Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viraemia and abnormalities in blood count and blood chemistry were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clinical use.
Previous studies have shown that formaldehyde (FA) could cause immunotoxicity by changing the number of T lymphocytes and that cytokines play a pivotal role in the regulation of T lymphocytes. However, the previously used cytokine detection methods are difficult to use in the measurement of several cytokines in a small amount of sample for one test. Therefore, the cytometric bead array (CBA) technique was used. CBA showed better analytical efficiency and sensitivity than the previous methods. C57BL/6 mice were exposed to the control (normal saline), low FA concentration (0.5 mg/kg), and high FA concentration (2 mg/kg) for 1 week or 1 month. The contents of cytokines, including Th1-related cytokines (IL-2, IFN-?, and tumor necrosis factor), Th2-related cytokines (IL-4, IL-6, and IL-10), and Th17-related cytokines (IL-17A), were measured by using the BD FACS Canto II Flow Cytometer and analyzed by FCAP ArrayTM Software. Th1/Th2/Th17-related cytokines showed a slightly decreasing trend after low FA exposure. Conversely, a significantly increasing trend was found after high FA exposure. Th1/Th2/Th17-related cytokines all serve important functions in the immune reactions in mice after FA exposure.
Formaldehyde (FA) is a ubiquitous compound used in a wide variety of industries, and is also a major indoor pollutant emitted from building materials, furniture, etc. Because FA is rapidly metabolized and endogenous to many materials, specific biomarkers for exposure have not been identified. In this study, we identified small metabolite biomarkers in urine that might be related FA exposure. Mice were allowed to inhale FA (0, 4, 8 mg/m3) 6 h per day for 7 consecutive days, and urine samples were collected on the 7th day of exposure. Liquid chromatography coupled with time of flight-mass spectrometry and principal component analysis (PCA) was applied to determine alterations of endogenous metabolites in urine. Additionally, immune toxicity studies were conducted to ensure that any resultant toxic effects could be attributed to inhalation of FA. The results showed a significant decrease in the relative rates of T lymphocyte production in the spleen and thymus of mice exposed to FA. Additionally, decreased superoxide dismutase activity and increased reactive oxygen species levels were found in the isolated spleen cells of exposed mice. A total of 12 small molecules were found to be altered in the urine, and PCA analysis showed that urine from the control and FA exposed groups could be distinguished from each other based on the altered molecules. Hippuric acid and cinnamoylglycine were identified in urine using exact mass and fragment ions. Our results suggest that the pattern of metabolites found in urine is significantly changed following FA inhalation, and hippuric acid and cinnamoylglycine might represent potential biomarker candidates for FA exposure.
Classically activated macrophages (M1) or alternatively activated macrophages (M2) have different functions during helminth infections including Trichinella spiralis (T. spiralis). The excretory/secretory antigens (ESA) of T. spiralis can inhibit macrophage pro-inflammatory cytokines production. However, the specific molecules of ESA that regulate macrophages have not been identified. We previously reported that recombinant T. spiralis derived molecule 53-kDa protein (rTsP53) had protected mice from colitis. Furthermore, in the present study in vitro, we investigated rTsP53 showed anti-inflammatory function by inducing peritoneal macrophages to M2 with expressing M2 molecules of mannose receptor (MR), a novel mammalian lectin (Ym1), arginase-1 (Arg1), and interleukin (IL)-10. Next, we found the effect of rTsP53 on M2 independently of IL-4R?. But rTsP53 can act dependently on signal transducers and activators of transcription 6 (STAT6). These results further imply that rTsP53 has potential as prospective immuno-therapeutics for inflammatory disorders.
The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus. Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V. parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 10(5) colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5?g of IMNPs within 30min. Although a very low level of non-specific binding was seen among 8 non-V. parahaemolyticus Vibrio spp. and 5 non-Vibrio strains, the IMS-LAMP method identified 133 V. parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4×10(2)CFU/mL in pure culture and was unaffected by the presence of 10(8)CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9×10(3)CFU/g without enrichment. After enrichment for 6-8h, the limit of detectability could be improved to 1.9 to 0.19CFU/g. Hence, the IMS-LAMP assay provided a rapid, simple, and cost-effective method for total V. parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution.
