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1.
Ultramicroscopy ; 261: 113964, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38579523

RESUMO

Compressive sensing (CS) can reconstruct the rest information almost without distortion by advanced computational algorithm, which significantly simplifies the process of atomic force microscope (AFM) scanning with high imaging quality. In common CS-AFM, the partial measurements randomly come from the whole region to be measured, which easily leads to detail loss and poor image quality in regions of interest (ROIs). Consequently, important microscopic phenomena are missed probably. In this paper, we developed an adaptive under-sampling strategy for CS-AFM to optimize the process of sampling. Under a certain under-sampling ratio, the weight coefficient of ROIs and regions of base (ROBs) were set to control the distribution of under-sampling points and corresponding measurement matrix. A series of simulations were completed to demonstrate the relationship between the weight coefficient of ROIs and image quality. After that, we verified the effectiveness of the method on our homemade AFM. Through a lot of simulations and experiments, we demonstrated how the proposed method optimized the sampling process of CS-AFM, which speeded up the process of AFM imaging with high quality.

2.
Sci Total Environ ; 923: 171332, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38447716

RESUMO

The synergy between bacteria and fungi is a key determinant of soil health and have a positive effect on plant development under drought conditions, with the potentially enhancing the sustainability of amending soil with natural materials. However, identifying how soil amendments influence plant growth is often difficult due to the complexity of microorganisms and their links with different soil amendment types and environmental factors. To address this, we conducted a field experiment to examine the impact of soil amendments (biochar, Bacillus mucilaginosus, Bacillus subtilis and super absorbent polymer) on plant growth. We also assessed variations in microbial community, links between fungi and bacteria, and soil available nutrients, while exploring how the synergistic effects between fungus and bacteria influenced the response of soil amendments to plant growth. This study revealed that soil amendments reduced soil bacterial diversity but increased the proportion of the family Enterobacteriaceae, Nitrosomonadaceae, and also increased soil fungal diversity and the proportion of the sum of the family Lasiosphaeriaceae, Chaetomiaceae, Pleosporaceae. Changes in soil microbial communities lead to increase the complexity of microbial co-occurrence networks. Furthermore, this heightened network complexity enhanced the synergy of soil bacteria and fungi, supporting bacterial functions related to soil nutrient cycling, such as metabolic functions and genetic, environmental, and cellular processes. Hence, the BC and BS had 3.0-fold and 0.5-fold greater root length densities than CK and apple tree shoot growth were increased by 62.14 %,50.53 % relative to CK, respectively. In sum, our results suggest that the synergistic effect of bacteria and fungi impacted apple tree growth indirectly by modulating soil nutrient cycling. These findings offer a new strategy for enhancing the quality of arable land in arid and semi-arid regions.


Assuntos
Microbiota , Solo , Bactérias/metabolismo , Nutrientes , Fungos/metabolismo , Microbiologia do Solo
3.
J Sci Food Agric ; 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38363126

RESUMO

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

4.
J Biochem ; 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38382634

RESUMO

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn-antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn-antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn-antigen showed a significant increase, whereas the expression of the T-antigen (galactose-ß1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn-antigen modification.

5.
Front Biosci (Landmark Ed) ; 29(1): 3, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-38287800

RESUMO

BACKGROUND: Colorectal cancer liver metastasis (CRLM) and hepatocellular carcinoma (HCC) are both high incidence tumors in China. In certain poorly differentiated cases they can exhibit comparable imaging and pathological characteristics, which impedes accurate clinical diagnosis. The use of protein-based techniques with tissue slides offers a more precise means to assess pathological changes and has the potential to assist with tumor diagnosis. METHODS: A simple in situ protein digestion protocol was established for protein fingerprint analysis of paraffin-embedded tissue slide samples. Additionally, machine learning techniques were employed to construct predictive models for CRLM and HCC. The accuracy of these models was validated using tissue slides and a clinical database. RESULTS: Analysis of differential protein expression between CRLM and HCC groups reliably identified 977 proteins. Among these, 53 were highly abundant in CRLM samples and 57 were highly abundant in HCC samples. A prediction model based on the expression of six proteins (CD9, GSTA1, KRT20, COL1A2, AKR1C3, and HIST2H2BD) had an area under curve (AUC) of 0.9667. This was further refined to three proteins (CD9, ALDH1A1, and GSTA1) with an AUC of 0.9333. CONCLUSIONS: Tissue slide proteomics can facilitate accurate differentiation between CRLM and HCC. This methodology holds great promise for improving clinical tumor diagnosis and for identifying novel markers for challenging pathological specimens.


