Enhancement of protein production in Aspergillus niger by engineering the antioxidant defense metabolism.

BACKGROUND: Research on protein production holds significant importance in the advancement of food technology, agriculture, pharmaceuticals, and bioenergy. Aspergillus niger stands out as an ideal microbial cell factory for the production of food-grade proteins, owing to its robust protein secretion capacity and excellent safety profile. However, the extensive oxidative folding of proteins within the endoplasmic reticulum (ER) triggers ER stress, consequently leading to protein misfolding reactions. This stressful phenomenon results in the accelerated generation of reactive oxygen species (ROS), thereby inducing oxidative stress. The accumulation of ROS can adversely affect intracellular DNA, proteins, and lipids. RESULT: In this study, we enhanced the detoxification of ROS in A. niger (SH-1) by integrating multiple modules, including the NADPH regeneration engineering module, the glutaredoxin system, the GSH synthesis engineering module, and the transcription factor module. We assessed the intracellular ROS levels, growth under stress conditions, protein production levels, and intracellular GSH content. Our findings revealed that the overexpression of Glr1 in the glutaredoxin system exhibited significant efficacy across various parameters. Specifically, it reduced the intracellular ROS levels in A. niger by 50%, boosted glucoamylase enzyme activity by 243%, and increased total protein secretion by 88%. CONCLUSION: The results indicate that moderate modulation of intracellular redox conditions can enhance overall protein output. In conclusion, we present a strategy for augmenting protein production in A. niger and propose a potential approach for optimizing microbial protein production system.

Study on the effects and mechanisms of Wenzhong Bushen Formula in improving ovarian reserve decline in mice based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: The Wenzhong Bushen Formula (WZBSF) is a traditional Chinese medicine empirical formula known for its effects in tonifying qi, strengthening the spleen, warming the kidneys, promoting yang, regulating blood circulation, and balancing menstruation. Clinical evidence has demonstrated its significant efficacy in treating Diminished Ovarian Reserve (DOR) by improving ovarian reserves. However, the specific pharmacological mechanisms of WZBSF remain unclear. AIM OF THE STUDY: This study aims to investigate the mechanisms by which WZBSF improves ovarian reserve decline through network pharmacology and animal experiments. METHODS AND MATERIALS: WZBSF was analyzed using a dual UPLC-MS/MS and GC-MS platform. Effective components and targets of WZBSF were obtained from the TCMSP database and standardized using UniProt. Disease targets were collected from GeneCard, OMIM, PHARMGKB, and DisGeNET databases, with cross-referencing between the two sets of targets. A PPI protein interaction network was constructed using Cytoscape3.9.1 and STRING database, followed by KEGG and GO enrichment analysis using the Metascape database. Finally, an ovarian reserve decline model was established in mice, different doses of WZBSF were administered, and experimental validation was conducted through serum hormone detection, H&E staining, immunofluorescence (IF), immunohistochemistry (IHC), and Western blot analysis (WB). RESULTS: WZBSF shares 145 common targets with ovarian reserve decline. GO enrichment analysis revealed involvement in biological processes such as response to hormone stimulation and phosphatase binding, while KEGG analysis implicated pathways including the PI3K-AKT signaling pathway and FoxO signaling pathway. In mice with ovarian reserve decline, WZBSF restored weight gain rate, increased ovarian index, normalized estrous cycles, reversed serum hormone imbalances, restored various follicle counts, and improved ovarian morphology. Additionally, WZBSF reduced p-AKT and p-FOXO3a levels, preventing excessive activation of primordial follicles and maintaining ovarian reserve. CONCLUSION: WZBSF can ameliorate cyclophosphamide and busulfan-induced ovarian reserve decline, and its mechanism may be associated with the inhibition of the PI3K/AKT/FOXO3a signaling pathway.

Molecular imaging-guided extracellular vesicle-based drug delivery for precise cancer management: Current status and future perspectives.

