Dose and DNA damage modelling of diffusing alpha-emitters radiation therapy using Geant4.

PURPOSE: Diffusing alpha-emitters radiation therapy (DaRT) is a brachytherapy technique using α-particles to treat solid tumours. The high linear energy transfer (LET) and short range of α-particles make them good candidates for the targeted treatment of cancer. Treatment planning of DaRT requires a good understanding of the dose from α-particles and the other particles released in the 224Ra decay chain. METHODS: The Geant4 Monte Carlo toolkit has been used to simulate a DaRT seed to better understand the dose contribution from all particles and simulate the DNA damage due to this treatment. RESULTS: Close to the seed α-particles deliver the majority of dose, however at radial distances greater than 4 mm, the contribution of ß-particles is greater. The RBE has been estimated as a function of number of double strand breaks (DSBs) and complex DSBs. A maximum seed spacing of 5.5 mm and 6.5 mm was found to deliver at least 20 Gy RBE weighted dose between the seeds for RBEDSB and RBEcDSB respectively. CONCLUSIONS: The DNA damage changes with radial distance from the seed and has been found to become less complex with distance, which is potentially easier for the cell to repair. Close to the seed α-particles contribute the majority of dose, however the contribution from other particles cannot be neglected and may influence the choice of seed spacing.

Co-Analysis of Serum and Urine Differentially Expressed Proteins in Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by the deficiency of the enzyme α-l-iduronidase (IDUA), typically leading to devastating secondary pathophysiological cascades. Due to the irreversible nature of the disease's progression, early diagnosis and interventional treatment has become particularly crucial. Considering the fact that serum and urine are the most commonly used specimens in clinical practice for detection, we conducted an analysis to identify the differential protein profile in the serum and urine of MPS I patients using the tandem mass tag (TMT) technique. A total of 182 differentially expressed proteins (DEPs) were detected in serum, among which 9 showed significant differences as confirmed by parallel reaction monitoring (PRM) analysis. The proteins APOA1 and LGFBP3 were downregulated in serum, while the expression levels of ALDOB, CD163, CRTAC1, DPP4, LAMP2, SHBG, and SPP2 exhibited an increase. In further exploratory studies of urinary proteomics, 32 identified DEPs were consistent with the discovered findings in serum tests, specifically displaying a high diagnostic area under the curve (AUC) value. Thus, our study demonstrates the value of serum-urine integrated proteomic analysis in evaluating the clinical course of MPS I and other potential metabolic disorders, shedding light on the importance of early detection and intervention in these conditions.

In-silico calculations of DNA damage induced by α-particles in the 224Ra DaRT decay chain for a better understanding of the radiobiological effectiveness of this treatment.

Diffusing alpha-emitters radiation Therapy (DaRT) is an interstitial brachytherapy technique using 224Ra seeds. For accurate treatment planning a good understanding of the early DNA damage due to α-particles is required. Geant4-DNA was used to calculate the initial DNA damage and radiobiological effectiveness due to α-particles with linear energy transfer (LET) values in the range 57.5-225.9 keV/µm from the 224Ra decay chain. The impact of DNA base pair density on DNA damage has been modelled, as this parameter varies between human cell lines. Results show that the quantity and complexity of DNA damage changes with LET as expected. Indirect damage, due to water radical reactions with the DNA, decreases and becomes less significant at higher LET values as shown in previous studies. As expected, the yield of complex double strand breaks (DSBs), which are harder for a cell to repair, increases approximately linearly with LET. The level of complexity of DSBs and radiobiological effectiveness have been found to increase with LET as expected. The quantity of DNA damage has been shown to increase for increased DNA density in the expected base pair density range of human cells. The change in damage yield as a function of base pair density is largest for higher LET α-particles, an increase of over 50% for individual strand breaks between 62.7 and 127.4 keV/µm. This change in yield shows that the DNA base pair density is an important parameter for modelling DNA damage particularly at higher LET where the DNA damage is greatest and most complex.

Serum Proteomic Analysis by Nanoflow LC-MS/MS-Based Proteomics in IgA Chronic Kidney Disease.

