Optimization of crystal violet technique for enhanced fingerprint detection on various surfaces.

The crystal violet (CV) staining technique represents a prevalent approach for the development of latent fingerprints, especially on adhesive tape surfaces. Nevertheless, the technique necessitates intricate formulations to augment its performance. In this investigation, an optimized CV staining protocol was developed, characterized by the absence of residual dye on the target substrates and the capability of facilitating fingerprint visualization under ambient light conditions. Four donors, comprising two males and two females, deposited natural fingerprints on various substrates, including glass microscope slides, aluminum foil, and 115 g glossy coated paper, without any specific guidelines. Fingerprints developed using cyanoacrylate fuming served as benchmarks and were contrasted with those generated through alternative methods: CV, ardrox, rhodamine 6G, powdering, and the optimized CV staining protocol. The fingerprint development experiment was replicated at seven distinct time intervals, encompassing 1 day, 1 week, 1, 3, 6, 9, and 12 months, resulting in a dataset of 420 fingerprints. The evaluation of fingerprint identifiability employed a scoring system established by the Home Office Centre for Applied Science and Technology. The results indicated that the optimized CV staining technique demonstrated superior performance, boasting a 92.9% rate of identifiable fingerprint development in contrast to other employed methodologies. Consequently, this optimized CV staining approach is recommended as an efficient, rapid, and straightforward critical dyeing method, applicable to a wide array of substrates in forensic investigations.

Evaluating Enterococcus faecium9 N-2 as a probiotic candidate from traditional village white cheese.

In this study, various functional and probiotic attributes of the Enterococcus faecium 9 N-2 strain isolated from village-style white cheese were characterized, while also assessing its safety. To achieve this, we conducted an in vitro analysis of several key probiotic properties exhibited by the 9 N-2 strain. Notably, this strain demonstrated robust resilience to low pH, high bile salt concentrations, lysozyme, pepsin, pancreatin, and phenol. Furthermore, this strain displayed exceptional auto-aggregation capabilities and moderate co-aggregation tendencies when interacting with Escherichia coli. The cell-free supernatant derived from strain 9 N-2 exhibited significant antimicrobial activity against the tested pathogens. The strain exhibited resistance to gentamicin, meropenem, and bacitracin, while remaining susceptible to vancomycin and various other antibiotics. Furthermore, it was found that E. faecium 9 N-2 possessed the capacity to produce the phytase enzyme. When all the results of this study are evaluated, it is thought that 9 N-2 strain has superior probiotic properties, and therefore it can be used as probiotic in food, medicine, and animal feed in the future. In addition, further in vivo tests should be performed to fully understand its effects and mechanisms of action and to confirm its safety and probiotic effects. Further research and clinical trials are also needed to identify new strains with potential probiotic properties.

Development of light and pH-dual responsive self-quenching theranostic SPION to make EGFR overexpressing micro tumors glow and destroy.

Drug resistant and undetectable tumors easily escape treatment leading metastases and/or recurrence of the lethal disease. Therefore, it is vital to diagnose and destroy micro tumors using simple yet novel approaches. Here, we present fluorescence-based detection and light-based destruction of cancer cells that are known to be resistant to standard therapies. We developed a superparamagnetic iron oxide nanoparticle (SPION)-based theranostic agent that is composed of self-quenching light activated photosensitizer (BPD) and EGFR targeting ligand (Anti-EGFR ScFv or GE11 peptide). Photosensitizer (BPD) was immobilized to PEG-PEI modified SPION with acid-labile linker. Prior to stimulation of the theranostic system by light its accumulation within cancer cells is vital since BPD phototoxicity and fluorescence is activated by lysosomal proteolysis. As BPD is cleaved, the system switches from off to on position which triggers imaging and therapy. Targeting, therapeutic and diagnostic features of the theranostic system were evaluated in high and moderate level EGFR expressing pancreatic cancer cell lines. Our results indicate that the system distinguishes high and moderate EGFR expression levels and yields up to 4.3-fold increase in intracellular fluorescence intensity. Amplification of fluorescence signal was as low as 1.3-fold in the moderate or no EGFR expressing cell lines. Anti-EGFR ScFv targeted SPION caused nearly 2-fold higher cell death via apoptosis in high EGFR expressing Panc-1 cell line. The developed system, possessing advanced targeting, enhanced imaging and effective therapeutic features, is a promising candidate for multi-mode detection and destruction of residual drug-resistant cancer cells.

Biochemical characterization and detection of antitumor activity of l-asparaginase from thermophilic Geobacillus kaustophilus DSM 7263T.

