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1.
BMC Anesthesiol ; 22(1): 22, 2022 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-35026992

RESUMO

BACKGROUND: Postoperative acute kidney injury (AKI) is associated with poor clinical outcomes. Early identification of high-risk patients of developing postoperative AKI can optimize perioperative renal management and facilitate patient survival. The present study aims to develop and validate a nomogram to predict postoperative AKI after liver resection in older patients. METHODS: A retrospective observational study was conducted involving data from 843 older patients scheduled for liver resection at a single tertiary high caseload general hospital between 2012 and 2019. The data were randomly divided into training (70%, n = 599) and validation (30%, n = 244) datasets. The training cohort was used to construct a predictive nomogram for postoperative AKI with the logistic regression model which was confirmed by a validation cohort. The model was evaluated by receiver operating characteristic (ROC) curve, calibration plot, and decision curve analysis in the validation cohort. A summary risk score was also constructed for identifying postoperative AKI patients. RESULTS: Postoperative AKI occurred in 155 (18.4%) patients and was highly associated with in-hospital mortality (5.2% vs. 0.7%, P <  0.001). The six predictors selected and assembled into the nomogram included age, preexisting chronic kidney disease (CKD), non-steroidal anti-inflammatory drugs (NSAIDs) usage, intraoperative hepatic inflow occlusion, blood loss, and transfusion. The predictive nomogram performed well in terms of discrimination with area under ROC curve (AUC) in training (0.73, 95% confidence interval (CI): 0.68-0.78) and validation (0.71, 95% CI: 0.63-0.80) datasets. The nomogram was well-calibrated with the Hosmer-Lemeshow chi-square value of 9.68 (P = 0.47). Decision curve analysis demonstrated a significant clinical benefit. The summary risk score calculated as the sum of points from the six variables (one point for each variable) performed as well as the nomogram in identifying the risk of AKI (AUC 0.71, 95% CI: 0.66-0.76). CONCLUSION: This nomogram and summary risk score accurately predicted postoperative AKI using six clinically accessible variables, with potential application in facilitating the optimized perioperative renal management in older patients undergoing liver resection. TRIAL REGISTRATION: NCT04922866 , retrospectively registered on clinicaltrials.gov on June 11, 2021.

2.
Genes Genomics ; 2022 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-35038160

RESUMO

BACKGROUND: Glutamine-fructose-6-phosphate aminotransferase (GFPT) is a key factor in the hexosamine metabolism pathway. It regulates the downstream factor O-GlcNAc to change cell function and plays an important role in the metabolism and immune process of tissues and organs. However, the evolutionary relationship of GFPT family proteins in vertebrates has not been elucidated. OBJECTIVE: To deduce and explore the evolution and function of vertebrate GFPT family. METHODS: 18 GFPT sequences were obtained from Homo sapiens (H. sapiens), Trachypithecus francoisi (T. francoisi), Mus musculus (M. musculus), Rattus norvegicus (R. norvegicus), Gallus gallus (G. gallus), Zootoca vivipara (Z. vivipara), Xenopus tropicalis (X. tropicalis), Danio rerio (D. rerio), Rhincodon typus (R. typus), Plasmodium relictum from National Center for Biotechnology Information (NCBI). The physical and chemical characteristics and molecular evolution of GFPT family proteins and nucleic acid sequences were analyzed by ClustalX2, Gene Doc, MEGA-X, SMART, Datamonkey, R etc. RESULTS: Based on the neighbor-joining (NJ) phylogenetic tree and evolution fingerprints, GFPT family members of vertebrates can be divided into two groups: the GFPT1 group and the GFPT2 group. Seven positive selection sites were identified by IFEL and integrated methods mixed effects model of evolution (MEME) and fixed effects likelihood (REL). Finally, we predicted 28 phosphorylation sites and 18 ubiquitousness sites in the human GFPT1 sequence, 10 phosphorylation sites, and five ubiquitousness sites in GFPT2. Gene ontology (GO) analyzes the protein molecules and KEGG signaling pathways of vertebrates interacting with GFPT family proteins. CONCLUSIONS: Our work confirmed that higher animals GFPT family may have differentiated GFPT1 and GFPT2, which meets their own functional needs. This knowledge answers the question what the origin and evolution of GFPT family in vertebrates and provided the basis for disease treatment and function research of GFPT protein.

