AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Mechanism of voltage gating in the voltage-sensing phosphatase Ci-VSP.

Conformational changes in voltage-sensing domains (VSDs) are driven by the transmembrane electric field acting on the protein charges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes as well as the major conformations visited during a complete functional gating cycle in the isolated VSD of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1 to S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated "Up" and resting "Down" states, the simulations predict two Ci-VSD conformations: a deeper resting state ("down-minus") and an extended activated ("up-plus") state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. The present results show that these four states are visited sequentially in a stepwise manner during voltage activation, each step translocating one arginine or the equivalent of ∼1 e0 across the membrane electric field, yielding a transfer of ∼3 e0 charges in total for the complete process.

Metabolic reprogramming contributes to radioprotection by protein kinase Cδ.

Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.

Phosphatase regulatory subunit MYPT2 knockout partially compensates for the cardiac dysfunction in mice caused by lack of myosin light chain kinase 3.

Cardiac contraction is modulated by the phosphorylation state of myosin regulatory light chain 2 (MLC-2v). The level of MLC-2v phosphorylation is dependent on the opposing activities of MLC kinases and phosphatases. The predominant MLC phosphatase found in cardiac myocytes contains Myosin Phosphatase Targeting Subunit 2 (MYPT2). Overexpression of MYPT2 in cardiac myocytes results in a decreased level of MLC phosphorylation, reduced left ventricular contraction, and induction of hypertrophy; however, the effect of knocking out MYPT2 on cardiac function is unknown. We obtained heterozygous mice containing a MYPT2 null allele from the Mutant Mouse Resource Center. These mice were produced in a C57BL/6N background which lack MLCK3, the main regulatory light chain kinase in cardiac myocytes. We found that mice null for MYPT2 were viable and had no obvious phenotypic abnormality when compared to WT mice. Additionally, we determined that WT C57BL/6N mice had a low basal level of MLC-2v phosphorylation, which was significantly increased when MYPT2 was absent. At 12-weeks, MYPT2 KO mice had smaller hearts and showed downregulation of genes involved in cardiac remodeling. Using cardiac echo, we found that 24-week-old male MYPT2 KO mice had decreased heart size with increased fractional shortening compared to their MYPT2 WT littermates. Collectively, these studies highlight the important role that MYPT2 plays in cardiac function in vivo and demonstrate that its deletion can partially compensate for the lack of MLCK3.

Targeting WIP1 phosphatase promotes partial remission in experimental collapsing glomerulopathy.

Collapsing focal segmental glomerulosclerosis (FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by a rapid progression to kidney failure. Understanding CG pathogenesis represents a key step for the development of targeted therapies. Previous work implicated the telomerase protein component TERT in CG pathogenesis, as transgenic TERT expression in adult mice resulted in a CG resembling that seen in human primary CG and HIV-associated nephropathy (HIVAN). Here, we used the telomerase-induced mouse model of CG (i-TERTci mice) to identify mechanisms to inhibit CG pathogenesis. Inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocked disease initiation in i-TERTci mice. Repression of disease initiation upon WIP1 deficiency was associated with senescence enhancement and required transforming growth factor-ß functions. The efficacy of a pharmacologic treatment to reduce disease severity in both i-TERTci mice and in a mouse model of HIVAN (Tg26 mice) was then assessed. Pharmacologic inhibition of WIP1 enzymatic activity in either the telomerase mice with CG or in the Tg26 mice promoted partial remission of proteinuria and ameliorated kidney histopathologic features. Histological as well as high-throughput sequencing methods further showed that selective inhibition of WIP1 does not promote kidney fibrosis or inflammation. Thus, our findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG.

Mechanisms of CH4 activation over oxygen-preadsorbed transition metals by ReaxFF and AIMD simulations.

The chemisorbed oxygen usually promotes the CH bond activation over less active metals like IB group metals but has no effect or even an inhibition effect over more active metals like Pd based on the static electronic structure study. However, the understanding in terms of dynamics knowledge is far from complete. In the present work, methane dissociation on the oxygen-preadsorbed transition metals including Au, Cu, Ni, Pt, and Pd is systemically studied by reactive force field (ReaxFF). The ReaxFF simulation results indicate that CH4 molecules mainly undergo the direct dissociation on Ni, Pt, and Pd surfaces, while undergo the oxygen-assisted dissociation on Au and Cu surfaces. Additionally, the ab initio molecular dynamics (AIMD) simulations with the umbrella sampling are employed to study the free-energy changes of CH4 dissociation, and the results further support the CH4 dissociation pathway during the ReaxFF simulations. The present results based on ReaxFF and AIMD will provide a deeper dynamic understanding of the effects of pre-adsorbed oxygen species on the CH bond activation compared to that of static DFT.

