Applied Biochemistry and Biotechnology最新文献

The oceanic actinobacteria have strong potential to secrete novel enzymes with unique properties useful for biotechnological applications. The Nocardiopsis dassonvillei strain VCS-4, associated with seaweed Caulerpa scalpeliformis, was a halo-alkaline protease producer. Further investigation focuses on medium optimization and the use of agro-industrial waste for economically feasible, high-yield protease production. A total of 12 experimental runs were designed using Minitab-20 software and Placket-Burman design. Among the 7 physicochemical parameters analyzed, incubation time and gelatin were detected as significant factors responsible for higher protease production. Incubation time and gelatin were further analyzed using OVATs. Optimal protease production was achieved with 2% gelatin, 0.1% yeast extract, 0.1% bacteriological peptone, 7% NaCl, pH 8, 5% inoculum, and a 7-day incubation period, resulting in a maximum protease activity (Pmax) of 363.97 U/mL, generation time of 11.9 h, specific growth rate of 0.161 g/mL/h, and protease productivity (Qp) of 61.65 U/mL/h. Moreover, utilizing groundnut cake as an agro-industrial waste led to enhanced production parameters: Pmax of 408.42 U/mL, generation time of 9.74 h, specific growth rate of 0.361 g/mL/h, and Qp of 68.07 U/mL/h. The immobilization of crude protease was achieved using Seralite SRC 120 as a support matrix resulting in 470.38 U/g immobilization, 88.20% immobilization yield, and 28.90% recovery activity. Characterization of both crude and immobilized proteases revealed optimal activity at pH 10 and 70 °C. Immobilization enhanced the shelf-life, reusability, and stability of VCS-4 protease under extreme conditions.

The biosynthesis of metal oxide nanoparticles using leaf extract of medicinal plants is a promising substitute for the traditional chemical method. This work aimed to synthesize zinc oxide nanoparticles using a green approach from local "Dholkolmi" (Ipomoea carnea) leaf extract which is a medicinal plant growing outside the roads of different regions of Bangladesh. The biosynthesized zinc oxide nanoparticles (ZnONPs) were characterized using ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, particle size analyzer, zeta-potential, scanning electron microscopy-energy dispersive spectroscopy, field emission scanning electron microscopy, and transmission electron microscopy. The results of UV-visible spectrophotometers observed an absorption peak at 373 nm wavelength, which confirmed the synthesis of ZnONPs in the solution. ZnONP sizes determined by XRD, DLS, and TEM are approximately ~37 nm, 105.61 nm, and 19.66 nm, respectively. ZnONPs were present because of the strong oxygen and zinc signals in the EDX profile. Additionally, this research assessed the antifungal activity of the biosynthesized ZnONPs and as well as folicur-incorporated ZnONPs against Rhizoctonia solani by the poison bait technique. According to the result of this study, ZnONPs synthesized from Ipomoea carnea leaf extract showed no promising result against Rhizoctonia solani mycelial growth reduction. But folicur-incorporated ZnONPs revealed a significant finding with a maximum 100% inhibition of mycelial growth at 1:1 and 3:1 ratio of ZnONPs with folicur fungicide under in vitro conditions. In the net house experiment, folicur-incorporated ZnONPs at a 1:1 ratio of ZnONPs with folicur showed considerable disease inhibition (26.96% RLH) as compared to disease control (52.83% RLH). In the case of rainfed transplanted Aus (March-June), the highest percentage of RLH was recorded in disease control (64.61%), and the lowest RLH was found in folicur (24.79%) followed by a 1:1 ratio of ZnONPs with folicur (32.10%) in field condition. On the other hand, the highest percentage of RLH was recorded in disease control (65.31%) and the lowest RLH was found in folicur (18.14%) followed by a 1:1 ratio of ZnONPs with folicur (21.39%) in rainfed transplanted Aman (July-November) season. The findings of the in vitro and in vivo studies provided evidence that ZnONPs and folicur had a strong synergistic antifungal impact and may be employed as a possible rice sheath blight disease management agent.

Non-invasive monitoring of glucose levels in tears and saliva is crucial for diagnosing and predicting various illnesses, such as diabetic nephropathy. However, the capability of the current glucose detection methods to identify small amounts of glucose with a high sensitivity remains a significant obstacle. This study proposes a simple, visual technique for sensitively detecting glucose levels from tears and saliva using glucose oxidase (GOx) to catalyze glucose and pistol-like DNAzyme (PLDz) to enhance the signal. In particular, the β-D-glucose present in the samples serves as the initial molecule that GOx identifies and catalyzes to generate gluconic acid and hydrogen peroxide (H₂O₂). The H₂O₂ induces the self-cleavage of PLDz, activating the "part b" sequence. This activation initiates catalytic hairpin assembly (CHA) and releases the DNAzyme section in the H1 probe. The DNAzyme acts as a peroxidase analog, facilitating the catalysis of the 3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide (H₂O₂) system and resulting in color changes. The proposed method exhibits a broad detection range of six orders of magnitude and a low limit of 0.32 μM for glucose detection. Furthermore, the proposed method was highly effective in detecting glucose in saliva and tears, suggesting that it could potentially diagnose hyperglycemia-related disorders in clinical environments.

