Efficient Fermentative Production of d-Alanine and Other d-Amino Acids by Metabolically Engineered Corynebacterium glutamicum.

d-Amino acids (d-AAs) have wide applications in industries such as pharmaceutical, food, and cosmetics due to their unique properties. Currently, the production of d-AAs has relied on chemical synthesis or enzyme catalysts, and it is challenging to produce d-AAs via direct fermentation from glucose. We observed that Corynebacterium glutamicum exhibits a remarkable tolerance to high concentrations of d-Ala, a crucial characteristic for establishing a successful fermentation process. By optimizing meso-diaminopilmelate dehydrogenases in different C. glutamicum strains and successively deleting l-Ala biosynthetic pathways, we developed an efficient d-Ala fermentation system. The d-Ala titer was enhanced through systems metabolic engineering, which involved strengthening glucose assimilation and pyruvate supply, reducing the formation of organic acid byproducts, and attenuating the TCA cycle. During fermentation in a 5-L bioreactor, a significant accumulation of l-Ala was observed in the broth, which was subsequently diminished by introducing an l-amino acid deaminase. Ultimately, the engineered strain DA-11 produced 85 g/L d-Ala with a yield of 0.30 g/g glucose, accompanied by an optical purity exceeding 99%. The fermentation platform has the potential to be extended for the synthesis of other d-AAs, as demonstrated by the production of d-Val and d-Glu.

Clinical value of alveolar lavage supernatant specimens in the detection of the EGFR gene mutation in patients with non-small cell lung carcinoma.

Background: This study sought to compare the consistency of the epidermal growth factor receptor (EGFR) gene mutation detection results in the supernatant of alveolar lavage specimens to the tissue sample results, and the consistency of the blood EGFR gene mutation detection results to the tissue detection results. Methods: In total, 29 patients with non-small cell lung carcinoma (NSCLC) were selected, and their bronchoalveolar lavage fluid (BALF) was collected. The supernatant and precipitate were separated by centrifugation. Deoxyribonucleic acid (DNA) was extracted from the supernatant, and blood and tumor tissues were collected to detect patients' EGFR gene mutation status. Results: Of the 29 enrolled patients, 12 of the 23 tissue-biopsy patients (52.2%) were positive for EGFR mutations, 11 of the 28 blood-test patients (39.2%) were positive for EGFR mutations, and 13 of the 29 cases of the BALF-test patients (44.8%) were positive for EGFR mutations. The most common mutations were the exon 19 deletion mutation and the L858R point mutation. The EGFR gene mutation rate was higher in female, young, non-smoker, and stage IIIB patients (than stage IV patients), but the differences were not statistically significant (all P>0.05). Of the 29 NSCLC patients tested for the EGFR gene mutation, the BALF supernatant and blood results were the same for 27 patients (coincidence rate: 93.10%). Of the 23 of the 29 enrolled patients tested for the EGFR gene mutation, the BALF supernatant and tissue test results were the same for 21 patients (coincidence rate: 91.30%). Further, the blood-test and the tissue test results were the same for 20 patients (coincidence rate: 86.96%). Conclusions: The EGFR gene mutation rate was high in NSCLC patients. The coincidence rate of the EGFR gene mutation detection results between BALF supernatant and tumor tissues was slightly higher than that of the blood and tumor tissue EGFR gene mutation detection results.

Colloidal characteristics, emulsifying activities, and interfacial properties of α-lactalbumin-chitosan electrostatic complexes: effects of mass ratio and pH.

