The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor.

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration.

Investigation of the Binding Interaction of Mfsd2a with NEDD4-2 via Molecular Dynamics Simulations.

Major facilitator superfamily domain-containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine cotransporter that plays an important role in maintaining the integrity of the blood-brain barrier and neurological function. Abnormal degradation of Mfsd2a often leads to dysfunction of the blood-brain barrier, while upregulation of Mfsd2a can retrieve neurological damage. It has been reported that Mfsd2a can be specifically recognized and ubiquitinated by neural precursor cell-expressed developmentally downregulated gene 4 type 2 (NEDD4-2) ubiquitin ligase and finally degraded through the proteasome pathway. However, the structural basis for the specific binding of Mfsd2a to NEDD4-2 is unclear. In this work, we combined deep learning and molecular dynamics simulations to obtain a Mfsd2a structure with high quality and a stable Mfsd2a/NEDD4-2-WW3 interaction model. Moreover, molecular mechanics generalized Born surface area (MM-GBSA) methods coupled with per-residue energy decomposition studies were carried out to analyze the key residues that dominate the binding interaction. Based on these results, we designed three peptides containing the key residues by truncating the Mfsd2a sequences. One of them was found to significantly inhibit Mfsd2a ubiquitination, which was further validated in an oxygen-glucose deprivation (OGD) model in a human microvascular endothelial cell line. This work provides some new insights into the understanding of Mfsd2a and NEDD4-2 interaction and might promote further development of drugs targeting Mfsd2a ubiquitination.

Metabolomics profiling to characterize cerebral ischemia-reperfusion injury in mice.

Cerebral ischemia, resulting from compromised blood flow, is one of the leading causes of death worldwide with limited therapeutic options. Potential deleterious injuries resulting from reperfusion therapies remain a clinical challenge for physicians. This study aimed to explore the metabolomic alterations during ischemia-reperfusion injury by employing metabolomic analysis coupled with gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and ultraperformance liquid chromatography quadrupole (UPLC/Q)-TOF-MS. Metabolomic data from mice subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion (MCAO/R) were compared to those of the sham and MCAO groups. A total of 82 simultaneously differentially expressed metabolites were identified among each group. The top three major classifications of these differentially expressed metabolites were organic acids, lipids, and organooxygen compounds. Metabolomics pathway analysis was conducted to identify the underlying pathways implicated in MCAO/R. Based on impactor scores, the most significant pathways involved in the response to the reperfusion after cerebral ischemia were glycerophospholipid metabolism, linoleic acid metabolism, pyrimidine metabolism, and galactose metabolism. 17 of those 82 metabolites were greatly elevated in the MCAO/Reperfusion group, when compared to those in the sham and MCAO groups. Among those metabolites, glucose-6-phosphate 1, fructose-6-phosphate, cellobiose 2, o-phosphonothreonine 1, and salicin were the top five elevated metabolites in MCAO/R group, compared with the MCAO group. Glycolysis, the pentose phosphate pathway, starch and sucrose metabolism, and fructose and mannose degradation were the top four ranked pathways according to metabolite set enrichment analysis (MSEA). The present study not only advances our understanding of metabolomic changes among animals in the sham and cerebral ischemia groups with or without reperfusion via metabolomic profiling, but also paves the way to explore potential molecular mechanisms underlying metabolic alteration induced by cerebral ischemia-reperfusion.

Investigation of an artifact during non-reduced capillary electrophoresis with sodium dodecyl sulfate analysis utilizing N-ethylmaleimide as an alkylation reagent.

This manuscript describes the formation of an artifact shoulder peak with a slightly larger retention time than the main peak under the standard non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS) analysis of a therapeutic recombinant protein X, and clarifies the formation mechanism of the artifact caused by N-ethylmaleimide (NEM) during the sample preparation procedure. A design of experiment (DoE) approach was used to investigate the impact of the factors on the formation of the impurity. Additionally, orthogonal analytical experiments were performed to study the root cause of this phenomenon. The results consistently suggested that the Michael addition reaction between NEM and lysine residues in protein X, and decreased electrophoretic mobility due to increased molecular weight, was the root cause for the artifact, which could be partially inhibited by modifications of incubation conditions. Thus, before performing the nrCE-SDS method, the effects of alkylation reagents and sample preparation procedure on analytical results need to be considered seriously.

Identification and characterization of chemical and physical stability of insulin formulations utilizing degraded glycerol after repeated use and storage.

