CSF1R antagonism results in increased supraspinal infiltration in EAE.

BACKGROUND: Colony stimulating factor 1 receptor (CSF1R) signaling is crucial for the maintenance and function of various myeloid subsets. CSF1R antagonism was previously shown to mitigate clinical severity in experimental autoimmune encephalomyelitis (EAE). The associated mechanisms are still not well delineated. METHODS: To assess the effect of CSF1R signaling, we employed the CSF1R antagonist PLX5622 formulated in chow (PLX5622 diet, PD) and its control chow (control diet, CD). We examined the effect of PD in steady state and EAE by analyzing cells isolated from peripheral immune organs and from the CNS via flow cytometry. We determined CNS infiltration sites and assessed the extent of demyelination using immunohistochemistry of cerebella and spinal cords. Transcripts of genes associated with neuroinflammation were also analyzed in these tissues. RESULTS: In addition to microglial depletion, PD treatment reduced dendritic cells and macrophages in peripheral immune organs, both during steady state and during EAE. Furthermore, CSF1R antagonism modulated numbers and relative frequencies of T effector cells both in the periphery and in the CNS during the early stages of the disease. Classical neurological symptoms were milder in PD compared to CD mice. Interestingly, a subset of PD mice developed atypical EAE symptoms. Unlike previous studies, we observed that the CNS of PD mice was infiltrated by increased numbers of peripheral immune cells compared to that of CD mice. Immunohistochemical analysis showed that CNS infiltrates in PD mice were mainly localized in the cerebellum while in CD mice infiltrates were primarily localized in the spinal cords during the onset of neurological deficits. Accordingly, during the same timepoint, cerebella of PD but not of CD mice had extensive demyelinating lesions, while spinal cords of CD but not of PD mice were heavily demyelinated. CONCLUSIONS: Our findings suggest that CSF1R activity modulates the cellular composition of immune cells both in the periphery and within the CNS, and affects lesion localization during the early EAE stages.

Identification of transporters involved in aromatic compounds tolerance through screening of transporter deletion libraries.

Aromatic compounds are used in pharmaceutical, food, textile and other industries. Increased demand has sparked interest in exploring biotechnological approaches for their sustainable production as an alternative to chemical synthesis from petrochemicals or plant extraction. These aromatic products may be toxic to microorganisms, which complicates their production in cell factories. In this study, we analysed the toxicity of multiple aromatic compounds in common production hosts. Next, we screened a subset of toxic aromatics, namely 2-phenylethanol, 4-tyrosol, benzyl alcohol, berberine and vanillin, against transporter deletion libraries in Escherichia coli and Saccharomyces cerevisiae. We identified multiple transporter deletions that modulate the tolerance of the cells towards these compounds. Lastly, we engineered transporters responsible for 2-phenylethanol tolerance in yeast and showed improved 2-phenylethanol bioconversion from L-phenylalanine, with deletions of YIA6, PTR2 or MCH4 genes improving titre by 8-12% and specific yield by 38-57%. Our findings provide insights into transporters as targets for improving the production of aromatic compounds in microbial cell factories.

Ongoing Laboratory Performance Study on Chemical Analysis of Hydrophobic and Hydrophilic Compounds in Three Aquatic Passive Samplers.

The quality of chemical analysis is an important aspect of passive sampling-based environmental assessments. The present study reports on a proficiency testing program for the chemical analysis of hydrophobic organic compounds in silicone and low-density polyethylene (LDPE) passive samplers and hydrophilic compounds in polar organic chemical integrative samplers. The median between-laboratory coefficients of variation (CVs) of hydrophobic compound concentrations in the polymer phase were 33% (silicone) and 38% (LDPE), similar to the CVs obtained in four earlier rounds of this program. The median CV over all rounds was 32%. Much higher variabilities were observed for hydrophilic compound concentrations in the sorbent: 50% for the untransformed data and a factor of 1.6 after log transformation. Limiting the data to the best performing laboratories did not result in less variability. Data quality for hydrophilic compounds was only weakly related to the use of structurally identical internal standards and was unrelated to the choice of extraction solvent and extraction time. Standard deviations of the aqueous concentration estimates for hydrophobic compound sampling by the best performing laboratories were 0.21 log units for silicone and 0.27 log units for LDPE (factors of 1.6 to 1.9). The implications are that proficiency testing programs may give more realistic estimates of uncertainties in chemical analysis than within-laboratory quality control programs and that these high uncertainties should be taken into account in environmental assessments.