A heat-inducible expression system for external control of gene expression in plastids.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

One-pot selective biosynthesis of Tyrian purple in Escherichia coli.

Tyrian purple (6,6'-Dibromoindigo) is an ancient precious dye, which possesses remarkable properties as a biocompatible semiconductor material. Recently, biosynthesis has emerged as an alternative for the sustainable production of Tyrian purple from a natural substrate. However, the selectivity issue in enzymatic tryptophan (Trp) and bromotryptophan (6-Br-Trp) degradation was an obstacle for obtaining high-purity Tyrian purple in a single cell biosynthesis. In this study, we present a simplified one-pot process for the production of Tyrian purple from Trp in Escherichia coli (E. coli) using Trp 6-halogenase from Streptomyces toxytricini (SttH), tryptophanase from E. coli (TnaA) and a two-component indole oxygenase from Providencia Rettgeri GS-2 (GS-C and GS-D). To enhance the in vivo solubility and activity of SttH and flavin reductase (Fre) fusion enzyme (Fre-L3-SttH), a chaperone system of GroEL/GroES (pGro7) was introduced in addition to the implementation of a set of optimization strategies, including fine-tuning the expression vector, medium, concentration of bromide salt and inducer. To overcome the selectivity issue and achieve a higher conversion yield of Tyrian purple with minimal indigo formation, we applied the λpL/pR-cI857 thermoinducible system to temporally control the bifunctional fusion enzyme of TnaA and monooxygenase GS-C (TnaA-L3-GS-C). Through optimization of the fermentation process, we were able to achieve a Tyrian purple titer of 44.5 mg L-1 with minimal indigo byproduct from 500 µM Trp. To the best of our knowledge, this is the first report of the selective production of Tyrian purple in E. colivia a one-pot process.

The response of mesophyll conductance to short-term CO2 variation is related to stomatal conductance.

The response of mesophyll conductance (gm) to CO2 plays a key role in photosynthesis and ecosystem carbon cycles under climate change. Despite numerous studies, there is still debate about how gm responds to short-term CO2 variations. Here we used multiple methods and looked at the relationship between stomatal conductance to CO2 (gsc) and gm to address this aspect. We measured chlorophyll fluorescence parameters and online carbon isotope discrimination (Δ) at different CO2 mole fractions in sunflower (Helianthus annuus L.), cowpea (Vigna unguiculata L.), and wheat (Triticum aestivum L.) leaves. The variable J and Δ based methods showed that gm decreased with an increase in CO2 mole fraction, and so did stomatal conductance. There were linear relationships between gm and gsc across CO2 mole fractions. gm obtained from A-Ci curve fitting method was higher than that from the variable J method and was not representative of gm under the growth CO2 concentration. gm could be estimated by empirical models analogous to the Ball-Berry model and the USO model for stomatal conductance. Our results suggest that gm and gsc respond in a coordinated manner to short-term variations in CO2, providing new insight into the role of gm in photosynthesis modelling.

Illuminating stomatal responses to red light: establishing the role of Ci-dependent vs -independent mechanisms in control of stomatal behaviour.

The stomatal response to red light appears to link stomatal conductance (gs) with photosynthetic rates. Initially, it was suggested that changes in intercellular CO2 (Ci) provide the main cue via a Ci-dependent response. However, evidence for Ci-independent mechanisms suggests an additional, more direct relationship with photosynthesis. While both Ci-dependent and -independent mechanisms clearly function in stomatal red-light responses, little is known regarding their relative contribution. The present study aimed to quantify the relative magnitude of Ci-dependent and -independent mechanisms on the stomatal red-light response, to characterise their interplay and to assess the putative link between plastoquinone (PQ) redox state and Ci-independent stomatal responses. Red light response curves measured at a range of Ci values for wild-type Arabidopsis thaliana (Col-0) and the CO2 hyposensitive mutant, ca1ca4, allowed deconvolution of Ci-dependent and -independent pathways. Surprisingly, we observed that both mechanisms contribute equally to stomatal red-light responses, but Ci-independent stomatal opening is suppressed at high Ci. The present data are also consistent with the involvement of PQ redox in coordinating the Ci-independent component. Overall, it seems that while Ci-independent mechanisms are distinct from responses to Ci, interplay between these two pathways is important to facilitate effective coordination between gs and photosynthesis.

