Incubation Time Research Articles

The effect of key factors on anaerobic co-digestion of leachate and crude glycerin by statistical design of experiments

Anaerobic digestion is a versatile biotechnology because of the energy recovery from different kinds of waste. In addition to anaerobic digestion, there is the anaerobic co-digestion process, which improves the energy recovery of many kinds of waste in a single installation. The association of landfill leachate and crude glycerin is promising, considering the respective characteristics of each one. However, data about the anaerobic co-digestion of these substrates is still scarce. In this context, the present study aimed to evaluate the effect of the factors: incubation time (20-40 days), pH (7-8), glycerol proportion (0.5-2.5% v/v) and substrate/inoculum ratio (0.2-0.6 g COD g-1 VSS) by Plackett-Burman statistical design of experiments. The advantages of anaerobic co-digestion were evaluated achieving the highest treatment performance of 98.1% efficiency of organic matter removal, and a high methane content in the biogas of approximately 79%. Furthermore, a positive effect of the key factor incubation time and glycerol proportion, instead of the substrate/inoculum ratio, which requires low values. Overall, the following parameters: incubation time, glycerol proportion and S/I ratio highlighted their significant effect in the bioprocess performance.

Chemical Composition, In vitro Digestibility and Gas Production of Selected Forages Preferred by Dromedary Camels in Peri Urban Area of Isiolo Town, Kenya

Inadequate feed resources in quantity and quality during dry season is the major factor affecting milk production in lactating dromedary camels under the peri-urban production system. This study aimed to evaluate the nutritive contribution of selected shrubs and fodder species to lactating camels in peri urban area of Isiolo town, Kenya during dry season. Nineteen samples of different browse species were collected based on palatability and analyzed for proximate composition, detergent fiber fractions, In vitro digestibility and gas production. Average dry matter (DM) percentage on all dried sampled shrubs was 90%. The crude protein (CP) content ranged between 4.98 - 26.66%. The neutral detergent fibre (NDF) and acid detergent fibre (ADF) values ranged from 24.90 - 72.85% and 19.02 - 55.85% respectively. Organic matter digestibility (OMD) and dry matter digestibility (DMD) mean were 47.32% and 41.25% respectively. Acacia nilotica had highest OMD (75.29%) and DMD (62.80%) while Prosopis cineraria recorded the lowest at 24.21% and 22.10% respectively. Cumulative gas production was high in Haloxylon salicornicum (0.00-70.43ml/200mg) and low in Zizyphus mucronata (23.31ml/200mg) with increasing incubation time from 2 to 15 hours. The results indicate that the nutritive values of the forages were not adequate hence supplementation is required for dromedary camels during dry season in peri urban production system. Proper management and conservation of the rangelands through reseeding will also improve the forages nutritional composition.

Protein Stacking on the APTES-Functionalized Pyrochlore Bi2Ru2O7 Clusters for Ultrasensitive and Selective Immunosensing.

With their unique physicochemical properties, such as metallic-like conductivity, favorable (electro)catalytic properties, electrochemical stability, and ease of functionalization, pyrochlores have found applications in various fields such as solid oxide fuel cells, batteries, thick film resistors, and temperature sensors; however, there are no reports on their application in electrochemical immunosensing. In this study, we exploited the (electro)catalytic nature and stability of the pyrochlore Bi2Ru2O7 clusters silanized with (3-aminopropyl)triethoxysilane (APTES) to demonstrate their potential for the effective stacking of functional proteins. Characterization of the clusters by XPS disclosed a dual environment of Bi, also indicating the presence of Bi2O3 alongside APTES-Bi2Ru2O7 clusters and, importantly, the predominant involvement of pyrochlore moieties in subsequent protein stacking. After stacking protein A and antibodies, the immunosensor revealed a nearly interference-free operation, high sensitivity, a detection limit of 118 fM SARS-CoV-2 spike protein, and operation in a wide examined concentration range of 10-5-10-1 μg mL-1 with an r2 of 0.98. In combination with a short incubation time of 30 min, the pyrochlore-based immunosensor provides a solid platform for future point-of-need applications.

