Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of ∼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.

Foxq1 Promotes Alveolar Epithelial Cell Death through Tle1-mediated Inhibition of the NF-κB Signaling Pathway.

Acute lung injury (ALI) is a common respiratory disease characterized by diffuse alveolar injury and interstitial edema, as well as a hyperinflammatory response, lung cell damage, and oxidative stress. Foxq1, a member of the FOX family of transcription factors, is expressed in various tissues, such as the lungs, liver, and kidneys, and contributes to various biological processes, such as stress, metabolism, cell cycle arrest, and aging-related apoptosis. However, the role of Foxq1 in ALI is unknown. We constructed ex vivo and in vivo ALI models by LPS tracheal perfusion of ICR mice and conditioned medium stimulation of injured MLE-12 cells. Foxq1 expression was increased, and its localization was altered, in our ALI model. In normal or injured MLE-12 cells, knockdown of Foxq1 promoted cell survival, and overexpression had the opposite effect. This regulatory effect was likely mediated by Tle1 and the NF-κB/Bcl2/Bax signaling pathway. These data suggest a potential link between Foxq1 and ALI, indicating that Foxq1 can be used as a biomarker for the diagnosis of ALI. Targeted inhibition of Foxq1 expression could promote alveolar epithelial cell survival and may provide a strategy for mitigating ALI.

Transcriptional adaptor 3 influences the proliferative and invasive phenotypes of non-small cell lung cancer cells via regulating EMT.

Transcriptional adaptor 3 (TADA3/ADA3) is a conserved transcriptional co-activator and is dysregulated in many aggressive tumors. However, the role of TADA3 in non-small cell lung cancer (NSCLC) remains unknown. It was previously demonstrated that TADA3 expression correlates with poor prognosis in patients with NSCLC. In the present study, the expression and function of TADA3 were investigated in cells in vitro and in vivo. TADA3 expression was evaluated in clinical specimens and cell lines using reverse transcription-quantitative PCR and western blot analysis. The TADA3 protein level was significantly higher in human NSCLC specimens compared with matched normal tissues. In human NSCLC cell lines, short hairpin RNA-mediated silencing of TADA3 suppressed their proliferative, migratory and invasive abilities in vitro, and delayed G1 to S phase progression through the cell cycle. Consistent with this, TADA3 silencing increased expression of the epithelial marker E-cadherin and reduced expression of the mesenchymal markers, N-cadherin, Vimentin, Snail, and Slug. To verify the effect of TADA3 on tumor formation and growth in vivo, a mouse tumor xenograft model was established. TADA3 silencing slowed the growth of NSCLC tumor xenografts in nude mice, and excised tumors showed a similarly altered pattern of epithelial-mesenchymal transition (EMT) marker expression. The present results demonstrated the significance of TADA3 in regulating the growth and metastasis of NSCLC and may provide a theoretical basis for early diagnosis and targeted therapy of NSCLC.

Whole-Genome Sequencing of 502 Individuals from Latvia: The First Step towards a Population-Specific Reference of Genetic Variation.

Despite rapid improvements in the accessibility of whole-genome sequencing (WGS), understanding the extent of human genetic variation is limited by the scarce availability of genome sequences from underrepresented populations. Developing the population-scale reference database of Latvian genetic variation may fill the gap in European genomes and improve human genomics research. In this study, we analysed a high-coverage WGS dataset comprising 502 individuals selected from the Genome Database of the Latvian Population. An assessment of variant type, location in the genome, function, medical relevance, and novelty was performed, and a population-specific imputation reference panel (IRP) was developed. We identified more than 18.2 million variants in total, of which 3.3% so far are not represented in gnomAD and dbSNP databases. Moreover, we observed a notable though distinct clustering of the Latvian cohort within the European subpopulations. Finally, our findings demonstrate the improved performance of imputation of variants using the Latvian population-specific reference panel in the Latvian population compared to established IRPs. In summary, our study provides the first WGS data for a regional reference genome that will serve as a resource for the development of precision medicine and complement the global genome dataset, improving the understanding of human genetic variation.

