Incubation Temperature Research Articles

Abstract 2063: Using the edge wells in 96-well plates without increasing variability in cytotoxicity assays through thermal controls

Abstract There are very few things as fundamental to cancer research as the 96-well-plate, yet edge effect has continued to cause problems since its introduction. The most common remedy for edge effect is to avoid using edge wells altogether, which eliminates 37% of the plate from the assay. If all of those wells could be used, an estimated USD $119M could be saved in research costs annually across the globe. We have shown previously that maintaining constant thermal control of plating conditions reduces variability in stem cell toxicity assays. Here, we ask the question: can we use the edge wells in cancer screening assays if we control thermal changes that happen in the traditional lab as cells are plated at room temperature and incubated at 37 degrees C? We had the null hypothesis that including the edge wells would add variability to an A549 cytotoxicity assay, even if the cell handling and incubation temperatures were matched and held constant. We plated A549 cells at either room temperature (like a BSC) or at 37 degrees C using the Xvivo System, which maintains constant temperature, oxygen, CO2, and relative humidity levels around cell handling operations. The cells were treated with serial dilutions of peracetic acid/hydrogen peroxide solution. After incubation for 3 days, the plates were stained with Crystal Violet and assessed for cell density (n=3 separate experiments). The null hypothesis was disproven. We found that if the plates were maintained at 37 degrees C during cell handling as well as incubation, that cell distribution was random and even across all wells, even edge wells. We found that there was no statistical difference between edge well and center well results in cytotoxicity assays. Edge wells were usable without increasing variability in the assays. This is consistent with earlier findings showing that when temperature changes between cell plating and incubation were averted, stem cell distribution across the plate was random and the edge effect was eliminated. Citation Format: Alicia D. Henn, Shannon Darou, Randy Yerden. Using the edge wells in 96-well plates without increasing variability in cytotoxicity assays through thermal controls [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2063.

Design of Incu Analyzer for IoT-Based Baby Incubator Calibration

An incubator analyzer, serving as a calibration tool, is utilized to measure diverse parameters such as temperature, mattress temperature, humidity, airflow, and noise in infant incubators. The present study focuses on the development of the Design Incubator Analyzer for IoT (Mattress Temperature and Humidity) with LCD and ThingSpeak display, specifically designed for calibrating baby incubators. The primary objective is to design and develop an Incubator Analyzer as a calibration device for assessing various parameters in infant incubators, encompassing temperature, mattress temperature, humidity, airflow, and noise. The design of this calibration device incorporates a Thermocouple Type-K sensor for baby incubator mattress temperature parameters, a DHT22 sensor for humidity parameters, and an ESP32 microcontroller. The ESP32 processes data from the Thermocouple Type-K and DHT22 sensors to generate values for mattress temperature (TM) and humidity (RH), which are then displayed on LCD and ThingSpeak displays. The device underwent rigorous testing against an established measuring device, the INCU II. In the study, the TM parameter or mattress temperature exhibited the smallest error of -0.0140% at 35°C and the largest error of 0.0584% at 36°C. Concerning the humidity parameter, the largest error was 0.0570% at 32°C, while the smallest error was 0.0207% at 35°C. Overall, the Incubator Analyzer Design for IoT-Based Baby Incubator Calibration device, or IoT-based Incubator Analyzer, demonstrates potential usability following the planning and execution phases, including a thorough review of existing literature. To enhance user experience during the calibration process, an IoT system was developed for data transmission over Wi-Fi, presenting results on the ThingSpeak platform in real-time

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Identification and Pathogenicity of Fusarium Fungi Associated with Dry Rot of Potato Tubers.

