Specific Proteins Research Articles

Localizing Protein Expression of Transporters in Epithelial Cell Membranes of Mosquitoes.

Identifying and localizing ion transport proteins in epithelia is important for understanding how these tissues can regulate salt and water balance in animals. Mosquitoes face distinct challenges regarding salt and water balance as larvae live in water of varying ionic composition while adult female mosquitoes must deal with periodic large blood meals. This protocol will explain how to localize ion transporters in epithelia of mosquitoes. Antibodies raised against specific proteins can be used to carry out immunohistochemistry to tag and visualize native proteins in tissues and cells. This technique may be used for whole tissues and organs or can also be used on histological thin sections of fixed tissue. This protocol will detail the use of immunohistochemistry to localize membrane proteins in mosquito organs.

Cold-shock proteins accumulate in centrosomes and their expression and primary cilium morphology are regulated by hypothermia and shear stress.

Primary cilia act as cellular sensors for multiple extracellular stimuli and regulate many intracellular signaling pathways in response. Here we investigate whether the cold-shock proteins (CSPs), CIRP and RBM3, are present in the primary cilia and the physiological consequences of such a relationship. R28, an immortalized retinal precursor cell line, was stained with antibodies against CIRP, RBM3, and ciliary markers. Both CSPs were found in intimate contact with the basal body of the cilium during all stages of the cell cycle, including migrating with the centrosome during mitosis. In addition, the morphological and physiological manifestations of exposing the cells to hypothermia and shear stress were investigated. Exposure to moderately cold (32°C) temperatures, the hypothermia mimetic small molecule zr17-2, or to shear stress resulted in a significant reduction in the number and length of primary cilia. In addition, shear stress induced expression of CIRP and RBM3 in a complex pattern depending on the specific protein, flow intensity, and type of flow (laminar versus oscillatory). Flow-mediated CSP overexpression was detected by qRT-PCR and confirmed by Western blot, at least for CIRP. Furthermore, analysis of public RNA Seq databases on flow experiments confirmed an increase of CIRP and RBM3 expression following exposure to shear stress in renal cell lines. In conclusion, we found that CSPs are integral components of the centrosome and that they participate in cold and shear stress sensing.

Beyond the Dusty Fog: Local Eye Drop Therapy and Potentially New Treatment Alternatives in Pseudoexfoliative Glaucoma.

Pseudoexfoliative glaucoma (PEG) is a type of secondary open-angle glaucoma characterized by the accumulation of whitish-gray material on the trabecular meshwork and lens, leading to an increase in intraocular pressure (IOP) and optic nerve damage. Local eye drop therapy is one of the first-line treatments for PEG, which include prostaglandin analogues, beta-blockers, and alpha-adrenergic agonists to lower IOP. New treatments beyond conventional techniques, however, are constantly being developed. One potential treatment proposed for PEG is based on magnetic phage display, which involves using magnetic nanoparticles conjugated to specific peptides or proteins selected using phage display techniques to remove aggregates in the anterior chamber of the eye or inflammatory cells and cytokines that contribute to PEG pathogenesis. Other potential treatments include microRNAs (miRNAs) that are involved in the regulation of gene expression at the post-transcription stages. Gene therapies, nanotechnology, immunotherapy and methods based on stem cells can also be potentially used to target and treat specific tissues and cells responsible for regulating IOP. In addition, photobiomodulation therapy (PBMT), a non-invasive procedure that utilizes low-level laser therapy to improve cellular function and promote tissue repair, can prove an interesting alternative in treating PEG. The aim of our mini-review is to provide a brief overview of these innovative methods that appear to offer potentially promising treatment options for PEG.

Geometry-Based Molecular Generation With Deep Constrained Variational Autoencoder.

Finding target molecules with specific chemical properties plays a decisive role in drug development. We proposed GEOM-CVAE, a constrained variational autoencoder based on geometric representation for molecular generation with specific properties, which is protein-context-dependent. In terms of machine learning, it includes continuous feature embedding encoder and molecular generation decoder. Our key contribution is to propose an efficient geometric embedding method, including the spatial structure representations of drug molecule (converting the 3-D coordinates into image) and the geometric graph representations of protein target (modeling the protein surface as a mesh). The 3-D geometric information is vital to successful molecular generation, which is different from previous molecular generative methods based on 1-D or 2-D. Our model framework generates specific molecules in two phases, by first generating special image with molecular 3-D information to learn latent representations and generating molecules with constrained condition based on geometric graph convolution for specific protein and then inputting the generated structural molecules into a parser network for obtaining Simplified Molecular Input Line Entry System (SMILES) strings. Our model achieves competitive performance that implies its potential effectiveness to enable the exploration of the vast chemical space for drug discovery.

