ABSTRACT
BACKGROUND: Chinese Nong-favor daqu, the presentative liquor starter of Baijiu, has been enriched with huge amounts of enzymes in degrading various biological macromolecules by openly man-made process for thousand years. According to previous metatranscriptomics analysis, plenty of α-glucosidases were identified to be active in NF daqu and played the key role in degrading starch under solid-state fermentation. However, none of α-glucosidases was characterized from NF daqu, and their actual functions in NF daqu were still unknown. RESULTS: An α-glucosidase (NFAg31A, GH31-1 subfamily), the second highest expressed α-glucosidases in starch degradation of NF daqu, was directly obtained by heterologous expression in Escherichia coli BL21 (DE3). NFAg31A exhibited the highest sequence identities of 65.8% with α-glucosidase II from Chaetomium thermophilum, indicating its origin of fungal species, and it showed some similar features with homologous α-glucosidase IIs, i.e., optimal activity at pH ~ 7.0 and litter higher temperature of 45 â, well stability at 41.3 â and a broad pH range of pH 6.0 to pH 10.0, and preference on hydrolyzing Glc-α1,3-Glc. Besides this preference, NFAg31A showed comparable activities on Glc-α1,2-Glc and Glc-α1,4-Glc, and low activity on Glc-α1,6-Glc, indicating its broad specificities on α-glycosidic substrates. Additionally, its activity was not stimulated by any of those detected metal ions and chemicals, and could be largely inhibited by glucose under solid-state fermentation. Most importantly, it exhibited competent and synergistic effects with two characterized α-amylases of NF daqu on hydrolyzing starch, i.e., all of them could efficiently degrade starch and malto-saccharides, two α-amylases showed advantage in degrading starch and long-chain malto-saccharides, and NFAg31A played the competent role with α-amylases in degrading short-chain malto-saccharides and the irreplaceable contribution in hydrolyzing maltose into glucose, thus alleviating the product inhibitions of α-amylases. CONCLUSIONS: This study provides not only a suitable α-glucosidase in strengthening the quality of daqu, but also an efficient way to reveal roles of the complicated enzyme system in traditional solid-state fermentation. This study would further stimulate more enzyme mining from NF daqu, and promote their actual applications in solid-state fermentation of NF liquor brewing, as well as in other solid-state fermentation of starchy industry in the future.
Subject(s)
Alcoholic Beverages , Fermentation , alpha-Glucosidases , alpha-Amylases , alpha-Glucosidases/genetics , Glucose , Starch , Substrate SpecificityABSTRACT
Three mixed-valence barium iron fluorides, Ba7Fe7F34, Ba2Fe2F9, and BaFe2F7, were prepared through hydrothermal redox reactions. The characteristic structures of these compounds feature diverse distributions of FeIIF6 octahedra and FeIIIF6 groups. Ba7Fe7F34 contained one-dimensional infinite ∞[FeIIFeIII6F34]14- double chains, comprising cis corner-sharing octahedra along the b direction; Ba2Fe2F9 contained one-dimensional ∞[Fe2F9]4- double chains, consisting of cis corner-sharing octahedra along the chain (a-axis direction) and trans corner-sharing octahedra vertical to the chain, while BaFe2F7 revealed three-dimensional (3D) frameworks that consist of isolated edge-sharing dinuclear FeII2F10 units linked via corners by FeIIIF6 octahedra. Magnetization and Mössbauer spectroscopy measurements revealed that Ba7Fe7F34 exhibits an antiferromagnetic phase transition at â¼11 K, where ferrimagnetic ∞[FeIIFeIII6F34]14- double chains are arranged in a paralleling manner, while Ba2Fe2F9 shows canted antiferromagnetic ordering at â¼32.5 K, leading to noncollinear spin ordering.
