_OSX software, respectively. The clinical outcomes of the patients were evaluated after long-term treatment. RESULTS: In 10 mutations of the 9 patients, 4 were novel mutations (G20V, V86D, G130S and A175R), 4 were reported by us previously, and 2 known mutations were identified. R221X was a hotspot mutation (27.7%) in our patients. Eight missense mutations probably had damage to protein. Six patients in this series were treated with a good control of phenylalanine level. The height and weight of the patients were normal at the age of 4 months to 7.5 years. Four patients, who underwent a neonatal screening and were treated early, showed a normal mental development. In 2 patients diagnosed late, neurological symptoms were significantly improved. CONCLUSIONS: The mutation spectrum of the QDPR gene is different in the Chinese population. Most mutations are related to severe phenotype. The determination of DHPR activity should be performed in patients with hyperphenylalaninemia. DHPR-deficient patients who were treated below the age of 2 months may have a near normal mental development.

[Analysis of GALNS gene mutation in thirty-eight Chinese patients with mucopolysaccharidosis type IVA].

OBJECTIVE: Mucopolysaccharidosis (MPS) type IVA (MPS IVA) is an autosomal recessive lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) needed to degrade glycosaminoglycanes (GAGs), accumulation of GAGs in the tissue resulting in disorder of function. So far, the small number of articles about clinical study of Chinese MPS IVA were published and only one paper about gene mutation analysis was published. This study aimed to investigate the mutation spectrum and characteristic of GALNS gene in Chinese patients with MPS IVA who were diagnosed in our hospital. METHOD: Thirty-eight patients from 36 families (male 17, female 21) were diagnosed as MPS IVA by GALNS activity determination [(0.85 ± 1.33) nmol/(17 h·mg)] and clinical symptoms during 2006-2012. The average age of diagnosis was (5.7 ± 3.6) years. Mutation analysis of GALNS gene performed performed by PCR-direct DNA sequencing for 38 patients. PCR-restriction fragment length polymorphism analysis was used for validating novel mutation, and also to assess amino acid conservation for novel missense variants in five different species. PolyPhen-2 tool was used to predict the possible impact of missense mutations on the structure and function of the human GALNS protein, etc. Analysis of GALNS activity and gene mutation in amniotic fluid were performed to provide the prenatal diagnosis for some families with MPS type IVA. RESULT: (1) Thirty-eight kinds of mutation in GALNS gene were identified in 38 patients of them, 71% were missense mutations. p. M318R was a hot-spot mutation (21%) tested. Five kinds of mutation i.e., p. P163H, p.G168L, p. A324E, p. L366P and p. F452L were only found in Chinese patients with MPS IVA. Eighteen kinds of novel mutation were detected including p. E315K, p.G304D, p.R251Q, p.Y240C, p.G161E, p.N32D, p.L390P, p. D60E, p. P420S, W403C/T404S, p.L454P, for p.W405X, p. M1I, c.409_ c.420del12, c.1176_1178del3, c.1046delG, c.1188delG and IVS9-2A>C. (2) The polymorphism of novel missense variants were ruled out by the PCR-restriction fragment length polymorphism analysis and no related mutations were found in 50 normal controls. A splice site mutation IVS9-2A>C had been validated by reverse transcription PCR direct sequencing. The amino acid of mutant position of 10 kinds of missense variants are highly conserved and only p. L454 is moderately conserved position. These missense variants were predicted to cause damage to the structure and function of human GALNS protein possibly according to the PolyPhen-2 tool, so these novel missense variants may be disease-causing mutations. (3) Prenatal diagnosis was provided for 7 families and three fetuses were diagnosed as MPS IVA. CONCLUSION: The GALNS gene mutation spectrum in Chinese patients with MPS IVA is really different from that in other countries, five kinds of mutation were only found in Chinese patients with MPS IVA. The reports of hot-spot mutation in Chinese patients were also different, and should be analyzed by more data of gene mutation analysis and epidemiological study.