Using small RNA sequences to diagnose, sequence, and investigate the infectivity characteristics of vegetable-infecting viruses

In a virus-infected plant, small interfering RNAs (siRNAs) corresponding to the viral genome form a large proportion of the small RNA population. It is possible to reassemble significant portions of the virus sequence from overlapping siRNA sequences and use these to identify the virus. We tested this technique with a resistance-breaking and a non-resistance-breaking strain of tomato spotted wilt virus (TSWV). We were able to assemble contigs covering 99% of the genomes of both viruses. The abundance of TSWV siRNAs allowed us to detect TSWV at early time points before the onset of symptoms, at levels too low for conventional detection. Combining traditional and bioinformatic detection methods, we also measured how replication of the resistance-breaking strain differed from the non-resistance-breaking strain in susceptible and resistant tomato varieties. We repeated this technique in identification of a squash-infecting geminivirus and also used it to identify an unspecified tospovirus.     

Mammalian target of rapamycin expression and laryngeal squamous cell carcinoma prognosis: novel preliminary evidence

Marioni G, Staffieri A, Giacomelli L, Lionello M, Guzzardo V, Busnardo A & Blandamura S
(2011) Histopathology  58, 1148–1156
Mammalian target of rapamycin expression and laryngeal squamous cell carcinoma prognosis: novel preliminary evidence Aims: The mammalian target of rapamycin (mTOR) has a key role in regulating cancer cell proliferation, apoptosis, cell migration, and angiogenesis. The aim of this study was to assess the relationships between mTOR and clinicopathological and prognostic parameters in laryngeal squamous cell carcinoma (SCC). Methods and results: Mammalian target of rapamycin expression was determined in 103 consecutive operable laryngeal SCCs. Among the mTOR‐positive cases, the locoregional recurrence rate was higher (P = 0.048) and the disease‐free survival (DFS) rate was shorter (P = 0.031) in patients with mTOR expression >50.7%. In the N₀ subgroup, the disease recurrence rate was higher (P = 0.034) and the DFS was shorter (P = 0.009) in patients with mTOR expression >50.7%. In mTOR‐positive patients, multivariate analysis showed that N stage (P = 0.0001) and mTOR status (P = 0.042) were independent indicators of a poor prognosis. Conclusions: mTOR appeared to be a significant predictor of DFS in univariate and multivariate models. mTOR expression in laryngeal SCC may be useful for the detection of patients at higher risk for recurrence, and N₀ patients at higher risk for early locoregional recurrence who might benefit from more aggressive therapy. The role of mTOR inhibitors in multimodality or multitarget strategies against laryngeal SCC warrants investigation.     

Preventing the ubiquitin-proteasome-dependent degradation of frataxin, the protein defective in Friedreich's ataxia

Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent frataxin ubiquitination and degradation, and increase frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the frataxin degradation pathway.     

(1)H-MRS can detect aberrant glycosylation in tumour cells: a study of the HeLa cell line

Glycosylation is the most abundant and diverse form of post-translational modification of proteins. Two types of glycans exist in glycoproteins: N-glycans and O-glycans often coexisting in the same protein. O-glycosylation is frequently found on secreted or membrane-bound mucins whose overexpression and structure alterations are associated with many types of cancer. Mucins have several cancer-associated structures, including high levels of Lewis antigens characterized by the presence of terminal fucose. The present study deals with the identification of MR signals from N-acetylgalactosamine and from fucose in HeLa cells by detecting a low-field signal in one-dimensional (1D) spectra assigned to the NH of N-acetylgalactosamine and some cross peaks assigned to fucose in two-dimensional (2D) spectra. The increase of Golgi pH by treatment with ammonium chloride allowed the N-acetylgalactosamine signal assignment to be confirmed. Behaviour of MR peak during cell growth and comparison with studies from literature taken together made it possible to have more insight into the relationship between aberrantly processed mucin and the presence of non-processed N-acetylgalactosamine residues in HeLa cells. Fucose signals, tentatively ascribed to residues bound to galactose and to N-acetylglucosamine, are visible in both intact cell and perchloric acid spectra. Signals assigned to fucose bound to galactose are more evident in ammonium chloride-treated cells where structural changes of mucin-related Lewis antigens are expected as a result of the higher Golgi pH. A common origin for the N-acetylgalactosamine and fucose resonances attributing them to aberrantly processed mucin can be inferred from the present results.     