Cancer-associated inflammation is a key determinant of disease progression and survival in most cancers. The aim of our study was to assess the predictive value of preoperative inflammatory markers, such as the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio, red cell distribution width (RDW), and mean platelet volume, for survival in breast cancer patients. In total, 608 breast cancer patients operated on between January 2009 and December 2011 were included in this observational study. The association between preoperative inflammatory markers and survival outcomes was analyzed. Patients with high NLR (>2.57) or high RDW (>13.45%) showed a significantly lower overall survival rate than those with lower NLR (?2.57) or lower RDW (?13.45%). NLR and RDW, along with node stage and molecular subtypes, were independent prognostic factors. There was a significant survival difference according to NLR in the luminal A and triple-negative subtypes (93.3% versus 99.3%, P=0.001; 68.8% versus 95.1%, P=0.000, respectively). The triple-negative subtype was the only subtype in which higher RDW patients showed significantly poor prognosis (81.3% versus 95.5%, P=0.025). Pre-operation NLR and RDW is a convenient, easily measured prognostic indicator for patients with breast cancer, especially in patients with the triple-negative subtype.
This study aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2 and standard FACHB 905). The LTH-1 isolated from Lake Taihu, near Wuxi City in China, was identified as Aeromonas sp. based on its morphological characteristic features and phylogenetic analysis by sequencing of 16S rDNA. Extracellular compounds produced by LTH-1 showed strong algaelysing activity, and they were water-soluble and heat-tolerant, with a molecular mass lower than 2 kDa. Two algae-lysing compounds were isolated and purified from extracellular filtrate using silica gel column chromatography. One of these was identified as phenylalanine (C9H11NO2, m/z 166.0862) and the other (C8H16N2O3, m/z 189.1232) was unidentified by hybrid ion trap/time-of-flight mass spectrometry coupled with a high-performance liquid chromatography (LC/MS-IT-TOF) system. The half maximal effective concentration (EC50) of phenylalanine produced by LTH-1 against FACHB 905 was 68.2 +/- 8.2 microg mL(-1) in 48h. These results suggest that the algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are also potentially useful for regulating blooms of the harmful M. aeruginosa.
Microcystin-LR (MC-LR) and microcystin-RR (MC-RR) are the two most common microcystins (MCs) present in fresh water posing a direct threat to public health because of their hepatotoxicity. A novel MC-degrading bacterium designated MC-LTH1 capable of degrading MC-LR and -RR was isolated, and the degradation rates and mechanisms of MC-LR and -RR for this bacterium were investigated. The bacterium was identified as Bordetella sp. and shown to possess a homologous mlrA gene responsible for degrading MCs. To the best of our knowledge, this is the first report of mlrA gene detection in Bordetella species. MC-LR and -RR were completely degraded separately at rates of 0.31 mg/(L h) and 0.17 mg/(L h). However, the degradation rates of MC-LR and -RR decreased surprisingly to 0.27 mg/(L h) and 0.12 mg/(L h), respectively, when both of them were simultaneously present. Degradation products were identified by high performance liquid chromatography coupled with time-of-flight mass spectrometry. Adda (m/z 332.2215, C20H29NO3) commonly known as a final product of MC degradation by isolated bacteria was detected as an intermediate in this study. Linearized MC-LR (m/z 1013.5638, C49H76N10O13), linearized MC-RR (m/z 1056.4970, C49H77N13O13), and tetrapeptide (m/z 615.3394, C32H46N4O8) were also detected as intermediates. These results indicate that the bacterial strain MC-LTH1 is quite efficient for the detoxification of MC-LR and MC-RR, and possesses significant bioremediation potential.