ABSTRACT
Circulating tumor cells (CTCs) play a vital role in the metastasis and recurrence of breast cancer. CTCs are highly heterogeneous at the stage of Epithelial-to-Mesenchymal Transition (EMT), but the phenotypic and biological characteristics in different EMT stages remain poorly defined. We conducted an orthotopic mouse (4T1) model of breast cancer to isolate CTCs and identified two phenotypes of CTCs: intermediate E/M and mesenchymal CTCs. MTT, Colony formation, Transwell migration and invasion assays were utilized to examined cell proliferation, colony forming, migration and invasion ability. Both the intermediate E/M and mesenchymal CTCs exhibited lower rates of proliferation, colony formation and invasion, as compared to primary tumor cells. The mesenchymal CTCs had a higher rate of invasion but lower rates of proliferation and colony formation than the intermediate E/M CTCs. They also exhibited lower rates of growth and metastasis than the primary tumor cells in vivo, but the mesenchymal CTCs had a higher rate of metastasis than the intermediate E/M CTCs. Fluid shear stress induced the EMT transition of CTCs. The comprehensive analysis of CTCs proteomics discovered proteins that differentially expressed in the two types of CTCs and their primary tumor cells.
Subject(s)
Neoplastic Cells, Circulating , Mice , Animals , Neoplastic Cells, Circulating/pathology , Biomarkers, Tumor/metabolism , Epithelial-Mesenchymal Transition/genetics , Cell Count , Neoplasm MetastasisABSTRACT
Chronic obstructive pulmonary disease (COPD) and asthma are chronic inflammatory diseases of the airways. Galectin-13 has recently been forwarded as a biomarker for airway eosinophilic inflammation in asthma. However, the association between galectin-13 and COPD remains unknown. To examine the changes in galectin-13 expression in acute exacerbations of COPD (AECOPD) and the stable phase of COPD and unveil the association between galectin-13 expression and eosinophilic inflammation in COPD, we measured plasma galectin-13 expression in different phases of COPD patients (n = 60, 44 AECOPD patients, and 16 stable COPD patients) and healthy controls (n = 15). Plasma levels of galectin-13 in 60 COPD patients were further analyzed and compared to systemic inflammation, airway eosinophilic inflammation, and lung function. The plasma galectin-13 level was markedly increased in subjects with AECOPD compared to stable COPD patients and healthy controls. Plasma galectin-13 levels in COPD subjects were positively correlated with serum CRP (rs = 0.46, p = 0.0003), peripheral blood eosinophilia count (rs = 0.57, p<0.0001), and FeNO (rs = 0.46, p = 0.0002). In addition, the level of galectin-13 was negatively correlated with FEV1 (rs = -0.43, p = 0.0001), FEV1 pred (%) (rs = -0.544, p<0.0001), as well as FEV1/FVC (rs = -0.46, p<0.0001). Multiple linear regression analysis suggested that plasma galectin-13 levels were affected by FEV1 pred (%), peripheral blood eosinophilia count, and FeNO. We concluded that galectin-13 levels were increased in COPD patients, and elevated galectin-13 expressions related to airway eosinophilic inflammation. Galectin-13 may facilitate the identification of COPD endotypes and may become a potential therapeutic target.
Subject(s)
Asthma , Eosinophilia , Pulmonary Disease, Chronic Obstructive , Humans , Asthma/complications , Inflammation , GalectinsABSTRACT
Time-to-event data such as time to death are broadly used in medical research and drug development to understand the efficacy of a therapeutic. For time-to-event data, right censoring (data only observed up to a certain point of time) is common and easy to recognize. Methods that use right censored data, such as the Kaplan-Meier estimator and the Cox proportional hazard model, are well established. Time-to-event data can also be left truncated, which arises when patients are excluded from the sample because their events occur before a specific milestone, potentially resulting in an immortal time bias. For example, in a study evaluating the association between biomarker status and overall survival, patients who did not live long enough to receive a genomic test were not observed in the study. Left truncation causes selection bias and often leads to an overestimate of survival time. In this tutorial, we used a nationwide electronic health record-derived de-identified database to demonstrate how to analyze left truncated and right censored data without bias using example code from SAS and R.
