Combined analysis of chromosomal instabilities and gene expression for colon cancer progression inference

Background

The human body plays host to a vast array of bacteria, found in oral cavities, skin, gastrointestinal tract and the vagina. Some bacteria are harmful while others are beneficial to the host. Despite the availability of many methods to identify bacteria, most of them are only applicable to specific and cultivable bacteria and are also tedious. Based on high throughput sequencing technology, this work derives 16S rRNA sequences of bacteria and analyzes probiotics and pathogens species.

Results

We constructed a database that recorded the species of probiotics and pathogens from literature, along with a modified Smith-Waterman algorithm for assigning the taxonomy of the sequenced 16S rRNA sequences. We also constructed a bacteria disease risk model for seven diseases based on 98 samples. Applicability of the proposed platform is demonstrated by collecting the microbiome in human gut of 13 samples.

Conclusions

The proposed platform provides a relatively easy means of identifying a certain amount of bacteria and their species (including uncultivable pathogens) for clinical microbiology applications. That is, detecting how probiotics and pathogens inhabit humans and how affect their health can significantly contribute to develop a diagnosis and treatment method.     

Resistance of p53 knockout cells to doxorubicin is related to reduced formation of DNA strand breaks rather than impaired apoptotic signaling

The anthracycline doxorubicin (adriamycin) is an important chemotherapeutic agent used in the treatment of solid epithelial and mesenchymal tumors as well as leukemias. A variety of mechanisms has been proposed to be involved in doxorubicin-induced cytotoxicity such as DNA intercalation, oxidative stress, DNA strand breakage by inhibition of topoisomerase II, activation of death receptors, and altered p53 expression. Concerning doxorubicin resistance and p53 status data reported are contradictory. Here, we show that mouse fibroblasts deficient in p53 (p53(-/-)) are more resistant to doxorubicin than p53 wild-type (p53 wt) cells. This is in contrast to other genotoxic agents (UV-light, alkylating drugs) for which p53(-/-) fibroblasts proved to be more sensitive. Resistance of p53(-/-) cells to doxorubicin is related to reduced induction of apoptosis. This is not likely to be due to altered apoptotic signaling since the expression of Bax and Bcl-2 was unchanged and the induction of Fas/CD95/APO-1 receptor and caspase-8 was the same in p53(-/-) and p53 wt cells on treatment with doxorubicin. However, we observed a clearly lower level of doxorubicin-induced DNA strand breaks in p53(-/-) cells compared to the wt. P170 glycoprotein was equally expressed and the accumulation and elimination of the drug occurred with identical kinetics in both cell types. p53 deficient cells were cross-resistant to another topoisomerase II inhibitor etoposide, which also provoked increased DNA strand breakage in p53 wt cells. Based on the data we conclude that the p53 status significantly impacts the generation of DNA strand breaks because of drug-induced topoisomerase inhibition rather than death receptor signaling. Since human tumors are frequently mutated in p53 the findings bear clinical implications.     

Pro-oxidant effect of dehydroepiandrosterone in rats is mediated by PPAR activation

DHEA-treatment exerts a dual effect, prooxidant or antioxidant, depending on the dosage and, therefore, on the tissue concentration reached. In agreement with previous studies showing a prooxidant effect of DHEA, here we show that pharmacological doses of DHEA produce increased H(2)O(2) levels and a marked reduction of GSH content in rat liver. DHEA, also increases both catalase (by 30%) and cytochrome-C-reductase (by 30%) activities in the liver cytosol. The effectiveness of the state of increased oxidative stress is also documented by changes in fatty acid pattern of the microsomal membranes. Moreover, DHEA, at high doses, enhances beta-oxidation, as demonstrated by an increase of acyl-CoA-oxidase activity and of cytochrome P450 4A content, confirming that it acts as a PPARs inducer. Both PPARs induction and proxidant effects completely disappear when DHEA is administered at lower doses. Seven days treatment (4 or 10 mg) is unable to affect either levels of proxidant species and of antioxidant molecules, or cytochrome P450 4A content and beta-oxidation. Prolonged DHEA treatment (4 mg/day) for three weeks not only is unable to affect PPARs activation and beta-oxidation, but it also exerts a protective effect against ADP/Fe(2+) induced lipid peroxidation. This latter result confirms the antioxidant effects of DHEA at low doses, as already previously documented.     

