Altered cogs of the clock: Insights into the embryonic etiology of spondylocostal dysostosis

Spondylocostal dysostosis (SCDO) is a rare heritable congenital condition, characterized by multiple severe malformations of the vertebrae and ribs. Great advances were made in the last decades at the clinical level, by identifying the genetic mutations underlying the different forms of the disease. These were matched by extraordinary findings in the Developmental Biology field, which elucidated the cellular and molecular mechanisms involved in embryo body segmentation into the precursors of the axial skeleton. Of particular relevance was the discovery of the somitogenesis molecular clock that controls the progression of somite boundary formation over time. An overview of these concepts is presented, including the evidence obtained from animal models on the embryonic origins of the mutant-dependent disease. Evidence of an environmental contribution to the severity of the disease is discussed. Finally, a brief reference is made to emerging in vitro models of human somitogenesis which are being employed to model the molecular and cellular events occurring in SCDO. These represent great promise for understanding this and other human diseases and for the development of more efficient therapeutic approaches.PTDC/BEX-BID/5410/2014, SFRH/BD/146043/2019, UID/BIM/04773/2019info:eu-repo/semantics/publishedVersio

Enzyme-inspired dry-powder polymeric catalyst for green and fast pharmaceutical manufacturing processes

Funding Information: The authors thank financial support from Fundação para a Ciência e a Tecnologia , Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES Portugal), through project PTDC/EQU-EQU/32473/2017 , a Principal Investigator contract IF/00915/2014 (T.C.), and a doctoral grant SFRH/BDE/51907/2012 , a partnership from FCT/MCTES and the pharmaceutical company HOVIONE (R.V.). L.B.M. would like to acknowledge for FCT/MCTES funding with reference CEECIND/03810/2017. The NMR spectrometers in LabNMR@Cenimat are part of the National NMR Facility, supported by FCT (ROTEIRO/0031/2013 - PINFRA/22161/2016 ), co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC ( POCI-01-0145-FEDER-007688 ; UID/CTM/50025/2020-2023 ). The Associate Laboratory Research Unit for Green Chemistry - Clean Technologies and Processes - LAQV is financed by national funds from FCT/MCTES ( UIDB/QUI/50006/2020 ) and cofunded by the ERDF under the PT2020 Partnership Agreement ( POCI-01-0145-FEDER-007265 ). We also acknowledge Dr. Luz Fernandes, REQUIMTE analytical services, for GC analysis. Publisher Copyright: © 2022Catalysis in pharma manufacturing processes is typically homogeneous, expensive and with hard catalyst recovery/regeneration. Herein an enzyme-inspired dry-powder molecularly imprinted polymeric (MIP) system was designed for fast, selective oxidation of a cholesterol derivative and easy catalyst regeneration. The strategy involved the synthesis of a template-monomer (T:M) complex followed by the crosslinked polymerization in supercritical carbon dioxide (scCO2). A 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)-MIP catalyst is obtained after the template cleavage from the matrix, and the oxidation of the N[sbnd]H groups turns available TEMPO moieties within the MIP. The oxidation of benzyl alcohol, 5α-cholestan-3β-ol and cholic acid was fast, in high yield and with selective oxidation capacity.publishersversionpublishe

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells

The outcome of cell-based therapies can benefit from carefully designed cell carriers. A multifunctional injectable vehicle for the co-delivery of human mesenchymal stem cells (hMSCs) and osteoinductive peptides is proposed, to specifically direct hMSCs osteogenic differentiation. The osteogenic growth peptide (OGP) inspired the design of two peptides, where the bioactive portion of OGP was flanked by a protease-sensitive linker, or its scrambled sequence, to provide faster and slower release rates, respectively. Peptides were fully characterized and chemically grafted to alginate. Both OGP analogs released bioactive fragments in vitro, at different kinetics, which stimulated hMSCs proliferation and osteogenesis. hMSCs-laden OGP-alginate hydrogels were tested at an ectopic site in a xenograft mouse model. After 4 weeks, OGP-alginate hydrogels were more degraded and colonized by vascularized connective tissue than the control (without OGP). hMSCs were able to proliferate, migrate outward the hydrogels, produce endogenous extracellular matrix and mineralize it. Moreover, OGP-groups stimulated hMSCs osteogenesis, as compared with the control. Overall, the ability of the proposed platform to direct the fate of transplanted hMSCs in loco was demonstrated, and OGP-releasing hydrogels emerged as a potentially useful system to promote bone regeneration

Peptide-modified dendrimer nanoparticles for targeted therapy of colorectal cancer

