Cell Function Research Articles

Intracellular Calcium Dynamics in Primary Human Adrenocortical Cells Deciphered with a Novel Pipeline.

The fluctuations of the intracellular Ca2+ concentration ([Ca2+]i) are key physiological signals for cell function under normal conditions and can undergo profound alterations in disease states, as high blood pressure due to endocrine disorders like primary aldosteronism (PA). However, when assessing such fluctuations several parameters in the Ca2+ signal dynamics need to be considered, which renders their assessmentchallenging. Aimto develop an observer-independent custom-made pipeline to analyze Ca2+ dynamics in terms of frequency and peak parameters, as amplitude, full width at half maximum (FWHM) and area under the curve (AUC). We applied a custom-made methodology to aldosterone-producing adenoma (APA) and APA adjacent cells (AAC) and found this pipeline to be suitable for monitoring and processing a wide-range of [Ca2+]i events in these cell types delivering reproducible results. The designed pipeline can provide a useful tool for [Ca2+]i signal analysis that allows comparisons of Ca2+ dynamics not only in PA, but in other cell phenotypes that are relevant for the regulation of blood pressure.

Cellular Patterning Alone Using Bioprinting Regenerates Articular Cartilage Through Native-Like Cartilagenesis.

Few studies have proved that bioprinting itself helps recapitulate native tissue functions mainly because the bioprinted macro shape can rarely, if ever, influence cell function. This can be more problematic in bioprinting cartilage, generally considered more challenging to engineer. Here a new method is shown to micro-pattern chondrocytes within bioprinted sub-millimeter micro tissues, denoted as patterned micro-articular-cartilages tissues (PA-MCTs). Under the sole influence of bioprinted cellular patterns. A pattern scoring system is developed after over 600 bioprinted cellular patterns are analyzed. The top-scored pattern mimics that of the isogenous group in native articular cartilage. Under the sole influence of this pattern during PA-MCTs bio-assembling into macro-cartilage and repairing cartilage defects, chondrogenic cell phenotype is preserved, and cartilagenesis is initiated and maintained. Neocartilage tissues from individual and assembled PA-MCTs are comparable to native articular cartilage and superior to cartilage bioprinted with homogeneously distributed cells in morphology, biochemical components, cartilage-specific protein and gene expression, mechanical properties, integration with host tissues, zonation forming and stem cell chondrogenesis. PA-MCTs can also be used as osteoarthritic and healthy cartilage models for therapeutic drug screening and cartilage development studies. This cellular patterning technique can pave a new way for bioprinting to recapitulate native tissue functions via tissue genesis.

GPR124 promotes trophoblast proliferation, migration, and invasion and inhibits trophoblast cell apoptosis and inflammation via JNK and P38 MAPK pathways.

Early-onset preeclampsia, which occurrs before 34 weeks of gestation, is the most dangerous classification of preeclampsia, which is a pregnancy-specific disease that causes 1% of maternal deaths. G protein-coupled receptor 124 (GPR124) is significantly expressed at various stages of the human reproductive process, particularly during embryogenesis and angiogenesis. Our prior investigation demonstrated a notable decrease in GPR124 expression in the placentas of patients with early-onset preeclampsia compared to that in normal pregnancy placentas. However, there is a lack of extensive investigation into the molecular processes that contribute to the role of GPR124 in placenta development. This study aimed to examine the mechanisms by which GPR124 affects the occurrence of early-onset preeclampsia and its function in trophoblast. Proliferative, invasive, migratory, apoptotic, and inflammatory processes were identified in GPR124 knockdown, GPR124 overexpression, and normal HTR8/SVneo cells. The mechanism of GPR124-mediated cell function in GPR124 knockdown HTR8/SVneo cells was examined using inhibitors of the JNK or P38 MAPK pathway. Downregulation of GPR124 was found to significantly inhibit proliferation, invasion and migration, and promote apoptosis of HTR8/SVneo cells when compared to the control and GPR124 overexpression groups. This observation is consistent with the pathological characteristics of preeclampsia. In addition, GPR124 overexpression inhibits the secretion of pro-inflammatory cytokines interleukin (IL)-8 and interferon-γ (IFN-γ) while enhancing the secretion of the anti-inflammatory cytokine interleukin (IL)-4. Furthermore, GPR124 suppresses the activation of P-JNK and P-P38 within the JNK/P38 MAPK pathway. The invasion, apoptosis, and inflammation mediated by GPR124 were partially restored by suppressing the JNK and P38 MAPK pathways in HTR8/SVneo cells. GPR124 plays a crucial role in regulating trophoblast proliferation, invasion, migration, apoptosis, and inflammation via the JNK and P38 MAPK pathways. Furthermore, the effect of GPR124 on trophoblast suggests its involvement in the pathogenesis of early-onset preeclampsia.

