205 research outputs found
STUDIES ON LINEAGE SHIFT RESPONSES OF HUMAN PERIPHERAL BLOOD MULTIPOTENT CELLS
Full text linkStem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. As embryonic stem cells pose a lot of ethical issues, adult stem cells, isolated from various sources like cord blood, bone marrow or adipose tissue, are being considered as a realistic option due to their well documented therapeutic potentials. In our lab, we have standardised a method to isolate fibroblastic multipotent stem cells (PBMCs) from human peripheral blood, that are able to sustain long term in vitro culture and differentiate towards adipogenic, chondrogenic and osteogenic lineage.
In this work, PBMCs were stimulated to obtain in vitro neuronal and myogenic-like cells. Moreover, their restorative potential in degenerative diseases of skeletal muscle and nervous tissue was evaluated using in vivo models. In order to test the neuronal differentiation potential, the cells were seeded (1x104) on gelatin coated dishes and cultured for 7 days in neurobasal medium with EGF and FGF followed by Retinoic acid and NGF for next 7 days. Myogenic induction was carried out using IGF and ascorbic acid for 14 days. At different time points, morphological studies were performed by SEM and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. PBMCs showed characteristic dendrite like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of PBMCs under stimulation with KCl 56 mM and the secretion of the neurotransmitter, noradrenalin, a precursor in the dopamine synthesis confirmed their ability to acquire a functional phenotype. When premarked by a cell tracker Qdot 800 and injected stearotactically into a rat brain, PBMCs showed to be migratory and proliferative as detected after 10 and 20 days of injection. No tumor mass was identified. The myogenic potential of PBMCs were confirmed by their ability to form syncitium like structures in in vitro culture and to express typical myogenic markers both at early and late phases of differentiation. PBMCs were showed to integrate within the host tissue and to take part in tissue repair as demonstrated in a bupivacaine induced muscle damage model
Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco
No full textSYNTHESIS AND BIOFUNCTIONALIZATION OF PLASMONIC NANOSTRUCTURES FOR EGFR-MARKED CANCER CELL TARGETING AND SERRS IMAGING
No full textPlasmonic nanoparticles are increasingly utilized in biomedical applications including imaging, diagnostics and therapy. Gold nanoparticles (AuNP), besides displaying useful optical properties, possess a facile surface chemistry and absence of inherent toxicity, an essential requirement for biological application. AuNP can passively target tumors by the enhanced permeability and retention effect, but active targeting by proteins, peptides or small molecules, can further improve the pharmacokinetic and pharmacodynamics profiles of these multifunctional agents. The dodecapeptide YHWYGYTPQNVI (GE11) was recently identified as a specific ligand for the Epidermal Growth Factor Receptor, which is overexpressed in many types of cancer[1]. In the present work we have employed the enormous sensitivity of the Surface Enhanced Raman Spectroscopy [2] to study the targeting activity of GE11-functionalized plasmonic nanostructures on different types of tumor cells. Nanoparticles were prepared, without stabilizing molecules, by laser ablation of a gold target in water, functionalized with a SERRS reporter [3], and conjugated with a number of ligands: GE11, mPEG, and different PEG-GE11 conjugates. Nanoaggregates covered with PEG or with the monoclonal antibody Cetuximab were used as negative and positive control, respectively. Targeting of nanostructures to EGFR was checked by incubation with cancer cells expressing or not the receptor and recording the SERRS signals for each cell. The influence of different aspects for EGFR recognition, such as peptide orientation and exposure on the NP surface, will be presented
Genetic variation of clock genes and cancer risk: a field synopsis and meta-analysis
No full textThe number of studies on the association between clock genes' polymorphisms and cancer susceptibility has increased over the last years but the results are often conflicting and no comprehensive overview and quantitative summary of the evidence in this field is available.
RESULTS:
Literature search identified 27 eligible studies comprising 96756 subjects (cases: 38231) and investigating 687 polymorphisms involving 14 clock genes. Overall, 1025 primary and subgroup meta-analyses on 366 gene variants were performed. Study distribution by tumor was as follows: breast cancer (n=15), prostate cancer (n=3), pancreatic cancer (n=2), non-Hodgkin's lymphoma (n=2), glioma (n=1), chronic lymphocytic leukemia (n=1), colorectal cancer (n=1), non-small cell lung cancer (n=1) and ovarian cancer (n=1).We identified 10 single nucleotide polymorphisms (SNPs) significantly associated with cancer risk: NPAS2 rs10165970 (mixed and breast cancer shiftworkers), rs895520 (mixed), rs17024869 (breast) and rs7581886 (breast); CLOCK rs3749474 (breast) and rs11943456 (breast); RORA rs7164773 (breast and breast cancer postmenopausal), rs10519097 (breast); RORB rs7867494 (breast cancer postmenopausal), PER3 rs1012477 (breast cancer subgroups) and assessed the level of quality evidence to be intermediate. We also identified polymorphisms with lower quality statistically significant associations (n=30).
CONCLUSIONS:
Our work supports the hypothesis that genetic variation of clock genes might affect cancer risk. These findings also highlight the need for more efforts in this research field in order to fully establish the contribution of clock gene variants to the risk of developing cancer.