Conformationally constrained ethylenediamines: synthesis and receptor binding of 6,8-diazabicyclo[3.2.2]nonanes

The synthesis and receptor affinity of 6,8-diazabicyclo[3.2.2]nonanes representing conformationally constrained ethylenediamines are described. The Dieckmann analogous cyclization of the (piperazin-2-yl)propionate 9 provided the bicyclononane 10 only, when the first cyclization product was trapped with chlorotrimethylsilane. 10 was stereoselectively transformed into the bicyclic amines 19a,b and amides 22a,b, which were investigated in competition experiments with radioligands for their sigma(1)-, sigma(2)-, kappa-, and mu-receptor affinities. The (2R)-configured dimethylamine 19a showed promising sigma(1)-receptor affinity (K(i)=23.8 nM) and selectivity, whereas the (2S)-configured (dichlorophenyl)acetamide 22b displayed a sigma-receptor binding profile (sigma(1): K(i)=184 nM; sigma(2): K(i)=263 nM) very similar to the binding profile of the atypical antipsychotic BMY-14802 (26).     

miRNA profiling in colorectal cancer highlights miR-1 involvement in MET-dependent proliferation

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression.     

The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447_Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447_Δc. Upon infection of the natural host, Vp447_Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.     

Alginate-PEG sponge architecture and role in the design of insulin release dressings

Wound healing is a natural process involving several signaling molecules and cell types over a significant period of time. Although current dressings help to protect the wound from debris or infection, they do little in accelerating the healing process. Insulin has been shown to stimulate the healing of damaged skin. We have developed an alginate sponge dressing (ASD) that forms a hydrogel capable of providing a moist and protective healing environment. By incorporating insulin-loaded poly(d,l-lactide-co-glycolide) (PLGA) microparticles into ASD, we successfully stabilized and released insulin for up to 21 days. Insulin release and water absorption and transfer through the ASD were influenced by altering the levels of poly(ethylene glycol) (PEG) in the dressing matrix. Bioactivity of released insulin can be maintained for at least 10 days, demonstrated using a human keratinocyte migration assay. Results showed that insulin-loaded PLGA microparticles, embedded within PEG-ASD, functioned as an effective long-term delivery platform for bioactive insulin.     

Inflammatory signals enhance piezo2-mediated mechanosensitive currents

Heightened nociceptor function caused by inflammatory mediators such as bradykinin (BK) contributes to increased pain sensitivity (hyperalgesia) to noxious mechanical and thermal stimuli. Although it is known that sensitization of the heat transducer TRPV1 largely subserves thermal hyperalgesia, the cellular mechanisms underlying mechanical hyperalgesia have been elusive. The role of the mechanically activated (MA) channel piezo2 (known as FAM38B) present in mammalian sensory neurons is unknown. We test the hypothesis that piezo2 activity is enhanced by BK, an algogenic peptide that induces mechanical hyperalgesia within minutes. Piezo2 current amplitude is increased and inactivation is slowed by bradykinin receptor beta 2 (BDKRB2) activation in heterologous expression systems. Protein kinase A (PKA) and protein kinase C (PKC) agonists enhance piezo2 activity. BDKRB2-mediated effects are abolished by PKA and PKC inhibitors. Finally, piezo2-dependent MA currents in a class of native sensory neurons are enhanced 8-fold by BK via PKA and PKC. Thus, piezo2 sensitization may contribute to PKA- and PKC-mediated mechanical hyperalgesia.     

LMNA Knock-Down Affects Differentiation and Progression of Human Neuroblastoma Cells

Background

Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma.

Methodology/Principal Findings

Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases.

Conclusions/Significance

We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.