Targeted Degradation of Cell-Surface Proteins via Chaperone-Mediated Autophagy by Using Peptide-Conjugated Antibodies.

Cell-surface proteins are important drug targets but historically have posed big challenges for the complete elimination of their functions. Herein, we report antibody-peptide conjugates (Ab-CMAs) in which a peptide targeting chaperone-mediated autophagy (CMA) was conjugated with commercially available monoclonal antibodies for specific cell-surface protein degradation by taking advantage of lysosomal degradation pathways. Unique features of Ab-CMAs, including cell-surface receptor- and E3 ligase-independent degradation, feasibility towards different cell-surface proteins (e.g., epidermal growth factor receptor (EGFR), programmed cell death ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)) by a simple change of the antibody, and successful tumor inhibition in vivo, make them attractive protein degraders for biomedical research and therapeutic applications. As the first example employing CMA to degrade proteins from the outside in, our findings may also shed new light on CMA, a degradation pathway typically targeting cytosolic proteins.

Integrating the analysis of human biopsies using post-translational modifications proteomics.

Proteome diversities and their biological functions are significantly amplified by post-translational modifications (PTMs) of proteins. Shotgun proteomics, which does not typically survey PTMs, provides an incomplete picture of the complexity of human biopsies in health and disease. Recent advances in mass spectrometry-based proteomic techniques that enrich and study PTMs are helping to uncover molecular detail from the cellular level to system-wide functions, including how the microbiome impacts human diseases. Protein heterogeneity and disease complexity are challenging factors that make it difficult to characterize and treat disease. The search for clinical biomarkers to characterize disease mechanisms and complexity related to patient diagnoses and treatment has proven challenging. Knowledge of PTMs is fundamentally lacking. Characterization of complex human samples that clarify the role of PTMs and the microbiome in human diseases will result in new discoveries. This review highlights the key role of proteomic techniques used to characterize unknown biological functions of PTMs derived from complex human biopsies. Through the integration of diverse methods used to profile PTMs, this review explores the genetic regulation of proteoforms, cells of origin expressing specific proteins, and several bioactive PTMs and their subsequent analyses by liquid chromatography and tandem mass spectrometry.

Abstract 3542: The Chemical Probes Portal: An essential resource providing expert advice on chemical tools for cancer research

Abstract Background: The Chemical Probes Portal (Portal) serves as a vital resource in the realm of cancer research and biomedical science generally - offering expert guidance on high-quality chemical tools, usually protein inhibitors, crucial for studying physiological and pathological processes and validating therapeutic targets. Researchers often grapple with selecting suitable chemical probes, inadvertently utilizing tools ill-suited for their purpose, and not following best practice, thereby contaminating the scientific literature and wasting valuable time and resources. To address this challenge, the Portal (https://www.chemicalprobes.org) was established as a non-profit, independent online platform, meticulously curating, annotating, and scoring the quality of chemical probes and providing associated information and advice. Results: The Portal stands apart as the sole expert-curated public resource evaluating the quality and utility of small-molecule chemical tool compounds for use in cancer research and wider biomedical research. More than 250 experts, comprising scientific leaders in chemical biology, medicinal chemistry, pharmacology, and related fields, contribute their experience and insights to assess featured chemical probes. Since 2021, a progressively updated and enhanced version of the Portal has been operational, housing over 1000 compounds targeting 500+ proteins across diverse families, now including PROTACs and molecular glues. We also flag 254 ‘Unsuitables’, which are compounds that should not be used to interrogate a specific protein target. Additionally, the Portal now offers informative pages, providing advice delineating guidelines for selecting well-validated compounds, including those suitable for rodent studies, necessitating specific pharmacokinetic and pharmacodynamic properties. Best practice use requires employing high-quality chemical probes from more than one distinct chemical class that target the same protein, incorporating an inactive control compound, and following the recommended concentration range - all aimed at mitigating off-target risks. Conclusion: The enhanced Chemical Probes Portal provides a vital platform for researchers to navigate and evaluate chemical probes, easily and effectively. It employs a rigorous expert review to ensure the quality of the probes listed. The resource not only aids high-quality probe selection but also provides advice through an Information Centre, offering guidelines, FAQs, and additional information on probe criteria and use. The continuous expansion and outreach efforts aim to make the Portal an indispensable tool for cancer researchers and the broader biomedical research community. Researchers are encouraged to participate in the Portal's development and contribute to the robustness of chemical probe use in scientific endeavors. Citation Format: Domenico Sanfelice, Albert Antolin, Alisa Crisp, Yi Chen, Paul Brennan, Susanne Müller, Bissan Al-Lazikani, Aled Edwards, Paul Workman. The Chemical Probes Portal: An essential resource providing expert advice on chemical tools for cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3542.

