With the development of genome-sequencing technologies, protein sequences are readily obtained by translating the measured mRNAs. Therefore predicting protein-protein interactions from the sequences is of great demand. The reason lies in the fact that identifying protein-protein interactions is becoming a bottleneck for eventually understanding the functions of proteins, especially for those organisms barely characterized. Although a few methods have been proposed, the converse problem, if the features used extract sufficient and unbiased information from protein sequences, is almost untouched.
In this study, we interrogate this problem theoretically by an optimization scheme. Motivated by the theoretical investigation, we find novel encoding methods for both protein sequences and protein pairs. Our new methods exploit sufficiently the information of protein sequences and reduce artificial bias and computational cost. Thus, it significantly outperforms the available methods regarding sensitivity, specificity, precision, and recall with cross-validation evaluation and reaches ~80% and ~90% accuracy in Escherichia coli and Saccharomyces cerevisiae respectively. Our findings here hold important implication for other sequence-based prediction tasks because representation of biological sequence is always the first step in computational biology.
By considering the converse problem, we propose new representation methods for both protein sequences and protein pairs. The results show that our method significantly improves the accuracy of protein-protein interaction predictions.
Enzymes are known as the largest class of proteins and their functions are usually annotated by the Enzyme Commission (EC), which uses a hierarchy structure, i.e., four numbers separated by periods, to classify the function of enzymes. Automatically categorizing enzyme into the EC hierarchy is crucial to understand its specific molecular mechanism.
In this paper, we introduce two key improvements in predicting enzyme function within the machine learning framework. One is to introduce the efficient sequence encoding methods for representing given proteins. The second one is to develop a structure-based prediction method with low computational complexity. In particular, we propose to use the conjoint triad feature (CTF) to represent the given protein sequences by considering not only the composition of amino acids but also the neighbor relationships in the sequence. Then we develop a support vector machine (SVM)-based method, named as SVMHL (SVM for hierarchy labels), to output enzyme function by fully considering the hierarchical structure of EC. The experimental results show that our SVMHL with the CTF outperforms SVMHL with the amino acid composition (AAC) feature both in predictive accuracy and Matthew’s correlation coefficient (MCC). In addition, SVMHL with the CTF obtains the accuracy and MCC ranging from 81% to 98% and 0.82 to 0.98 when predicting the first three EC digits on a low-homologous enzyme dataset. We further demonstrate that our method outperforms the methods which do not take account of hierarchical relationship among enzyme categories and alternative methods which incorporate prior knowledge about inter-class relationships.
Our structure-based prediction model, SVMHL with the CTF, reduces the computational complexity and outperforms the alternative approaches in enzyme function prediction. Therefore our new method will be a useful tool for enzyme function prediction community.
The development of small molecule chemical probes or therapeutics that target RNA remains a significant challenge despite the great interest in such compounds. The most significant barrier to compound development is a lack of knowledge of the chemical and RNA motif spaces that interact specifically. Herein, we describe a bioactive small molecule probe that targets expanded r(CGG) repeats, or r(CGG)exp , that causes Fragile X-associated Tremor Ataxia Syndrome (FXTAS). The compound was identified by using information on the chemotypes and RNA motifs that interact. Specifically, 9-hydroxy-5,11-dimethyl-2-(2-(piperidin-1-yl)ethyl)-6H-pyrido[4,3-b]carbazol-2-ium, binds the 5’CGG/3’GGC motifs in r(CGG)exp and disrupts a toxic r(CGG)exp -protein complex in vitro. Structure-activity relationships (SAR) studies determined that the alkylated pyridyl and phenolic side chains are important chemotypes that drive molecular recognition to r(CGG)exp . Importantly, the compound is efficacious in FXTAS model cellular systems as evidenced by its ability to improve FXTAS-associated pre-mRNA splicing defects and to reduce the size and number of r(CGG)exp -protein aggregates. This approach may establish a general strategy to identify lead ligands that target RNA while also providing a chemical probe to dissect the varied mechanisms by which r(CGG)exp promotes toxicity.
DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5′-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.
The role of mast cells (MCs) in Toxoplasma gondii infection is poorly known. Kunming outbred mice were infected intraperitoneally with RH strain T. gondii, either treated with compound 48/80 (C48/80, MC activator) or disodium cromoglycate (DSCG, MC inhibitor). Compared with infected controls, infected mice treated with C48/80 exhibited significantly increased inflammation in the liver (P < 0.01), spleen (P < 0.05), and mesentery (P < 0.05) tissues, higher parasite burden in the peritoneal lavage fluids (P < 0.01), and increased levels of mRNA transcripts of T. gondii tachyzoite surface antigen 1 (SAG1) gene in the spleen and liver tissues (P < 0.01), accompanied with significantly increased Th1 cytokine (IFN-γ, IL-12p40, and TNF-α) (P < 0.01) and decreased IL-10 (P < 0.01) mRNA expressions in the liver, and increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.01) but decreased TNF-α (P < 0.01) and IL-4 (P < 0.01) in the spleens of infected mice treated with C48/80 at day 9-10 p.i. Whereas mice treated with DSCG had significantly decreased tissue lesions (P < 0.01), lower parasite burden in the peritoneal lavage fluids (P < 0.01) and decreased SAG1 expressions in the spleen and liver tissues (P < 0.01), accompanied with significantly increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.05) in the liver, and decreased IFN-γ (P < 0.05) and TNF-α (P < 0.01) in the spleens; IL-4 and IL-10 expressions in both the spleen and liver were significantly increased (P < 0.01) in the infected mice treated with DSCG. These findings suggest that mediators associated with the MC activation may play an important role in modulating acute inflammatory pathogenesis and parasite clearance during T. gondii infection in this strain of mice. Thus, MC activation/inhibition mechanisms are potential novel targets for the prevention and control of T. gondii infection.
Francois’ langur (Trachypithecus francoisi) is an endangered primate and endemic to the limestone forests of the tropical and subtropical zone of northern Vietnam and South-west China with a population of about 2,000 individuals. Conservation efforts are hampered by limited knowledge of habitat preference in its main distribution area. We surveyed the distribution of Francois’ langur and modeled the relationship between the probability of use and habitat features in Mayanghe National Nature Reserve, Guizhou, China. The main objectives of this study were to provide quantitative information on habitat preference, estimating the availability of suitable habitat, and providing management guidelines for the effective conservation of this species. By comparing 92 used locations with habitat available in the reserve, we found that Francois’ langur was mainly distributed along valleys and proportionally, used bamboo forests and mixed conifer-broadleaf forests more than their availability, whereas they tended to avoid shrubby areas and coniferous forests. The langur tended to occur at sites with lower elevation, steeper slope, higher tree canopy density, and a close distance to roads and water. The habitat occupancy probability was best modeled by vegetation type, vegetation coverage, elevation, slope degree, distances to nearest water, paved road, and farmland edge. The suitable habitat in this reserve concentrated in valleys and accounted for about 25% of the total reserve area. Our results showed that Francois’ langur was not only restricted at the landscapes level at the regions with karst topography, limestone cliffs, and caves, but it also showed habitat preference at the local scale. Therefore, the protection and restoration of the langur preferred habitats such as mixed conifer-broadleaf forests are important and urgent for the conservation of this declining species.
Background and Aims
Transcatheter intraarterial techniques can effectively deliver chemotherapeutic agents to tumor and improve the efficacy of chemotherapy. The present study is designed to evaluate the effect of transcatheter intraarterial techniques on the distribution of doxorubicin in relation to blood vessels in liver cancer.