3.
Psychiatry Investig ; 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34986556

RESUMO

OBJECTIVE: Adolescents are at a special stage of physical and mental development, which is a susceptible period for mental disorders. Since the outbreak of coronavirus pneumonia in December 2019, long term stress may have negative effects on the mental health of the adolescents. In the context of the coronavirus disease 2019 (COVID-19), the study was designed to investigate the mental and psychological health of adolescents in China and its possible related factors. METHODS: A cross-sectional study design was adopted using a structured questionnaire which were distributed through the Internet to measure depression, anxiety, life events and stress related factors. Descriptive statistics and multiple regression analyses were conducted to process the data. RESULTS: The final sample comprised 795 adolescents. The total detection rate of depression was 76.48% and the total detection rate of anxiety was 33.08%. ANOVA showed that there were significant differences in depression scores in terms of gender, anxiety scores, history of mental disorders, COVID-19 knowledge reserve, family and social contradictions (p<0.05). And there were significant differences in anxiety scores in terms of gender, depression scores, mental health knowledge reserves, family and social contradictions (p<0.05). Multiple regression analysis showed that anxiety score, health status and COVID-19 knowledge reserve were positively associated with depression score (p<0.01), and history of psychosocial disorders was negatively associated with depression score (p<0.05); depression score, family and social contradictions were significantly positively correlated with anxiety score (p<0.01), and history of mental disorders was significantly negatively correlated with SDS score (p<0.01). CONCLUSION: During the outbreak of COVID-19, adolescent students with better understanding of the pandemic, more complete knowledge of mental health, and better family and social relationship had less impact on their mental health. Therefore, to ensure a sound social support system, elaborate health instruction, and family communication and mutual understanding are conducive to alleviating the psychological stress caused by the epidemic, and it is positive for adolescent students to maintain a good mental health.

4.
Genes Genomics ; 2022 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-35025083

RESUMO

BACKGROUND: Fam20c is intimately related to tissue development and diseases. At present, it has been reported that Fam20c regulates the mineralization of osteoblasts, but there are few reports on other effects. OBJECTIVE: To study the effect of Fam20c on osteoblasts by knocking out the Fam20c gene. METHODS: Fam20c knockout osteoblasts were constructed by transfecting mouse osteoblasts with lentivirus. The proliferation, migration and mineralization of Fam20c knockout cells were detected by CCK-8, scratch test and alizarin red staining assays. The subcellular structure was observed by transmission electron microscopy. RT-PCR was used to detect the differential expression of mesenchymal-to-epithelial transition (MET)-related marker genes and core transcription factors. The differential expression of MET-related proteins was detected by immunofluorescence or Western blot. Transcriptome analysis of Fam20c knockout osteoblasts was performed, and real-time PCR was used to verify transcriptome analysis related to MET. RESULTS: The proliferation ability of osteoblasts was not significantly changed after Fam20c deletion, but the migration ability and mineralization ability were significantly weakened. There were tight junctions between Fam20c knockout cells. The expression of mesenchymal cell marker genes and core transcription factors was significantly decreased, and the expression of epithelial cell marker genes was significantly increased. The expression of mesenchymal cell marker proteins was significantly decreased, and the expression of epithelial cell marker proteins was significantly increased. Multiple signalling molecules and pathways involved in MET have changed. CONCLUSIONS: Knockdown of Fam20c resulted in MET. Fam20c affects the transcription of key factors in osteoblast MET.

5.
J Biophotonics ; : e202100344, 2022 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-34978383

RESUMO

Saccharomyces cerevisiae is an attractive organism used in the fermentation industry and is an important model organism for virus research. The ability to sort yeast cells is important for diverse applications. Replicative aging of Saccharomyces Cerevisiae is accompanied by metabolic changes that are related to an essential coenzyme, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H). Here, a single cell sorting method based on fluorescence lifetime imaging microscopy (FLIM) and laser-induced forward transfer (LIFT) was implemented for the first time. The aging level of yeast was determined based on the FLIM by NAD(P)H, which was a label-free and noninvasive method for studying individual cells. Then, young and active yeast cells were sorted by the LIFT system at the single cell level. During the entire experiment, a sterile and humid environment was maintained to ensure the activity of cells. The high viability of sorted cells was achieved by the LIFT combining with FLIM.