Subject(s)
Models, Statistical , Humans , Proportional Hazards Models , Survival Analysis , Bias , Selection BiasABSTRACT
We performed a single-arm exploratory clinical trial that is ongoing and registered at ClinicalTrials.gov (NCT03093688). Patients were infused with autologous iNKT cells, PD-1 + CD8+ T cells, and dendritic cells every 3-5 weeks, which was considered 1 cycle. The primary endpoints were safety and objective tumor response. The preliminary results from the first three patients are reported here. The first patient received 16 cycles. Computed tomography (CT) examination revealed a stable disease (SD) response after 4 cycles and progressive disease (PD) response after 11 cycles. For the second patient that received 10 cycles, CT examination revealed an SD response after 4 cycles and a PD response after 9 cycles. For the third patient who was treated with 6 cycles, CT examination revealed an SD response after 4 cycles. The patients suffered from only grade 1-2 adverse events. iNKT cell and PD-1 + CD8+ T cell-based immunotherapy showed a manageable tolerability profile.
Subject(s)
Adenocarcinoma of Lung , Antineoplastic Agents, Immunological , Lung Neoplasms , Natural Killer T-Cells , Antineoplastic Agents, Immunological/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , CD8-Positive T-Lymphocytes/pathology , Feasibility Studies , Humans , Immunologic Factors/therapeutic use , Immunotherapy , Lung Neoplasms/drug therapy , Natural Killer T-Cells/pathology , Programmed Cell Death 1 ReceptorABSTRACT
State-to-state photodissociation of CO2(v2 = 0 and 1) at 157 nm via the O(1D) + CO(X1Σ+) channel was studied by using the sliced velocity map imaging technique. Both the O(1D) and CO(X1Σ+) products were detected by (2 + 1) resonance enhanced multiphoton ionization (REMPI). Detection of CO via the B1Σ+ ââ X1Σ+ transition allowed ro-vibrational state-selective detection, and combined with imaging, the fragment energy and angular distributions have been derived. For CO(v = 0 and 1|j) products from the CO2(v2 = 0) molecule, the angular distributions of low-j CO display positive anisotropic parameters (about 0.8); with j increasing, the product anisotropic parameters gradually reduce to zero. While for CO(v = 0 and 1|j) products from the vibrational excited CO2(v2 = 1) molecule, the angular distributions of low-j CO also display positive anisotropic parameters; with j increasing, the product anisotropic parameters first decrease to zero and then become negative (about -0.5). Experimental results show that the observed variation of the product angular distribution with the rotational quantum number of CO is consistent with trends predicted by a classical model for non-axial fragment recoil. The results support advanced theoretical predictions of a predominantly parallel transition to the bent 21A' excited state of CO2, where bending introduces torque during the direct dissociation process.
ABSTRACT
Purple sweet potato is considered an abundant, inexpensive, and ideal source of anthocyanins. Purple sweet potato anthocyanins (PSPAs) have been shown to possess high antimutagenicity and antitumor effects due to the abundance of acylated anthocyanins. However, the effect and underlying mechanism of PSPA effects in acute lymphoblastic leukemia (ALL), especially T-cell acute lymphoblastic leukemia (T-ALL), remain unclear. In this study, the antileukemic effects of PSPAs and the underlying molecular mechanisms were evaluated by inâ vitro and inâ silico assays. PSPAs extracted from ten cultivars were analyzed and quantified. Anthocyanins from Nanzi 018, which showed the best antileukemic effect, were selected to analyze the underlying mechanism. First, the PSPAs potently reduced cell viability and induced apoptosis. Additionally, the PSPAs sharply increased intracellular Ca2+ levels, which resulted in calcium overload in T-ALL cells. Furthermore, on the basis of bioinformatics analyses, we focused on an osmotically regulated transcription factor, NFAT5. Molecular docking preliminarily indicated that PSPA molecules bound and interacted with the NFAT5 protein. Western blot analyses confirmed that PSPAs elicited calcium overload by nonosmotic regulation of NFAT5/S100A4-S100A9 pathway activation. Moreover, pretreatment with a NFAT5 inducer confirmed that PSPAs targeted NFAT5 and affected p38/NF-κB/Bcl-2/Caspase-3 axis activation. This study demonstrates that PSPAs exert their antileukemic effects through calcicoptosis induction by targeting NFAT5.