An Improved Sporulation Method for Producing Asexual Zoospores of Pythium aphanidermatum on Culture Medium

An improved sporulation method for Pythium aphanidermatum on 10% V8-juice agar was developed to generate substantial quantities of zoospores. We conducted experiments using a 4 × 4 factorial design to evaluate the effects of temperature (20, 25, 30, and 35 °C) and incubation time (2, 3, 4, and 5 days) on zoospore production from isolates HSv05 and KACC 48066. The results indicated that a temperature of 30 °C consistently yielded the highest numbers of zoospores across all incubation periods. Applying the same temperature (30 °C) and all incubation periods to other isolates (CCp03, CCp04, CCp05, HSv10, and KACC 40156) also resulted in higher numbers of zoospores regardless of incubation times compared with that under the control condition (25 °C and 2 days). Further virulence tests of isolate HSv05 revealed that high concentrations of zoospores (5 × 102 − 105 zoospores/ml) induced severe rot symptoms; however, a low concentration of 102 zoospores/ml produced only considerably weak symptoms on inoculated potato tubers. No symptoms appeared in tubers inoculated with 0 (uninoculated control) or 101 zoospores/ml. These findings suggest that our sporulation method can help obtain adequate zoospores for various basic and applied studies, including pathogen identification, virulence assessment, resistance resource screening, and control strategy development.

Characteristics of Plasmodium vivax apicomplexan amino acid transporter 8 (PvApiAT8) in the cationic amino acid transport.

Plasmodium vivax is the most widespread malaria parasite affecting humans, and its eradication is challenging due to the spread of drug-resistant parasites and their ability to remain in liver as a dormant stage. These parasites invade and multiply extensively within hepatocytes and erythrocytes in the host, relying on nutrient acquisition for their growth and replication. A promising new treatment aimed at targeting P. vivax involves blocking cationic amino acid uptake, which is a biological source of nutrients for the parasite. Novel Putative Transporter 1 (NPT1), identified as a cationic amino acid transporter in Apicomplexan, has a homologue in Plasmodium species known as apicomplexan amino acid transporter 8 (ApiAT8). This study focuses on P. vivax ApiAT8 to understand its precise role. PvApiAT8 was expressed in Xenopus laevisoocytes and shown to selectively uptake cationic amino acids. The uptake activity of [3H] L-arginine was shown to depend on PvApiAT8 expression time and substrate incubation time. PvApiAT8 was sodium-independent and functioned at pH levels between 6.5 and 8.5, with no efflux activity observed. Kinetic analysis showed saturable uptake for L-arginine consistent with Michaelis-Menten kinetics, with a Km of 1.5 ± 0.3 µM and a Vmax of 25.0 ± 4.8 pmol/oocyte/hr. Inhibition assays further confirmed its selectivity for cationic amino acids such as L-arginine, L-lysine, L-histidine, and L-ornithine. Sequence and structural analyses revealed a conserved binding pocket for cationic amino acids in Plasmodium species, distinct from that in Toxoplasma gondii NPT1. These findings highlight the potential of targeting PvApiAT8 in developing new treatments for P. vivax malaria.

A Reporter Gene Assay for Measuring the Biological Activity of PEGylated Recombinant Human Growth Hormone

PEGylated recombinant human growth hormone (PEG-rhGH) has garnered significant interest in growth hormone research due to its prolonged half-life and improved patient compliance. An accurate evaluation of its biological activity is critical for ensuring the quality of PEG-rhGH-based therapeutics. In this study, we established an in vitro bioactivity assay using a reporter gene method based on the HepG2/IGF-1 cell line. Key assay parameters, including the initial concentration of PEG-rhGH, serial dilution ratios, cell density, and incubation time, were systematically optimized to generate robust dose–response curves. The assay demonstrated high sensitivity, precision, and reproducibility across multiple batches of PEG-rhGH. The validation results showed an excellent correlation with traditional in vivo animal studies and the Nb2-11 cell proliferation assay, highlighting its suitability for quality control. Furthermore, we developed an ion exchange chromatography (IEC) method to separate five positional isomers of PEG-rhGH, revealing significant differences in bioactivity depending on the PEG modification site. This study demonstrates that the optimized reporter gene assay is not only effective for quality control of PEG-rhGH but also serves as a valuable tool for evaluating and optimizing PEGylated long-acting growth hormone therapeutics.