6.
Front Endocrinol (Lausanne) ; 12: 752995, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867797

RESUMO

Background and Aims: The ankle-brachial index (ABI) is an efficient tool for objectively documenting the presence of lower-extremity peripheral arterial disease (PAD). The predictive factors of cardiovascular events and diabetic foot ulcer were not clear from the ABI examination in Taiwanese patients with type 2 diabetes mellitus (DM). Methods: We enrolled 482 patients with type 2 DM who regularly visited the outpatient department of Chang Gung Memorial Hospital and received ABI as well as brachial-ankle pulse wave velocity (ba-PWV) examinations from 2010 to 2017. Age, gender, PAD symptoms, comorbidities, family history of chronic diseases, lifestyle (smoking, alcohol consumption, and exercise), height, weight, waist circumference, monofilament testing and foot ulcer status were studied. Results: There were 104 (22%) patients (mean age, 67.8 years) with the ABI <1.0. These patients with low ABI (ABI<1.0) had a significantly older age (p=0.001), higher delta PWV (p<0.001), higher rates of stroke (p=0.007), myocardial infarction (p=0.016), and foot ulcer (p=0.039). In a multivariable analysis model, the adjusted odds ratio (aOR) for myocardial infarction, stroke, and foot ulcers associated with low ABI were 1.219 (0.397-3.743, p=0.729), 1.204 (0.556-2.610, p=0.638), and 2.712 (1.199-6.133, p=0.017), respectively. The patients with low PWV (PWV<1400 cm/s) were significantly younger (p<0.001) and had a lower rate of hypertension (p<0.001), and higher percentages of stroke (p=0.027) and dialysis (p=0.041) family history. Conclusions: Low ABI was associated with cardiovascular events and diabetic foot ulcer independently in patients with type 2 DM.

7.
Front Oncol ; 11: 764912, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868987

RESUMO

Therapeutic options for metastatic CRC (mCRC) have changed significantly in recent years, greatly increasing the complexity of therapeutic decision-making. Although oncology guidelines have helped improve the care process, guidelines may also limit the flexibility to individualize in-clinic decision-making. This consensus paper addresses specific gaps in the current international guidelines to assist Taiwanese colon and rectal experts make specific therapeutic choices. Over 3 years and three meetings with selected experts on "real-world" Taiwanese practice patterns for mCRC, consensus was achieved. The experts also discussed specific questions during in-depth one-on-one consultation. Outcomes of the discussion were then correlated with published evidence by an independent medical writer. The final consensus includes clinically implementable recommendations to provide guidance in treating Taiwanese mCRC patients. The consensus includes criteria for defining fit and unfit intensive treatment patients, treatment goals, treatment considerations of molecular profiles, treatment consideration, and optimal treatment choices between different patient archetypes, including optimal treatment options based on RAS, BRAF, and microsatellite instability (MSI) status. This consensus paper is the second in the Taiwan Society of Colon and Rectal Surgeons (TSCRS) Consensus series to address unmet gaps in guideline recommendations in lieu of Taiwanese mCRC management. Meticulous discussions with experts, the multidisciplinary nature of the working group, and the final drafting of the consensus by independent medical professionals have contributed to the strong scientific value of this consensus.