BACKGROUND: IgA nephropathy (IgAN) is the most frequently occurring primary glomerulonephritis. A lack of specific biomarkers hinders the early diagnosis and treatment of this disease. This study analyzes and validates potential serum biomarkers using mass spectrometry proteomics. METHODS: Global proteomics profiles of serum from 60 patients with IgAN and 43 healthy control subjects were compared to identify significantly changed proteins. These proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in an independent validation set consisting of samples from 67 different stage IgAN patients and 60 healthy controls. RESULTS: A total of 37 significantly changed proteins were found in the discovery set, among which 18 proteins were identified as potential biomarkers for IgAN through PRM assays in the validation set. Of these 18 proteins, IgGFc-binding protein, MS-A1 light chain variable region, transthyretin, ficolin-3, and myosin-reactive immunoglobulin light chain variable region were up-regulated in different IgAN stages, B cell receptor heavy chain variable region, rheumatoid factor RF-ET6, heavy chain Fab, cryocrystalglobulin CC1 heavy chain variable region, FLJ94213, lumican, and Q68CN4 (uncharacterized protein) were down-regulated in different IgAN stages. These proteins support previous findings that CKD is accompanied by altered immune response. CONCLUSIONS: This study lays the groundwork for additional research using biomarkers to clinically diagnose IgAN. These proteins are potential molecular markers that could help us understand the potential molecular mechanism of IgAN.

RNA-seq Analysis Reveals Mitochondrial and Cuticular Protein Genes Are Associated with Phosphine Resistance in the Rusty Grain Beetle (Coleoptera:Laemophloeidae).

The rusty grain beetle, Cryptolestes ferrugineus (Stephens), is a serious pest of stored grain, which has developed high levels of resistance to phosphine. In this study, five geographically distant populations of C. ferrugineus had been collected in China, specifically in granaries where phosphine fumigant is used for pest control, and they showed a high resistance ratio up to 1,907 (LC50 = 21.0 mg/liter). Then, a reference transcriptome was constructed to use as a basis for investigating the molecular mechanisms of phosphine resistance in this species, which consisted of 47,006 unigenes with a mean length of 1,090. Subsequently, the RNA-Seq analysis of individuals from the most susceptible and resistant populations led to the identification of 54 genes that are differentially expressed. GO and KEGG analysis demonstrated that genes associated with mitochondrial and respiration functions were significantly enriched. Also, the 'structural constituent of cuticle' term was annotated in the GO enrichment analysis and further qRT-PCR confirmed that the expression levels of nine cuticular protein genes were significantly increased in the resistant population. In conclusion, we present here a transcriptome-wide overview of gene expression changes between resistant and susceptible populations of C. ferrugineus, and this in turn documents that mitochondria and cuticular protein genes may play together a crucial role in phosphine resistance. Further gene function analysis should enable the provision of advice to expedite resistance management decisions.

Proteome profiling of gestational diabetes mellitus at 16-18 weeks revealed by LC-MS/MS.

BACKGROUND: The practices used to diagnose gestational diabetes mellitus (GDM) could only be carried out around the time of detectable symptoms, and predictive capacity is little. METHODS: LC-MS/MS was conducted to explore overview proteomics for GDM complicated pregnant woman at 16-18 gestation weeks, while normal pregnant for control. Enzyme-linked immunosorbent assay was further applied in an independent cohort of 15 GDM cases and 15 controls for verification. RESULTS: The results indicated that 24 protein expression levels were significantly changed in GDM group samples, and inflammation, oxidative stress, insulin resistance, blood coagulation, and lipid homeostasis were associated with GDM. The abnormal expression of CRP and IGFBP2 was verified in the first-trimester maternal plasma in women who subsequently developed GDM. CONCLUSIONS: This study not only identified 24 potential predictive biomarkers for GDM also provided a global overview of protein rearrangements induced by GDM.

Perfluorooctanoic acid induces migration and invasion and inhibits apoptosis through the PI3K/AKT signaling pathway in human rhabdomyosarcoma cells.