Assuntos
Carcinoma Hepatocelular , Neoplasias Colorretais , Neoplasias Hepáticas , Humanos , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/patologia , Proteômica , Neoplasias Colorretais/metabolismo , China
6.
Cell Oncol (Dordr) ; 47(1): 113-128, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37578594

RESUMO

BACKGROUND: Metabolic reprogramming is closely related to the development of gastric cancer (GC), which remains as the fourth leading cause of cancer-related death worldwide. As a tumor suppressor for GC, whether receptor for activated C-kinase 1 (RACK1) play a modulatory role in metabolic reprogramming remains largely unclear. METHODS: GC cell lines and cell-derived xenograft mouse model were used to identify the biological function of RACK1. Flow cytometry and Seahorse assays were applied to examine cell cycle and oxygen consumption rate (OCR), respectively. Western blot, real-time PCR and autophagy double fluorescent assays were utilized to explore the signaling. Immunohistochemistry was performed to detect the expression of RACK1 and other indicators in tissue sections. RESULTS: Loss of RACK1 facilitated the viability, colony formation, cell cycle progression and OCR of GC cells in a glutamine-dependent manner. Further investigation revealed that RACK1 knockdown inhibited the lysosomal degradation of Alanine-serine-cysteine amino acid transporter 2 (ASCT2). Mechanistically, depletion of RACK1 remarkably decreased PTEN expression through up-regulating miR-146b-5p, leading to the activation of AKT/mTOR signaling pathway which dampened autophagy flux subsequently. Moreover, knockdown of ASCT2 could reverse the promotive effect of RACK1 depletion on GC tumor growth both in vitro and in vivo. Tissue microarray confirmed that RACK1 was negatively correlated with the expression of ASCT2 and p62, as well as the phosphorylation of mTOR. CONCLUSION: Together, our results demonstrate that the suppressive function of RACK1 in GC is associated with ASCT2-mediated glutamine metabolism, and imply that targeting RACK1/ASCT2 axis provides potential strategies for GC treatment.


Assuntos
Neoplasias Gástricas , Humanos , Animais , Camundongos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glutamina/metabolismo , Linhagem Celular Tumoral , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Receptores de Quinase C Ativada/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
7.
J Agric Food Chem ; 71(51): 20826-20837, 2023 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-38096130

RESUMO

Extracellular vesicles (EVs) are membrane-bound vesicles released by living cells. As vesicles for macromolecule transmission and intercellular communication, EVs are broadly applied in clinical diagnosis and biomimetic drug delivery. Milk-derived EVs (MEVs) are an ideal choice for scale-up applications because they exhibit biocompatibility and are easily obtained. Herein, intact glycopeptides in MEVs from bovines, caprines, porcines, and humans were comprehensively analyzed by high-resolution mass spectrometry using the sceHCD, followed by the EThcD fragment method, revealing that protein glycosylation is abundant and heterogeneous in MEVs. The dominant glycans in all MEVs were sialic acid-modified N-linked glycans (over 50%). A couple of species-specific glycans were also characterized, which are potentially markers of different original EVs. Interestingly, the Neu5Gc-modified glycans were enriched in caprine milk-derived EVs (58 ± 2%). Heterogeneity of MEV protein glycosylation was observed for glycosites and glycan compositions, and the structural heterogeneity of protein glycosylation was also identified and validated. The glycosignatures of EV biogenesis- and endocytosis-related proteins (CD63 and MFGE8) were significantly different in these four species. Overall, we comprehensively characterized the glycosylation signature of MEVs from four different species and provided insight into protein glycosylation related to drug target delivery.