Insulin treatment is currently considered to be the main strategy for controlling diabetes. Although the recombinant insulin formulation is relatively mature, we found that a batch of insulin formulation exhibited an unusual degradation rate in the stability experiment. The main purposes of this article are to identify the root cause for this phenomenon and characterize of chemical and physical degradation products. We compared the chemical and physical stability of two batches of insulin formulations prepared separately with simulated repeated use and freshly opened glycerol. The chemical stability of insulin was identified by liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Micro-flow imaging (MFI), far-ultraviolet circular dichroism (Far-UV CD) and Thioflavin T (ThT) fluorescent assays were used to reveal protein aggregation and fibrosis. The chemical and physical stability of the insulin formulation with newly opened glycerol was much better than that with degraded glycerol, and both groups of formulations were extremely sensitive to light. The results indicated that the original batch insulin formulation with abnormal stability was indeed caused by the excipient glycerol after long-term storage and repeated usage. More attention should be paid to the quality changes of excipients during repeated usage and storage of excipients for the practical purpose. Moreover, we have discovered a novel degradation pathway for insulin and peptides in general. In addition, LC-MS/MS results suggested that the N-terminus of insulin B-chain was prone to chemical degradation which enlightens that it could be potentially modified to improve the stability of insulin formulations.

Redistribution of Histone Marks on Inflammatory Genes Associated With Intracerebral Hemorrhage-Induced Acute Brain Injury in Aging Rats.

The contribution of histone mark redistribution to the age-induced decline of endogenous neuroprotection remains unclear. In this study, we used an intracerebral hemorrhage (ICH)-induced acute brain injury rat model to study the transcriptional and chromatin responses in 13- and 22-month-old rats. Transcriptome analysis (RNA-seq) revealed that the expression of neuroinflammation-associated genes was systematically upregulated in ICH rat brains, irrespective of age. Further, we found that interferon-γ (IFN-γ) response genes were activated in both 13- and 22-month-old rats. Anti-IFN-γ treatment markedly reduced ICH-induced acute brain injury in 22-month-old rats. At the chromatin level, ICH induced the redistribution of histone modifications in the promoter regions, especially H3K4me3 and H3K27me3, in neuroinflammation-associated genes in 13- and 22-month-old rats, respectively. Moreover, ICH-induced histone mark redistribution and gene expression were found to be correlated. Collectively, these findings demonstrate that histone modifications related to gene expression are extensively regulated in 13- and 22-month-old rats and that anti-IFN-γ is effective for ICH treatment, highlighting the potential of developing therapies targeting histone modifications to cure age-related diseases, including brain injury and neuroinflammation.

RING-box proteins regulate leaf senescence and stomatal closure via repression of ABA transporter gene ABCG40.

Plant hormone abscisic acid (ABA) plays an indispensable role in the control of leaf senescence, during which ABA signaling depends on its biosynthesis. Nevertheless, the role of ABA transport in leaf senescence remains unknown. Here, we identified two novel RING-box protein-encoding genes UBIQUITIN LIGASE of SENESCENCE 1 and 2 (ULS1 and ULS2) involved in leaf senescence. Lack of ULS1 and ULS2 accelerates leaf senescence, which is specifically promoted by ABA treatment. Furthermore, the expression of senescence-related genes is significantly affected in mature leaves of uls1/uls2 double mutant (versus wild type (WT)) in an ABA-dependent manner, and the ABA content is substantially increased. ULS1 and ULS2 are mainly expressed in the guard cells and aging leaves, and the expression is induced by ABA. Further RNA-seq and quantitative proteomics of ubiquitination reveal that ABA transporter ABCG40 is highly expressed in uls1/uls2 mutant versus WT, though it is not the direct target of ULS1/2. Finally, we show that the acceleration of leaf senescence, the increase of leaf ABA content, and the promotion of stomatal closure in uls1/usl2 mutant are suppressed by abcg40 loss-of-function mutation. These results indicate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Pericytes Regulate Cerebral Perfusion through VEGFR1 in Ischemic Stroke.

Neurons in the penumbra (the area surrounding ischemic tissue that consists of still viable tissue but with reduced blood flow and oxygen transport) may be rescued following stroke if adequate perfusion is restored in time. It has been speculated that post-stroke angiogenesis in the penumbra can reduce damage caused by ischemia. However, the mechanism for neovasculature formation in the brain remains unclear and vascular-targeted therapies for brain ischemia remain suboptimal. Here, we show that VEGFR1 was highly upregulated in pericytes after stroke. Knockdown of VEGFR1 in pericytes led to increased infarct area and compromised post-ischemia vessel formation. Furthermore, in vitro studies confirmed a critical role for pericyte-derived VEGFR1 in both endothelial tube formation and pericyte migration. Interestingly, our results show that pericyte-derived VEGFR1 has opposite effects on Akt activity in endothelial cells and pericytes. Collectively, these results indicate that pericyte-specific expression of VEGFR1 modulates ischemia-induced vessel formation and vascular integrity in the brain.

