Final Concentration Research Articles

Improving antibiotic removal and anaerobic digestion performance of discarded cefradine pellets through thermo-alkaline pretreatment

Discarded cefradine pellets (DCP) as the hazardous wastes contain lots of bioavailable sucrose. Anaerobic digestion (AD) may be a promising technology for treating DCP, achieving dual goals of waste treatment and resource recovery. However, high concentration of cefradine will inhibit the AD process. This study applied thermo-alkaline pretreatment (TAP) to remove cefradine and improve the AD performance of DCP. Around 95% cefradine could be degraded to different intermediate degradation products (TPs) in TAP at optimal condition, and hydrolysis and hydrogenation were the main degradation pathways. Quantitative structure-activity relationship analysis indicated that the main TPs exhibited lower toxicity than cefradine, and DCP residues after TAP were almost not toxic to E. coli K12 and B. subtilis growth by antibacterial activity analysis. Therefore, TAP promoted the biomethane yield in AD of DCP residues (274.74 mL/g COD), which was 1.91 times that of control group. Besides, compared to control group, final cefradine concentrations in liquids and sludge were significantly decreased in AD system with TAP, lowering environmental risk and indicating stronger prospect for process application. Microbiological analysis revealed that acidogens (Macellibacteroides, Bacteroides), syntrophs (Syntrophobacter, Syntrophorhabdus), and acetoclastic Methanosaeta were enriched in AD system with TAP, which contributed to improving AD performance of DCP.

Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid.

A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic bead-based (MB) isolation. Furthermore, we modified both methods, including pre- and post-elution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P<0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.

Efficient Production of Succinic Acid from Sugarcane Bagasse Hydrolysate by Actinobacillus succinogenes GXAS137

Sugarcane bagasse (SCB) is an abundant agricultural waste, rich in cellulose and hemicellulose, that could be used as an ideal raw material for succinic acid (SA) production. A two-step chemical pretreatment, involving alkali extraction and alkaline hydrogen peroxide treatment, was utilized to treat SCB, followed by multi-enzyme hydrolysis to obtain a reducing sugar hydrolysate mainly composed of glucose and xylose. Optimization of the multi-enzyme hydrolysis of pretreated SCB resulted in a final reducing sugar concentration of 78.34 g/L. In order to enhance the bioconversion of SCB to SA and to reduce the production costs, the initial reducing sugar concentration, nitrogen source, and MgCO3 content were further optimized. The results demonstrated that the inexpensive corn steep liquor powder (CSLP) could be utilized as an alternative nitrogen source to yeast extract for the production of SA; and the optimal concentrations of initial reducing sugar, CSLP, and MgCO3 were 70 g/L, 18 g/L, and 60 g/L, respectively. When fed-batch fermentation was conducted in a 2 L stirred bioreactor, approximately 72.9 g/L of SA was produced, with a yield of 83.2% and a productivity of 1.40 g/L/h. The high SA concentration, yield, and productivity achieved in this study demonstrate the potential of SCB, an agricultural waste, as a viable alternative substrate for Actinobacillus succinogenes GXAS137 to produce SA. This lays a solid foundation for the resource utilization of agricultural waste and cost-effective industrial-scale production of SA in the future.

Microalgae technology for polishing chemically-treated fish processing wastewater

The development of cost-effective treatment methods for nutrient-rich effluents is of great importance. In this study, microalgal-based technology was investigated as a final polishing stage for fish processing wastewater (FPWW) after fat and oil removal, with the purpose of facilitating wastewater reuse and nutrient recovery. First, the effect of FPWW filtration on the growth of two microalgal species (Chlorella sorokiniana and Selenastrum sp.) was investigated under batch mode. For both microalgae, the final biomass concentration and nutrient removal levels were higher in unfiltered FPWW than in filtered FPWW. Chlorella sorokiniana growth was notably greater than that of Selenastrum sp. Chlorella sorokiniana was further cultivated in a semi-continuous mode, which lead to stable microalgae growth and high nutrient uptake in unfiltered wastewater with no need to eliminate indigenous microorganisms and contamination. However, based on biochemical analysis, filtration increased the availability of essential fatty acids and carotenoids from Chlorella sorokiniana, thus increasing the commercial value of the algal biomass.

