Conformation-selective rather than avidity-based binding to tumor associated antigen derived peptide-MHC enables targeting of WT1-pMHC low expressing cancer cells by anti-WT1-pMHC/CD3 T cell engagers

T cell engagers, a category of T cell-retargeting immunotherapy, are rapidly transforming clinical cancer care. However, the lack of tumor-specific targets poses a significant roadblock for broad adaptation of this therapeutic modality in many indications, often resulting in systemic on-target off-tumor toxicity. Though various tumor-derived intracellular mutations provide a massive pool of potential tumor-specific antigens, targeting them is extremely challenging, partly due to the low copy number of tumor associated antigen (TAA)-derived pMHC on tumor cell surface. Further, the interplay of binding geometry and format valency in relation to the capacity of a T cell engager to efficiently target low density cell-surface pMHC is not well understood. Using the Wilms’ tumor 1 (WT1) oncoprotein as a proof-of-principle TAA, combined with an array of IgG-like T cell engager modalities that differ in their anti-TAA valency and binding geometry, we show that the ability to induce an immunological synapse formation, resulting in potent killing of WT1 positive cancer cell lines is primarily dependent on the distinct geometrical conformations between the Fab arms of anti-WT1-HLA-A*02:01 and anti-CD3. The augmented avidity conferred by the binding of two anti-WT1-HLA-A*02:01 Fab arms has only minimal influence on cell killing potency. These findings demonstrate the need for careful examination of key design parameters for the development of next-generation T cell engagers targeting low density TAA-pMHCs on tumor cells

Unveiling macrophage diversity in myocardial ischemia-reperfusion injury: identification of a distinct lipid-associated macrophage subset

Background and objectiveMacrophages play a crucial and dichotomous role cardiac repair following myocardial ischemia-reperfusion, as they can both facilitate tissue healing and contribute to injury. This duality is intricately linked to environmental factors, and the identification of macrophage subtypes within the context of myocardial ischemia-reperfusion injury (MIRI) may offer insights for the development of more precise intervention strategies.MethodsSpecific marker genes were used to identify macrophage subtypes in GSE227088 (mouse single-cell RNA sequencing dataset). Genome Set Enrichment Analysis (GSEA) was further employed to validate the identified LAM subtypes. Trajectory analysis and single-cell regulatory network inference were executed using the R packages Monocle2 and SCENIC, respectively. The conservation of LAM was verified using human ischemic cardiomyopathy heart failure samples from the GSE145154 (human single-cell RNA sequencing dataset). Fluorescent homologous double-labeling experiments were performed to determine the spatial localization of LAM-tagged gene expression in the MIRI mouse model.ResultsIn this study, single-cell RNA sequencing (scRNA-seq) was employed to investigate the cellular landscape in ischemia-reperfusion injury (IRI). Macrophage subtypes, including a novel Lipid-Associated Macrophage (LAM) subtype characterized by high expression of Spp1, Trem2, and other genes, were identified. Enrichment and Progeny pathway analyses highlighted the distinctive functional role of the SPP1+ LAM subtype, particularly in lipid metabolism and the regulation of the MAPK pathway. Pseudotime analysis revealed the dynamic differentiation of macrophage subtypes during IRI, with the activation of pro-inflammatory pathways in specific clusters. Transcription factor analysis using SCENIC identified key regulators associated with macrophage differentiation. Furthermore, validation in human samples confirmed the presence of SPP1+ LAM. Co-staining experiments provided definitive evidence of LAM marker expression in the infarct zone. These findings shed light on the role of LAM in IRI and its potential as a therapeutic target.ConclusionIn conclusion, the study identifies SPP1+ LAM macrophages in ischemia-reperfusion injury and highlights their potential in cardiac remodeling

Relatório de estágio em farmácia comunitária

Relatório de estágio realizado no âmbito do Mestrado Integrado em Ciências Farmacêuticas, apresentado à Faculdade de Farmácia da Universidade de Coimbr

