Correlational analysis of PLIN1 with inflammation in diabetic foot ulcer wounds.

BACKGROUND: The persistent presence of inflammation is a recognized pathogenic mechanisms of diabetic foot ulcers (DFUs). We aimed to investigate the expression of PLIN1 in tissues from DFU patients and assess its potential association with inflammation-induced damage. METHODS: We performed transcriptome sequencing and correlation analysis of the foot skin from patients with or without DFUs. Additionally, we examined the correlation between PLIN1 and related inflammatory indicators by analyzing PLIN1 expression in tissue and serum samples and through high-glucose stimulation of keratinocytes (HaCaT cells). RESULTS: PLIN1 is upregulated in the tissue and serum from DFU patients. Additionally, PLIN1 shows a positive correlation with leukocytes, neutrophils, monocytes, C-reactive protein, and procalcitonin in the serum, as well as IL-1ß and TNF-α in the tissues. Experiments with Cells demonstrated that reduced expression of PLIN1 leads to significantly decreased expression of iNOS, IL-1ß, IL-6, IL-18, and TNF-α. PLIN1 may mediate wound inflammatory damage through the NF-κB signaling pathway. CONCLUSION: Our findings suggest that PLIN1 mediates the inflammatory damage in DFU, offering new prospects for the treatment of DFU.

The composition of the gut microbiota is altered in biliary atresia with cholangitis.

Aim: To detect the composition of the gut microbiota in biliary atresia after Kasai surgery. Methods: Infants within six months after the Kasai operation who were diagnosed by cholangiography at Shanghai Children's Hospital were enrolled in the study. Fecal samples were collected from diapers, placed into sterile tubes in the inpatient department or outpatient department and frozen at -80°C within half an hour. The gut microbiota was detected by 16S rRNA sequences. Then, the patients that were followed up to one year after the Kasai operation who suffered from cholangitis at least one time were grouped into the BAcho group, and the others were grouped into the BAnoncho group. Results: Nine of 18 BA patients were grouped into the BAcho group, and the others were grouped into the BAnoncho group. In the BAcho group, AST, ALT and GGT were significantly increased compared to the BAnoncho group. The number of total OTUs (operational taxonomic units) in feces was more elevated in the BAnoncho group than in the BAcho group. In the BAnoncho group, the Chao index at the OTU level was significantly increased compared to that in the BAcho group (66.37 ± 21.5 vs. 45.64 ± 11.25, p = 0.02 < 0.05). Bifidobacterium was the most abundant genus in the BAnoncho group, accounting for 22.14%, and Klebsiella accounted for 22.74% in the BAcho group. Compared with the BAnoncho group, Bacteroides was significantly decreased in the BAcho group (p = 0.037). Conclusion: The composition of the gut microbiota was different between BA with cholangitis and BA without cholangitis.

Reduced peroxisome proliferator-activated receptor-α and bile acid nuclear receptor NR1H4/FXR may affect the hepatic immune microenvironment of biliary atresia.

