Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

Synthesis of 12,12'-azo-13,13'-diepi-Ritterazine N.

A synthesis of the 12,12'-azo-analogue of ritterazine N from hecogenin is reported. Ring contraction of two 6/5 bicyclic ring systems, one trans-fused and another spiro, to 5/5 spiro ring systems is accomplished with excellent stereochemical control. Key transformations include an abnormal Baeyer-Villiger oxidation, a Norrish type I cleavage, an intramolecular dipolar [3 + 2] cycloaddition, and an intramolecular oxymecuration. Failing to uncover the ß-OH ketone from the isoxazoline ring, we end up with a synthesis of a cyclic analogue of ritterazine N.

Constructing 24(23→22)-abeo-Cholestane from Tigogenin in a 20(22→23)-abeo-Way via a PhI(OAc)2-mediated Favorskii Rearrangement.

Transforming tigogenin, a steroidal sapogenin, to a 24(23→22)-abeo-cholestane, which is an unusual structural feature shared by the aglycons of saundersiosides and candicanoside A, is described. The spiroketal of tigogenin was unfolded and the resulting C22-ketone was subjected to Favorskii rearrangement mediated by PhI(OAc)2/KOH/MeOH to squeeze out the C22 from the side chain, thus reaching the 24(23→22)-abeo-cholestane structure.

Tanshinone IIA therapeutically reduces LPS-induced acute lung injury by inhibiting inflammation and apoptosis in mice.

AIM: To study the effects of tanshinone IIA (TIIA) on lipopolysaccharide (LPS)-induced acute lung injury in mice and the underlying mechanisms. METHODS: Mice were injected with LPS (10 mg/kg, i.p.), then treated with TIIA (10 mg/kg, i.p.). Seven hours after LPS injection, the lungs were collected for histological study. Protein, LDH, TNF-α and IL-1ß levels in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in lungs were measured. Cell apoptosis and Bcl-2, caspase-3, NF-κB and HIF-1α expression in lungs were assayed. RESULTS: LPS caused marked histological changes in lungs, accompanied by significantly increased lung W/D ratio, protein content and LDH level in BALF, and Evans blue leakage. LPS markedly increased neutrophil infiltration in lungs and inflammatory cytokines in BALF. Furthermore, LPS induced cell apoptosis in lungs, as evidenced by increased TUNEL-positive cells, decreased Bcl-2 content and increased cleaved caspase-3 content. Moreover, LPS significantly increased the expression of NF-κB and HIF-1α in lungs. Treatment of LPS-injected mice with TIIA significantly alleviated these pathological changes in lungs. CONCLUSION: TIIA alleviates LPS-induced acute lung injury in mice by suppressing inflammatory responses and apoptosis, which is mediated via inhibition of the NF-κB and HIF-1α pathways.

Postoperative jaundice related to UGT1A1 and ABCB11 gene mutations: A case report and literature review.

BACKGROUND: Patients with obstructive jaundice caused by intrahepatic bile duct stones can be effectively managed by surgery. However, some patients may develop postoperative complications, liver failure, and other life-threatening situations. Here, we report a patient with mutations in the uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and bile salt export pump (adenosine triphosphate-binding cassette subfamily B member 11, ABCB11) genes who presented multiple intrahepatic bile duct stones and cholestasis, and the jaundice of the patient increased after partial hepatectomy. CASE SUMMARY: A 52-year-old male patient admitted to the hospital on October 23, 2021, with a progressive exacerbation of jaundice, was found to have multiple intrahepatic bile duct stones with the diagnoses of obstructive jaundice and acute cholecystitis. Subsequently, the patient underwent left hepatectomy with biliary exploration, stone extraction, T-tube drainage, and cholecystectomy without developing any intraoperative complications. The patient had a dark urine color with worsening jaundice postoperatively and did not respond well to plasma exchange and other symptomatic and supportive treatments. Since the progressive increase in postoperative bilirubin could not be clinically explained with any potential reason, including, if not at all, viral infection, cholangitis, autoimmune liver disease, and other causes, the patient underwent whole-exon screening for any genetic diseases, which surprisingly identified UGT1A1 and ABCB11 gene mutations related to glucuronidation of indirect bilirubin as well as bile acid transport in hepatocytes, respectively. Thus, we hypothesized that postoperative refractory cholestasis might result from UGT1A1 and ABCB11 gene mutations and further recommended liver transplantation to the patient, who eventually declined it and died from liver failure six months later. CONCLUSION: Surgery may aggravate cholestasis in patients with multiple intrahepatic bile duct stones and cholestasis associated with UGT1A1 and ABCB11 gene mutations. A liver transplant may be the best option if active medical treatment fails.