Occurrence and concentrations of traditional and emerging contaminants in onsite wastewater systems and water supply wells in eastern North Carolina, USA.

Onsite wastewater treatment systems (OWTSs) and private wells are commonly used in Eastern North Carolina, USA. Water from private wells is not required to be tested after the initial startup, and thus persons using these wells may experience negative health outcomes if their water is contaminated with waste-related pollutants including bacteria, nitrate or synthetic chemicals such as hexafluoropropylne oxide dimer acid and its ammonium salt (GenX). Water samples from 18 sites with OWTSs and groundwater wells were collected for nitrate, Escherichia coli (E. coli), total coliform, and GenX concentration analyses. Results showed that none of the 18 water supplies were positive for E. coli, nitrate concentrations were all below the maximum contaminant level of 10 mg L-1, and one well had 1 MPN 100 mL-1 of total coliform. However, GenX was detected in wastewater collected from all 18 septic tanks and 22% of the water supplies tested had concentrations that exceeded the health advisory levels for GenX. Water supplies with low concentrations of traditionally tested for pollutants (nitrate, E. coli) may still pose health risks due to elevated concentrations of emerging contaminants like GenX and thus more comprehensive and routine water testing is suggested for this and similar persistent compounds.

Nitrate increases the capacity of an aerobic moving-bed biofilm reactor (MBBR) for winery wastewater treatment.

We used bench-scale tests and mathematical modeling to explore chemical oxygen demand (COD) removal rates in a moving-bed biofilm reactor (MBBR) for winery wastewater treatment, using either urea or nitrate as a nitrogen source. With urea addition, the COD removal fluxes ranged from 34 to 45 gCOD/m2-d. However, when nitrate was added, fluxes increased up to 65 gCOD/m2-d, twice the amount reported for aerobic biofilms for winery wastewater treatment. A one-dimensional biofilm model, calibrated with data from respirometric tests, accurately captured the experimental results. Both experimental and modelling results suggest that nitrate significantly increased MBBR capacity by stimulating COD oxidation in the deeper, oxygen-limited regions of the biofilm. Our research suggests that the addition of nitrate, or other energetic and broadly used electron acceptors, may provide a cost-effective means of covering peak COD loads in biofilm processes for winery or another industrial wastewater treatment.

Deciphering feedback regulation of prostaglandin F2α in blood stasis syndrome using nitrogen-doped porous transition metal carbides.

Blood stasis syndrome (BSS) has persistent health risks; however, its pathogenesis remains elusive. This obscurity may result in missed opportunities for early intervention, increased susceptibility to chronic diseases, and reduced accuracy and efficacy of treatments. Metabolomics, employing the matrix-assisted laser desorption/ionization (MALDI) strategy, presents distinct advantages in biomarker discovery and unraveling molecular mechanisms. Nonetheless, the challenge is to develop efficient matrices for high-sensitivity and high-throughput analysis of diverse potential biomarkers in complex biosamples. This work utilized nitrogen-doped porous transition metal carbides and nitrides (NP-MXene) as a MALDI matrix to delve into the molecular mechanisms underlying BSS pathogenesis. Structural optimization yielded heightened peak sensitivity (by 1.49-fold) and increased peak numbers (by 1.16-fold) in clinical biosamples. Validation with animal models and clinical serum biosamples revealed significant differences in metabolic fingerprints between BSS and control groups, achieving an overall diagnostic efficacy of 0.905 (95% CI, 0.76-0.979). Prostaglandin F2α was identified as a potential biomarker (diagnostics efficiency of 0.711, specificity = 0.7, sensitivity = 0.6), and pathway enrichment analysis disclosed disruptions in arachidonic acid metabolism in BSS. This innovative approach not only advances comprehension of BSS pathogenesis, but also provides valuable insights for personalized treatment and diagnostic precision.