Structured lipids from virgin coconut oil and omega‐3 fatty acids: Process optimization

AbstractStructured lipids (SLs) containing docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and DHA + EPA were synthesized via enzymatic acidolysis using virgin coconut oil (VCO) as the substrate in n‐hexane. Commercially available enzymes Lipozyme TL IM (produced from Thermomyces lanuginosus, a 1,3‐specific lipase), Lipozyme IM60 (produced from Rhizomucor miehei, a 1,3‐specific lipase), and non‐specific lipase from Candida rugosa (powder) were used as biocatalysts. The T. lanuginosus lipase was chosen to evaluate the effects of various parameters on the incorporation of PUFAs into VCO and to optimize the process. As the enzyme load increased from 1% to 4%, the incorporation of omega‐3 PUFAs also increased; however, it decreased when the enzyme load was further increased to 6%. The incorporation of these fatty acids increased with reaction time from 12 to 36 h but decreased at 48 h. Similarly, the incorporation increased with temperature from 35 to 45 °C, but decreased at 55 and 65 °C. The highest incorporation rates of DHA (18.91%), EPA (30.38%), and DHA + EPA (34.64%) were achieved at a mole ratio of 1:3 (VCO to DHA or EPA) or 1:3:3 (VCO to DHA + EPA), with a 4% enzyme load, 36 h incubation time, and a temperature of 45 °C. A central composite design (CCD) with three levels and three factors—reaction temperature (35, 45, and 55 °C), enzyme amount (2%, 4%, and 6%), and reaction time (24, 36, and 48 h)—was used to model and optimize the reaction conditions via response surface methodology (RSM). Under optimal conditions of 3.3% T. lanuginosus enzyme, 42.22 °C, and 33.38 h, the incorporation rates were 32.92% for DHA, 44.48% for EPA, and 47.04% for DHA + EPA in VCO.

Gold Nanorod Nanolayer-Mediated Photothermal Transfection Promotes DNA Nanomachine Crossing of Cell Membranes for Sensitive Cell Imaging.

The transmembrane efficiency of DNA nanomachines greatly affects the performance of cell imaging. The current transmembrane methods suffer from long time incubation, low efficiency, and potential cyto-toxicity. Here, we report on a novel gold nanorod nanolayer-mediated photothermal transfection (AuNRs-NMPT) method to deliver a DNA nanomachine across the cell membrane. Due to good photothermal effects, AuNRs nanolayer-functionalized slide (AuNRs-NFS) converted light energy into local heat. Local heat disrupted the fluidity of the cell membrane for perforation. The DNA nanomachine penetrates the cell membrane rapidly and efficiently via those holes under the optimal laser power and irradiation time. The AuNRs-NMPT exhibited a 12-fold lower incubation time and 13-fold higher fluorescent intensity compared to that of common direct incubation. The cells also exhibited a high cell viability after being treated with the AuNRs-NMPT, due to cell membrane self-repair. Due to efficient, rapid, and low cytotoxic transmembrane transfection, the AuNRs-NMPT ensures sensitive cell imaging of miRNA-21. The proposed AuNRs-NMPT will promote the application of DNA nanomachines for clinical cancer diagnosis in the future.

Determination of hepatitis E virus inactivation during manufacturing of spreadable pork liver sausage and salami-like raw pork sausage.

The zoonotic hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Meat from domestic pigs, which represent a major animal reservoir of HEV, plays a key role in HEV transmission. Although pork meat products can contain HEV-RNA, it is unknown whether infectious HEV is still present after their manufacturing process. Here, we used a newly developed method for virus extraction from sausages and a quantitative method for detecting HEV infectivity in artificially contaminated sausages to investigate the HEV inactivation during production of spreadable pork liver sausage and salami-like raw pork sausage. The cell culture-adapted HEV genotype 3c strain 47832c was used to contaminate meat preparations intended for production of sausages, which were manufactured based on recipes commonly used in Germany. According to these recipes, spreadable liver sausages of a certain diameter are to be held in a water bath at 70°C for 30min. Therefore, the HEV inoculated liver sausage preparations were filled into conical tubes and heated in a 70°C water bath. After 21min, the sausages reached a core temperature of 70°C and samples were taken after further incubation for up to 18min. For the raw sausages, the HEV inoculated meat preparation was filled into natural casings and sausages were cured at 18°C and 80% relative humidity. Samples were taken for up to 21days. HEV was extracted from all samples, which were quantitatively analyzed for infectious virus and viral RNA using cell culture and RT-qPCR, respectively. During liver sausage production, infectious HEV decreased by four log10 immediately after reaching the core temperature of 70°C and was completely inactivated (>4.3 log10 decrease) 2min later (23min heat treatment). In contrast, the HEV-RNA amount decreased only marginally (<0.6 log10) throughout the whole incubation time. During raw sausage manufacturing, infectious HEV decreased only slightly (<1.3 log10) over three weeks of curing, while the HEV-RNA amount remained unchanged. It can be concluded that the intended heating regime during production of spreadable liver sausages leads to an inactivation of HEV, indicating a low risk of HEV infection for consumers if these sausages are manufactured properly. In contrast, HEV was only slightly inactivated during production of salami-like raw pork sausage. Therefore, raw sausage can contain infectious HEV if starting material with a high HEV amount was used for production. Viral RNA testing cannot be used to predict infectivity of HEV in meat products.

