Target Proteins Research Articles

Activation of fibroblast growth factor 21 by Canagliflozin reduces high-fat diet associated obesity-related cardiac injury: a translational study from bench to bedside

Abstract Aims/hypothesis Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are indicated for the treatment of type 2 diabetes, and evidence shows that they may confer a cardioprotective effect to reduce the incidence of cardiovascular conditions in obese type 2 diabetes patients. The mechanisms underlying this effect remain unclear, but an SGLT2i, canagliflozin (CANA), has been reported to increase levels of fibroblast growth factor-21 (FGF21) and enhance activation of its downstream target proteins. We thus aimed to study whether the effect on FGF21 by CANA can reduce obesity-related cardiac injury. Methods 1) Animal study: Sixteen-week-old normal diet and high-fat diet male mice were treated with or without 30 mg/kg CANA once a day for 24 weeks, and were subsequently examined for body weight, blood glucose levels, adipose tissue changes, lipid accumulation in the liver, and cardiac function. 2) Cell study: An in vitro study that treated H9c2 cardiomyocyte cells cultured in a simulated high-lipid environment containing palmitic acid (PA) with FGF21 recombinant protein was also conducted to examine protein expression profiles. Furthermore, the effect of CANA on FGF21 expression. 3) Human clinical trial: A randomized double-blinded control trial (NCT 05764811) was performed to test effect of CANA on MRI liver fibrosis scores of obese type 2 diabetes patients (n = 40). Results 1) High-fat diet mice treated with CANA for 24 weeks had lower average body weight than untreated controls (34 g vs. 40 g, p < 0.001). In addition, CANA treatment stimulated FGF21 protein production in liver tissue and plasma at higher levels for both normal diet and high-fat diet mice, and reduced expression levels of proteins associated with fibrosis and apoptosis. 2) H9c2 cardiomyocyte culture studies suggested that a simulated high-lipid environment with PA caused higher expression of apoptotic proteins, but the addition of FGF21 recombinant protein significantly reduced apoptosis signalling. 3) MRI before and after 4 weeks of CANA treatment in obese type 2 diabetes patients showed a significant reduction in liver fibrosis scores for those receiving CANA treatment. Conclusion CANA treatment stimulated FGF21 protein expression and reduced fibrosis and apoptosis signalling protein expression. Moreover, reductions in liver fibrosis scores were observed in obese type 2 diabetes patients treated with CANA. Taken together, this suggests that CANA-induced FGF21 expression may exert a cardioprotective effect against fibrosis and apoptosis and can potentially reduce the risk of obesity-related cardiac injury.

Genistein inhibited endocytosis and fibrogenesis in keloid via CTGF signaling pathways

BackgroundThis study aimed to evaluate soy isoflavones' effect and potential use—specifically genistein—in treating human keloid fibroblast cells (KFs) and in a keloid tissue culture model.MethodsTo investigate the effects of genistein on keloid, a wound-healing assay was performed to detect cell migration. Flow cytometry was used to measure apoptosis. Western blotting and immunofluorescence staining were performed to detect the expression of target proteins. KF tissues were isolated, cultured, and divided into the control, silenced connective tissue growth factor (CTGF) proteins, and shNC (negative control) groups.ResultsGenistein suppressed cell proliferation and migration, triggering the cell cycle at the G2/M phase and increasing the expression of p53 dose-dependent in keloids. Genistein inhibited the expression of COL1A1, FN, and CTGF mRNA and protein. Knockdown CTGF reduced the migrated ability in KFs. Genistein also abated TGF-β1-induced keloid fibrosis through the endocytosis model. Separated and cultured the keloid patient’s tissues decreased the cell migration ability by genistein treatment and was time-dose dependent.ConclusionsThis study indicated that genistein-induced p53 undergoes cell cycle arrest via the CTGF pathway-inhibited keloid cultured cells, and genistein suppressed the primary keloid cell migration, suggesting that our research provides a new strategy for developing drugs for treating keloids.

