Blue Method Research Articles

Preosteoblast Adhesion and Viability Study of Freeze-Dried Bovine Bone Block Scaffold Coated with Human Umbilical Cord Mesenchymal Stem Cell Secretome.

Combining a three-dimensional scaffold with growth factors before implantation is one method used to increase scaffold bioactivity in bone tissue engineering. The mesenchymal stem cell (MSC)-conditioned medium (CM), called secretome, contains many proteins and growth factors required for tissue repair and growth. This study evaluated the bioactivity of a bovine bone scaffold combined with the secretome of human umbilical cord MSCs (hUC-MSCs) by analyzing MC3T3-E1 cell adhesion and viability on the scaffold. This in vitro laboratory study evaluated the effect of hUC-MSC secretome applied to bovine bone scaffolds processed using various techniques on MC3T3-E1 cell adhesion and viability. The three experimental groups included deproteinized bovine bone mineral-secretome (DBBM-CM), freeze-dried bovine bone-secretome (FDBB-CM), and decellularized FDBB-CM, whereas the control group was treated with DBBM alone. The cell adhesion test was performed using the centrifugation method after 6 and 24 hours, whereas the cell viability test was conducted using the trypan blue exclusion method after 24, 48, and 72 hours. Cell attachment was visualized after 4',6-diamidino-2-phenylindole staining and viewed under inverted fluorescence microscopy. Statistical analyses were performed using one-way analysis of variance, followed by a post hoc test in cases of significant differences. Statistical analyses showed significantly greater adhesion of the preosteoblasts to the FDBB-CM scaffold at 6 hours (p = 0.002). The results of the adhesion test at 24 hours and the viability tests at all observation times showed no significant differences (p > 0.05). This study found that the average MC3T3-E1 cell adhesions and viabilities were highest for the FDBB-CM and DBBM-CM scaffolds. DBBM scaffolds with the secretome had better cell adhesion and viability than those without the secretome. The addition of MSC secretome increased bovine bone scaffold bioactivity especially in DBBM and FDBB scaffolds.

Doxorubicin Loaded Nano-Gel Preoperative Application Effect as Adjuvant Chemotherapy on Osteosarcoma

This paper aimed to observe the effect of adjuvant chemotherapy with adriamycin loaded nano-gel on osteosarcoma. 120 patients with osteosarcoma who visited the hospital from January 2018 to February 2022 were selected as control group and nano group by the red blue ball method. The control group received conventional doxorubicin combined with ifosfamide chemotherapy. The nano group was treated with doxorubicin loaded nano-gel as adjuvant chemotherapy before surgery. Both groups received limb salvage surgery. Serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were compared between two groups before, after, and 1 day after chemotherapy. Follow up records were kept for 12 months to obtain adverse reactions to chemotherapy, postoperative clinical efficacy, and progression free survival time (PFS). No significant difference existed in ALP, LDH, VEGF, and bFGF levels between two groups of patients before chemotherapy (P > 0.05). For the overall comparison, inter group, time, and interaction effects had statistical significance (P < 0.05). For the comparison of two groups, the average ALP, LDH, VEGF, and bFGF in the nano group were lower after chemotherapy. The incidence of grade III–IV adverse reactions in the nano group was 10% lower than the controlling group. The CR+PR rate in the nano group was higher. The preoperative application of adriamycin loaded nano-gel as adjuvant chemotherapy can make the patients’ prognosis with osteosarcoma improved. It can enhance the chemotherapy effect, control the degree of adverse reactions of chemotherapy, and improve the clinical efficiency, thus extending the progression free survival period of patients with osteosarcoma.

Validation of an automated iron stain process for use with bone marrow aspirate smear slides.

The assessment of bone marrow iron stores is typically performed on an aspirate smear slide that has been manually stained by a technologist using a commercially available kit. This approach can contribute to inconsistent results and limit the broad use of iron staining in bone marrow specimens, particularly when laboratories have low staffing and/or high specimen volumes. Here, we describe the adaptation and validation of the Ventana Benchmark automated stainer and iron stain kit for routine clinical use of staining iron in bone marrow aspirate smear slides. We assessed accuracy and precision of the Ventana automated iron staining protocol compared to the Perls Prussian blue manual iron staining index method. Hematopathologists assigned Gale scores and enumerated the percentages of erythroid sideroblasts on paired patient bone marrow aspirate smear slides stained by the automated method and the manual iron staining method. We found a similar level of performance of the Ventana automated iron stain relative to the index manual method (as assessed by Pearson correlation and Bland-Altman analyses). In addition, there was low imprecision between replicates performed via the automated iron stain protocol. We also report superior qualitative findings of the automated method in ease of localization of iron storage, visualization of sideroblasts, and counterstain consistency. Automated iron staining of bone marrow aspirate smear slides performed similarly to the manual method and may allow for accurate routine evaluation of bone marrow iron stores as part of bone marrow analysis.