8.
Microb Pathog ; 162: 105297, 2021 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-34883227

RESUMO

Duodenal microbiota may have impact in Functional Dyspepsia. The aim of this study was to explore the difference of microbiota on duodenal mucosa between patients with Functional Dyspepsia and normal subjects. The duodenal mucosa of the subjects were collected under upper gastrointestinal endoscope and the contents of the descending duodenal intestine were extracted with cell brushes in 20 patients with Functional Dyspepsia and 5 healthy subjects. The microbiome on duodenal was studied by 16SrDNA gene sequencing. The differences of duodenal flora were analyzed and compared by LEfSe, FAPROTAX, SPSS and other software. There were significant differences in ACE index, shannon index and observedspecies index between patients with functional dyspepsia and healthy people (P < 0.05). PCoA analysis of the structure of bacteria between two groups found that the duodenal microbiome showed a separate trend. In further study, Amova analysis showed a significant difference (P < 0.05). We found that the there are obvious differences in the composition of duodenal microbiome in functional dyspepsia and healthy people. At the genus level, there were significant differences in Alloprevotella, Peptostreptococcus,Sutterella, Corynebacteriurn,Catonella, Faecalibacterium,Staphylococcus,Eubacteriumnodatumgro-up, Lachnoclostridiurn and Lautropia between the two groups (P < 0.05). The prediction results of Microflora function from FAPROTAX showed that the urea decomposing (ureolysis) and fumaric acid respiratory (fumaraterespiration) function of duodenal bacteria in patients with functional dyspepsia were significantly different from those in healthy people (P < 0.05). In conclusion, there is a significant difference in mucosal microflora of duodenum between patients with functional dyspepsia and healthy groups. It includes greater microflora diversity, different microflora structure, different microflora composition, specific taxa and specific microbiome function. The disorder of duodenal microecology may be the formation mechanism of functional dyspepsia.

9.
Materials (Basel) ; 14(23)2021 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-34885422

RESUMO

The objective of this study was to investigate the physico-chemical properties of concrete made with recycled brick-concrete aggregate, which was the mixture from waste concrete and waste clay brick in a 7:3 ratio. Specifically, this paper investigated the mechanical properties, freeze-thaw resistance, and distribution of water-soluble chloride ions of concrete containing RBCA and fly ash (FA) against combined freeze-thaw and sodium chloride attack. Concrete containing RBCA replacement of natural coarse aggregate and fly ash replacement of Portland cement was subjected to 45 freeze-thaw cycles containing sodium chloride solution. It was discovered that the mechanical properties and freeze-thaw resistance to sodium chloride attack gradually decreased with increasing RBCA content. At the same time, a replacement level of 15% FA by weight resulted in significant improvements in compressive strength and resistance to combined freeze-thaw and chloride attack. Furthermore, using a replacement of 30% FA by weight markedly improved the resistance to chloride ion penetration of concrete due to the lowest water-soluble chloride content.

10.
Front Endocrinol (Lausanne) ; 12: 801324, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34966361

RESUMO

Background and Aims: The long-term survival in people with type 2 diabetes following first diagnosis of diabetic foot complications (FDDFC) is unclear. The object is to evaluate the mortality rate in subjects with type 2 diabetes following FDDFC and the impacts of the major cardiovascular comorbidities. Methods: Nationwide data were analyzed for subjects with T2D and DFC between 2003 and 2017 according to ICD-9 coding. DFC was defined with the codes of ulcers, infections, or severe peripheral artery disease that required intervention (PAD) to mimic the real world diagnosis. Criteria of FDDFC were preceded by a period without any DFC for at least 5 years. Major cardiovascular comorbidities: established PAD and cardiovascular diseases (CVD: including coronary heart disease (CHD), stroke, or heart failure) before the index date as well as lower-extremity amputations (LEA) at the index episode were analyzed. Results: Among 300,115 subjects with DFC, a total of 103,396 patients had FDDFC. The mean 5-year survival rate of these subjects was 81.05%. Using subjects without associated major cardiovascular comorbidity as baseline, the adjusted hazard ratios (aHR) were1.43 (95% confidence interval 1.38-1.49) in group PAD-/CVD+, followed by 1.70 (1.59-1.80) in PAD+/CVD- and 1.98 (1.89-2.08) in PAD+/CVD+. The aHR was further increased in patients with PAD who additionally had heart failure (3.77, 3.50-4.05), stroke (2.06, 1.95-2.18), or CHD (1.89, 1.79-2.00). Subjects with PAD rather than other CVD were associated with LEA at FDDFC. Patients with major LEA (above the ankle) at FDDFC episode had lower 5-year survival rate (65.01%) followed by those with minor LEA (72.24%) and without LEA (81.61%). Conclusions: Cardiovascular comorbidity as well as LEA status at the event of FDDFCs were both associated with patient survival outcomes. Earlier identification of this large population could lead to higher survival rates.