Targeted protein degradation via cellular trafficking of nanoparticles.

Strategies that selectively bind proteins of interest and target them to the intracellular protein recycling machinery for targeted protein degradation have recently emerged as powerful tools for undruggable targets in biomedical research and the pharmaceutical industry. However, targeting any new protein of interest with current degradation tools requires a laborious case-by-case design for different diseases and cell types, especially for extracellular targets. Here we observe that nanoparticles can mediate specific receptor-independent internalization of a bound protein and further develop a general strategy for degradation of extracellular proteins of interest by making full use of clinically approved components. This extremely flexible strategy aids in targeted protein degradation tool development and provides knowledge for targeted drug therapies and nanomedicine design.

Design and Synthesis of Ester Linked 1,2,3‐Triazole Quinoline Derivatives with Pancreatic β Cells Protective Activity against Oxidative Stress and Endoplasmic Reticulum Stress for Better Management of Type II Diabetes Mellitus

AbstractUnresolved endoplasmic reticulum (ER) stress and oxidative stress lead to the pathogenesis of type II diabetes mellitus (DM). Connecting links between the ER stress and oxidative stress pathways might play an important role in treatment and pathogenesis of diabetes. Here, we used network pharmacology approach to find the target proteins linking the ER stress and oxidative stress pathways. CHOP emerged as a hub protein from our results and was targeted by 16 newly designed and synthesized ester linked 1,2,3‐triazole quinoline derivatives by molecular docking. Drug likeliness prediction studies revealed all compounds as good druggable candidates. Compounds 5b (R = H, R1 = F), 5j (R = CH3, R1 = F), 5m (R = OCH3, R1 = H), and 5n (R = OCH3, R1 = F) were found to have considerable binding energies and active site amino acid interactions with CHOP. We further showed that compounds 5b, 5m, and 5n exhibited low toxicity with more than 50% cell survival and low IC50 values. These were therefore subjected to evaluate their effects on ER stress and oxidative stress. Finally, compound 5n (R = OCH3, R1 = F) was found to alleviate ER stress and is also a good antioxidant. Identification of such targets and compounds effective in improving ER stress and oxidative stress may provide new insights in the development of therapies and management of T2DM.

High-Level Production of a Recombinant Protein in Nicotiana benthamiana Leaves Through Transient Expression Using a Double Terminator

Various bio-based recombinant proteins have been produced for industrial, medical, and research purposes. Plants are potential platforms for recombinant protein production because of several advantages. Therefore, establishing a system with high target gene expression to compensate for the low protein yield of plant systems is crucial. In particular, selecting and combining strong terminators is essential because the expression of target genes can be substantially enhanced. Here, we aimed to quantify the enhancement in the fluorescence intensity of the turbo green fluorescence protein (tGFP) caused by the best double-terminator combinations compared to that of the control vector using agroinfiltration in Nicotiana benthamiana leaves. tGFP fluorescence increased by 4.1-fold in leaf samples infiltrated with a vector containing a double terminator and markedly increased by a maximum of 23.7-fold when co-infiltrated with the geminiviral vector and P19 compared to that in constructs containing an octopine synthase terminator. Polyadenylation site analysis in leaf tissues expressing single or dual terminators showed that the first terminator influenced the polyadenylation site determination of the second terminator, resulting in different polyadenylation sites compared with when the terminator is located first. The combination of the high-expression terminators and geminiviral vectors can increase the production of target proteins.

Selective bioorthogonal probe for N-glycan hybrid structures.