Subject(s)
Ipomoea batatas , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Anthocyanins/metabolism , Anthocyanins/pharmacology , Calcium/metabolism , Caspase 3/metabolism , Humans , Molecular Docking Simulation , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , S100 Calcium-Binding Protein A4/metabolism , Transcription FactorsABSTRACT
OBJECTIVES: Electroacupuncture (EA) at Zusanli (ST36) can attenuate inflammation in different rodent models. However, the therapeutic mechanisms underlying its action in inhibiting intestinal barrier destruction and liver injury in cholestasis mice have not been clarified. This study aimed at investigating whether EA at ST36 could activate the cholinergic anti-inflammatory pathway to inhibit intestinal barrier destruction and liver injury in cholestasis mice. MATERIALS AND METHODS: Male Hmox1floxp/floxp C57BL/6 mice were randomized and subjected to a sham or bile duct ligation (BDL) surgery. The BDL mice were randomized and treated with, or without (BDL group), sham EA at ST36 (BDL+sham-ST36) or EA at ST36 (BDL+ST36), or received α-bungarotoxin (α-BGT), a specific inhibitor of nicotinic acetylcholine receptor α7 subunit (α7nAChR), before stimulation (BDL+ST36+α-BGT). These mice, together with a group of intestine-specific heme oxygenase-1 (HO-1) knockout (KO) Villin-Cre-HO-1-/- mice, were monitored for their body weights before and 14 days after BDL. The levels of plasma cytokines and liver injury-related alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by enzyme-linked immunoassay, and pathological changes in the intestinal mucosa and liver fibrosis as well as intestinal barrier permeability in individual mice were examined by histology and immunohistochemistry. The levels of α7nAChR, HO-1, ZO-1, Occludin, Claudin-1, and NF-κBp65 expression and NF-κBp65 phosphorylation in intestinal tissues were quantified. RESULTS: Compared with the sham group, BDL significantly increased the levels of plasma interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor α, ALT, and AST and caused intestinal mucosal damages, high permeability, and liver fibrosis in mice, which were remarkably mitigated, except for further increased levels of plasma IL-10 in the BDL+ST36 group of mice. Similarly, EA at ST36 significantly up-regulated α7nAChR and HO-1 expression; mitigated the BDL-decreased ZO-1, Occludin, and Claudin-1 expression; and attenuated the BDL-increased NF-κBp65 phosphorylation in intestinal tissues of mice. The therapeutic effects of EA at ST36 were significantly abrogated by pretreatment with α-BGT or HO-1 KO. CONCLUSION: EA at ST36 inhibits the BDL-induced intestinal mucosal damage and liver fibrosis by activating the HO-1 cholinergic anti-inflammatory pathway in intestinal tissues of mice.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Cholestasis , Electroacupuncture , Rats , Mice , Male , Animals , Interleukin-10 , Rats, Sprague-Dawley , Occludin , Neuroimmunomodulation , alpha7 Nicotinic Acetylcholine Receptor , Claudin-1 , Mice, Inbred C57BL , Intestines , Liver Cirrhosis , Bile Ducts/surgeryABSTRACT
BACKGROUND: The immune protective mechanisms during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection remain to be deciphered for the development of an effective intervention approach. METHODS: We examined early responses of interleukin 37 (IL-37), a powerful anti-inflammatory cytokine, in 254 SARS-CoV-2-infected patients before any clinical intervention and determined its correlation with clinical prognosis. RESULTS: Our results demonstrated that SARS-CoV-2 infection causes elevation of plasma IL-37. Higher early IL-37 responses were correlated with earlier viral RNA negative conversion, chest computed tomographic improvement, and cough relief, consequently resulted in earlier hospital discharge. Further assays showed that higher IL-37 was associated with lower interleukin 6 and interleukin 8 (IL-8) and higher interferon α responses and facilitated biochemical homeostasis. Low IL-37 responses predicted severe clinical prognosis in combination with IL-8 and C-reactive protein. In addition, we observed that IL-37 administration was able to attenuate lung inflammation and alleviate respiratory tissue damage in human angiotensin-converting enzyme 2-transgenic mice infected with SARS-CoV-2. CONCLUSIONS: Overall, we found that IL-37 plays a protective role by antagonizing inflammatory responses while retaining type I interferon, thereby maintaining the functionalities of vital organs. IL-37, IL-8, and C-reactive protein might be formulated as a precise prediction model for screening severe clinical cases and have good value in clinical practice.