Tumor heterogeneity assessed by sequencing and fluorescence in situ hybridization (FISH) data.

MOTIVATION: Computational reconstruction of clonal evolution in cancers has become a crucial tool for understanding how tumors initiate and progress and how this process varies across patients. The field still struggles, however, with special challenges of applying phylogenetic methods to cancers, such as the prevalence and importance of copy number alteration (CNA) and structural variation events in tumor evolution, which are difficult to profile accurately by prevailing sequencing methods in such a way that subsequent reconstruction by phylogenetic inference algorithms is accurate. RESULTS: In this work, we develop computational methods to combine sequencing with multiplex interphase fluorescence in situ hybridization to exploit the complementary advantages of each technology in inferring accurate models of clonal CNA evolution accounting for both focal changes and aneuploidy at whole-genome scales. By integrating such information in an integer linear programming framework, we demonstrate on simulated data that incorporation of FISH data substantially improves accurate inference of focal CNA and ploidy changes in clonal evolution from deconvolving bulk sequence data. Analysis of real glioblastoma data for which FISH, bulk sequence and single cell sequence are all available confirms the power of FISH to enhance accurate reconstruction of clonal copy number evolution in conjunction with bulk and optionally single-cell sequence data. AVAILABILITY AND IMPLEMENTATION: Source code is available on Github at https://github.com/CMUSchwartzLab/FISH_deconvolution. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chronic Pain Causes Peripheral and Central Responses in MIA-Induced TMJOA Rats.

Chronic pain is the predominant symptom that drives temporomandibular joint osteoarthritis (TMJOA) patients to seek medical care; however, currently used treatment modalities remain less effective. This study aimed to investigate chronic pain and the peripheral and central responses in monoiodoacetate (MIA)-induced TMJOA rats. First, the appropriate dose of MIA was determined based on pain behavior assessment in rats. Alterations of the condylar structure in TMJOA rats were evaluated by histological staining and micro-computed tomography (micro-CT). Second, the period of TMJOA chronic pain was further explored by assessing the numbers of glial fibrillary acidic protein (GFAP)-positive astrocytes and ionized calcium-binding adaptor molecule 1 (IBA-1)-positive microglia in the trigeminal spinal nucleus (TSN) and performing nonsteroidal anti-inflammatory drug (NSAID) efficacy experiments. Finally, the expression of neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4) in the trigeminal ganglion (TG) and TSN was assessed by immunofluorescence. MIA at 4 mg/kg was considered an appropriate dose. Gradual MIA-induced alterations of the condylar structure were correlated with temporomandibular joint (TMJ) pain. The numbers of GFAP- and IBA-1-positive cells were increased at 2, 3, and 4 weeks after MIA injection. NSAIDs failed to alleviate pain behavior 10 days after MIA injection. CGRP and IB4 levels in the TG and TSN were upregulated at 2 and 4 weeks. These results suggest that TMJOA-related chronic pain emerged 2 weeks after MIA injection. CGRP- and IB4-positive afferents in both the peripheral and central nervous systems may be involved in MIA-induced TMJOA-related chronic pain in rats.

STAT3 Promotes Cell Proliferation by Potentiating the CCL4 Transcriptional Activity in Diffuse Large B-Cell Lymphoma.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and a candidate therapeutic option for human cancers. However, the underlying mechanism of STAT3 in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) is yet to be established. We studied here whether STAT3 contributes to C-C motif chemokine ligand (CCL4) transcription elevation in DLBCL. Our established protein-protein interactions network revealed the overexpression of STAT3 and CCL4 in DLBCL. Mechanistically, STAT3 activated CCL4 transcription to induce the Wnt/ß-catenin pathway. The prognostic analysis exhibited that the overall survival of patients with high STAT3 and CCL4 were poorer than those with low STAT3 and CCL4 expression. In addition, silencing of STAT3 reverted the malignant phenotype in DLBCL cells. CCL4 overexpression partly weakened the si-STAT3-mediated antitumor effects on DLBCL cells. Tumor xenograft models showed that si-STAT3 inhibited tumor growth in vivo and decreased proliferative and mitogenic activities in tumor tissues, which was consistent with the in vitro data. Hence, this study provides new evidence that STAT3 and CCL4 may be new prognostic biomarkers and therapeutic targets for treating DLBCL.