Metabolic incorporation of chemically tagged monosaccharides is a facile means of tagging cellular glycoproteins and glycolipids. However, since the monosaccharide precursors are often shared by several pathways, selectivity has been difficult to attain. For example, N-linked glycosylation is a chemically complex and ubiquitous posttranslational modification, with three distinct classes of GlcNAc-containing N-glycan structures: oligomannose, hybrid and complex. Here we describe the synthesis of 1,3-Pr2-6-OTs GlcNAlk (MM-JH-1) as a next-generation metabolic chemical reporter for the selective labeling of hybrid N-glycan structures. We first developed a general strategy for defining the selectivity of labeling with chemically tagged monosaccharides. We then applied this approach to establish that MM-JH-1 is selectively incorporated into hybrid N-glycans. Using this metabolic chemical reporter as a detection tool, we performed imaging and fractionation to define features of the intracellular localization and trafficking of target proteins bearing hybrid N-glycan structures.

Proteogenomic analysis in population biobanks identifies new therapeutic targets across heart failure subtypes

Abstract Background Heart failure (HF) represents a multifactorial clinical syndrome with differential response to existing therapies across its subtypes. Proteogenomic analysis of genetic variants associated with changes in circulating protein level and risk of heart failure subtypes provides an opportunity for a systematic identification of proteins with subtype-specific causal effects as novel therapeutic targets. Purpose To identify proteins as potential therapeutic targets for specific heart failure subtypes by leveraging proteomics and genomics data in population biobanks. Methods We evaluated the causal effects of 2,773 proteins on 5 HF-related outcomes, including overall HF, non-ischaemic HF (ni-HF), ni-HF with reduced / preserved ejection fraction (ni-HFrEF / ni-HFpEF), and dilated cardiomyopathy (DCM). For each protein-phenotype pair, we performed a two-sample Mendelian randomisation (MR) analysis using protein-specific genetic association estimates derived from protein quantitative trait locus (pQTL) studies in 114,145 individuals from deCODE, UK Biobank, Fenland study, and SCALLOP consortium and genome-wide association studies (GWAS) of HF outcomes in 145,795 individuals by HERMES Consortium. We further prioritised candidate protein targets based on replication of results across datasets, model parameters, assays, and colocalization of causal genetic variants. Finally, we profiled the causal effects of identified proteins on 32 related cardiac traits and risk factors to explore potential mechanisms of actions and on-target side effects. Results We identified 493 proteins associated with at least one HF outcome at a false discovery rate (FDR) < 1% in the primary MR analysis. We further prioritised 44 proteins with concordant effect direction across datasets, model parameters, and assays or colocalization in at least one dataset. All 44 identified proteins showed cross association with at least one of 35 tested traits other than HF or cardiomyopathies at FDR <1%, including coronary artery disease (18 proteins), body mass index (11 proteins), type 2 diabetes (6 proteins), systolic / diastolic blood pressure (13 proteins), and chronic kidney disease (9 proteins). Conclusion We characterised the causal associations of 2,773 circulating proteins across HF-related outcomes and prioritised 44 proteins by integrating proteomics and genomics data in population biobanks. These findings extend our understanding of the molecular mechanism underlying different forms of HF and may inform the development of new therapeutic strategies.Study design

Oleanolic acid purified from the stem bark of Olax subscorpioidea Oliv. inhibits the function and catalysis of human 17β-hydroxysteroid dehydrogenase 1

Cancer is a leading cause of global death. Medicinal plants have gained increasing attention in cancer drug discovery. In this study, the stem bark extract of Olax subscorpioidea, which is used in ethnomedicine to treat cancer, was subjected to phytochemical investigation leading to the isolation of oleanolic acid (OA). The structure was elucidated by 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopic (NMR) data, and by comparing its data with previously reported data. Molecular docking was used to investigate the interactions of OA with nine selected cancer-related protein targets. OA docked well with human 17β-hydroxysteroid dehydrogenase type-1 (17βHSD1), caspase-3, and epidermal growth factor receptor tyrosine kinase (binding affinities: −9.8, −9.3, and −9.1 kcal/mol, respectively). OA is a triterpenoid compound with structural similarity to steroids. This similarity with the substrates of 17βHSD1 gives the inhibitor candidate an excellent opportunity to bind to 17βHSD1. The structural and functional dynamics of OA-17βHSD1 were investigated by molecular dynamics simulations at 240 ns. Molecular mechanics/Poisson-Boltzmann surface area (MMPBSA) studies showed that OA had a binding free energy that is comparable with that of vincristine (–52.76, and −63.56 kcal/mol, respectively). The average C-α root mean square of deviation (RMSD) value of OA (1.69 Å) compared with the unbound protein (2.01 Å) indicated its high stability at the protein’s active site. The binding energy and stability at the active site of 17βHSD1 recorded in this study indicate that OA exhibited profound inhibitory potential. OA could be a good scaffold for developing new anti-breast cancer drugs.