The effects of essential hydrophobic carrier properties of α-lactalbumin (α-LA) : chitosan (CHI, Mw 100 kDa, degree of deacetylation 75-85%) including mass ratio and pH on the characteristics, emulsifying activities, and interfacial properties of α-LA-CHI complexes were investigated. The interaction between α-LA and CHI was dominated by electrostatic interactions. Soluble α-LA-CHI complexes can be formed at both pH 6.0, and 6.5 at mass ratios of 1 : 1, 2 : 1, and 5 : 1, as shown by their optical appearance. UV-vis spectra demonstrated that the interaction between α-LA and CHI is highly CHI concentration, and pH-dependent. AFM graphs confirmed that α-LA-CHI complexes were spherical in shape and homogeneously dispersed in solution. The diameters of α-LA-CHI complexes were found to be around 100-250 nm, which was in accordance with DLS results, even though some aggregates were observed for the 1 : 1 α-LA-CHI complex. The emulsifying activity of α-LA was enhanced by complexation with CHI. Both the emulsifying activity index (EAI) and emulsifying stability index (ESI) of α-LA-CHI complexes were associated with mass ratio and pH values. The interfacial tension of α-LA-CHI complexes was negatively exponentially correlated with the EAI. The lower the interfacial tension of α-LA-CHI complexes on the oil-water interface, the higher the EAI. The results obtained from this study were complementary and provided insights into the interaction between α-LA and CHI, and suggested that α-LA-CHI complexes can be utilized as effective food ingredients for stabilizing, protecting, and delivering hydrophobic nutraceuticals.

Fabrication of curcumin-loaded pea protein-pectin ternary complex for the stabilization and delivery of ß­carotene emulsions.

There is an ever-increasing need to protect health-beneficial ß-carotene (BC) from degradation with novel ingredients. Natural antioxidant-loaded protein-polysaccharide ternary complex has great potential for BC emulsions stabilization. In this study, curcumin (CUR)-loaded pea protein isolate (PPI), and high methoxyl pectin (HMP) ternary complex (804.0 nm) was fabricated by a self-assembly approach for BC emulsions stabilization. Highest CUR loading amount (LA, 33.19 µg/mg) was obtained in CUR-PPI-HMP complex. Hydrophobic interaction and hydrogen bonding were the prime driving forces for ternary complex formation. XRD results showed that CUR was amorphous. BC emulsion with PPI-HMP and CUR-PPI-HMP possessed higher droplet sizes (357.8 and 360.2 nm) than that with PPI and CUR-PPI (325.6, and 313.5 nm). Excellent physical stability with PPI-HMP and CUR-PPI-HMP was observed. BC retention with CUR-PPI-HMP was highest exposure to UV light (76.15%, 8 h), or heat treatment at 25 (91.50%) and 50 °C (74.35%) for 30 days.

α-Lactalbumin and chitosan core-shell nanoparticles: resveratrol loading, protection, and antioxidant activity.

Resveratrol (RES)-loaded protein-polysaccharide nanoparticles were fabricated through simple electrostatic interactions with oppositely charged α-lactalbumin (ALA) and chitosan (CHI) with a mass ratio of 5 : 1 without the addition of NaCl at pH 6.5. The Z-average diameter and zeta-potential values of RES-ALA-CHI nanoparticles were 211.0 nm and 13.23 mV, respectively. Both TEM and AFM graphs confirmed that RES-ALA-CHI nanoparticles had a spherical shape, and were dispersed homogeneously at the nanoscale. The encapsulation efficiency (EE) and loading amount (LA) of RES in RES-ALA-CHI nanoparticles were 58.86% and 196.2 µg mg-1, respectively, in the presence of 400 µg mL-1 RES. XRD results confirmed that RES was in amorphous form in ALA-CHI nanoparticles. The interaction between RES and ALA-CHI nanoparticles was mainly driven by hydrophobic interaction and hydrogen bonding. Compared to RES (free), the UV light and heat stability, in vitro bioaccessibility, and antioxidant activity of RES in RES-ALA-CHI nanoparticles were pronouncedly enhanced. The information provided in this study should be of interest to the food industry to fabricate robust nanoscale delivery systems with ALA-CHI nanoparticles for RES and other hydrophobic bioactive compounds.

Fabrication of Resveratrol-Loaded Whey Protein-Dextran Colloidal Complex for the Stabilization and Delivery of ß-Carotene Emulsions.

