Inhibitory effects and mechanisms of the anti-covid-19 traditional Chinese prescription, Keguan-1, on acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Keguan-1, a new traditional Chinese medicine (TCM) prescription contained seven Chinese herbs, is developed to treat coronavirus disease 19 (COVID-19). The first internationally registered COVID-19 randomised clinical trial on integrated therapy demonstrated that Keguan-1 significantly reduced the incidence of ARDS and inhibited the severe progression of COVID-19. AIM OF THE STUDY: To investigate the protective mechanism of Keguan-1 on ARDS, a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model was used to simulate the pathological state of ARDS in patients with COVID-19, focusing on its effect and mechanism on ALI. MATERIALS AND METHODS: Mice were challenged with LPS (2 mg/kg) by intratracheal instillation (i.t.) and were orally administered Keguan-1 (low dose, 1.25 g/kg; medium dose, 2.5 g/kg; high dose, 5 g/kg) after 2 h. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h and 24 h after i.t. administration of LPS. The levels of inflammatory factors tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, keratinocyte-derived chemokine (KC or mCXCL1), macrophage inflammatory protein 2 (MIP2 or mCXCL2), angiotensin II (Ang II), and endothelial cell junction-associated proteins were analysed using ELISA or western blotting. RESULTS: Keguan-1 improved the survival rate, respiratory condition, and pathological lung injury; decreased the production of proinflammatory factors (TNF-α, IL-6, IL-1ß, KC, and MIP2) in BALF and the number of neutrophils in the lung tissues; and ameliorated inflammatory injury in the lung tissues of the mice with LPS-induced ALI. Keguan-1 also reduced the expression of Ang II and the adhesion molecule ICAM-1; increased tight junction proteins (JAM-1 and claudin-5) and VE-cadherin expression; and alleviated pulmonary vascular endothelial injury in LPS-induced ALI. CONCLUSION: These results demonstrate that Keguan-1 can improve LPS-induced ALI by reducing inflammation and pulmonary vascular endothelial injury, providing scientific support for the clinical treatment of patients with COVID-19. Moreover, it also provides a theoretical basis and technical support for the scientific use of TCMs in emerging infectious diseases.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.).

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1ß, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1ß, and nitric oxide.

Purification, identification, and computational analysis of xanthine oxidase inhibitory peptides from kidney bean.

Hyperuricemia is related to plenty of diseases, seriously damaging human health. Current clinical drugs used to treat hyperuricemia have many adverse effects. In this study, kidney bean hydrolysate (KBH) was found to exert high xanthine oxidase inhibitory (XOI) activity. Compared to KBH (50.31 ± 2.73%), XOI activities of three fractions (Mw <5 kDa, Mw <3 kDa, Mw  < 1 kDa) by ultrafiltration were higher and increased to 58.58 ± 3.57%, 59.34 ± 1.78%, and 55.05 ± 5.00%, respectively (P < 0.05). A total of 69 peptides were identified by HPLC-ESI-MS/MS and analyzed binding affinities with XO with the help of molecular docking. AVDSLVPIGR, DWYDIK, LDNLLR, ISPIPVLK, ISSLEMTR showed well binding affinities with XO and DWYDIK presented the highest XOI activity (68.63 ± 5.07%) among five synthetic peptides (P < 0.05). Additionally, visual analysis results indicated that DWYDIK was pushed into the hydrophobic channel and formed hydrogen bonds with pivotal amino acids of xanthine oxidase. Overall, KBH could be a promising candidate as anti- hyperuricemia functional food. PRACTICAL APPLICATION: This research initially revealed that kidney bean peptides could significantly inhibit the activity of xanthine oxidase, indicating kidney bean peptides could be a treatment for hyperuricemia. Kidney bean peptides may have commercial potentials as a safer alternative with few side effects to drugs.

Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

Protein fingerprinting with digital sequences of linear protein subsequence volumes: a computational study.

