Exploring Honeybee Abdominal Anatomy through Micro-CT and Novel Multi-Staining Approaches.

Continuous improvements in morphological and histochemical analyses of Apis mellifera could improve our understanding of the anatomy and physiology of these insects at both the cellular and tissue level. In this work, two different approaches have been performed to add new data on the abdomen of worker bees: (i) Micro-computed tomography (Micro-CT), which allows the identification of small-scale structures (micrometers) with adequate/optimal resolution and avoids sample damage and, (ii) histochemical multi-staining with Periodic Acid-Schiff-Alcian blue, Lactophenol-Saphranin O and pentachrome staining to precisely characterize the histological structures of the midgut and hindgut. Micro-CT allowed high-resolution imaging of anatomical structures of the honeybee abdomen with particular emphasis on the proventriculus and pyloric valves, as well as the connection of the sting apparatus with the terminal abdominal ganglia. Furthermore, the histochemical analyses have allowed for the first-time description of ventricular telocytes in honeybees, a cell type located underneath the midgut epithelium characterized by thin and long cytoplasmic projections called telopodes. Overall, the analysis of these images could help the detailed anatomical description of the cryptic structures of honeybees and also the characterization of changes due to abiotic or biotic stress conditions.

Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles.

Research into stress and stability is essential during the development of therapeutic proteins to ensure quality and safety of the final medicine. Greater knowledge of the effects of stress on aggregation can help avoid undesirable conformational and colloidal instabilities. With this in mind we investigated five marketed therapeutic monoclonal antibodies (mAbs) namely bebacizumab (BVZ), cetuximab (CTX), infliximab (IFX), rituximab (RTX) and tratuzumab (TTZ) in their innovative medicines. These were submitted to different controlled stresses, to freeze/thaw cycles and used for long-term stability studies once the vials were opened. Aggregate formation was tracked by analyzing the mAbs chromatographic profiles by size-exclusion liquid chromatography coupled with diode array detection. Results indicated that the tendency to aggregate depends on the particular stress conditions and on the concentration and nature of the mAb, even though all share similar IgG1-structure. Fragmentation of the mAb produced by the stress was probably due to the rupture of cystines between the two heavy chains. Regarding stability study, BVZ, RTX and TTZ proved to be the most stable when stored at 4 °C and in freeze/thaw cycles with no tendency to form aggregates. INF tends to form aggregates at 0.5 mg/mL, while in CTX, the most unstable, degradation was detected.

Untargeted LC-HRMS-Based Metabolomics for Searching New Biomarkers of Pancreatic Ductal Adenocarcinoma: A Pilot Study.

Pancreatic ductal adenocarcinoma is one of the most lethal tumors since it is usually detected at an advanced stage in which surgery and/or current chemotherapy have limited efficacy. The lack of sensitive and specific markers for diagnosis leads to a dismal prognosis. The purpose of this study is to identify metabolites in serum of pancreatic ductal adenocarcinoma patients that could be used as diagnostic biomarkers of this pathology. We used liquid chromatography-high-resolution mass spectrometry for a nontargeted metabolomics approach with serum samples from 28 individuals, including 16 patients with pancreatic ductal adenocarcinoma and 12 healthy controls. Multivariate statistical analysis, which included principal component analysis and partial least squares, revealed clear separation between the patient and control groups analyzed by liquid chromatography-high-resolution mass spectrometry using a nontargeted metabolomics approach. The metabolic analysis showed significantly lower levels of phospholipids in the serum from patients with pancreatic ductal adenocarcinoma compared with serum from controls. Our results suggest that the liquid chromatography-high-resolution mass spectrometry-based metabolomics approach provides a potent and promising tool for the diagnosis of pancreatic ductal adenocarcinoma patients using the specific metabolites identified as novel biomarkers that could be used for an earlier detection and treatment of these patients.

Cytoskeletal proteins in the cerebrospinal fluid as biomarker of multiple sclerosis.