Exploring the interaction of graphene-based nanomaterials with atherosclerosis-related protein targets: insights from molecular docking and dynamics simulations

Exploring the interaction of graphene-based nanomaterials with atherosclerosis-related protein targets: insights from molecular docking and dynamics simulations

STRAP Knockdown Inhibits Migration and Growth of Non-Small Cell Lung Cancer.

Serine-threonine kinase receptor-associated protein (STRAP) regulates cell proliferation and apoptosis by binding to many target proteins and plays an important regulatory role in tumor development. We studied the effects of STRAP on non-small cell lung cancer (NSCLC) in vivo and in vitro in order to elucidate possible mechanisms underlying the regulatory effects of this protein. The levels of STRAP in NSCLC tissues and cells were determined by quantitative reverse transcription PCR, immunohistochemical staining, and Western blotting. In in vitro experiments, A549 and HCC827 cells were transfected with small interfering RNA (siRNA) to knockdown STRAP (si-STRAP) or with negative control sequence; cell migration and invasion were detected by scratch and Transwell assays, respectively. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), caspase-3, and caspase-9 were determined by Western blotting. In addition, we analyzed changes of tumor volume in a nude mouse NSCLC model. STRAP was highly expressed in NSCLC tissues and cells, but its expression was significantly suppressed in A549 and HCC827 cells transfected with si-STRAP. STRAP knockdown resulted in a significant inhibition of migration and invasion of A549 and HCC827 cells. It also significantly reduced the expression of XIAP and elevated expression of caspase-3 and caspase-9. In nude mice with tumor originated from transplanted A549 cells, inhibition of STRAP expression retarded the tumor growth. Overall, these findings indicate that STRAP is overexpressed in NSCLC, while knockdown of STRAP gene inhibits the growth of NSCLC.

Novel Perspectives in the Management of Colorectal Cancer: Mechanistic Investigations Into the Reversal of Drug Resistance via Active Constituents Derived From Herbal Medicine.

The high incidence and mortality rate of colorectal cancer have become a significant global health burden. Chemotherapy has been the traditional treatment for colorectal cancer and has demonstrated promising antitumor effects, leading to significant improvements in patient survival. However, the development of chemoresistance poses a major challenge during chemotherapy in colorectal cancer, significantly impeding treatment efficacy and affecting patient prognosis. Despite the development of a variety of novel anticolorectal cancer chemotherapy agents, their effectiveness and side effects vary, possibly due to the complex mechanisms of resistance in colorectal cancer. Abnormal drug metabolism or protein targets are the most direct causes of resistance. Further studies have revealed that these resistance mechanisms involve biochemical processes such as altered protein expression, autophagy, and epithelial-mesenchymal transitions. Herbal active ingredients offer an alternative treatment option and have shown promise in reversing colorectal cancer drug resistance. This paper aims to summarize the role of various biochemical processes and key protein targets in the occurrence and maintenance of resistance mechanisms in colorectal cancer. Additionally, it elaborates on the mechanisms of action of herbal active ingredients in reversing colorectal cancer drug resistance. The article also discusses the limitations and opportunities in developing novel anticolorectal cancer drugs based on herbal medicine.