Iron Promotes the Retention of Terrigenous Dissolved Organic Matter in Subtidal Permeable Sediments.

Marine permeable sediments are important sites for organic matter turnover in the coastal ocean. However, little is known about their role in trapping dissolved organic matter (DOM). Here, we examined DOM abundance and molecular compositions (9804 formulas identified) in subtidal permeable sediments along a near- to offshore gradient in the German North Sea. With the salinity increasing from 30.1 to 34.6 PSU, the DOM composition in bottom water shifts from relatively higher abundances of aromatic compounds to more highly unsaturated compounds. In the bulk sediment, DOM leached by ultrapure water (UPW) from the solid phase is 54 ± 20 times more abundant than DOM in porewater, with higher H/C ratios and a more terrigenous signature. With 0.5 M HCl, the amount of leached DOM (enriched in aromatic and oxygen-rich compounds) is doubled compared to UPW, mainly due to the dissolution of poorly crystalline Fe phases (e.g., ferrihydrite and Fe monosulfides). This suggests that poorly crystalline Fe phases promote DOM retention in permeable sediments, preferentially terrigenous, and aromatic fractions. Given the intense filtration of seawater through the permeable sediments, we posit that Fe can serve as an important intermediate storage for terrigenous organic matter and potentially accelerate organic matter burial in the coastal ocean.

Changes of soil nutrients and organic carbon fractions in Caragana korshinskii forests with different restoration years in mountainous areas of southern Ningxia, China.

Vegetation restoration can effectively enhance soil quality and soil organic carbon (SOC) sequestration. In this study, the distribution characteristics of soil nutrients and SOC along soil profile (0-100 cm), and their responses to restoration years (16, 28, 38 years) were studied in Caragana korshinskii plantations in the southern mountainous area of Ningxia, compared with cropland and natural grassland. The results showed that: 1) the contents of SOC, soil total nitrogen (TN), total phosphorus (TP), particulate organic carbon (POC), mineral-associated organic carbon (MAOC) and the proportion of particulate organic carbon to total organic carbon (POC/SOC) all decreased with increasing soil depth. The ratio of mineral-associated organic carbon to total organic carbon (MAOC/SOC) exhibited an opposite trend. 2) The contents of SOC, TN, TP, C:P, N:P, POC and MAOC gra-dually decreased as the restoration years increased. However, the C:N ratio showed no significant change. The POC/SOC ratio initially increased and then decreased, while the MAOC/SOC ratio decreased initially and then increased. 3) In three different types of vegetation, POC, MAOC, and SOC showed a highly significant positive linear correlation, with the increase in SOC mainly depended on the increase in MAOC. The SOC, TN, TP, POC and MAOC contents in natural grassland and C. korshinskii plantations were significantly higher than those in cropland. In conclusion, soil nutrients and POC and MAOC contents of C. korshinskii plantations gradually decreased with the increases in restoration years. However, when compared with cropland, natural grassland and C. korshinskii plantations demonstrated a greater capacity to maintain and enhance soil nutrient and carbon storage.

Strategies for the synthesis of the osmolyte glucosylglycerate and its precursor glycerate.

Glycosidic osmolytes are widespread natural compounds that protect microorganisms and their macromolecules from the deleterious effects of various environmental stresses. Their protective properties have attracted considerable interest for industrial applications, especially as active ingredients in cosmetics and healthcare products. In that regard, the osmolyte glucosylglycerate is somewhat overlooked. Glucosylglycerate is typically accumulated by certain organisms when they are exposed to high salinity and nitrogen starvation, and its potent stabilizing effects have been demonstrated in vitro. However, the applications of this osmolyte have not been thoroughly explored due to the lack of a cost-efficient production process. Here, we present an overview of the progress that has been made in developing promising strategies for the synthesis of glucosylglycerate and its precursor glycerate, and discuss the remaining challenges. KEY POINTS: • Bacterial milking could be explored for fermentative production of glucosylglycerate • Glycoside phosphorylases of GH13_18 represent attractive alternatives for biocatalytic production • Conversion of glycerol with alditol oxidase is a promising strategy for generating the precursor glycerate.