Biological Control of Chili Anthracnose Disease using Talaromyces flavus Bodhi001

Background: Anthracnose disease of chili caused by Colletotrichum spp is one of the most destructive diseases affecting chili fruits in Thailand and significantly reduces fruit quality and chili production. Currently, this disease is managed primarily with synthetic fungicides that may affect public health and the environment adversely. Consequently, there is a need for biological management options. In vitro and in vivo evaluations of the antagonistic activity of Talaromyces flavus Bodhi001, Talaromyces trachyspermus Bodhi002, Talaromyces flavus Bodhi003 and Neosartorya fischeri Bodhi004 against Colletotrichum capsici, the causal agent of chili anthracnose disease, were conducted in the present study. Methods: The activity of antagonistic fungi against C. capsici was determined using PDA plate by dual culture method. The spore suspensions of C. capsici and antagonistic fungi were prepared in sterile water and adjusted using a hemocytometer to achieve a final concentration of about 106 spores mL-1. Result: The most effective antagonistic strains were T. flavus Bodhi001 and N. fischeri Bodhi004, which inhibited the mycelial growth of C. capsici by 68.99% and 70.76%, respectively. Interestingly, the antagonistic T. flavus Bodhi001 strain was the most effective at reducing the severity of chili anthracnose in vivo by up to 80%. The biological control activity of T. flavus Bodhi001 was to produce antibiosis against C. capsici; therefore, testing can be recommended to confirm its field trial stability. The results indicate that the application of the antagonistic fungi T. flavus Bodhi001 may be quite effective in biological control of chili anthracnose.

Quantitative risk ranking of mycotoxins in milk under climate change scenarios

Mycotoxins are toxic fungal metabolites that may occur in crops. Mycotoxins may carry-over into bovine milk if bovines ingest mycotoxin-contaminated feed. Due to climate change, there may be a potential increase in the prevalence and concentration of mycotoxins in crops. However, the toxicity to humans and the carry-over rate of mycotoxins from feed to milk from bovines varies considerably. This research aimed to rank emerging and existing mycotoxins under different climate change scenarios based on their occurrence in milk and their toxicity to humans. The quantitative risk ranking took a probabilistic approach, using Monte-Carlo simulation to take account of input uncertainties and variabilities. Mycotoxins were ranked based on their hazard quotient, calculated using estimated daily intake and tolerable daily intake values. Four climate change scenarios were assessed, including an Irish baseline model in addition to best-case, worst-case, and most likely scenarios, corresponding to equivalent Intergovernmental Panel on Climate Change (IPCC) scenarios. This research prioritised aflatoxin B1, zearalenone, and T-2 and HT-2 toxin as potential human health hazards for adults and children compared to other mycotoxins under all scenarios. Relatively lower risks were found to be associated with mycophenolic acid, enniatins, and deoxynivalenol. Overall, the carry-over rate of mycotoxins, the milk consumption, and the concentration of mycotoxins in silage, maize, and wheat were found to be the most sensitive parameters (positively correlated) of this probabilistic model. Though climate change may impact mycotoxin prevalence and concentration in crops, the carry-over rate notably affects the final concentration of mycotoxin in milk to a greater extent. The results obtained in this study facilitate the identification of risk reduction measures to limit mycotoxin contamination of dairy products, considering potential climate change influences.

Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol.

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 106 per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 106 exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 106 (49.9% and 19.7%) and 35 × 106 (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration.