Isolation, physicochemical, and structure-function relationship of the hydrophobic variant of Fc-fusion proteins that bind to TNF-α receptor, HS002 and HS002A.

HS002 is the recombinant human tumor necrosis factor-α receptor Ⅱ: IgG Fc fusion protein licensed in China to treat rheumatism and psoriasis. The aim of this study was to isolate and characterize the hydrophobic freeze-dried powder injection (HS002) and ampoule injection (HS002A) variants derived from proteins of the same sequence and then to explore the structure-function relationship. Extensive physicochemical and structural testing was performed during a side-by-side comparison of the monomer peak and variant. Then the TNF-α-related binding activity, cell biological activity and affinity with FcRn were analyzed. Finally, a transformation study of the hydrophobic variant was performed under serum-like redox conditions. This research revealed that HS002A has similar physicochemical and structure-function relationship profiles to those of HS002. The hydrophobic variant exhibited the presence of new incorrect disulfide bridging. At the same time, this novel disulfide scrambled species structure-function relationship was found to be the molecular basis for reduced TNF-α binding and cell biological activities. In addition, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring TNF-α binding and cell biological activities to almost normal levels, all of which indicate that the variant is probably irrelevant to clinical efficacy once the drug enters the bloodstream.

Association Between Time to Endovascular Therapy and Outcomes in Patients With Acute Basilar Artery Occlusion.

OBJECTIVE: To characterize the association of onset to puncture time (OPT) with clinical outcomes among patients with acute basilar artery occlusion receiving endovascular therapy (EVT) in clinical practice. METHODS: Using the EVT for Acute Basilar Artery Occlusion (BASILAR) study, we identified consecutive patients with acute basilar artery occlusion receiving EVT in 47 comprehensive stroke centers in China from January 2014 to May 2019. The primary outcome was favorable functional outcome (defined as modified Rankin Scale score [mRS] 0-3) at 90 days. Secondary outcomes included function independence (mRS 0-2), mortality, and symptomatic intracerebral hemorrhage. The associations of OPT with clinical outcomes were analyzed using multivariable logistic regression (OPT as a categorical variable) and restricted cubic spline regression (OPT as a continuous variable). RESULTS: Among 639 eligible patients, the median age was 64 years, and median OPT was 328 minutes (interquartile range 220-490). Treatment within 4-8 hours and 8-12 hours was associated with lower rates of favorable outcome (adjusted odds ratio, 0.63 [95% confidence interval (CI), 0.40-0.98] and 0.47 [95% CI, 0.23-0.93], respectively) compared with treatment within 4 hours. Restricted cubic spline regression analysis showed that the OPT had L-shaped associations with favorable outcome (p nonlinearity = 0.028) and functional independence (p nonlinearity = 0.025), with significant benefit loss throughout the first 9 hours, but then appeared relatively flat. The odds of mortality increased relatively for OPT up to 9 hours, but then leveled off (p nonlinearity = 0.042). The association between symptomatic intracerebral hemorrhage and OPT was not significant. CONCLUSION: Among patients with acute basilar artery occlusion in routine practice, earlier treatment with EVT was associated with better outcomes throughout the first 9 hours after onset, but benefit may sustain unchanged afterwards. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with acute ischemic stroke due to basilar artery occlusion, earlier EVT is associated with better outcomes.

A novel twin-column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants.

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin-column continuous chromatography (called N-rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled-up batch-mode cation exchange (CEX) chromatography was conducted. For the N-rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22-cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled-up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N-rich and scaled-up CEX processes, the results indicated that the N-rich method displays a remarkable advantage on the product yield, i.e. 1.46-fold increment without the loss of productivity and purity. Generally, twin-column N-rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.

Isolation and characterization of charge variants of infliximab biosimilar HS626.

Charge variants are the most commonly observed sources of heterogeneity in the routine manufacturing of monoclonal antibodies. To gain further insight into the structural foundation of charge heterogeneity and its influence on biological functions, an infliximab biosimilar HS626 from a biopharmaceutical facility was isolated by semipreparative cation exchange chromatography (CEX) to obtain fractions of acidic and basic charge variants and determine the main species. It was assessed again by CEX to ensure purities. Through a series of structural and physicochemical characterizations, we concluded that the acidic variants were caused by fragments, Met oxidation, Asn deamidation, higher levels of sialylation and galactosylation of N-linked glycans, and less high mannose. The basic variants resulted mainly from aggregates, fragments, and Met oxidation. Through further analysis of antigen binding affinity, cell death inhibitory activity, ADCC, and CDC, as well as FcRn, FcγRIIIa, and C1q affinity, we demonstrated that the charge heterogeneity did not affect biological functions. This research enhances the understanding of charge variants, which are usually effective components that should not be intentionally reduced unless biological functions are affected.

Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein.

PURPOSES: The main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon. METHODS: A series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques. RESULTS: The formation of the impurity as observed in CE-SDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LC-MS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS. CONCLUSIONS: Our study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.

Quantitative Profiling of Oxylipins in Acute Experimental Intracerebral Hemorrhage.

Oxylipins are a series of bioactive lipid metabolites derived from polyunsaturated fatty acids that are involved in cerebral homeostasis and the development of intracerebral hemorrhage (ICH). However, comprehensive quantification of the oxylipin profile in ICH remains unknown. Therefore, an ICH mouse model was constructed and liquid chromatography tandem mass spectrometry was then performed to quantify the change in oxylipins in ICH. The expression of the oxylipin relative enzymes was also reanalyzed based on RNA-seq data from our constructed ICH dataset. A total of 58 oxylipins were quantifiable and the levels of 17 oxylipins increased while none decreased significantly in the first 3 days following ICH. The most commonly increased oxylipins in ICH were derived from AA (10/17) and EPA (4/17) followed by LA (2/17) and DHA (1/17). 18-HEPE from EPA was the only oxylipin that remained significantly increased from 0.5 to 3 days following ICH. Furthermore, 14 of the increased oxylipins reached a peak level on the first day of ICH, and soon decreased while five oxylipins (PGJ2, 15-oxo-ETE, 12-HEPE, 18-HEPE, and 5-oxo-ETE) had increased 3 days after ICH suggesting that the profile shifted with the progression of ICH. In our constructed RNA-seq dataset based on ICH rats, 90 oxylipin relative molecules were detected except for COX. Among these, Cyp4f18, Cyp1b1, Cyp2d3, Cyp2e1, Cyp1a1, ALOX5AP, and PLA2g4a were found up-regulated and Cyp26b1 was found to decrease in ICH. In addition, there was no significant change in sEH in ICH. This study provides fundamental data on the profile of oxylipins and their enzymes in ICH. We found that the profile shifted as the progression of ICH and the metabolism of arachidonic acid and eicosapentaenoic acid was highly affected in ICH, which will help further studies explore the functions of oxylipins in ICH.

Ethylene promotes seed iron storage during Arabidopsis seed maturation via ERF95 transcription factor.

Because Iron (Fe) is an essential element, Fe storage in plant seeds is necessary for seedling establishment following germination. However, the mechanisms controlling seed Fe storage during seed development remain largely unknown. Here we reveal that an ERF95 transcription factor regulates Arabidopsis seed Fe accumulation. We show that expression of ERF95 increases during seed maturation, and that lack of ERF95 reduces seed Fe accumulation, consequently increasing sensitivity to Fe deficiency during seedling establishment. Conversely, overexpression of ERF95 has the opposite effects. We show that lack of ERF95 decreases abundance of FER1 messenger RNA in developing seed, which encodes Fe-sequestering ferritin. Accordingly, a fer1-1 loss-of-function mutation confers reduced seed Fe accumulation, and suppresses ERF95-promoted seed Fe accumulation. In addition, ERF95 binds to specific FER1 promoter GCC-boxes and transactivates FER1 expression. We show that ERF95 expression in maturing seed is dependent on EIN3, the master transcriptional regulator of ethylene signaling. While lack of EIN3 reduces seed Fe content, overexpression of ERF95 rescues Fe accumulation in the seed of ein3 loss-of-function mutant. Finally, we show that ethylene production increases during seed maturation. We conclude that ethylene promotes seed Fe accumulation during seed maturation via an EIN3-ERF95-FER1-dependent signaling pathway.

Prdx1 Reduces Intracerebral Hemorrhage-Induced Brain Injury via Targeting Inflammation- and Apoptosis-Related mRNA Stability.

