[Analysis of genetic variants in a patient with Familial hemophagocytic lymphohistiocytosis].

OBJECTIVE: To explore the genetic basis for a patient with Familial hemophagocytic lymphohistiocytosis (FHL). METHODS: A 35-day-old male infant who was admitted to the Oriental Hospital Affiliated to Xiamen University on August 3, 2021 due to fever for over 7 hours was selected as the study subject. Whole exome sequencing (WES) was carried out for the proband and his parents, and candidate variants were selected based on the clinical phenotypes of the proband and confirmed by Sanger sequencing. RESULTS: WES and Sanger sequencing results revealed that the proband had harbored compound heterozygous c.67_71delinsGCCC and c.65delC variants of the PRF1 gene, which were respectively inherited from his mother and father. The c.67_71delinsGCCC variant was unreported previously. Based on the guidelines of American College of Medical Genetics and Genomics and clinical manifestations, it was classified as pathogenic (PVS1+PM2_Supporting+PM3+PP4). c.65delC was a known pathogenic variant (PVS1+PM2_Supporting+PM3_Strong+PP4). CONCLUSION: The compound heterozygous variants of c.67_71delinsGCCC and c.65delC of the PRF1 gene probably underlay the disease in the proband. The identification of the novel variant has expanded the mutational spectrum of the PRF1 gene.

Correlation analysis of potential factors influencing graft maturity based on MRI after lateral meniscal allograft transplantation.

The aim of this respective study was to assess the graft signal/noise quotient (SNQ) value and associated factors based on magnetic resonance imaging (MRI) after lateral meniscal allograft transplantation (LMAT). Patients with LMAT were included. The SNQ, width of the anterior horn (WAH), width of the midbody (WMB), width of the posterior horn (WPH) of each lateral meniscus, coronal graft extrusion (CGE), the anterior cartilage meniscus distance (ACMD) and the posterior cartilage meniscus distance (PCMD) were measured using MRI and tested by multivariate stepwise regression analysis. The relative percentage of extrusion (PRE) was calculated. Seventy-one male patients were examined, and 7 patients were lost to follow-up. The SNQ of the meniscus increased from immediately after surgery to 6 months postoperatively, decreased from 6 to 12 months, increased from 12 to 24 months, and increased from 24 to 36 months. The mean SNQ had a significant negative association with the WPH and CGE at 6 months (p < 0.05), the WPH at 1 year (p < 0.05), the PRE of CGE (CPRE) at 2 years (p < 0.05), and the PCMD, CPRE, and PRE of the PCMD (PPRE) at 3 years (p < 0.01) postoperatively. Multivariate stepwise regression analysis showed that the WPH at 6 months, WPH at 1 year, WMD and PCMD at 2 years, and WMD, ACMD and CGE at 3 years were significant independent factors correlated with the mean SNQ of grafts in different periods. Maturation of meniscal grafts fluctuated with time. The maturation process occupied the main role before 1 year postoperatively, but after the maturation process, tearing of the meniscal allograft played the leading role. Changes in an allograft's location had an obvious association with the SNQ. The WPH influenced the graft SNQ value at 6 months and 1 year postoperatively, but after the maturation process, the WMB and graft extrusion played the same roles.

Biallelic mutations in DCDC2 cause neonatal sclerosing cholangitis in a Chinese family.

BACKGROUND: Neonatal sclerosing cholangitis (NSC) is a severe cholestatic liver disease, which often develops into end-stage liver disease in childhood and requires liver transplantation. Mutations in CLDN1 and DCDC2 are confirmed to be the main pathogenic mechanism of NSC. METHODS: Whole exon sequencing (WES) was performed to find the possible disease-causing mutations of this family. The mutation was confirmed by Sanger sequencing, and large fragment copy number variation was confirmed by qPCR. RESULTS: We found novel biallelic mutations c.[705-2A>G];[923_1023del] in the DCDC2 gene of the proband. The proband's father had the heterozygous mutation c.705-2A>G, and his mother had a heterozygous c.923_1023del. The proband's younger brother, who had similar clinical manifestations, was found the same biallelic mutations with the proband. CONCLUSION: Novel biallelic mutations were identified in DCDC2 of this Chinese family, according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants, both mutations were classified as pathogenic, which might be the cause of NSC in this family.

[Genetic analysis of a pedigree affected with congenital high myopia caused by a novel splice site variant of COL11A1 gene].