AMPK-a key factor in crosstalk between tumor cell energy metabolism and immune microenvironment?

Immunotherapy has now garnered significant attention as an essential component in cancer therapy during this new era. However, due to immune tolerance, immunosuppressive environment, tumor heterogeneity, immune escape, and other factors, the efficacy of tumor immunotherapy has been limited with its application to very small population size. Energy metabolism not only affects tumor progression but also plays a crucial role in immune escape. Tumor cells are more metabolically active and need more energy and nutrients to maintain their growth, which causes the surrounding immune cells to lack glucose, oxygen, and other nutrients, with the result of decreased immune cell activity and increased immunosuppressive cells. On the other hand, immune cells need to utilize multiple metabolic pathways, for instance, cellular respiration, and oxidative phosphorylation pathways to maintain their activity and normal function. Studies have shown that there is a significant difference in the energy expenditure of immune cells in the resting and activated states. Notably, competitive uptake of glucose is the main cause of impaired T cell function. Conversely, glutamine competition often affects the activation of most immune cells and the transformation of CD4+T cells into inflammatory subtypes. Excessive metabolite lactate often impairs the function of NK cells. Furthermore, the metabolite PGE2 also often inhibits the immune response by inhibiting Th1 differentiation, B cell function, and T cell activation. Additionally, the transformation of tumor-suppressive M1 macrophages into cancer-promoting M2 macrophages is influenced by energy metabolism. Therefore, energy metabolism is a vital factor and component involved in the reconstruction of the tumor immune microenvironment. Noteworthy and vital is that not only does the metabolic program of tumor cells affect the antigen presentation and recognition of immune cells, but also the metabolic program of immune cells affects their own functions, ultimately leading to changes in tumor immune function. Metabolic intervention can not only improve the response of immune cells to tumors, but also increase the immunogenicity of tumors, thereby expanding the population who benefit from immunotherapy. Consequently, identifying metabolic crosstalk molecules that link tumor energy metabolism and immune microenvironment would be a promising anti-tumor immune strategy. AMPK (AMP-activated protein kinase) is a ubiquitous serine/threonine kinase in eukaryotes, serving as the central regulator of metabolic pathways. The sequential activation of AMPK and its associated signaling cascades profoundly impacts the dynamic alterations in tumor cell bioenergetics. By modulating energy metabolism and inflammatory responses, AMPK exerts significant influence on tumor cell development, while also playing a pivotal role in tumor immunotherapy by regulating immune cell activity and function. Furthermore, AMPK-mediated inflammatory response facilitates the recruitment of immune cells to the tumor microenvironment (TIME), thereby impeding tumorigenesis, progression, and metastasis. AMPK, as the link between cell energy homeostasis, tumor bioenergetics, and anti-tumor immunity, will have a significant impact on the treatment and management of oncology patients. That being summarized, the main objective of this review is to pinpoint the efficacy of tumor immunotherapy by regulating the energy metabolism of the tumor immune microenvironment and to provide guidance for the development of new immunotherapy strategies.

Role of m6A modifications in immune evasion and immunotherapy.

RNA modification has garnered increasing attention in recent years due to its pivotal role in tumorigenesis and immune surveillance. N6-methyladenosine (m6A) modification is the most prevalent RNA modification, which can affect the expression of RNA by methylating adenylate at the sixth N position to regulate the occurrence and development of tumors. Dysregulation of m6A affects the activation of cancer-promoting pathways, destroys immune cell function, maintains immunosuppressive microenvironment, and promotes tumor cell growth. In this review, we delve into the latest insights into how abnormalities in m6A modification in both tumor and immune cells orchestrate immune evasion through the activation of signaling pathways. Furthermore, we explore how dysregulated m6A modification in tumor cells influences immune cells, thereby regulating tumor immune evasion via interactions within the tumor microenvironment (TME). Lastly, we highlight recent discoveries regarding specific inhibitors of m6A modulators and the encapsulation of m6A-targeting nanomaterials for cancer therapy, discussing their potential applications in immunotherapy.

Polypeptide N-Acetylgalactosaminyl transferase 14 is a novel mediator in pancreatic β-cell function and growth.