The effects of resveratrol (RES)-loaded whey protein isolate (WPI)-dextran nanocomplex on the physicochemical stability of ß-carotene (BC) emulsions were evaluated. WPI-dextran was prepared by Maillard-based glycation and confirmed with gel electrophoresis and OPA assay. WPI-RES and WPI-dextran-RES nanoparticles were prepared with a simple nanocomplexation protocol. Fluorescence spectra indicated that hydrophobic interaction was the main driving force for the WPI-dextran-RES nanocomplex. Spherical and uniformly dispersed structures as well as nanoscale Z-average size (<100 nm) were confirmed for WPI-RES and WPI-dextran-RES nanocomplex with DLS and TEM. The Z-average diameter of emulsions with WPI-dextran conjugate was remarkably lower than that with WPI. Environmental stress (ionic strength, heat, and pH) and storage stability were pronouncedly improved. The chemical stability of BC with WPI-dextran-RES and WPI-RES was also remarkably enhanced when exposed to UV light and thermal treatment. The advantages of the WPI-dextran-RES colloidal complex may provide a better alternative to effectively protect and deliver hydrophobic nutraceuticals.

Enhanced pH and thermal stability, solubility and antioxidant activity of resveratrol by nanocomplexation with α-lactalbumin.

The health-promoting benefits of resveratrol (RES) have attracted significant attention. Poor water solubility and chemical stability, however, hindered its application. In this study, α-lactalbumin (ALA)-RES nanoparticles were prepared by a simple nanocomplexation protocol. The particle sizes of ALA and the ALA-RES complex were 87.8, and 95.3 nm, respectively. AFM confirmed that both nanoparticles were spherical. XRD results confirmed that RES in ALA nanoparticles was amorphous. Fluorescence spectroscopy and FTIR showed that the ALA-RES nanocomplex was formed mainly by hydrophobic interactions. The water solubility increased by 32 times, compared to that of free RES. ALA nanocomplexation also appreciably improved the chemical stability of RES under all storage conditions, especially at pH 8.0 and high temperature. The ALA-RES nanocomplex showed significantly higher in vitro antioxidant activity than free RES. The results showed that the simple ALA-RES nanocomplex has the potential to be used as an effective antioxidant. The information obtained may enable the expanded application of ALA as an effective nanoscale carrier for delivering RES or other lipophilic nutraceuticals in the functional food, biomedical, and pharmaceutical products.

Oxidative stability and in vitro digestion of menhaden oil emulsions with whey protein: Effects of EGCG conjugation and interfacial cross-linking.

The goal of this study was to improve the chemical stability of menhaden oil and control the lipolysis in emulsions with whey protein during in vitro digestion through EGCG conjugation and genipin-mediated interfacial cross-linking (CL). WPI-EGCG conjugate was successfully synthesized, confirmed by SDS-PAGE, ESI-MS, and phenolic group quantifications (125.3 mg/g), and characterized with far UV CD and ATR-FTIR. Emulsion particle diameter with WPI-EGCG is lower than with WPI. Compared to the native emulsion, WPI CL increased particle diameter and physical stability. Higher oxidative stability was observed for emulsions stabilized with WPI-EGCG conjugate than that with interfacial cross-linking due to the great antioxidant activity. Whereas, WPI CL is more effective than WPI-EGCG conjugate in hindering the rate and extent of lipolysis. The combination of EGCG conjugation and interfacial CL showed both the highest protection of menhaden oil against degradation and highest inhibition on the rate and extent of lipolysis of menhaden oil.

Improved chemical stability and cellular antioxidant activity of resveratrol in zein nanoparticle with bovine serum albumin-caffeic acid conjugate.

In this study, bovine serum albumin (BSA)-caffeic acid (CA) conjugate was prepared with free radical-induced grafting method. The CA to BSA ratio of the conjugate was 115.7 mg/g. In vitro antioxidant activity assays suggested that BSA-CA conjugates had stronger antioxidant activity than BSA. Resveratrol-loaded zein encapsulated with BSA and BSA-CA conjugate core-shell nanoparticles were prepared with antisolvent method. Particle sizes were 206.3 nm, and 217.2 nm for BSA and BSA-CA, respectively. The encapsulation efficiencies (EEs) were 85.3% and 86.5% for zein-BSA and zein-BSA-CA nanoparticles, respectively. SEM results indicated that both nanoparticles were spherical with mean diameter approximately 200 nm and smooth surfaces. Both thermal and UV light stability of resveratrol was significantly improved after nanoencapsulation. BSA-CA conjugate showed remarkably greater protection than BSA against resveratrol degradation. Cellular antioxidant activity (CAA) study confirmed that resveratrol in both zein-BSA and zein-BSA-CA nanoparticles had significant higher antioxidant activities than resveratrol alone.