Extracellular vesicles (EVs), the mediators of intercellular communication, have attracted the attention of researchers for the important roles they play in cancer treatment. Compared with other inorganic nano-materials, EVs possess the advantages of higher biocompatibility, better physiochemical stability, easier surface modification, and excellent biosafety. They can be used as an advanced drug delivery system with an improved therapeutic index for various therapeutic agents. Engineered EV-based imaging and therapeutic agents (engineered EVs) have emerged as useful tools in targeted cancer diagnosis and therapy. Non-invasive tracing of engineered EVs contributes to a better evaluation of their functions in cancer progression, in vivo dynamic biodistribution, therapeutic response, and drug-loading efficiency. Recent advances in real-time molecular imaging (MI), and innovative EV labeling strategies have led to the development of novel tools that can evaluate the pharmacokinetics of engineered EVs in cancer management, which may accelerate further clinical translation of novel EV-based drug delivery platforms. Herein, we review the latest advances in EVs, their characteristics, and current examples of EV-based targeted drug delivery for cancer. Then, we discuss the prominent applications of MI for tracing both natural and engineered EVs. Finally, we discuss the current challenges and considerations of EVs in targeted cancer treatment and the limitations of different MI modalities. In the coming decades, EV-based therapeutic applications for cancer with improved drug loading and targeting abilities will be developed, and better anti-cancer effects of drug delivery nanoplatform will be achieved.

Tunable Interfacial Electronic Pd-Si Interaction Boosts Catalysis via Accelerating O2 and H2O Activation.

Engineering the interfacial structure between noble metals and oxides, particularly on the surface of non-reducible oxides, is a challenging yet promising approach to enhancing the performance of heterogeneous catalysts. The interface site can alter the electronic and d-band structure of the metal sites, facilitating the transition of energy levels between the reacting molecules and promoting the reaction to proceed in a favorable direction. Herein, we created an active Pd-Si interface with tunable electronic metal-support interaction (EMSI) by growing a thin permeable silica layer on a non-reducible oxide ZSM-5 surface (termed Pd@SiO2/ZSM-5). Our experimental results, combined with density functional theory calculations, revealed that the Pd-Si active interface enhanced the charge transfer from deposited Si to Pd, generating an electron-enriched Pd surface, which significantly lowered the activation barriers for O2 and H2O. The resulting reactive oxygen species, including O2 -, O2 2-, and -OH, synergistically facilitated formaldehyde oxidation. Additionally, moderate electronic metal-support interaction can promote the catalytic cycle of Pd0 ⇆ Pd2+, which is favorable for the adsorption and activation of reactants. This study provides a promising strategy for the design of high-performance noble metal catalysts for practical applications.

Exploration of the Strategy for Improving the Expression of Heterologous Sweet Protein Monellin in Aspergillus niger.

Aspergillus niger is a primary cell factory for food-grade protein (enzyme) production due to its strong protein secretion capacity and unique safety characteristics. The bottleneck issue for the current A. niger expression system is the difference in expression yield of heterologous proteins of non-fungal origin compared to those of fungal origin, which is about three orders of magnitude. The sweet protein monellin, derived from West African plants, has the potential to become a food-grade sweetener due to its high sweetness and the benefit of not containing sugar itself, but it is extremely difficult to establish a research model for heterologous expression in A. niger, owing to extremely low expression, a small molecular weight, and being undetectable with conventional protein electrophoresis. HiBiT-Tag was fused with low-expressing monellin in this work to create a research model for heterologous protein expression in A. niger at ultra-low levels. We increased monellin expression by increasing the monellin copy number, fusing monellin with the endogenous highly expressed glycosylase glaA, and eliminating extracellular protease degradation, among other strategies. In addition, we investigated the effects of overexpression of molecular chaperones, inhibiting the ERAD pathway, and enhancing the synthesis of phosphatidylinositol, phosphatidylcholine, and diglycerides in the biomembrane system. Using medium optimization, we finally obtained 0.284 mg/L of monellin in the supernatant of the shake flask. This is the first time recombinant monellin has been expressed in A. niger, with the goal of investigating ways to improve the secretory expression of heterologous proteins at ultra-low levels, which can serve as a model for the expression of other heterologous proteins in A. niger.

Fibronectin-targeting and metalloproteinase-activatable smart imaging probe for fluorescence imaging and image-guided surgery of breast cancer.

Residual lesions in the tumor bed have been a challenge for conventional white-light breast-conserving surgery. Meanwhile, lung micro-metastasis also requires improved detection methods. Intraoperative accurate identification and elimination of microscopic cancer can improve surgery prognosis. In this study, a smart fibronectin-targeting and metalloproteinase-activatable imaging probe CREKA-GK8-QC is developed. CREKA-GK8-QC possesses an average diameter of 21.7 ± 2.5 nm, excellent MMP-9 protein responsiveness and no obvious cytotoxicity. In vivo experiments demonstrate that NIR-I fluorescence imaging of CREKA-GK8-QC precisely detects orthotopic breast cancer and micro-metastatic lesions (nearly 1 mm) of lungs with excellent imaging contrast ratio and spatial resolution. More notably, fluorescence image-guided surgery facilitates complete resection and avoids residual lesions in the tumor bed, improving survival outcomes. We envision that our newly developed imaging probe shows superior capacity for specific and sensitive targeted imaging, as well as providing guidance for accurate surgical resection of breast cancer.