Cytotoxic lanostane triterpenoids from the ethanol extract of Schisandra viridis.

Three new lanostane triterpenoids, designated as 6-hydroxyl schiglausin A (1), 29-hydroxyl schiglausin D (2), and 6-hydroxyl schiglausin G (3), were isolated from the ethanol extract of the stems of Schisandra viridis. Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic activities. As a result, compound 1 exhibited cytotoxic activities for all six tested human lung cancer cell lines with IC50 values less than 10 µM.

The Ideal Insertion Site for the Flexor Digitorum Profundus Tendon in Jersey Finger Repair: A Biomechanical Analysis.

PURPOSE: Most jersey finger repair techniques involve reattaching the tendon to an approximate location corresponding to the tendon's native attachment. This study aimed to determine the biomechanical effect on the distal interphalangeal joint flexion forces and range of motion when the flexor digitorum profundus (FDP) tendon attachment site on the distal phalanx is altered within its broad footprint. METHODS: We fixed 14 fresh-frozen cadaveric fingers to a wooden block with an attached pulley and weights system. A pressure mapping sensor placed under the fingertip measured the contact force and area in response to FDP tendon loading for the intact tendon and 3 repair sites along the FDP footprint. Two-way repeated-measures analysis of variance test using mixed-effect model was performed to test the influences of attachment location (intact, proximal, central, and distal) and digit (index, middle, and ring) on the outcomes. RESULTS: Mean ± SD contact force under 45 N tendon loading force was 43.5 ± 7.2 N for the intact tendon, 34.6 ± 7.4 N for the proximal insertion, 38.0 ± 7.1 N for the central insertion, and 43.1 ± 6.3 N for the distal insertion. Compared with the intact tendon, the proximal group generated notably less contact force. No significant difference was detected between the intact tendon and the central or distal repairs. Comparisons among the 3 repair groups show that the distal group generated significantly higher force than the proximal group. There was no difference between contact areas across all groups. CONCLUSIONS: The FDP tendon inserted at the distal edge of its footprint conferred significantly greater distal interphalangeal joint flexion force compared with the proximal insertion site and most closely resembled the intact FDP tendon. CLINICAL RELEVANCE: Biomechanically, distal reattachment of the FDP most closely approximates the contact force of the native anatomy and may help guide intraoperative placement of the repair footprint.

Exosomes from gingival mesenchymal stem cells enhance migration and osteogenic differentiation of pre-osteoblasts.

Gingival mesenchymal stem cells (GMSCs) have great potential in bone tissue regeneration. However, it is not well known how on exosomes derived from GMSCs affect the functions of bone-related cells. In this study, we explored the impact of GMSCs-derived exosomes (GMSCs-Exos) on pre-osteoblast MC3T3-E1 proliferation, migration and osteogenic differentiation. Results of CCK-8 assay showed that GMSCs-Exos had no effect on proliferation of pre-osteoblasts. Further, we found that GMSCs-Exos promoted the migration of pre-osteoblasts and osteogenic differentiation of MC3T3-E1 as revealed by enhanced Alizarin red staining, elevated alkaline phosphatase (ALP) activity and upregulated expression of osteogenic genes. This study provides new insights into the potential exosome-mediated paracrine mechanism of GMSCs in bone regeneration.

Association study of a functional variant of TNF-α gene and serum TNF-α level with the susceptibility of congenital heart disease in a Chinese population.