VX2 tumors were implanted in the livers of 32 rabbits. The animals were divided into 4 groups of 8 animals each. Group 1 (doxo iv) animals received doxorubicin intravenous injection; group 2 (doxo ia) received doxorubicin hepatic intraarterial infusion; group 3 (doxo ia + E) received doxorubicin hepatic intraarterial infusion followed by embolization; group 4 (doxo + L ia + E) received hepatic intraarterial infusion of doxorubicin mixed with Lipiodol followed by embolization. Ten minutes or 4 hours after treatment, the animals were sacrificed and tumors were sampled. Immunofluorescence techniques were used to evaluate the distribution of doxorubicin in relation to blood vessels.
Doxorubicin fluorescence was distributed around tumor blood vessels and decreased with distance from the blood vessels. Tumor cells in avascular and adjacent regions were not exposed to detectable concentrations of doxorubicin. Tumors in the group 2, 3 and 4 had a significant increase in doxorubicin penetration compared with the group 1 tumors (P<0.05). Among the three groups of transcatheter therapies, doxorubicin penetration distance in group 3 was significantly larger than that in group 2 and 4 (P<0.05), and no significant difference was found between group 2 and 4 tumors (P>0.05) at 10 minutes. In contrast, at 4 hours and in total, both group 3 and 4 tumors had significant increases in drug penetration compared with group 2 (P<0.05), and no significant difference was noted between group 3 and 4 tumors (P>0.05).
Transcatheter intraarterial therapies improve doxorubicin penetration in liver cancer; nevertheless their effect on drug distribution is somewhat limited.
We report a new type of memory device based on insulating LaAlO3/SrTiO3 (LAO/STO) hetero-interface. The microstructures of the LAO/STO interface are characterized by Cs-corrected scanning transmission electron microscopy, which reveals the element intermixing at the interface. The inhomogeneous element distribution may result in carrier localization, which is responsible for the insulating state. The insulating state of such interface can be converted to metallic state by light illumination and the metallic state maintains after light off due to giant persistent photoconductivity (PPC) effect. The on/off ratio between the PPC and the initial dark conductance is as large as 105. The metallic state also can be converted back to insulating state by applying gate voltage. Reversible and reproducible resistive switching makes LAO/STO interface promising as a nonvolatile memory. Our results deepen the understanding of PPC phenomenon in LAO/STO, and pave the way for the development of all-oxide electronics integrating information storage devices.
Magnetic resonance imaging; coronary artery disease
Multiple canonical or classic transient receptor potential (TRPC) molecules are expressed in animal and human airway smooth muscle cells (SMCs). TRPC3, but not TRPC1, is a major molecular component of native non-selective cation channels (NSCCs) to contribute to the resting [Ca2+]i and muscarinic increase in [Ca2+]i in freshly isolated airway SMCs. TRPC3-encoded NSCCs are significantly increased in expression and activity in airway SMCs from ovalbumin-sensitized/challenged “asthmatic” mice, whereas TRPC1-encoded channel activity, but not its expression, is largely augmented. The upregulated TRPC3- and TRPC1-encoded NSCC activity both mediate “asthmatic” membrane depolarization in airway SMCs. Supportively, tumor necrosis factor-α (TNFα), an important asthma mediator, increases TRPC3 expression, and TRPC3 gene silencing inhibits TNFα-mediated augmentation of acetylcholine-evoked increase in [Ca2+]i in passaged airway SMCs. In contrast, TRPC6 gene silencing has no effect on 1-oleoyl-2-acetylsn-glycerol (OAG)-evoked increase in [Ca2+]i in primary isolated cells. These findings provide compelling information indicating that TRPC3-encoded NSCCs are important for physiological and pathological cellular responses in airway SMCs. However, continual studies are necessary to further determine whether, which, and how TRPC-encoded channels are involved in cellular responses in normal and diseased (e.g., asthmatic) airway SMCs.
Association between parity and colorectal cancer (CRC) risk has been investigated by several epidemiological studies but results are controversial, yet a comprehensive and quantitative assessment of this association has not been reported so far.