Proteins in a proteome can be identified from a sequence of K integers equal to the digitized volumes of subsequences with L residues from the primary sequence of a stretched protein. Exhaustive computations on the proteins of Helicobacter pylori (UniProt id UP000000210) with L and K in the range 4-8 show that approx. 90% of the proteins can be identified uniquely in this manner. This computational result can be translated into practice with a nanopore, an emerging technology that does not require analyte immobilization, proteolysis or labeling. Unlike other methods, most of which focus on a specific target protein, nanopore-based methods enable the identification of multiple proteins from a sample in a single run. Recent work by Kennedy, Kolmogorov and associates shows that the blockade current due to a protein molecule translocating through a nanopore is roughly proportional to one or more contiguous residues. The present study points to a modified version in which the volumes of subsequences (rather than of single residues) may be obtained by integrating the blockade current due to L contiguous residues. The advantages arising from this include lower detector bandwidth, elimination of the homopolymer problem and reduced noise. Because an identifier is based on near as well as distant (up to 2KL-L) residues, this approach uses more global information than an approach based on single residues and short-range correlations. The results of the study, which are available in a data supplement, are discussed in detail. Potential implementation issues are addressed.

Iron Supplementation Improves Cardiovascular Outcomes in Patients with Heart Failure.

BACKGROUND: Iron deficiency is prevalent in patients with heart failure. This meta-analysis was performed to evaluate the therapeutic effects of iron in patients with systolic heart failure and iron deficiency. METHODS: We searched PubMed, Embase, and Cochrane databases through March 2018 and included 10 randomized controlled trials involving 1404 heart failure patients who underwent iron or placebo treatment. Odds ratio (OR) and weighted mean differences (WMD) were calculated using fixed- or random-effects models. RESULTS: Our results showed that iron supplementation significantly reduced hospitalization for worsening heart failure (OR 0.39; 95% confidence interval [CI], 0.19-0.80) and the combined endpoint of death and heart failure hospitalization (OR 0.47; 95% CI, 0.32-0.69). In addition, iron treatment was found to improve New York Heart Association class, 6-minute walk distance, left ventricular ejection fraction, and peak oxygen consumption. Iron therapy was also associated with improvements in Patient Global Assessment, Kansas City Cardiomyopathy Questionnaire score, European Quality of Life-5 Dimensions score, and Minnesota Living with Heart Failure Questionnaire score. Moreover, serum levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and C-reactive protein (CRP) were markedly decreased in patients with iron repletion compared with placebo treatment (WMD: -332.48 pg/mL; 95% CI, -497.48 to -167.47; WMD: -4.64 mg/L; 95% CI, -6.12 to -3.17, respectively). CONCLUSIONS: Our meta-analysis suggests that iron therapy can reduce heart failure hospitalization, increase cardiac function, improve quality of life, and decrease serum levels of NT-proBNP and CRP in patients with heart failure.

Lysine Propionylation To Boost Sequence Coverage and Enable a "Silent SILAC" Strategy for Relative Protein Quantification.

Quantification in proteomics largely relies on the incorporation of stable isotopes, with protocols that either introduce the label through metabolic incorporation or chemical tagging. Most methods rely on the use of trypsin and/or LysC to generate labeled peptides. Although alternative proteases can enhance proteome coverage, generic quantitative methods that port over to such enzymes are lacking. Here we describe a quantification strategy amenable to most proteases, which involves propionylation of metabolically labeled lysine, using a "silent stable isotope labeling by amino acids in cell culture (SILAC)" strategy that reveals isotopic labels on second-stage mass spectrometry (MS2) fragmentation in a tandem mass tag (TMT)-like manner. We selectively propionylated lysine residues prior to digestion to generate pure ArgC-like digestion for trypsin and novel ArgN-like digestions for LysargiNase, by restricting digestion at lysine. The modification offers highly complementary sequence coverage, and even enhanced protein identification rates in certain situations (GluC digestion). Propionylated lysine residues were present in the majority of identified peptides generated from digests of cell lysates and led to the consistent release of an intense cyclic imine reporter ion at mass-to-charge ratio ( m/ z) 140 using higher-energy collisional dissociation. We grew A549 cells in media containing either l-1-13C-lysine or l-6-13C-lysine, to generate proteins that share the same accurate mass when paired. Peptides were indistinguishable on the first-stage mass spectrometry (MS1) level and, upon fragmentation, released reporter ions at m/ z 140 and m/ z 141, without otherwise affecting sequence ion mass. The quantification approach is independent of the number of peptide lysines and offers a new strategy for quantitative proteomics.