Dry rot of potato tubers is a harmful disease caused by species of the Fusarium genus. Studies on the composition and features of Fusarium spp. that cause the disease in Russia are limited. Thirty-one Fusarium strains belonging to the F. sambucinum species complex (FSAMSC) and F. solani species complex (FSSC) were accurately identified using multilocus phylogenetic analysis of the tef and rpb2 loci, and their physiological characteristics were studied in detail. As a result, 21 strains of F. sambucinum s. str. and 1 strain of F. venenatum within the FSAMSC were identified. Among the analyzed strains within the FSSC, one strain of F. mori, four strains of F. noneumartii, and two strains of both F. stercicola and F. vanettenii species were identified. This is the first record of F. mori on potato as a novel host plant, and the first detection of F. noneumartii and F. stercicola species in Russia. The clear optimal temperature for the growth of the strains belonging to FSAMSC was noted to be 25 °C, with a growth rate of 11.6-15.0 mm/day, whereas, for the strains belonging to FSSC, the optimal temperature range was between 25 and 30 °C, with a growth rate of 5.5-14.1 mm/day. The distinctive ability of F. sambucinum strains to grow at 5 °C has been demonstrated. All analyzed Fusarium strains were pathogenic to potato cv. Gala and caused extensive damage of the tuber tissue at an incubation temperature of 23 °C for one month. Among the fungi belonging to the FSAMSC, the F. sambucinum strains were more aggressive and caused 23.9 ± 2.2 mm of necrosis in the tubers on average compared to the F. venenatum strain-17.7 ± 1.2 mm. Among the fungi belonging to the FSSC, the F. noneumartii strains were the most aggressive and caused 32.2 ± 0.8 mm of necrosis on average. The aggressiveness of the F. mori, F. stercicola, and especially the F. vanettenii strains was significantly lower: the average sizes of damage were 17.5 ± 0.5 mm, 17.2 ± 0.2 mm, and 12.5 ± 1.7 mm, respectively. At an incubation temperature of 5 °C, only the F. sambucinum strains caused tuber necroses in the range of 6.7 ± 0.5-15.9 ± 0.8 mm.

Influence of incubation temperature, maternal effects, and paternity on quality of olive ridley hatchlings (Lepidochelys olivacea) from a mass-nesting beach in the Mexican Pacific.

Future climate change scenarios project that the increase in surface temperatures will affect ocean temperatures, inducing shifts in marine biodiversity. Sea turtles are species that are particularly vulnerable to the effects of climate change because temperature is a factor that influences embryonic development. We collected clutches of olive ridley turtles from a mass-nesting beach in the Mexican Pacific, which were incubated in ex situ conditions. When the hatchlings emerged, we measured the body condition index-which evaluates the weight-length relationship-and swim thrust, both were considered traits associated with fitness, termed "fitness proxies," and evaluated the effects of incubation temperature, maternal effects, and paternity on these fitness proxies. The body condition index was correlated positively and significantly with the arribada month and temperature during the last third of the incubation period but showed an inverse relationship with the maternal effect. While swim thrust was positively correlated with the maternal effect and the arribada month, there was an inverse relationship with incubation temperature during the first third of the period. Paternity, whether single or multiple, did not have a significant effect on either fitness proxies; however, it may have effects on the average fitness of a population of hatchlings. These results underscore the need to expand research on the sublethal effects of high incubation temperatures on the adaptation and survival of sea turtles, particularly in scenarios of rapid climate change.

Effect of moderate heat stress on tuberization of potato genotypes: Morphological and physiological changes using in vitro and ex vitro propagation

Potato plants are often subjected to heat stress during growth and development, resulting in loss of yield and quality. However, the possibility exists that commercially important genotypes show tolerance to heat stress. In this study, we aim to investigate the morphological and physiological effects of moderate conditions of heat stress on tuberization in nine potato genotypes, and how it affects propagation using an ex vitro and in vitro system. The plant material came from the In Vitro Germplasm Bank of the International Potato Center (CIP). The incubation temperatures of the cultures in in vitro and ex vitro conditions were the ambient in the laboratory and greenhouse, respectively. In in vitro system a randomized complete block design was employed in nine genotypes, two treatments: multiplication culture medium and the same culture medium supplemented with 8.0% sucrose and 500 mg/L-1 CCC, six flasks and five explants per experimental unit. Among all genotypes evaluated, Leona and Liberteña did not form tubers under ex vitro treatment conditions. Under in vitro conditions, Amarilis, Capiro, Perricholi and Tacna showed no evidence of tuberization, and Canchán was the only genotype where 100% formation of tuberous structures were produced in all evaluations. The findings concluded in this study showed that of all the potatoes evaluated, Canchán best responded to the evaluations under both in ex vitro and in vitro conditions, at maximum temperatures between 22.4 to 26.9 and 27.0 to 28.2 oC, respectively. Therefore, it is vitally important to consider its development potential in different producing locations around the world, which are directly and indirectly affected by climate change.