Peptides have recently emerged as a promising class of targeting ligands forspecific drug delivery in cancer treatment, which avoid undesirable side effectsof the systemic administration of chemotherapeutics. Their conjugation withnanoparticles has been demonstrated to improve the functionality of peptidesresulting in a versatile platform for biomedical applications. In this work, thedevelopment of carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM)dendrimer nanoparticles functionalized with YIGSR laminin receptor bindingpeptide for the active targeting and specific delivery of therapeutic agents intocolorectal cancer cells is described. The successful functionalization isconfirmed by several physico-chemical characterization techniques. Theselectivity of the YIGSR-CMCht/PAMAM dendrimer nanoparticles is firstvalidated in vitro using a micropatterned array of 67 kDa laminin receptor.Next, the specificity of YIGSR-CMCht/PAMAM dendrimers nanoparticlestoward laminin receptor is further confirmed both in 2D and 3D settings usingHCT-116 colorectal cancer cells and L929 fibroblasts in co-culture. Finally,gemcitabine-loaded YIGSR-CMCht/PAMAM dendrimer nanoparticles inducea targeted mortality on HCT-116 cancer cells in a co-culture scenario. Overall,the study shows solid evidence that YIGSR laminin receptor binding peptidecoupled to CMCht/PAMAM dendrimer nanoparticles may be employed as ananticancerous target for the specific and intracellular delivery ofchemotherapeutic agents.This work was financially supported through the project FROnTHERA (NORTE-01-0145-FEDER-000023), Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); M.R.C. for her Ph.D. scholarship NORTE-08-5369-FSE-000044, funded by Programa Operacional Regional do Norte, Fundo Social Europeu, Norte 2020 TERM&SC and EMBO Short-Term Fellowship 7232. J.M.O. for his distinction attributed under the FCT Investigator program (IF/00423/2012 and IF/01285/2015; F.R.M. acknowledges FCT for her work contract under the Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)). D.C. acknowledges the financial support from the Portuguese Foundation for Science and Technology (FCT) under the program CEEC Individual 2017 (CEECIND/00352/2017). D.C. and S.C.K. for the Portuguese Foundation for Science and Technology (FCT) under the scope of the project 2MATCH (PTDC/BTMORG/28070/2017) funded by the Programa Operacional Regional do Norte supported by European Regional Development Funds (ERDF). This work is also partially supported by the IET Harvey Engineering Research Award 2018 (ENG ThE CANCER) and the European Union Framework Program for Research and Innovation Horizon 2020 on FoReCaST project under Grant Agreement No. 668983

Organ-on-a-chip platforms for drug screening and delivery in tumor cells: a systematic review

The development of cancer models that rectify the simplicity of monolayer or static cell cultures physiologic microenvironment and, at the same time, replicate the human system more accurately than animal models has been a challenge in biomedical research. Organ-on-a-chip (OoC) devices are a solution that has been explored over the last decade. The combination of microfluidics and cell culture allows the design of a dynamic microenvironment suitable for the evaluation of treatments’ efficacy and effects, closer to the response observed in patients. This systematic review sums the studies from the last decade, where OoC with cancer cell cultures were used for drug screening assays. The studies were selected from three databases and analyzed following the research guidelines for systematic reviews proposed by PRISMA. In the selected studies, several types of cancer cells were evaluated, and the majority of treatments tested were standard chemotherapeutic drugs. Some studies reported higher drug resistance of the cultures on the OoC devices than on 2D cultures, which indicates the better resemblance to in vivo conditions of the former. Several studies also included the replication of the microvasculature or the combination of different cell cultures. The presence of vasculature can influence positively or negatively the drug efficacy since it contributes to a greater diffusion of the drug and also oxygen and nutrients. Co-cultures with liver cells contributed to the evaluation of the systemic toxicity of some drugs metabolites. Nevertheless, few studies used patient cells for the drug screening assays.This work has been supported by the projects NORTE-01-0145-FEDER-030171 (project reference PTDC/EME-SIS/30171/2017), NORTE-01-0145-FEDER-029394 (project reference PTDC/EMDEMD/29394/2017), through the COMPETE2020, the Lisb@2020, the Programa Operacional Regional do Norte–Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (FEDER) and by Fundação para a Ciência e Tecnologia (FCT), and through FEDER and FCT, project references EXPL/EMD-EMD/0650/2021 and PTDC/EEI-EEE/2846/2021. The authors also acknowledge the partial financial support within the R&D Units Project Scope: UIDB/00319/2020, UIDB/04077/2020, UIDB/00690/2020, UIDB/04436/2020. This work was also funded by AMED-CREST Grant Number JP20gm1310001h0002. Raquel O. Rodrigues (R.O.R.) thanks FCT for her contract funding provided through 2020.03975.CEECIND

Quimioterapia Neoadjuvante em Câncer de Mama Localmente Avançado: Análise Imunohistoquímica é Preditiva da Resposta à Quimioterapia