OBJECTIVE: To analyze genetic variant in a pedigree affected with congenital high myopia. METHODS: Whole exome sequencing (WES) was carried out for the proband. Suspected variation was verified with Sanger sequencing. The pedigree was also subjected to co-segregation analysis. RESULTS: WES has identified a novel splice site heterozygous variant (c.2556+1G>A) in the COL11A1 gene in the proband. Co-segregation analysis of the pedigree showed that the affected mother and two daughters of the proband have carried the same variant(c.2556+1G>A), while his unaffected father and sister did not. Based on the ACMG Standards and Guidelines for the Interpretation of Sequence Variants, the variant was classified as "likely pathogenic" (PVS1+PM2). CONCLUSION: A novel splice variant (c.2556+1G>A) of the COL11A1 gene has been identified in a pedigree affected with congenital high myopia, which probably underlies the disease.

MicroRNA­192 inhibits cell proliferation and induces apoptosis in human breast cancer by targeting caveolin 1.

It has been demonstrated that microRNA­192 (miR­192) serves important roles in different cancer types, including breast cancer, prostate cancer and colorectal cancer. However, its biological role and function in breast cancer remains largely unknown. The present study aimed to determine the role of miR­192 in breast cancer. In the present study, one normal breast and two breast tumor cells lines were used, which included the normal mammary fibroblast cell line Hs578Bst, a more aggressive breast tumor cell line MDA­MB­231 and a less aggressive breast tumor cell line MCF­7. The effect of miR­192 on proliferation of breast cancer cells was detected with an MTT assay. Western blot analysis was performed to determine protein expression of caveolin 1 (CAV1). A lentiviral vector that overexpresses pre­miR­192 and control lentiviral packaging plasmids were used in the present study. The Student's t­test was performed to analyze the significance of differences between samples. In the present study, it was determined that the expression of miR­192 is downregulated in breast cancer, compared with the adjacent normal tissues. Overexpression of miR­192 significantly inhibited cell proliferation, and induced cell apoptosis and cell cycle arrest in MCF7 and MDA­MB­231 cells. Using a bioinformatics method, CAV1 was considered a potential target of miR­192. Furthermore, it was demonstrated that CAV1 is a direct target of miR­192 and its protein expression is negatively regulated by miR­192. Therefore, the present study demonstrated that miR­192 serves an important role as a regulator in breast cancer and the miR­192/CAV1 axis has a potential as a therapeutic target for treatment of breast cancer.

The clinical application of preimplantation genetic diagnosis for X-linked retinitis pigmentosa.

OBJECTIVE: To investigate the usefulness of preimplantation genetic diagnosis (PGD) based on mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) for a pedigree with X-linked retinitis pigmentosa (XLRP). METHODS: One pathogenic mutation (c.494G > A) of the retinitis pigmentosa GTPase regulator (RPGR) gene was identified in a pedigree affected by XLRP. Then, PGD was carried out for the couple, of which the wife was an XLRP carrier. Three blastocysts were biopsied and then MARSALA was performed by next-generation sequencing (NGS). Prenatal diagnosis was also carried out to confirm the PGD results. RESULTS: Three blastocysts were all unaffected. Then, one of the embryos was chosen randomly to be transferred, and the pregnancy was acquired successfully. The results of prenatal diagnosis were consistent with the PGD results. The fetus did not carry RPGR mutation (c.494G > A) and had normal chromosome karyotype. As a result, a healthy baby free of XLRP condition was born. CONCLUSION: The PGD method based on MARSALA was established and applied to a family with XLRP successfully. MARSALA will be a valid tool, not only for XLRP families but also for families affected with other monogenetic disorders, to prevent transmission of the genetic disease from parents to offspring.

Robotic single-site surgery for mature cyst teratoma cystectomy: an initial case series study in a single medical center in China.

OBJECTIVE: To report the first case series of robotic single-site (RSS) surgery via the da Vinci Si Surgical System for mature cyst teratoma cystectomy in China. MATERIALS AND METHODS: The study was devised as a retrospective study in a single medical center. Five patients with mature cyst teratomas requested a minimally invasive surgical treatment. These patients were treated with RSS surgery for mature cyst teratoma between January 2014 and January 2015. RSS mature cyst teratoma cystectomies were performed with the da Vinci single-site platform in the Hainan branch of PLA General Hospital. Data regarding patient characteristics, surgical approach, and perioperative clinical outcomes were collected and analyzed in a retrospective study. RESULTS: All RSS procedures were completed successfully in the five patients. No instrument failure was noted during the procedures. The median operating time was 65 minutes (range 45-100 minutes). The median docking time was 20 minutes (range 18-28 minutes). No instrument failure was noted during any surgical procedures. The median blood loss was 30 mL (range 10-70 mL). No patient had massive intraoperative bleeding nor required a transfusion. No extra trocar was placed during the surgery. None of the patients had bladder or rectal injury. The median length of stay in hospital was 2.8 days. All patients were followed up until 6 months postoperatively, and no surgical complication occurred. CONCLUSION: RSS mature cyst teratoma cystectomy using the wristed semirigid instrumentation is feasible. Randomized controlled trials with a larger number of patients and longer postoperative follow-up should be conducted to further evaluate the effect of this therapeutic strategy.