Case-control study on the association between qi-stagnation and insomnia.

OBJECTIVE: To investigate the relationship between insomnia and qi-stagnation by using the international standardized measurement of sleep quality and the Traditional Chinese Medicine (TCM) Constitution Scales. METHODS: A survey by means of the TCM Constitution Scales, the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS) and the Deep Sleep Scale (DSS) in 169 participants aged between 16 and 80 years old was conducted. Comparison was made to examine the sleep quality and insomnia symptoms in the qi-stagnation group and other-constitution group. RESULTS: Univariate analysis found that the qi-stagnation group had a significantly increased risk of difficulty in falling asleep (OR=3.012, and 95% CI 1.310 to 6.923 for PSQI; OR=3.016, and 95% CI 1.358 to 6.709 for DSS) and early waking (OR=3.545, and 95% CI 1.229 to 10.232 for PSQI; OR=2.742, and 95% CI 1.072 to 7.014 for DSS), while the other-constitution group had a significant risk of dreaminess (OR=2.419, and 95% CI 1.154 to 5.072 for PSQI; OR=2.561, and 95% CI 1.116 to 5.880 for DSS). A dose-effect relationship existed between insomnia symptoms and qi-stagnation. Qi-stagnation significantly increased the risk of difficulty in falling asleep and early waking. CONCLUSION: This case-control study revealed that there is a statistically significant association between qi-stagnation and insomnia. Based on this study, we recommend that further research should be conducted for the rehabilitative care and cure of insomnia from the perspective of TCM constitution.

Feedback loop between hepatocyte nuclear factor 1α and endoplasmic reticulum stress mitigates liver injury by downregulating hepatocyte apoptosis.

Hepatocyte nuclear factor alpha (HNF1α), endoplasmic reticulum (ER) stress, and hepatocyte apoptosis contribute to severe acute exacerbation (SAE) of liver injury. Here, we explore HNF1α-ER stress-hepatocyte apoptosis interaction in liver injury. LO2, HepG2 and SK-Hep1 cells were treated with thapsigargin (TG) or tunicamycin (TM) to induce ER stress. Carbon tetrachloride (CCl4) was used to induce acute liver injury in mice. Low-dose lipopolysaccharide (LPS) exacerbated liver injury in CCl4-induced mice. Significant apoptosis, HNF1α upregulation, and nuclear factor kappa B (NF-κB) activation were observed in human-derived hepatocytes during ER stress. Knockdown of Rela, NF-κB p65, inhibited the HNF1α upregulation. Following CCl4 treatment ER stress, apoptosis, HNF1α expression and RelA phosphorylation were significantly increased in mice. HNF1α knockdown reduced activating transcription factor 4 (ATF4) expression, and aggravated ER stress as well as hepatocyte apoptosis in vivo and in vitro. The double fluorescent reporter gene assay confirmed that HNF1α regulated the transcription of ATF4 promoter. LPS aggravated CCl4-induced liver injury and reduced HNF1α, and ATF4 expression. Therefore, in combination, HNF1α and ER stress could be mutually regulated forming a feedback loop, which helps in protecting the injured liver by down-regulating hepatocyte apoptosis. Low-dose LPS aggravates hepatocyte apoptosis and promotes the SAE of liver injury by interfering with the feedback regulation of HNF1α and ER stress in acute liver injury.

FMR1 allele frequencies in 51,000 newborns: a large-scale population study in China.

BACKGROUND: Fragile X syndrome (FXS), caused by CGG-repeat expansion in FMR1 promoter, is one of the most common causes of mental retardation. Individuals with full mutation and premutation alleles have a high risk of psychophysiological disorder and of having affected offspring. Frequencies of FMR1 alleles in general newborns have been reported in Caucasians but have not been investigated in the large-scale population in  the mainland of China. METHODS: The sizes of FMR1 CGG-repeats were analyzed in 51,661 newborns (28,114 males and 23,547 females) and also in a cohort of 33 children diagnosed with developmental delay using GC-rich polymerase chain reaction (PCR) and triple repeat primed PCR. RESULTS: The frequency of CGG repeats > 100 was 1/9371 in males and 1/5887 in females, and the frequency of CGG repeats > 54 was 1/1561 in males and 1/1624 in females. FMR1 full mutation and premutation were identified in 27.27% of children who had Ages and Stages Questionnaire scores less than two standard deviations from the cutoff value. CONCLUSIONS: Our study revealed the prevalence of FXS in China and improved the sample databases of FXS, suggesting that the prevalence of FXS in Chinese is higher than estimated previously and that FXS screening can be advised to high-risk families.

