Incubation Method Research Articles

Effects of winter water quality conditions on Atlantic Salmon embryo mortality and deformity rates in the Serpentine River (New Brunswick, Canada)

In-stream incubation of Atlantic Salmon (Salmo salar) embryos within artificial redds using Jordan/Scotty fish egg incubator boxes is an alternative to traditional hatchery-based incubation methods. Winter water quality conditions, such as temperature and dissolved oxygen vary within natural redds and influence wild embryo development and survival. The differences in landscape characteristics and in-stream habitat features at catchment-, reach-, and site spatial scales were investigated to determine how they influenced water quality conditions within the Serpentine River, New Brunswick. A total of 14,400 hatchery-fertilized salmon embryos were incubated within river substrates from November 2020 to January 2021. The proportion of embryo mortalities within our experiment was high (44.9%), but still within the range observed in the literature from other incubation experiments, and the proportion of embryo deformities between our sites was very low (2.7%). Despite variations in average water temperature, cumulative degree days, and coefficient of variation in dissolved oxygen (DO) concentration, none were statistically significant for explaining embryo mortalities or deformities. Patterns were, however, evident across sites showing trends towards a decrease in mortality with cumulative degree days, and an increase in deformities with greater variation in DO. Improving our knowledge on Atlantic Salmon incubation habitat supports future research and restoration opportunities for adaptation under a changing climate.

#253 : Administration of Umbilical Cord-Derived Mesenchymal Stem Cells Secretome to Sperm Preparation Medium: A Pilot Study

Background and Aims: As previously known, umbilical cord-derived mesenchymal stem cells (UC-MSCs) secretome has antioxidant potential that can reduce oxidative and nitrosative stress. This study is very important because understanding the effect of the UC-MSCs secretome on spermatozoa can assist in the development of sperm quality improvement techniques. This study aims to examine the sperm concentration and velocity before and after the addition of UC-MSCs secretome as a modification in sperm preparation with time duration and concentration variation. Method: A pilot study used a secretome produced at Dr. Cipto Mangunkusumo Hospital. Ejaculate samples were collected from married couples who already had children and the results of the semen analysis showed normozoospermia and DNA fragmentation index examination at a good level (15%). Sperm preparation was done using the Swim-up method. Samples were divided into 7 treatments with different ratios of secretions and ejaculate and washing medium (1:1 and 1:2) with incubation time durations (30, 45, and 60 minutes) to find optimal conditions to be used in further studies. The sperm concentration and velocity were observed before and after incubation. Results: Based on the results, it can be observed differences in sperm concentration and velocity from various incubation methods using UC-MSCs secretome. The 1:1 (v/v) secretome treatment had the highest spermatozoa concentration (68 million/mL), while the 1:2 (v/v) secretome treatment had the fastest spermatozoa velocity (30.51 μm/s), with the same incubation time of 45 minutes. The treatment group that was given UC-MSCs secretome 1:1 (60 minutes) and 1:2 (60 minutes) had slower spermatozoa velocity (22.91 μm/s and 21.45 μm/s, respectively) compared to the control group (26.39 μm/s). Conclusion: The administration of UC-MSCs secretome to the sperm preparation medium could increase the concentration and velocity of spermatozoa after incubation. However, this conclusion is provisional and needs further study to support this technique’s development.

Will the endophytic fungus Phomopsis liquidambari increase N-mineralization in maize soil?

Endophytes can be developed into biocontrol agents and can be fungi, bacteria, or archaea that live inside plant tissues without causing symptoms of disease. Phomopsis liquidambari is an endophytic fungus that plays an important ecosystem role as a biofertilizer by helping its host obtain soil nitrogen. How this fungus impacts N mineralization and microbial communities is little known. Our understanding of soil nutrient transformations and soil-plant-microbe interactions in Phomopsis liquidambari-crop versus conventional crop systems is incomplete. This study provided a better understanding of the effect of Phomopsis liquidambari on nitrogen mineralization and investigated the interaction between P. liquidambari and nitrogen, which in turn will be helpful to the farmer in reducing the required amount of soil N fertilizer. This change in N availability in maize soil will have significant implications for soil productivity and plant N utilization, especially in N-limited soils, and significantly reduce the required amount of soil N fertilizer. The effect of P. liquidambari on N mineralization in maize soil was investigated by treating it with four levels of N (urea) at rates of 0, 1.25, 2.5, and 3.75 g of nitrogen. N-mineralization was determined by the anaerobic incubation method. Were stored for 7 days in an incubator at a constant 37 C. A colorimetric microplate procedure was used for NH4+-N analysis. A significant increase in the available NH4+-N contents was reported in soil maize (Zea mays L.) inoculated with P. liquidambari, which increased by 80%. A significant increase in N-mineralization was observed under all N conditions. This work highlighted the importance of the fungal endophyte for soil N-mineralization with lower N input. Using this fungal agent will almost certainly help reduce fertilizer input.