The present study aimed to investigate the effects of perfluorooctanoic acid (PFOA) on tumor cell migration, invasion and apoptosis by activation of the PI3K/AKT signaling pathway in human rhabdomyosarcoma (RMS). PFOA is a persistent, synthetic organic environment pollutant, which has been previously associated with multiple diseases, including cancer. The present study aimed to confirm whether PFOA can elicit cell growth in the RD subline of RBS. RD cells were treated with different concentrations of PFOA. Cell proliferation was evaluated using Cell Counting Kit­8 and cell cycle assays. Cell migration and invasion were determined using wound healing and Transwell assays. Apoptotic rates were estimated by Annexin V­FITC/propidium iodide staining. The expression levels of vimentin, serum/glucocorticoid­regulated kinase 1 (SGK1), cyclin E2, cyclin dependent kinase (CDK)2, p53, p21, p27, phosphatidylinositol­3 kinase (PI3K) and protein kinase B (AKT), and apoptosis­associated genes and proteins (including Bcl­2 and Bax) were detected by reverse transcription­PCR and western blot analyses. The results showed that PFOA significantly promoted RD cell proliferation, migration and invasion and significantly inhibited RD cell apoptosis. Exposure to PFOA also induced the expression of vimentin, SGK1, cyclin E2, CDK2, AKT, PI3K and Bcl­2, but suppressed the expression of Bax in the RD cells. The treatment of RD cells with BEZ235, a PI3K inhibitor, antagonized the effects of PFOA on metastatic formation and apoptosis. The results obtained show that the PI3K/AKT signaling pathway is implicated in mediating the pro­neoplastic effects of PFOA. The data suggests that PFOA is a carcinogen capable of promoting RD cell migration and invasion and inhibiting apoptosis through the PI3K/AKT signaling pathway.

Proteomic approaches in the discovery of potential urinary biomarkers of mucopolysaccharidosis type II.

Mucopolysaccharindosis type II (MPS II) is a rare lysosomal storage disorder caused by deficient or absent activity of the iduronate-2-sulfatase (IDS) enzyme, which leads to pathological accumulation of the glycosaminoglycans(GAGs). The absence of early diagnosis can result in irreversible developmental, neurological, and physiological damage. The lack of clear understanding of the etiology of physiological dysfunction in MPS II has been a major obstacle to the development of new treatment. Therefore, a reliable biomarker for early diagnosis and exploration of pathogenic mechanism are of great importance. Proteomics provides powerful tool for protein expression alterations and study of complicated pathological process. This study was performed to identify the differential protein profile in urine of MPS II patients using two-dimensional gel electrophoresis(2D-PAGE)combining with MALDI-TOF/TOF and a total of 15 differentially expressed proteins were identified. Content of alpha1-antitrypsin, Gm2 activator and lipocalin-type prostaglandin D synthase was measured by ELISA method. The value of urinary α1-AT/Cr in MPS II group was 0.79 ±â€¯0.10 mg/mmol, significantly higher than 0.42 ±â€¯0.05 mg/mmol in healthy control group; whereas the value of GM2A/Cr and L-PGDS/Cr in MPS II group was 1.30 ±â€¯0.12 µg/mmol and 9.86 ±â€¯1.16 ng/mmol respectively, which was significantly lower than 2.19 ±â€¯0.19 µg/mmol and 13.98 ±â€¯1.48 ng/mmol in healthy control group. The proteins can be considered as accessory diagnostic biomarkers for MPS II. This approach helped to discover early diagnostic markers and provided a better understanding of the pathogenic mechanism of MPS II.

Exploration of Acute Phase Proteins and Inflammatory Cytokines in Early Stage Diagnosis of Acute Mountain Sickness.