The deposition of proteins as insoluble amyloid aggregates is a characteristic feature of more than 20 degenerative conditions. A growing body of evidence indicates that the oligomeric species formed by proteins, but not the mature fibrils, are inherently toxic and are associated with clinical diseases. The N-terminal and middle region of Sup35 (Sup35NM), a yeast prion, can assemble into oligomers and fibrils. Here we analyze the cytotoxicity of different aggregates of Sup35NM and its variant, the proteins that is not associated with clinical disease. Our results showed that prefibrillar aggregates generated from Sup35NM and its variant Sup35NM-1 were toxic to cultured mammalian cells. In addition, the activation of caspase-3, 8, and 9 were detected, suggesting that apoptosis was involved in the observed cytotoxicity. Our findings provide evidence for the underlying mechanism of amyloid aggregate-induced cytotoxicity and suggest that it may arise from common structural features of the aggregates rather than from primary amino acid sequences.
Acute mixed-lineage leukemia (AMLL) is characterized as the acute leukemia involved with acute myeloid cells and lymphoid cells at the same time. The AMLL is easily misdiagnosed because of a dual character involved with lymphoid and myeloid cells. At present, researches of AMLL in adults are more common. Only some are reported for children. Therefore, our aim was to study clinical characteristics of the childhood AMLL.
Following the studies of urea denaturation of ?-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of ?-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of ?-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.
To investigate the sequence dependence in the molecular mechanism of urea induced denaturation, molecular dynamics denaturing simulations of two beta-hairpin peptides, a fast folding peptide 1 (SESYINPDGTWTVTE) and a slow folding TRPZIP4 (GEWTWDDATKTWTWTE), were performed in urea aqueous solutions. It was found that beta-hairpin denaturation by urea is highly dependent on the hydrophobicity of the side chains. The two beta-hairpin peptides studied here and the GB1 studied previously display three different denaturant processes in urea solution by which the breaking of backbone native hydrogen bonds takes different orders. The variation of their denaturing mechanism is well correlated to the variation in their structural properties. In peptide 1, which has only a loosely packed hydrophobic core formed by residues Trp11 and Ile5, all backbone native hydrogen bonds (1 to 5) are broken in a short period of time. Whereas for TRPZIP4 with a compact hydrophobic core of four tryptophan residues, the backbone native hydrogen bonds (1 to 6) are considerably more stable, with the middle hydrogen bonds protected well by the hydrophobic core being the most stable. The comparison of different beta-hairpin peptides shows that the side-chain packing on each face of the strands plays a major role in the stability of the backbone native hydrogen bonds in urea solution, and indicates that protein denaturation by urea can be highly sequence dependent.
Southeast Asian macaques are hosts of a number of Plasmodium infections, some of which are transmittable to humans. During examination of blood films of five wild-caught long-tailed macaques Macaca fascicularis from South China, malaria infection was detected in one of the monkeys. In order to isolate this parasite for identification and characterization, we experimentally passed this parasite through both Assamese (M. assamensis) and rhesus (M. mulatta) monkeys by intravenous injection of infected blood. This parasite morphologically resembled Plasmodium inui, and had a typical 72 h quartan periodicity. This parasite was infective to Anopheles dirus mosquitoes, and salivary gland sporozoites appeared 13 days post feeding. Feeding by 20 infected An. dirus mosquitoes on another Assamese monkey produced infection with a prepatent period of 8 days. Molecular analysis of the small subunit rRNA genes and the mitochondrial genome confirmed this parasite as an isolate of P. inui. In spleen-intact macaques, the infection had a protracted duration with parasites being detected during the rearing of the infected monkeys for over two years. In summary, this study identified a P. inui isolate and successfully passed this parasite through Assamese monkeys by both intravenous inoculation and mosquito transmission.
Key elements of ?-structure folding include hydrophobic core collapse, turn formation, and assembly of backbone hydrogen bonds. In the present folding simulations of several ?-hairpins and ?-sheets (peptide 1, protein G B1 domain peptide, TRPZIP2, TRPZIP4, 20mer, and 20mer(D)P6D), the folding free-energy landscape as a function of several reaction coordinates corresponding to the three key elements indicates apparent dependence on turn stability and side-chain hydrophobicity, which demonstrates different folding mechanisms of similar ?-structures of varied sequences. Turn stability is found to be the key factor in determining the formation order of the three structural elements in the folding of ?-structures. Moreover, turn stability and side-chain hydrophobicity both affect the stability of backbone hydrogen bonds. The three-stranded ?-sheets fold through a three-state transition in which the formation of one hairpin always takes precedence over the other. The different stabilities of two anti-parallel hairpins in each three-stranded ?-sheet are shown to correlate well with the different levels of their hydrophobic interactions.