Background: Biliary atresia (BA) is a childhood liver disease characterized by fibrous obstruction and obstruction of the extrahepatic biliary system and is one of the most common and serious biliary disorders in infants. Significant inflammation and fibrosis of the liver and biliary tract are the most prominent features, regardless of the initial damage to the BA. Abnormalities in innate or adaptive immunity have been found in human patients and mouse models of BA. We previously reported that children with BA had abnormal lipid metabolism, including free serum carnitine. Objective: To study gene and protein expression levels of the hepatic peroxisome proliferator-activated receptor-α (PPARα) signaling pathway and farnesoid X receptor (FXR) in BA and BA fibrosis, and assess their clinical values. Methods: Low expression of PPARα and NR1H4 (FXR) in BA were validated in the Gene Expression Omnibus database. Functional differences were determined by gene set enrichment analysis based on of PPARα and NR1H4 expression. BA patients from GSE46960 were divided into two clusters by using consensus clustering according to PPARα, NR1H4, and SMAD3 expression levels, and immunoinfiltration analysis was performed. Finally, 58 cases treated in our hospital were used for experimental verification. (IHC: 10 Biliary atresia, 10 choledochal cysts; PCR: 10 Biliary atresia, 14 choledochal cysts; WB: 10 Biliary atresia, 4 choledochal cysts). Results: Bioinformatics analysis showed that the expression of PPARα, CYP7A1 and NR1H4 (FXR) in the biliary atresia group was significantly lower than in the control group. More BA-specific pathways, including TGFß signaling pathway, P53 signaling pathway, PI3K-AKT-mTOR signaling pathway, etc., are enriched in BA patients with low PPARα and NR1H4 expression. In addition, low NR1H4 expression is abundant in inflammatory responses, IL6/STAT3 signaling pathways, early estrogen responses, IL2 STAT5 signaling pathways, and TGFß signaling pathways. The TGFß signaling pathway was significant in both groups. According to the expression of PPARα, NR1H4 and SMAD3, a key node in TGFß pathway, BA patients were divided into two clusters using consensus clustering. In cluster 2, SMAD3 expression was high, and PPARα and NR1H4 expression were low. In contrast to cluster 1, immune cell infiltration was higher in cluster 2, which was confirmed by immunohistochemistry. The mRNA and protein levels of PPARα and NR1H4 in BA patients were lower than in the control group by immunohistochemistry, Western blot analysis and real-time PCR. Conclusions: The downregulation of PPARα and NR1H4 (FXR) signaling pathway may be closely related to biliary atresia.

LncRNA NEAT1 Targets miR-342-3p/CUL4B to Inhibit the Proliferation of Cutaneous Squamous Cell Carcinoma Cells.

Objective: This study investigated whether lncRNA NEAT1 could inhibit the proliferation of cutaneous squamous cell carcinoma (CSCC) cells by targeting miR-342-3p/CUL4B, thereby affecting the occurrence and development of CSCC. Methods: Fluorescence quantitative PCR was used to detect the expression of lncRNA NEAT1 and miR-42-3p in skin squamous cell carcinoma and adjacent tissues. Bioinformatics software and luciferase reporter gene assay were used to analyze the association of lncRNA NEAT1 and miR-342-3p. The effect of overexpression or knockdown of miR-342-3p on the proliferation of CSCC cells was examined by MTT and colony formation assays. Western blotting was used to detect the proteins of the miR-342-3p/CUL4B signaling axis. Results: The lncRNA NEAT1 is abnormally overexpressed in CSCC tissues and cell lines. The expression of lncRNA NEAT1 and miR-342-3p in CSCC was negatively correlated. Bioinformatics prediction analysis revealed that lncRNA NEAT1 regulates the expression of miR-342-3p. The results of MTT and plate colony formation experiments showed that the transfection of miR-342-3p mimics significantly inhibited the proliferation and plate colony formation of CSCC cells, while the transfection of miR-342-3p inhibitor significantly promoted the proliferation and plate colony-forming ability of CSCC cells. Western blot results showed that lncRNA NEAT1 affected CSCC cell proliferation through miR-342-3p/CUL4B/PI3K-Akt signaling pathway. Conclusion: The expression of lncRNA NEAT1 and miR-342-3p in CSCC tissues was negatively correlated. This study is the first to demonstrate that the lncRNA NEAT1, as a ceRNA, affects the proliferation of skin squamous cell carcinoma cells through the miR-342-3p/CUL4B/PI3K-Akt signaling pathway. Therefore, lncRNA NEAT1 could be a biological marker or target for CSCC diagnosis or treatment.

Downregulation of lncRNA NEAT1 inhibits the proliferation of human cutaneous squamous cell carcinoma in vivo and in vitro.