Temporal shifts in the phosphate mobilising bacterial community reveal contrasting succession patterns in response to different phosphorus sources.

Phosphate mobilising bacteria (PMB), such as phoD- and pqqC-harbouring bacteria, play a crucial role in mobilising insoluble phosphorus (P) in soil through the secretion of alkaline phosphatase and organic acids. To evaluate the succession pattern of PMB communities in response to different insoluble P sources. Five P source treatments, including the addition of KH2PO4 (PDP), FePO4 (FEP), Ca3(PO4)2 (TCP), lecithin (LEC), and a negative control, were established in the soil in a microcosmic system. Subsequently, phoD- and pqqC-harbouring bacterial communities were investigated by sequential sampling and high-throughput sequencing. In addition, PMB strains were isolated from these five treatments, and their phosphate mobilising activity was further analysed. The effect of the insoluble organic P source (LEC) on the succession of PMB communities consistently exceeded that of insoluble inorganic P (Pi) sources (FEP and TCP). A successively enhanced succession and a successively diminishing succession were observed in PMB communities in FEP and TCP, respectively. Furthermore, the soil AP content significantly increased with incubation time in LEC and FEP. Most of the variation in phoD- and pqqC-harbouring bacterial communities was explained by all P fractions, respectively, while Fe-P and stable organic P fractions determined the PMB communities. Insoluble Pi sources, particularly FEP, tended to enrich more high-performance PMB strains for Fe-P. This study firstly reveals the dynamic response of PMB communities to different insoluble P sources at the community level in a short time scale and identifies key PMB taxa that respond to different insoluble P sources, particularly Fe-P. It is also the first to provide community evidence and a feasible method for obtaining high-performance PMB strains for Fe-P in subtropical acidic red soils through directed enrichment culture.

Polyphenol autoxidation and prooxidative activity induce protein oxidation and protein-polyphenol adduct formation in model systems

Polyphenols are well-known for their antioxidant properties, but their prooxidative activity remain less understood. This study quantitatively examined the formation of hydrogen peroxide (H2O2) during the autooxidation of nine different polyphenols in model systems, investigating how it impacts protein oxidation and protein-polyphenol covalent adduct formation. Polyphenols (4 mM) generated H2O2 in the range of 0.2–242 μM, depending on type of polyphenol, incubation time, temperature, and pH, but no clear relationship between polyphenol structure and H2O2 production was observed. The presence of free amino acids and proteins (bovine serum albumin and β-lactoglobulin) inhibited H2O2 formation, with Cys completely scavenging H2O2. Met was highly susceptible to oxidation with a 25–75 % loss, forming methionine sulfoxide through a two-electron oxidation pathway. Trp and Tyr were oxidized to produce dioxindolyl-L-alanine, kynurenine, 3,4-dihydroxyphenylalanine, N′-formylkynurenine, and 5-hydroxytryptophan in the nmol/mol-mmol/mol amino acid range. Furthermore, autoxidation of polyphenols resulted in >177 distinct amino acid/protein-polyphenol adducts as identified using LC-Orbitrap-MS/MS analysis.