Minimizing Carbon Dioxide Emissions with Clinoptilolite Zeolite in Moris Pineapple Cultivation on Drained Sapric Soils

Drained tropical peat soils for agriculture emit more carbon dioxide (CO2) into the atmosphere from their stored carbon compared with their pristine state. Field and laboratory experiments were conducted to assess whether the natural zeolite of the clinoptilolite (ZeoC) species could be included in the pineapple fertilization program to decrease the CO2 emissions from tropical sapric soils. The static closed-chamber and laboratory incubation methods were used to determine the effects of ZeoC on the CO2 emitted from a drained sapric soil planted with Moris pineapple. The treatments assessed were as follows: (a) suggested ratio of ZeoC (5 g, 10 g, 14 g, and 20 g of ZeoC) and 20 g compound NPK 30:1:32 fertilizer, enumerated based on the pineapple plant requirement; (b) 20 g of compound NPK 30:1:32 fertilizer only; and (c) unfertilized sapric soils. The drained sapric soils amended with ZeoC (rate of 5 g to 20 g/plant) minimized the CO2 emissions compared with those without the ZeoC, because of the physical and chemical sorption of organic compounds and polar CO2 onto the lattices of ZeoC, which inhibited organic matter decomposition. ZeoC fertilization reduces sapric soil acidity and improves Moris pineapple fruit quality attributes and yield. Monthly pineapple fertilization with ZeoC at the vegetative and flowering phases is an alternative agronomic strategy to reduce CO2 emissions. This approach does not reduce pineapple yield on drained tropical sapric soils.

Sediment respiration dynamics and its contribution to carbon emissions in stratified reservoirs

Sediment respiration, the relation between dissolved oxygen (DO) attribution and carbon dioxide (CO2) emission, is an important index of the aquatic ecosystem and a key concern in the evaluation of reservoir cleanliness performance. To understand the sediment respiration dynamics regulated by the thermal stratification that is common in deep-water reservoirs, this study conducted in-situ measurements of thermal structures and benthic environments in the Daheiting Reservoir for 16 months. Then, the variations of DO and CO2 fluxes at the sediment-water interface (SWI) were obtained based on the aquatic eddy correlation method and the newly proposed virtual incubator method, respectively. The results show that the SWI fluxes dynamics can be decomposed into tendency variations dominated by thermal stratification and impulse variations induced by extreme events. The annual average SWI DO and CO2 fluxes of 3.80–6.62 and 1.92–3.15 mmol m−2·d−1 are estimated in the study site through the respiration dynamics, respectively, which CO2 flux is at a moderate level among the nearly 50 lakes and reservoirs worldwide but accounts for less than 15% of the total CO2 emission of this reservoir. Moreover, the sediment respiration quotient in stratified reservoirs is lower than in other aquatic environments, suggesting that the permanently flooded area is a weak net carbon source, while most carbon emissions from stratified reservoirs are carbon displacement or net carbon generated within the water. Sediment net carbon emissions correspond to human benefits such as flood control, power generation, and fisheries, whereas water net carbon emissions are usually not beneficial. Therefore, reducing net carbon emissions generated in the water may become an important way to achieve low-carbon operation of deep-water reservoirs.