Subject(s)
COVID-19/immunology , Cytokine Release Syndrome/virology , Interleukin-1/blood , Adult , Animals , C-Reactive Protein/metabolism , COVID-19/blood , Female , Humans , Inflammation/immunology , Inflammation/virology , Interleukin-8/blood , Male , Mice , Mice, Transgenic , Middle AgedABSTRACT
BACKGROUND: Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. RESULTS: In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. CONCLUSIONS: This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.
Subject(s)
Alcoholic Beverages/microbiology , Fungi/enzymology , alpha-Amylases/isolation & purification , Hydrolysis , Starch/metabolismABSTRACT
Three new alkali-metal manganese fluoride selenates, A2Mn(SeO4)F3 (A = K, Rb, Cs), were prepared through hydrothermal redox reactions. The products consisted of one-dimensional polymeric anionic ∞[Mn(SeO4)F3]2- chains, where the A+ cations are connected by O and/or F atoms to form blocks with two-dimensional layers. A2Mn(SeO4)F3 (A= Rb, Cs) is isostructural with the monoclinic space group P21/c, while K2Mn(SeO4)F3 crystallizes in the orthorhombic space group Pbcn. A2Mn(SeO4)F3 (A = K, Rb, Cs) forms spin chains of Mn3+ with different Mn-F-Mn bridges, which showed canting antiferromagnetic behaviors. Single-crystal magnetic measurements revealed that the magnetic moments of the Mn ions were more canted for larger alkali-metal compounds in an antiferromagnetically ordered state.
ABSTRACT
OBJECTIVE: To investigate the pathologic features and differential diagnosis of a pediatric dendritic fibromyxolipoma (DFML). METHODS: We collected one case of dendritic fibromyxolipoma in the back of a pediatric patient (6 years old, female) diagnosed at the First Affiliated Hospital of Dalian Medical University. The case of DFML was studied by H&E and MaxVision immunohistochemical staining. The clinical and pathological features were analyzed with review of the literatures RESULTS: The tumor was round or ovoid in shape and appeared in the surface of back. Microscopically, the tumor was mainly consisted of stellate to spindle cells, rope-like collagen fibers and varying amount of mature lipocytes embedded in myxoid stroma. Immunohistochemical staining showed that spindle cells and stellate cells were positive for vimentin, CD34 and BCL-2. CONCLUSION: DFML is a rare special variant of lipoma. It is benign, and it seems to be misdiagnosed as myxoid liposarcoma prior to the admission to our hospital. The pathological histomorphology and immunohistochemistry phenotypes are helpful to the diagnosis and differential diagnosis. Given that DFML mainly occurs in the elderly, this case is rare and worthy to be reported since it occurs in children.