Relevant published studies of parity and CRC were identified using MEDLINE, EMBASE and Web of Science databases through end of April 2013. Two authors independently assessed eligibility and extracted data. Eleven prospective studies reported relative risk (RR) estimates and 95% confidence intervals (CIs) of CRC risk associated with parity. We pooled the RR from individual studies using fixed- or random-effects models and carried out heterogeneity and publication bias analyses.
The summary RR for the ever parity vs. nulliparous was 0.95 (95% CI: 0.88–1.02), with no heterogeneity (Q = 9.04, P = 0.443, I2 = 0.5%). Likewise, no significant association was yielded for the highest vs. lowest parity number (RR = 1.02, 95% CI: 0.89–1.17), with moderate heterogeneity (Q = 17.48, P = 0.094, I2 = 37.1%). Dose-response analysis still indicated no effect of parity on CRC risk and the summary RR of per one livebirth was 0.99 (95% CI: 0.96–1.02), with moderate of heterogeneity (Q = 16.50, P<0.021, I2 = 57.6%). Similar results were observed among all the subgroup analyses. No evidence of publication bias and significant heterogeneity between subgroups were detected by meta-regression analyses.
Results of this dose-response meta-analysis of prospective studies found that there was little evidence of an association between parity and CRC risk.
Ryanodine receptor 1 (RyR1) is well-known to be expressed in systemic and pulmonary vascular smooth muscle cells (SMCs); however, its functional roles remain largely unknown. In the present study, we attempted to determine the potential importance of RyR1 in membrane depolarization-, neurotransmitter-, and hypoxia-induced Ca2+ release and contraction in pulmonary artery SMCs (PASMCs) using RyR1 homozygous and heterozygous gene deletion (RyR1−/− and RyR1+/−) mice. Our results indicate that spontaneous local Ca2+ release and caffeine-induced global Ca2+ release are significantly reduced in embryonic RyR1−/− and adult RyR+/− cells. An increase in [Ca2+]i following membrane depolarization with high K+ is markedly attenuated in RyR1−/− and RyR1+/− PASMCs in normal Ca2+ or Ca2+-free extracellular solution. Similarly, muscle contraction evoked by membrane depolarization is reduced in RyR1+/− pulmonary arteries in the presence or absence of extracellular Ca2+. Neurotransmitter receptor agonists and inositol 1,4,5-triphosphate elicit a much smaller increase in [Ca2+]i in both RyR1−/− and RyR1+/− cells. We have also found that neurotransmitter-evoked muscle contraction is significantly inhibited in RyR1+/− pulmonary arteries. Hypoxia-induced increase in [Ca2+]i and contraction are largely blocked in RyR1−/− and/or RyR1+/− PASMCs. Collectively, our findings provide genetic evidence for the functional importance of RyR1 in spontaneous local Ca2+ release, and membrane depolarization-, neurotransmitter-, as well as hypoxia-induced global Ca2+ release and attendant contraction in PASMCs.
Ryanodine receptor; Calcium release; Membrane depolarization; Hypoxia; Pulmonary artery smooth muscle cell
Algorithms designed to identify canonical yeast prions predict that ~250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here, we define pathogenic mutations in PrLDs of hnRNPA2/B1 and hnRNPA1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and a case of familial ALS. Wild-type hnRNPA2 and hnRNPA1 display an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Importantly, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant ‘steric zipper’ motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs must be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.
Oxyma and an oxyma derivative, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (5b), displayed remarkable effect on selective esterifications of primary alcohols. A wide range of carboxylic acids could be esterified with primary alcohols by using EDCI, NaHCO3, and Oxyma or an Oxyma derivative 5b in 5% H2O-CH3CN. An Oxyma derivative 5b is particularly useful since it could be removed after the reaction via a simple basic or an acidic aqueous work-up procedure.