Differences in Methanogenic Pathways and Communities in Paddy Soils Under Three Typical Cropping Modes

AbstractThe methane (CH4) production process in paddy fields varies depending on different cropping modes. However, there is limited knowledge on the changes in anaerobically produced CH4 isotopic ratios (δ13CH4), the methanogenic pathways, and the dominant communities of methanogens in paddy soils under different modes. To address this, we conducted experiments using anaerobic incubation methods, stable carbon isotope fractionation through inhibition studies with fluoromethane, and high‐throughput sequencing. These methods allowed us to measure the CH4 production potential (MPP), the relative contribution of acetoclastic methanogenesis to CH4 production (fac), and the composition of the methanogen community in paddy soils under three typical cropping modes: Rice‐Wheat (RW), Rice‐Fallow (RF), and Double‐Rice (DR) in China. The results showed that MPP was 30.7 μg CH4 g−1 d−1 in DR soil, around 57% and 66% higher than that in RW and RF soils, respectively, possibly due to the lower pH, clay content, and higher abundance of the mcrA gene. Moreover, RF soil had the highest value of produced δ13CH4 (−43.9‰) but the lowest value of produced δ13CO2 (−26.3‰). Based on the carbon isotope fractionation associated with methanogenesis via H2/CO2 reduction (1.049–1.062), the values of fac estimated in the RF soil (80%–98%) were much higher than that in the RW (39%–60%) and DR soils (52%–75%). This could be supported by the finding showing that Methanosarcina, the acetoclastic methanogens, were dominant in RF soil, while Methanosarcina and Methanobacterium (the hydrogenotrophic methanogens) dominated in RW and DR soils. Redundancy analysis revealed that the community structure of methanogens was significantly affected by soil pH. The findings suggest that differences in the abundance and composition of methanogens, possibly driven by soil pH, play a key role in regulating methanogenesis in the paddy soils, and further provide valuable insights into the process of CH4 production in different cropping modes.

Effect of Metal Cations with Different Valences on the Humus Composition of Dark-Brown Soil Mixed with Tilia Wood Shavings

To clarify the contribution of the bridging effect from three metal cations (K+, Mn2+, and Fe3+) on the humification of lignin-rich Tilia wood shavings and further enrich the theory of lignin humification, an indoor incubation method with constant temperature and humidity was adopted. K+, Mn2+, and Fe3+ served as additives, with CK as the control for studying the differential influence of metal cations with different valences on the humus composition of dark-brown soil mixed with Tilia wood shavings. The change in the C contents of water-soluble substance (CWSS), humic-extracted acid (CHE) and humin (CHu), ∆logK value of HE, atomic ratio and FTIR spectra of humic acid (HA), and the ratio of C content of humic acid to fulvic acid (CHA/CFA) of dark-brown soil mixed with Tilia wood shavings were analyzed after 0, 30, 80, and 150 days of incubation, and the following conclusions were reached: (1) The addition of metal cations, regardless of their valence, could effectively improve the microbial utilization and consumption of WSS, and the effect was as follows: Fe3+ > Mn2+ > K+. The addition of three metal cations could effectively inhibit mineralization and reduce the loss of TOC, and the effect could be seen as follows: Fe3+ > Mn2+ > K+. (2) Although the CHE content first decreased and then increased with incubation, the addition of Fe3+ and Mn2+ ions increased the CHE content, showing that Fe3+ > Mn2+, and K+ ions had no significant effect. Throughout the incubation, the structure of HE molecules changed first via a complex process and then through a simple process. Comparing the change before and after the incubation, the overall structure of HE molecules tended to be simpler with the CK control, and HE became more complicated with the addition of Fe3+ and Mn2+; however, the addition of K+ had little effect on the structure of HE molecules. (3) At the end of the incubation, the addition of Fe3+, Mn2+, and K+ ions strengthened the molecular condensation of HA and its aromatization degree, while the CK control without any added metal cations caused HA molecules to decompose and obtain a greater aliphatic degree. In addition, the number of O-containing functional groups and N-containing compounds in HA molecules increased to varying degrees regardless of which metal cation was added. The decomposition of Tilia wood chips led to a partial entry of the decomposition products into the HA component, which was then reconsumed by continuous mineralization. After incubation, the polysaccharides in HA molecules were consumed only with the addition of Mn2+ ions. Fe3+ and Mn2+ ions had greater advantages in increasing the CHA/CFA ratio and improving the humus quality than K+ ions. (4) The addition of metal cations could effectively inhibit the mineralization and decomposition of the Hu component, among which Fe3+ ions had the most significant effect, followed by Mn2+ ions. Compared to monovalent cations (K+), polyvalent cations (Fe3+ and Mn2+) had the advantage of a bridging effect, and their addition promoted the microbial utilization of WSS, effectively reduced the loss of TOC, increased the CHE content, complicated its molecular structure, improved the humus quality, and inhibited the decomposition of Hu. Regardless of which metal cation was added, the degree of molecular polycondensation and aromatization of HA was enhanced, and the number of O-functional groups and N-containing compounds in HA molecules increased.