METHODS:
We conducted a systematic review and meta-analysis of the evidence on the association between clock genes' germline variants and the risk of developing cancer. To assess result credibility, summary evidence was graded according to the Venice criteria and false positive report probability (FPRP) was calculated to further validate result noteworthiness. Subgroup meta-analysis was also performed based on participant features and tumor type. The breast cancer subgroup was further stratified by work conditions, estrogen receptor/progesterone receptor status and menopausal status, conditions associated with the risk of breast cancer in different studies
Associations of clock genes polymorphisms with soft tissue sarcoma susceptibility and prognosis
Full text linkBACKGROUND:
Dysfunction of the circadian clock and polymorphisms of some circadian genes have been linked to cancer development and progression. We investigated the relationship between circadian genes germline variation and susceptibility or prognosis of patients with soft tissue sarcoma.
PATIENTS AND METHODS:
We considered the 14 single nucleotide polymorphisms (SNPs) of 6 core circadian genes that have a minor allele frequency > 5% and that are known to be associated with cancer risk or prognosis. Genotyping was performed by q-PCR. Peripheral blood and clinic-pathological data were available for 162 patients with liposarcoma or leiomyosarcoma and 610 healthy donors. Associations between the selected clock genes polymorphisms and sarcoma susceptibility or prognosis were tested assuming 3 models of inheritance: additive, recessive and dominant. Subgroup analysis based on sarcoma histotype was performed under the additive genetic model. Multivariate logistic regression and multivariate Cox proportional hazard regression analyses were utilized to assess the association between SNPs with patient susceptibility and survival, respectively. Pathway variation analysis was conducted employing the Adaptive Rank Truncated Product method.
RESULTS:
Six out of the 14 analyzed SNPs were statistically significantly associated with susceptibility or prognosis of soft tissue sarcoma (P < 0.05). The present analysis suggested that carriers of the minor allele of the CLOCK polymorphism rs1801260 (C) or of PER2 rs934945 (T) had a reduced predisposition to sarcoma (26% and 35% respectively with the additive model) and liposarcoma (33% and 41% respectively). The minor allele (A) of NPAS2 rs895520 was associated with an increased predisposition to sarcoma of 33% and leiomyosarcoma of 44%. RORA rs339972 C allele was associated with a decreased predisposition to develop sarcoma assuming an additive model (29%) and leiomyosarcoma (36%). PER1 rs3027178 was associated with a reduced predisposition only in liposarcoma subgroup (32%). rs7602358 located upstream PER2 was significantly associated with liposarcoma survival (HR: 1.98; 95% CI 1.02-3.85; P = 0.04). Germline genetic variation in the circadian pathway was associated with the risk of developing soft tissue sarcoma (P = 0.035).
CONCLUSIONS:
Genetic variation of circadian genes appears to play a role in the determinism of patient susceptibility and prognosis. These findings prompt further studies to fully dissect the molecular mechanisms
Gold Nanoparticle Aggregates Functionalized with Cyclic RGD Peptides for Targeting and Imaging of Colorectal Cancer Cells
No full textTheactivetargetingstrategyhasemergedasapromising approach to achieve selectivity in nanobiotechnologyapplications. Peptides are particularly suited as targeting moietiesbecause the multivalent presentation of these small molecules on ananoparticle provides high avidity for the target. However, to achievean efficient targeting activity, the presentation of the peptide on thenanostructure has to be supported by an appropriate design. Tooptimize the targeting to colorectal cancer cells, we have performed aligand design study of plasmonic nanostructures covered with a cyclicRGD peptide, a known targeting moiety for theαvβ3-integrin. Wefindthat to achieve a good targeting activity, the RGD peptide has to belinked to plasmonic nanostructures through a long PEG chain and ashort oligolysine spacer. When the cyclic RGD peptide is directly linked to the PEG chains of the nanostructures, their targetingability is lost. Molecular dynamics calculations make possible to understand the difference of the peptide organization in twotargeted nanosystems, unveiling an effect of the spacer on the orientation of the active component, which very likely positivelyaffects the targeting properties of the investigated plasmonic nanostructures
MOESM2 of miRNA deregulation targets specific pathways in leiomyosarcoma development: an in silico analysis
Full text linkAdditional file 2: Table S2. miRNAs targets predicted by TargetScan with cumulative weighted context score cut-off level of ââ1.0 or smaller
Understanding the good and poor cell targeting activity of gold nanostructures functionalized with molecular units for the epidermal growth factor receptor
No full textNanostructures can strongly interact with cells or other biological structures; furthermore when they are functionalized with targeting units, they are of great interest for a variety of applications in the biotechnology field like those for efficient imaging, diagnosis and therapy and in particular for cancer theranostics. Obtaining targeting with good specificity and sensitivity is a key necessity, which, however, is affected by the complexity of the interactions between the nanostructures and the biological components. In this work we report the study of specificity and sensitivity of gold nanoparticles functionalized with the peptide GE11 for the targeting of the epidermal growth factor receptor, expressed on many cells and, in particular, on many types of cancer cells. We show how a combination of spectroscopic measurements and molecular dynamics simulations allows the comprehension of the targeting activity of peptides linked to the surface of gold nanostructures and how the targeting is tuned by the presence of polyethylene glycol chains
Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs
No full textHaemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44+/CD49c+/CD49e+/CD151+/CD73+/CD49f-/CD26- resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia
In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration
No full textIn regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury
- …