An integrated method with adaptive decomposition and machine learning for renewable energy power generation forecasting.

In recent years, traditional energy sources have caused a variety of negative impacts on the environment, and reducing carbon emissions is a top priority. The development of renewable energy technology is the key to transform the energy structure. Renewable energy represented by wind energy and photovoltaics has abundant reserves so they are connected to the grid system on a large scale. However, because of natural energy's randomness, renewable energy power generation poses potential risks to energy production and grid security. By making short-term forecasts of renewable energy generation power, the uncertainty of energy generation can be reduced, and it is crucial to study renewable energy forecasting techniques. This paper proposes an integrated forecasting system for renewable energy sources. Firstly, ensemble empirical mode decomposition is used for data preprocessing, and stationarity analysis is used for modal identification; then, support vector regression optimized by sparrow search algorithm and statistical methods are combined to make forecast according to different characteristics of the series respectively; finally, the feasibility of this method in renewable energy time series prediction is verified by experiments. The experiments prove that the proposed model effectively improves the accuracy and prediction performance on ultra-short-term renewable energy forecasting; and it has good applicability and competitiveness with different forecasting scenarios and characteristics, which satisfy the actual forecasting requirements in terms of operational efficiency and accuracy, thus providing a technical basis for the effective utilization of renewable energy.

Non-invasive molecular imaging for precision diagnosis of metastatic lymph nodes: opportunities from preclinical to clinical applications.

Lymph node metastasis is an indicator of the invasiveness and aggressiveness of cancer. It is a vital prognostic factor in clinical staging of the disease and therapeutic decision-making. Patients with positive metastatic lymph nodes are likely to develop recurrent disease, distant metastasis, and succumb to death in the coming few years. Lymph node dissection and histological analysis are needed to detect whether regional lymph nodes have been infiltrated by cancer cells and determine the likely outcome of treatment and the patient's chances of survival. However, these procedures are invasive, and tissue biopsies are prone to sampling error. In recent years, advanced molecular imaging with novel imaging probes has provided new technologies that are contributing to comprehensive management of cancer, including non-invasive investigation of lymphatic drainage from tumors, identifying metastatic lymph nodes, and guiding surgeons to operate efficiently in patients with complex lesions. In this review, first, we outline the current status of different molecular imaging modalities applied for lymph node metastasis management. Second, we summarize the multi-functional imaging probes applied with the different imaging modalities as well as applications of cancer lymph node metastasis from preclinical studies to clinical translations. Third, we describe the limitations that must be considered in the field of molecular imaging for improved detection of lymph node metastasis. Finally, we propose future directions for molecular imaging technology that will allow more personalized treatment plans for patients with lymph node metastasis.

Invasive micropapillary breast carcinoma: A retrospective study on the clinical imaging features and pathologic findings.

Purpose: To describe the clinical imaging and pathological features of invasive micropapillary breast carcinoma (IMPC), including breast mammography, sonography, magnetic resonance imaging (MRI), and molecular imaging findings. Patients and methods: We retrospectively reviewed our institution's surgical pathology database and identified 65 patients with pathologically proven IMBC; 63/65 patients had available imaging results. Two radiologists retrospectively reviewed all imaging evaluations according to the Breast Imaging Reporting / Data System (BI-RADS) Lexicon. Clinical and histopathologic features, receptor statuses, and clinical follow-up data were recorded. Results: Sixty-three patients were admitted with palpable abnormalities; one patient's mammogram revealed no abnormality (3.3%, 1/32), whereas 31 had abnormal mammograms (31/32, 96.8%) demonstrating 37 lesions. Twenty-four had irregular, spiculated masses, 12 had microcalcifications, and 19 had architectural distortion. Sonography detected 69 masses (54 patients), characterized by irregular shapes (61/69, 88.4%), hypoechoic structures (50/69, 72.4%), angular or spiculated margins (38/69, 55.1%; 30/69, 43.4%), echogenic halo (8/69, 11.5%), and abnormal vascularity (52/69, 75.3%). MRI detected 68 lesions (52 patients); 59/68 (86.8%) appeared as masses with angular or spiculated margins (32/68, 47.1%; 35/68, 51.4%), 58 exhibited irregular or lobulated shapes (58/68, 89.7%), 29 displayed heterogeneous internal enhancement (29/68, 42.5%), and 64 demonstrated type II or III washout kinetic curves (37/68, 55%; 27/68, 40%). Breast molecular imaging showed mild-to-moderate radiotracer uptake in 15 focal areas among 13 patients. Thirty-two, 38, and 43 patients had abnormal lymph nodes identified mammographically, by breast sonography, and by MRI, respectively. Immunohistochemistry revealed high estrogen receptor (90.5%), high progesterone receptor (71.6%), and low HER-2 (26.4%) positivity. Conclusion: IMPC mammography, sonography, and MRI clinical imaging features highly suggest malignancy. Breast molecular imaging also contributed to the diagnosis. IMPC's invasiveness correlated well with regional lymph node metastasis. Radiologists and surgeons should be more attentive to these imaging findings and additional clinical and pathological IMPC features.