Nutritive value and in-vitro digestibility of peels and pomaces of different citrus species

The nutritive value and invitro digestibility of agro-industrial byproducts of Citrus aurantium, Citrus paradisi, Citrus reticulata, Citrus limon and Citrus sinensis were assessed for their feed modification potential. Nutritive value assessment was done through chemical analysis while the ANKOM Daisy II incubator method was used to determine the invitro digestibility. At p>0.05, there was no statistical significance for nutritive value of the citrus byproducts for the five species. The agro-industrial by-products had significantly different phenolic content. Invitro digestibility of the peels significantly differed from the pomaces (p<0.001). The byproducts of the five citrus species showed potential to be considered as non-forage energy sources in ruminant feed. Lemon byproducts had high protein but not sufficient as a single protein source while bitter orange pomace may be considered for antioxidant capacity and high digestibility. Abundance of citrus agro-industrial byproducts, nutritive value potentials and their digestibility makes it a good strategy for ruminant feed modification and utilization.

Antibacterial and antivirulence factor activities of protein hydrolysates from Phatthalung Sangyod rice (Oryza sativa L.) seeds against zoonotic and foodborne pathogens.

Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals. After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 μg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli, P. aeruginosa, B. cereus, and S. aureus, using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum. Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 μg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa, B. cereus, and E. coli, at 256 and >256 μg/mL, respectively. We observed that at least 256 μg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16-128 μg/mL, it exhibited antibiofilm activity against S. aureus. Furthermore, at 256 μg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 μg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 μg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 μg/mL. Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals.

Microplate-based incubation method for quality assessment of eggs of Indian major carp Catla catla

Incubation of fertilised eggs from the Indian major carp species, Catla catla was carriedout in tissue culture microplates to assess the quality in terms of hatching rate and posthatchsurvival rate. Eggs from five females (F1, F2, F3, F5 and F6) were strip spawned andfertilised with milt from a single male. The fertilised eggs were distributed at one egg perwell of 24 well microplates, with three plates for each female. The plates were incubated at30oC in an incubator with continuous shaking and complete hatching was observed by 36 h.The hatching rate varied within a range of 0 to 52.8% showing the maximum of 5% for F5followed by F1 (50%) which were significantly higher than the other three females (F2-18.1,F6-5.6 and F3-0%). The eggs from these two females (F1 and F5) thus could be consideredas good. The survival percentage of hatchlings recorded for a period of 8 days post-hatchingwas found to vary from 58.3 to 83.3 and there was low variability among different females onany observed day. Between these two egg quality determinants, the hatching rate was foundto be more suitable considering the wide variability shown by different females indicatingthe quality difference. The microplate method seems to be an appropriate tool to assess theegg quality in catla and can be extended to other species as well. Keywords:Catla catla, Egg quality, Hatching rate, Microplate

A Simple Method of Plant Sectioning Using Agarose Embedding Technique for Screening Intracellular Green Fluorescent Protein

The transformed plant tissue sections through the agroinfiltration method are difficult to be observed under a fluorescent microscope. It is due to the softness of the post-transformation explant. This research was conducted to optimize the sectioning of the transformed plant using the agarose embedding technique. The optimization method was carried out in various agarose concentrations: 2%, 4%, and 6%, followed by five minutes of incubation in various temperatures: -80 oC, 4 oC, and 25 oC. The results showed that the embedding method using 6% agarose produced a transformed plant section was better than 2% and 4% agarose. Meanwhile, the incubation method at 25 oC was more suitable for the transformed plant tissue than 4 oC and -80 oC. Green Fluorescent Protein (GFP) could be determined using these methods under a fluorescent microscope. Thus, the optimum method for making sections of transformed plants by embedding was using 6% agarose then incubating at 25 oC for 5 minutes.