Wang, Chi, Hui Jiang, Jinyan Duan, Jingwen Chen, Qi Wang, Xiaoting Liu, and Chengbin Wang. Exploration of acute phase proteins and inflammatory cytokines in early stage diagnosis of acute mountain sickness. High Alt Med Biol. 19:170-177, 2018. BACKGROUND: Early diagnosis of acute mountain sickness (AMS) is currently based on personal appreciation of the severity of symptoms. A more objective method to diagnose AMS is required. Inflammatory cytokines and acute phase proteins have been reported to be different at high altitude. METHODS: A total of 104 male soldiers rapidly ascending from Beijing (20-60 m) to Germu, Qinghai (3200 m), were divided into AMS group and non-AMS group according to the Lake Louis Score system. Blood pressure, pulse rate, and oxygen saturation were measured. Forty-nine blood samples were collected before and on the 3rd day after ascending to the high altitude. Serum haptoglobin (Hp), transferrin (Tf), and complement C3 were detected by immune scattered nephelometry, whereas serum interleukin-1beta (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) were detected by chemical luminescence immunity analyzer. The sensitivity, specificity, and receiver operating characteristic curve were evaluated. Youden index with the maximum value was used to determine cutoff values of each parameter. Logistic regression was performed to determine the diagnostic efficiency of combination of three cytokines. RESULTS: Differences of physical indexes between AMS group and non-AMS group were of no statistical significance. In AMS group, serum Tf significantly increased while Hp decreased when compared with non-AMS group. Serum IL-1ß, IL-6, and TNF-α were higher in the AMS group than in the non-AMS group. The cutoff values for Tf, Hp, IL-1ß, IL-6, and TNF-α were 263.5 mg/dL, 119.35 mg/dL, 6.2 pg/mL, 15.05 pg/mL, and 18.35 pg/mL, respectively. Area under the curve (AUC) of combining three cytokines together was higher than AUC of each cytokine separately. CONCLUSIONS: Acute phase proteins and inflammatory cytokines (IL-1ß, IL-6, and TNF-α) show significant changes between the AMS group and the non-AMS group. Combination of inflammatory cytokines or acute phase proteins improves the specificity for diagnosis of AMS. This might provide objective indexes for scanning and screening individuals susceptible to AMS in the early stage of rapid ascending.

Reference gene selection to determine differences in mitochondrial gene expressions in phosphine-susceptible and phosphine-resistant strains of Cryptolestes ferrugineus, using qRT-PCR.

Cryptolestes ferrugineus is a serious pest of stored grain and has developed high levels of resistance to phosphine fumigants in many countries. Measuring differences in expression levels of certain 'resistant' genes by quantitative real-time PCR (qRT-PCR) may provide insights into molecular mechanisms underlying resistance to phosphine in C. ferrugineus, but reliable qRT-PCR results depend on suitable reference genes (RGs). We evaluated the stability of nine candidate RGs across different developmental stages and phosphine strains of C. ferrugineus, using four softwares. The results showed that RPS13 and EF1α were the most stable RGs, whereas α-TUB was the least under developmental stages. Across the different strains, RPS13 and γ-TUB were the most stable RGs, whereas CycA and GAPDH were the least. We confirmed the reliability of the selected RGs by qRT-PCR analyses of the mitochondrial cox1 gene. Expression of cox1 was not significantly different in the phosphine-resistant strain compared with the phosphine-susceptible strain, but three mitochondrial genes (nad3, atp6 and cob) were significantly down-regulated. These results suggest that alterations in the expressions of these three genes may be associated with phosphine resistance in C. ferrugineus. The findings will facilitate future functional genomics studies on the development and phosphine resistance in C. ferrugineus.

α-Synuclein binds to cytoplasmic vesicles in U251 glioblastoma cells.

α-Synuclein is the major component of Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions. It plays an important role in neurodegenerative diseases such as Parkinson's disease, multiple system atrophy, and other synucleinopathies. However, the pathogenesis and neurodegenerative effects of α-synuclein remain unknown. In this study, we established an α-synuclein and an α-synuclein-EGFP overexpressing U251 cell line. α-Synuclein overexpression increases oxidative stress and alters the cell surface and mitochondrial morphologies. We provide fluorescent-protein tagging, immunofluorescence and ultrastructural evidence showing that α-synuclein accumulations are associated with clusters of cytoplasmic vesicles and the diameter of these vesicles increases by H2O2 in a time- and dose-dependent manner.

Leukocyte Esterase as a Biomarker in the Diagnosis of Periprosthetic Joint Infection.