Optimization of a carbon adsorption/HS-GC–MS method for ignitable liquids classification from fire debris

The main goal of any fire investigation is to find the origin and cause of fire. Fire is commonly initiated or accelerated by using ignitable liquids (ILs). The analysis of fire debris can contribute to the determination of the origin of the fire. In this study, the thermal desorption of IL residues (ILRs) from activated charcoal strips (ACS) was optimized prior to headspace- gas chromatography- mass spectrometry (HS-GC–MS) analysis for the identification and classification of ILRs in fire debris samples. A Box-Behnken Design (BBD) coupled with Response Surface Methodology (RSM) were used to determine the optimized conditions in the following ranges: incubation temperature (80–140 ˚C), incubation time (30–60 min), and agitation speed (250–750 rpm). The sum of the differences between the total ion spectrum (TIS) of gasoline and diesel was used as dependent variables. The quadratic model was significant with a p- value of 0.0061 and a coefficient of determination equals to 0.911. RSM revealed that an increase in incubation temperature and agitation speed increases the response. The optimized conditions were 140 °C and 750 rpm. Further optimization of the incubation time was studied by analyzing fire debris samples at different times (5–30 min) and 5 min was determined as the optimum condition. The final optimized conditions were applied in combination with chemometrics for the characterization of fire debris samples burned with different ILs. The obtained results demonstrate the feasibility of the method in fire debris analysis which avoids the use of organic solvents providing a greener and faster alternative method.

An integrated approach to explore the microbial biodiversity of natural milk cultures for cheesemaking

The use of natural milk culture (NMC) represents a key factor in PDO Montasio cheeses, contributing to its distinctive sensory profile. The complex microbial ecosystem of NMCs is the result of heat treatment and incubation conditions, which can vary considerably among different production plants. In this study, the microbiota of NMCs collected from 10 PDO Montasio cheese dairies was investigated employing colony counts and metagenomic analysis. Furthermore, residual sugars, organic acids, and volatile profiles were quantitatively investigated. Results showed that Streptococcus thermophilus was the dominant species in all NMCs, and a subdominant population made of other streptococci and L. salivarius was also present. The incubation temperature appeared to be the main driver of biodiversity in NMCs. Metagenomics allowed us to evidence the presence of minor species involving safety (e.g., Staph. aureus) as well as possible functional aspects (Next Generation Probiotics). Statistical analysis based on residual sugars, organic acids, and volatiles' content allowed to correlate the presence of specific microbial groups with metabolites of great technological and sensory relevance, which can contribute to giving value to the artisanal production procedures of NMCs and clarify their role in the creation of the characteristics of PDO Montasio cheese.

Exploring the impact of biocontrol and temperature variations on the population dynamics of Paracoccus marginatus

The mealybug (Paracoccus marginatus) is one of the most important pests of papaya (Carica papaya L.). The high potential damage of this pest threatens papaya production. The objective of this paper is to explore the impact of biocontrol and temperature variations on the population dynamics of Paracoccus marginatus (P. marginatus). We first propose and study a mathematical model for the dynamics of P. marginatus within a papaya field. This model consists of a time-delayed non-autonomous system of differential equations that takes into account the egg incubation period, temperature variations, plant compensation and predators that are natural enemies of the papaya pest. We provide the basic properties of the model, compute the basic offspring number N0 and show that there exists two threshold parameters N_0 and ξ0 such that N_0≤N0≤ξ0≤1 and N0≥N_0>1 provide conditions for the extinction and persistence of the pest within the papaya field, respectively. After, we investigate the impact of biocontrol on the pest population. We found that the use of a control strategy based on constant releases of predators could reduce the pest population by 35.62 percent and increase the biomass by 77.50 percent, while the use of impulsive releases of predators result in a decrease of 96.31 percent of the pest population and an increases of 55.74 percent of the biomass. We also combine the use of continuous predator releases and bio-pesticides and discovered that a spray applied 90 days after germination could completely eliminate the pest population and boost the biomass by 81.58 percent.