Higher microbial C use efficiency in paddy than in adjacent upland soils: evidence from continental scale

Regularly flooded rice paddies usually show greater soil organic C and microbial-derived C contents than adjacent upland counterparts, but the soil microbial physiological traits under these two different land uses spanning regions remain unclear. Here, we collected 40 pairs of adjacent paddy and upland soils from four different climates (mid temperate, warm temperate, subtropics, and tropics) across eastern China to determine the microbial growth, respiration, and C use efficiency using the 18O-H2O incubation method. Upland soils from warmer climates exhibited lower microbial growth but higher respiration normalized to microbial biomass C (qGrowth and qRespiration, respectively) than those from cooler climates, since the lower soil pH and higher clay content in warmer climates induced a shift from microbial growth to respiration. Whereas, paddy soils from warmer climates had consistently lower qGrowth and qRespiration than cooler climates, probably due to the long term water-logged condition decreased the sensitivity of microbial metabolism in response to lower pH. Paddy soils had higher qGrowth, but lower qRespiration than upland soils, resulting in a greater C use efficiency regardless of climate zones. The difference in microbial C use efficiency between paddy and upland soils was positively correlated to their difference in soil organic C content. From the perspective of microbial C metabolism, the greater organic C accumulation in paddy than that in upland soils is attributed to the weaker microbial uptake of organic C and stronger microbial anabolism under the water-logged condition.

A Bacterial Nanomedicine Combines Photodynamic-Immunotherapy and Chemotherapy for Enhanced Treatment of Oral Squamous Cell Carcinoma.

Bacterial therapy is an emerging hotspot in tumor immunotherapy, which can initiate antitumor immune activation through multiple mechanisms. Porphyromonas gingivalis (Pg), a pathogenic bacterium inhabiting the oral cavity, contains a great deal of pathogen associated molecular patterns that can activate various innate immune cells to promote antitumor immunity. Owing to the presence of protoporphyrin IX (PpIX), Pg is also an excellent photosensitizer for photodynamic therapy (PDT) via the in situ generation of reactive oxygen species. This study reports a bacterial nanomedicine (nmPg) fabricated from Pg through lysozyme degradation, ammonium chloride lysis, and nanoextrusion, which has potent PDT and immune activation performances for oral squamous cell carcinoma (OSCC) treatment. To further promote the tumoricidal efficacy, a commonly used chemotherapeutic drug doxorubicin (DOX) is efficiently encapsulated into nmPg through a simple incubation method. nmPg/DOX thus prepared exhibits significant synergistic effects on inhibiting the growth and metastasis of OSCC both in vitro and in vivo via photodynamic-immunotherapy and chemotherapy. In summary, this work develops a promising bacterial nanomedicine for enhanced treatment of OSCC.

Effects of Ionized Water Irrigation on Organic Nitrogen Mineralization in Saline-Alkali Soil in China

The application of ionized water to irrigation, as a new type of water treatment technology, can improve the spatial distribution of water in soil and increase water utilization efficiency, which may affect the microbiological processes involved in nitrogen transformation and alter soil nitrogen supply capability. However, the effects of ionized water technology on soil organic nitrogen mineralization are still in need of further research. In this study, we investigated the soil organic nitrogen mineralization process with four different water additions: non-ionized fresh water (CK), ionized fresh water (DE), non-ionized brackish water (BCK), and ionized brackish water (BDE). By using a short-term laboratory incubation method, we monitored the changes of the inorganic nitrogen concentration in each treatment during the incubation process. We compared the net nitrogen mineralization and nitrogen mineralization rates in different treatments, and fitted the organic nitrogen mineralization process with three models (One-pool model, Special model, and EATM model). We divided the whole incubation process into three periods based on the differences of the organic nitrogen mineralization trends. The results demonstrated that when DE was compared with CK, the net nitrogen mineralization increased by 21.97% and the nitrogen mineralization rate increased by 20.42% in the latter incubation period. When BDE was compared with BCK, the net nitrogen mineralization decreased by 3.63%, and the nitrogen mineralization rate increased by 21.86% in the latter incubation period. When BCK was compared with CK, brackish water irrigation reduced the organic nitrogen mineralization intensity to a certain extent, with the net nitrogen mineralization decreased by 11.62% and the nitrogen mineralization rate decreased by 41.07% in the whole incubation process. When BDE was compared with DE, the net nitrogen mineralization decreased by 30.09% and the nitrogen mineralization rate decreased by 53.39% in the whole incubation process. The simulation model of the soil organic nitrogen mineralization process showed that the special model and EATM model are superior to the One-pool model. This study provides a theoretical basis for the popularization and application of ionized water irrigation in agricultural production.