[Construction of VEGF gene-targeted small interfering RNA mediated by CMV promoter and its silencing effect].

OBJECTIVE: To construct VEGF gene-targeted small interfering RNA (siRNA) and its expression vector driven by CMV promoter and to investigate its interference effect. METHODS: The VEGF gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strands were synthesized. After annealing, two-strand oligonucleotide was inserted into pDC311-SV40-RC vector, which was then identified by PCR and sequenced. Then human U-2 OS cell line was transfected with the vector using lipofectamine method. Finally, ELISA was performed to evaluate the expression of VEGF protein. RESULTS: PCR-identification of positive clone and sequencing confirmed the vector containing the target siRNA. ELISA showed that compared with the control group, the expression levels of VEGF protein in transfected U-2 OS cells were decreased significantly (P<0.05). CONCLUSION: VEGF gene-targeted siRNA and its vector mediated by CMV promoter were successfully constructed, which can reduce the VEGF protein expression after transfecting.

A genetic map constructed using a doubled haploid population derived from two elite Chinese common wheat varieties.

Genetic mapping provides a powerful tool for the analysis of quantitative trait loci (QTLs) at the genomic level. Herein, we report a new genetic linkage map developed from an F(1)-derived doubled haploid (DH) population of 168 lines, which was generated from the cross between two elite Chinese common wheat (Triticum aestivum L.) varieties, Huapei 3 and Yumai 57. The map contained 305 loci, represented by 283 simple sequence repeat (SSR) and 22 expressed sequence tag (EST)-SSR markers, which covered a total length of 2141.7 cM with an average distance of 7.02 cM between adjacent markers on the map. The chromosomal locations and map positions of 22 new SSR markers were determined, and were found to distribute on 14 linkage groups. Twenty SSR loci showed different chromosomal locations from those reported in other maps. Therefore, this map offers new information on the SSR markers of wheat. This genetic map provides new opportunities to detect and map QTLs controlling agronomically important traits. The unique features of this map are discussed.

[Genetic polymorphism of dendritic cell-specific ICAM-3 grabbing nonintegrin and DC-SIGNR's exon 4 in Chinese hepatitis C patients].

OBJECTIVE: To study into the genetic polymorphism of DC-SIGN and DC-SIGNR's exon 4 in Chinese hepatitis C patients and its relationship with HCV infection susceptibility. METHODS: Patients with hepatitis C (n=300, group A) and healthy subjects (n=520, group B) were genotyped and analysed for the repeat sequence of polymorphism of DC-SIGN and DC-SIGNR's exon 4 using PCR and DNA sequencing. RESULTS: The distribution of genotypes and alleles in DC-SIGN's exon 4 in the two groups did not differ significantly (P > 0.05). The difference of allele frequency in DC-SIGNR's exon 4 between the two groups was also not significant (P > 0.05). However, 9/5 genotype distribution frequency of DC-SIGNR's exon 4 in patients with hepatitis C was significantly higher than that in the healthy subjects (P < 0.05). CONCLUSION: There is no significant correlation between the genetic polymorphism of DC-SIGN's exon 4 and HCV infection susceptibility. 9/5 genotype distribution frequency of DC-SIGNR's exon 4 in patients with hepatitis C is significantly higher and may be associated with HCV infection susceptibility.

[Inhibition of invasiveness and expression of heparanase-mRNA in human malignant melanoma cell line A375 by matrine].

OBJECTIVE: To investigate the effect of matrine on the invasiveness and expression of heparanase-mRNA in human malignant melanoma cell line A375. METHODS: The A375 cells were treated by matrine in different concentration. The total RNAs were extracted from the cells 48 hours after treatment and then semi-quantitative RT-PCR were performed to evaluate the heparanase-mRNA expression levels. Effect of matrine on adhesion of treated A375 cells was tested by cell-Matrigel adhesion assay. The invasiveness of treated A375 cells was measured by Matrigel invasion assay. RESULTS: The hepanase-mRNA expression, adhesion and invasiveness of A375 cells treated with matrine of different final concentrations significantly decreased compared with that of the controls (p < 0.01). Besides, the inhibitory effects were signifcantly different when the cells treated with matrine of different concentrations (P < 0.01). CONCLUSION: By down-regulating the expression of heparanase-mRNA, matrine has a significant inhibitory effect on the adhesion and invasiveness of human malignant melanoma cell line in a dose-dependent manner.