Immune system-related plasma extracellular vesicles in healthy aging.

Objectives: To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. Methods: EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Results: Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1ß, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1ß, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Conclusion: Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

Resting cells of Skeletonema marinoi assimilate organic compounds and respire by dissimilatory nitrate reduction to ammonium in dark, anoxic conditions.

Diatoms can survive long periods in dark, anoxic sediments by forming resting spores or resting cells. These have been considered dormant until recently when resting cells of Skeletonema marinoi were shown to assimilate nitrate and ammonium from the ambient environment in dark, anoxic conditions. Here, we show that resting cells of S. marinoi can also perform dissimilatory nitrate reduction to ammonium (DNRA), in dark, anoxic conditions. Transmission electron microscope analyses showed that chloroplasts were compacted, and few large mitochondria had visible cristae within resting cells. Using secondary ion mass spectrometry and isotope ratio mass spectrometry combined with stable isotopic tracers, we measured assimilatory and dissimilatory processes carried out by resting cells of S. marinoi under dark, anoxic conditions. Nitrate was both respired by DNRA and assimilated into biomass by resting cells. Cells assimilated nitrogen from urea and carbon from acetate, both of which are sources of dissolved organic matter produced in sediments. Carbon and nitrogen assimilation rates corresponded to turnover rates of cellular carbon and nitrogen content ranging between 469 and 10,000 years. Hence, diatom resting cells can sustain their cells in dark, anoxic sediments by slowly assimilating and respiring substrates from the ambient environment.

Do Chemformers Dream of Organic Matter? Evaluating a Transformer Model for Multistep Retrosynthesis.

Synthesis planning of new pharmaceutical compounds is a well-known bottleneck in modern drug design. Template-free methods, such as transformers, have recently been proposed as an alternative to template-based methods for single-step retrosynthetic predictions. Here, we trained and evaluated a transformer model, called the Chemformer, for retrosynthesis predictions within drug discovery. The proprietary data set used for training comprised ∼18 M reactions from literature, patents, and electronic lab notebooks. Chemformer was evaluated for the purpose of both single-step and multistep retrosynthesis. We found that the single-step performance of Chemformer was especially good on reaction classes common in drug discovery, with most reaction classes showing a top-10 round-trip accuracy above 0.97. Moreover, Chemformer reached a higher round-trip accuracy compared to that of a template-based model. By analyzing multistep retrosynthesis experiments, we observed that Chemformer found synthetic routes, leading to commercial starting materials for 95% of the target compounds, an increase of more than 20% compared to the template-based model on a proprietary compound data set. In addition to this, we discovered that Chemformer suggested novel disconnections corresponding to reaction templates, which are not included in the template-based model. These findings were further supported by a publicly available ChEMBL compound data set. The conclusions drawn from this work allow for the design of a synthesis planning tool where template-based and template-free models work in harmony to optimize retrosynthetic recommendations.

Nitrate denitrification rate response to temperature gradient change during river bank infiltration.

Nitrate attenuation during river bank infiltration is the key process for reducing nitrogen pollution. Temperature is considered to be an important factor affecting nitrate attenuation. However, the magnitude and mechanism of its impact have not been clear for a long time. In this study, the effects of temperature and temperature gradient on the nitrate denitrification rate were investigated via static batch and dynamic soil column simulation experiments. The results showed that temperature had a significant effect on the denitrification rate. Temperature effects were first observed in denitrifying bacteria. At low temperatures, the microorganism diversity was low, resulting in a lower denitrification rate constant. The static experimental results showed that the denitrification rate at 19 °C was approximately 2.4 times that at 10 °C. The dynamic soil column experiment established an exponential positive correlation between the nitrate denitrification decay kinetic constant and temperature. The affinity of denitrifying enzymes for nitrate in the reaction substrate was ordered as follows: decreasing temperature gradient (30 °C → 10 °C) > zero temperature gradient (10 °C) > increasing temperature gradient condition (0 °C → 10 °C). This study provides a theoretical basis for the biogeochemical processes underlying river bank infiltration, which will help aid in the development and utilization of groundwater resources.