Design of a lipid nano-delivery system containing recombinant Candida albicans chitinase 3 as a potential vaccine against fungal infections

Opportunistic fungi cause lethal systemic infections and impose high medical costs to health systems. The World Health Organization has recognized the importance of fungal infections, including them in its global priority list guiding research, development, and discovery of new therapeutic approaches. Fungal vaccine development has been proposed as one of the treatment and prevention strategies in the last decade. In this study, we present the design of a lipid antigen delivery system based on Dioctadecyldimethylammonium bromide: Monoolein (DODAB: MO) containing recombinant Candida albicans Chitinase 3 (Cht3) for modulation the immune response against fungal infections. Several DODAB:MO liposomes containing Cht3 were prepared and those prepared by the incubation method and containing 5 µg/mL Cht3 were selected due to their favorable size, ζ-potential and stability, suited for antigen delivery applications. The encapsulation of Cht3 in these liposomes resulted in a significant increase in cellular uptake compared to empty liposomes, demonstrating their efficacy in delivering the antigen. Moreover, the liposomes proved to be safe for use in immunization procedures. Subcutaneous administration of Cht3 liposomes elicited a Th1/Th17 immune response profile, associated with the production of high levels of antibodies against Cht3. These antibodies recognized both the native and the recombinant forms of the protein, opsonizing mother-yeast at the cell scars, which has the potential to disrupt cell separation and hinder yeast growth. The findings suggest that the designed lipid antigen delivery system shows promise as a potential candidate for enhancing immune responses against fungal infections, offering a valuable strategy for future fungal vaccine development.

Utilization of Hot-Spring-Water-Bound CO2 for Horticulture Plants Using Incubation Method

This study focused on free carbon dioxide (H2CO3) contained in volcanic hot spring water. It was clarified whether free carbon dioxide can be emitted into the atmosphere and increase the CO2 concentration in greenhouses by using the incubation method. Factors influencing the increase in CO2 concentration were identified based on implementation time in a demonstration experiment, temperature and humidity in the incubator, the amount of hot spring water, and the contact area between the hot spring water and the air. An incubator with an inner volume of 2.17 × 107 mm3 was used in this study. The results showed that unrelated factors were the implementation time in the demonstration experiment, temperature, and humidity. There was a relationship with the amount of hot spring water. The increment of CO2 concentration in the air by free carbon dioxide increased proportionally by increasing the amount of water. Free carbon dioxide contained in hot spring water can be utilized by considering the volume of facility horticulture and plant factories. The ideal methods for utility are to replace the hot spring water every few minutes and to increase the contact area between the air and the hot spring water.

Impact of the Drug Loading Method on the Drug Distribution and Biological Efficacy of Exosomes.

Exosomes are biological nanovesicles that are intrinsically loaded with thousands of biomacromolecules and are principally responsible for cell-to-cell communication. Inspired by the natural payload, they have been extensively investigated as drug delivery vehicles; however, the drug distribution, whether into or onto exosomes, is still debatable. In the present work, we have tried to investigate it systemically by selecting 5-fluorouracil (5-FU) (hydrophilic) and paclitaxel (PAC) (hydrophobic), drugs with very different physicochemical characteristics, for the loading to the exosomes. Exosomes were obtained from bovine milk, and the drugs were loaded using three different methods: incubation, sonication, and triton x-100. The particle size was found to be approximately 100 nm in all the cases; however, the highest drug loading was found in the sonication method. Fluorescence spectrophotometer, EDX analysis, EDX mapping, XPS, and XRD analysis indicated the possible presence of more drugs over the surface in the case of the incubation method. Drugs loaded by the sonication method had more controlled release than simple incubation and triton x-100. The method of drug loading had an insignificant effect on the cytotoxicity while in line with our previous observation, the combination (PAC and 5-FU) exhibited synergism as evidenced by ROS assay, colony formation assay, and mitochondrial membrane potential assay.