Bottom-Up Two-Dimensional Electron-Capture Dissociation Mass Spectrometry of Calmodulin.

Two-dimensional mass spectrometry (2D MS) is a tandem mass spectrometry technique that allows data-independent fragmentation of all precursors in a mixture without previous isolation, through modulation of the ion cyclotron frequency in the ICR-cell prior to fragmentation. Its power as an analytical technique has been proven particularly for proteomics. Recently, a comparison study between 1D and 2D MS has been performed using infrared multiphoton dissociation (IRMPD) on calmodulin (CaM), highlighting the capabilities of the technique in both top-down (TDP) and bottom-up proteomics (BUP). The goal of this work is to expand this study on CaM using electron-capture dissociation (ECD) 2D MS as a single complementary BUP experiment in order to enhance the cleavage coverage of the protein under analysis. By adding the results of the BUP 2D ECD MS to the 2D IRMPD MS analysis of CaM, the total cleavage coverage increased from ~40% to ~68%. Graphical abstract ᅟ.

Antioxidant activity measurement and potential antioxidant peptides exploration from hydrolysates of novel continuous microwave-assisted enzymolysis of the Scomberomorus niphonius protein.

A novel continuous microwave-assisted enzymatic digestion (cMAED) method is proposed for the digestion of protein from Scomberomorus niphonius to obtain potential antioxidant peptides. In this study, bromelain was found to have a high capacity for the digestion of the Scomberomorus niphonius protein. The following cMAED conditions were investigated: protease species, microwave power, temperature, bromelain content, acidity of the substrate solution, and incubation time. At 400W, 40°C, 1500U·g-1 bromelain, 20% substrate concentration, pH 6.0 and 5min incubation, the degree of hydrolysis and total antioxidant activity of the hydrolysates were 15.86% and 131.49µg·mL-1, respectively. The peptide analyses showed that eight of the potential antioxidant peptide sequences, which ranged from 502.32 to 1080.55Da with 4-10 amino acid residues, had features typical of well-known antioxidant proteins. Thus, the new cMAED method can be useful to obtain potential antioxidant peptides from protein sources, such as Scomberomorus niphonius.

Effect of Chinese herbal medicines for nourishing yin, supplementing qi, and activating blood on reproductive endocrine activity and immune functions in patients with primary Sjogren's syndrome.

OBJECTIVE: To investigate the effect of Chinese herbal medicines for nourishing yin, supplementing qi, and activating blood on the reproductive endocrine-immune network and its mechanisms in patients with primary Sjogren's syndrome (pSS). METHODS: Seventy pSS patients were randomly assigned to two groups using a randomized digital table: the integrative therapy group (36 cases) and the control group (34 cases). Thirty healthy subjects were taken as a normal group. The control group was treated with hydroxychloroquine sulfate tablets alone, and the integrative therapy group was treated by Chinese herbal medicines for nourishing yin, supplementing qi, and activating blood combined with hydroxychloroquine sulfate tablets. The treatment course was 6 months for both groups. Before and after treatment, serum estradiol (E2), testosterone (T), luteinizing hormone (LH), prolactin (PRL) by radioimmunoassay and immunoglobulin (IgG) by immunodiffusion, erythrocyte sedimentation rate (ESR) by Westergren, interferon-γ (IFN-γ) and interleukin-4 (IL-4) by enzyme linked immunosorbent assay were determined. RESULTS: E2 and T levels in all patients were lower than those of normal subjects before treatment (P<0.05) and were increased significantly after 6-month treatment (P<0.05). ESR, FSH, LH, IgG, IFN - γ, IL - 4 and ratios of E2/T, and IFN -γ/IL in the patients were higher than those of normal subjects before the treatments (P<0.05), and were reduced significantly after the treatments (P<0.05). The T and IFN - γ levels and E2/T ratio in the patients treated with integrative therapy were reduced significantly compared with the control group (P<0.05). However, the PRL levels before and after treatment were not significantly changed in the two groups (P>0.05). The ratios of E2/T and IFN -γ/IL-4, and levels of IgG and ESR were positively correlated before and after treatment (P<0.05). CONCLUSIONS: The ratios of E2/T and IFN -γ/IL-4 might be used as indicators of pSS activity. Chinese herbal medicines for nourishing yin, supplementing qi, and activating blood combined with Western medicine could improve the therapeutic effect by regulating the reproductive endocrine-immune network in pSS patients.