A Special Phenotype of Aconidial Aspergillus niger SH2 and Its Mechanism of Formation via CRISPRi.

The complex morphological structure of Aspergillus niger influences its production of proteins, metabolites, etc., making the genetic manipulation and clonal purification of this species increasingly difficult, especially in aconidial Aspergillus niger. In this study, we found that N-acetyl-D-glucosamine (GlcNAc) could induce the formation of spore-like propagules in the aconidial Aspergillus niger SH2 strain. The spore-like propagules possessed life activities such as drug resistance, genetic transformation, and germination. Transcriptomic analysis indicated that the spore-like propagules were resting conidia entering dormancy and becoming more tolerant to environmental stresses. The Dac1 gene and the metabolic pathway of GlcNAc converted to glycolysis are related to the formation of the spore-like propagules, as evidenced by the CRISPRi system, qPCR, and semi-quantitative RT-PCR. Moreover, a method based on the CRISPR-Cas9 tool to rapidly recycle screening tags and recover genes was suitable for Aspergillus niger SH2. To sum up, this suggests that the spore-like propagules are resting conidia and the mechanism of their formation is the metabolic pathway of GlcNAc converted to glycolysis, particularly the Dac1 gene. This study can improve our understanding of the critical factors involved in mechanisms of phenotypic change and provides a good model for researching phenotypic change in filamentous fungi.

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger.

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.

Application of Noninvasive Imaging to Combined Immune Checkpoint Inhibitors for Breast Cancer: Facts and Future.

With the application of mono-immunotherapy in cancer, particularly immune checkpoint inhibitors, improved outcomes have been achieved. However, there are several limitations to immunotherapy, such as a poor response to the drugs, immune resistance, and immune-related adverse events. In recent years, studies of preclinical animal models and clinical trials have demonstrated that immune checkpoint inhibitors for breast cancer can significantly prolong the overall survival and quality of patients' lives. Meanwhile, combined immune checkpoint inhibitor treatment has attracted researchers' attention and showed great potential in the comprehensive treatment of breast cancer patients. Additionally, noninvasive imaging enables physicians to predict response to combined immunotherapeutic drugs, achieve treatment efficacy, and lead to better clinical management. Herein, we review the background of combined immune checkpoint inhibitor therapy and summarize its targeted imaging as well as progress in noninvasive imaging aimed at evaluating therapeutic outcomes. Finally, we describe several factors that may influence the outcome of this combined immunotherapy, the future direction of medical imaging, and the potential application of artificial intelligence in breast cancer. With further development of noninvasive imaging for the guidance of combined immune checkpoint inhibitors, cures for this disease may be achieved.

Co-metabolic degradation of the antibiotic ciprofloxacin by the enriched bacterial consortium XG and its bacterial community composition.

Ciprofloxacin is a broad spectral and highly refractory antibiotic. It is an emerging pollutant. This study aimed to utilise co-metabolism as a means to degrade ciprofloxacin by a bacterial consortium. The stable bacterial consortium XG capable of efficiently degrading ciprofloxacin was successfully established through successive acclimation of indigenous microorganisms. The consortium XG was primarily consisted of Achromobacter, Bacillus, Lactococcus, Ochrobactrum, and Enterococcus as well as at least other five minor genera. A novel strain YJ17 with CIP-degrading ability was isolated from the consortium and identified as Ochrobactrum sp. The consortium XG utilised amino acids, carbohydrates, and carboxylic acids at a rate approximately 16.6-243-fold greater than the other carbon substrates, but only slow utilisation of ciprofloxacin as a sole carbon source. Ciprofloxacin can be co-metabolized along with many carbon sources, attaining degradation rates up to 63%. Glycyl-l-glutamic acid, d-cellobiose, and itaconic acid are among the substrates most favourable for co-metabolism. The metabolites of ciprofloxacin were identified by LC-QTOF-MS. Co-metabolic degradation of ciprofloxacin by consortium XG led to the removal of essential functional groups from parent compound, thus resulting in formation of metabolites with less bioactive potency. Finally, a possible biochemical pathway for the degradation of ciprofloxacin was proposed. Consortium XG possesses high potential for bioremediation of ciprofloxacin-contaminated environments in the presence of a co-substrate.