Assuntos
Vesículas Extracelulares , Leite Humano , Humanos , Animais , Bovinos , Suínos , Glicosilação , Leite Humano/metabolismo , Cabras/metabolismo , Vesículas Extracelulares/metabolismo , Polissacarídeos/metabolismo
8.
Technol Cancer Res Treat ; 22: 15330338231218163, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38112409

RESUMO

PURPOSE: Collectin subfamily member 12, a transmembrane scavenger receptor C-type lectin, is aberrantly expressed in various cancers. However, its physiological role in gastric cancer remains somewhat unclear. This study aimed to investigate the Collectin subfamily member 12 expression pattern in human gastric cancer and its role in gastric cancer progression. METHODS: The Kaplan-Meier method was used for survival analysis. The univariate and multivariate Cox proportional hazards regression models were used to identify independent predictors for progression-free survival and overall survival. The effects of Collectin subfamily member 12 on gastric cancer cell proliferation, migration, invasion, and apoptosis were detected through the cell counting kit-8 assay, colony formation assay, wound healing assay, transwell assay, and flow cytometry analysis, respectively. Additionally, the correlation between Collectin subfamily member 12 expression and immune cell infiltration was analyzed through bioinformatics. RESULTS: Collectin subfamily member 12 was highly expressed in advanced gastric cancer (T3-T4, pathologic stage III-IV). High Collectin subfamily member 12 expression was correlated with a worse progression-free survival and overall survival in the gastric cancer patients. In vitro, cell line studies revealed that Collectin subfamily member 12 promoted gastric cancer cell proliferation, migration, and invasion and inhibited gastric cancer cell apoptosis. The bioinformatics analysis further demonstrated that the Collectin subfamily member 12 expression level positively correlated with infiltration of several immune cells, such as M2 macrophages, dendritic cells, neutrophils, and regulatory T cells, suggesting that Collectin subfamily member 12 may also play a role in suppressing tumor immune response in gastric cancer. CONCLUSIONS: Collectin subfamily member 12 was identified as a novel predictive marker and target for the clinical treatment of gastric cancer.


Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/metabolismo , Prognóstico , Biomarcadores Tumorais/metabolismo , Análise de Sobrevida , Colectinas , Linhagem Celular Tumoral , Proliferação de Células/genética , Receptores Depuradores
9.
Cell Death Dis ; 14(12): 839, 2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38110356

RESUMO

Imatinib (IM) has significantly improved the prognosis of gastrointestinal stromal tumor (GIST) patients, but some patients still have primary resistance to IM, and approximately half of patients develop acquired drug resistance within 2 years of treatment, necessitating exploration of new treatment strategies. Targeting ferroptosis as a novel approach to tumor treatment has gained attention. Yet, there is limited research on ferroptosis in GIST, and the underlying mechanism remains unclear. In this study, we revealed that IM increased lipid reactive oxygen species and intracellular Fe2+ levels, and decreased glutathione levels in GIST. This effect could be partially inhibited by Ferrostatin-1. Additionally, knocking down STUB1 and overexpressing GPX4 reversed the IM-induced ferroptosis effect. Moreover, STUB1 was identified as a novel E3 ubiquitin ligase of GPX4, promoting the ubiquitination at site K191 of GPX4. The combination of the GPX4 inhibitor RSL3 and IM synergistically induces ferroptosis, inhibiting GIST proliferation both in vivo and in vitro. Furthermore, STUB1 and GPX4 expression serve as independent prognostic factors for GIST. In conclusion, This study is the first to demonstrate that IM induces ferroptosis by promoting STUB1-mediated GPX4 ubiquitination in GIST, and the combination of RSL3 and IM emerges as a promising therapeutic strategy for GIST.