In this study, we focused on the relationship between aldosterone and NOX1 expression in vascular smooth muscle cells (VSMCs). For the first time, with the use of specific inhibitors of protein kinase C (PKC), we report that PKCdelta mediates upregulation of NOX1 induced by 10 nM aldosterone in cultured VSMCs. Participation of PKC in the mediation of NOX1 regulation was further confirmed by the effect of diacylglycerol, a PKC agonist, on the NOX1 RNA in A7r5 cells with Northern blot analysis. To establish cause and effect, we next silenced the PKCdelta gene partly by RNA interference and found knockdown of PKCdelta gene attenuated aldosterone-induced NOX1 expression, generation of superoxide, as well as protein synthesis in VSMCs. Taken together, these data indicated PKCdelta might mediate aldosterone-dependent NOX1 upregulation in VSMCs. In addition, we showed that the cascade from aldosterone to PKCdelta activation had the participation of the mineralocorticoid receptor.
The present review focuses primarily on the studies we made in recent years to improve the understanding of the molecular mechanisms of PGF2alpha-induced hypertrophy of Vascular Smooth Muscle Cells (VSMC). In this review, we will summarize the recent findings in the context of the PGF2alpha signaling pathway in three parts: PGF2alpha binding to its receptor, transactivation of EGF receptor, two independent signaling transduction pathways increasing the expression of NOX1 gene.
Separation of the through-space (TS) from the through-bond (TB) interactions between the two atomic orbitals at C(1) and C(3) of 1,3-dehydrobenzene (1) has been achieved by carrying out ab initio, valence-bond, self-consistent-field (VBSCF) calculations. The results indicate that, at the CCSD(T)/cc-pVTZ optimized geometry of the singlet state of 1, the stabilization provided by TB interactions contributes 10% more than the stabilization provided by the TS interactions to the adiabatic singlet-triplet energy difference. The highest occupied MO of 1 contains a contribution from a hybrid AO at C(2), which has the same phase as the smaller lobes of the AOs at C(1) and C(3). Consequently, TB interactions in 1 increase with decreasing values of the C(1)-C(3) distance. The origin of this hybrid AO at C(2) and the contributions of hyperconjugation to TB and TS interactions in 1 are described and discussed.
A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5 end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ?100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.
In this study, both experimental and theoretical approaches, including absorption spectra, fluorescence emission spectra, 1H- and 31P-NMR, electrospray ionization mass spectrometry (ESI-MS), pH-potentiometry and theoretical approaches using the BEST & SPE computer programs were applied to study the competitive complexation between ciprofloxacin (CIP) and b-nicotinamide adenine dinucleotide phosphate (NADP) with aluminum (III) in aqueous solutions. Rank annihilation factor analysis (RAFA) was used to analyze the absorption and fluorescence emission spectra of the ligands, the binary complexes and the ternary complexes. It is found, at the mM total concentration level and pH = 7.0, the bidentate mononuclear species [Al(CIP)]2+ and [Al(NADP)] predominate in the aqueous solutions of the Al(III)-CIP and Al(III)-NADP systems, and the two complexes have similar conditional stability constants. However, the pH-potentiometry results show at the mM total concentration level and pH = 7.0, the ternary species [Al(CIP)(HNADP)] predominates in the ternary complex system. Comparing predicted NMR spectra with the experimental NMR results, it can be concluded that for the ternary complex, CIP binds to aluminum ion between the 3-carboxylic and 4-carbonyl groups, while the binding site of oxidized coenzyme II is through the oxygen of phosphate, which is linked to adenosine ribose, instead of pyrophosphate. The results also suggested CIP has the potential to be a probe molecular for the detection of NADP and the Al(III)-NADP complexes under physiological condition.
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