MicroRNAs have been implicated in the regulation of gene expression of various biological processes in a post-transcriptional manner under physiological and pathological conditions including host responses to viral infections. The 2009 pandemic H1N1 influenza virus is an emerging reassortant strain of swine, human and bird influenza virus that can cause mild to severe illness and even death. To further understand the molecular pathogenesis of the 2009 pandemic H1N1 influenza virus, we profiled cellular microRNAs of lungs from BALB/c mice infected with wild-type 2009 pandemic influenza virus A/Beijing/501/2009 (H1N1) (hereafter referred to as BJ501) and mouse-adapted influenza virus A/Puerto Rico/8/1934 (H1N1) (hereafter referred to as PR8) for comparison. Microarray analysis showed both the influenza virus BJ501 and PR8 infection induced strain- and temporal-specific microRNA expression patterns and that their infection caused a group of common and distinct differentially expressed microRNAs. Characteristically, more differentially expressed microRNAs were aroused on day 5 post infection than on day 2 and more up-regulated differentially expressed microRNAs were provoked than the down-regulated for both strains of influenza virus. Finally, 47 differentially expressed microRNAs were obtained for the infection of both strains of H1N1 influenza virus with 29 for influenza virus BJ501 and 43 for PR8. Among them, 15 microRNAs had no reported function, while 32 including miR-155 and miR-233 are known to play important roles in cancer, immunity and antiviral activity. Pathway enrichment analyses of the predicted targets revealed that the transforming growth factor-β (TGF-β) signaling pathway was the key cellular pathway associated with the differentially expressed miRNAs during influenza virus PR8 or BJ501 infection. To our knowledge, this is the first report of microRNA expression profiles of the 2009 pandemic H1N1 influenza virus in a mouse model, and our findings might offer novel therapy targets for influenza virus infection.
Hybrid materials made from all inorganic components are intriguing in many fields, because they have shown in-depth potential use for electronic and optoelectronic applications including solar cells, gas sensors, photodetectors, and field effect transistors. Hybrid materials made from SnO2 nanoparticles on SnSe nanosheets have been synthesized via a facile, lost-cost and safe solution method, and have been demonstrated as promising multifunctional materials in various prototype devices, including gas sensors, photodetectors, and field effect transistors.
AIM: To examine transforming growth factor-β1 (TGF-β1) promoter methylation in gastric cancer and to determine if Helicobacter pylori (H. pylori) or interleukin (IL)-1β could induce TGF-β1 hypermethylation in vitro.
METHODS: We examined the frequency and extent of TGF-β1 promoter methylation using methylation-specific PCR in the gastric tissues from 47 gastric cancer patients and 39 non-gastric cancer subjects. H. pylori infection was confirmed by a positive result from either a serological test, histological analysis or C13 urea breath test. GES-1 and MKN-45 cells co-cultured with H. pylori or treated with IL-1β for 12, 24 and 48 h in vitro tested the effects of H. pylori or IL-1β on TGF-β1.
RESULTS: Twenty-four/forty-seven (51%) cases of gastric cancer (GC) tissues showed TGF-β1 promoter methylation, 15/47 (31.9%) cases of matched non-cancerous gastric mucosa tissues from the GC patients, and 11/39 (28%) case of the normal gastric mucosa tissues from non-GC subjects showed TGF-β1 promoter methylation (51% vs 28%, P < 0.05). Significantly higher levels of methylation of TGF-β1 were found in the tumor tissues than in non-tumor tissues from GC patients (0.24 ± 0.06 vs 0.17 ± 0.04, P < 0.05) and normal gastric tissues from non-GC subjects (0.24 ± 0.06 vs 0.15 ± 0.03, P < 0.05). TGF-β1 methylation was found in 48.3% of H. pylori-positive gastric mucosal tissues whereas only 23.1% of H. pylori-negative gastric mucosal tissues showed TGF-β1 methylation (48.3% vs 23.1%, P < 0.05). IL-1β appeared to induce a dose-dependent methylation of TGF-β1 and the strongest methylation was observed in GES-1 cells treated with 2.5 ng/mL of IL-1β for 48 h. Further studies showed that pre-treatment of GES-1 cells with 20 ng/mL IL-1RA for 1 h could partially abolish the effect of IL-1β on TGF-β1 methylation. Infection of GES-1 cells by H. pylori was not found to induce significant TGF-β1 promoter methylation.