Association between Delayed Lactogenesis â ¡ and Early Milk Volume among Mothers of Preterm Infants.

PURPOSE: This study aimed to evaluate the effect of delayed lactogenesis Ⅱ on early milk volume in mothers expressing milk for their preterm infants. METHODS: 142 mothers with preterm infants participated in a longitudinal cohort study, the milk volumes over 14 days postpartum between mothers with delayed lactogenesis Ⅱ (≥ 72 hours) and mothers with non-delayed lactogenesis Ⅱ(< 72 hours) were compared using Wilcoxon's rank sum tests. RESULTS: The prevalence of delayed lactogenesisⅡ among mothers of preterm infants was 36.0% (36/100). There existed negative correlations between the onset of lactogenesis Ⅱ and the daily milk volumes( rs = -0.525∼-0.354, p = .002 ∼ p < .001). The milk volumes in every 24-hour of the 14 days postpartum in delayed group were significantly less than that in non-delayed group (p = .002 ∼ p < .001). After controlling for the covariates, pregnancy-induced hypertension syndrome, delayed expression initiation, shorter daily sleeping time were found to be the risk factors for delayed lactogenesis Ⅱ. CONCLUSION: Delayed lactogenesis Ⅱ was associated with lower milk volume in early postpartum period. Women who were at risk for delayed lactogenesis Ⅱ need targeted interventions and additional support during pregnancy and postpartum.

[Familial Y chromosome microdeletion with pericentric inversion of chromosome 19].

OBJECTIVE: To investigate the familial cytomolecular genetics of an infertile male. METHODS: We analyzed the clinical phenotypes and karyotypes of three males from the family of an infertile man, detected the sequence-tagged sites (STS) in the AZF deletions of the Y chromosome by multiplex polymerase chain reaction (PCR), and identified the target genes by multiplex ligation-dependent probe amplification (MLPA). RESULTS: The karyotypes of the proband and his brother were 46, XY, inv (19) (p13.3q13.1) and that of his father was 46, XY. The three males were all carriers of AZFc deletion of the Y chromosome, and all found with the same reduction of the gene copy number in the AZFb and AZFc regions. CONCLUSIONS: Combined use of karyotype analysis, Y chromosome STS PCR, and MLPA revealed the genetic causes of the male infertile family.

CQ synergistically sensitizes human colorectal cancer cells to SN-38/CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk.

Autophagy plays a key role in supporting cell survival against chemotherapy-induced apoptosis. In this study, we found the chemotherapy agent SN-38 induced autophagy in colorectal cancer (CRC) cells. However, inhibition of autophagy using a small molecular inhibitor 3-methyladenine (3-MA) and ATG5 siRNA did not increase SN-38-induced cytotoxicity in CRC cells. Notably, another autophagy inhibitor chloroquine (CQ) synergistically enhanced the anti-tumor activity of SN-38 in CRC cells with wild type (WT) p53. Subsequently, we identified a potential mechanism of this cooperative interaction by showing that CQ and SN-38 acted together to trigger reactive oxygen species (ROS) burst, upregulate p53 expression, elicit the loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm). In addition, ROS induced by CQ plus SN-38 upregulated p53 levels by activating p38, conversely, p53 stimulated ROS. These results suggested that ROS and p53 reciprocally promoted each other's production and cooperated to induce CRC cell death. Moreover, we showed induction of ROS and p53 by the two agents provoked the loss of LMP and ∆ψm. Altogether, all results suggested that CQ synergistically sensitized human CRC cells with WT p53 to SN-38 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk. Lastly, we showed that CQ could enhance CRC cells response to CPT-11 (a prodrug of SN-38) in xenograft models. Thus the combined treatment might represent an attractive therapeutic strategy for the treatment of CRC.

Breastfeeding Evaluation Indicators System is a Promising Evaluation Tool for Preterm Infants in Neonatal Intensive Care Units (NICU).