Background: The incidence of cutaneous squamous cell carcinoma (CSCC), a malignant tumor that threatens human life, is increasing every year, and yet its pathogenesis is still unclear. This study found that long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) was abnormally expressed in CSCC. However, the biochemical mechanisms of lncRNA NEAT1 in carcinogenesis and the development of cancer remain unclear. Methods: Fluorescence quantitative polymerase chain reaction (qPCR) was conducted to determine lncRNA NEAT1 expression in CSCC and paracarcinoma tissues and investigate the correlation between NEAT1 levels and patients' clinicopathological features. The invasion, proliferation, and migration of CSCC cells were measured using colony formation, Cell Counting Kit-8, and Transwell assays. Western blot assay was conducted to test whether NEAT1 knockdown affected invasion and migration-related proteins. In addition, a nude mouse subcutaneous tumorigenesis experiment was performed to determine whether the knockdown of NEAT1 affected the proliferation ability of CSCC cells. Results: Changes in lncRNA NEAT1 expression in CSCC tissues were correlated with the degree of lymph node metastasis and the tumor, regional lymph nodes, and distant metastasis (TNM) grade of patients. The downregulation of NEAT1 lncRNA significantly impeded cell invasion, proliferation, and migration in CSCC. Through lncRNA NEAT1 knockdown, significant reductions in metalloproteinase-2, metalloproteinase-9, N-cadherin, and vimentin expression were observed, and the level of E-cadherin increased. In vivo experiments in nude mice revealed that knockdown of lncRNA NEAT1 greatly inhibited cell proliferation in CSCC. Conclusions: In CSCC tissues, NEAT1 lncRNA was expressed at high levels and correlated with lymph node metastasis and TNM stage. The knockdown of NEAT1 lncRNA could significantly impede CSCC proliferation, metastasis, and invasion. Additionally, by measuring the expression level of lncRNA NEAT1, we may be able to detect the clinical and pathological characteristics of CSCC.

Association between fibrinogen/albumin ratio and arterial stiffness in patients with type 2 diabetes: A cross-sectional study.

Background: Fibrinogen albumin ratio (FAR) is significantly correlated with the severity and prognosis of cardiovascular disease (CVD). Arterial stiffness is an early lesion of CVD, but no studies have examined the correlation between arterial stiffness and FAR. This study aimed to examine the relationship between FAR and arterial stiffness in patients with type 2 diabetes (T2D), as measured by brachial-ankle pulse wave velocity (baPWV). Methods: In this cross-sectional investigation, patients with T2D were enrolled between January 2021 and April 2022. In each patient, the levels of fibrinogen and albumin in the serum, and baPWV in the serum were measured. A baPWV greater than 1800 cm/s was utilized to diagnose arterial stiffness. Results: The study included 413 T2D patients. The mean age of these participants was 52.56 ± 11.53 years, 60.8% of them were male, and 18.6% of them had arterial stiffness. There were significant differences in baPWV level and proportion of arterial stiffness (p < .001) between the four subgroups categorized by the FAR quartile. The relationships between the FAR and baPWV and arterial stiffness were significantly favorable in the overall population and subgroups of elderly men and non-elderly men (p < .01), while they were insignificant in subgroups of elderly and non-elderly women (p > .05). To investigate the correlation between the FAR and baPWV, the arterial stiffness and the FAR in male T2D patients, respectively, multivariable logistic regression analysis and multiple linear regression analysis were developed. The lnFAR and lnbaPWV had a significant relationship in the multiple linear regression analysis fully adjusted model. After adjusting for potential covariables, multivariable logistic regression analysis revealed that the FAR was independently associated with arterial stiffness [OR (95% CI), 1.075 (1.031-1.120)]. In addition, receiver operating characteristic analysis indicated that the best FAR cutoff value for detecting arterial stiffness in male T2D patients was 76.67 mg/g. Conclusion: The level of FAR had an independent and positive correlation with baPWV and arterial stiffness in male patients with T2D, but not in female patients.

Integrative Analysis of Minichromosome Maintenance Proteins and Their Prognostic Significance in Melanoma.