Microvessel isolation protocol for RNA visualization and profiling

Disruptions in pericyte and endothelial cell communication can compromise the integrity of the blood-brain barrier (BBB), leading to neurovascular dysfunction and the development of neurological disorders. However, the evaluation of microvessel RNAs has been limited to tissue homogenates, with spatial visualization only available for protein targets. The aim of the present study is the development of an innovative microvessel isolation technique that is RNA-friendly for the purpose of coupling with in situ hybridization RNAscope analysis. RNA-friendly microvessel isolation combined with RNAscope analysis enables the visualization of cell-specific RNA within the spatial and histological context of the BBB. Using this approach, we have gained valuable insights into the structural and functional differences associated with the microvessels of 5xFAD mice, a mouse model of Alzheimer’s disease (AD). RNAscope analysis revealed a decrease in pericytes from microvessels isolated from 5xFAD mice in comparison to wild-type mice. Additionally, the microvessels of 5xFAD mice exhibited an increase in TYRO protein tyrosine kinase binding protein (TYROBP) mRNA expression. These findings significantly advance our understanding of neurovascular interactions and hold great promise for guiding the development of targeted therapeutic interventions. This innovative approach enables visualization of cell RNA while preserving the spatial and histological context of the BBB, shedding light on the mechanisms underlying neurovascular unit communication.

Essential Oils from the Leaves and Stem Barks of Pluchea Indica (L.) Less.: Chemical Analysis, Cytotoxicity, Anti-inflammation, Antimicrobial Activity, Molecular Docking, and ADMET Profiling.

Pluchea indica (L.) Less. is a medicinal plant native to Asia. Traditionally, it is known for numerous traditional uses, such as treatments for fever, cough, and digestive issues. The present investigation aims to determine the chemical compositions of essential oils from its fresh leaves and stem barks. By using hydro-distillation and the GC-FID/MS (gas chromatography-flame ionization detection/mass spectrometry) analysis, the studied samples were dominated by sesquiterpene hydrocarbons (76.8-82.2 %) and their oxygenated derivatives (8.4-19.0 %). β-Selinene (42.0-43.5 %) and silphinene (21.1-22.9 %) were the main compounds. Significantly, the stem bark essential oil strongly monitored the growth of four cancer cell lines K562, HeLa, HepG2, and MCF-7 with IC50 values of 2.89-7.34 μg/mL. Both studied samples showed strong anti-inflammatory activity against NO (nitric oxide) production with IC50 values of 21.81-23.18 μg/mL. The studied sample also exhibited antimicrobial activity at different levels. The molecular docking study revealed that β-selinene exhibited the strongest binding affinity for all four cancer-related protein targets: epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Abelson tyrosine-protein kinase 1 (ABL1), and phosphatidylinositol 3-kinase (PI3 K-α). The ADMET profiles of the major compounds were also predicted to provide insights for further research considerations.

Cell‐Permeable Nicotinamide Adenine Dinucleotides for Exploration of Cellular Protein ADP‐Ribosylation

AbstractPosttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene‐encoded variations. One intriguing PTM is ADP‐ribosylation, which utilizes nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell‐permeable NAD+ analogs and demonstrate their utility for investigating cellular ADP‐ribosylation. Using a desthiobiotin‐labelled analog for affinity enrichment of proteins that are ADP‐ribosylated in living cells under oxidative stress, we identified protein targets associated with host‐virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP‐ribosylome studies.

Integrating network pharmacology, molecular docking, and experimental validation to reveal the mechanism of Radix Rehmanniae in psoriasis.