BACKGROUND: Congenital heart disease (CHD) is among the leading causes of infant death worldwide. Although shortage of folate has been found potentially to contribute to CHD in the embryo, the aetiology of CHD was not completely understood. Inflammation and altered immune processes are involved in all forms of cardiac malformation, including CHD. Tumour necrosis factor-α (TNF-α), was involved in the pathogenesis of multiple kinds of heart diseases. However, no studies have systematically evaluated the associations of genetic variants of TNF-α with susceptibility of CHD. METHODS: A case-control study was conducted to evaluate the associations between tagSNPs of TNF-α and CHD susceptibility. Serum level of TNF-α was assessed using ELISA. The dual luciferase reporter assay was used to evaluate the functional significance of variant rs1800629 on TNF-α transcriptional activity. RESULTS: We found rs1800629 was significantly correlated with increased CHD susceptibility (OR: 1.72, 95% CI 1.26 to 2.36, p=0.001). Serum levels of TNF-α were significantly higher in CHD group (9.09±1.90 pg/mL) than that in control group (6.12±1.56 pg/mL, p<0.001). The AA genotype and AG genotype of rs1800629 was associated with higher serum TNF-α level, compared with GG genotype. The dual luciferase reporter assay showed that promoter activity was significantly increased by 57% and 76% for plasmids containing the minor A allele compared with the major G allele in H9c2 and HEK 293T, respectively. CONCLUSION: These results indicate that higher level of serum TNF-α increases risk of CHD, while TNF-α rs1800629 A allele might contribute to higher risk for CHD due to the increase in TNF-α expression.

Vascularized Tissue Reconstruction in Previously Irradiated Sarcoma Defects.

INTRODUCTION: Radiation therapy (RT) is recommended for appropriately selected sarcoma patients to minimize the risk of local recurrence and to maximize outcomes of disease-free survival and function. The purpose of this study was to confirm the safety of vascularized tissue reconstruction in recently irradiated sarcoma defects. METHODS: A retrospective review of all patients treated by the senior author for sarcoma reconstruction from January 2005 to July 2017 was performed. Two independent reviewers collected data from both electronic and paper medical records. Patients were included if they underwent flap reconstruction (pedicled or free) following sarcoma resection. The safety of neoadjuvant RT was compared with a control group with no previous irradiation using χ(2) analysis. RESULTS: Fifty-seven patients were included in the study; 35 patients were included in the preoperative RT group, and 22 patients were included in the control group (no previous irradiation). There was no significant difference in wound complications between the 2 groups (infection, dehiscence, hematoma, and seroma). Microvascular complications (arterial thrombosis, venous thrombosis, partial/total flap loss) were also comparable in the free tissue transfer subgroup. CONCLUSIONS: The current study demonstrates the safety of both pedicled and free flap reconstruction in previously irradiated sarcoma defects. Judicious selection of reconstructive technique and recipient vessels is crucial in obtaining optimal outcomes given the devastating effects of RT on native tissues.

Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1N and KshA2N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2N to be a redundant form of kshA1N Through a combined modification of kshA1N, kshA2N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter-1 ADD from the transformation of 20 g liter-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter-1 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1N and KshA2N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.

Enhanced Red Emission in Er3+-Sensitized NaLuF4 Upconversion Crystals via Energy Trapping.

Luminescence efficiency of trivalent lanthanide-doped upconversion (UC) materials is significantly limited by luminescence concentration quenching. In this work, red UC emission is dramatically enhanced in Er3+-sensitized NaLuF4 UC crystals through energy trapping under multiple excitation wavelengths. Cross-relaxation quenching and the energy migration to internal lattice defects are simultaneously suppressed by confining the excitation energy in the Er3+ activator after introducing the Tm3+ or Ho3+ energy trapping center. The enhanced red UC emission (Er3+: 660 nm) mainly comes from the effective excitation energy confinement by Tm3+ and Ho3+ trapping centers through an easy energy transfer between Er3+ and Tm3+/Ho3+: 4I11/2 (Er3+) → 3H5 (Tm3+) → 4I13/2 (Er3+) and 4I11/2 (Er3+) → 5I6 (Ho3+) → 4I13/2 (Er3+). It is found that the confining efficiency of excitation energy in Er3+-sensitized NaLuF4 crystals is higher than that in Yb3+/Er3+ cosensitized NaLuF4 crystals, and the luminescence efficiency of Er3+-sensitized NaLuF4 crystals is much higher than that of Er3+-based host sensitization UC crystals (NaErF4). Moreover, Er3+-sensitized UC particles can be efficiently excited by three different wavelengths (808, 980, and 1532 nm), indicating huge advantages for applications in bioimaging, anticounterfeiting, and solar cells.