Subject(s)
Lipoma/diagnosis , Aged , Child , Female , Humans , ImmunohistochemistryABSTRACT
The interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligands on a target tumour may limit the capacity of chimeric antigen receptor (CAR) T cells to eradicate solid tumours. PD-1 blockade could potentially enhance CAR T cell function. Here, we show that mesothelin is overexpressed in human triple-negative breast cancer cells and can be targeted by CAR T cells. To overcome the suppressive effect of PD-1 on CAR T cells, we utilized CRISPR/Cas9 ribonucleoprotein-mediated editing to disrupt the programmed cell death-1 (PD-1) gene locus in human primary T cells, resulting in a significantly reduced PD-1hi population. This reduction had little effect on CAR T cell proliferation but strongly augmented CAR T cell cytokine production and cytotoxicity towards PD-L1-expressing cancer cells in vitro. CAR T cells with PD-1 disruption show enhanced tumour control and relapse prevention in vivo when compared with CAR T cells with or without αPD-1 antibody blockade. Our study demonstrates a potential advantage of integrated immune checkpoint blockade with CAR T cells in controlling solid tumours and provides an alternative CAR T cell strategy for adoptive transfer therapy.
Subject(s)
CRISPR-Cas Systems/physiology , GPI-Linked Proteins/genetics , Programmed Cell Death 1 Receptor/genetics , Receptors, Antigen, T-Cell/genetics , Animals , Cell Line, Tumor , Cytotoxicity, Immunologic , Humans , Immunotherapy, Adoptive , Lymphocyte Activation , Mesothelin , Mice , Neoplasms, Experimental/therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Receptors, Antigen, T-Cell/immunologyABSTRACT
The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8-9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.
Subject(s)
Agrobacterium tumefaciens/genetics , Alismatales/genetics , Genetic Engineering/methods , Alismatales/metabolism , Bioreactors , Cytochrome P-450 Enzyme System/genetics , Genetic Vectors/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Polymerase Chain Reaction , Stigmasterol/metabolism , Transformation, Genetic/geneticsABSTRACT
BACKGROUND: Chinese Nong-flavor (NF) liquor is continuously and stably produced by solid-state fermentation technology for 1000 years, resulting in enrichment of special microbial community and enzymes system in its starter. Based on traditional culture-dependent methods, these functional enzymes are hardly obtained. According to our previous metatranscriptomic analysis, which identifies plenty of thermostable carbohydrate-active enzymes in NF liquor starter, the aim of this study is to provide a direct and efficient way to mine these thermostable enzymes. RESULTS: In present study, an alpha-amylase (NFAmy13A) gene, which showed the highest expression level of enzymes in starch degradation at high temperature stage (62 °C), was directly obtained by functional metatranscriptomics from Chinese Nong-flavor liquor starter and expressed in Pichia pastoris. NFAmy13A had a typical signal peptide and shared the highest sequence identity of 64% with α-amylase from Aspergillus niger. The recombinant enzyme of NFAmy13A showed an optimal pH at 5.0-5.5 and optimal temperature at 60 °C. NFAmy13A was activated and stabilized by Ca2+, and its half-lives at 60 and 70 °C were improved significantly from 1.5 and 0.4 h to 16 and 0.7 h, respectively, in the presence of 10 mM CaCl2. Meanwhile, Hg2+, Co2+ and SDS largely inhibited its activity. NFAmy13A showed the maximum activity on amylopectin, followed by various starches, amylose, glycogen, and pullulan, and its specificity activity on amylopectin was 200.4 U/mg. Moreover, this α-amylase efficiently hydrolyzed starches (from corn, wheat, and potato) at high concentrations up to 15 mg/ml. CONCLUSIONS: This study provides a direct way to mine active enzymes from man-made environment of NF liquor starter, by which a fungal thermostable α-amylase (NFAmy13A) is successfully obtained. The good characteristics of NFAmy13A in degrading starch at high temperature are consistent with its pivotal role in solid-state fermentation of NF liquor brewing. This work would stimulate mining more enzymes from NF liquor starter and studying their potentially synergistic roles in NF liquor brewing, thus paving the way toward the optimization of liquor production and improvement of liquor quality in future.