Evaluation of White Rot Fungus to Control Growth of Escherichia coli in Cattle Manure

Biological soil amendments of animal origin, such as aqueous dairy manure, may be contaminated with microbial pathogens that can subsequently result in contaminated soil, water runoff, and crops. Multiple mitigation strategies are being evaluated to reduce these risks. Inclusion of filamentous fungus in a biofiltration system to inactivate pathogenic bacteria in aqueous dairy manure prior to land application is explored in this study as a preharvest preventative method. Of the microbes used to remediate biologically contaminated sites, ligninolytic white-rot fungi have been previously studied for their ability to degrade a wide variety of toxic or persistent environmental contaminants. Reduction of two E. coli strains (E. coli TVS355 and E. coli O157:H7 4407) was evaluated in aqueous dairy manure and PBS and in the presence of white-rot fungi Pleurotus ostreatus on three different nutrient sources (woodchips (WC), spent mushroom compost (SMC), and reticulated polyurethane foam (RPF)). Overall, E. coli TVS355 was more persistent in aqueous dairy manure and PBS, surviving for 50 days in the presence of P. ostreatus, with a final concentration of 4 log CFU/g in aqueous manure and 7 log CFU/g in PBS. However, greater (p < 0.0001) reduction of E. coli O157:H7 was observed, surviving for 50 days at an average of 4 log CFU/g in aqueous dairy manure and an average of 3 log CFU/g in PBS. Therefore, P. ostreatus has the potential to result in bacterial decay, with potential reduction observed in E. coli O157:H7. The RPF matrix showed positive results as a potential model for a nutrient limiting resource for P. ostreatus and could be the key to increased bacterial reductions if resulting in ligninolytic activity in order to seek other nutrient sources.

Low molecular weight heparin decreases pro-coagulant activity in clinical MSC products

Background aimsMesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT). MethodsClinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load. ResultsPresence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher. DiscussionAll MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE.

The effect of thalidomide on the invasive ability of gastric cancer cells by regulating miR-524-5p/FSTL1.

This study aimed to investigate the effect of thalidomide (Thal) regulating microRNA (miR)-524-5p/follistatin-like protein 1 (FSTL1) on the invasion ability of gastric cancer cells. For this purpose, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the level of miR-524-5p in GES1, SUN-16, MGC-803, SGC-7901, MKN-28, and MKN-45 cells. Then the MGC-803 and MKN-45 cells would proceed to the next research. The MGC-803 and MKN-45 cells were cultured in vitro and added Thal to the final concentration of (6.25, 12.5, 25, 50, 100) μg/mL. The blank control group only added 0.1% dimethyl sulfoxide (DMSO) culture medium, and cultured for 48 hours. CCK8 was used to detect cell proliferation, and Transwell was used to detect cell invasion. The experiment was divided into a blank control group, Thal group (25 μg/mL Thal-treated cells), Thal+inhibitor NC group, and Thal+miR-524-5p inhibitor group (transfected with inhibitor NC and miR-524-5p inhibitor respectively on the basis of Thal group), cultivated for 48 h. The level of miR-524-5p in the cells was detected by RT-qPCR; the cell invasion was detected by Transwell; the expression of matrix metalloproteinase (MMP)-2, MMP-9, FSTL1 protein in the cells was detected by Western blot. The targeting relationship between miR-524-5p and FSTL1 was verified by dual luciferase. Results showed that compared with GES1 cells, the level of miR-524-5p in SUN-16, MGC-803, SGC-7901, MKN-28, and MKN-45 cells decreased (P<0.05). In MGC-803 and MKN-45 cells, compared with the blank control group, the cell proliferation rate and the number of invasions in the (50, 100) μg/mL Thal treatment groups, and the number of invasions in (6.25, 12.5, 25) μg/mL Thal treatment groups decreased (P<0.05). Compared with the blank control group, the level of miR-524-5p in the cells of the Thal group, Thal+inhibitor NC group, and Thal+miR-524-5p inhibitor group increased (P<0.05), the number of invasions, the levels of MMP-2, MMP-9 and FSTL1 proteins in cells decreased (P<0.05); compared with the Thal group and the Thal+inhibitor NC group, the level of miR-524-5p in the cells of the Thal+miR-524-5p inhibitor group decreased (P<0.05), the number of invasions, the levels of MMP-2, MMP-9, and FSTL1 proteins in the cells increased (P<0.05). Dual luciferase verification revealed that there was a targeting relationship between miR-524-5p and FSTL1. In conclusion, that can up-regulate the expression of miR-524-5p to reduce the expression of FSTL1 protein, inhibit the invasion of gastric cancer cells, and achieve alleviation of the disease.