Polypeptide N-Acetylgalactosaminyl transferase 14 (GALNT14) plays important roles in cancer progression and chemotherapy response. Here, we show that GALNT14 is highly expressed in pancreatic β cells and regulates β cell function and growth. We found that the expression level of Ganlt14 was significantly decreased in the primary islets from three rodent type-2 diabetic models. Single-Cell sequencing defined that Galnt14 was mainly expressed in β cells of mouse islets. Galnt14 knockout (G14KO) INS-1 cell line, constructed by using CRISPR/Cas9 technology were growth normal, but showed blunt shape, and increased basal insulin secretion. Combined proteomics and glycoproteomics demonstrated that G14KO altered cell-to-cell junctions, communication, and adhesion. Insulin receptor (IR) and IGF1-1R were indirectly confirmed for GALNT14 substrates, contributed to diminished IGF1-induced p-AKT levels and cell growth in G14KO cells. Overall, this study uncovers that GALNT14 is a novel modulator in regulating β cells biology, providing a missing link of β cells O-glycosylation to diabetes development.

Classical and non-classical effects of angiotensin converting enzyme: how increased ACE enhances myeloid immune function.

As part of the classical renin-angiotensin system, the peptidase angiotensin converting enzyme (ACE) makes angiotensin II which has myriad effects on systemic cardiovascular function, inflammation, and cellular proliferation. Less well known is that macrophages and neutrophils make ACE in response to immune activation which has marked effects on myeloid cell function independent of angiotensin II. Here, we discuss both classical (angiotensin) and non-classical functions of ACE and highlight mice called ACE 10/10 in which genetic manipulation increases ACE expression by macrophages and makes these mice much more resistant to models of tumors, infection, atherosclerosis, and Alzheimer's disease. In another model called NeuACE mice, neutrophils make increased ACE and these mice are much more resistant to infection. In contrast, ACE inhibitors reduce neutrophil killing of bacteria in mice and humans. Increased expression of ACE induces a marked increase in macrophage oxidative metabolism, particularly mitochondrial oxidation of lipids, secondary to increased PPARα expression, and results in increased myeloid cell ATP. ACE present in sperm has a similar metabolic effect and the lack of ACE activity in these cells reduces both sperm motility and fertilization capacity. These non-classical effects of ACE are not due to the actions of angiotensin II but to an unknown molecule, probably a peptide, that triggers a profound change in myeloid cell metabolism and function. Purifying and characterizing this peptide could offer a new treatment for several diseases and prove potentially lucrative.

Identification and Functional Analysis of a de novo IKZF3 Mutation in a Pediatric Patient with Combined Immunodeficiency.

AIOLOS, a vital member of the IKAROS protein family, plays a significant role in lymphocyte development and function through DNA binding and protein-protein interactions. Mutations in the IKZF3 gene, which encodes AIOLOS, lead to a rare combined immunodeficiency often linked with infections and malignancy. In this study, we evaluated a 1-year-4-month-old female patient presenting with recurrent infections, diarrhea, and failure to thrive. Laboratory investigations revealed decreased T lymphocyte and immunoglobulin levels. Through whole-exome and Sanger sequencing, we discovered a de novo mutation in IKZF3 (NM_012481; exon 5 c.571G > C, p.Gly191Arg), corresponding to the third DNA-binding zinc finger region of the encoded protein AIOLOS. Notably, the patient with the AIOLOS G191R mutation showed reduced recent thymic emigrants in naïve CD4+T cells compared to healthy counterparts of the same age, while maintaining normal levels of Th1, Th2, Th17, Treg, and Tfh cells. This mutation also resulted in decreased switched memory B cells and lower CD23 and IgM expression. In vitro studies revealed that AIOLOS G191R does not impact the expression of AIOLOS but compromises its stability, DNA binding and pericentromeric targeting. Furthermore, AIOLOS G191R demonstrated a dominant-negative effect over the wild-type protein. This case represents the first reported instance of a mutation in the third DNA-binding zinc finger region of AIOLOS highlighting its pivotal role in immune cell functionality.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Tumor derived exosomal ENTPD2 impair CD8+ T cell function in colon cancer through ATP-adenosine metabolism reprogramming.

Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer. Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

Micro/nano-plastics and microalgae in aquatic environment: Influence factor, interaction, and molecular mechanisms.