The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity to steady-state approaches in half the time.

The net CO2 assimilation (A) response to intercellular CO2 concentration (Ci) is a fundamental measurement in photosynthesis and plant physiology research. The conventional A/Ci protocols rely on steady-state measurements and take 15-40 min per measurement, limiting data resolution or biological replication. Additionally, there are several CO2 protocols employed across the literature, without clear consensus as to the optimal protocol or systematic biases in their estimations. We compared the non-steady-state Dynamic Assimilation Technique (DAT) protocol and the three most used CO2 protocols in steady-state measurements, and tested whether different CO2 protocols lead to systematic differences in estimations of the biochemical limitations to photosynthesis. The DAT protocol reduced the measurement time by almost half without compromising estimation accuracy or precision. The monotonic protocol was the fastest steady-state method. Estimations of biochemical limitations to photosynthesis were very consistent across all CO2 protocols, with slight differences in Rubisco carboxylation limitation. The A/Ci curves were not affected by the direction of the change of CO2 concentration but rather the time spent under triose phosphate utilization (TPU)-limited conditions. Our results suggest that the maximum rate of Rubisco carboxylation (Vcmax), linear electron flow for NADPH supply (J), and TPU measured using different protocols within the literature are comparable, or at least not systematically different based on the measurement protocol used.

Precision matters: the value of PET/CT and PET/MRI in the clinical management of cervical cancer.

The incidence of cervical cancer has been increasing recently, becoming an essential factor threatening patients' health. Positron emission computed tomography (PET/CT) and positron emission tomography/magnetic resonance imaging (PET/MRI) are multimodal molecular imaging methods that combine functional imaging (PET) and anatomical imaging (CT) with MRI fusion technology. They play an important role in the clinical management of patients with cervical cancer. Precision radiotherapy refers to the use of advanced intensive modulated radiotherapy (IMRT) to give different doses of radiation to different treatment areas to achieve the purpose of killing tumors and protecting normal tissues to the greatest extent. At present, pelvic target delineation is mostly based on CT and MRI, but these mostly provide anatomical morphological information, which is difficult to show the internal metabolism of tumors. PET/CT and PET/MRI combine information on biological function, metabolism and anatomical structure, thereby more accurately distinguishing the boundaries between tumor and non-tumor tissues and playing a positive guiding role in improving radiotherapy planning (RTP) for cervical cancer and evaluating treatment effect.

Targeting of immune checkpoint regulator V-domain Ig suppressor of T-cell activation (VISTA) with 89Zr-labelled CI-8993.

BACKGROUND: CI-8993 is a fully human IgG1κ monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Phase I safety has been established in patients with advanced cancer (NCT02671955). To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop 89Zr-labelled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial. METHODS: CI-8993 and human isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabelling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [89Zr]Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabelled CI-8993, as well as isotype control [89Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsirtm1.1(VSIR)Geno, huVISTA KI) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumours; and in BALB/c nu/nu mice bearing pancreatic Capan-2 tumours. RESULTS: Stable constructs with an average chelator-to-antibody ratio of 1.81 were achieved. SDS-PAGE and SE-HPLC showed integrity of CI-8993 was maintained after conjugation; and ELISA indicated no impact of conjugation and radiolabelling on binding to human VISTA. PET imaging and biodistribution in MB49 tumour-bearing huVISTA KI female mice showed specific localisation of [89Zr]Zr-Df-CI-8993 to VISTA in spleen and tumour tissues expressing human VISTA. Specific tumour uptake was also demonstrated in Capan-2 xenografted BALB/c nu/nu mice. CONCLUSIONS: We radiolabelled and validated [89Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.

Mitochondria complex I deficiency in Candida albicans arrests the cell cycle at S phase through suppressive TOR and PKA pathways.