BACKGROUND Total joint arthroplasty (TJA) has been one of the most rewarding interventions for treating patients suffering from joint disorders. However, periprosthetic joint infection (PJI) is a serious complication that frequently accompanies TJA. Our study aimed to investigate the application of the leukocyte esterase (LE) strip in the diagnosis of PJI. MATERIAL AND METHODS From October 2014 to July 2015, 72 patients who had undergone joint puncture after arthroplasty in our hospital were enrolled in this trial. One drop of synovial fluid from each available patient was applied to the LE strip, and the results were observed after 1-3 min. If the color turned to dark purple, we recognized this as a positive result, while other colors were regarded as negative results. Centrifugation was used when the synovial fluid was mixed with blood. The Musculoskeletal Infection Society (MSIS) definition was used as the standard reference to identify whether PJI was found in patients or not. The results of diagnosis and LE strips test were compared, and indicators reflecting diagnostic value were calculated. Correlation of the LE data with erythrocyte sedimentation rate (ESR), elevated C-reactive protein (CRP), synovial white blood cell (WBC) counts, and polymorphonuclear neutrophil (PMN) percentage was calculated. RESULTS By MSIS criteria, 38 patients were diagnosed with PJI and 34 patients were not infected. Two types of LE strip presented the same results with sensitivity of 84.21% (95% confidence interval [CI]: 68.75~93.98%), specificity of 97.06% (95% CI: 84.67~99.93%), positive predictive value (PPV) of 96.97% (95% CI: 84.24~99.92%), and negative predictive value (NPV) of 84.62% (95% CI: 69.47~94.14%). There were one false-positive case and six false-negative cases in this trial. There is a strong correlation between LE strip and synovial fluid PMN percentage. CONCLUSIONS The sensitivity and specificity of the LE strip in the diagnosis of PJI are quite high, which means the LE strip might be used as an alternative to diagnose PJI in clinical practice.

Synovial Fluid C-reactive Protein as a Diagnostic Marker for Periprosthetic Joint Infection: A Systematic Review and Meta-analysis.

BACKGROUND: Periprosthetic joint infection (PJI) is the main cause of failure following total joint arthroplasty. Until now, the diagnosis of PJI is still confronted with technical limitations, and the question of whether synovial fluid biomarker, C-reactive protein (CRP), can provide high value in the diagnosis of PJI remains unanswered and, therefore, was the aim of the study. METHODS: First, we conducted a systematic review on CRP in the diagnosis of PJI by searching online databases using keywords such as "periprosthetic joint infection", "synovial fluid", and "C-reactive protein". Eligible studies providing sufficient data to construct 2 × 2 contingency tables were then selected based on the list of criteria and the quality of included studies was assessed subsequently. Finally, the reported sensitivity, specificity, diagnostic odds ratio (DOR), summary receiver operating characteristic (SROC) curve, and the area under the SROC (AUSROC) were pooled together and used to evaluate overall diagnostic performance. RESULTS: Seven studies were included in our review, six of which comprising a total of 456 participants were further investigated in our meta-analysis. The pooled sensitivity, specificity, and DOR were 0.92 (95% confidence interval [CI]: 0.86-0.96), 0.90 (95% CI: 0.87-0.93), and 101.40 (95% CI: 48.07-213.93), respectively. The AUSROC was 0.9663 (standard error, 0.0113). CONCLUSIONS: Synovial fluid CRP is a good biomarker for the diagnosis of PJI with high sensitivity and specificity.

Variation of Circulating Inflammatory Mediators in Staphylococcus aureus and Escherichia coli Bloodstream Infection.

BACKGROUND: The aim of this study was to examine the behavior of circulating inflammatory mediators and to exclude gram-positive from gram-negative bloodstream infections. Results may be helpful in selection of optimal specific antibiotic therapies. MATERIAL/METHODS: Mice (25-27 g) were randomized to 3 groups infected with Staphylococcus aureus (S. aureus) ATCC 25923, Escherichia coli (E. coli) ATCC 25922, or phosphate-buffered saline (PBS). The white blood cell count (WBC) and the concentrations of serum C-reactive protein (CRP), procalcitonin (PCT), interleukin (IL)-1α, IL-1ß, IL-6, IL-10, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) were detected in blood samples at different time intervals after intravenous tail injection. RESULTS: The results showed that compared to the control mice, infected animals exhibited significantly higher levels of all mediators after bacterial infection. Moreover, compared to the mice that received S. aureus, animals with E. coli infection showed significantly greater increases in serum IL-1α, IL-1ß, IL-6, MCP-1, and MIP-1α levels. CONCLUSIONS: These results suggest that the use of the analyzed serum markers at an early stage of bloodstream infection may give useful information for the clinician to distinguish gram-negative from gram-positive infections.