Assuntos
Ferroptose , Tumores do Estroma Gastrointestinal , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo
10.
ACS Synth Biol ; 12(12): 3743-3753, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37991716

RESUMO

Soil microbial communities with reduced complexity are emerging as model systems for studying consortia-scale phenotypes. To establish synthetic biology tools for studying these communities in hard-to-image environmental materials, we evaluated whether a single member of a model soil consortium (MSC) can be programmed to report on gene expression without requiring matrix disruption. For these studies, we targeted a five-membered MSC that includes Dyadobacter fermentans, Ensifer adhaerens, Rhodococcus sp003130705, Streptomyces sp001905665, and Variovorax beijingensis. By coupling the expression of a methyl halide transferase to a constitutive promoter, we show that V. beijingensis can be programmed to synthesize methyl halides that accumulate in the soil headspace at levels that are ≥24-fold higher than all other MSC members across a range of environmentally relevant hydration conditions. We find that methyl halide production can report on an MSC promoter that is activated by changes in water potential, and we demonstrate that a synthetic gas signal can be read out directly using gas chromatography and indirectly using a soil-derived Methylorubrum that is programmed to produce a visual output in response to methyl halides. These tools will be useful for future studies that investigate how MSC responds to dynamic hydration conditions, such as drought and flood events induced by climate change, which can alter soil water potential and induce the release of stored carbon.


Assuntos
Hidrocarbonetos Bromados , Solo , Solo/química , Água , Transdução de Sinais
11.
Sheng Wu Gong Cheng Xue Bao ; 39(10): 4135-4149, 2023 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-37877396

RESUMO

The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.


Assuntos
Quitosana , Spirulina , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Quitosana/farmacologia , Biofilmes , Quitina/farmacologia
12.
Nat Commun ; 14(1): 6371, 2023 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-37821460

RESUMO

Soil contamination is an environmental issue due to increasing anthropogenic activities. Existing processes for soil remediation suffer from long treatment time and lack generality because of different sources, occurrences, and properties of pollutants. Here, we report a high-temperature electrothermal process for rapid, water-free remediation of multiple pollutants in soil. The temperature of contaminated soil with carbon additives ramps up to 1000 to 3000 °C as needed within seconds via pulsed direct current input, enabling the vaporization of heavy metals like Cd, Hg, Pb, Co, Ni, and Cu, and graphitization of persistent organic pollutants like polycyclic aromatic hydrocarbons. The rapid treatment retains soil mineral constituents while increases infiltration rate and exchangeable nutrient supply, leading to soil fertilization and improved germination rates. We propose strategies for upscaling and field applications. Techno-economic analysis indicates the process holds the potential for being more energy-efficient and cost-effective compared to soil washing or thermal desorption.

13.
Cardiovasc Res ; 119(16): 2607-2622, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-37713664

RESUMO

AIMS: Endurance exercise is associated with an increased risk of atrial fibrillation (AF). We previously established that adverse atrial remodelling and AF susceptibility induced by intense exercise in mice require the mechanosensitive and pro-inflammatory cytokine tumour necrosis factor (TNF). The cellular and mechanistic basis for these TNF-mediated effects is unknown. METHODS AND RESULTS: We studied the impact of Tnf excision, in either atrial cardiomyocytes or endothelial cells (using Cre-recombinase expression controlled by Nppa or Tie2 promoters, respectively), on the cardiac responses to six weeks of intense swim exercise training. TNF ablation, in either cell type, had no impact on the changes in heart rate, autonomic tone, or left ventricular structure and function induced by exercise training. Tnf excision in atrial cardiomyocytes did, however, prevent atrial hypertrophy, fibrosis, and macrophage infiltration as well as conduction slowing and increased AF susceptibility arising from exercise training. In contrast, endothelial-specific excision only reduced the training-induced atrial hypertrophy. Consistent with these cell-specific effects of Tnf excision, inducing TNF loss from atrial cardiomyocytes prevented activation of p38MAPKinase, a strain-dependent downstream mediator of TNF signalling, without affecting the atrial stretch as assessed by atrial pressures induced by exercise. Despite TNF's established role in innate immune responses and inflammation, neither acute nor chronic exercise training caused measurable NLRP3 inflammasome activation. CONCLUSIONS: Our findings demonstrate that adverse atrial remodelling and AF vulnerability induced by intense exercise require TNF in atrial cardiomyocytes whereas the impact of endothelial-derived TNF is limited to hypertrophy modulation. The implications of the cell autonomous effects of TNF and crosstalk between cells in the atria are discussed.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Cardiomiopatias , Animais , Camundongos , Fibrilação Atrial/etiologia , Fibrilação Atrial/prevenção & controle , Fibrilação Atrial/patologia , Miócitos Cardíacos/metabolismo , Células Endoteliais/metabolismo , Átrios do Coração , Fator de Necrose Tumoral alfa/metabolismo , Cardiomiopatias/metabolismo , Hipertrofia/complicações , Hipertrofia/metabolismo
14.
Yi Chuan ; 45(8): 669-683, 2023 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-37609818