How mutations in mitochondrial electron transport chain (ETC) proteins impact the cell cycle of Candida albicans was investigated in this study. Using genetic null mutants targeting ETC complexes I (CI), III (CIII), and IV (CIV), the cell cycle stages (G0/G1, S phase, and G2/M) were analyzed via fluorescence-activated cell sorting (FACS). Four CI null mutants exhibited distinct alterations, including extended S phase, shortened G2/M population, and a reduction in cells size exceeding 10 µM. Conversely, CIII mutants showed an increased population in G1/G0 phase. Among four CI mutants, ndh51Δ/Δ and goa1Δ/Δ displayed aberrant cell cycle patterns correlated with previously reported cAMP/PKA downregulation. Specifically, nuo1Δ/Δ and nuo2Δ/Δ mutants exhibited increased transcription of RIM15, a central hub linking cell cycle with nutrient-dependent TOR1 and cAMP/PKA pathways and Snf1 aging pathway. These findings suggest that suppression of TOR1 and cAMP/PKA pathways or enhanced Snf1 disrupts cell cycle progression, influencing cell longevity and growth among CI mutants. Overall, our study highlights the intricate interplay between mitochondrial ETC, cell cycle, and signaling pathways.

Asymptotic Confidence Interval, Sample Size Formulas and Comparison Test for the Agreement Intra-Class Correlation Coefficient in Inter-Rater Reliability Studies.

The agreement intra-class correlation coefficient (ICCa) is a suitable statistical index for inter-rater reliability studies. With balanced Gaussian data, we prove the explicit form of ICCa asymptotic normality (ASN), valid both with analysis of variance (ANOVA), maximum likelihood (ML), or restricted ML (REML) estimates. An asymptotic confidence interval is then derived and its performances are examined by simulation compared to the most commonly used methods, under small, moderate and large sample size designs. Then, we deduce sample size calculation formulas, for the number of subjects and observers needed, to achieve a desired confidence interval width or an acceptable ICCa value test power and give concrete examples of their use. Finally, we propose a likelihood ratio test (LRT) to compare two ICCa's from two distinct subpopulations of patients (or raters) and study by simulation its first order risk and power properties. These methods are illustrated using data from two inter-rater reliability studies, one in physiotherapy with 42 patients and 10 raters and the second in neonatology with 80 subjects and 14 raters. In conclusion, we made recommendations to employ the proposed confidence interval for medium to large samples combined with the quantification of the minimal required sample size at the planning step, or the posterior-power at the analysis step, using simple dedicated formulas. Furthermore, with sufficient sizes, the proposed LRT seems suitable to compare inter-rater reliability between two patient subpopulations. Used wisely, this proposed methods toolbox can remedy common current issues in inter-rater reliability studies.

Complex I, V, and MDH2 deficient human skin fibroblasts reveal distinct metabolic signatures by 1 H HR-MAS NMR.

In this study, we investigated the metabolic signatures of different mitochondrial defects (two different complex I and complex V, and the one MDH2 defect) in human skin fibroblasts (HSF). We hypothesized that using a selective culture medium would cause defect specific adaptation of the metabolome and further our understanding of the biochemical implications for the studied defects. All cells were cultivated under galactose stress condition and compared to glucose-based cell culture condition. We investigated the bioenergetic profile using Seahorse XFe96 cell analyzer and assessed the extracellular metabolic footprints and the intracellular metabolic fingerprints using NMR. The galactose-based culture condition forced a bioenergetic switch from a glycolytic to an oxidative state in all cell lines which improved overall separation of controls from the different defect groups. The extracellular metabolome was discriminative for separating controls from defects but not the specific defects, whereas the intracellular metabolome suggests CI and CV changes and revealed clear MDH2 defect-specific changes in metabolites associated with the TCA cycle, malate aspartate shuttle, and the choline metabolism, which are pronounced under galactose condition.

Panax quinquefolium saponins attenuates microglia activation following acute cerebral ischemia-reperfusion injury via Nrf2/miR-103-3p/TANK pathway.