Increased expression of the kynurenine pathway in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection

Angiostrongylus cantonensis is the major cause of eosinophilic meningitis worldwide. The imbalance of neurotoxic and neuroprotective metabolites in the kynurenine pathway (KP) have been suggested to contribute to the pathogenesis of central nervous system (CNS) infection. We hypothesized that KP may also be involved in parasitic eosinophilic meningitis. BALB/c mice were orally infected with 40 A. cantonensis L3, intraperitoneal dexamethasone at a dose of 500 µg/kg/day was administered from the seventh day of infection until the end of the study. The Evans blue method was used to analyze blood-brain barrier (BBB) dysfunction, and indoleamine 2,3-dioxygenase (IDO) proteins levels was measured by Western blot, immunohistochemistry (IHC), and immunofluorescence. Tryptophan and kynurenine concentrations were analyzed by IHC and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of Evans blue, IDO, tryptophan and kynurenine in the different groups of mice were compared using the nonparametric Kruskal-Wallis test. BBB dysfunction was found in mice with eosinophilic meningitis. The administration of dexamethasone significantly decreased the amount of Evans blue. An increased IDO expression was shown in Western blot, IHC and immunofluorescence following 2–3 weeks infection. Increased tryptophan and kynurenine expressions in the brain and cerebrospinal fluid (CSF) were also found in IHC and LC-MS/MS studies. The administration of dexamethasone significantly decreased the amount of IDO, tryptophan and kynurenine. In conclusion, A. cantonensis infection inducing BBB damage, then increased the influx of tryptophan into CSF. The administration of dexamethasone significantly decreased the amount of IDO, tryptophan and kynurenine.

P-chromophore stability of Mehlich-1 and Mehlich-3 under Braga & Defelipo or Murphy & Riley dosing methods

ABSTRACT The performance of colorimetric methods for P quantification has been overlooked by researchers for decades. We investigated the performance of two blue colorimetric methods for P quantification, Braga and Defelipo (B&D) and Murphy and Riley (M&R), using two commonly soil P extractants, Mehlich-1 (M1) and Mehlich-3 (M3). Specifically, we evaluated the color development time and its stability in different soil extractant solution proportions (1:1 or 1:4 v/v), the optimum wavelength, limits of detection, and element quantification. Our results indicate that M3 leads to lower limits of detection and quantification for both colorimetric methods, particularly for B&D. For M1, the volumetric ratio (1:1 or 1:4) did not influence color development and both B&D and M&R methods showed a fast color development. However, B&D showed greater color stability (from 5 to 600 min) and an optimum wavelength of 711 nm, while M&R was stable from 27 to 600 min an optimum wavelength of 889 nm. For M3 soil extractant, there are important issues, such as M&R being unstable and B&D presenting slow color development. In addition, the spectral profile obtained from soil extractant was different from the one obtained by the calibration curve without soil for both M&R and B&D. Therefore, the adoption of original methods on P quantification in M3 extracting solution represents a potential source of error, leading to wrong P fertilizer recommendations. Thus, the best option for P quantification in M3 extracts seems to be the use of Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES).

Autocalibration based on dilution of a single concentrated standard is used for the determination of silicate in sea water by the modified molybdenum blue method

The silicate (Si) molybdenum blue method was modified by combining oxalate and ascorbic acid into a single reagent and was used for determining Si in sea water samples. The first step of this automated assay protocol was designed to perform either a calibration by a single Si standard prepared in deionized (DI) water, or to dilute samples in the range of 0–160 μM Si to fit into 0–20 μM Si calibration range using a 20 cm flow cell. By designing the assay protocol to function in batch mode, the influence of salinity on calibration was eliminated, thus making the method suitable for analysis of samples collected in the open ocean, coastal areas, or rivers. Reproducibility and accuracy of this method were evaluated by analysis of certified sea water reference materials. Phosphate (P) does not interfere significantly if the Si:P ratio is 4:1 or larger. The limit of detection was 514 nM Si, r.s.d. 2.1 %, sampling frequency 40 s/h, reagent consumption 700 μL/sample, and using deionized water as the carrier solution.

Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24h with 50μg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

Systems design and techno-economic analysis of a novel cryogenic carbon capture process integrated with an air separation unit for autothermal reforming blue hydrogen production system

In the transition of the global energy paradigm toward decarbonization, hydrogen is expected to play a crucial role as an energy vector. To enhance the sustainability of hydrogen as an energy source, it is important to reduce costs and carbon emissions in the hydrogen production. Among various hydrogen production technologies, the autothermal reforming (ATR)-based blue hydrogen production method can stand out as a promising option in terms of both economic and environmental considerations. However, there are several bottlenecks: (i) ATR requires an air separation unit that increases the cost burden, and (ii) conventional amine-based carbon capture method consumes a significant amount of energy. To address these bottlenecks, this study proposes a novel cryogenic carbon capture process for ATR-based blue hydrogen production. In the proposed process, air separation unit are utilized not only for oxygen production but also for carbon capture. The compressed air from the air separation unit provides cooling energy to the mixture gas, leading to solidification and separation of carbon dioxide. Despite imposing an additional burden on the air separation unit, it significantly reduces the energy and cost associated with carbon capture. Consequently, there exists a trade-off relationship between the increased load on the air separation unit and the decreased load in carbon capture. This trade-off was evaluated by comparing it with the conventional absorption-based carbon capture process in terms of energy and economic aspects. The proposed process exhibits a 36.4% reduction in total energy consumption, with a particularly approximately 49.5% decrease in the energy consumed for carbon capture (0.278 kW h/kg-CO2). The economic performance indicates an 11% decrease in the levelized cost of hydrogen (1.62$/kg-H2), and a 45.3% decrease in CO2 avoidance cost (40.1$/kg-CO2). The captured solid CO2 can offer advantages during storage and transportation. Furthermore, the potential utilization of cooling energy of solid CO2 from an end-user perspective is expected to contribute to the establishment of a new industrial value chain.

Determination the Anti –Oxidant Capacity, Total Phenols, Minerals and Evaluation the Anti- Bacteria Activity of Leafs and Stems of Gaper Plant Extracts

In this study, Caper plant were collected from Al-Jabal Al-Akhdar region (Libya) during the spring season (2023), the leaves and stems of the plant were separated and dried in the open air. The contents of minerals and metals (Na, K, Ca, Fe and Ni) were determined. The strength of antioxidants was determined using the Prussian blue method. Also, phenol compounds were estimated by the Folin Ciacalteu Method. The results showed that the metal values were showed higher contents in the leaves comparing with their contents in the stems, where a relative increase was found in the leaves of capers with a value of (1.83 ppm) compared to its value in the stems (0.94 ppm), the phenol contents were recorded in leaves (0.530 ppm), and stems (0.362 ppm). The anti-bacterial activity using different concentrations of the plant extract concentrations of (25,50,75 and100%) on two types of bacteria (Bacilli and Escherichia Coli) was applied for the extracts. The results showed that the inhibition zone of the leave extracts was recorded in all concentrations of Bacilli, in the stems the high inhibitaion zone were observed for concentrations (75%, &100%). The leaves and stems extract didn't showed any effect on Escherichia coli except for high concentrations of leaves (100%).

Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system.

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.

Development and characterization of stable proanthocyanidin‐loaded PLAROsomes as a potential drug carrier system for augmenting anticancer activity

AbstractThe aim of the present study was to develop potential drug carrier system formulations, namely PLAROsomes for the delivery of Proanthocyanidin (PACY), isolated from the extract of dried grape seeds. The PLAROsomes loaded with PACY were prepared using the thin film hydration method. The influence of various process parameters and material attributes was investigated using the design of experiments (DoE). The PACY‐loaded PLAROsomes were characterized using hyphenated tools. The in‐vitro anticancer activities of PACY‐loaded PLAROsomes were confirmed using the Trypan blue method, MTT method, and flow cytometric analysis on MCF‐7 cells. The polymer‐to‐lipid ratio, among various process parameters and material attributes, significantly influenced the average particle size. Additionally, it played a crucial role in determining the percentage of PACY released from PACY‐loaded PLAROsomes. The size of the PACY‐loaded PLAROsomes ranged from 40 to 300 nm, and the optimized batch demonstrated a drug entrapment efficiency of 86.38±0.22%. PACY‐loaded PLAROsomes exhibited improved in vitro anticancer activities against MCF‐7 breast cancer cell lines compared to PACY. PACY‐loaded PLAROsomes showed greater activity at lower concentrations in cytotoxicity studies, as supported by apoptosis analysis. Therefore, PLAROsomes could present a promising drug carrier system for delivering PACY in breast cancer treatment, offering a sustained release effect.