Micro/nano-plastics, as emerging persistent pollutant, are frequently detected in aquatic environments together with other environmental pollutants. Microalgae are the major primary producers and bear an important responsibility for maintaining the balance of aquatic ecosystems. Numerous studies have been conducted on the influence of micro/nano-plastics on the growth, photosynthesis, oxidative stress, gene expression and metabolites of microalgae in laboratory studies. However, it is difficult to comprehensively evaluate the toxic effects of micro/nano-plastics on microalgae due to different experimental designs. Moreover, there is a lack of effective analysis of the aforementioned multi-omics data and reports on shared biological patterns. Therefore, the purpose of this review is to compare the acute, chronic, pulsed, and combined effect of micro/nano-plastics on microalgae and explore hidden rules in the molecular mechanisms of the interaction between them. Results showed that the effect of micro/nano-plastics on microalgae was related to exposure mode, exposure duration, exposure size, concentration, and type of micro/nano-plastics. Meanwhile, the phenomenon of poisoning and detoxification between micro/nano-plastics and microalgae was found. The inhibitory mechanism of micro/nano-plastics on algal growth was due to the micro/nano-plastics affected the photosynthesis, oxidative phosphorylation, and ribosome pathways of algal cells. This brought the disruption of the functions of chloroplasts, mitochondria, and ribosome, as well as impacted on energy metabolism and translation pathways, eventually leading to impairment of cell function. Besides, algae resisted this inhibitory effect by regulating the alanine, aspartate, and glutamate metabolism and purine metabolism pathways, thereby increasing the chlorophyll synthesis, inhibiting the increase of reactive oxygen species, delaying the process of lipid peroxidation, balancing the osmotic pressure of cell membrane.

The potential of proteomics for in-depth bioanalytical investigations of satellite cell function in applied myology.

Regenerative myogenesis plays a crucial role in mature myofibers to counteract muscular injury or dysfunction due to neuromuscular disorders. The activation of specialized myogenic stem cells, called satellite cells, is intrinsically involved in proliferation and differentiation, followed by myoblast fusion and the formation of multi-nucleated myofibers. This report provides an overview of the role of satellite cells in the neuromuscular system and the potential future impact of proteomic analyses for biomarker discovery, as well as the identification of novel therapeutic targets in muscle disease. The article reviews the ways in which the systematic analysis of satellite cells, myoblasts and myocytes by single cell proteomics can help to better understand the process of myofiber regeneration. In order to better comprehend satellite cell dysfunction in neuromuscular disorders, mass spectrometry-based proteomics is an excellent large-scale analytical tool for the systematic profiling of pathophysiological processes. The optimized isolation of muscle-derived cells can be routinely performed by mechanical/enzymatic dissociation protocols, followed by fluorescence-activated cell sorting in specialized flow cytometers. Ultrasensitive single cell proteomics using label-free quantitation methods or approaches that utilize tandem mass tags are ideal bioanalytical approaches to study the pathophysiological role of stem cells in neuromuscular disease.

Lipid Variability Induces Endothelial Dysfunction by Increasing Inflammation and Oxidative Stress.

This study investigates the impact of fluctuating lipid levels on endothelial dysfunction. Human aortic and umbilical vein endothelial cells were cultured under varying palmitic acid (PA) concentrations: 0, 50, and 100 μM, and in a variability group alternating between 0 and 100 μM PA every 8 hours for 48 hours. In the lipid variability group, cells were exposed to 100 μM PA during the final 8 hours before analysis. We assessed inflammation using real-time polymerase chain reaction, Western blot, and cytokine enzyme-linked immunosorbent assay (ELISA); reactive oxygen species (ROS) levels with dichlorofluorescin diacetate assay; mitochondrial function through oxygen consumption rates via XF24 flux analyzer; and endothelial cell functionality via wound healing and cell adhesion assays. Cell viability was evaluated using the MTT assay. Variable PA levels significantly upregulated inflammatory genes and adhesion molecules (Il6, Mcp1, Icam, Vcam, E-selectin, iNos) at both transcriptomic and protein levels in human endothelial cells. Oscillating lipid levels reduced basal respiration, adenosine triphosphate synthesis, and maximal respiration, indicating mitochondrial dysfunction. This lipid variability also elevated ROS levels, contributing to a chronic inflammatory state. Functionally, these changes impaired cell migration and increased monocyte adhesion, and induced endothelial apoptosis, evidenced by reduced cell viability, increased BAX, and decreased BCL2 expression. Lipid variability induce endothelial dysfunction by elevating inflammation and oxidative stress, providing mechanistic insights into how lipid variability increases cardiovascular risk.

Post-Penetrating Keratoplasty Assessment of Endothelial Function With a Scleral Lens Challenge.