BACKGROUND Breast feeding can enhance preterm infants' neurodevelopmental outcome, regulate immune function development. This study aims to develop breastfeeding evaluation indicators system in neonatal intensive care units (NICU) and to provide theoretical basis for all-round evaluation of breast feeding quality for hospitalized preterm infants. MATERIAL AND METHODS This study was performed based on Avedis Donabedian's theory of medical care quality. Preterm infant breast feeding evaluation indicators system frame was initially formed by using literature review, clinical on-spot observation and expert consultation methods. By using specialists meeting method and Delphi method, evaluation indicators system for preterm infants breastfeeding was verified and established. Breastfeeding evaluation indicators system were performed in NICU of hospitals in Binzhou and Shanghai. Feasibility and usability of indicators system were examined. RESULTS Breastfeeding evaluation indicators system for preterm infants comprise 3 levels, including level 1 (3 indicators), level 2 (7 indicators), and level 3 (18 indicators). Recognition rates of importance for level 2 and 3 range from 94.4% to 100.0% and 80.6% to 100.0%, respectively. Mean of Likert rating for level 2 and 3 range from 3.31 to 3.89 and 3.03 to 3.97, which are all higher than the average value of 2.50. Kendall's coefficient and its significance test showed that consistency of experts' opinion for indicators' importance is high (P<0.001). This strategy of combining qualitative and quantitative methods could be used in overall evaluation of the breastfeeding quality in NICUs. CONCLUSIONS Indicators system is feasible and is a promising evaluation tool for continuously improving breastfeeding quality for preterm infants in NICUs.

The possible role of CD4⁺CD25(high)Foxp3⁺/CD4⁺IL-17A⁺ cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis.

Hashimoto thyroiditis (HT) is a prototypic organ-specific autoimmune thyroid disease, for which the exact etiology remains unclear. The aim of this study was to investigate dynamic changes in regulatory T cell (Treg) and T helper 17 cell (Th17) populations in patients with HT at different stages of thyroid dysfunction, as well as to analyze the possible correlation between the Treg/Th17 cell axis and autoimmune status in HT. We assessed thyroid function and autoantibody serology both in HT patients and in healthy controls (HCs) and divided HT patients into three subgroups according to thyroid function. We then determined the percentages of Treg and Th17 cells in peripheral blood mononuclear cells and analyzed mRNA expression of the Treg and Th17 cell-defining transcription factors Foxp3 and RORγt. In addition, serum levels of TGF-ß and IL-17A were assessed. We found that the percentage of Treg cells, Foxp3 mRNA levels, and the ratio of Treg/Th17 cells were all significantly lower in HT patients, while Th17 cell percentages and RORγt mRNA levels were significantly higher. Interestingly, we also observed significant differences in these measurements between HT patient subgroups. Serum IL-17A levels were markedly increased in HT patients, while serum concentrations of TGF-ß were lower, compared to HCs. The ratio of Treg/Th17 cells was negatively correlated with the levels of serum thyroperoxidase antibody, thyroglobulin antibody, and thyrotropin (TSH) in HT patients. Taken together, our data suggest that the balance between Treg and Th17 cells shifts in favor of Th17 cells during clinical progression of HT, which is negatively correlated with levels of thyroid-specific autoantibodies and TSH, implying that Treg/Th17 cell imbalance may contribute to thyroid damage in HT.

A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover.

BACKGROUND: In a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production. RESULTS: An on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEX(TM)), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation. CONCLUSIONS: Compositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.

Combination of gefitinib and DNA methylation inhibitor decitabine exerts synergistic anti-cancer activity in colon cancer cells.

Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR) and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor) synergized with gefitinib (an EGFR inhibitor) to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1) which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA) depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon cancer.

Effect of hyperoxia on the viability and proliferation of the primary type II alveolar epithelial cells.

To observe the effect of hyperoxia on the growth of type II alveolar epithelial cells (AEC II). The lungs of 19-day gestation fetal rats were primary cultured and the AEC II were purified by differential adhesion method. The cells were divided into control (normoxia) group and hyperoxia group. The cell growth, cell viability, cell apoptosis, and cell cycle were examined at 2, 4, 6, and 8 days of normoxia or hyperoxia exposure. The number of cells in hyperoxia-exposed group significantly decreased as compared to those of air control group. Number of cells in hyperoxia group was the highest at day 2 of exposure and gradually decreased with time. The viability of cells exposed to hyperoxia was substantially reduced compared with cells exposed to air. Percentage of cells in G1 phase and S phase in hyperoxia group increased gradually with increase in exposure duration and significant differences were seen at day 4 and day 6 compared with either the preceding time points and also with corresponding air-exposed cells. The percentage of both early apoptotic cells (Annexin-V(+)/PI(-)) and late apoptotic cells and necrotic cells (Annexin-V(+)/PI(+)) increased significantly in cells exposed to hyperoxia compared with cells exposed to air. Hyperoxia inhibits proliferation, viability and growth of AEC II and promotes apoptosis.

Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH-ST).

High productivity processes are critical for commercial production of cellulosic ethanol. One high productivity process-continuous hydrolysis and fermentation-has been applied in corn ethanol industry. However, little research related to this process has been conducted on cellulosic ethanol production. Here, we report and compare the kinetics of both batch SHF (separate hydrolysis and co-fermentation) and SSCF (simultaneous saccharification and co-fermentation) of AFEX™ (Ammonia Fiber Expansion) pretreated corn stover (AFEX™-CS). Subsequently, we designed a SSCF process to evaluate continuous hydrolysis and fermentation performance on AFEX™-CS in a series of continuous stirred tank reactors (CSTRs). Based on similar sugar to ethanol conversions (around 80% glucose-to-ethanol conversion and 47% xylose-to-ethanol conversion), the overall process ethanol productivity for continuous SSCF was 2.3- and 1.8-fold higher than batch SHF and SSCF, respectively. Slow xylose fermentation and high concentrations of xylose oligomers were the major factors limiting further enhancement of productivity.

Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides.

BACKGROUND: High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides. RESULTS: The core fungal cellulases in the optimal cocktail include cellobiohydrolase I [CBH I; glycoside hydrolase (GH) family 7A], cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and ß-glucosidase (ßG; GH family 3). Hemicellulases tested along with the core cellulases include xylanases (LX1, GH family 10; LX2, GH family 10; LX3, GH family 10; LX4, GH family 11; LX5, GH family 10; LX6, GH family 10), ß-xylosidase (LßX; GH family 52), α-arabinofuranosidase (LArb, GH family 51) and α-glucuronidase (LαGl, GH family 67) that were cloned, expressed and/or purified from different bacterial sources. Different combinations of these enzymes were tested using a high-throughput microplate based 24 h hydrolysis assay. Both family 10 (LX3) and family 11 (LX4) xylanases were found to most efficiently hydrolyze AFEX pretreated corn stover in a synergistic manner. The optimal mass ratio of xylanases (LX3 and LX4) to cellulases (CBH I, CBH II and EG I) is 25:75. LßX (0.6 mg/g glucan) is crucial to obtaining monomeric xylose (54% xylose yield), while LArb (0.6 mg/g glucan) and LαGl (0.8 mg/g glucan) can both further increase xylose yield by an additional 20%. Compared with Accellerase 1000, a purified cocktail of cellulases supplemented with accessory hemicellulases will not only increase both glucose and xylose yields but will also decrease the total enzyme loading needed for equivalent yields. CONCLUSIONS: A diverse set of accessory hemicellulases was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (~20 mg protein/g glucan) using an in-house developed cocktail compared to commercial enzymes.

[Expression of MnSOD mRNA and protein in esophageal squamous cell carcinoma and its clinical significance].

OBJECTIVE: To investigate the expression of manganese superoxide dismutase (MnSOD) and to determine the relationship between MnSOD expression and clinicopathological features, biological behaviors in esophageal carcinoma. METHODS: Immunohistochemistry (SP) and RT-PCR were respectively used to detect the expression of MnSOD in 45 specimens of esophageal carcinoma tissues and normal esophageal mucosa (5 cm distant from the margin of cancer). RESULTS: The positive rate of MnSOD protein expression was 31.1% in esophageal carcinoma tissues, significantly lower than 86.7% in the normal tissues (P < 0.05). The expressions of MnSOD mRNA and protein were significantly correlated with the lesion length, depths of invasion and histological grade (P < 0.05), but not with lymph node metastasis, lesion site and gross type of the tumor (P > 0.05). The relative content of MnSOD mRNA was (0.310 ± 0.036) and (0.482 ± 0.053) in the cancer and normal tissues, respectively, with a significant difference between the two groups (P < 0.05). The relative content of MnSOD mRNA was significantly related to lesion length, depths of invasion and histological grade (P < 0.05), but not correlated with lymph node status, lesion site and gross type of the tumor (P > 0.05). CONCLUSION: The expression of MnSOD protein and mRNA is decreased in esophageal carcinoma, suggesting that MnSOD gene may be closely associated with the carcinogenesis and the degree of malignancy. Detection of MnSOD expression may be useful in diagnosis, treatment and prognosis of esophageal carcinoma.