Potential Mechanisms Involved in the Anticonvulsant Effect of Recombinant Human VEGF on Maximal Electroshock-induced Seizure

Background: Vascular endothelial growth factor (VEGF) signaling pathway plays an important role in the pathogenesis of seizure. The oxidant/antioxidant factors and miRNA expression in the brain are differentially regulated in seizure. Objectives: We aim to investigate the potential mechanism of action for the recombinant human VEGF (rhVEGF) in mice with maximal electroshock (MES)-induced seizure. Materials & Methods: A total of 40 male mice (weight: 20-25 g) were treated intraperitoneally with normal saline, or rhVEGF (50, 100, and 150 µg/kg, daily for 4 consecutive days). One hour after the last injection, seizures were induced in each animal by MES. The latency for the onset of the first clonus and the duration of hind limb extension (HLE) were recorded. The levels of nitric oxide (NO), total antioxidant capacity (TAC), and micoRNA-142-5p expression were determined in the hippocampus of mice. Blood-brain barrier (BBB) permeability was also estimated by Evans blue dye extravasation method. Results: The administration of rhVEGF at all doses significantly reduced the HLE duration. However, latency for the seizure onset increased after administration of 50 and 150 μg/kg rhVEGF and decreased after administration of 100 μg/kg rhVEGF. In the brain, the NO level decreased, while TAC level and microRNA-142-5p expression increased by rhVEGF treatment in mice with MES-induced seizure. Pretreatment with rhVEGF at doses of 100 and 150 μg/kg reversed the increase in BBB leakage induced by MES-induced seizures. Conclusion: The rhVEGF administration can prevent MES-induced seizures by regulating NO, TAC, and miR-142-5 expression levels in the hippocampus and reducing BBB leakage in mice.

Rapid screening of inorganic arsenic in groundwater on-site by a portable three-channel colorimeter

Rapid screening of inorganic arsenic (iAs) in groundwater used for drinking by hundreds of millions of mostly rural residents worldwide is crucial for health protection. Most commercial field test kits are based on the Gutzeit reaction that uses mercury-based reagents for color development, an environmental concern that increasingly limits its utilization. This study further improves the molybdenum blue (MB) colorimetric method to allow for faster screening with more stable reagents. More importantly, a portable three-channel colorimeter is developed for screening iAs relative to the WHO drinking water guideline value (10 µg/L). Adding the reducing reagents in sequence not only prolongs the storage time to > 7 days, but also accelerates the color development time to 6 min in conjunction with lowering the H2SO4 concentration in chromogenic reagents. The optimal pH ranges from 1.2 to 1.3 and is achieved by acidifying groundwater to 1% (V/V) HCl. With detection limits of 3.7 µg/L for inorganic arsenate (iAs(V)) and 3.8 µg/L for inorganic arsenite (iAs(III)), testing groundwater with ∼10 µg/L of As has a precision < 20%. The method works well for a range of phosphate concentrations of 48–950 µg/L (0.5–10 µmol/L). Concentrations of total_iAs (6–300 µg/L), iAs(V) (6–230 µg/L) and iAs(III) (0–170 µg/L) for 14 groundwater samples from Yinchuan Plain, Pearl River Delta, and Jianghan Plain, are in excellent agreements (linear regression slope: 0.969–1.029) with the benchmark methods. The improved chemistry here lays the foundation for the MB colorimetric method to become a commercially viable screening tool, with further engineering and design improvement of the colorimeter.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P&lt;0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P&lt;0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P&lt;0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Cytotoxic Activity of Cambodian Leaves Extract (Plumeria acuminate) on Breast Cancer Cells and COX-2 Targeted Prediction of Its Chemical Contents