Complete sequence of a hepatic cirrhosis inbred C57BL/6 mice model mitochondrial genome.

In the present work we undertook the complete mitochondrial genome sequencing of an important hepatic cirrhosis model inbred C57BL/6 strain for the first time. Its mitogenome was 16,312 bp and coding 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes. A total of 96 SNPs were examined when compared to reference BN sequence.

Concise synthesis of the core structures of saundersiosides.

A divergent synthesis of three core pentacyclic lactones of nine rearranged cholestane sapogenins, saundersiosides A-H (1-8) and candicanoside A (9), is reported. Key features include a one-flask CBS reduction/Brown hydroboration-oxidation, a SmI2-mediated intramolecular Reformatskii reaction, and an intramolecular transesterification. This synthesis provides a general strategy and key precursors for the collective synthesis of natural and designed saundersiosides. An efficient formal synthesis of candicanoside A is also achieved.

Attenuated or absent HRV response to postural change in subjects with primary insomnia.

Previous studies have compared rest heart rate variability (HRV) between insomniacs and good sleepers, but the results have not been consistent. The altered HRV behavior in response to postural change was considered useful as another sensitive measure for evaluating the autonomic nervous function, however, to our knowledge, no study was found using HRV response to postural change in primary insomnia. Our study aimed to examine HRV response to postural change maneuver (PCM) in both primary insomniacs and controls between 22 and 39 years of age to gain insights into the characteristics of the autonomic nervous system (ANS) function in primary insomnia subjects. HRV was recorded for 5 min at seated rest, and then, the subjects quickly stood up from a seated position in up to 3s and remained standing for 15 min. HRV was recorded at the following times: seated rest and 0-5 min, 5-10 min and 10-15 min in the standing position. In primary insomnia subjects, attenuated or absent HRV response to postural change was identified, the increase in LF/HF ratio and the decrease in HF and SD1 from seated to standing were much slower than in the normal controls. In conclusion, this study provided evidence of the possible bi-directional relationship between insomnia and autonomic nervous system (ANS) function, which will move us closer to developing a new sensitive method for measuring autonomic impairment and early sympathetic damage in primary insomnia subjects.

A blunted sympathetic and accentuated parasympathetic response to postural change in subjects with depressive disorders.

BACKGROUND: In recent years, the bi-directional relationship between depression and ANS dysfunction has received considerable attention, but findings remain inconclusive. In this study, we aimed to examine the spectral HRV response to postural change in subjects with depressive disorders and in healthy controls, in order to gain insight into the characteristics of autonomic nervous system (ANS) response to postural change in subjects with depressive disorders. METHODS: We compared HRV response to postural change between subjects with depressive disorders and healthy controls aged 20-37 years. Depression severity was assessed by the self-reported Beck Depression Inventory-II (BDI-II). Spectral HRV was analyzed at two moments: 10 min seated rest and 10 min at standing position, with spontaneous breathing. RESULTS: No significant differences existed in the resting spectral HRV indices between subjects with depressive disorders and controls, however, following postural change, the increasing level of LF and LF/HF was lower and the decreasing level of HF power was higher, in the individuals with depression than that in healthy subjects. The differences in the LF power, HF power and the LF/HF ratio between seated rest before standing up and after postural change were found negatively correlated with depression severity. CONCLUSION: We found a blunted sympathetic and accentuated parasympathetic response to postural change in subjects with depressive disorder, suggesting that the autonomic impairment and early ANS dysfunction may exist among depressed individuals. These findings indicated that spectral analysis of HRV associated with postural change may be a more sensitive method than resting HRV analysis for detecting ANS dysfunction in depressive disorders. LIMITATIONS: Further studies are needed to expand the sample size and to clarify the mechanisms responsible for the autonomic dysfunction observed in individuals with depressive disorders.

Islet transplantation in the twenty-first century.

Isolated islet transplantation is poised for clinical application to treat insulin-dependent diabetes. Unlike exogenous insulin therapy, islet transplantation has promise for preventing and/or reversing the dismal secondary complications of diabetes. Islet transplants are arguably the most unique type of allografts, and we discuss their properties, limitations, and potential in this overview. The induction of immunologic tolerance to allow islet grafts to endure and prevail, without the hardship of chronic immunosuppressive therapy, is a major goal in this field. In this context, we discuss our successful results in preclinical models of primate allogeneic and xenogeneic islet graft tolerance.