Transplantation of SNAP-treated adipose tissue-derived stem cells improves cardiac function and induces neovascularization after myocardium infarct in rats

Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30 days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p = 0.008). Immunohistochemical reactions showed an increased expression of troponin T–C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.     

Ganglioside-exposed dendritic cells inhibit T-cell effector function by promoting regulatory cell activity

Tumour pathogenesis is characterized by an immunosuppressive microenvironment that limits the development of effective tumour‐specific immune responses. This is in part the result of tumour‐dependent recruitment and activation of regulatory cells, such as myeloid‐derived suppressor cells and regulatory T cells in the tumour microenvironment and draining lymph nodes. Shedding of gangliosides by tumour cells has immunomodulatory properties, suggesting that gangliosides may be a critical factor in initiating an immunosuppressive microenvironment. To better define the immunomodulatory properties of gangliosides on antigen‐specific T‐cell activation and development we have developed an in vitro system using ganglioside‐treated murine bone‐marrow‐derived dendritic cells to prime and activate antigen‐specific CD4⁺ T cells from AND T‐cell receptor transgenic mice. Using this system, ganglioside treatment promotes the development of a dendritic cell population characterized by decreased CD86 (B7‐2) expression, and decreased interleukin‐12 and interleukin‐6 production. When these cells are used as antigen‐presenting cells, CD4 T cells are primed to proliferate normally, but have a defect in T helper (Th) effector cell development. This defect in Th effector cell responses is associated with the development of regulatory T‐cell activity that can suppress the activation of previously primed Th effector cells in a contact‐dependent manner. In total, these data suggest that ganglioside‐exposed dendritic cells promote regulatory T‐cell activity that may have long‐lasting effects on the development of tumour‐specific immune responses.     

Motive-oriented therapeutic relationship in brief psychodynamic intervention for patients with depression and personality disorders

Motive-Oriented Therapeutic Relationship (MOTR, also called Complementary Therapeutic Relationship) has already shown itself to be related to therapeutic outcome in several studies. The present study aims to test MOTR in a 4-session Brief Psychodynamic Intervention for patients presenting with major depressive disorder (MDD) and comorbid personality disorder (PD). In total, N = 20 patients were selected; n = 10 had MDD, n = 10 had MDD with comorbid PD. The first therapy session was videotaped and analyzed by means of Plan Analysis and the MOTR scale. Results suggest a differential effect on outcome: only the nonverbal component of MOTR is related to symptomatic change in patients presenting with MDD and comorbid PD; no such effect was found for patients with MDD alone. These results are discussed in line with the generalization and refinement of the conclusions of previous findings on the MOTR.     

Botulinum Toxin Injection in Long-Standing Facial Paralysis Patients: Improvement of Facial Symmetry Observed up to 6 Months

Background  Despite modern reanimation surgical techniques, facial paralysis presents with functional and aesthetic deficits. We evaluated facial symmetry after treating with botulinum toxin the healthy side of the face of 25 patients with long-standing facial paralysis who had previously been treated by surgical methods, with 6 months follow-up. Methods  Evaluation consisted of a clinical score, the two subscales of the Facial Disability Index, and surface electromyography. The mean botulinum toxin dose was 38 ± 5 U (range = 15–69 U). Results  The clinical score showed significant reduction of asymmetry of 48.4% at 1 month and 16.8% after 6 months. The initial result was a consequence of reduced motion on the treated side combined with better motion on the paralyzed side. At 6 months, the treated side returned to basal scores. The residual effect seen in symmetry was due to an increase (18%) of motion in the paralyzed side. There was a significant decrease in the action potential of muscles on the nonparalyzed side 1 month post injection but completely reverted after 6 months. The Physical Function Index increased, but not significantly. The Social/Well-Being Function Index showed a significant increase at 6 months compared to pretreatment. Conclusion  The proposed treatment improved facial symmetry for up to 6 months. Even after the end of the clinical effect of the drug, the paralyzed side’s clinical score was 18% higher than pretreatment, with an increased quality of life.