Advances in genetic engineering of marine algae

Algae are a component of bait sources for animal aquaculture, and they produce abundant valuable compounds for the chemical industry and human health. With today's fast growing demand for algae biofuels and the profitable market for cosmetics and pharmaceuticals made from algal natural products, the genetic engineering of marine algae has been attracting increasing attention as a crucial systemic technology to address the challenge of the biomass feedstock supply for sustainable industrial applications and to modify the metabolic pathway for the more efficient production of high-value products. Nevertheless, to date, only a few marine algae species can be genetically manipulated. In this article, an updated account of the research progress in marine algal genomics is presented along with methods for transformation. In addition, vector construction and gene selection strategies are reviewed. Meanwhile, a review on the progress of bioreactor technologies for marine algae culture is also revisited. (c) 2012 Elsevier Inc. All rights reserved.Algae are a component of bait sources for animal aquaculture, and they produce abundant valuable compounds for the chemical industry and human health. With today's fast growing demand for algae biofuels and the profitable market for cosmetics and pharmaceuticals made from algal natural products, the genetic engineering of marine algae has been attracting increasing attention as a crucial systemic technology to address the challenge of the biomass feedstock supply for sustainable industrial applications and to modify the metabolic pathway for the more efficient production of high-value products. Nevertheless, to date, only a few marine algae species can be genetically manipulated. In this article, an updated account of the research progress in marine algal genomics is presented along with methods for transformation. In addition, vector construction and gene selection strategies are reviewed. Meanwhile, a review on the progress of bioreactor technologies for marine algae culture is also revisited. (c) 2012 Elsevier Inc. All rights reserved

Analysis of Psychosomatic Disorders Perceptions and Healthcare Seeking Behavior Characteristics Based on Patient-physician Multi-party Interviews

Background Psychosomatic disorders present high prevalence and disease burden in the community. Currently, different disciplines of medical institutions possess unclear functions and inadequate process strategies for the diagnosis and treatment of psychosomatic disorders, resulting in differences in the perceptions of disease and behavioral characteristics of doctors and patients, which affects the management of disease in the community. Objective To understand and analyze the cognitive and behavioral characteristics as well as the influencing factors of psychosomatic disorders from multiple perspectives of doctors and patients, and then provide strategic directions to improve the diagnosis and management of psychosomatic patients in the community. Methods During the period from 2022-02-01 to 06-31, 5 internists from a general hospital (H1) in Yangpu District, 12 general practitioners from 2 community health centers (H2, H3) in Yangpu District, and 2 community health centers (H4, H5) in Jiading and Pudong New Districts, 2 healthcare professionals from a mental health center hospital in Yangpu District, and 10 patients and with psychosomatic disorders who had been treated in the above hospitals or their relatives. The interview was performed by WeChat, face-to-face as well as telephone and other methods, with the content including perceptions of psychosomatic disorders, healthcare seeking behaviors, disease attitudes, problems and suggestions, and lasted 15-30 min. The interview content was recorded and transcribed into text, coded, summarized to generate themes, and finally quoted from representative interviewees. Results (1) There was a high level of disease awareness among general practitioners and psychiatrists, a low level of awareness among internists, and a general lack of awareness among patients. (2) General practitioners and psychiatrists possessed more experience in the diagnosis and treatment of psychosomatic disorders, but lacking sufficient work and effective treatment results. (3) The attitudes of multiple doctors and patients were varied with an overall lack of positivity. General practitioners had the most positive attitudes toward the diagnosis and treatment of psychosomatic disorders. (4) Doctors of different disciplines faced difficulties, including large patient bases, insufficient mental health resources, lack of solid psychological perceptions, lack of standardized guidelines for diagnosis and treatment, and busy practices, etc. (5) Most patients had not received psychotherapy, but they didn't exclude it either, while there were problems such as distrust of general practitioners, fear of treatment side effects, and health seeking behavior affected by many factors. (6) Both doctors and patients offered constructive suggestions, which included strengthening publicity and policy guidance to the public, promoting the continuous development of psychosomatic medicine, improving doctors' identification of psychological disorders, opening joint clinic for chronic disease and psychology, improving the experience of medical treatment, setting up a comfortable treatment environment, and coordinating referrals, etc. Conclusion Barriers such as differences in perceptions and attitudes between doctors and patients, unequal medical resources, and irregularities in behavior exist between doctors and patients. In the community, it is necessary to strengthen the advantages and functions of general practitioners on the management of psychosomatic disorders; fully implement and utilize relevant resources; and improve the negative attitudes of both doctors and patients, so that patients can seek proper medical treatment and improve their mental health

Fatty Acid-Binding Protein 4 in Endometrial Epithelium Is Involved in Embryonic Implantation