11.
Insects ; 12(12)2021 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-34940198

RESUMO

Hydrogen sulfide (H2S) has been recognized for its beneficial influence on physiological alterations. The development (body weight) and economic characteristics (cocoon weight, cocoon shell ratio, and cocoon shell weight) of silkworms were increased after continuous 7.5 µM H2S treatment. In the present study, gene expression changes in the fat body of silkworms at the 5th instar larvae in response to the H2S were investigated through comparative transcriptome analysis. Moreover, the expression pattern of significant differentially expressed genes (DEGs) at the 5th instar larvae was confirmed by quantitative real-time PCR (qRT-PCR) after H2S exposure. A total of 1200 (DEGs) was identified, of which 977 DEGs were up-regulated and 223 DEGs were down-regulated. Most of the DEGs were involved in the transport pathway, cellular community, carbohydrate metabolism, and immune-associated signal transduction. The up regulated genes under H2S exposure were involved in endocytosis, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), and the synthesis of fibroin, while genes related to inflammation were down-regulated, indicating that H2S could promote energy metabolism, the transport pathway, silk synthesis, and inhibit inflammation in the silkworm. In addition, the expression levels of these genes were increased or decreased in a time-dependent manner during the 5th instar larvae. These results provided insight into the effects of H2S on silkworms at the transcriptional level and a substantial foundation for understanding H2S function.

12.
Vaccines (Basel) ; 9(12)2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34960188

RESUMO

Infectious bursal disease (IBD), caused by the infectious bursal disease virus (IBDV), is a highly contagious and immunosuppressive disease in chickens worldwide. The novel variant IBDV (nvIBDV) has been emerging in Chinese chicken farms since 2017, but there are no available vaccines that can provide effective protection. Herein, the capsid protein VP2 from nvIBDV strain FJ-18 was expressed in Kluyveromyces marxianus with the aim to produce nvIBDV subviral particles (SVPs). Two recombinant strains constructed for expression of nvIBDV VP2 (nvVP2) and His-tagged VP2 (nvHVP2) formed two types of nvIBDV subviral particles (SVPs), namely nvVP2-SVPs and nvHVP2-SVPs. TEM scans showed that both SVPs were about 25 nm in diameter, but there was a large portion of nvVP2-SVPs showing non-spherical particles. Molecular dynamics simulations indicate that an N-terminal His tag strengthened the interaction of the nvHVP2 monomer and contributed to the assembly of SVPs. Vaccination of chicks with the nvHVP2-SVPs provided 100% protection against novel variant IBDV infection when challenged with the FJ-18 strain, as well as the classical strain BC6/85. By contrast, vaccination with the nvVP2-SVPs only provided 60% protection against their parent FJ-18 strain, suggesting that the stable conformation of subviral particles posed a great impact on their protective efficacy. Our results showed that the nvHVP2-SVPs produced by the recombinant K. marxianus strain is an ideal vaccine candidate for IBDV eradication.

13.
JAMA Netw Open ; 4(12): e2138775, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34905004

RESUMO

Importance: Patients with type 1 diabetes (T1D) and a family history of type 2 diabetes (T2D) appear to be at a high risk of diabetes complications and other cardiovascular diseases. However, estimates of individual risks in patients in Taiwan are largely unavailable or unreliable. Objective: To evaluate the risk of diabetes complications and major adverse cardiovascular events (MACEs) in patients with T1D with a family history of T2D. Design, Setting, and Participants: A population-based cohort study used the Taiwan National Health Insurance Research Database. Participants included all individuals registered in that database on December 31, 2017, and followed up since March 1, 1995. The data were analyzed from December 6, 2018, to December 5, 2019. Exposure: Patients with T1D and a family history of T2D were evaluated. Main Outcomes and Measures: The prevalence and hazard ratios (HRs) of diabetes complications and other cardiovascular diseases in patients with T1D were analyzed. The MACEs were identified by diagnostic or procedural codes and heritability was formulated by the registry data of beneficiaries. Results: Of 27 370 965 individuals included in the database, 11 237 (mean [SD] age, 22.7 [14.4] years; 54% were female) had T1D. The crude prevalence of T1D was 0.04%, with a female to male ratio of 1.22: 1. The adjusted HRs in individuals who had a first-degree relative with T2D were 2.61 (95% CI, 1.32-5.16) for MACEs at an age at diagnosis of less than 20 years. Adjusted HRs were 1.44 (95% CI, 1.27-1.64) for diabetic neuropathy, 1.28 (95% CI, 1.12-1.47) for retinopathy, and 1.24 (95% CI, 1.06-1.47) for neuropathy at all ages of diagnosis. Conclusions and Relevance: In this study of patients in Taiwan with T1D, having relatives with T2D was associated with an increase in the individual risks of developing diabetes complications. Patients with T1D and a family history of T2D might have more complications and require close management.