EnZolv delignification of cotton spinning mill waste and optimization of process parameters using response surface methodology (RSM)

BackgroundEnZolv is a novel enzyme-based, eco-friendly biomass pretreatment process that has shown great potential in the field of textile engineering and biotechnology. It employs laccase from Hexagonia hirta MSF2 and 2% ethanol in the process of delignification. The process is designed to evaluate optimal conditions to remove lignin and other impurities from cotton spinning mill waste (CSMW), without compromising the quality and strength of the fibers. CSMW is a low-cost and readily available source of cellulose, making it an ideal candidate for delignification using EnZolv. By optimizing the pretreatment conditions and harnessing the potential of enzymatic delignification, this research aims to contribute to more sustainable and efficient ways of utilizing lignocellulosic biomass in various industries for the production of biochemical and bioproducts.ResultsThe present study emphasizes the EnZolv pretreatment in the delignification of cotton spinning mill wastes irrespective of the cellulose content. EnZolv process parameters such as, moisture content, enzyme load, incubation time, incubation temperature, and shaking speed were optimized. Under pre-optimized conditions, the percent lignin reduction was 61.34%, 61.64%, 41.85%, 35.34%, and 35.83% in blowroom droppings (BD), flat strips (FS), lickerin fly (LF), microdust (MD) and comber noils (CN), respectively. Using response surface methodology (RSM), the statistically optimized EnZolv pretreatment conditions showed lignin reduction of 59.16%, 62.88%, 48.26%, 34.64%, and 45.99% in BD, FS, LF, MD, and CN, respectively.ConclusionTraditional chemical-based pretreatment methods often involve harsh chemicals and high energy consumption, which can have detrimental effects on the environment. In contrast, EnZolv offers a greener approach by utilizing enzymes that are biodegradable and more environmentally friendly. The resulting fibers from EnZolv treatment exhibit improved properties that make them suitable for various applications. Some of the key properties include enhanced cellulose recovery, reduced lignin content, and improved biophysical and structural characteristics. These improvements can contribute to the fiber's performance and processability in different industries and future thrust for the production of cellulose-derived and lignin-derived bioproducts.Graphical

Magnetic self-assembled label-free electrochemical biosensor based on Fe3O4/α-Fe2O3 heterogeneous nanosheets for the detection of Tau proteins

A type of electrochemical biosensors based on magnetic Fe3O4/α-Fe2O3 heterogeneous nanosheets was constructed to detect Tau proteins for early diagnosis and intervention therapy of Alzheimer's disease (AD). Firstly, Fe3O4/α-Fe2O3 heterogeneous nanosheets were fabricated as the substrate to realize magnetic self-assembly and magnetic separation to improve current response, and Fe3O4/α-Fe2O3@Au-Apt/ssDNA/MCH biosensors were successfully constructed through the reduction process of chloroauric acid, the immobilizations of aptamer (Apt) and ssDNA, and the intercept process of 6-Mercapto-1-hexanol (MCH); the construction process of the electrochemical biosensor was monitored using Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and the factors affecting the current response of this sensor (concentration of Fe3O4/α-Fe2O3@Au and Apt/ssDNA, incubation temperature and time of Tau) were explored and optimized using differential pulse voltammetry (DPV). Analyzing the performance of this sensor under optimal conditions, the linear range was finally obtained to be 0.1 pg/mL–10 ng/mL, the limit of detection (LOD) was 0.08 pg/mL, and the limit of quantification (LOQ) was 0.28 pg/mL. The selectivity, reproducibility and stability of the biosensors were further investigated, and in a really sample analysis using human serum, the recoveries were obtained in the range of 93.93 %–107.39 %, with RSD ranging from 1.05 % to 1.94 %.

Geographic variation in incubation temperatures promoting viable offspring production in broadly co-distributed turtles.