L-asparaginases, which are oncolytic enzymes, have been used in clinical applications for many years. These enzymes are also important in food processing industry due to their potential in acrylamide-mitigation. In this study, the gene for l-asparaginase (GkASN) from a thermophilic bacterium, Geobacillus kaustophilus, was cloned and expressed in E. coli Rosetta™2 (DE3) cells utilizing the pET-22b(+) vector. The 6xHis-tag attached enzyme was purified and analyzed both biochemically and structurally. The molecular mass of GkASN was determined as ∼36 kDa by SDS-PAGE, Western Blotting, and MALDI-TOF MS analyses. Optimum temperature and pH for the enzyme was determined as 55 °C and 8.5, respectively. The enzyme retained 89% of its thermal stability at 37 °C and 75% at 55 °C after 6 h of incubation. The enzyme activity was inhibited in the presence of Cu2+, Fe3+, Zn2+, and EDTA, while the activity was enhanced in the presence of Mn2+, Mg2+, and thiol group protective agents such as 2-mercaptoethanol and DTT. The structural modeling analysis demonstrated that the catalytic residues of the enzyme were partially similar to other asparaginases. The therapeutic potential of GkASN was tested on hepatocellular carcinoma cells, a solid cancer type with high mortality rate and rapidly increasing incidence in recent years. We showed that the GkASN-induced asparagine deficiency effectively reduced the metastatic synergy in HCC SNU387 cells on a xCELLigence system with differentiated epithelial Hep3B and poorly differentiated metastatic mesenchymal HCC SNU387 cells.

Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope.

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263 T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43 kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50 °C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22 mM and 417.1 min-1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

Highly-stable Madurella mycetomatis laccase immobilized in silica-coated ZIF-8 nanocomposites for environmentally friendly cotton bleaching process.

In this study, a laccase from Madurella mycetomatis (MmLac) was produced heterologously in Pichia pastoris; the initial immobilization in a metal-organic framework (MOF) (MmLac/ZIF-8) was achieved using zinc nitrate and 2-methylimidazole. Due to the instability of MmLac/ZIF-8 in an acidic medium, a silica layer was created on the surface of MmLac/MOF-8. The immobilized laccase composite (silica@MmLac/ZIF-8) obtained was further treated with glutaraldehyde (silica@Glu-MmLac/ZIF-8) to increase stability of composite. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of MmLac and to investigate the morphology of the immobilized laccase samples. The MmLac samples were also characterised in terms of optimum pH, temperature and thermal stability. The optimum pH of all the MmLac samples was determined to be 4.0. The free MmLac showed maximum activity at 55 °C, whereas both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were maximumly active at 65 °C. The silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were 9.3- and 11.8-fold higher in stability, respectively, than the free MmLac at 65 °C. Furthermore, both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 showed a higher bleaching performance than free MmLac on cotton woven fabric. According to these results, silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 may be promising candidates for biocatalysts in laccase-based biotechnological applications.

From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18.

BACKGROUND: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. OBJECTIVES: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. METHODS: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. RESULTS: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. CONCLUSION: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent.

Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these "green catalysis" enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0-6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50-60 °C); and is stable for long-term storage at - 20 °C and - 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies.

Investigation of Diagnostic Proteins by 2D Electrophoresis in Major Depression Model Induced by Forced Swim Test in Rats.

BACKGROUND: Aside from its pervasiveness, whereby it affects as much as 20% of the world's population, depression continues to be one of the most crucial psychiatric problems due to the loss of power it causes by disrupting daily life functioning, containing economic consequences, and having a high suicidal tendency. Major depression (MD) is a systemic and multifactorial disorder involving complex interactions between genetic predisposition and disturbances of various molecular pathways. OBJECTIVES: In our current study, we aimed to identify the proteins obtained from serum samples that change during depression with the MD model. METHODS: The MD model was applied through the forced swim test in rats. 14 Winstar Albino male rats were divided into two equal groups as follows: depression and control groups. Serum samples were separated by chromatographic methods and then compared with two-dimensional (2D) electrophoresis. RESULTS: A total of 9 potential diagnostic protein sequences were identified, which were distinguished with computer software. During the last phase of the study, the Matrix-Assisted Laser Desorption/ Ionization - Time of Flight (MALDI-TOF) analysis, the previous expression sequences identified among the groups were determined and classified. By comparing protein expressions, it was concluded that 9 different points could be used together as a potential biomarker. CONCLUSION: Results can help us identify a new diagnostic system that can be used to diagnose MD.