Contribution of upregulated dipeptidyl peptidase 9 (DPP9) in promoting tumoregenicity, metastasis and the prediction of poor prognosis in non-small cell lung cancer (NSCLC).

Dipeptidyl peptidase 9 (DPP9) is encoded by DPP9, which belongs to the DPP4 gene family. Proteins encoded by these genes have unique peptidase and extra-enzymatic functions that have been linked to various diseases including cancers. Here, we describe the expression pattern and biological function of DPP9 in non-small-cell lung cancer (NSCLC). The repression of DPP9 expression by small interfering RNA inhibited cell proliferation, migration, and invasion. Moreover, we explored the role of DPP9 in regulating epithelial-mesenchymal transition (EMT). The epithelial markers E-cadherin and MUC1 were significantly increased, while mesenchymal markers vimentin and S100A4 were markedly decreased in DPP9 knockdown cells. The downregulation of DPP9 in the NSCLC cells induced the expression of apoptosis-associated proteins both in vitro and in vivo. We investigated the protein expression levels of DPP9 by tissue microarray immunohistochemical assay (TMA-IHC) (n = 217). Further we found mRNA expression levels of DPP9 in 30 pairs of clinical NSCLC tissues were significantly lower than in the adjacent non-cancerous tissues. Survival analysis showed that the overexpression of DPP9 was a significant independent factor for poor 5-year overall survival in patients with NSCLC (p = 0.003). Taken together, DPP9 expression correlates with poor overall survival in NSCLC.

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes.

BACKGROUND: The strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols. In this study, a combined strategy of improving the cell envelope permeability as well as the intracellular sterol metabolism efficiency was investigated to increase the productivity of 4-HBC. RESULTS: MmpL3, encoding a transmembrane transporter of trehalose monomycolate, is an important gene influencing the assembly of mycobacterial cell envelope. The disruption of mmpL3 in Mycobacterium neoaurum ATCC 25795 significantly enhanced the cell permeability by 23.4% and the consumption capacity of sterols by 15.6%. Therefore, the inactivation of mmpL3 was performed in a 4-HBC-producing strain derived from the wild type M. neoaurum and the 4-HBC production in the engineered strain was increased by 24.7%. Subsequently, to enhance the metabolic efficiency of sterols, four key genes, choM1, choM2, cyp125, and fadA5, involved in the sterol conversion pathway were individually overexpressed in the engineered mmpL3-deficient strain. The production of 4-HBC displayed the increases of 18.5, 8.9, 14.5, and 12.1%, respectively. Then, the more efficient genes (choM1, cyp125, and fadA5) were co-overexpressed in the engineered mmpL3-deficient strain, and the productivity of 4-HBC was ultimately increased by 20.3% (0.0633 g/L/h, 7.59 g/L 4-HBC from 20 g/L phytosterol) compared with its original productivity (0.0526 g/L/h, 6.31 g/L 4-HBC from 20 g/L phytosterol) in an industrial resting cell bio-transformation system. CONCLUSIONS: Increasing cell permeability combined with the co-overexpression of the key genes (cyp125, choM1, and fadA5) involved in the conversion pathway of sterol to 4-HBC was effective to enhance the productivity of 4-HBC. The strategy might also be useful for the conversion of sterol to other steroidal intermediates by mycobacteria.

iNuc-PseKNC: a sequence-based predictor for predicting nucleosome positioning in genomes with pseudo k-tuple nucleotide composition.