BACKGROUND: Minichromosome maintenance (MCM) is known for participating in cell cycle progression, as well as DNA replication. While the diverse expression patterns and prognostic values of MCMs in melanoma still remained unclear. METHODS: In the present study, the transcriptional and clinical profiles of MCMs were explored in patients with melanoma from multiple databases, including GEO, TCGA, ONCOMINE, GEPIA, UALCAN, cBioPortal, and TIMER databases. RESULTS: We found that the elevated expressions of MCM2-6 and MCM10 were significantly expressed in melanoma compared to normal skin. High mRNA levels of MCM4, MCM5, and MCM10 were closely related to worse prognosis in patients with melanoma. GSEA showed hallmark pathways were most involved in mTORC1 signaling, G2M checkpoint, E2F targets, and mitotic spindle. Furthermore, we found potential correlations between the MCM expression and the immune cell infiltration, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells. CONCLUSION: Upregulated MCM gene expression in melanoma probably played a crucial part in the development and progression of melanoma. The upregulated MCM4/5/10 expressions could be used as potential prognostic markers to improve the poor outcome and prognostic accuracy in patients with melanoma. Our study might shed light on the selection of prognostic biomarkers as well as the underlying molecular pathogenesis of melanoma.

SphK1-targeted miR-6784 inhibits functions of skin squamous cell carcinoma cells.

Sphingosine kinase 1 (SphK1) is overexpressed in skin squamous cell carcinoma (SCC). It has emerged as a novel therapeutic oncotarget. The current study identified a novel SphK1-targeting microRNA, microRNA-6784 (miR-6784). Here, we show that miR-6784 is located at the cytoplasm of A431 skin SCC cells. It directly binds to SphK1 mRNA. Ectopic overexpression of miR-6784 inhibited SphK1 3'-untranslated region (UTR) luciferase activity and downregulated its expression. Moreover, miR-6784 overexpression caused ceramide accumulation in skin SCC cells. Functional studies in established (A431 and SCC9) and primary skin SCC cells revealed that miR-6784 overexpression inhibited cell viability, proliferation, migration, and invasion. It also simultaneously provoked apoptosis activation. Conversely, miR-6784 silencing by antagomiR-6784 induced SphK1 elevation and augmented A431 cell proliferation, migration, and invasion. miR-6784 overexpression-induced anti-A431 cell activity was inhibited by the expression of an UTR-null SphK1 construct. CRISPR/Cas9-induced SphK1 knockout inhibited A431 cell growth. Importantly, miR-6784 was completely ineffective when treating SphK1-knockout A431 cells. Collectively, miR-6784 silences SphK1 and inhibits skin SCC cell progression.

Parameterized design and fabrication of porous bone scaffolds for the repair of cranial defects.

In bone tissue engineering, the structure of a scaffold is very important for cell growth and bone regeneration. It is better to make the scaffold resemble the native cancellous bone because natural cancellous bone can promote scaffold revascularization, which then accelerates cell proliferation. This study presents a parameterized design and fabrication method for cranial scaffold construction. A native human cranial sample was first scanned using micro computed tomography (CT), followed by 3D reconstruction, after which the internal structure of the bone trabecula was created. Based on an extracted negative bone trabecula model, the design components of "cavity", "connecting pipe" and "spatial framework" were proposed to describe the scaffold model. Then, by using the parameterized component model and an assembly and deformation algorithm, the bionic scaffold was designed. Its porous distribution, connection, porosity and area size were easily controlled. Finally, a biomaterial scaffold case was fabricated using a gelcasting process, and cell culture testing was performed in vitro to verify the scaffold's biocompatibility. The results show that the scaffold can promote cell growth and that cells accumulate in the form of a mass within three days.

virB11 gene potentially involves in ATP metabolism to provide energy in H. pylori infection.