Linagliptin improves diabetic kidney disease in rats by promoting mitochondrial biogenesis through the AMPK/PGC-1α/TFAM pathway

To investigate whether linagliptin improves diabetic kidney disease (DKD) by promoting mitochondrial biosynthesis via activating adenosine monophosphate activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1α/mitochondrial transcription factor A (AMPK/PGC-1α/TFAM) pathway. With 6 male SD rats feeding normal chow as the control group, 16 SD rat models of DKD induced by intraperitoneal injection of 45 mg/kg STZ and high-fat and high-glucose feeding for 4 weeks were randomized into DKD model group and linagliptin treatment group. The rats in the latter two groups were subjected to daily intragastric administration of vehicle or 5 mg/kg linagliptin (dissolved in 5 g/L sodium carboxymethylcellulose, final concentration of 2 mg/mL) for 12 weeks with further high-fat and high-glucose feeding. After the treatments, the rats were sacrificed and blood samples from the abdominal aorta and kidney tissues were collected for testing blood glucose, liver function and lipid metabolism; HE, PAS, Masson, Sirius red staining and electron microscopy were used to observe renal tissue damage. Renal expressions of transforming growth factor β1 (TGF-β1), fibronectin (FN) and collagen I (Col I) were detected by immunohistochemistry, and the changes in membrane potential (ΔψM) and ATP enzyme content were analyzed to assess mitochondrial damage; The expressions of AMPK/PGC-1α/TFAM pathway proteins were detected using Western blotting. Compared with DKD model rats, the rats receiving linagliptin treatment showed significantly decreased blood glucose level (P < 0.01) and improved proteinuria (P < 0.05) with obviously alleviated renal ultrastructural damage and fibrosis, increased ATPase content and ΔψM (P < 0.0001), and enhanced renal expressions of P-AMPK/AMPK, PGC-1α and TFAM (P < 0.05). Linagliptin improves proteinuria and renal fibrosis in rat models of DKD possibly by activating the AMPK/PGC-1α/TFAM pathway to promote mitochondrial biosynthesis.

PROTEIN-PROTEIN INTERACTION ANALYSIS TO IDENTIFY NUCLEAR FACTOR-ERYTHROID-2 FACTOR 2 (NRF2) INHIBITION BY EXTRACELLULAR ENZYMES FROM WATER KEFIR ORGANISMS