Intratumoral injection of dendritic cells in combination with local hyperthermia induces systemic antitumor effect in patients with advanced melanoma.

Dendritic cells (DC) are potent antigen-presenting cells that can present tumor antigens chaperoned by heat shock proteins (HSPs), while local hyperthermia (LHT) can increase the expression of HSPs. In this study, we determine if intratumoral injection of immature DC after LHT (LHT+IT-DC) induces systemic antitumor immunity in patients with advanced melanoma, and investigate the potential immunological mechanisms involved in the treatments. Patients were randomly assigned to intratumoral administration of autologous immature DC triweekly, with (LHT+IT-DC, arm A, n = 9) or without (IT-DC, arm B, n = 9) LHT. Our results showed that there were no grade 3/4 toxicities. The time to progress (TTP) of arm A was 5 months, significantly longer than that in arm B (2 months, p < 0.05). However, the overall survival time had no statistical difference (13 months vs. 6 months, p > 0.05) between the 2 groups. Our ELISPOT assay showed a significantly increased melanoma-specific IFN-gamma production in arm A, suggesting that LHT+IT-DC was more effective in the induction of cytotoxic T lymphocytes (CTL) than IT-DC alone. Furthermore, we detected an increased HSPs expression 4 hr after the first LHT, an enhanced Th1/Th2 chemokines production 24 hr after the first LHT+IT-DC treatment, a promoted migration of DC to afferent lymph nodes, and a decreased infiltration of regulatory T cells (CD4(+)CD25(+)) and an increased infiltration of active CTL (CD8(+)CD28(+)) 48 hr after the third DC injection in arm A patients. Therefore, LHT+IT-DC can induce effective specific antitumor immunity and facilitate a Th1-polarized immune response in patients with advanced melanoma.

Expression profile of cancer-testis genes in 121 human colorectal cancer tissue and adjacent normal tissue.

PURPOSE: Among tumor antigens identified to date, cancer-testis (CT) antigens, which are coded by CT genes, are identified as a group of highly attractive targets for cancer vaccines. This study is the first to analyze the mRNA expression and possible correlation with pathologic characteristics of multiple CT genes in a large cohort of colorectal cancer (CRC) patients. EXPERIMENTAL DESIGN: The expression of 10 individual CT genes in 121 CRC and adjacent tissues were analyzed by RT-PCR method. The presence of autologous antibodies against NY-ESO-1 was examined in serum samples by ELISA. To confirm the protein expression, immunohistochemistry was done for detecting the NY-ESO-1 antigen in mRNA-positive CRC tissues. RESULTS: The CT genes were detected with various frequencies in CRC tissue, SCP-1, 1.7%; SSX-2, 2.5%; SSX-4, 2.5%; SSX-1, 5.0%; CT10, 6.6%; NY-ESO-1, 9.9%; MAGE-1, 11.6%; LAGE-1, 15.7%; MAGE-4, 22.3%; and MAGE-3, 27.3%. In 56.2% of tumor tissues examined in this study, at least one CT gene was detected. In contrast, no CT gene expression was found in cancer adjacent tissues. Among 10 CT genes investigated, NY-ESO-1 and LAGE-1 are of particular interest because their mRNA expression in CRC was rarely reported before. In our study, NY-ESO-1 mRNA was found to express in 9.9% of the samples, and also correlated significantly with stages (P = 0.041) and local lymph node metastasis (P = 0.002). In addition, we also identified one NY-ESO-1 antibody-positive serum sample. MAGE-4 mRNA was expressed at a high frequency in tumor tissues with vessel emboli samples (P = 0.025). CONCLUSIONS: These results suggested that CT genes, especially NY-ESO-1 and LAGE-1, do express in CRC. More than 50% of the CRC patients in this study express at least one CT gene, making them eligible for CT vaccination. NY-ESO-1 gene may serve as a marker for local metastasis and advanced disease. MAGE-4 gene is significantly associated with the vessel emboli.