Helicobater pylori (H. pylori) is the most important bacteria known to be associated with various gastroduodenal diseases. virB11 gene is a structural gene of tfs3a genes cluster in the plasticity region of H. pylori. In this study, the structure and biology of virB11 gene were analyzed and elucidated with bioinformatics analysis. After cloning, expression and purification, VirB11 protein was generated for the cytotoxicity to GES-1 cells and the anti-VirB11 protein antibody production for localization and interaction proteins analysis. The results showed that VirB11 protein is a hydrophilic protein, mainly locates in cell membrane. IL-8 productions from GES-1 cells co-culture with VirB11 protein were increased gradually with time (p < 0.001). The interaction proteins of VirB11 protein were F0F1 ATP synthase subunit alpha, ATP synthase subunit beta and isocitrate dehydrogenase. We demonstrate that VirB11 protein possesses cytotoxicity and potentially plays important roles in ATP metabolism to provide energy in the course of H. pylori infection.

Can Free Carnitine or Bilirubin in Blood Be Used in Neonatal Screening for Biliary Atresia?

OBJECTIVE: To investigate the efficiency of free carnitine, unconjugated bilirubin (UBIL), bilirubin monoglucuronide (BMG), and bilirubin diglucuronide (BDG) in dry blood spots (DBSs) measured using tandem mass spectrometry (MS/MS) for screening biliary atresia (BA). MATERIALS AND METHODS: All the patients with BA, residing in Shanghai, were collected from four different children's hospitals in Shanghai from January 1, 2015, to June 30, 2017. UBILMS, BMG, BDG, and free carnitine were measured in the DBS samples of 48 patients with BA, 10,008 pediatric patients, and 52,862 newborns using MS/MS. Conjugated bilirubin was measured by MS/MS (CBMS) = BMG + BDG, and total bilirubin was measured by MS/MS (TBMS) = UBILMS + CBMS. Four hundred pediatric patients' direct bilirubin (DB) and total bilirubin (TB), measured by the clinical laboratory and MS/MS, were used as a control. RESULTS: The total number of births at the registered permanent residences in Shanghai was 233,000; among them, the occurrence of BA was in 33 patients in 2 years. Therefore, the incidence of BA in Shanghai was 1:7,060. The ratio of DB/TB and CBMS/TBMS of most patients with BA was elevated gradually in the neonatal period and higher than the normal range after 1 month after birth. The area under the receiver operating characteristic curve of DB, DB/TB, CBMS/TBMS, CBMS, and free carnitine for predicting BA was 0.98, 0.95, 0.73, 0.57, and 0.92, respectively. Using the 95% percentile as a cutoff, the sensitivity of DB and free carnitine to predict BA was 100 and 85%, respectively, and the specificity was 52 and 85%, respectively. CONCLUSION: In free carnitine, DB, and CBMS/TBMS tests, blood concentrations are elevated in all infants with BA. However, they may not be elevated while they are newborns. These tests will result in high false negatives or positives. Thus, they should not be used as newborn screening tests for BA due to their lower sensitivity and specificity.

miRNA-221 promotes cutaneous squamous cell carcinoma progression by targeting PTEN.

BACKGROUND: Cutaneous squamous cell carcinoma (CSCC) is a common type of skin malignancy. MicroRNA-221 (miRNA-221) is a critical non-coding RNA in tumor initiation and progression. However, the molecular mechanisms of miRNA-221 in the development of CSCC remain unknown. This study investigated the expression of miRNA-221 in CSCC and its potential tumor biological functions. METHODS: MTT assay, colony assay, PCR, and Western blot were adopted. RESULTS: In this study, miRNA-221 expression was significantly higher in CSCC tissues and cell lines than in normal tissues and cells (P < 0.05). Further functional experiments indicated that miRNA-221 knockdown inhibited the proliferation and cell cycle, while upregulation of miRNA-221 presented the opposite role. The dual reporter gene assays indicated that PTEN is a direct target gene of miRNA-221. PTEN protein or mRNA levels were decreased after the cells were transfected with miR-221 mimics. CONCLUSIONS: Taken together, the obtained results indicated that miR-221 plays an oncogenic function in CSCC by targeting PTEN and further suggest that miR-221 may be a potential target for CSCC diagnosis and treatment.