Radix Rehmanniae (RR) plays an important role in treating psoriasis. However, the active compounds of RR and potential mechanisms are unclear. The current study was designed to investigate the potential active ingredients, targets, and mechanisms of RR in treating psoriasis through network pharmacology, molecular docking, and vitro experiments. Initially, the TCMSP database and literature retrieval were used to access the active ingredients of RR. The psoriasis target proteins were obtained from Therapeutic Target Database, OMIM, GeneCards, and DrugBank databases. The target proteins were then converted into target genes using Uniprot. Secondly, overlapping genes were obtained through Venn online tool. Then, protein-protein interactions network diagram is finished by STRING database. Next, Cytoscape software was used to acquire the top 10 hub proteins; gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis were then used to predict possible mechanisms. Afterwards, molecular docking validation of the active ingredients with the main targets was performed by AutoDock software. Finally, lipopolysaccharides induced RAW264.7, to assess the effects and molecular mechanisms by MTT, RT-qPCR, and Western blot assays. Overall, there are 20 effective compounds and 33 targets involved in biological processes including apoptosis, intracellular signaling, vasodilation, and mitogen-activated protein kinase (MAPK) signaling cascade. The docking results showed strong binding capacity between the active ingredients and targets. We verified aucubin as the key active ingredient, tumor necrosis factor α, and IL6 as the core targets, and focused on the p38MAPK protein pathway. Cellular experiments showed that aucubin down-regulated the phosphorylated p38MAP protein and reduced the expression of tumor necrosis factor α mRNA, IL6 mRNA, and IL1βmRNA. In summary, RR is featured with multicomponent, multi-target, and multi-pathway in treating psoriasis; the preliminary mechanism may be associated with the down-regulation of p38MAPK phosphorylation and curbing the expression of inflammatory factor by aucubin. This paper provides the scientific basis for Traditional Chinese medicine treating psoriasis.

Aligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein

Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N6-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli. We used the orthogonal pyrrolysyl-tRNA synthetase/suppressor tRNACUAPyl pair from Methanosarcina mazei to incorporate Azk site-specifically. We characterized Azk uptake and Fab production at bench-scale under different fermentation conditions, varying timing and mode of Azk addition, Azk-to-cell ratio and induction time. Our results indicate that Azk uptake is comparatively efficient in the batch phase. We discovered that the time between Azk uptake and inducing its incorporation into the Fab must be kept short, which suggests that intracellular Azk is consumed and/or degraded. The results obtained in this study are an important step towards the development of efficient production methods for Azk-incorporated proteins in E. coli. The developed process is scalable and provides excellent yields of 2.95 mg functionalized Fab per g CDM, which corresponds to 80% of yield obtained with the wild type Fab. We also identified the cellular uptake of Azk being dependent on the physiological state of the cell as a potential bottleneck in production.

Effect of acupuncture on AMPK/ULK1 signaling pathway of hippocampus in chronic stress-induced depression rats

To observe the effect of manual acupuncture stimulation on changes of behavior and hippocampal signaling pathways of AMP-activated kinase (AMPK)/UNC-51-like kinase 1 (ULK1) in rats with chronic unpredictable mild stress (CUMS)-induced depression, so as to explore its molecular mechanism underlying improvement of depression. Thirty-two male SD rats were randomly divided into normal control, model, acupuncture and medication (fluoxetine) groups, with 8 rats in each group. The depression model was established by using CUMS (fasting, water depredation, restraint, swimming in cold water, crowding, stroboscope stimulation, and tail clipping, each of which was used once a week) for 6 weeks. Manual acupuncture stimulation was applied to "Baihui"(GV20) and "Yintang"(EX-HN3) for 10 min, once daily, 6 times a week for 6 weeks before every-day modeling. Rats of the medication group received gavage of fluoxetine (10 mg/kg) once a day for 6 weeks before every-day modeling. The percentage of sucrose preference, and the percentages of open arm times and open arm time in the elevated plus maze experiments were detected. The expression levels of AMPK, phosphorylated (p)-AMPK, ULK1, p-ULK1, mammalian target of rapamycin (mTOR) and p-mTOR proteins in the hippocampus tissue were detected by Western blot, and the expression of autophagy effecting protein (Beclin-1) and the selective autophagy receptors (p62) mRNA in the hippocampal tissue was detected using real-time fluorescence quantitative PCR, respectively. Compared with the normal group, the percentage of sucrose preference, percentage of times of entering the open arm and percentage of open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1 /ULK1 ratio of proteins, and Beclin-1 mRNA were significantly decreased (P<0.01, P<0.05), and the expression levels of p-mTOR /mTOR ratio and p62 mRNA were significantly increased (P<0.01, P<0.05) in the model group. In comparison with the model group, the percentage of sucrose preference, percentage of open arm times and open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1/ULK1 ratio of proteins, and Beclin-1 mRNA were significantly increased in both acupuncture and medication groups (P<0.01, P<0.05), while the expression levels of p-mTOR /mTOR ratio of proteins, and p62 mRNA were markedly decreased only in the medication group (P<0.05). Acupuncture treatment can alleviate depression-like behavior in CUMS rats, which may be related to its functions in up-regulating AMPK/ULK1 signaling pathway to induce cellular autophagy in the hippocampus.