RESUMO

In human cells, there are more than 146 glycosylphosphatidylinositol-anchored proteins (GPI-APs), including receptors, ligands, adhesion molecules and enzymes. The proteins are associated with membrane microdomains called lipid rafts through GPI, and plays a variety of important biological functions. At present, plenty of studies have been carried out on the biosynthesis of GPI-APs. The biosynthesis of GPI-APs requires at least 20 steps, and more than 40 GPI biosynthetic genes have been identified. However, it remains unclear how expression of GPI-AP related genes is regulated in normal and cancer tissues. In this study, we utilized gene expression data from both the TCGA database and GTEx portal to analysis the gene expression involved in GPI-AP biosynthesis and encoding GPI-APs in normal and cancer tissues. In order to perform a comprehensive analysis, we employed the GlycoMaple, a tool that is specifically designed to analyze glycosylation pathways. The results showed that compared with normal tissues, the expression of genes involved in GPI-AP biosynthesis in cancer tissues such as early glioma, glioblastoma multiforme, pancreatic cancer, testicular germ cell carcinoma, skin primary cutaneous melanoma and skin metastatic cutaneous melanoma, was changed significantly. Particularly, the expression of PIGY in these six cancers was increased. In addition, the expression of CD14, a GPI-AP gene, was increased in these six cancers. The expression of GAS1, GPC2 and GPC4 was increased only in early glioma and glioblastoma multiforme indicating that some GPI-APs such as GAS1 can be used as biomarkers of glioma. This study provides new insights into the expression of GPI-AP related genes in normal and cancer tissues, and lays a solid foundation for the development of GPI-APs as biomarkers.


Assuntos
Glioblastoma , Glioma , Melanoma , Neoplasias Cutâneas , Humanos , Glicosilfosfatidilinositóis/genética
15.
J Biosci Bioeng ; 136(3): 166-172, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37393188

RESUMO

In humans, almost all the cell surface and secreted glycoproteins are modified with complex-type N-glycans. Thus, it is essential to obtain complex-type N-glycans to fully understand the biological properties of glycoproteins. Here, human ß-1,2-N-acetylglucosaminyltransferase II (hGnT-II), a Golgi-localized enzyme integral to complex-type N-glycan biosynthesis, was cloned as a truncated transmembrane form (GnT-II-ΔTM) and heterologously overexpressed in Escherichia coli. Our results showed that hGnT-II could be overexpressed in its soluble form by fusing the truncated enzyme with a thioredoxin (Trx)-tag in the Rosetta-Gami 2 strain. Using the optimized induction conditions, the expression level of recombinant protein was enhanced to yield approximately 4 mg per liter culture after affinity purification. The enzyme exhibited appropriate glycosyltransferase activity, and the calculated Km value was 52.4 µM, similar to the protein expressed in mammalian cells. Furthermore, the effect of MGAT2-CDG mutations on enzyme activity was also measured. These results suggested that the E. coli expression system was capable of the large-scale production of bioactive hGnT-II, which can be used for functional study and effective synthesis of complex-type N-glycans.


Assuntos
Escherichia coli , N-Acetilglucosaminiltransferases , Animais , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , N-Acetilglucosaminiltransferases/metabolismo , Glicoproteínas , Polissacarídeos/metabolismo , Mamíferos/metabolismo
16.
Biotechnol J ; 18(11): e2300259, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37470505

RESUMO

BACKGROUND: Human lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited. RESULTS: To achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co-expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL-1 , which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL-1 in a 5 L fermenter. CONCLUSION: This research provides a very useful and cost-effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins.