Calcium Channel Blockers, Progression to Dementia, and Effects on Amyloid Beta Peptide Production.

Previous epidemiologic studies suggest that antihypertensive drugs may be protective against cognitive decline. To determine if subjects enrolled in the University of Kentucky longitudinal aging study who used antihypertensive drugs showed diminished progression to dementia, we used a 3-parameter logistic regression model to compare the rate of progression to dementia for subjects who used any of the five common categories of antihypertensive drugs to those with similar demographic characteristics but who did not use antihypertensives. Regression modeling showed that subjects who used calcium channel blockers (CCBs) but not the other classes of antihypertensives showed a significant decrease in the rate of progression to dementia. Significantly, use of CCBs ameliorated the negative effects of the presence of APOE-4 alleles on cognitive decline. To determine if CCBs could minimize amyloid beta peptide (Aß(1-42)) production, H4 neuroglioma cultures transfected to overexpress APP were treated with various CCBs and Aß(1-42) levels and levels of proteins involved in Aß production were quantified. Results show that treatment with nifedipine led to a significant decrease in levels of Aß(1-42), with no significant decrease in cell viability. Collectively, these data suggest that use of CCBs significantly diminishes the rate of progression to dementia and may minimize formation of Aß(1-42).

Peri-procedural use of rivaroxaban in elective percutaneous coronary intervention to treat stable coronary artery disease. The X-PLORER trial.

Patients on rivaroxaban requiring percutaneous coronary intervention (PCI) represent a clinical conundrum. We aimed to investigate whether rivaroxaban, with or without an additional bolus of unfractionated heparin (UFH), effectively inhibits coagulation activation during PCI. Stable patients (n=108) undergoing elective PCI and on stable dual antiplatelet therapy were randomised (2:2:2:1) to a short treatment course of rivaroxaban 10 mg (n=30), rivaroxaban 20 mg (n=32), rivaroxaban 10 mg plus UFH (n=30) or standard peri-procedural UFH (n=16). Blood samples for markers of thrombin generation and coagulation activation were drawn prior to and at 0, 0.5, 2, 6-8 and 48 hours (h) after start of PCI. In patients treated with rivaroxaban (10 or 20 mg) and patients treated with rivaroxaban plus heparin, the levels of prothrombin fragment 1 + 2 at 2 h post-PCI were 0.16 [0.1] nmol/l (median) [interquartile range, IQR] and 0.17 [0.2] nmol/l, respectively. Thrombin-antithrombin complex values at 2 h post-PCI were 3.90 [6.8]µg/l and 3.90 [10.1] µg/l, respectively, remaining below the upper reference limit (URL) after PCI and stenting. This was comparable to the control group of UFH treatment alone. However, median values for thrombin-antithrombin complex passed above the URL with increasing tendency, starting at 2 h post-PCI in the UFH-alone arm but not in rivaroxaban-treated patients. In this exploratory trial, rivaroxaban effectively suppressed coagulation activation after elective PCI and stenting.

Simultaneous detection of polar and nonpolar compounds by ambient mass spectrometry with a dual electrospray and atmospheric pressure chemical ionization source.