Objective: This research was conducted to screen the anticancer activity of bitter herbs that contains Andrographis paniculata (Brum. f) leaves (AP) and Tinospora crispa L. stems (TC) in form of fresh materials and extracts using a mechanism-based yeast bioassay.&#x0D; Methods: Samples tested by mechanism-based yeast bioassay (MBYB) were single extract, mixed extract, and jamu gendong pahitan from a traditional market and made in the laboratory. Fresh sample of jamu gendong pahitan from the market and a single extract (AP and TC) was tested at one dose. While fresh jamu gendong pahitan made in the laboratory and the mixed extract (AP: TC) was tested at three different doses, doses 1 (3:10), dose 2 (1:1), and dose 3 (10:3). The leaves and stems were extracted by 70% ethanol for 3x24 h, each day the solvent was changed then every macerate was evaporated using a rotavapor and water bath. By this MBYB method, noted that the active sample must have an IC12 value of&lt;8000µg/ml, so all the samples or doses were tested using final concentration varying at around 8,000; 4,000; 2,000; 1,000; 500, 250, and 125µg/ml.&#x0D; Results: The percentage yield of Andrographis paniculata (Brum. f) leaves was 11.2% and Tinospora crispa L. stems was 19.%. The activity assay for jamu gendong pahitan from the traditional market was inactive as a topoisomerase inhibitor (IC12&gt;8000µg/ml). Samples showed topoisomerase I inhibitor activity were jamu gendong pahitan made in laboratory doses 1 and 2. While samples showed topoisomerase I and II inhibitor activities were jamu gendong pahitan made in laboratory dose 3, single and mixed extracts.&#x0D; Conclusion: The fresh material of jamu gendong pahitan (bitter herbs) bought from the market is inactive, while the fresh material of samples of jamu gendong pahitan made in laboratory doses 1 and 2 have topoisomerase I inhibitor activity. Based on the IC12, value, it is known that the sample that gave the best activity was the mixed extract of bitter herbs dose 3 that contain extract of A. paniculata and T. crispa (10:3), with IC12 values in strains 1138, 1140, and 1353 were 926.28±173, 576.75±42, and 865.5±135µg/ml respectively.

Protocol Development for Yielding High Quality DNA and RNA from Archived Formalin-Fixed Paraffin Embedded Tissues

Introduction: While genetic analysis of archived formalin-fixed paraffin embedded (FFPE) tissue specimens would be a significant research resource, extraction of high-quality DNA and RNA from these specimens is a significant challenge. Storage duration, tissue handling and tissue preservation processes as well as their interactions with the nucleic acids could influence the quality and integrity of the DNA or RNA required for genetic analysis.&#x0D; Objectives: The goal of this study was to establish a protocol to extract high quality DNA and RNA from the FFPE tissues for further use in quantitative PCR analysis.&#x0D; Methods: For the purposes of this study, human FFPE biopsy tissues from lung, liver, colon and kidney were obtained from a single center biorepository. GeneJET Genomic DNA Purification Kit (Catalog # K0722) obtained from Thermo Fisher Scientific and RecoverAll Total Nucleic Acid Isolation Kit obtained from Life Technologies were modified and used for extraction of DNA and RNA, respectively. Briefly, modifications involved extending reagent incubation times, increasing sample volumes and wash steps, and increased final nucleic acids recovery and concentration steps. Eight sections around 8-10 ?m thick were microtomed for each tissue sample and used for extraction. The purity of the nucleic acids obtained was verified using Nanodrop Spectrophotometer.&#x0D; Results: The average DNA yield from eight sections for each of the tissues was 270±184 ng/?l and for RNA was 296±188 ng/?l. Nucleic acid quality was assessed by measuring the 260nm/280nm absorbance ratio for protein contamination as well as the 260nm/230nm absorbance ratio for salt contamination. Both were found to be within acceptable ranges. RNA was reverse transcribed to cDNA and qPCR was successfully performed on both DNA and cDNA samples.&#x0D; Conclusions: These results indicate that protocols using the silica-based membrane technology can yield high quality DNA and RNA that can be successfully used for downstream genetic analysis.

First Report of Wilt Disease of Ginger Caused by Achromobacter xylosoxidans in China