EVALUASI NILAI KECERNAAN IN VITRO DAUN KELAPA SAWIT TERFERMENTASI MA-11 SEBAGAI PAKAN TERNAK

ABSTRACT This research to determine the effect of fermentation incubation using MA-11 on the in vitro digestibility value of dry matter and organic matter of palm leaves. The design used is a Complete Random Design (RAL) unidirectional pattern with 4 treatments and and 3 replicates. The treatment is T0: oil palm leaves without fermentation; T1: oil palm leaves fermented using MA-11, incubated for 3 days, T2: oil palm leaves fermented using MA-11, incubated for 6 days. T3: palm leaves are fermented using MA-11, incubated for 9 days. The variables observed included the digestibility value of dry materials and organic materials. The data obtained was then analyzed using Variance Analysis (Anova) with a unidirectional pattern RAL and if there was a difference between the treatments, further tests were carried out using the Duncan't Multiple Region Test (DMRT). The results showed that fermentation using MA-11 with an incubation period of 0 – 9 days had a very significant effect on increasing the digestibility value of dry and organic materials (P&lt;0.01). This research can conclude that the fermentation of oil palm leaves using MA-11 with an incubation period of 0 – 9 days can increase the digestibility value of dry matter and organic matter, and the longer the incubation time in fermentation, the more it increases the digestibility value of dry matter and organic matter.

Consolidated Microscale Interferon-γ Release Assay with Tip Optofluidic Immunoassay for Dynamic Parallel Diagnosis of Tuberculosis Infection.

Interferon-γ release assay (IGRA) is one of the most important diagnostic tools for tuberculosis (TB) infection. Despite its high accuracy, conventional IGRA has several drawbacks, including complicated procedures, large blood volume requirements, lengthy incubation times, and difficulties in parallel testing. Efforts have been made to develop miniaturized and highly sensitive biosensors for interferon-γ or to evaluate the specific immune response through microfluidic platforms. However, the need for sophisticated consumables and equipment, as well as the partial experimental design, has limited the application of these advanced techniques in TB diagnosis and disease control. Here, we report the development of a tip optofluidic immunoassay (TOI)-based consolidated microscale IGRA (CM-IGRA) for the dynamic and parallel evaluation of TB infection, refining both the blood incubation and interferon-γ quantification processes. The TOI system comprises 12 microfluidic immuno-reactors and a portable chemiluminescent imaging station, capable of quantifying interferon-γ with high sensitivity (8.00 pg/mL in plasma) and a wide detection range (∼104). The results generated with CM-IGRA achieved 98.39% agreement with the standard IGRA while reducing blood sample consumption to 50 μL per assay (20-fold reduction) and significantly shortening the incubation time from 20 to 10 h. This diagnostic method simplifies operations and improves efficiency for the parallel assays required in IGRA, providing a promising solution for TB screening in patients for whom current methods are inconvenient, such as children and older adults.

Revisiting the Effect of Trypsin Digestion Buffers on Artificial Deamidation.

Deamidation of asparagine and glutamine residues occurs spontaneously, is influenced by pH, temperature, and incubation time, and can be accelerated by adjacent amino acid residues. Incubation conditions used for proteolytic digestion in bottom-up proteomic studies can induce significant deamidation that affects results, either knowingly or unknowingly. This has prompted studies into modifications to common trypsin digestion protocols to minimize chemical deamidation, including shorter incubation times and specific lysis buffers. Prior work suggested ammonium acetate at pH 6 to minimize chemical deamidation, but this buffer has compatibility issues with trypsin digestion and common assays (e.g., bicinchoninic acid assays). Here, we re-evaluated former comparisons of Tris-HCl, ammonium bicarbonate, and triethylammonium bicarbonate buffers for the amount of artificial, chemically induced deamidation generated in a standard bottom-up proteomics workflow, and we added an evaluation of three commonly used and biologically compatible buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), EPPS (3-[4-(2-Hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid), and PBS (phosphate buffered saline). Our findings show that HEPES exhibited the least amount of artificial deamidation and is a reasonable choice for general proteomic experiments, especially for studies considering N-glycosylation.

Production and characterization of levans with different molecular weights synthesized by Bacillus licheniformis ZM107.

Levan, a fructan-type polysaccharide with diverse applications in food, pharmaceuticals, and biotechnology, has garnered significant attention for its functional properties, such as prebiotic, immunomodulatory, antioxidant, and antimicrobial activities. In this study, the effects of fermentation temperature, sucrose concentration, and incubation time on the molecular weight of levan produced by Bacillus licheniformis fermentation were investigated. The results showed that as the temperature (37-50°C) and sucrose concentration (300-500g l-1) increased, the molecular weight of levan produced by the strain during fermentation decreased. At the same time, the molecular weight initially increased and then decreased with longer culture times, indicating that levan within a specific size range could be produced by controlling the fermentation conditions. In addition, the properties and characteristics of levan produced by fermentation at two molecular weights (HML and LML) were compared. The results showed that molecular weight significantly affected the micromorphology, thermal behavior, rheological properties, and prebiotic activity of levan. Therefore, this study demonstrated that Bacillus licheniformis SFLV-ZM107 can produce low molecular weight levan, and its molecular weight can be controlled through fermentation conditions. Consequently, the properties and functions of levan can be influenced, allowing the production of levans with the desired characteristics.