Treatment of First-Time Perianal Abscess in Childhood, Balance Recurrence and Fistula Formation Rate with Medical Intervention.

PURPOSE: Because of differences in therapy for first-time perianal abscess, a wide range of recurrences and/or development of fistula-in-ano (RF) rates have been reported. The indication for determining when surgical intervention is needed remains obscure and controversial. This retrospective study sought to compare outcomes of conservative treatment with those after incision and drainage (ID) to determine the optimal time for surgical intervention. METHODS: A total of 697 patients with first-time perianal abscess were included in this study. The median patient age at the time of onset was 129 days (range: 5-5,110 days). The median follow-up period was 395 days (range: 120-760 days). RESULTS: Of the 697 patients with first-time perianal abscess, 355 (50.9%) patients who received conservative treatment had 12.7% (45/355) RF rate, which is less than that of abscesses treated with ID (24.6%, 72/297; p < 0.001). The median course was 23 (2,466) days, which did not differ significantly from that of abscesses with ID (18 [3,510] days) (p = 0.609). Forty-six (6.6%) patients with abscesses that perforated spontaneously had 10.9% (5/46) RF rate, which was less than that of abscesses with ID (p = 0.019), and the median course was 9 (3,316) days, which was shorter than that of abscesses with ID (p = 0.04). CONCLUSION: Conservative treatment is a safe and effective technique for most first-time perianal abscesses with less recurrence and a lower fistula formation rate. Incision must be performed when an abscess is likely to spread or shows no sign of spontaneous perforation.

Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is commonly detected during mass screening for neonatal disease. We developed a method to measure reduced glutathione (GSH) and glutathione disulfide (GSSG) using tandem mass spectrometry (MS/MS) for detecting G6PD deficiency. METHODS: The concentration of GSH and the GSH/GSSG ratio in newborn dry-blood-spot (DBS) screening and in blood plus sodium citrate for test confirmation were examined by MS/MS using labeled glycine as an internal standard. RESULTS: G6PD-deficient newborns had a lower GSH content (242.9 ± 15.9 µmol/L)and GSH/GSSG ratio (14.9 ± 7.2) than neonatal controls (370.0 ± 53.2 µmol/L and 46.7 ± 19.6, respectively). Although the results showed a significance of P < 0.001 for DBS samples plus sodium citrate that were examined the first day after preparation, there were no significant differences in the mean GSH concentration and GSH/GSSG ratio between the G6PD deficiency-positive and negative groups when examined three days after sample preparation. CONCLUSION: The concentration of GSH and the ratio of GSH/GSSG in blood measured using MS/MS on the first day of sample preparation are consistent with G6PD activity and are helpful for diagnosing G6PD deficiency.

Fn14 hepatic progenitor cells are associated with liver fibrosis in biliary atresia.

PURPOSE: The liver in biliary atresia (BA) is characterized by progressing fibrosis which is promoted by unclear reasons. We aimed to understand the factors influencing liver fibrosis. This study hypothesized that HPCs (hepatic progenitor cells) are activated and associated with liver fibrosis in biliary atresia. METHODS: Liver samples from biliary atresia patients are as BA group, and the normal liver derived from hepatoblastoma infants during operation are control group. The extent of fibrosis in liver samples was blindly evaluated by two experienced pathologists depending on Ishak system. The BA liver samples were divided into mild liver fibrosis group (grade I-IV, BAa) and severe liver fibrosis group (grade V-VI, BAb) to detect Fn14 protein expression. RESULTS: In mRNA level, Fn14 expression was 21.23 ± 8.3 vs. 1.00 ± 0.17, p = 0.023 < 0.05 and CD133 expression was 6.02 ± 2.16 vs. 1.14 ± 0.75, p = 0.008 < 0.01 between BA group and control group. Fn14 cells co-expressed the progenitor marker CD133 in liver, and activated in BA. Fn14 andα-SMA were co-location in fibrous area in liver. Compared to the control group, Fn14, CD133, and α-SMA protein expression were 2.10 ± 0.53 vs. 0.97 ± 0.2, p = 0.001, 2.23 ± 0.57 vs. 1.00 ± 0.03, p = 0.000, 4.96 ± 2.4 vs. 1.00 ± 0.22, p = 0.001. The Fn14 protein expression was 2.60 ± 0.35 vs. 1.86 ± 0.42, p = 0.012, between BAb and BAa group. CONCLUSION: Fn14 cells, which co-express the progenitor marker CD133 in liver, are HPCs and activated in BA. Fn14 + HPCs are associated with liver fibrosis in BA.