A dual ionization source combining electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was developed to simultaneously ionize both polar and nonpolar compounds. The source was constructed by inserting a fused silica capillary into a stainless steel column enclosed in a glass tube. A high dc voltage was applied to a methanol solution flowing in the fused silica capillary to generate an ESI plume at the capillary tip. A high ac voltage was applied to a ring electrode attached to the glass tube to generate plasma from the nitrogen gas flowing between the glass tube and the stainless steel column. The concentric arrangement of the ESI plume and the APCI plasma in the source ensured that analytes entering the ionization region interacted with both ESI and APCI primary ion species generated in the source. Because the high voltages required for ESI and APCI were independently applied and controlled, the dual ion source could be operated in ESI-only, APCI-only, or ESI+APCI modes. Analytes were introduced into the ESI and/or APCI plumes by irradiating sample surfaces with a continuous-wavelength laser or a pulsed laser beam. Analyte ions could also be produced by directing the dual ESI+APCI source toward sample surfaces for desorption and ionization. The ionization mechanisms involved in the dual ion source include Penning ionization, ion molecule reactions, and fused-droplet electrospray ionization. Standards of polycyclic aromatic hydrocarbons, angiotensin I, lidocaine, ferrocene, diesel, and rosemary oils were used for testing. Protonated analyte ions were detected in ESI-only mode, radical cations were detected in APCI-only mode, and both types of ions were detected in ESI+APCI mode.

LuciPHOr2: site localization of generic post-translational modifications from tandem mass spectrometry data.

UNLABELLED: We present LuciPHOr2, a site localization tool for generic post-translational modifications (PTMs) using tandem mass spectrometry data. As an extension of the original LuciPHOr (version 1) for phosphorylation site localization, the new software provides a site-level localization score for generic PTMs and associated false discovery rate called the false localization rate. We describe several novel features such as operating system independence and reduced computation time through multiple threading. We also discuss optimal parameters for different types of data and illustrate the new tool on a human skeletal muscle dataset for lysine-acetylation. AVAILABILITY AND IMPLEMENTATION: The software is freely available on the SourceForge website http://luciphor2.sourceforge.net. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Protein enrichment of an Opuntia ficus-indica cladode hydrolysate by cultivation of Candida utilis and Kluyveromyces marxianus.

BACKGROUND: The cladodes of Opuntia ficus-indica (prickly pear cactus) have a low protein content; for use as a balanced feed, supplementation with other protein sources is therefore desirable. We investigated protein enrichment by cultivation of the yeasts Candida utilis and Kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass. RESULTS: Dilute acid pretreatment and enzymatic hydrolysis of sun-dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Even though K. marxianus had a much higher growth rate and utilized l-arabinose and d-galactose more completely than C. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. Yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains. CONCLUSIONS: This K. marxianus strain was weakly Crabtree positive. Despite its low biomass yield, its performance compared well with C. utilis. This is the first report showing that the protein content and quality of O. ficus-indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of C. utilis and K. marxianus.

Early Detection of Autism (ASD) by a Non-invasive Quick Measurement of Markedly Reduced Acetylcholine & DHEA and Increased ß-Amyloid (1-42), Asbestos (Chrysotile), Titanium Dioxide, Al, Hg & often Coexisting Virus Infections (CMV, HPV 16 and 18), Bacterial Infections etc. in the Brain and Corresponding Safe Individualized Effective Treatment.