Studies on Seed Borne Mycoflora of Soybean Seeds by Incubation Methods

The present study was conducted to detect and identify seed-borne pathogenic fungi in some soybean varieties and their effect on seed germination. Six varieties of soybean seed samples viz.JS 95-60, JS 97-52, RSC 10-46, RSC 10-71, CG Soya-1 and local variety were selected for the experiment. Twelve seed-borne mycoflora viz. Aspergillus flavus, Aspergillus niger, Alternaria spp., Cladosporium spp., Curvularia spp., Rhizopus spp., Trichoderma spp., Rhizoctonia spp., Nigrospora spp., Macrophomina spp., Fusarium spp. and Penicillium spp. were detected and identified from the seeds of six soybean varieties by using the standard blotter method, agar plate method, 2,4-D blotter method, roll paper towel method and deep freeze method. The seed sample of local variety recorded the highest percentage of mycoflora with the lowest germination percentage, whereas CG Soya-1 variety found as the least percentage of mycoflora with the highest germination percentage compared to other varieties of soybean taken in the study. Among all of the incubation methods, the agar plate was found as the most effective in detecting seed-related mycoflora, on soybean seeds.

The effect of bovine milk lactoferrin-loaded exosomes (exoLF) on human MDA-MB-231 breast cancer cell line

BackgroundCancer is still the most challenging disease and is responsible for many deaths worldwide. Considerable research now focuses on targeted therapy in cancer using natural components to improve anti-tumor efficacy and reduce unfavorable effects. Lactoferrin is an iron-binding glycoprotein found in body fluids. Increasing evidence suggests that lactoferrin is a safe agent capable of inducing anti-cancer effects. Therefore, we conducted a study to evaluate the effects of the exosomal form of bovine milk lactoferrin on a human MDA-MB-231 breast cancer cell line.MethodsThe exosomes were isolated from cancer cells by ultracentrifugation and incorporated with bovine milk lactoferrin through the incubation method. The average size of the purified exosome was determined using SEM imaging and DLS analysis. The maximum percentage of lactoferrin-loaded exosomes (exoLF) was achieved by incubating 1 mg/ml of lactoferrin with 30 µg/ml of MDA-MB-231 cells-derived exosomes. Following treatment of MDA-MB-231 cancer cells and normal cells with 1 mg/ml exoLF MTT assay applied to evaluate the cytotoxicity, PI/ annexin V analysis was carried out to illustrate the apoptotic phenotype, and the real-time PCR was performed to assess the pro-apoptotic protein, Bid, and anti-apoptotic protein, Bcl-2.ResultsThe average size of the purified exosome was about 100 nm. The maximum lactoferrin loading efficiency of exoLF was 29.72%. MTT assay showed that although the 1 mg/ml exoLF treatment of MDA-MB-231 cancer cells induced 50% cell growth inhibition, normal mesenchymal stem cells remained viable. PI/ annexin V analysis revealed that 34% of cancer cells had late apoptotic phenotype after treatment. The real-time PCR showed an elevated expression of pro-apoptotic protein Bid and diminished anti-apoptotic protein Bcl-2 following exoLF treatment.ConclusionThese results suggested that exoLF could induce selective cytotoxicity against cancer cells compared to normal cells. Incorporating lactoferrin into the exosome seems an effective agent for cancer therapy. However, further studies are required to evaluate anti-tumor efficacy and the underlying mechanism of exoLF in various cancer cell lines and animal models.

Development and evaluation of a medium-free incubation method for hatching Chinese soft-shelled turtle (Pelodiscus sinensis) eggs

In aquaculture, the traditional method for hatching Pelodiscus sinensis eggs needs further investigation. Here, we developed a medium-free incubation method for hatching P. sinensis eggs and evaluated its differences from the traditional vermiculite incubation method and the effects of light on it. We found that medium-free incubation had no significant effects on hatching success, body weight, carapace length, carapace width, plastron length, plastron width, and righting response time of P. sinensis compared with those under traditional vermiculite incubation, irrespective of thermostatic or fluctuating temperature conditions. Furthermore, light treatment significantly shortened the incubation period and negatively affected body weight, carapace length, carapace width, plastron length, and righting response time during medium-free incubation. Additionally, this study is the first to report the phenomenon of egg-hatching synchrony in P. sinensis. Overall, our results provide basic data and novel insights for improving the method for hatching P. sinensis eggs.