Organisms whose early life stages are environmentally sensitive produce offspring within a relatively narrow range of suitable abiotic conditions. In reptiles, development rate and survival are often maximized if incubation temperatures remain under 31°C, though this upper bound may vary within and among species. We addressed this expectation by comparing responses to egg incubation at 30°C versus33°C in congeneric turtle species pairs with broad syntopic geographic distributions. In the two softshell turtles (Apalone spp.), the greatest changes in development rate and phenotypic variance were observed in the northernmost population, which had a low survival rate (40%) at 33°C. The presumably suboptimal temperature (33°C) for northern populations otherwise yielded 76%-93% survival rates and fast swimming speeds in more southern populations. Still, in one species, northern hatchlings incubated at 33°C matched the elevated speeds of their southern counterparts, revealing a countergradient response. In northern populations of the two map turtles (Graptemys spp.), survival was also reduced (28%-60%) at 33°C and the development rate (relative to 30°C) increased by up to 75%. Our experiments on divergent taxa with similar nesting ecologies substantiate that the optimal thermal range for offspring production is variable. These findings encourage further work on how population- and species-level differences relate to local adaptation in widely distributed oviparous species.

Increased antibacterial activity of silver nanoparticles synthesized from leaf extract of Isodon coetsa

Application of nanotechnology for the synthesis of silver nanoparticles using plant extract can be a promising alternative to traditional chemical methods. Products of biological origin have less side effects, thus are safe for human health besides being cost-effective and pollution-free. Keeping in view this, the current work was designed for the environment- friendly production of silver nanoparticles (AgNPs) utilising Isodon coetsa acetone leaf extract. The effect of some important variables including incubation time, pH, AgNO3 concentration, temperature and volume of plant extract were examined for the optimum production of AgNPs. Synthesized nanoparticles were further characterized using atomic force microscopy (AFM), X-ray diffraction spectroscopy (XRD), and UV-visible absorption spectroscopy. In addition, the antibacterial potential of nanoparticles was also determined by agar-well diffusion method. The optimal conditions for synthesis of AgNPs achieved after 24 h of incubation at pH 9.0, 2mM AgNO3, 60°C temperature, and using 10% I. coetsa leaf extract. X-ray diffraction spectroscopy revealed that the size of the strongest peak for AgNPs was 9.5 nm, while atomic force microscopy confirmed their spherical shape with smooth surface without any pinholes or cracks. AgNPs showed excellent antibacterial action against Gram-positive and Gram-negative bacteria. The current study has contributed to the development of a simple and fast bioprocess for the synthesis of AgNPs of the appropriate size and form which also have increased antibacterial activity in comparison to crude extract. Therefore, the process of biogenically synthesised AgNPs from plants is a straightforward, commercially feasible, and environmentally benign approach that can be used in disease control while utilizing their important role as therapeutic agent for treating various ailments caused by pathogenic bacteria.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity

BackgroundLignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process.ResultsThe current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities.ConclusionThe results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Characteristics of Bacterial Biofilm Formation in Nasolacrimal Silicone Tubes Post-dacryocystorhinostomy.

To examine the biofilm formation characteristics of bacteria identified at the genus level in samples obtained from silicone tubes after dacryocystorhinostomy surgery. In the study involving consecutive patients who underwent dacryocystorhinostomy surgery at Ankara Bilkent City Hospital and whose silicone tubes were removed six months after surgery, between January 2023 and May 2023; the tubes were placed in glycerol-PBS (phosphate buffered saline) solution and cultured on descriptive selective media at the genus level. The biofilm-forming properties of the obtained isolates were examined in solid-air and liquid-air interphases. Salmonella Typhimurium ATCC SL1344 strain was used as the control bacterium. As a result of the analysis of the samples taken from the patients, Pseudomonas spp. was identified in three of the samples, Staphylococcus spp. in five of the samples, and Streptococcus spp. in one of the samples. Among these samples, except for the bacteria identified in samples one and five, the rest were found to be strong biofilm producers. In all strong biofilm producers, the maximum biofilm production time was determined as 72 h and the incubation temperature was 37°C. The presence of cellulose and amyloid proteins in biofilm matrix structures is identified. Swimming and swarming motilities were observed in all bacterial samples. Since biofilms are considered potential factors in the pathogenesis of infectious and inflammatory diseases, they are a subject that needs to be thoroughly investigated. In our study, although there were no clinical infections in any of the patients, biofilm formation was detected in the patient samples. The fact that the bacteria exhibited moderate to strong biofilm formation characteristics suggests that these microorganisms could be persistent infectious agents.