Optimized Protocol For DNA Extraction from Human Whole Blood

Background/Aims: DNA isolation is the initial process in genetic research. The product is used in many PCR reactions (PCR-RFLP, Real-Time PCR, multiplex PCR). That is why it is important to optimize DNA isolation protocol to obtain a good quality of DNA. Our first attempts at isolation, conducted using Purification Kit, did not result in sufficient concentration (6.414 ng*μL-1) and purity (A-260/280) of 0.764 of isolated DNA. Methods: We used twice the recommended amount of tissue and cell lysis solution to get more effective cell lysis. We extend the time of vortexing, centrifugation and incubation at critical steps. We manipulated the speed and temperatures of centrifugation. We used cold iso-propanol to get white strands of DNA faster. When rinsing with ethanol we used cold alcohol. We tested efficiency of two methods of drying of ethanol to achieve optimal DNA pureness. We leave the isolated DNA for 20 minutes to evaporate the ethanol and then resuspend nucleic acid in TE Buffer. Results: Our modifications resulted in the improvement of isolation efficiency. After optimization we achieved DNA concentration (in range of 50-150 ng*μL-1) and purity (A 260/280) of 1.735. Similar results for DNA parameters were achieved from the whole blood frozen for 2-3 months (concentration in the range of 125.762 ng*μL-1, pureness: 1.761) and from blood frozen for 18 months (117.94 ng*μL-1 and 1.7194, respectively). We performed electrophoresis after each isolation to confirm the effectiveness of optimized procedure. The refinements we used in DNA isolation are more efficient than those recommended in DNA Purification Kits. Conclusion: Our results confirm that optimized DNA protocol fulfills the conditions of good extraction technique: it is relatively fast and easy to perform yet it guarantees a high reproducibility, specificity and sensitivity. There are also no dangerous or harmful steps. Our paper demonstrates innovative and effective approach. It confirms a high effectiveness of method regardless of duration of sample freezing, as well as introduce important modifications (timing, temperature conditions, drying details, absence of K-proteinase) that make overall procedure more productive and relatively fast.

Biofabrication of zinc oxide nanoparticles using Moringa oleifera, characterization and statistical optimization for their application in crystal violet adsorption

Crystal violet (Cry) is an essential textile dye belonging to the triphenylmethane group, that is widely used in the textile industry. It is also applied for paper printing and Gram staining. Previously, it was significant as a topical antiseptic due to its antibacterial, antifungal, and anthelmintic properties. Despite its various applications, crystal violet has been recognized as a biohazard dye due to its toxic and carcinogenic properties. It persists in the environment with long-lasting effects and has detrimental impacts. In this research, water extract from Moringa oleifera leaves is employed as environmentally friendly methods to synthesize zinc oxide nanoparticles (Mo/ZnO-NPs), and characterized by TEM, EDX, FT-IR, and Zeta potential. Mo/ZnO-NPs exhibit a Zeta potential of − 21.9 mV, and X-ray diffraction (XRD) analysis confirms their crystallographic structure. The size of the biogenic Mo/ZnO-NPs ranges from 5.52 to 41.59 nm. This study was designed to estimate and maximize the ability of Mo/ZnO-NPs to remove crystal violet using Central Composite Design (CCD), considering pH (ranging from 3 to 11), incubation time (ranging from 30 to 150), nanoparticles concentrations (ranging from 0.2 to 1.8 mg/mL), and crystal violet concentrations (ranging from 25 to 125 ppm). The maximum percentage value of removal of crystal violet by Mo/ZnO-NPs was 97.26 with optimal conditions of pH 9, incubation time 120 min, Mo/ZnO-NPs 1.4 mg/mL, and crystal violet concentration of 50 ppm. The best-predicted conditions that caused the highest removal of crystal violet (97.8%) were determined using the desirability function as pH 10, incubation time of 140 min, Mo/ZnO-NPs concentrations of 1.3 mg/mL, and a concentration of crystal violet of 80 ppm. Under these optimal conditions, the maximum experimental crystal violet removal% by Mo/ZnO-NPs was (98.7%) was verified. Mo/ZnO-NPs synthesized by Moringa oleifera can be a promising candidate for the adsorption of crystal violet.