The axonal cytoskeleton is a finely organized system, essential for maintaining the integrity of the axon. Axonal degeneration is implicated in the pathogenesis of unremitting disability of multiple sclerosis (MS). Purpose of this study is to evaluate levels of cytoskeletal proteins such as neurofilament light protein (NFL), glial fibrillary acidic protein (GFAP), and ß-tubulin (ß-Tub) isoforms II and III in the cerebrospinal fluid (CSF) of MS patients and their correlation with MS clinical indices. CSF levels of cytoskeletal proteins were determined in 51 patients: 33 with MS and 18 with other neurological diseases (OND). NFL, GFAP and ß-Tub II proteins were significantly higher (p < 0.0001) in MS than in OND group; no significant difference (p > 0.05) was found between MS and OND with regard to ß-Tub III. Interestingly, levels of ß-Tub III and NFL were higher in progressive than in remitting MS forms; on the contrary, higher levels of ß-Tub II and GFAP were found in remitting MS forms. However, with the exception of ß-Tub III, all proteins tend to decrease their CSF levels concomitantly with the increasing disability (EDSS) score. Overall, our results might indicate ß-Tub II as a potential candidate for diagnostic and ß-Tub III as a possible prognostic biomarker of MS. Therefore, further analyses are legitimated and desirable.

The chemotherapeutic drug 5-fluorouracil promotes PKR-mediated apoptosis in a p53-independent manner in colon and breast cancer cells.

The chemotherapeutic drug 5-FU is widely used in the treatment of a range of cancers, but resistance to the drug remains a major clinical problem. Since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in 5-FU-induced apoptosis is of main clinical interest. We have identified the ds-RNA-dependent protein kinase (PKR) as a key molecular target of 5-FU involved in apoptosis induction in human colon and breast cancer cell lines. PKR distribution and activation, apoptosis induction and cytotoxic effects were analyzed during 5-FU and 5-FU/IFNα treatment in several colon and breast cancer cell lines with different p53 status. PKR protein was activated by 5-FU treatment in a p53-independent manner, inducing phosphorylation of the protein synthesis translation initiation factor eIF-2α and cell death by apoptosis. Furthermore, PKR interference promoted a decreased response to 5-FU treatment and those cells were not affected by the synergistic antitumor activity of 5-FU/IFNα combination. These results, taken together, provide evidence that PKR is a key molecular target of 5-FU with potential relevance in the clinical use of this drug.

Indirect immunofluorescence study on the cytoskeleton of normal (FG) and neoplastic (SGS/3A) cadmium treated fibroblasts.

Cadmium is a heavy metal dangerous for the environment and for the human health, with well shown carcinogenic potentiality. Many studies have related the professional exposure of the Cadmium with human pulmonary, prostatic and renal tumors, and it would be a role also in the tumors of the liver, of the hemopoietic system, of the bladder and of the stomach. The aim of the current study is to examine in normal and neoplastic fibroblasts culture cells the modifications induced by the Cadmium at cellular level, in particular on the cytoskeleton, responsible not only of the intracellular transport of vesicles and cell organules, but also of their positioning and of the cellular integrity. Two fibroblastic cellular strains, normal (FG) and neoplastic (SGS/3A), have been incubated in a 5 microM Cadmium acetate added medium for 1, 8, 24 hours and studied by indirect immunofluorescence methods, particularly for the following proteins: Actin, Tubulin and Vimentin. The observations show in normal and neoplastic fibroblasts comparables modifications and anomalies of cytoskeletal shape. In both the cases the cellular morphology suffers drastic modifications, gradually evolving through intermediary shapes: from triangular and spindle-shaped in the normal fibroblasts to irregular, star-shaped, and globular in the neoplastic ones. The Cadmium action on the morphology of the normal and tumoral cells changes according to the time of incubation, producing structural alterations of the cytoskeletal. The modifications that start to be observable at the first hour of incubation are more evident after the eighth hour of exposure, reaching the maximum expression at the twenty-fourth hour, often with reduction of the total volume of the cells and loss of their ability to adhere to the substratum. Such modifications can be related to great alterations of the cellular membrane, producing the change of shape and the progressive partial separation from the substratum. The intermediary filaments seem to be less sensitive, from a morphological but not functional point of view, to the action of the Cadmium in comparison to the Actin and the microtubules that, on the contrary, seem to lose their proper morphological characteristics.