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The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.
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BACKGROUND: Acute myeloid leukemia (AML) is a hematological malignancy that remains a therapeutic challenge due to the high incidence of disease relapse. To better understand resistance mechanisms and identify novel therapies, robust preclinical models mimicking the bone marrow (BM) microenvironment are needed. This study aimed to achieve an automated fabrication process of a three-dimensional (3D) AML disease model that recapitulates the 3D spatial structure of the BM microenvironment and applies to drug screening and investigational studies. METHODS: To build this model, we investigated a unique class of tetramer peptides with an innate ability to self-assemble into stable hydrogel. An automated robotic bioprinting process was established to fabricate a 3D BM (niche-like) multicellular AML disease model comprised of leukemia cells and the BM's stromal and endothelial cellular fractions. In addition, monoculture and dual-culture models were also fabricated. Leukemia cell compatibility, functionalities (in vitro and in vivo), and drug assessment studies using our model were performed. In addition, RNAseq and gene expression analysis using TaqMan arrays were also performed on 3D cultured stromal cells and primary leukemia cells. RESULTS: The selected peptide hydrogel formed a highly porous network of nanofibers with mechanical properties similar to the BM extracellular matrix. The robotic bioprinter and the novel quadruple coaxial nozzle enabled the automated fabrication of a 3D BM niche-like AML disease model with controlled deposition of multiple cell types into the model. This model supported the viability and growth of primary leukemic, endothelial, and stromal cells and recapitulated cell-cell and cell-ECM interactions. In addition, AML cells in our model possessed quiescent characteristics with improved chemoresistance attributes, resembling more the native conditions as indicated by our in vivo results. Moreover, the whole transcriptome data demonstrated the effect of 3D culture on enhancing BM niche cell characteristics. We identified molecular pathways upregulated in AML cells in our 3D model that might contribute to AML drug resistance and disease relapse. CONCLUSIONS: Our results demonstrate the importance of developing 3D biomimicry models that closely recapitulate the in vivo conditions to gain deeper insights into drug resistance mechanisms and novel therapy development. These models can also improve personalized medicine by testing patient-specific treatments.
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62Articular cartilage is a nonvascularized and poorly cellularized tissue with a low self-repair capacity. Therefore, damage to this tissue due to trauma or degenerative joint diseases such as osteoarthritis needs a high-end medical intervention. However, such interventions are costly, have limited healing capacity, and could impair patients' quality of life. In this regard, tissue engineering and three-dimensional (3D) bioprinting hold great potential. However, identifying suitable bioinks that are biocompatible, with the desired mechanical stiffness, and can be used under physiological conditions is still a challenge. In this study, we developed two tetrameric self-assembling ultrashort peptide bioinks that are chemically well-defined and can spontaneously form nanofibrous hydrogels under physiological conditions. The printability of the two ultrashort peptides was demonstrated; different shape constructs were printed with high shape fidelity and stability. Furthermore, the developed ultrashort peptide bioinks gave rise to constructs with different mechanical properties that could be used to guide stem cell differentiation toward specific lineages. Both ultrashort peptide bioinks demonstrated high biocompatibility and supported the chondrogenic differentiation of human mesenchymal stem cells. Additionally, the gene expression analysis of differentiated stem cells with the ultrashort peptide bioinks revealed articular cartilage extracellular matrix formation preference. Based on the different mechanical stiffness of the two ultrashort peptide bioinks, they can be used to fabricate cartilage tissue with different cartilaginous zones, including the articular and calcified cartilage zones, which are essential for engineered tissue integration.
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PURPOSE: To study the use of autologous platelet lysate prepared in a standardized method for the healing of persistent corneal epithelial defects (PED). STUDY DESIGN: Clinical and experimental investigation. METHODS: In this prospective pilot study (ClinicalTrials.gov identifier NCT02979912), ten patients with a PED duration of a minimum 14 days were included. Autologous platelet lysate was prepared in a standardized methodology. Repeated freeze-thaw cycles were used to lyse the platelets. Patients were advised to apply the eye drops four times a day and were evaluated at baseline and on days 7, 14, 21, 28. RESULTS: No adverse events were reported due to the use of undiluted autologous platelet lysate. A total of 70% of patients had complete re-epithelialization within 28 days. Of these, 40% healed within 14 days (effective group) and 30% within 28 days (partially effective group). CONCLUSIONS: Undiluted autologous platelet lysate, prepared according to a standardized methodology, is a safe and effective adjunct therapy for the treatment of PED.