Assuntos
Muramidase , Saccharomycetales , Humanos , Muramidase/genética , Pichia , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
17.
J Biochem ; 174(2): 109-123, 2023 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-37279648

RESUMO

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.


Assuntos
Fenda Labial , Fissura Palatina , Humanos , Glicosilfosfatidilinositóis/metabolismo , Proteínas Ligadas por GPI/genética , Inositol/metabolismo
18.
Micromachines (Basel) ; 14(6)2023 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-37374828

RESUMO

For the orbit operation of spacecraft, due to environmental factors, a star sensor installed on the spacecraft must have data censoring, which greatly reduces the attitude determination ability of the traditional combined-attitude-determination algorithm. To address this problem, this paper proposes an algorithm for high-precision attitude estimation based on a Tobit unscented Kalman filter. This is on the basis of establishing the nonlinear state equation of the integrated star sensor and gyroscope navigation system. The measurement update process of the unscented Kalman filter is improved. The Tobit model is used to describe the gyroscope drift when the star sensor fails. The latent measurement values are calculated using the probability statistics, and the measurement error covariance expression is derived. The proposed design is verified via computer simulations. When the star sensor fails for 15 min, the accuracy of the Tobit unscented Kalman filter based on the Tobit model is improved by approximately 90% compared to the unscented Kalman filter. Based on the results, the proposed filter can effectively estimate the error caused by the gyro drift, and the method is effective and feasible, provided there is theoretical support for the engineering practice.

19.
Opt Express ; 31(13): 20740-20749, 2023 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-37381190

RESUMO

We proposed and demonstrated a highly efficient sub-microscale focusing from a GaN green laser diode (LD) integrated with double-sided asymmetric metasurfaces. The metasurfaces consist of two nanostructures in a GaN substrate: nanogratings on one side and a geometric phase based metalens on the other side. When it was integrated on the edge emission facet of a GaN green LD, linearly polarized emission was firstly converted to the circularly polarized state by the nanogratings functioning as a quarter-wave plate, the phase gradient was then controlled by the metalens on the exit side. In the end, the double-sided asymmetric metasurfaces achieve a sub micro-focusing from linearly polarized states. Experimental results show the full width at half maximum of the focused spot size is about 738 nm at the wavelength 520 nm and the focusing efficiency is about 72.8%. Our results lay a foundation for the multi-functional applications in optical tweezers, laser direct writing, visible light communication, and biological chip.

20.
Genes (Basel) ; 14(6)2023 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-37372385

RESUMO

Aythya marila is a large diving duck belonging to the family Anatidae. However, the phylogenetic relationship among these Aythya species remains unclear due to the presence of extensive interspecific hybridization events within the Aythya genus. Here, we sequenced and annotated the complete mitochondrial genome of A. marila, which contained 22 tRNAs, 13 protein-coding genes (PCGs), 2 ribosomal RNAs, and 1 D-loop, with a length of 16,617 bp. The sizes of the PCGs ranged from 297 to 1824 bp and were all, except for ND6, located on the heavy chain (H). ATG and TAA were the most common start and termination codons of the 13 PCGs, respectively. The fastest- and slowest-evolving genes were ATP8 and COI, respectively. Codon usage analysis indicated that CUA, AUC, GCC, UUC, CUC, and ACC were the six most frequent codons. The nucleotide diversity values indicated a high level of genetic diversity in A. marila. FST analysis suggested a widespread gene exchange between A. baeri and A. nyroca. Moreover, phylogenetic reconstructions using the mitochondrial genomes of all available Anatidae species showed that, in addition to A. marila, four major clades among the Anatidae (Dendrocygninae, Oxyurinae, Anserinae, and Anatinae) were closely related to A. fuligula. Overall, this study provides valuable information on the evolution of A. marila and new insights into the phylogeny of Anatidae.


Assuntos
Patos , Genoma Mitocondrial , Animais , Patos/genética , Filogenia , Genoma Mitocondrial/genética , Sequência de Bases , Organismos Aquáticos/genética
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