Subject(s)
Alcoholic Beverages/analysis , alpha-Amylases/metabolism , Asian People , Flavoring Agents , HumansABSTRACT
BACKGROUND: Prospective study protocols and registrations can play a significant role in reducing incomplete or selective reporting of primary biomedical research, because they are pre-specified blueprints which are available for the evaluation of, and comparison with, full reports. However, inconsistencies between protocols or registrations and full reports have been frequently documented. In this systematic review, which forms part of our series on the state of reporting of primary biomedical, we aimed to survey the existing evidence of inconsistencies between protocols or registrations (i.e., what was planned to be done and/or what was actually done) and full reports (i.e., what was reported in the literature); this was based on findings from systematic reviews and surveys in the literature. METHODS: Electronic databases, including CINAHL, MEDLINE, Web of Science, and EMBASE, were searched to identify eligible surveys and systematic reviews. Our primary outcome was the level of inconsistency (expressed as a percentage, with higher percentages indicating greater inconsistency) between protocols or registration and full reports. We summarized the findings from the included systematic reviews and surveys qualitatively. RESULTS: There were 37 studies (33 surveys and 4 systematic reviews) included in our analyses. Most studies (n = 36) compared protocols or registrations with full reports in clinical trials, while a single survey focused on primary studies of clinical trials and observational research. High inconsistency levels were found in outcome reporting (ranging from 14% to 100%), subgroup reporting (from 12% to 100%), statistical analyses (from 9% to 47%), and other measure comparisons. Some factors, such as outcomes with significant results, sponsorship, type of outcome and disease speciality were reported to be significantly related to inconsistent reporting. CONCLUSIONS: We found that inconsistent reporting between protocols or registrations and full reports of primary biomedical research is frequent, prevalent and suboptimal. We also identified methodological issues such as the need for consensus on measuring inconsistency across sources for trial reports, and more studies evaluating transparency and reproducibility in reporting all aspects of study design and analysis. A joint effort involving authors, journals, sponsors, regulators and research ethics committees is required to solve this problem.
Subject(s)
Biomedical Research/standards , Databases, Bibliographic , Research Design/standards , Research Report/standards , Biomedical Research/methods , Biomedical Research/statistics & numerical data , Clinical Trials as Topic/methods , Clinical Trials as Topic/standards , Clinical Trials as Topic/statistics & numerical data , Humans , Outcome Assessment, Health Care/methods , Outcome Assessment, Health Care/standards , Outcome Assessment, Health Care/statistics & numerical data , Prospective StudiesABSTRACT
Heme oxygenase-1 (HO-1) is reported to protect against liver injury, but little is known about its effect on the intestinal barrier in cholestatic liver injury. In this study, we investigated the effects of HO-1 and its enzymatic by-product on intestinal barrier dysfunction in bile duct ligation (BDL) rats and explored the possible mechanism. The HO-1 inducer cobalt protoporphyrin (CoPP) and carbon monoxide-releasing molecule-2 (CORM-2) were used; the expression levels of tight junction (TJ) proteins, intestinal inflammation and NF-κB p65 were measured. For an in vitro experiment, stable Caco-2 cell lines were constructed, one overexpressed the HO-1 gene and another with that gene knocked down, and the specific NF-κB inhibitor JSH-23 was used. CoPP and CORM-2 treatment alleviated liver and intestinal mucosa injury in BDL rats; improved ZO-1, claudin-1 and PCNA expression; and reduced cell apoptosis and intestinal interleukin-6 (IL-6) expression. In vitro studies confirmed that HO-1, ZO-1 and occludin were overexpressed in HO-1-transfected Caco-2 cells, while decreased in the sh-HO-1 group. JSH-23 significantly increased occludin expression in both the HO-1 overexpression and sh-HO-1 groups, compared with their respective controls. HO-1 overexpression also inhibited the nuclear translocation of NF-κB p65 after lipopolysaccharide (LPS) treatment. Additionally, phospho-p65 expression in sh-HO-1 cells was significantly increased compared with that of the HO-1 overexpression group. In conclusion, HO-1 and CORM-2 improved intestinal epithelial barrier function in BDL-induced cholestatic liver injury mainly by restoring TJ, reducing cell apoptosis and intestinal inflammation. HO-1 exerts a protective effect, which is partially correlated with the regulation of NF-κB.