Assuntos
Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/etiologia , Retinopatia Diabética/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Cardiovasculares/epidemiologia , Bases de Dados Factuais , Nefropatias Diabéticas/epidemiologia , Retinopatia Diabética/epidemiologia , Feminino , Humanos , Modelos Logísticos , Masculino , Anamnese , Pessoa de Meia-Idade , Prevalência , Modelos de Riscos Proporcionais , Fatores de Risco , Taiwan
14.
Molecules ; 26(24)2021 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-34946594

RESUMO

Moringa oleifera (M. oleifera), which belongs to the Moringaceae family, is a common herb, rich in plant compounds. It has a variety of bioactive compounds that can act as antioxidants, antibiotics, anti-inflammatory and anti-cancer agents, etc., which can be obtained in different body parts of M. oleifera. Isothiocyanates (ITCs) from M. oleifera are one class of these active substances that can inhibit cancer proliferation and promote cancer cell apoptosis through multiple signaling pathways, thus curbing cancer migration and metastasis, at the same time they have little adverse effect on normal cells. There are multiple variants of ITCs in M. oleifera, but the predominant phytochemical is 4-(α-L-rhamnosyloxy)benzyl isothiocyanate, also known as moringa isothiocyanate (MIC-1). Studies have shown that MIC-1 has the possibility to be used clinically for the treatment of diabetes, neurologic diseases, obesity, ulcerative colitis, and several cancer types. In this review, we focus on the molecular mechanisms underlying the anti-cancer and anti-chronic disease effects of MIC-1, current trends, and future direction of MIC-1 based treatment strategies. This review combines the relevant literature of the past 10 years, in order to provide more comprehensive information of MIC-1 and to fully exploit its potentiality in the clinical settings.

15.
Biotechnol Biofuels ; 14(1): 236, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34906221

RESUMO

BACKGROUND: Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far. RESULTS: In this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2-Ire1-Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells. CONCLUSIONS: Our results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

16.
Yeast ; 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34791694

RESUMO

Kluyveromyces marxianus is a promising host for the production of heterologous proteins, chemicals, and bioethanol. One superior feature of this species is its capacity to assimilate lactose, which is rendered by the LAC12-LAC4 gene pair encoding a lactose permease and a ß-galactosidase enzyme. Little is known about the regulation of LAC4 in K. marxianus. In this study, we showed the presence of weak glucose repression in the regulation of LAC4 and that might contribute to the leaky expression of LAC4 in the glucose medium. In a mutagenesis screen of 1000-bp LAC4 upstream region, one mutant region, named H1, drove low-leakage expression of a URA3 reporter gene in glucose medium. Two mutations inside a polyadenosine stretch (poly(A)) of 5' UTR were major contributors to the low-leakage phenotype of H1. H1 directed low-leakage expression of GFP on a plasmid and that of LAC4 in situ in the glucose medium, which was not due to the reduction of mRNA levels. Meanwhile, H1 did not affect the induction of GFP or LAC4 by lactose. Cre recombinase expressed by H1 caused lower toxicity in the repressive condition and achieved higher yield after induction, compared with that expressed by a wild-type LAC4 upstream region or a strong INU1 promoter. Our study suggested that poly(A) inside 5' UTR played a role in regulating the expression of LAC4 in the repressive condition. Meanwhile, H1 provided a base for the development of a strict inducible system for expressing industrial proteins, especially toxic proteins.