An anti-infection and biodegradable TFRD-loaded porous scaffold promotes bone regeneration in segmental bone defects -experimental studies.

Addressing segmental bone defects remains a complex task in orthopedics, and recent advancements have led to the development of novel drugs to enhance the bone regeneration. However, long-term oral administration can lead to malnutrition and poor patient compliance. Scaffolds loaded with medication are extensively employed to facilitate the restoration of bone defects. Inspired by the local use of total flavonoids of Rhizoma Drynariae (TFRD) in the treatment of fracture, a novel 3D-printed HA/CMCS/PDA/TFRD scaffold with anti-infection, biodegradable and induced angiogenesis was designed, and to explore its preclinical value in segmental bone defect of tibia. The scaffold exhibited good degradation and drug release performance. In vitro, the scaffold extract promoted osteogenesis by enhancing bone-related gene/protein expression and mineral deposition in BMSCs. It also stimulated endothelial cell migration and promoted angiogenesis through the upregulation of specific genes and proteins associated with cell migration and tube formation. This may be attributed to the activation of the PI3k/AKT/HIF-1α pathway, facilitating the processes of osteogenesis and angiogenesis. Furthermore, the HA/CMCS/PDA/TFRD scaffold was demonstrated to alleviate infection, enhance angiogenesis, promote bone regeneration, and increase the maximum failure force of new formed bone in a rat model of segmental bone defects. Porous scaffolds loaded with TFRD can reduce infection, be biodegradable, and induce angiogenesis, presenting a novel approach for addressing tibial segmental bone defects.

Retinol-binding protein 4 (RBP4) circulating levels and gestational diabetes mellitus: a systematic review and meta-analysis.

Gestational diabetes mellitus (GDM) is a prevalent condition where diabetes is diagnosed during pregnancy, affecting both maternal and fetal outcomes. Retinol-binding protein 4 (RBP4) is a circulating adipokine which belongs to the lipocalin family and acts as a specific carrier protein that delivers retinol (vitamin A) from the liver to the peripheral tissues. Growing data indicate that circulating RBP4 levels may positively correlate with GDM. Thus, this systematic review and meta-analysis aimed to investigate the potential relationship between circulating RBP4 levels and GDM when measured at various stages of pregnancy. MEDLINE, CINAHL, EMCARE, EMBASE, Scopus, and Web of Science databases were searched to identify studies comparing pregnant women with and without GDM, whose circulating RBP4 levels were measured in at least one pregnancy trimester. Findings were reported using standardized mean difference (SMD) and random-effects models were used to account for variability among studies. Furthermore, the risk of bias was assessed using the RoBANS tool. Out of the 34 studies identified, 32 were included in the meta-analysis (seven with circulating RBP4 levels measured in the first trimester, 19 at 24-28 weeks, and 14 at >28 weeks of pregnancy). RBP4 levels were statistically higher in the GDM group than in controls when measured during all these pregnancy stages, with the noted RBP4 SMD being 0.322 in the first trimester (95% CI: 0.126-0.517; p < 0.001; 946 GDM cases vs. 1701 non-GDM controls); 0.628 at 24-28 weeks of gestation (95% CI: 0.290-0.966; p < 0.001; 1776 GDM cases vs. 1942 controls); and 0.875 at >28 weeks of gestation (95% CI: 0.252-1.498; p = 0.006; 870 GDM cases vs. 1942 non-GDM controls). Significant study heterogeneity was noted for all three pregnancy timepoints. The present findings indicate consistently higher circulating RBP4 levels in GDM cases compared to non-GDM controls, suggesting the potential relevance of RBP4 as a biomarker for GDM. However, the documented substantial study heterogeneity, alongside imprecision in effect estimates, underscores the need for further research and standardization of measurement methods to elucidate whether RBP4 can be utilized in clinical practice as a potential GDM biomarker. PROSPERO (CRD42022340097: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022340097).

Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1.

Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.

Person-specific differences in ubiquitin-proteasome mediated proteostasis in human neurons.

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in abnormal protein accumulation in Alzheimer's disease. It remains unclear if genetic variation affects the intrinsic properties of neurons that render some individuals more vulnerable to UPS impairment. Induced pluripotent stem cell (iPSC)-derived neurons were generated from over 50 genetically variant and highly characterized participants of cohorts of aging. Proteomic profiling, proteasome activity assays, and Western blotting were employed to examine neurons at baseline and in response to UPS perturbation. Neurons with lower basal UPS activity were more vulnerable to tau accumulation following mild UPS inhibition. Chronic reduction in proteasome activity in human neurons induced compensatory elevation of regulatory proteins involved in proteostasis and several proteasome subunits. These findings reveal that genetic variation influences basal UPS activity in human neurons and differentially sensitizes them to external factors perturbing the UPS, leading to the accumulation of aggregation-prone proteins such as tau. Polygenic risk score for AD is associated with the ubiquitin-proteasome system (UPS) in neurons. Basal proteasome activity correlates with aggregation-prone protein levels in neurons. Genetic variation affects the response to proteasome inhibition in neurons. Neuronal proteasome perturbation induces an elevation in specific proteins involved in proteostasis. Low basal proteasome activity leads to enhanced tau accumulation with UPS challenge.

Neat plasma proteomics: getting the best out of the worst

Plasma proteomics holds immense potential for clinical research and biomarker discovery, serving as a non-invasive "liquid biopsy" for tissue sampling. Mass spectrometry (MS)-based proteomics, thanks to improvement in speed and robustness, emerges as an ideal technology for exploring the plasma proteome for its unbiased and highly specific protein identification and quantification. Despite its potential, plasma proteomics is still a challenge due to the vast dynamic range of protein abundance, hindering the detection of less abundant proteins. Different approaches can help overcome this challenge. Conventional depletion methods face limitations in cost, throughput, accuracy, and off-target depletion. Nanoparticle-based enrichment shows promise in compressing dynamic range, but cost remains a constraint. Enrichment strategies for extracellular vesicles (EVs) can enhance plasma proteome coverage dramatically, but current methods are still too laborious for large series. Neat plasma remains popular for its cost-effectiveness, time efficiency, and low volume requirement. We used a test set of 33 plasma samples for all evaluations. Samples were digested using S-Trap and analyzed on Evosep One and nanoElute coupled to a timsTOF Pro using different elution gradients and ion mobility ranges. Data were mainly analyzed using library-free searches using DIA-NN. This study explores ways to improve proteome coverage in neat plasma both in MS data acquisition and MS data analysis. We demonstrate the value of sampling smaller hydrophilic peptides, increasing chromatographic separation, and using library-free searches. Additionally, we introduce the EV boost approach, that leverages on the extracellular vesicle fraction to enhance protein identification in neat plasma samples. Globally, our optimized analysis workflow allows the quantification of over 1000 proteins in neat plasma with a 24SPD throughput. We believe that these considerations can be of help independently of the LC–MS platform used.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Beneficial Effects of Fibroblast Growth Factor-1 on Retinal Pigment Epithelial Cells Exposed to High Glucose-Induced Damage: Alleviation of Oxidative Stress, Endoplasmic Reticulum Stress, and Enhancement of Autophagy.