Subject(s)
Cholestasis/metabolism , Heme Oxygenase-1/metabolism , Intestinal Mucosa/metabolism , Liver Cirrhosis/metabolism , NF-kappa B/metabolism , Animals , Caco-2 Cells , Heme Oxygenase-1/genetics , Humans , Intestinal Mucosa/drug effects , Male , NF-kappa B/antagonists & inhibitors , Organometallic Compounds/pharmacology , Protoporphyrins/pharmacology , Rats, Sprague-DawleyABSTRACT
Despite several publications on venous ulcers, there is still a lack of evidence from randomized controlled trials (RCTs) to support certain treatments for patients with this disorder. Well-designed research questions using the PICOT (Population; Intervention; Comparator; Outcome; Time-frame) format in RCTs can improve the quality of research. The objectives of this study were to assess how the PICOT format is used to frame research questions in RCTs published on venous ulcer disease and to determine the factors associated with better adherence to the PICOT format. We conducted a systematic survey of RCTs on venous ulcers published in the PubMed database between January 2009 and May 2016. All RCTs published in English addressing therapeutic interventions for venous ulcer disease in human subjects were included. We examined whether the five elements of the PICOT format were used in formulating the research question and scored them between 0 and 5. The primary outcome of this systematic survey was the percentage of studies that adequately reported all five PICOT elements. Eighty-five (85) RCTs were included with median PICOT score of 3 (IQR = 1.5). Four elements of PICOT were present in 28 reports (32.9%) and only 2 RCTS (2.3%) reported all the PICOT elements. Population and intervention were often appropriately described, in (70/85) 82.4% and (83/85) 97.6% of the studies, respectively; however, comparison intervention and outcome were presented in only (53/85) 62.3% and (48/85) 56.5% of studies, respectively. Very few RCTs (7.1%; 6/85) reported the study time frame. No journal or RCT characteristics were found to be significantly associated with better reporting. Use of the PICOT format to frame research questions in RCTs published on venous ulcers is suboptimal, and our study reinforces the importance of framing a good research question to improve the design of trials and quality of evidence in venous ulcer disease.
Subject(s)
Population Surveillance/methods , Randomized Controlled Trials as Topic , Varicose Ulcer/therapy , Wound Healing , HumansABSTRACT
BACKGROUND: Evidence shows that research abstracts are commonly inconsistent with their corresponding full reports, and may mislead readers. In this scoping review, which is part of our series on the state of reporting of primary biomedical research, we summarized the evidence from systematic reviews and surveys, to investigate the current state of inconsistent abstract reporting, and to evaluate factors associated with improved reporting by comparing abstracts and their full reports. METHODS: We searched EMBASE, Web of Science, MEDLINE, and CINAHL from January 1st 1996 to September 30th 2016 to retrieve eligible systematic reviews and surveys. Our primary outcome was the level of inconsistency between abstracts and corresponding full reports, which was expressed as a percentage (with a lower percentage indicating better reporting) or categorized rating (such as major/minor difference, high/medium/low inconsistency), as reported by the authors. We used medians and interquartile ranges to describe the level of inconsistency across studies. No quantitative syntheses were conducted. Data from the included systematic reviews or surveys was summarized qualitatively. RESULTS: Seventeen studies that addressed this topic were included. The level of inconsistency was reported to have a median of 39% (interquartile range: 14% - 54%), and to range from 4% to 78%. In some studies that separated major from minor inconsistency, the level of major inconsistency ranged from 5% to 45% (median: 19%, interquartile range: 7% - 31%), which included discrepancies in specifying the study design or sample size, designating a primary outcome measure, presenting main results, and drawing a conclusion. A longer time interval between conference abstracts and the publication of full reports was found to be the only factor which was marginally or significantly associated with increased likelihood of reporting inconsistencies. CONCLUSIONS: This scoping review revealed that abstracts are frequently inconsistent with full reports, and efforts are needed to improve the consistency of abstract reporting in the primary biomedical community.