Ginger (Zingiber officinale) is an important commercial crop that has been widely cultivated in China for more than 2500 years. One variety, Tongling white ginger, has been grown in the Yi'an District of Tongling city, Anhui province (30°45 N, 117°43 E), China. In August 2022, symptoms of yellowing and wilting were observed on ginger plants, with a disease incidence rate exceeding 20% in the field. The stem base of the diseased plants became soft, brown and water-soaked. Additionally, the rhizomes displayed symptoms of browning and water-soaked rot, resembling those caused by Ralstonia solanacearum and Enterobacter cloacae (Yu et al. 2003; Nishijima et al. 2004; Liu et al. 2021). Consequently, ginger bacterial wilt disease may potentially emerge from a combination of infections by diverse pathogenic bacteria. To identify novel pathogens causing the wilt disease, stem tissues of the diseased plants from different locations were sterilized with 1% sodium hypochlorite (NaOCl) for 10 min, followed by at least three time rinses with sterile water. The sterilized samples were then ground with 0.9% saline solution and left at room temperature for 30 min. A 20 μL aliquot of the suspension was serially diluted and cultured on Luria-Bertani (LB) medium at 28°C. A total of 217 isolates was picked and purified for taxonomic identification by 16S rRNA gene analyses with the primer 27F/1492R (Weisburg et al. 1991). Among these isolates, 23 single colony isolates were identified as A. xylosoxidans through NCBI BLASTn analyses. Furthermore, three single isolates from different locations were randomly selected for further experiments. The growing colonies appeared opaque white and round. Microscopic evaluation revealed that cells were rod-shaped with an average length of 1.95 µm and average width of 0.46 µm. The three isolates shared identical 16S rRNA sequences, displayed 99.72% identity with the sequence from A. xylosoxidans strain SeqID2 (GenBank accession NO. MH266081.1). The glutamate synthase (gltB), GTP-binding membrane protein (lepA), NADH:ubiquinone oxidoreductase subunit L (nuoL), RNA polymerase beta-subunit (rpoB), and the enolase (eno) genes of the three isolates were amplified by PCR using primer pairs gltB-F/gltB-R, lepA-F/lepA-R, nuoL-F/nuoL-R, rpoB-F/rpoB-R and eno-F/eno-R, respectively (Spilker et al. 2012; Vandamme et al. 2016). The gene sequences of 16S rRNA (OQ711945, OQ740153 and OR230037), gltB (OR242732, OQ737692 and OR262112), lepA (OR233727, OQ737693 and OR262113), nuoL (OR233726, OQ737694 and OR262114), ropB (OR233725, OQ737695 and OR262115) and eno (OR242733, OQ737696 and OR262116) for the isolates ZOR02, ZOR05 and ZOR12 were deposited in GenBank database. The gltB, lepA, nuoL, rpoB and eno sequences of the isolates ZOR02, ZOR05 and ZOR12 showed 98.66-99.16%, 98.9-100%, 96.28%-97.34%, 98.47-99.44% and 99.27-99.82% similarity to A. xylosoxidans strain AX27, respectively. Phylogenetic trees were constructed based on the 16S rRNA and gltB-lepA-nuoL-rpoB-eno multilocus using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA11.0 software (Álvarez et al. 2018). For pathogenicity tests, bacterial suspensions were initially prepared in sterile water at a final concentration of 108 CFU mL-1. Subsequently, 10 μL of bacterial suspensions was injected into the stem base of two-month-old ginger plants, while sterile water was used as a control (Wang et al. 2022). These plants were then incubated at 28°C and 70% relative humidity. There were three replicates for each treatment, and each replicate contained five plants. After six days of inoculation, the ginger plants injected with bacterial suspensions alone exhibited severe wilting symptoms similar to those observed in the field. However, water-soaked symptoms were not observed on rhizome tissues from the pathogen-infected plants. Bacterial pathogens were re-isolated from the diseased plants and identified using the morphological and molecular methods to meet Koch's hypothetical tests. To our knowledge, this is the first report of A. xylosoxidans causing wilt disease of ginger in China. In 2022, the average yield loss due to wilt disease in the Yi'an District of Tongling exceeded 20%, posing a major threat to local ginger cultivation. Effective disease management strategies are needed to develop for the control and prevention of the disease.