[High-flux detection of HBV lamivudine resistant strains by genechip and analysis of mutant hotspots in those strains].

OBJECTIVE: Lamivudine resistant HBV strains in Shenzhen were detected at multiple sites and in large amounts to understand further the distribution of lamivudine resistant mutants. METHODS: 552 Hepatitis B patients's sera were examined using genechip method. Among them, 192 samples of lamivudine resistant mutant were further analyzed. RESULTS: In those 192 lamivudine resistant samples, 191 were YMDD mutants, 124 mutants of codon 528 and 9 mutants of codon 555. 88% YMDD mutants were multi-mutants of YVDD and codon 528; single mutants of YIDD; multi-mutants of YIDD and codon 528. 91% codon of YMDD mutants were GTG, ATT; the other 9% were ATA, ATC. CONCLUSIONS: These results suggest that mutants of codon 552 (YMDD) are core mutants. Mutants of codon 528 and 555 are incidental mutants, YVDD mutants always emerge with mutants of codon 528, but YIDD mutants appear differently. 9% YMDD mutants's codons are ATA or ATC. This may be the reason for the low positive rate shown by using the conventional PCR methods.

[Clinical features of 23 cases of AIDS complicated by tuberculosis].

OBJECTIVE: To discuss the clinical features, treatment and prognosis of patients with AIDS complicated by tuberculosis(TB). METHODS: The clinical features of 23 patients with AIDS complicated by tuberculosis admitted from 1997 to July 2004 were retrospectively analyzed. RESULTS: Of the 23 patients, most (94.3%) were young or middle-aged, and 11 (47.8%) died within half a year. The main HIV transmission was via sexual contact in 15 (65.2%) patients. Loss of body weight by 5 - 15 kg was present in all patients, cough for over 1 month in 15 (65.2%), and multiple opportunistic infections were complicated in most cases. Out of the 23 cases, 14 (60.9%) showed only pulmonary TB, and 8 (34.8%) showed lymph node TB. In 12 cases with infiltrated pulmonary TB, X-ray showed bilateral infiltration and no cavity formation was found. Slightly positive PPD test was found in 2 (8.7%) cases, and positive acid-fast bacilli was detected in sputum in 1 case (4.4%). The pre-treatment CD(4)(+) cell number in 23 patients was much lower than that in AIDS patients without complicated TB (P < 0.05). The pre-treatment CD(4)(+) cell number in patients died shortly after diagnosis was much lower than that in survived patients (P < 0.05). The HIV RNA level in the 23 patients was much higher than that in patients without complication of TB (P < 0.05). The mortality in patients treated with therapy against both TB and HIV was much lower than that in patients untreated or treated only with anti-TB therapy (P < 0.05). CONCLUSIONS: For patients with AIDS complicated by TB, the high negative rate in PPD test and the atypical chest X-ray manifestations are common. However, lymph node TB is quite common with high mortality. The pre-treatment CD(4)(+) level decreases significantly, and is associated with mortality. The TB bacilli may accelerate HIV virus duplication. It is suggested that the patients be treated with a combination of anti-TB and anti-HIV therapies.

[Early indexes to predict the therapeutic effect of interferon on chronic hepatitis B].

OBJECTIVE: To summarize the clinical changing characters of the clinical markers after interferon treatment in chronic hepatitis B (CHB) and make out practical indexes to predict the effect. METHODS: 150 CHB patients were randomly divided into two groups: therapeutic group (90) and control group (60) in the prospective controlled trial. The levels of endogenous interferon before treatment, interferon antibody at the end of the second month and fourth month after treatment, alanine aminotransferase (ALT) and HBV DNA in the serum were detected. Then the data was analysed to find out indexes for predicting the effect. RESULTS: (1) The clearance rate of HBeAg had no significant difference in age except for 20 - 30 and 30 - 40 (t > 2.331 2, P < 0.01). (2) It was more effective if ALT level was higher than 400 U/L before treatment and it decreased more than 50% two months after treatment. (3) The patients whose HBV DNA was negative (dot hybridization) or less than 10(6) copies/ml before treatment had higher rate of HBeAg clearance. (4) There was no effect on patients whose interferon antibody turned positive at the end of the second month. (5)A predictive method of comprehensive factors was made out, whose sensitivity, specificity, and accuracy were 80%, 100% and 90%, respectively. CONCLUSION: The clinical characters of these Chinese patients are different from those of the westerners and the effects of interferon have close relation to the levels of ALT, HBV DNA and interferon antibody.