An upregulated histocompatibility minor 13 (HM13) has been studied in various tumors, yet the exact mechanism of HM13 in non-small cell lung cancer (NSCLC) is unclear. In view of same, the present study investigates crucial role and action mechanism of HM13 in human NSCLC. HM13 expression was higher in NSCLC tissue and cells through the Western blotting technique along with qRT-PCR. As per data from The Cancer Genome Atlas (TCGA), NSCLC patients having high HM13 expression show lower overall survival. 5-ethynyl-2-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and transwell tests were assessed for NSCLC cell growth, and invasion, and we found that silencing of HM13 inhibited the NSCLC cell proliferation, invasion. Additionally, to investigate the effects of HM13 on THP-1 macrophage polarization, a co-culture model of NSCLC and THP-1 macrophages were used. The CD206 + macrophages were examined using flow cytometry. As the markers of M2 macrophage, the mRNA levels of IL-10 and TGF-β of THP-1 cells were also detected by qRT-PCR. Knockdown of HM13 could inhibit the M2 polarization. Further experiments demonstrated that downregulated HM13 could inhibit the JAK2/STAT3 signaling pathway. RO8191 (activator of JAK/STAT3 pathway) influenced the invasion, proliferation, and expression of JAK2/STAT3 signaling pathway and Epithelial-mesenchymal transition (EMT) markers induced by HM13 silencing. HM13 knockdown also inhibited the tumor growth in vivo by xenograft nude mouse model. By inhibiting JAK2/STAT3 signaling pathway, HM13 knockdown inhibited the NSCLC cell proliferation, metastasis tumor growth, and tumor-associated macrophage M2 polarization. In NSCLC, HM13 could be a therapeutic target to treat the NSCLC.

Osteoarthritis (OA) is the most common degenerative joint disease affecting millions of people worldwide. Garlic-derived exosomes (GDEs) are nanoparticles extracted from garlic that exhibit anti-inflammatory effects on other diseases, but the effect of GDEs on OA has not been elucidated. In this study, GDEs were extracted and characterized. Chondrocytes were treated with IL-1β and incubated with GDEs in vitro, and the expression of cartilage matrix components (collagen II and aggrecan) and matrix degrading enzymes (MMP3 and MMP9) was evaluated via Western blotting. Changes in the MAPK pathway was also examined using Western blotting. The transcriptomic changes associated with GDE intervention were evaluated using high-throughput RNA-seq method. In vivo, we used anterior cruciate ligament transection (ACLT) combined with destabilization of the medial meniscus (DMM) surgery to establish a mouse OA model, and GDEs was intraarticularly injected into the joint cavity. The therapeutic effect of GDE was evaluated by behavioral and histopathological analysis. The results showed that IL-1β treatment inhibited the expression of collagen II and aggrecan, and upregulated the expression of MMP3 and MMP9, while GDE intervention alleviated these effects. GDEs also inhibited the phosphorylation of ERK, JNK, and P38. In vivo, GDE alleviated the sensitivity to heat stimulation and altered walking gait in a mouse OA model. Histopathological analysis indicated that GDE intervention ameliorated joint destruction in the knee joint without obvious toxicity. The results proved that GDEs alleviated the progression of OA in vitro and in vivo, and may be a potential disease-modifying drug for OA.

Colorectal cancer (CRC) is one of the most common malignancies all over the world. Increasing evidence has revealed that circular RNAs (circRNAs) are involved in the progression of CRC. In this study, we aimed to investigate the role and underlying mechanism of circ_0006174 in the development and radiosensitivity of CRC. Circ_0006174, microRNA-940 (miR-940), and insulin-like growth factor 1 receptor (IGF1R) expression levels were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). The radiosensitivity of cells also was assessed using colony formation assay. Besides, cell proliferation, apoptosis, migration, and invasion were detected by cell counting kit-8 (CCK-8), flow cytometry, and transwell assays. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the relationship between miR-940 and circ_0006174 or IGF1R. IGF1R protein level was examined using western blot. A xenograft tumor model was used to verify the function of circ_0006174 in CRC tumor growth in vivo. Circ_0006174 and IGF1R levels were elevated and miR-940 expression was decreased in CRC tissues and cells. Circ_0006174 knockdown enhanced the radiosensitivity of CRC cells by regulating cell proliferation, apoptosis, migration, and invasion in vitro. In mechanism, circ_0006174 served as a sponge for miR-940 to upregulate IGF1R expression. Moreover, circ_0006174 silencing suppressed CRC growth in vivo. Circ_0006174 boosts radioresistance of CRC cells at least partly through upregulating IGF1R expression by sponging miR-940, providing a novel theoretical basis for CRC therapy.