MOTIVATION: Nucleosome positioning participates in many cellular activities and plays significant roles in regulating cellular processes. With the avalanche of genome sequences generated in the post-genomic age, it is highly desired to develop automated methods for rapidly and effectively identifying nucleosome positioning. Although some computational methods were proposed, most of them were species specific and neglected the intrinsic local structural properties that might play important roles in determining the nucleosome positioning on a DNA sequence. RESULTS: Here a predictor called 'iNuc-PseKNC' was developed for predicting nucleosome positioning in Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster genomes, respectively. In the new predictor, the samples of DNA sequences were formulated by a novel feature-vector called 'pseudo k-tuple nucleotide composition', into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on the three stringent benchmark datasets that the overall success rates achieved by iNuc-PseKNC in predicting the nucleosome positioning of the aforementioned three genomes were 86.27%, 86.90% and 79.97%, respectively. Meanwhile, the results obtained by iNuc-PseKNC on various benchmark datasets used by the previous investigators for different genomes also indicated that the current predictor remarkably outperformed its counterparts. AVAILABILITY: A user-friendly web-server, iNuc-PseKNC is freely accessible at http://lin.uestc.edu.cn/server/iNuc-PseKNC.

Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols.

3-Ketosteroid-△(1)-dehydrogenase (KstD) is a key enzyme involved in the microbial catabolism of sterols. Here, three homologues of KstD were characterized from Mycobacterium neoaurum ATCC 25795, showing distinct substrate preferences and transcriptional responses to steroids. Single deletion of any MN-kstD failed to result in a stable and maximum accumulation of 9-OHAD due to residual KstD activities. To develop stable 9-OHAD producers, all of these MN-KstDs were inactivated, which led to about 6.02g l(-1) of 9-OHAD from 15g l(-1) of phytosterols. However, the product was mixed with 1.55g l(-1) of AD as a major by-product. To transform AD, the oxygenase component of 3-ketosteroid-9α-hydroxylase (KSH), encoded by kshA, was overexpressed. As a result, the yield of 9-OHAD increased to 7.33g l(-1) with less than 0.31g l(-1) of AD and the selectivity of 9-OHAD production was improved to 95-97% among metabolites.

Health-related quality of life of children with physical disabilities: a longitudinal study.

BACKGROUND: Outcomes of health and rehabilitation services for children and youth with disabilities increasingly include assessments of health-related quality of life (HRQoL). The purpose of this research was to 1) describe overall patterns of HRQoL, 2) examine changes in parent's perceptions of child's HRQoL across 18 months and 3) explore factors that predict these changes. METHODS: Participants in this study included 427 parents of children (229 boys and 198 girls) with a physically-based disability between the ages of 6 to 14 years. The Child Health Questionnaire (CHQ) was administered three times, at nine month intervals. Comparisons to the CHQ normative data were analyzed at Time 1 using t-tests, and change over time was examined using linear mixed-effects models. Possible predictors were modeled: 1) child's factors measured by the Activities Scale for Kids, Strengths and Difficulties Questionnaire, and general health measured by SF-36, 2) family characteristics measured by the Impact on Family Scale and 3) environmental barriers measured by the Craig Hospital Inventory of Environmental Factors. RESULTS: CHQ scores of the study's participants demonstrated significantly lower summary scores from the normative sample for both CHQ Physical and Psychosocial summary scores. On average, children did not change significantly over time for physical summary scores. There was an average increase in psychosocial health that was statistically significant, but small. However, there was evidence of heterogeneity among children. Environmental barriers, behavioral difficulties, family functioning/impact, general health and child physical functioning had negative and significant associations with physical QoL at baseline. Change in physical QoL scores over time was dependent on children's behavioral difficulties, family functioning and environmental barriers. Environmental barriers, behavioral difficulties, family functioning/impact and general health had significant associations with psychosocial scores at baseline, but none served as predictors of change over time. CONCLUSIONS: Children with physical disabilities differ from the normative group on parent ratings of their physical and psychosocial health. While there was little average change in CHQ scores over 18 months, there is evidence of heterogeneity among children. Factors such as environmental barriers, family functioning/impact, child physical functioning and behavioral difficulties and general health significantly influence QoL scores as measured by the CHQ.