[Analysis of the gene mutation in an inherited FVII deficiency patient].

OBJECTIVE: The identify the gene defect of an inherited FVII deficiency patient. METHODS: The promoter, all the exons and exon-intron boundaries and 3' UTR of F7 gene of the proband were analyzed by direct sequencing. The defected mutations were confirmed by sequencing the complementary strand. The mutations would be screened in the related database and 150 healthy donors to identify the SNP. By splice site prediction, we analyzed the pathogenesis of defected mutations. RESULTS: Genetic analysis revealed G to A transition at 15975 in the intron 6 of F7 gene (IVS6-1G>A) and A to G transition at 16813 in the intron 7 of F7 gene (IVS7+7 A>G). According to the fruitfly, the acceptor site could not be recognized when a G to A substitution took place. The closest candidate splice site was located 132 bp downstream. The distance was probably too far to allow the use of the cryptic splice site and resulted in the skipping of exon6. A to G transition at 16813 in the intron 7 of F7 gene could not change the splice site, but modify a different molecular interaction that is important for the splice process. CONCLUSION: The heterozygous mutation of IVS6-1G>A combined with polymorphism of IVS7+7 A>G in F7 gene relates to the FVII deficiency.

Puerarin attenuates ovalbumin-induced lung inflammation and hemostatic unbalance in rat asthma model.

Aim. We aimed to investigate and evaluate the preventive activity of puerarin on the ovalbumin-induced asthma rat model. Materials and Methods. Male Wistar rats were sensitized intraperitoneally on days 0, 7, and 14 and challenged to ovalbumin intratracheally on day 21. Groups of sensitized rats were treated randomly either with placebo, puerarin, dexamethasone, or puerarin combined with dexamethasone, from days 15 to 20. Inflammatory markers, including cell counts in bronchoalveolar lavage fluid (BALF), inflammatory cytokines, histopathology, and coagulation parameters, such as coagulation tests and the activity of coagulation factors, were analyzed. Results. Puerarin significantly inhibited the recruitment of inflammatory cells in BALF and lung tissue. At the same time, the release of IL-4, IL-10, and IFN- γ in serum and the expression of mRNAs in lung tissue homogenate were changed by puerarin. Administration of puerarin also effectively rectified the coagulation disorder in asthmatic rats, such as prothrombin time (PT) (P < 0.01), thrombin time (TT) (P < 0.05), fibrinogen (FIB) (P < 0.01),the activity of factor II (FII) (P < 0.01), the activity of factor V (FV) (P < 0.05), the activity of factor VII (FVII) (P < 0.05), the activity of factor X (FX) (P < 0.05), the activity of factor VIII (FVIII) (P < 0.01), the activity of factor IX (FIX) (P < 0.05), and the activity of factor XII (FXII) (P < 0.05). Conclusions. Our results provide a clue that puerarin was useful for the preventive of allergic airway disease in rodents.

Increased vulnerability of parkin knock down PC12 cells to hydrogen peroxide toxicity: the role of salsolinol and NM-salsolinol.

Dopamine-derived neurotoxins, 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (NM-salsolinol) are the two most possible 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-like endogenous neurotoxin candidates that involved in the pathogenesis of Parkinson's disease (PD). The levels of endogenously synthesized salsolinol and NM-salsolinol are increased in the cerebrospinal fluid (CSF) of PD patients. Both of them lead to neurotoxicity in dopaminergic cells by inhibiting mitochondrial electron transport chain. To study the role of salsolinol and NM-salsolinol in Parkin deficiency-induced dopaminergic cell damage, we determined the cellular level of oxidative stress, the formation of salsolinol and NM-salsolinol, the level of mitochondrial damage and cell viability with/without the presence of exogenous H2O2 using differentiated dopaminergic PC12 cells. Our data show that parkin knock down elevates cellular oxidative stress, salsolinol and NM-salsolinol levels, which are responsible for the higher cell mortality in Parkin-deficient cells upon exposure to exogenous H2O2. The level of mitochondrial membrane potential loss, cristae disruption and the release of cytochrome c increased significantly along with the increased level of salsolinol and NM-salsolinol, whereas compared to parkin knock down cells in the presence of H2O2, the mitochondrial damage and higher cell mortality were both diminished when the levels of salsolinol and NM-salsolinol was reduced. The results not only indicate the elevated level of salsolinol and NM-salsolinol, but also reveal the potential role of salsolinol and NM-salsolinol in parkin knock down-induced cell vulnerability. We assume that parkin deficiency is the trigger of excessive oxidative stress, elevated endogenous neurotoxin levels and mitochondrial damage, which eventually results in cell death of dopaminergic cells.