In this study, we used Fe-MIL-101 nanozyme to convert lactose into lactitol, and it was proved that Fe-MIL-101 nanozyme has lactase-like activity. Due to the potential health effects of nanomaterials, we evaluated the cytotoxicity of Fe-MIL-101 nanozyme. To reduce the potential toxicity of the nanozyme, we applied centrifugation and membrane filtration. When the membrane aperture size was 100 nm, the residual content of Fe-MIL-101 nanozyme was 14.09 μg/mL. The residual content of Fe-MIL-101 nanozyme was reduced by optimizing time, temperature, and Fe-MIL-101 nanozyme-to-substrate ratio. It was showed that the concentration of Fe was 38.47 mg/kg and the concentration of H₂BDC was 0 mg/kg under optimized conditions (110℃, 2 h of reaction and the ratio of Fe-MIL-101 nanozyme to substrate is 1:20). The result met the national standard of China. Experiments measuring cytotoxicity, oxidative stress, and cell membrane damage revealed that less than 20 μg/mL Fe-MIL-101 nanozyme had no significant cytotoxicity. Our study findings showed that Fe-MIL-101 nanozyme reduced lactose content in milk.

The squid, Sepioteuthis lessoniana, is a remarkable fishery product which is exported by many nations for use in industrial production or human consumption. This study focused on the synthesis of silver nanoparticles (AgNPs) from squid ink (SI) and its wide range of applications. The formation of the nanoparticles was confirmed through UV-Visible spectroscopy, FTIR, XRD, SEM with EDX, DLS, and zeta potential analysis. The results showed a strong absorbance peak at 407 nm, the presence of various functional groups, a nanocrystalline structure with a crystalline size of 17.56 nm, spherical-shaped particles with an average size of 76 nm, and the presence of the highest % mass of Ag and uniformly dispersed particles, respectively. The bioactivity of the synthesized squid ink silver nanoparticles was analyzed through antibacterial, antioxidant, anticancer, and toxicity studies. The dye degradation assay was also analyzed as a means of wastewater treatment for different industrial dyes. The antibacterial activity showed the highest zone of inhibition of 24 mm at a concentration of 100 μg/ml against Escherichia coli, followed by other tested strains. The nitric oxide radical scavenging assay showed the highest antioxidant activity (92%) at a concentration of 100 μg/ml. The cytotoxic ability of SI-AgNPs against the MDA-MB-231 breast cancer cell line revealed an IC₅₀ value of 4.52 μg/ml. The toxicity study revealed a dose and time-dependent activity with the LC₅₀ value of 5.090 and 3.303 mg/ml for 24 and 48 h, respectively. The successful degradation of dyes by SI-AgNPs is attributed to the cooperative action of the electron relay system with Ag as a catalyst and SI as a catalytic support. These findings indicate that SI-AgNPs are a novel potential product that should be further studied to improve its pharmacological, biomedical, and environmental applications.

IGFLR1 is a novel biomarker, and some evidences suggested that is involved in the immune microenvironment of CRC. Here, we explored the expression of IGFLR1 and its association with the prognosis as well as immune cell infiltration in CRC, with the aim to provide a basis for further studies on IGFLR1. Immunohistochemical staining for IGFLR1, TIM-3, FOXP3, CD4, CD8, and PD-1 was performed in eligible tissues to analyze the expression of IGFLR1 and its association with prognosis and immune cell infiltration. Then, we screened colon cancer samples from TCGA and grouped patients according to IGFLR1-related genes. We also evaluated the co-expression and immune-related pathways of IGFLR1 to identify the potential mechanism of it in CRC. When P < 0.05, the results were considered statistically significant. IGFLR1 and IGFLR1-related genes were associated with the prognosis and immune cell infiltration (P < 0.05). In stage II and III CRC tissue and normal tissue, we found (1) IGFLR1 was expressed in both the cell membrane and cytoplasm and which was differentially expressed between cancer tissue and normal tissue. IGFLR1 expression was associated with the expression of FOXP3, CD8, and gender but was not associated with microsatellite instability. (2) IGFLR1 was an independent prognostic factor and patients with high IGFLR1 had a better prognosis. (3) A model including IGFLR1, FOXP3, PD-1, and CD4 showed good prognostic stratification ability. (4) There was a significant interaction between IGFLR1 and GATA3, and IGFLR1 had a significant co-expression with related factors in the INFR pathway. IGFLR1 has emerged as a new molecule related to disease prognosis and immune cell infiltration in CRC patients and showed a good ability to predict the prognosis of patients.