Green synthesis of silver nanoparticles using olive mill wastewater and olive stones extract and testing their antimicrobial activities against Escherichia coli and Staphylococcus epidermidis

Abstract Plant mediated synthesis of silver nanoparticles is eco-friendly and of low cost. The synthesis involves a reduction of silver ions and is controlled by several independent factors. In this work, silver nanoparticles (AgNPs) were successfully synthesized using olive stone extract (OSE) and olive mill wastewater (OMW) extract. The nanoparticle synthesis was monitored using the plasmon resonance observed in the UV–Vis absorption spectrum, in which a Voigt profile was fitted. The peak wavelength (λ 0), the peak area (A), and the Full Width at Half Maximum (FWHM) were the fitting parameters and were used as the response. The independent factors were the incubation temperature, the incubation time, the silver nitrate, extract, and sodium hydroxide concentrations. The influence of these factors was investigated ‘two factor at a time’, using interaction plots. Strong interaction was observed between all factors, with sodium hydroxide to have a crucial role. The optimum conditions for silver nanoparticle formation were a) OSE (1% v/v), AgNO3 (2 mM), and NaOH (0.2 mM), and b) OMW (2% v/v), AgNO3 (1 mM), and NaOH (7.9 mM), showing an absorption maximum at 414 nm, and 410 nm, respectively. The mean diameter of AgNPs using OMW, measured with Transmission Electron Microscopy was 12.87 ± 4.84 nm. Both types of AgNPs showed antibacterial action against Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli), using the broth microdilution assay. Both nanoparticle types inhibited bacterial growth up to one dilution higher than reference samples.

Factors regulating incubation temperature and thermal stress in hawksbills in St. Croix, USVI

The hawksbill sea turtle is listed as Critically Endangered under the IUCN Red List and has been slow to recover in the Caribbean due to historical exploitation and ongoing anthropogenic threats. In turtles, sex and reproductive success are determined by incubation temperatures, whereby lower temperatures produce male hatchlings and higher temperatures produce female hatchlings. As incubation temperatures increase due to climate change, nests are predicted to produce predominantly female hatchlings, threatening sex ratios, reproductive success, and species persistence. One of the largest remaining nesting hawksbill populations within the USA nests on Buck Island. This study aimed to (1) assess the factors driving incubation temperatures and (2) identify the relationship between hatch success and the proportion of time exposed to increasing incubation temperatures. Nest incubation temperatures, beach sector, habitat type, deposition month, percent soil composition, hatch success, and emergence success data were collected from 2019 to 2021. Differences in incubation temperatures across these factors confirmed that the absence of vegetative cover is increasing incubation temperatures on Buck Island, leading to reduced hatchling survival. Results from this study indicate that declines in hatch success may be driven by long-term exposure to temperatures that were previously considered in the literature to be non-lethal to embryos. These findings emphasize the continued need for conservation interventions to protect the future of hawksbills.

Exploitation of Selected Fungal Endophytes of Andrographis paniculata (Burm. f.) Wall. ex Nees for the Production and Optimisation of Tannase and Screening for their Associated Hydrolysing Enzymes; Amylase, Protease, Lipase, and Laccase

Endophytes represent a diverse domain of microorganisms with immense biotechnological potentialities. Metabolites from endophytes, especially fungi, are useful in industrial as well as pharmaceutical aspects. Here, endophytic fungal isolates of Andrographis paniculata collected from the Tapobon, Junglemahal region of West Bengal, India, have been studied for their hydrolyzing enzyme production abilities. Out of eighty-one isolates, twenty-one were positive for amylase, protease, lipase, laccase, and tannase action. Microscopic features of positive isolates revealed that the enzyme producers were Aspergillus sp., Fusarium sp., Alternaria sp., Trichoderma sp., Exerohilum sp., Nigrospora sp., Curvularia sp., Cladosporium sp., Cochliobolus sp., Tricothecium sp., Penicillium sp., Verticillium sp., and Cephalosporium sp. The amylolytic activity was remarkable in the case of endophytic Aspergillus sp. and Fusarium sp. Also, Aspergillus sp. and Alternaria sp. had proteolytic activity. Aspergillus sp., Mycelia sterilia-2, and Trichoderma sp. were potent lipase producers. Along with Exerohilum sp. endophytic Aspergillus sp. had positive laccase activity. The tannic acid degrading activity was highest for Aspergillus sp. APL11 followed by Helicosporium sp. and Fusarium sp. Tannase producing ability of Aspergillus sp. was optimized and an incubation time- 96h, incubation temperature- 29°C, initial medium pH- 6.5, carbon source- glucose, and nitrogen source- NaNO3 exhibited a two-fold scale-up of (2.83±0.05 u mL-1) in tannase action. These enzymes offer eco-friendly and efficient solutions across a wide range of industrial processes including food production, textile processing, and pharmaceutical synthesis. Fungal endophytes of medicinal plants act as an alternative source for hydrolysing enzymes.