Association of plasminogen activator inhibitor-1 and vitamin D receptor expression with the risk of keloid disease in a Chinese population.

Keloid disease (KD) is a benign fibroproliferative scarring condition of unknown etiopathogenesis. Plasminogen activator inhibitor-1 (PAI-1) and vitamin D receptor (VDR) have been shown to play important roles in the progression of tissue fibrosis; therefore, both these genes are potential susceptibility genes for KD. We aimed to determine whether the gene expression levels of PAI-1 and VDR are altered in Chinese KD patients. We measured the expression of PAI and VDR in human peripheral blood lymphocytes in 236 patients with keloid and 219 age- and sex-matched healthy controls by quantitative real-time polymerase chain reaction. We found that PAI-1 expression in peripheral blood lymphocytes was significantly higher in patients with KD than in control individuals (p < 0.0001), while VDR expression was significantly lower in KD patients than in control individuals (p < 0.0001). High levels of PAI-1 and low levels of VDR expression were significantly associated with an increased risk for KD. PAI-1 and VDR might play important roles in keloid development. Gene expression levels of PAI-1 and VDR may, therefore, be used as potential markers for the prediction of keloid development after scarring.

Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency Differentiated from Biliary Atresia.

Purpose The aim of this article is to differentiate neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) from biliary atresia (BA) by total hexose. Methods A total of 11 patients with NICCD, 29 patients with BA, and 4,898 children as controls were involved in this study. The blood concentration of amino acids, carnitine, acylcarnitines, and total hexose were measured in dry blood spots (DBS) using tandem mass spectrometry (MS/MS). Results In the patients with NICCD, the blood concentration of the total hexose (15.3 ± 9.0 mmol/L vs. 7.3 ± 2.7 mmol/L; p < 0.001), citrulline (Cit) (197.9 ± 93.7 µmol/L vs. 17.5 ± 7.4 µmol/L; p < 0.001) were higher than those of patients with BA. Using total hexose (> 10 mmol/L), Cit (> 55 µmol/L) to diagnose NICCD, the sensitivity and specificity were 66.7 and 97.8% and 90.0 and 99.1%, respectively, and all of the areas under the receiver-operating characteristic curves were greater than 0.85. Conclusion Elevated total hexose in DBS measured by MS/MS associated with elevated amino acids, especially Cit can be used to diagnose NICCD and differentiate it from BA.

Cloning of mating-type gene MAT1-1 from the caterpillar medicinal mushroom, Cordyceps militaris (Ascomycetes) using TAIL-PCR technology.