Nitrate and oxygen significantly changed the abundance rather than structure of sulphate-reducing and sulphur-oxidising bacteria in water retrieved from petroleum reservoirs.

Sulphate-reducing bacteria (SRB) are the main culprits of microbiologically influenced corrosion in water-flooding petroleum reservoirs, but some sulphur-oxidising bacteria (SOB) are stimulated when nitrate and oxygen are injected, which control the growth of SRB. This study aimed to determine the distributions of SRB and SOB communities in injection-production systems and to analyse the responses of these bacteria to different treatments involving nitrate and oxygen. Desulfovibrio, Desulfobacca, Desulfobulbus, Sulfuricurvum and Dechloromonas were commonly detected via 16S rRNA gene sequencing. Still, no significant differences were observed for either the SRB or SOB communities between injection and production wells. Three groups of water samples collected from different sampling sites were incubated. Statistical analysis of functional gene (dsrB and soxB) clone libraries and quantitative polymerase chain reaction showed that the SOB community structures were more strongly affected by the nitrate and oxygen levels than SRB clustered according to the sampling site; moreover, both the SRB and SOB community abundances significantly changed. Additionally, the highest SRB inhibitory effect and the lowest dsrB/soxB ratio were obtained under high concentrations of nitrate and oxygen in the three groups, suggesting that the synergistic effect of nitrate and oxygen level was strong on the inhibition of SRB by potential SOB.

Partial substitution of chemical fertilizer by organic fertilizer increases yield, quality and nitrogen utilization of Dioscorea polystachya.

This field experiment aimed to investigate the effects of different ratios of organic and inorganic fertilizers with maintaining equal nitrogen application rates on the yield, quality, and nitrogen uptake efficiency of Dioscorea polystachya (yam). Six treatments were set, including a control without fertilizer (CK), sole application of chemical fertilizer (CF), sole application of organic fertilizer (OM), 25% organic fertilizer + 75% chemical fertilizer (25%OM + 75%CF), 50% organic fertilizer + 50% chemical fertilizer (50%OM + 50%CF), and 75% organic fertilizer + 25% chemical fertilizer (75%OM + 25%CF). The experiment followed a randomized complete block design with three replications. Various yield parameters, morphology, quality indicators, and nitrogen utilization were analyzed to assess the differences among treatments. The results indicated that all fertilizer treatments significantly increased the yield, morphology, quality indicators, and nitrogen utilization efficiency compared to the control. Specifically, 25%OM + 75%CF achieved the highest yield of 31.96 t hm-2, which was not significantly different from CF (30.18 t hm-2). 25%OM + 75%CF exhibited the highest values at 69.23 cm in tuber length and 75.86% in commodity rate, 3.14% and 1.57% higher than CF respectively. Tuber thickness and fresh weight of 25%OM + 75%CF showed no significant differences from CF, while OM and 50%OM+50%CF exhibited varying degrees of reduction compared to CF. Applying fertilizer significantly enhanced total sugar, starch, crude protein, total amino acid, and ash contents of D. polystachya (except ash content between CK and OM). Applying organic fertilizer increased the total sugar, starch, crude protein, total amino acid, and ash contents in varying degrees when compared with CF. The treatment with 25%OM+75%CF exhibited the highest increases of 6.31%, 3.78%, 18.40%, 29.70%, and 10%, respectively. Nitrogen content in different plant parts followed the sequence of tuber > leaves > stems > aerial stem, with the highest nitrogen accumulation observed in 25%OM + 75%CF treatment. Nitrogen harvest index did not show significant differences among treatments, fluctuating between 0.69 and 0.74. The nitrogen apparent utilization efficiency was highest in 25%OM + 75%CF (9.89%), followed by CF (9.09%), both significantly higher than OM (5.32%) and 50%OM + 50%CF (6.69%). The nitrogen agronomic efficiency varied significantly among treatments, with 25%OM + 75%CF (33.93 kg kg-1) being the highest, followed by CF (29.68 kg kg-1), 50%OM + 50%CF (21.82 kg kg-1), and OM (11.85 kg kg-1). Nitrogen partial factor productivity was highest in 25%OM + 75%CF treatment (76.37 kg kg-1), followed by CF (72.11 kg kg-1), both significantly higher than 50%OM + 50%CF (64.25 kg kg-1) and OM (54.29 kg kg-1), with OM exhibiting significantly lower values compared to other treatments. In conclusion, the combined application of organic and inorganic fertilizers can effectively enhance the yield, quality, and nitrogen utilization efficiency of D. polystachya. Particularly, the treatment with 25% organic fertilizer and 75% chemical fertilizer showed the most promising results.