Simulated microgravity affects semicarbazide-sensitive amine oxidase expression in recombinant Escherichia coli by HPLC-ESI-QQQ analysis.

Simulated microgravity has been reported to affect the gene, protein expression, and its function in the cells. Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in vascular cells, smooth muscle cells, and adipocytes. It is noteworthy whether the expression of SSAO is affected under simulated microgravity or not. In this study, an SSAO-transformed Escherichia coli BL21 was constructed firstly. Then, a sensitive, selective, and accurate method based on high-performance liquid chromatography electrospray ionization triple quadrupole (HPLC-ESI-QQQ) was developed to determine the amount of SSAO in the E. coli BL21. The limit of detection and limit of quantification were 5.0 and 10 fmol, respectively. Finally, SSAO expression in the recombinant E. coli BL21 was evaluated with various gravity and temperature conditions by HPLC-ESI-QQQ analysis. It is interesting that the tendency in the alteration of SSAO under simulated microgravity showed temperature difference. At 18 °C, the amount of SSAO in the inclusion bodies and soluble fractions under the simulated microgravity increased by 83% and 116%, respectively, compared with normal gravity. However, the decrease by 38% and 49% in the inclusion bodies and soluble fractions under the simulated microgravity was observed at 37 °C. Results obtained here indicate that the SSAO expression under simulated microgravity is dramatically sensitive to the temperature. On the other hand, a novel bioreactor from this study may also be useful for the recombinant protein expression in the field of gene engineering.

Assessment of salsolinol N-methyltransferase activity in rat peripheral lymphocytes by liquid chromatography-electrospray time-of-flight mass spectrometry.

Salsolinol N-methyltranseferase (SNMT), which may play a crucial role in the pathogenesis of Parkinson's diseases (PD), is a key enzyme to metabolize salsolinol into N-methylsalsolinol that is a neurotoxin specific to dopaminergic neurons. A sensitive method for the quantitative determination of SNMT activity in rat peripheral lymphocytes was developed and validated using liquid chromatography-electrospray with time-of-flight mass spectrometry (LC-ESI-TOF). The calibration curve was linear over the range of 7.40-368.80 nM, with 7.40 nM of the lower limit of quantification. The inter-day and intra-day precisions and accuracy for all samples were acceptable. The validated method was successfully applied for the determination of SNMT activity in both the substantia nigra (SN) and peripheral lymphocytes of a unilateral 6-hydroxydopamine-lesion model of Parkinson's disease in rats. The SNMT activity in the peripheral lymphocytes treated with the 6-hydroxydopamine was significantly increased compared with the control and sham-operated groups, which was coincident with the alteration of SNMT activity in the SN. Our results might indicate that SNMT activity may become a potential clinical marker for PD.

Rice endophyte Pantoea agglomerans YS19 forms multicellular symplasmata via cell aggregation.

Pantoea agglomerans is characterized by the formation of multicellular symplasmata. One unanswered question regarding this bacterium is how these structures are formed. In this study, the rice diazotrophic endophyte P. agglomerans YS19 was selected for exploration of this theme. YS19 was labeled with green fluorescent protein and the resulting recombinant YS19::gfp was observed to grow only slightly more slowly (a decrease of 5.5%) than the wild-type strain, and to show high GFP label stability (label loss rate 8.9218 x 10(-6) per generation, nearly reaching the generally accepted spontaneous mutation rate for most bacteria). YS19::gfp resembled the wild-type YS19 in symplasmata formation and growth profiles. Based on associated cultivation of both strains by mixing their individually cultivated single cells, symplasmata were formed and composed of both YS19::gfp and YS19, suggesting that YS19 formed symplasmata via aggregation, not proliferation, of the original single cells.