A quimioterapia é tratamento padrão inicial para câncer de mama localmente avançado. A correlação entre a resposta à quimioterapia neoadjuvante e fatores prognósticos pode ser útil nesta doença. De setembro 1996 a dezembro de 1997, 25 pacientes portadoras de câncer de mama localmente avançado (UICC - estádio IIIA, IIIB e inflamatório (1), foram submetidas a 4 ciclos de quimioterapia neoadjuvante com doxorrubucina 60mg/m² e ciclotosfamida 600mg/m² a cada 21 dias, mastectomia à Patey e tratamento adjuvante. A reposta clínica e patológica foi correlacionada com marcadores obtidos através de análise imunohistoquímica da biópsia do tumor. Os marcadores analisados foram; receptores hormonais, p53, HER/neu (cerb-B2), MIB, grau nuclear, PCNA. A resposta clínica objetiva foi de 74%. Vinte e um de 23 pacientes (91%) analisadas foram submetidas à cirurgia. Quatro pacientes não apresentavam doença microscópica na mama (19%). Destas pacientes, 2 também não apresentavam doença em linfonodos axilares, enquanto 4 apresentavam doença residual na mama de até 2 cm (19%). Todos os marcadores apresentaram positividade em percentuais elevados. A positividade do p53 e do MIB apresentou correlação com a resposta ao tratamento quimioterápico neoadjuvante, porém não alcançou significância estatística. Os resultados iniciais sugerem uma relação entre a positividade do p53 com a resposta clínica e com a resposta patológica, relação esta que não é demonstrada em estudos anteriores. A presença do MIB positivo também esteve associada com uma resposta patológica favorável

3D printing techniques and their applications to organ-on-a-chip platforms: a systematic review

Three-dimensional (3D) in vitro models, such as organ-on-a-chip platforms, are an emerging and effective technology that allows the replication of the function of tissues and organs, bridging the gap amid the conventional models based on planar cell cultures or animals and the complex human system. Hence, they have been increasingly used for biomedical research, such as drug discovery and personalized healthcare. A promising strategy for their fabrication is 3D printing, a layer-by-layer fabrication process that allows the construction of complex 3D structures. In contrast, 3D bioprinting, an evolving biofabrication method, focuses on the accurate deposition of hydrogel bioinks loaded with cells to construct tissue-engineered structures. The purpose of the present work is to conduct a systematic review (SR) of the published literature, according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses, providing a source of information on the evolution of organ-on-a-chip platforms obtained resorting to 3D printing and bioprinting techniques. In the literature search, PubMed, Scopus, and ScienceDirect databases were used, and two authors independently performed the search, study selection, and data extraction. The goal of this SR is to highlight the importance and advantages of using 3D printing techniques in obtaining organ-on-a-chip platforms, and also to identify potential gaps and future perspectives in this research field. Additionally, challenges in integrating sensors in organs-on-chip platforms are briefly investigated and discussed.The authors are grateful for the funding of FCT through the projects NORTE-01-0145- FEDER-029394, NORTE-01-0145-FEDER-030171 funded by COMPETE2020, NORTE2020, PORTUGAL2020, and FEDER. This work was also supported by Fundação para a Ciência e a Tecnologia (FCT) under the strategic grants UIDB/04077/2020, UIDB/00319/2020, UIDB/04436/2020 and UIDB/00532/2020. This work was also funded by AMED-CREST Grant Number JP20gm1310001h0002.Violeta Carvalho acknowledges the PhD scholarship UI/BD/151028/2021 attributed by FCT. Inês Gonçalves acknowledges the PhD scholarship BD/08646/2020 attributed by FCT

Novel deletions and unusual genetic mechanisms underlying alpha-thalassemia

Hemoglobin (Hb) is a protein responsible for oxygen transportation from lungs to the entire body. It is composed by four globular subunits - the globins - each with a central core containing a heme molecule. Globins are encoded by the α- and β-globin gene clusters located at 16p13.3 and 11p15.5, respectively. The pattern of globin genes expression during development is precisely controlled by the interaction of cis-regulatory genomic regions (located in close proximity to and far from genes) with trans-activating/silencing factors within permissive chromatin domains. In fact, approximately 25-65 kb upstream of the α-globin genes there are four multispecies conserved sequences (MCS-R1 to R4) which are critical for the expression regulation of the downstream globin genes. The main objectives of this work were to characterize the molecular lesions underlying eight unusual cases of α-thalassemia or Hb H disease, and to understand their origin and functional consequences. Deletions were detected by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140B HBA kit (MCR-Holland). Additionally, specifically designed synthetic MLPA probes, as well as Gap-PCR and Sanger sequencing were performed for fine deletion breakpoint mapping. We have found seven different deletions (ranging from 3.3 to ≈323 kb), four of them not previously described. The four largest deletions removed all the α-globin genes, whereas the other three deletions removed one or more of the distal regulatory elements keeping the globin genes structurally intact. In one case, only the MCS-R2 (also known as HS-40) was removed and replaced by a 39 nt DNA fragment possibly resulting from a complex rearrangement that introduces new pieces of DNA (probably from Chrs. 3 and 7) bridging the two deletion breakpoints. In the remaining case, no deletion was found and the patient revealed to be a very unusual case of acquired alpha-thalassemia-myelodysplastic syndrome. It is important to detect individuals with this type of uncommon deletions as there is a 25% risk of having a child with Hb Bart’s hydrops fetalis or Hb H disease if their partner is a carrier of an α0-thal or α+-thal allele, respectively. Moreover, further investigation is currently being developed on one of these natural mutants which is bringing new insights into the long-range regulation mechanism of the globin gene expression and to the pathophysiology of the α-thalassemia.N/