Diabetic retinopathy (DR) severely affects vision in individuals with diabetes. High glucose (HG) induces oxidative stress in retinal cells, a key contributor to DR development. Previous studies suggest that fibroblast growth factor-1 (FGF-1) can mitigate hyperglycemia and protect tissues from HG-induced damage. However, the specific effects and mechanisms of FGF-1 on DR remain unclear. In our study, FGF-1-pretreated adult retinal pigment epithelial (ARPE)-19 cells were employed to investigate. Results indicate that FGF-1 significantly attenuated HG-induced oxidative stress, including reactive oxygen species, DNA damage, protein carbonyl content, and lipid peroxidation. FGF-1 also modulated the expression of oxidative and antioxidative enzymes. Mechanistic investigations showed that HG induced high endoplasmic reticulum (ER) stress and upregulated specific proteins associated with apoptosis. FGF-1 effectively alleviated ER stress, reduced apoptosis, and restored autophagy through the adenosine monophosphate-activated protein kinase/mammalian target of the rapamycin signaling pathway. We observed that the changes induced by HG were dose-dependently reversed by FGF-1. Higher concentrations of FGF-1 (5 and 10 ng/mL) exhibited increased effectiveness in mitigating HG-induced damage, reaching statistical significance (p < 0.05). In conclusion, our study underscores the promising potential of FGF-1 as a safeguard against DR. FGF-1 emerges as a formidable intervention, attenuating oxidative stress, ER stress, and apoptosis, while concurrently promoting autophagy. This multifaceted impact positions FGF-1 as a compelling candidate for alleviating retinal cell damage in the complex pathogenesis of DR.

Proteomic changes of botulinum neurotoxin injection on muscle growth in children with spastic cerebral palsy.

The study aims to explore the proteomic profile and specific target proteins associated with muscle growth in response to botulinum neurotoxin A (BoNT-A) treatment, in order to improve spasticity management in children with cerebral palsy (CP). A total of 54 participants provided 60 plasma samples for proteomic analysis. Among them, six children were sampled before and after receiving their first BoNT-A injection. In addition, 48 unrelated children were enrolled, among whom one group had never received BoNT-A injections and another group was sampled after their first BoNT-A injection. Differentially expressed proteins were identified using the data-independent acquisition (DIA) mass spectrometry approach. Gene Ontology (GO), protein-protein interaction network, and Kyoto Encyclopedia of Genes and Genome analysis were conducted to explore the function and relationship among differentially expressed proteins. The expression levels of target proteins were verified by quantitative real-time PCR and western blotting. Analysis identified significant differential expression of 90 proteins across two time points, including 48 upregulated and 42 downregulated proteins. The upregulated thioredoxin, α-actinin-1, and aggrecan, and the downregulated integrin beta-1 may affect the growth of muscles affected by spasticity 3months after BoNT-A injection. This effect is potentially mediated through the activation or inhibition of PI3K-Akt, focal adhesion, and regulation of actin cytoskeleton signaling pathways. BoNT-A injection could lead to a disruption of protein levels and signaling pathways, a condition subsequently associated with muscle growth. This finding might aid clinicians in optimizing the management of spasticity in children with CP.

State of knowledge on ammonia handling by the kidney.

The disposal of ammonia, the main proton buffer in the urine, is important for acid-base homeostasis. Renal ammonia excretion is the predominant contributor to renal net acid excretion, both under basal condition and in response to acidosis. New insights into the mechanisms of renal ammonia production and transport have been gained in the past decades. Ammonia is the only urinary solute known to be produced in the kidney and selectively transported through the different parts of the nephron. Both molecular forms of total ammonia, NH3 and NH4+, are transported by specific proteins. Proximal tubular ammoniagenesis and the activity of these transport processes determine the eventual fate of total ammonia produced and excreted by the kidney. In this review, we summarized the state of the art of ammonia handling by the kidney and highlighted the newest processes described in the last decade.