Cambodian leaves are suspected to contain stigma sterol which may target Cyclo-Oxygenase-2 (COX-2) or Estrogen Receptor (ER) to contribute to its cytotoxic activity on breast cancer cells. This study aimed to determine the potential of Cambodian leaf compounds and extracts as chemopreventive agents for luminal breast cancer with a molecular target of COX-2. Ethanol was used to extract the active compound of Cambodian leaves. The study on chemical activity against COX-2 employed molecular docking with Molecular Operating Environment (MOE) and the cytotoxic property of Cambodian leaf extract (CLE) on T47D was determined using the trypan blue exclusion method. The extraction yielded as 4.87% w/w CLE. Thin layer chromatography showed that Cambodian leaves contain sterol. Molecular docking confirmed that several sterol compounds have greater affinity to COX-2 than native ligands indicating that they are potent as COX-2 inhibitors. They are Stigmast-7-en-3-ol, Lupeol Acetate, and Lupeol carboxylic acid with docking scores of -14.3874, -13.8098, and -14.1045 kcal/mol respectively. The CLE exhibited cytotoxic activity on T47D cells with an IC50 value of 18 μg/mL. Therefore, CLE has a potential effect as a chemopreventive agent for breast cancer and potentially as a COX-2 inhibitor.Keywords: Cambodian leaf extract, breast cancer, COX-2 inhibitor, chemopreventive.

Integrative leaf anatomy structure, physiology, and metabolome analyses revealed the response to drought stress in sainfoin at the seedling stage.

Sainfoin (Onobrychis viciaefolia) is a vital legume forage, and drought is the primary element impeding sainfoin growth. The anatomical structure, physiological indexes, and metabolites of the leaves of sainfoin seedlings with a drought-resistant line of P1 (DRL) and a drought-sensitive material of 2049 (DSM) were analyzed under drought (-1.0 MPa) with polyethylene glycol-6000 (PEG-6000). The leaf anatomy was studied by the paraffin section method. The related physiological indexes were measured by the hydroxylamine oxidation method, titanium sulfate colorimetric method, thiobarbituric acid method, acidic ninhydrin colorimetric method, and Coomassie brilliant blue method. The metabolomics analysis was composed of liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS). The results revealed that the thickness of the epidermis, palisade tissue, and sponge tissue of DRL were significantly greater than those of DSM. The leaves of DRL exhibited lower levels of superoxide anion (O2 •-) production rate, hydrogen peroxide (H2O2) content, and malondialdehyde (MDA) content compared with DSM, while proline (Pro) content and soluble protein (SP) content were significantly higher than those of DSM. A total of 391 differential metabolites were identified in two samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that the primary differential metabolites were concentrated into the tyrosine metabolism; isoquinoline alkaloid biosynthesis; ubiquinone and other terpenoid quinone biosynthesis; neomycin, kanamycin, and gentamicin biosynthesis; and anthocyanin biosynthesis metabolic pathways. Compared with DSM, DRL had more complete anatomical structure, lower active oxygen content, and higher antioxidant level. The results improved our insights into the drought-resistant mechanisms in sainfoin.

Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Clostridioides difficile infection promotes gastrointestinal dysfunction in human and mice post-acute phase of the disease

ObjectivesIn the US, Clostridioides difficile (C. difficile) infection (CDI) is the 8th leading cause of hospital readmission and 7th for mortality among all gastrointestinal (GI) disorders. Here, we investigated GI dysfunction post-CDI in humans and mice post-acute infection. Materials and methodsFrom March 2020 to July 2021, we reviewed the clinical records of 67 patients referred to the UVA Complicated C. difficile clinic for fecal microbiota transplantation (FMT) eligibility. C57BL/6 mice were infected with C. difficile and clinical scores were determined daily. Stool samples from mice were collected to measure the shedding of C. difficile and myeloperoxidase (MPO) levels. On day 21 post-infection, Evans's blue and FITC-70kDa methods were performed to evaluate GI motility in mice. ResultsOf the 67 patients evaluated at the C. difficile clinic, 40 patients (59.7%) were confirmed to have CDI, and 22 patients (32.8%) with post-CDI IBS (diarrhea-type, constipation-type, and mixed-type). In infected mice, levels of MPO in stools and clinical score were higher on day 3. On day 21, mice recovered from body weight loss induced by CDI, and fecal MPO was undetectable. The total GI transit time (TGITT) and FITC-70kDa levels on the proximal colon were increased in infected mice (p = 0.002), suggesting a constipation phenotype post-acute phase of CDI. A positive correlation intestinal inflammation on day 3 and TGITT on day 21 was observed. ConclusionIn conclusion, post-infection intestinal dysfunction occurs in humans and mice post-CDI. Importantly, we have validated in the mouse model that CDI causes abnormal GI transit in the recovery phase of the disease, indicating the potential utility of the model in exploring the underlying mechanisms of post-infectious IBS in humans.