RNA-binding proteins (RBPs) have been shown to be involved in posttranscriptional regulation, which plays an important role in the pathophysiology of intracerebral hemorrhage (ICH). Peroxiredoxin 1 (Prdx1), an RBP, plays an important role in regulating inflammation and apoptosis. On the basis that inflammation and apoptosis may contribute to ICH-induced brain injury, in this study, we used ICH models coupled with in vitro experiments, to investigate the role and mechanism of Prdx1 in regulating inflammation and apoptosis by acting as an RBP after ICH. We first found that Prdx1 was significantly up-regulated in response to ICH-induced brain injury and was mainly expressed in astrocytes and microglia in ICH rat brains. After overexpressing Prdx1 by injecting adeno-associated virus (AAV) into the striatum of rats at 3 weeks, we constructed ICH models and found that Prdx1 overexpression markedly reduced inflammation and apoptosis after ICH. Furthermore, RNA immunoprecipitation combined with high-throughput sequencing (RIP-seq) in vitro revealed that Prdx1 affects the stability of inflammation- and apoptosis-related mRNA, resulting in the inhibition of inflammation and apoptosis. Finally, overexpression of Prdx1 significantly alleviated the symptoms and mortality of rats subjected to ICH. Our results show that Prdx1 reduces ICH-induced brain injury by targeting inflammation- and apoptosis-related mRNA stability. Prdx1 may be an improved therapeutic target for alleviating the brain injury caused by ICH.

Young plasma ameliorates aging-related acute brain injury after intracerebral hemorrhage.

Aging has been shown to contribute to both the declined biofunctions of aging brain and aggravation of acute brain damage, and the former could be reversed by young plasma. These results suggest that young plasma treatment may also reduce the acute brain damage induced by intracerebral hemorrhage (ICH). In the present study, we first found that the administration of young plasma significantly reduced the mortality and neurological deficit score in aging ICH rodents, which might be due to the decreased brain water content, damaged neural cells, and increased survival neurons around the perihematomal brain tissues. Then, proteomics analysis was used to screen out the potential neuroprotective circulating factors and the results showed that many factors were changed in health human plasma among young, adult, and old population. Among these significantly changed factors, the plasma insulin-like growth factor 1 (IGF-1) level was significantly decreased with age, which was further confirmed both in human and rats detected by ELISA. Additionally, the brain IGF-1 protein level in aging ICH rats was markedly decreased when compared with young rats. Interestingly, the relative decreased brain IGF-1 level was reversed by the treatment of young plasma in aging ICH rats, while the mRNA level was non-significantly changed. Furthermore, the IGF-1 administration significantly ameliorated the acute brain injury in aging ICH rats. These results indicated that young circulating factors, like IGF-1, may enter brain tissues to exert neuroprotective effects, and young plasma may be considered as a novel therapeutic approach for the clinical treatment of aging-related acute brain injury.

[Determination of trace element silver in animal serum, tissues and organs by microwave digestion-ICP-MS].

Nowadays, the silver is widely used in the biological field and its biological safety catches great attention. It is important to know the distribution of silver ions within the biological organism and the toxic threshold concentration in the tissue. Therefore, a highly sensitive method for measurement of trace amount of silver ion in the medical biological samples is needed. With its high sensitivity for detection of metal ions, inductively coupled plasma mass spectrometry (ICP-MS) method is well suited for quantification of trace amount of silver ion in such samples, but method development is still in its infancy. Consequently, a simple and convenient method for determination of trace amount of silver in the animal serum, tissues or organs was developed, in which the samples were subjected to the microwave digestion, followed by the ICP-MS analysis. To begin with, the samples of serum, muscle, bone marrow, bone, heart, liver, spleen, and kidney were sequently processed in 5 mL of HNO3 and 2 mL of H2O2 solution. Then the samples were completely digested by microwave with the power of 2 000 watts. The temperature was raised gradually by 3-step program. Moreover, the data achieved were reproducible and the method was time saving and especially for large amounts of sample processing. Then the digested solutions were diluted to constant volume. Finally, the concentration of 107Ag in the samples was analyzed by the method of ICP-MS under the optimized conditions. Element yttrium (Y) was used as the internal standard to compensate for matrix suppression effect and improve the accuracy of measurement. For one thing, the analytical results showed that the detection limit of the trace element 107Ag was 0.98 µg · kg(-1), and furthermore, the correlation coefficient of standard curve was 0.999 9. For another thing, the recovery rate of the silver element ranged from 98% to 107%, which was calculated according to measured quantity before adding standard, adding standard and measured quantity after adding standard. At the same time, the relative standard deviation (RSD) of the method was in the range of 2.0%-4.3%. The concentrations of element silver in animal serum, tissues and organs were determined by the aboved method. The obtained results showed that silver ions were mainly accumulated in the liver after they were intaken into the body. The results suggested that the microwave digestion-ICP-MS method could accurately determine the trace element Ag in the body. The method developed has good feasibility and is suitable for the determination of trace element Ag in various types of medical and biological samples, especially for large quantities of biological samples. The process has the advantages of easysample processing and it is simple and convenient. In addition, the accurate results could be obtained in a short time with high sensitivity. Last but not least, the method provides the guidance for the determination of trace elements in other biological samples.