Ischemic stroke is one of the leading causes of death and disability among adults worldwide. Intravenous thrombolysis is the only approved pharmacological treatment for acute ischemic stroke. However, reperfusion by thrombolysis will lead to the rapid activation of microglia cells which induces interferon-inflammatory response in the ischemic brain tissues. Panax quinquefolium saponins (PQS) has been proven to be effective in acute ischemic stroke, but there is no unified understanding about its specific mechanism. Here, we will report for the first time that PQS can significantly inhibit the activation of microglia cells in cerebral of MCAO rats via activation of Nrf2/miR-103-3p/TANK axis. Our results showed that PQS can directly bind to Nrf2 protein and inhibit its ubiquitination, which result in the indirectly enhancing the expression of TANK protein via transcriptional regulation on miR-103-3p, and finally to suppress the nuclear factor kappa-B dominated rapid activation of microglial cells induced by oxygen-glucose deprivation/reoxygenation  vitro and cerebral ischemia-reperfusion injury in vivo. In conclusion, our study not only revealed the new mechanism of PQS in protecting against the inflammatory activation of microglia cells caused by cerebral ischemia-reperfusion injury, but also suggested that Nrf2 is a potential target for development of new drugs of ischemic stroke. More importantly, our study also reminded that miR-103-3p might be used as a prognostic biomarker for patients with ischemic stroke.

Antioxidant anthocyanin synergistic immune enhancer nanopeptide C-I20 remarkably enhances the protective effect of largemouth bass against largemouth bass ranavirus.

Largemouth bass ranavirus (LMBV) infection results in huge economic losses in largemouth bass (Micropterus salmoides) industry. Nanopeptide C-I20 and anthocyanins have a positive effect on promoting immune responses and antioxidant mechanisms in several aquatic organisms, and are therefore used to inhibit LMBV infection. In this study, we developed an LMBV immersion challenge model using three different viral concentrations (1 × 104 copies/mL, 1 × 105 copies/mL, and 1 × 106 copies/mL) to infect largemouth bass, and LMBV-MCP mRNA expression was detected in infected fish. Following infection, the fish exhibited severe external ulceration, redness swelling, and darkening of the skin. Histopathological examination revealed significant necrosis and inflammation in muscle tissue, epithelial cell shedding in renal tubules, macrophage aggregation centers and cellular vacuolization in spleen and head kidney, and cellular hypertrophy in liver. To mitigate LMBV infection, we explored the protective effects of a combined treatment strategy involving C-I20 and anthocyanin. Overall, the combination of anthocyanin and C-I20 demonstrated the highest protective efficacy, significantly reducing viral loads in muscle, liver, spleen, and head kidney. Moreover, this treatment regimen enhanced antioxidant enzyme activities (T-AOC, TSOD, GSH-Px, CAT) and modulated important immune genes (IL-1, IL-8, TNF-α, IL-10, Mx, and IgM) expression. In conclusion, the synergistic application of anthocyanin and C-I20 demonstrates significant efficacy in mitigating LMBV infection. This research introduces a novel and promising approach to managing infectious diseases in aquaculture settings.

Resolving Atmospheric Oxygenated Organic Molecules in Urban Beijing Using Online Ultrahigh-Resolution Chemical Ionization Mass Spectrometry.

Gaseous oxygenated organic molecules (OOMs) are crucial precursors of atmospheric organic aerosols. OOMs in urban atmospheres have complex compositions, posing challenges to understanding their formation, evolution, and influences. In this study, we identify 2403 atmospheric gaseous OOMs in urban Beijing using online nitrate-based chemical ionization Orbitrap mass spectrometry based on one-year atmospheric measurements. We find that OOMs in urban atmospheres can be identified with higher accuracy and wider coverage, compared to previously used online mass spectrometry. With optimized OOM resolving capabilities, previous knowledge of OOMs in urban atmospheres can be expanded. First, clear homologous and oxygen-addition characteristics of the OOMs are revealed. Second, OOMs with lower concentrations or higher masses are identified and characterized with high confidence, e.g., OOMs with masses above 350 Da. In particular, dimers of OOMs (e.g., C20H32O8-15N2), crucial species for organic nucleation, are identified. During four seasons, nitrogen-containing OOMs dominate the total concentration of OOMs, and OOMs are mainly from aromatic and aliphatic oxidation. Additionally, radicals with similar composition as OOMs, intermediates for OOM formation, are identified with clear diurnal variation, e.g., CnH2n-5O6 radicals (n = 8-10) and CmH2m-4NO9 radicals (m = 9-10), peak during the daytime and nighttime, respectively, previously having scarce measurement evidence in urban atmospheres.