Cordyceps militaris and Ophiocordyceps sinensis (syn. Cordyceps sinensis), 2 well-known traditional Chinese medicines, contain the same bioactive components and share a similar developmental process. In this study, one C. militaris strain preserved in our laboratory was proven to be a MAT1 mating-type strain using a polymerase chain reaction-based mating-type assay. A 5000-bp nucleotide sequence of the mating-type MAT1-1 from C. militaris was amplified by thermal asymmetric interlaced polymerase chain reaction, but genes within the mating-type MAT1-2 remain undetectable. Sequence analysis shows that the mating-type gene MAT1-1 idiomorph contains 2 genes, MAT1-1-1 and MAT1-1-2. The MAT1-1-1 gene consists of 1480-bp nucleotides that encode 456 amino acids and contain the conserved a-box domain interrupted by 2 introns; the MAT1-1-2 gene consists of 1066 nucleotides that encode 377 amino acids interrupted by one intron. The intervening distance between MAT1-1-1 and MAT1-1-2 is 778 bp. The C. militaris MAT1-1 idiomorph organization is the same as that of Cordyceps takaomontana. The MAT1-1 mating-type idiomorph of both Cordyceps species lacks the MAT1-1-3 gene, which is typically present in Pyrenomycetes. These studies provide some insights for further study of the morphological development of C. militaris and will eventually benefit the domestication of O. sinensis.

Isolation and analysis of a very virulent Marek's disease virus strain in China.

BACKGROUND: A severe MD was broken out at a farm in Shandong, China, despite FC126 vaccination of the chickens at 1-day-old. The mortality of the flocks reached up to 38.3%. The infected chickens were found to have MD pathological changes, including enlargement of spleens, livers and kidneys, and tumors occured on organs later. Samples were collected from the chickens for diagnosis. METHODS: The collected samples were inoculated into primary duck embryo fibroblast (DEF) cells, and the MDV strain named SD2012-1 was isolated. In order to identify the isolate, amplification by PCR and sequencing of oncogenic Meq and vIL-8 gene were processed, the obtained sequences were compared with the sequences of reference strains, and SD2012-1 was used to challenge immunized SPF chickens. RESULTS: A very virulent MDV isolate strain, SD2012-1, was isolated from a chicken flock in Shandong Province, China, the isolate had the characteristics of very virulent MDV-1, nucleotide and deduced amino acid sequence comparisons of Meq and vIL-8 gene of SD2012-1 with those of reference strains showed SD2012-1 had high homology with MDV strains isolated from China, SD2012-1 could break through the protection provided by HVT vaccine and HVT + SB-1 vaccine immunization and caused the mortality of SPF chickens over 60%. The immune failure occured at the farm could be due to the improper selection of vaccines. SD2012-1 produced death later and the gross postmortem lesions of chickens died early and later were different. CONCLUSIONS: MDV strain SD2012-1 isolated from Shandong Province, China was found to have the characteristics of very virulent MDV-1, which could break through the protection provided by HVT vaccine and HVT + SB-1 vaccine, the virus seemed to have a long latent period, and cause different gross postmortem lesions of chickens between chickens died early and later. A better immunization way should be chosen to prevent infection of this MDV strain in field.

Quantification of total hexose on dry blood spot by tandem mass spectrometry.

BACKGROUND: Because hypoglycemia and hyperglycemia are harmful and not always associated with overt clinical signs, it is necessary to have methods available to screen for glucose levels to detect hypoglycemia and diabetes as early as possible. A new method for such screening and the clinical determination of blood total hexose on a dry blood spot (DBS) using tandem mass spectrometry (MS/MS) was developed. METHODS: The serum glucose controls and blood were prepared as DBS and then extracted into a methanol solution containing isotope-labeled internal standards. The methanolic extraction was subjected to HPLC, followed by MS/MS in positive ion mode. Multiple-reaction monitoring of m/z 203.1→23 was used to detect hexose, and m/z 209.0→23 was used for 13C6-D-glucose. RESULTS: The recoveries of blood glucose by MS/MS were 90%-102% with an R(2) value of 0.999 after linear regression (p<0.001). The controls were within an acceptable range, and the coefficients of variation were less than 10%. The blood total hexose in neonates aged 3-7 days (6.41±1.46 mmol/L) was lower than that in neonates aged 8-30 days (6.66±1.38 mmol/L), and it was lower in neonates than in children aged 1-72 months (7.19±1.87 mmol/L). CONCLUSION: Quantification of total hexose on a dry blood spot by MS/MS is accurate, reliable and feasible for screening and clinical tests.