Exploring the Role of Serum Hydrogen Sulphide (H2S) Levels in Manic Depressive Psychosis in Terms of Its Association, Diagnostic Ability, and Severity Prediction: Findings From a Tertiary Care Center in North Bengal.

Manic depressive psychosis (MDP) or bipolar disorder, a prevalent psychiatric condition globally and in the Indian population, has been attributed to various pathological mechanisms. Hydrogen sulphide (H2S), a member of the gasotransmitter family, may be linked to the development of bipolar disorder because it plays a crucial role in maintaining proper neuronal function in terms of excitability, plasticity, and homeostatic functions. There is very little data regarding the role of the gasotransmitter H2S in MDP in terms of its association, diagnostic ability, and severity prediction, which led us to conduct this study among MDP patients in the Sub-Himalayan region of West Bengal. This was an observational case-control study performed in the Department of Biochemistry, North Bengal Medical College and Hospital, Siliguri, West Bengal, India, from January 2022 to December 2022. Fifty diagnosed MDP patients and 50 healthy age- and sex-matched control subjects satisfying the inclusion and exclusion criteria were studied. The H2S level in the blood was assayed using the standardised spectrophotometric methylene blue method. The severity of depression was assessed by Hamilton Depression Rating Scale (HAM-D) scoring. Of the 50 MDP patients, 45 (90%) were in the depressive phase, and five (10%) were in the manic phase. Of the 45 depressive patients, eight (17.8%) had mild depression, 12 (26.7%) had moderate depression, 19 (42.2%) had severe depression, and six (13.3%) had very severe depression. The mean H2S level in MDP patients (41.98±18.88 μmol/l) was significantly (P<0.05) lower than that in control subjects (99.20± 15.20 μmol/l). It was also observed that the mean H2S level in MDP patients decreased with the duration of the disease but was not statistically significant. The mean H2S levels in the different depression severity groups were found to be significantly different (P<0.001). Receiver operating characteristic (ROC) curve analysis revealed that a cut-off value of H2S <78.5 μmol/l was associated with MDP, with a sensitivity of 96%and a specificity of 88%,and a cut-off value of H2S < 53 μmol/lpredicted the severity of depression with a sensitivity of 89.3% and a specificity of 76.5%. The significant association of the gasotransmitter H2S in MDP patients and its role as a diagnostic and severity predictive marker can help us to employ proper measures for better management of MDP and improving quality of life.

Effects of common dissolved anions on the efficiency of Fe0-based remediation systems

Quiescent batch experiments were conducted to evaluate the influences of Cl−, F−, HCO3−, HPO42−, and SO42− on the reactivity of metallic iron (Fe0) for water remediation using the methylene blue (MB) method. Strong discoloration of MB indicates high availability of solid iron corrosion products (FeCPs). Tap water was used as an operational reference. Experiments were carried out in graduated test tubes (22 mL) for up to 45 d, using 0.1 g of Fe0 and 0.5 g of sand. Operational parameters investigated were (i) equilibration time (0–45 d), (ii) 4 different types of Fe0, (iii) anion concentration (10 values), and (iv) use of MB and Orange II (O-II). The degree of dye discoloration, the pH, and the iron concentration were monitored in each system. Relative to the reference system, HCO3− enhanced the extent of MB discoloration, while Cl−, F−, HPO42−, and SO42− inhibited it. A different behavior was observed for O-II discoloration: in particular, HCO3− inhibited O-II discoloration. The increased MB discoloration in the HCO3− system was justified by considering the availability of FeCPs as contaminant scavengers, pH increase, and contact time. The addition of any other anion initially delays the availability of FeCPs. Conflicting results in the literature can be attributed to the use of inappropriate experimental conditions. The results indicate that the application of Fe0–based systems for water remediation is a highly site-specific issue which has to include the anion chemistry of the water.