Bioremediation and Accelerated Phytoremediation of a Soil Impacted by a Hydrocarbons Mixture

Waste motor oil (WMO), without adequate final confinement, is spill in agricultural soil, there, it easily exceeds 4400 ppm according to the Mexican standard NOM-138-SEMARNAT/SSA1-2012; a concentration that causes loss of fertility. The aims of this research were: i) biostimulation of agricultural soil impacted by 39,000 ppm of WMO with Triton X-100/Tween 80 at 0.5% and mineral solution: ii) phytoremediation by Phaseolus vulgaris enhanced with Methylobacterium symbioticum and Xanthobacter autotrophicus to reduce WMO at lower concentration than the maximum accepted by NOM-138-SEMARNAT/SSA1-2021. The response variables were: a) the initial and final concentration of WMO by Soxhlet, and b) P. vulgaris: germination percentage; phenology and biomass at seedling and pre-flowering stages. The experimental data were analyzed by ANOVA/Tukey 0.05%, with the program Statgraphics Centurion XVI. II.&#x0D; The results showed that biostimulation of soil impacted by 39,000 ppm of WMO with Triton X-100/Tween 80 at 50%, and a mineral solution, decreased to 26,990 ppm in 25 days. Subsequently, phytoremediation by sowing P. vulgaris with M. symbioticum and X. autotrophicus, at pre-flowering reduced WMO to 1233 ppm in 50 days, a concentration lower than the maximum, accepted by NOM-138-SEMARNAT/SSA1-2012. This supports that is possible to accelerate biostimulation and phytoremediation reduced WMO in the agriculture soil to recover its productive capacity.

Elevated methane alters dissolved organic matter composition in the Arctic Ocean cold seeps

Cold seeps release methane (CH4) from the seafloor to the water column, which fuels microbially mediated aerobic methane oxidation (MOx). Methane-oxidising bacteria (MOB) utilise excess methane, and the MOB biomass serves as a carbon source in the food web. Yet, it remains unclear if and how MOx modifies the composition of dissolved organic matter (DOM) in cold seeps. We investigated MOx rates, DOM compositions and the microbial community during ex-situ incubations of seawater collected from a cold seep site at Norskebanken (north of the Svalbard archipelago) in the Arctic Ocean. Samples were incubated with and without methane amendments. Samples amended with methane (∼1 µM final concentration) showed elevated rates of MOx in both seep and non-seep incubations. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses showed that the number of DOM formulas (i.e., molecular diversity) increased by up to 39% in these incubations. In contrast, the number of formulas decreased by 20% in samples not amended with methane, both from non-seep and seep locations. DOM composition was thus altered towards a more diverse and heterogeneous composition along with elevated methanotrophic activity in methane-amended conditions. In addition to microbial DOM production, abating microbial diversity indicates that elevated DOM diversity was potentially related to grazing pressure on bacteria. The diversity of DOM constituents, therefore, likely increased with the variety of decaying cells contributing to DOM production. Furthermore, based on a principal coordinate analysis, we show that the final DOM composition of non-seep samples amended with methane became more resemblant to that of seep samples. This suggests that methane intrusions will affect water column DOM dynamics similarly, irrespective of the water column’s methane history.

LC-MS/MS methods for direct measurement of sepiapterin and tetrahydrobiopterin in human plasma and clinical applications.

Aim: Tetrahydrobiopterin (BH4), a natural cofactor of aromatic amino acid hydroxylases, and sepiapterin, a natural precursor of BH4, are endogenously present in human plasma. This is the first report on methods for direct quantification of sepiapterin and BH4 in human plasma by LC-MS/MS for pharmacokinetic assessment. Materials & methods: The analytes in plasma were harvested from blood that were treated with 10% ascorbic acid (AA) to a final concentration of 1% AA. Results & conclusion: The quantification methods were validated for calibration ranges of 0.75-500ng/ml and 0.5-500ng/ml for sepiapterin and BH4, respectively. Quantification of analytes was challenging due to their susceptibility to redox reactions. The validated methods were utilized successfully to support clinical development of sepiapterin.