A brief historical background on Autism & some of the important symptoms associated with Autism are summarized. Using strong Electro Magnetic Field Resonance Phenomenon between 2 identical molecules with identical weight (which received U.S. Patent) non-invasively & rapidly we can detect various molecules including neurotransmitters, bacteria, virus, fungus, metals & abnormal molecules. Simple non- invasive measurement of various molecules through pupils & head of diagnosed or suspected Autism patients indicated that in Autism patients following changes were often found: 1) Acetylcholine is markedly reduced; 2) Alzheimer's disease markers (i.e. ß-Amyloid (1-42), Tau Protein, Apolipoprotein (Apo E4)) are markedly increased; 3) Chrysotile Asbestos is increased; 4) Titanium Dioxide (TiO2) is moderately increased; 5) Al is moderately increased; 6) Hg is moderately increased; 7) Dopamine, Serotonin & GABA are significantly reduced (up to about 1/10 of normal); 8) Often viral infections (such as CMV, HHV-6, HPV-16, HPV-18, etc.), and Bacterial infections (such as Chlamydia trachomatis, Mycobacterium TB, Borrelia Burgdorferi, etc.) coexist. Research by others on Autism spectrum disorder (ASD) shows that it is a group of complex neurodevelopmental disorders, with about 70% of ASD patients also suffering from gastro-intestinal problems. While Alzheimer disease (AD) is characterized by formation of 1) Amyloid plaques, 2) Neurofibrillary tangles inside of neurons, and 3) Loss of connections between neurons. More than 90% of AD develops in people over the age of 65. These 3 characteristics often progressively worsen over time. Although Autism Spectrum Disorder and Alzheimer's disease are completely different diseases they have some similar biochemical changes. Eight examples of such measurement & analysis are shown for comparison. Most of Autism patients improved significantly by removing the source or preventing intake of Asbestos, TiO2, Al & Hg or enhancing urinary output of above abnormal substances & coexisting infections, if treatment is given early. When HPV-16 & HPV-18 coexist, at triangular central area of the top of head, in addition to inability to talk, severe neuromuscular problems of lower extremity were found to also exist. However, if treatment is given 3-4 years after onset of Autism symptoms, even when successful biochemical reduction of above abnormal substances occurs, clinical improvement is less significant, since permanent damage in brain tissue seems to already exist. Therefore, early diagnosis & early treatment is very important for both Autism & Alzheimer's disease. In addition the optimal doses of Vitamin D3 and Taurine may play an important role in the future treatment of Autism, Alzheimer's Disease and memory disturbances by significantly increasing Acetylcholine and DHEA levels, enhancing the excretion of toxic substances in the urine, as well as having an anticancer effect.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Proteomic characterization of pediatric craniopharyngioma intracystic fluid by LC-MS top-down/bottom-up integrated approaches.

The combination of top-down and bottom-up platforms was utilized for the LC-MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high-resolution LC-ESI-LTQ-Orbitrap-MS analysis while low-resolution LC-ESI-IT-MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top-down analyses were applied to liquid/liquid extracted samples while bottom-up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A-I, A-II, C-I and J, hemoglobin fragments, ubiquitin, α-2-HS-glycoprotein or fetuin A, α-1-antichymotrypsin, vitamin D binding protein, and α-1-acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments.

ß-Asarone prevents autophagy and synaptic loss by reducing ROCK expression in asenescence-accelerated prone 8 mice.

ß-Asarone is an active component of the Acori graminei rhizome that is a traditional Chinese medicine clinically used in treating dementia in China. However, the cognitive effect of ß-asarone and its mechanism has remained elusive. Here, we used asenescence-accelerated prone 8 (SAMP8) mice, which mimic many of the salient features of Alzheimer׳s disease (AD), to further investigate whether modulation of the ROCK signaling pathway and/or autophagy, synaptic loss is involved in the effects of ß-asarone on learning and memory. SAMP8 mice at the age of 6 months were intragastrically administered by ß-asarone or a vehicle daily for 2 months. Senescence-accelerated-resistant (SAMR1) mice were used as the control. Our results demonstrate that autophagy and ROCK expression were increased significantly in 8 months SAMP8 mice, which were concomitant with that SAMP8 mice at the same age displayed a significant synaptic loss and cognitive deficits. The up-regulation of ROCK expression and autophage in the hippocampus of SAMP8 were significantly reduced by ß-asarone, and prevents synaptic loss and improved cognitive function of the SAMP8 mice. ß-asarone decreased neuronophagia and lipofuscin in the hippocampus of SAMP8 mice, but did not reduce Aß42 levels and malondialdehyde levels and superoxide dismutase activities. Moreover, suppression of ROCK2 by siRNA significantly reduced the effects of ß-asarone on the autophage and synaptic proteins expression in PC12 cells damage induced by Aß1-40. Taken together, ß-asarone prevents autophagy and synaptic loss by reducing ROCK expression in SAMP8 mice.