Comparison of Three Preharvest Sampling Strategies to Monitor Pathogens in Cattle Lairage Areas

The objective of this study was to compare preharvest monitoring strategies by evaluating three different sampling methods in the lairage area to determine pathogen recovery for each sampling method and incoming pathogen prevalence from the cattle to inform in-plant decision making. Samples were gathered over a 5-month period, from February to June 2022, at a harvesting and processing facility located in Eastern Nebraska. Sampling methods included (i) fecal pats, (ii) boot swabs, and (iii) MicroTally swab. A total of 329 samples were collected over the study period (fecal pats: n = 105, boot swabs: n = 104, and MicroTally swabs: n = 120). Specific media combinations, an incubation temperature of 42°C, and incubation timepoints (18–24 h) were utilized for each matrix and the prevalence of Salmonella, Escherichia coli O157:H7, and six non-O157 Shiga-toxin producing E. coli (STEC) was evaluated using the BAX system Real-Time PCR assay. Overall, results from the study concluded that boot swabs were an effective sampling method for pathogen detection in the cattle lairage area. Boot swabs (97.1%) were statistically more likely to detect for Salmonella (p < 0.05) when compared to fecal pats (67.6%) and MicroTally swab (77.5%) methods. For E. coli O157:H7 and STEC – O26, O121, O45, and O103 prevalence, boot swabs were significantly better at detecting for these pathogens (p < 0.05) than MicroTally swabs (OR = 3.16 – 11.95) and a comparable sampling method to fecal pats (OR = 0.93 – 2.01, p > 0.05). Lastly, all three sampling methods detected a very low prevalence for E. coli O111 and O145; therefore, no further analysis was conducted. The boot swab sampling method was strongly favored because they require little training to implement, are inexpensive, and they do not require much sampling labor; therefore, would be a simple and effective sampling method to implement within the industry to evaluate pathogen prevalence preharvest.

Development of a composite microbial agent beneficial to improve drought and salt tolerance of Glycyrrhiza uralensis Fisch.

Plant growth is enhanced by microbiological agents. In order to develop environment friendly composite microbial agent that can also improve the salt and drought tolerance of plants, the orthogonal experiment and response surface methodology were used to screen nutrients and fermentation conditions of composite microbial agent, and pot experiment was conducted to validate the application effect of optimized and complex microbial agent. Compatibility test showed that G2 (Bacillus cereus) and G5 (Bacillus pumilus) did not show any inhibitory effect on each other’s growth in nutrient agar, moreover, in both the growth promotion characteristics and the seed germination tests, G2+G5 showed a synergistic effect. The process optimization results showed that the optimal carbon and nitrogen sources for the medium nutrients was maltose and yeast extract, and the optimal carbon-nitrogen ratio was 5:1; the optimal fermentation conditions included initial pH at 5.17, inoculation amount of 2.28%, liquid volume of 105.96 mL(250 mL)−1, and incubation temperature at 30.06℃. Pot experiment showed that application with composite microbial agent was able to increase antioxidant enzyme activities, ascorbat, proline content, and reduce reactive oxygen species accumulation and membrane peroxidation of G. uralensis seedlings under salt-drought stress. In conclusion, the composite G2+G5 agent possessed remarkable synergistic effect for improve plant tolerance to drought combined with salt stress, suggesting a noticeble practical potential in agricultural practice, and it can be used to develop newtype environment friendly microbial fertilizer.