Dominant B cell epitope from NY-ESO-1 recognized by sera from a wide spectrum of cancer patients: implications as a potential biomarker.

Monitoring the spontaneous antibody (Ab) response against a panel of relevant tumor-associated antigens (TAA) in cancer patients may provide useful information regarding the clinical status of cancer. However, current Ab detection approaches require the purification of recombinant proteins, which is often difficult to achieve. In order to bypass the purification of recombinant proteins, we identified a dominant B-cell epitope from a shared tumor antigen NY-ESO-1. A synthetic peptide of the epitope, ESO:1-40, was as sensitive as the recombinant protein for detecting Ab against NY-ESO-1 in most patients. NY-ESO-1 specific Ab present in the sera of patients with melanoma, prostate cancer, nonsmall cell lung cancer, esophageal cancer, gastric cancer and hepatocellular carcinoma reacted with the dominant peptide at a similar frequency as the recombinant protein. To our knowledge, ESO:1-40 is the first peptide epitope recognized by sera from a wide spectrum of cancer patients but not healthy donors. This simple and straightforward approach may allow the investigation of the clinical significance of spontaneous Ab responses against multiple TAA and their correlation with the clinical course of malignant diseases in the future.

[Expression of multiple cancer-testis antigen genes in non-small cell lung cancer treated by chemotherapy prior surgery].

OBJECTIVE: To investigate the possibility of utilizing cancer-testi (CT) antigens as targets for immunotherapy of non-small cell lung cancer (NSCLC) with vaccines. METHODS: Tissues from 51 NSCLC patients who had chemotherapy prior surgery were assayed for the expression of 11 different CT antigens by RT-PCR. RESULTS: Of the 11 CT antigens analyzed, MAGE-3 was found to be expressed most frequently in NSCLC tissues and CT7 the least frequently. The frequencies of CT antigen expression was: MAGE-3 (38%), NY-ESO-1 (21%), CT10 (17%), LAGE-1 (15%), MAGE-4 (13%), SCP-1, SSX1 and SSX4 (12%), MAGE-1 (10%), SSX2 (6%), and CT7 (2%). Among these cases, 34 (67%) expressed at least one CT antigen gene. 13 of the 17 cases in which no CT antigen expression was found in the tumor tissue, the tumors were classified as at the stage I. MAGE-3 and CT10 were found to be expressed more frequently in tissues from patients with late stage diseases while SCP-1 was found more frequently in earlier stages of NSCLC. CT expression was more frequently found in squamous cell carcinoma than in adenocarcinoma. CONCLUSIONS: (1) Cancer vaccines with CT antigens including MAGE-3, NY-ESO-1, LAGE-1, etc, are suitable for immunotherapy of NSCLC after chemotherapy and surgery. (2) NSCLC patients at different stages of disease may be treated with vaccines of different CT antigen composition. (3) CT antigen vaccines are most attractive for patients with late stage NSCLC and/or squamous cell carcinoma of NSCLC.

[Expression of NY-ESO-1/LAGE-1 genes in hepatocellular carcinoma and autologous humoral responses induced thereby].

OBJECTIVE: To investigate the potential of utilizing NY-ESO-1/LAGE-1 antigens in hepatocellular carcinoma (HCC) vaccines. METHODS: RT-PCR method was used to detect the expression of NY-ESO-1/LAGE-1 mRNA in the cancerous tissues and adjacent tissues resected from 34 patients with HCC. ELISA assay was adopted to analyze the NY-ESO-1 specific antibodies in 37 serum samples of HCC patients, 1 positive control sample, and 8 samples of normal persons. RESULTS: Nine (26.5%) out of the 34 HCC samples were NY-ESO-1 mRNA positive, while 12 (35.3%) were LAGE-1 positive. Among them, seven HCC samples expressed both genes, and 14 (41.6%) expressed at least one of the genes. Among the 37 serum samples tested six contained high titer of anti-NY-ESO-1 antibodies. Five of the samples were from stage III or later stage HCC patients; one was from a stage II patient. CONCLUSION: NY-ESO-1/LAGE-1 mRNA is expressed in a high frequency in HCC tissue samples, and induces autologous humoral responses in HCC patients. Both of the antigens can be considered as candidates for HCC vaccines.