Evaluating the In Situ Effects of Whole Protein Coronas on the Biosensing of Antibody-Immobilized Nanoparticles Using Two-Color Fluorescence Nanoparticle Tracking Analysis

The formation of protein coronas around engineered nanoparticles (ENPs) in biological environments is critical in nanomedicine, as these coronas significantly influence the biological behavior of ENPs. Despite extensive research on protein coronas, understanding the in situ influence of whole (soft plus hard) protein coronas has remained challenging. In this study, we demonstrate a strategy to assess the in situ effects of whole coronas on the model biosensing of anti-IgG using IgG-conjugated gold nanoparticles (IgG-AuNPs) through fluorescence nanoparticle tracking analysis (F-NTA), which enables the selective tracking of fluorescent particles within complex media. In our approach, anti-IgG and IgG-AuNPs were labeled with distinct fluorescent dyes. The accordance in hydrodynamic diameter distributions observed at two different wavelengths verifies the successful capture of anti-IgG on the IgG-AuNPs. The counting of fluorescent anti-IgG within the size distribution allows for a quantitative assessment of biosensing efficiency. This method was applied to evaluate the effects of four protein coronas—human serum albumin, high-density lipoproteins, immunoglobulin G, and fibrinogen—as well as their mixture across varying incubation times and concentrations. The results suggest that the physical presence of whole protein coronas surrounding the IgG-AuNPs may assist the biosensing interaction in situ rather than screening it.

Temperature-Enhanced Purine Metabolism-Based Versatile SERS Platform for Rapid Clinical Pathogens Diagnosis and Drug-Resistant Assessment.

Label-free surface-enhanced Raman spectroscopy (SERS) combined with machine learning (ML) techniques presents a promising approach for rapid pathogen identification. Previous studies have demonstrated that purine degradation metabolites are the primary contributors to SERS spectra; however, generating these distinguishable spectra typically requires a long incubation time (>10 h) at room temperature. Moreover, the lack of attention to spectral variations between strains of the same bacterial species has limited the generalizability of ML models in real-world applications. To address these issues, we investigated temperature-induced alterations in bacterial purine metabolism and found that robust SERS spectra could be obtained within just 1 h by heating samples to 60 °C. Our study further revealed that pathogens exhibit multiple fingerprint patterns across strains, rather than a uniform spectral signature. To enhance practicality, we optimized ML models by training them on data sets capturing all relevant SERS fingerprints and validated them on separate bacterial strains. The SoftMax classifier achieved 100% accuracy in identifying both laboratory and clinical specimens within 17 h. Additionally, the platform demonstrated over 91% accuracy in distinguishing drug-resistant strains, such as methicillin-resistant Staphylococcus aureus and carbapenem-resistant Klebsiella pneumoniae, and achieved 99.66% accuracy in differentiating specific strains within a species, such as enterohemorrhagic Escherichia coli. This accelerated, purine metabolism-based SERS platform offers a highly promising alternative for the rapid diagnosis of bacterial infections.

Arsenic efflux and bioremediation potential of Klebsiella oxytoca via the arsB gene.

Arsenic-resistant Klebsiella oxytoca strain AT-02 was isolated from the ground water of the Multan region of Pakistan. The strain displayed high arsenite and arsenate resistance as minimal inhibitory concentration (MIC) was 600ppm and 10,000ppm respectively. The high tolerance of the isolated strain towards arsenate can be postulated due to significant increase in biofilm in response to arsenate. The bacterial strain exposed to 1/2 and 3/4 MIC showed a significant 10 and 12 folds increase in expression of the arsenite efflux gene arsB. Sequential and structural comparison of the arsB gene showed the presence of conserved arsenic binding residues. Arsenic remediation by AT-02 biomass was 50% after 0.5 hours of incubation and 66% in 2 hours. the increase in remediation efficiency with the increase in incubation time indicates its biosorption potential. the arsenic sensitive strain NK11 showed only 4-5% arsenic remediation. Fourier transform infrared spectroscopy (FTIR) analysis confirmed interaction of arsenate and arsenite with functional groups (aromatic amino acid residues) on the cell surface leading to characteristic peak shifts. Thus, the isolated AT-02 has the potential to remediate both arsenite and arsenate from contaminated environmental sites.