CONCLUSION: Our data revealed that TGF-β1 promoter is methylated in GC patients. IL-1β may be an important mediator for H. pylori induced gene methylation during GC development.
Transforming growth factor-β1; Interleukin-1β; Methylation; Helicobacter pylori; Gastric cancer
In some cross-sectional studies, hypertriglyceridemic waist (HTGW) has been recommended as an alternative to metabolic syndrome (MetS) for screening individuals at high risk for diabetes mellitus (DM). However, little information is about the predictive power of HTGW for future DM. The aims of the study were to assess the DM predictive power of HTGW compared with MetS based on the follow-up data over 15 years collected from a general Chinese population.
And Findings: The data were collected in 1992 and then again in 2007 from the same group of 687 individuals without DM in 1992. For the whole population (n =687), multivariate analysis showed presence of HTGW was associated with a 4.1-fold (95%CI: 2.4-7.0, p < 0.001) increased risk and presence of MetS was associated with a 3.7-fold (95%CI: 2.2-6.2, p < 0.001) increased risk for future DM. For the population without elevated fasting plasma glucose (n = 650), multivariate analysis showed presence of HTGW was associated with a 3.9-fold (95%CI: 2.2-7.0, p < 0.001) increased risk and presence of MetS was associated with a 3.7-fold (95%CI: 2.1-6.6, p < 0.001) increased risk for future DM.
HTGW could predict future DM independently, and the predictive power was similar to MetS. HTGW might be an alternative to MetS for predicting future DM. For simpler and fewer components, HTGW might be more practical than MetS, and it might be recommended in most clinical practices. This finding might be more useful for the individuals who only have elevated WC and TG. Although these individuals are without MetS, they are still at high risk for future DM, similarly to the individuals with MetS.
This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n=52) with high-purified Serva NB1 collagenase (Serva NB1, n=42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to eliminate endotoxin contamination and impurities; in addition this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The difference of the digestion efficacy between Sigma V and Serva NB1 was very small, however, statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p <0.05). Islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p>0.05) between groups. No significant purity differences were observed for fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p>0.05), and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p>0.05) were similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. However, we were intrigued to find that the endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 EU/ml vs. 0.052 EU/ml, p<0.05). In addition, we found that there was minimal lot to lot variation after filtration and no significant loss of enzymatic activity. These finding indicate that using this or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all.
Collagenase; Human islet isolation; Islet transplantation; Diabetes
A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca2+]i) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL/min, without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay.
Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase.
Antimonite; aquaglyceroporin; LmjAQP1; Leishmania; volume regulation; metalloid permeability; MAP kinase
Plumbagin, a quinonoid constituent isolated from the root of Plumbago zeylanica L., has been proven to possess anti-tumor activity both in vitro and in vivo. However, its anti-tumor properties for human tongue carcinoma have not been reported. This study aimed to investigate the inhibitory effect and the underlying mechanism of plumbagin on the growth of human tongue carcinoma cells.
Cell proliferation ability was detected by EdU incorporation assay and colony formation assay. Cell-cycle distribution was determined by flow cytometric analysis using propidium iodide (PI) staining. Cellular apoptosis was then evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blotting was applied to assay the expression of Bax and Bcl-2.
Plumbagin inhibited the growth and proliferation of Tca8113 cells in vitro in a concentration- and time-dependent manner. The cell cycles of plumbagin-treated Tca8113 cells were arrested at the G2/M phase. Cells treated with plumbagin presented the characteristic morphological changes of apoptosis. The ratio of Bax/Bcl-2 was raised by plumbagin in a concentration-dependent manner.