Investigation of the influence of minor components and fatty acid profile of oil on properties of beeswax and stearic acid-based oleogels.

Understanding the impact of minor components and the fatty acid profile of oil on oleogel properties is essential for optimizing their characteristics. Considering the scarcity of literature addressing this aspect, this study aimed to explore the correlation between these factors and the properties of beeswax and stearic acid-based oleogels derived from rice bran oil and sesame oil. Minor oil components were modified by stripping the oil, heating the oil with water, and adding ß-sitosterol. Oleogels were then prepared using a mixture of beeswax and stearic acid (3:1, w/w) at a concentration of 11.74 % (w/w). The properties of oils and oleogels were evaluated. The findings indicated that minor components and fatty acid composition of the oils substantially influence the oleogel properties. Removing minor components by stripping resulted in smaller and less uniformly distributed crystals and less oil binding capacity compared to the oleogels prepared from untreated oils. A moderate amount of minor components exhibited a significant influence on oleogel properties. The addition of ß-sitosterol did not show any influence on oleogel properties except for the oleogel made from untreated oil blend added with ß-sitosterol which had more uniform crystals in the microstructure and demonstrated better rheological stability when stored at 5 °C for two months. The oil composition did not show any influence on the thermal and molecular properties of oleogels. Consequently, the oleogel formulation derived from the untreated oil blend enriched with ß-sitosterol was identified as the optimal formula for subsequent development. The findings of this study suggest that the physical and mechanical properties as well as the oxidative stability of beeswax and stearic acid-based oleogels are significantly affected by the minor constituents and fatty acid composition of the oil. Moreover, it demonstrates that the properties of oleogels can be tailored by modifying oil composition by blending different oils.

Semiconducting polymer dots based l-lactate sensor by enzymatic cascade reaction system.

BACKGROUND: l-lactate detection is important for not only assessing exercise intensity, optimizing training regimens, and identifying the lactate threshold in athletes, but also for diagnosing conditions like L-lactateosis, monitoring tissue hypoxia, and guiding critical care decisions. Moreover, l-lactate has been utilized as a biomarker to represent the state of human health. However, the sensitivity of the present l-lactate detection technique is inadequate. RESULTS: Here, we reported a sensitive ratiometric fluorescent probe for l-lactate detection based on platinum octaethylporphyrin (PtOEP) doped semiconducting polymer dots (Pdots-Pt) with enzymatic cascade reaction. With the help of an enzyme cascade reaction, the l-lactate was continuously oxidized to pyruvic and then reduced back to l-lactate for the next cycle. During this process, oxygen and NADH were continuously consumed, which increased the red fluorescence of Pdots-Pt that responded to the changes of oxygen concentration and decreased the blue fluorescence of NADH at the same time. By comparing the fluorescence intensities at these two different wavelengths, the concentration of l-lactate was accurately measured. With the optimal conditions, the probes showed two linear detection ranges from 0.5 nM to 5.0 µM and 5.0 µM-50.0 µM for l-lactate detection. The limit of detection was calculated to be 0.18 nM by 3σ/slope method. Finally, the method shows good detection performance of l-lactate in both bovine serum and artificial serum samples, indicating its potential usage for the selective analysis of l-lactate for health monitoring and disease diagnosis. SIGNIFICANCE: The successful application of the sensing system in the complex biological sample (bovine serum and artificial serum samples) demonstrated that this method could be used for sensitive l-lactate detection in practical clinical applications. This detection system provided an extremely low detection limit, which was several orders of magnitude lower than methods proposed in other literatures.