Antibody blockade of the PSGL-1 immune checkpoint enhances T-cell responses to B-cell lymphoma.

Despite advancements in cancer immunotherapy, most lymphomas remain unresponsive to checkpoint inhibitors. P-selectin glycoprotein ligand-1 (PSGL-1), recently identified as a promoter of T-cell exhaustion in murine melanoma models, has emerged as a novel immune checkpoint protein and promising immunotherapeutic target. In this study, we investigated the potential of PSGL-1 antibody targeting in B-cell lymphoma. Using allogeneic co-culture systems, we demonstrated that targeted antibody interventions against human PSGL-1 enhanced T-cell activation and effector cytokine production in response to lymphoma cells. Moreover, in vitro treatment of primary lymphoma cell suspensions with PSGL-1 antibody resulted in increased activation of autologous lymphoma-infiltrating T cells. Using the A20 syngeneic B-cell lymphoma mouse model, we found that PSGL-1 antibody treatment significantly slowed tumor development and reduced the endpoint tumor burden. This antitumoral effect was accompanied by augmented tumor infiltration of CD4+ and CD8+ T cells and reduced infiltration of regulatory T cells. Finally, anti-PSGL-1 administration enhanced the expansion of CAR T cells previously transferred to mice bearing the aggressive Eμ-Myc lymphoma cells and improved disease control. These results demonstrate that PSGL-1 antibody blockade bolsters T-cell activity against B-cell lymphoma, suggesting a potential novel immunotherapeutic approach for treating these malignancies.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

ABSTRACTChlamydomonas incerta, a genetically close relative of the model green algaChlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship betweenC. incertaandC. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly withC. reinhardtii,C. incertaexhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed thatC. incertaproduced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared toC. reinhardtii. For xylanase expression and secretion, bothC. incertaandC. reinhardtiishowed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally,C. incertashowed comparable PHL7 activity levels toC. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids betweenC. incertaandC. reinhardtiias a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasizeC. incerta’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.GRAPHICAL ABSTRACT

Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins.

Complex integral membrane proteins, which are embedded in the cell surface lipid bilayer by multiple transmembrane spanning polypeptides, encompass families of proteins that are important target classes for drug discovery. These protein families include G protein-coupled receptors, ion channels, transporters, enzymes, and adhesion molecules. The high specificity of monoclonal antibodies and the ability to engineer their properties offers a significant opportunity to selectively bind these target proteins, allowing direct modulation of pharmacology or enabling other mechanisms of action such as cell killing. Isolation of antibodies that bind these types of membrane proteins and exhibit the desired pharmacological function has, however, remained challenging due to technical issues in preparing membrane protein antigens suitable for enabling and driving antibody drug discovery strategies. In this article, we review progress and emerging themes in defining discovery strategies for a generation of antibodies that target these complex membrane protein antigens. We also comment on how this field may develop with the emerging implementation of computational techniques, artificial intelligence, and machine learning.