The incorporation of chlorine and cosmogenic 36Cl into speleothem carbonate

AbstractCave carbonate mineral deposits (speleothems) contain trace elements that are intensively investigated for their significance as palaeoclimate and environmental proxies. However, chlorine, which is abundant in marine and meteoric waters, has been overlooked as a potential palaeo-proxy, while cosmogenic 36Cl could, in principle, provide a solar irradiance proxy. Here, total Cl concentrations analysed from various speleothems were low (3–14 mg/kg), with variations linked to crystal fabrics. High-resolution synchrotron radiation micro X-ray fluorescence (μ-XRF) trace element mapping showed Cl often associated with Na, Si, and Al. We propose that speleothems incorporate Cl in two fractions: (1) water soluble (e.g., fluid inclusions) and (2) water insoluble and strongly bound (e.g., associated with detrital particulates). However, disparities indicated that alternate unidentified mechanisms for Cl incorporation were present, raising important questions regarding incorporation of many trace elements into speleothems. Our first measurements of 36Cl/Cl ratios in speleothems required large samples due to low Cl concentrations, limiting the potential of 36Cl as a solar irradiance proxy. Critically, our findings highlight a knowledge gap into how Cl and other trace elements are incorporated into speleothems, how the incorporation mechanisms and final elemental concentrations are related to speleothem fabrics, and the significance this may have for how trace elements in speleothems are interpreted as palaeoclimate proxies.

Optimizing Diffusion Time and other Resources by Using the Diffusion Rate and Number of Stages Concepts

Considering that velocity of diffusion in a system solute-porous material-solvent depends on several factors, (among them, the concentration differential between “solute” in the porous material and “solute” in the solvent) the diffusion process will finish only when maximum entropy is achieved. Thus, the solute concentration will be equal in the matrix and in the solvent (equilibrium concept). On the other hand, if the velocity of diffusion depends on the differential of concentration, then, the amount of materials transferred per time unit (diffusion rate) will diminish as the process goes on. Moreover, when the final concentration of solute in the porous material is desired to be lower than that of the one-stage-equilibrium, then n-more stages must be added. Thus, the decision to choose a process with one or more stages, as well as the end point in each stage (as close or as far as equilibrium) will determine processing time and the use of other resources, i.e. amount of solvent, installation size, financial investment and so on. Therefore, the objective of this study is to develop a tool that helps to optimize these decisions by using a numerical approach.

An experimental universal swine influenza a virus (IAV) vaccine candidate based on the M2 ectodomain (M2e) peptide does not provide protection against H1N1 IAV challenge in pigs

Swine flu is a common disease problem in North American pig populations and swine influenza A viruses (IAV) are extremely diverse and the lack of cross protection between heterologous strains is impacting vaccine efficacy in the field. The objective of this study was to design and test a novel swine flu vaccine targeting the M2 ectodomain (M2e) of IAV, a highly conserved region within the IAV proteome. In brief, an M2e peptide was designed to match the predominant swine IAV M2 sequence based on global analysis of sequences from pigs and humans. The resulting sequence was used to synthesize the M2e peptide coupled to a carrier protein. The final vaccine concentration was 200 µg per dose, and a commercial, microemulsion-based aqueous adjuvant was added. Nine 3-week-old IAV negative piglets were randomly assigned to three groups and rooms including non-vaccinated pigs (NEG-CONTROLs) and vaccinated pigs using the intramuscular (M2e-IM) or the intranasal route (M2e-IN). Vaccinations were done at weaning and again at 2 weeks later. An in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated to study the M2e IgG antibody response and demonstrated M2e-IM pigs had a higher systemic antibody response compared to M2e-IN pigs. Subsequently, an IAV challenge study was conducted. The results indicated that M2e-IM vaccinated pigs were not protected from H1N1 (US pandemic clade, global clade 1A.3.3.2) challenge despite having a strong humoral anti-M2e immune response. In conclusion, while the experimental IAV vaccine was able to induce anti-M2e antibodies, when challenged with H1N1, the vaccinated pigs were not protected, perhaps indicating that reactivity to the M2e antigen alone is not sufficient to reduce clinical signs, lesions or shedding associated with experimental IAV challenge.