These results indicate that plumbagin induces the apoptosis of Tca8113 cells through mitochondria-mediated pathway.
plumbagin; human tongue carcinoma; proliferation; cell cycle arrest; apoptosis
Microencapsulation may allow for immunosuppression free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3±2.5 and 87.5±2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4±0.5 and 6.3±0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30–55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca2+/Ba2+ microbeads allows for long-term in vivo human islet graft function, despite long-distance shipment.
To search for more effective tuberculosis (TB) subunit vaccines, antigens expressed in different growth stages of Mycobacterium tuberculosis (M. tuberculosis), such as RpfE (Rv2450c) produced in the stage of resuscitation, Mtb10.4 (Rv0288), Mtb8.4 (Rv1174c), ESAT6 (Rv3875), Ag85B (Rv1886c) mainly secreted by replicating bacilli, and HspX (Rv2031c) highly expressed in dormant bacilli, were selected to construct six fusion proteins: ESAT6-Ag85B-MPT64190-198-Mtb8.4 (EAMM), Mtb10.4-HspX (MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6-Ag85B, and ESAT6-RpfE. The six fusion proteins were separately emulsified in an adjuvant composed of N,N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA), polyribocytidylic acid (poly I:C) and gelatin to construct subunit vaccines, and their protective effects against M. tuberculosis infection were evaluated in C57BL/6 mice. Furthermore, the boosting effects of EAMM and MH in the adjuvant of DDA plus trehalose 6,6'-dimycolate (TDM) on BCG-induced immunity were also evaluated. It was found that the six proteins were stably produced in E. coli and successfully purified by chromatography. Among them, EAMM presented the most effective protection against M. tuberculosis. Interestingly, the mice that received EAMM+MH had significantly lower bacterial counts in the lungs and spleens than the single protein vaccinated groups, and had the same effect as those that received BCG. In addition, EAMM and MH could improve BCG-primed protective efficacy against M. tuberculosis infection in mice. In conclusion, the combination of EAMM and MH containing antigens from both replicating and dormant stages of the bacilli could induce robust immunity against M. tuberculosis infection in mice and may serve as promising subunit vaccine candidate.
To compare the effects of transcatheter arterial chemoembolization (TACE) with transcatheter arterial embolization (TAE) on liver function, hepatic damage, and hepatic fibrogenesis in a rabbit tumor model.
Materials and Methods
Thirty-nine New Zealand white rabbits implanted with VX2 tumors in the left liver lobes were randomly divided into three groups: TAE, TACE, and control group. In the TAE group (n = 15), polyvinyl alcohol particles (PVAs) were used for left hepatic artery embolization. In the TACE group (n = 15), the tumors were treated with left hepatic arterial infusions of a suspension of 10-hydroxycamptothecin and lipiodol, followed by embolization with PVAs. In the control group (n = 9), the animals received sham treatment with distilled water. Serum and liver samples were collected at 6 hours, 3 days and 7 days after treatment. Liver damage was measured using a liver function test and histological analyses. Liver fibrogenesis and hepatic stellate cell (HSC) activation were evaluated using Sirius Red and anti-alpha-smooth muscle actin (α-SMA) immunohistochemical stains.
TACE caused liver injury with greater increases in serum alanine aminotransferase and aspartate aminotransferase levels on day 3 (P<0.05). Histological analyses revealed increased hepatic necrosis in adjacent non-tumorous liver tissue from day 3 compared to the TAE group (Suzuki score of 2.33±1.29 versus 1.13±1.18, P = 0.001). HSC activation and proliferation were significantly increased in the TACE group compared to the control group at 3 and 7 days after treatment (0.074±0.014 vs. 0.010±0.006, and 0.088±0.023 vs. 0.017±0.009, P<0.05). Sirius Red staining demonstrated a statistically significant increase in collagen deposition in the livers in the TACE group 7 days after embolization compared to the control group (0.118±0.012 vs. 0.060±0.017, P = 0.05).
The results of this animal study revealed that TACE induced prominent hepatocellular damage and hepatic fibrogenesis, which compromised liver function and may be responsible for chronic liver decompensation.