EXPLORATION OF ANTI-MELANOMA POTENTIAL OF PHYTOCHEMICALS FROM NYCTANTHES ARBORTRISTIS THROUGH COMPUTATIONAL STUDIES

Objective: The goal of the current research is to identify the dominant phytochemical from the plantNyctanthesarbor-tristis Linn. and to investigate their binding affinities against the proteins BRaf Kinase mutant (3OG7) and Hsp90 Chaperone (2VCJ) that causesmelanoma. Methods: In this work, Schrodinger software was utilized to investigate the anti-cancer potential of phytochemicals Nyctanthesarbor-tristis against specific target proteins, namely BRaf Kinase mutant (3OG7) and Hsp90 Chaperone (2VCJ) Inhibitors. Results: Based on the outcome of the docking investigation, phytochemicals that exhibited highest binding affinity to the specified protein targets were subjected to induced fit docking and MM-GBSA computations using the Schrodinger Maestro version 2021.2 in prime module. According to the analysis, the compounds with the highest binding affinities for 2VCJ and 3OG7 are Arbortristoside D and Nicotiflorin respectively. The compound that interacted with both the proteins wasArbortristoside B. These phytochemicals appear to be more effective to the FDA-approved V600E-BRaf inhibitor Vemurafenib and Hsp90 Chaperone Inhibitor Diclonine. Conclusion: One of the most common, deadly, and dangerous malignant diseases with a high global prevalence rate is melanoma (skin cancer). The present study may prove more helpful in developing an ideal targeted drug delivery system of phytochemicals obtained from plant Nyctanthesarbor-tristisfor treatment of melanoma. This suggests that these substances could be evolved into highly effective anti-melanoma drugs.

Comparison of the mesodermal differentiation potential between embryonic stem cells and scalable induced pluripotent stem cells.

Mesenchymal stem cells (MSCs) have promising potential in clinical application, whereas their limited amount and sources hinder their bioavailability. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have become prominent options in regenerative medicine as both possess the ability to differentiate into MSCs. Recently, our research team has successfully developed human leukocyte antigen (HLA)-homozygous iPSC cell lines with high immune compatibility, covering 13.5% of the Taiwanese population. As we deepen our understanding of the differences between these ESCs and HLA-homozygous iPSCs, our study focused on morphological observations and flow cytometry analysis of specific surface marker proteins during the differentiation of ESCs and iPSCs into MSCs. The results showed no significant differences between the two pluripotent stem cells, and both of them demonstrated the equivalent ability to further differentiate into adipose, cartilage, and bone cells. Our research revealed that these iPSCs with high immune compatibility exhibit the same differentiation potential as ESCs, enhancing the future applicability of highly immune-compatible iPSCs.

Using Chromatin Immunoprecipitation (ChIP) to Study the Chromatin State in Drosophila.

The chromatin state plays an important role in regulating gene expression, which affects organismal development and plasticity. Proteins, including transcription factors, chromatin modulatory proteins, and histone proteins, usually with modifications, interact with gene loci involved in cellular differentiation, function, and modulation. One molecular method used to characterize protein-DNA interactions is chromatin immunoprecipitation (ChIP). ChIP uses antibodies to immunoprecipitate specific proteins cross-linked to DNA fragments. This approach, in combination with quantitative PCR (qPCR) or high-throughput DNA sequencing, can determine the enrichment of a certain protein or histone modification around specific gene loci or across the whole genome. ChIP has been used in Drosophila to characterize the binding pattern of transcription factors and to elucidate the roles of regulatory proteins in gene expression during development and in response to environment stimuli. This review outlines ChIP procedures using tissues from the Drosophila nervous system as an example and discusses all steps and the necessary optimization.

Performing Quantitative PCR after Chromatin Immunoprecipitation (ChIP) of Drosophila Antennal and Brain Samples.

Chromatin immunoprecipitation (ChIP) is a technique used to study specific protein-DNA interaction. Briefly, in this technique, antibodies to proteins of interest are used to isolate regions of DNA where these proteins bind. ChIP samples can be processed and analyzed in different ways. One of the approaches for assessing the results of ChIP experiments is quantitative PCR (qPCR). qPCR is used to quantitatively measure the amount of DNA fragments that have been isolated, reflecting the signal of specific proteins interacting with these fragments. This protocol describes both the "percent input" method and the "fold enrichment" method for ChIP-qPCR data analysis using Drosophila tissues as an example. The "percent input" method measures signals of DNA fragments against the input measurement. In contrast, the "fold enrichment" method quantifies the amplified signal strength relative to a background control. Because the quality of primers is critical for the reliability of ChIP-qPCR results, this protocol also describes how to measure primer amplification efficiency using Drosophila genomic DNA.