A Study on Prevalence and Characterization of Bacillus cereus in Ready-to-Eat Foods in China.

Bacillus cereus is widely distributed in different food products and can cause a variety of symptoms associated with food poisoning. Since ready-to-eat (RTE) foods are not commonly sterilized by heat treatment before consumption, B. cereus contamination may cause severe food safety problems. In this study, we investigated the prevalence of B. cereus in RTE food samples from different regions of China and evaluated the levels of bacterial contamination, antibiotic resistance, virulence gene distribution, and genetic polymorphisms of these isolates. Of the tested retail RTE foods, 35% were positive for B. cereus, with 39 and 83% of the isolated strains harboring the enterotoxin-encoding hblACD and nheABC gene clusters, respectively. The entFM gene was detected in all B. cereus strains. The cytK gene was present in 68% of isolates, but only 7% harbored the emetic toxin-encoding gene cesB. Antimicrobial susceptibility testing revealed that the majority of the isolates were resistant not only to most ß-lactam antibiotics, but also to rifamycin. Multilocus sequence typing (MLST) revealed that the 368 isolates belonged to 192 different sequence types (STs) including 93 new STs, the most prevalent of which was ST26. Collectively, our study indicates the prevalence, bacterial contamination levels, and biological characteristics of B. cereus isolated from RTE foods in China and demonstrates the potential hazards of B. cereus in RTE foods.

Novel phosphate-solubilising bacteria isolated from sewage sludge and the mechanism of phosphate solubilisation.

A great amount of insoluble phosphate in agricultural soils is not available for crops. Three strains of bacteria (Bacillus megaterium YLYP1, Pseudomonas prosekii YLYP6 and Pseudomonas sp. YLYP29) isolated from activated sludge and soil could efficiently solubilise tricalcium phosphate. In particular, the novel strain P. prosekii YLYP6 produced 716 mg L-1 of available phosphate within 6 days under the optimal culture conditions [20 °C, pH 7.9, inoculum size of 0.5% (v:v)] determined by response surface methodology. P. prosekii YLYP6 demonstrated efficient phosphate solubilisation in response to broad variations in pH (5-9) and temperature (15-35 °C). The phosphate solubilisation curves of the strains fit well with a first-order kinetic model (R2 > 0.939), with a half-life of 1.51-5.94 d for 5.0 g L-1 calcium phosphate. Continuous culture experiments combined with scanning electron microscopic observations and gas chromatography-mass spectrometry analysis revealed that 2,3-dimethylfumaric acid, gluconic and N-butyl-tert-butylamine that were produced by P. prosekii YLYP6 were responsible for phosphate solubilisation by supplying H+ ions and organic anions. Efficient phosphate solubilisation in actual soil by P. prosekii YLYP6 demonstrated the strong application potential to reduce the use of chemical P fertilisers and the resulting agricultural nonpoint pollution.

Genotypic variation and mechanism in uptake and translocation of perfluorooctanoic acid (PFOA) in lettuce (Lactuca sativa L.) cultivars grown in PFOA-polluted soils.

The cultivation of crop cultivars with low pollutant accumulation is an important strategy to reduce the potential health risks of food produced from polluted soils. In this study, we identified three loose-leaf lettuce cultivars with low accumulation of perfluorooctanoic acid (PFOA), a highly toxic and persistent organic pollutant. PFOA concentrations in the shoots of low-PFOA cultivars were 3.7-5.5-fold lower than those of high-PFOA cultivars. The identification of low-PFOA cultivars could contribute to ensuring food safety despite cultivation in highly polluted soils (1 mg/kg) based on the tolerable daily PFOA intake (1.5 µg/kg/d). We detected lower desorbing fractions of PFOA in rhizosphere soil, lower bioconcentration factors, and higher distribution in the cell walls and organelles of roots in low-PFOA cultivars, all of which are key factors in limiting PFOA uptake and translocation from soil to shoots, than in high-PFOA cultivars. This study reveals the mechanism of PFOA uptake from soil to crop and lays a foundation for establishing a cost-effective strategy to plant crops in polluted soil and reduce exposure risk due to persistent organic pollutants in crops.