17.
MAbs ; 13(1): 1991256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34747330

RESUMO

Human/humanized IgG4 antibodies have reduced effector function relative to IgG1 antibodies, which is desirable for certain therapeutic purposes. However, the developability and biophysical properties for IgG4 antibodies are not well understood. This work focuses on the head-to-head comparison of key biophysical properties, such as self-interaction and viscosity, for 14 human/humanized, and chimeric IgG1 and IgG4 S228P monoclonal antibody pairs that contain the identical variable regions. Experimental measurements showed that the IgG4 S228P antibodies have similar or higher self-interaction and viscosity than that of IgG1 antibodies in 20 mM sodium acetate, pH 5.5. We report sequence and structural drivers for the increased viscosity and self-interaction detected in IgG4 S228P antibodies through a combination of experimental data and computational models. Further, we applied and extended a previously established computational model for IgG1 antibodies to predict the self-interaction and viscosity behavior for each antibody pair, providing insight into the structural characteristics and differences of these two isotypes. Interestingly, we observed that the IgG4 S228P swapped variants, where the CH3 domain was swapped for that of an IgG1, showed reduced self-interaction behavior. These domain swapped IgG4 S228P molecules also showed reduced viscosity from experiment and coarse-grained simulations. We also observed that experimental diffusion interaction parameter (kD) values have a high correlation with computational diffusivity prediction for both IgG1 and IgG4 S228P isotypes.Abbreviations: AHc, constant region Hamaker constant; AHv, variable region Hamaker constant; CDRs, Complementarity-determining regions; CG, Coarse-grained model; CH1, Constant heavy chain 1; CH2 Constant heavy chain 2; CH3 Constant heavy chain 3; chgCH3 Effective charge on the CH3 region; CL Constant light chain; cP, Centipoise; DLS, Dynamic light scattering; Fab, Fragment antigen-binding; Fc, Fragment crystallizable; Fv, Variable domaing; (r) Radial distribution function; H1 CDR1 of Heavy Chain; H2 CDR2 of Heavy Chain; H3 CDR3 of Heavy Chain; HVI, High viscosity index; IgG1 human immunoglobulin of IgG1 subclass; IgG4 human immunoglobulin of IgG4 subclass; kD, Diffusion interaction parameter; L1 CDR1 of Light Chain; L2 CDR2 of Light Chain; L3 CDR3 of Light Chain; mAb, Monoclonal antibody; MD, Molecular dynamics; PPI Protein-protein interactions; SCM, Spatial charge map; UP-SEC, Ultra-high-performance size-exclusion chromatography; VH, Variable domain of Heavy Chain; VL, Variable domain of Light Chain.

19.
Brief Funct Genomics ; 2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34755827

RESUMO

Breast cancer is a kind of malignant tumor that occurs in breast tissue, which is the most common cancer in women. Cellular metabolism is a critical determinant of the viability and function of cancer cells in tumor microenvironment. In this study, based on the gene expression profile of metabolism-related genes, the prognostic value of 20 metabolic pathways in patients with breast cancer was identified. A universal risk stratification signature that relies on 20 metabolic pathways was established and validated in training cohort, two testing cohorts and The Cancer Genome Atlas pan cancer cohort. Then, the relationship between metabolic risk score subtype, prognosis, immune infiltration level, cancer genotypes and their impact on therapeutic benefit were characterized. Results demonstrated that the patients with the low metabolic risk score subtype displayed good prognosis, high level of immune infiltration and exhibited a favorable response to neoadjuvant chemotherapy and immunotherapy. Taken together, the work presented in this study may deepen the understanding of metabolic hallmarks of breast cancer, and may provide some valuable information for personalized therapies in patients with breast cancer.

20.
Biotechnol Biofuels ; 14(1): 220, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34809677

RESUMO

BACKGROUND: Hemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs. RESULTS: Two polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a ß-mannanase gene M330 (without the stop codon), a P2A sequence and a ß-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular ß-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular ß-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and ß-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of ß-xylanase and ß-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, ß-mannanase, ß-xylanase, and ß-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs. CONCLUSIONS: The FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

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