Aims: To clarify the role of fatty acid-binding protein 4 (FABP4) of endometrial epithelial cell in the establishment and maintenance of pregnancy and the involvement in the pathogenesis of pregnancy loss. Methods: The expression of FABP4 and uterine receptive factor (LIF, Integrin-β3 and Claudin 4) was determined by Western blotting or quantitative PCR. FABP4 siRNA was used to silence FABP4 while FABP4 inhibitor was used to inhibit the function of FABP4 in endometrial epithelial cell. ICR mice were raised to evaluate the effect of FABP4 silence or inhibition on embryo implantation in vivo after FABP4 siRNA mixture or inhibitor was injected into uterus, and an embryonic adhesion system using trophoblast spheroids mimicking embryos was set up to assess the effect of FABP4 silence or inhibition on embryonic adhesion onto endometrial cell in vitro. Results: The expression of FABP4 mRNA was significantly decreased in the deciduas of women with pregnancy loss compared with that of women with normal pregnancy. FABP4 siRNA significantly reduced the number of embryos implanted and FABP4 expression in ICR mice. FABP4 inhibition also significantly decreased the number of embryos implanted. Either silence or inhibition of FABP4 in endometrial epithelial cell abolished the expression of uterine receptive factors induced by the combination of estrogen and progesterone-induced, and reduced the number of trophoblast spheroids adhered onto endometrial cell. Conclusions: FABP4 regulates embryo implantation via altering uterine receptivity and decreased expression of FABP4 in endometrium may be linked with pregnancy loss, indicating FABP4 has biological role in the establishment and maintenance of pregnancy and subsequently is involved in pathogenesis of pregnancy loss

Genetic transformation of marine cyanobacterium Synechococcus sp CC9311 (Cyanophyceae) by electroporation

Synechococcus sp. CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation (CA). A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA. The results show that Synechococcus sp. CC9311 cells were sensitive to four commonly used antibiotics: ampicillin, kanamycin, spectinomycin, and chloramphenicol. An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV, using a kanamycin resistance gene as selectable marker, was constructed by recombinant polymerase chain reaction. The plasmid was then transformed into Synechococcus sp. CC9311 via electroporation. High transformation efficiency was achieved at a field strength of 2 kV/cm. DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin. In addition, the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.Synechococcus sp. CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation (CA). A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA. The results show that Synechococcus sp. CC9311 cells were sensitive to four commonly used antibiotics: ampicillin, kanamycin, spectinomycin, and chloramphenicol. An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV, using a kanamycin resistance gene as selectable marker, was constructed by recombinant polymerase chain reaction. The plasmid was then transformed into Synechococcus sp. CC9311 via electroporation. High transformation efficiency was achieved at a field strength of 2 kV/cm. DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin. In addition, the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium

Biosynthesis of a stable allophycocyanin beta subunit in metabolically engineered Escherichia coli

Allophycocyanin (APC) is widely used as a fluorescent tag for fluorescence detection techniques. In this study, the alpha pcB gene from a thermophilic cyanobacterium strain was cloned and ligated into an expression vector to construct a pathway for the biosynthesis of an allophycocyanin beta subunit (holo-apcBT) in Escherichia coli. Isopropyl beta-D-1-thiogalactopyranoside induction successfully reconstituted holo-apcBT in E. coli. The recombinant holo-apcB showed spectroscopic properties similar to native APC. The stability and spectroscopic properties of this protein were then compared with another recombinant allophycocyanin beta subunit (holo-apcBM) whose apcB gene was cloned from mesophilic cyanobacterium. At high temperatures and during the course of storage, holo-apcBT was significantly more stable than holo-apcBM. In addition, holo-apcBT had an unexpectedly higher extinction coefficient and fluorescence quantum yield than holo-apcBM, suggesting that holo-apcBT would be a promising fluorescent tag and serve as a substitute for native APC. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.Allophycocyanin (APC) is widely used as a fluorescent tag for fluorescence detection techniques. In this study, the alpha pcB gene from a thermophilic cyanobacterium strain was cloned and ligated into an expression vector to construct a pathway for the biosynthesis of an allophycocyanin beta subunit (holo-apcBT) in Escherichia coli. Isopropyl beta-D-1-thiogalactopyranoside induction successfully reconstituted holo-apcBT in E. coli. The recombinant holo-apcB showed spectroscopic properties similar to native APC. The stability and spectroscopic properties of this protein were then compared with another recombinant allophycocyanin beta subunit (holo-apcBM) whose apcB gene was cloned from mesophilic cyanobacterium. At high temperatures and during the course of storage, holo-apcBT was significantly more stable than holo-apcBM. In addition, holo-apcBT had an unexpectedly higher extinction coefficient and fluorescence quantum yield than holo-apcBM, suggesting that holo-apcBT would be a promising fluorescent tag and serve as a substitute for native APC. (c) 2012, The Society for Biotechnology, Japan. All rights reserved