Delineating the Spectrum of Pituitary Adenoma Based on the WHO 2017 Classification.

BACKGROUND: The WHO 2017 classification of endocrine tumors incorporates lineage-specific transcription factors (TF) and hormone expression for the classification of pituitary adenoma (PA). There is paucity of reports describing the spectrum of PA based on this classification. OBJECTIVE: The aim of this study was to delineate the spectrum of PA based on WHO 2017 classification of endocrine tumors. MATERIALS AND METHODS: PA diagnosed in the year 2018 were studied. H and E and hormonal immunohistochemistry (IHC) for GH, PRL, ACTH, TSH, FSH, LH, CK, T-Pit and MIB-1 were performed and the results were analyzed. RESULTS: The cohort included 88 cases. M: F ratio was 2:1. Clinically, 22 (25%) were functional and 66 (75%) were non-functional adenomas. Amongst the clinically functional adenomas, GH secreting adenomas were the commonest (68%). Majority (83%) of non-functional adenomas were hormone positive with gonadotroph adenomas being the commonest (72.7%). Eleven (12.5%) PA were clinically and hormonally silent. Three of these showed intense nuclear T-Pit positivity, classifying them under silent corticotroph adenoma. Lineage of the remaining eight adenomas remained undetermined, since, IHC for Pit-1 and SF-1 was not performed. The aggressive adenomas identified by IHC included sparsely granulated somatotroph adenoma, Crooke cell adenoma, silent corticotroph adenoma, densely granulated lactotroph adenoma in men and constituted 17% of the PA. Four (4/88) cases were clinically invasive. CONCLUSION: A large majority of PA including aggressive adenomas can be identified by IHC. Addition of T-Pit helped to identify silent corticotroph adenoma. Pit -1 and SF-1 TF would help identify plurihormonal Pit-1 PA and null cell adenomas.

Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.

FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography-tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002-10 µg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at -20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5-5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies.

Regulation of the Nur77-P2X7r Signaling Pathway by Nodakenin: A Potential Protective Function against Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is the main factor that induces liver-related death worldwide and represents a common chronic hepatopathy resulting from binge or chronic alcohol consumption. This work focused on revealing the role and molecular mechanism of nodakenin (NK) in ALD associated with hepatic inflammation and lipid metabolism through the regulation of Nur77-P2X7r signaling. In this study, an ALD model was constructed through chronic feeding of Lieber-DeCarli control solution with or without NK treatment. Ethanol (EtOH) or NK was administered to AML-12 cells, after which Nur77 was silenced. HepG2 cells were exposed to ethanol (EtOH) and subsequently treated with recombinant Nur77 (rNur77). Mouse peritoneal macrophages (MPMs) were treated with lipopolysaccharide/adenosine triphosphate (LPS/ATP) and NK, resulting in the generation of conditioned media. In vivo, histopathological alterations were markedly alleviated by NK, accompanied by reductions in serum triglyceride (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels and the modulation of Lipin-1, SREBP1, and Nur77 levels in comparison to the EtOH-exposed group (p < 0.001). Additionally, NK reduced the production of P2X7r and NLRP3. NK markedly upregulated Nur77, inhibited P2X7r and Lipin-1, and promoted the function of Cytosporone B, a Nur77 agonist (p < 0.001). Moreover, Nur77 deficiency weakened the regulatory effect of NK on P2X7r and Lipin-1 inhibition (p < 0.001). In NK-exposed MPMs, cleaved caspase-1 and mature IL-1ß expression decreased following LPS/ATP treatment (p < 0.001). NK also decreased inflammatory-factor production in primary hepatocytes stimulated with MPM supernatant. NK ameliorated ETOH-induced ALD through a reduction in inflammation and lipogenesis factors, which was likely related to Nur77 activation. Hence, NK is a potential therapeutic approach to ALD.