Patients who have undergone penetrating keratoplasty may have corneal edema because of endothelial cell dysfunction. Scleral lens wear may exacerbate edema, particularly if lens fit is suboptimal. Distinguishing between edema because of inherent endothelial cell dysfunction and swelling because of scleral lens-related hypoxia can be challenging. It is necessary, however, to identify the most likely cause of increased corneal thickness to determine whether the patient simply needs refitting for a different lens design or needs additional surgical intervention. This case report describes the utility of corneal tomographic imaging before and after scleral lens wear both to estimate endothelial cell function and to direct decisions when designing a scleral lens for a post-transplant eye.

Tissue-specific sex differences in pediatric and adult immune cell composition and function

Sex-based differences in immune cell composition and function can contribute to distinct adaptive immune responses. Prior work has quantified these differences in peripheral blood, but little is known about sex differences within human lymphoid tissues. Here, we characterized the composition and phenotypes of adaptive immune cells from male and female ex vivo tonsils and evaluated their responses to influenza antigens using an immune organoid approach. In a pediatric cohort, female tonsils had more memory B cells compared to male tonsils direct ex vivo and after stimulation with live-attenuated but not inactivated vaccine, produced higher influenza-specific antibody responses. Sex biases were also observed in adult tonsils but were different from those measured in children. Analysis of peripheral blood immune cells from in vivo vaccinated adults also showed higher frequencies of tissue homing CD4 T cells in female participants. Together, our data demonstrate that distinct memory B and T cell profiles are present in male vs. female lymphoid tissues and peripheral blood respectively and suggest that these differences may in part explain sex biases in response to vaccines and viruses.

Posttranslational protein modifications as gatekeepers of cancer immunogenicity.

Triple-negative breast cancer (TNBC) presents a formidable challenge in oncology due to its aggressive phenotype and the immunosuppressive nature of its tumor microenvironment (TME). In this issue of the JCI, Zhu, Banerjee, and colleagues investigated the potential of targeting the OTU domain-containing protein 4 (OTUD4)/CD73 axis to mitigate immunosuppression in TNBC. They identified elevated CD73 expression as a hallmark of immunosuppression in TNBC. Notably, the CD73 expression was regulated by OTUD4-mediated posttranslational modifications. Using ST80, a pharmacologic inhibitor of OTUD4, the authors demonstrated the restoration of cytotoxic T cell function and enhanced efficacy of anti-PD-L1 therapy in preclinical models. These findings underscore the therapeutic potential of targeting the OTUD4/CD73 axis in TNBC.

The Role of the Gap Junction Protein Connexin in Adrenal Gland Tumorigenesis

Gap junctions (GJs) are important in the regulation of cell growth, morphology, differentiation and migration. However, recently, more attention has been paid to their role in the pathogenesis of different diseases as well as tumorigenesis, invasion and metastases. The expression pattern and possible role of connexins (Cxs), as major GJ proteins, under both physiological and pathological conditions in the adrenal gland, were evaluated in this review. The databases Web of Science, PubMed and Scopus were searched. Studies were evaluated if they provided data regarding the connexin expression pattern in the adrenal gland, despite current knowledge of this topic not being widely investigated. Connexin expression in the adrenal gland differs according to different parts of the gland and depends on ACTH release. Cx43 is the most studied connexin expressed in the adrenal gland cortex. In addition, Cx26, Cx32 and Cx50 were also investigated in the human adrenal gland. Cx50 as the most widespread connexin, along with Cx26, Cx29, Cx32, Cx36 and Cx43, has been expressed in the adrenal medulla with distinct cellular distribution. Considerable effort has recently been directed toward connexins as therapeutically targeted molecules. At present, there exist several viable strategies in the development of potential connexin-based therapeutics. The differential and hormone-dependent distribution of gap junctions within adrenal glands, the relatively large gap junction within this gland and the increase in the gap junction size and number following hormonal treatment would indicate that gap junctions play a pivotal role in cell functioning in the adrenal gland.

Deconvolution analysis identified altered hepatic cell landscape in primary sclerosing cholangitis and primary biliary cholangitis

IntroductionPrimary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are characterized by ductular reaction, hepatic inflammation, and liver fibrosis. Hepatic cells are heterogeneous, and functional roles of different hepatic cell phenotypes are still not defined in the pathophysiology of cholangiopathies. Cell deconvolution analysis estimates cell fractions of different cell phenotypes in bulk transcriptome data, and CIBERSORTx is a powerful deconvolution method to estimate cell composition in microarray data. CIBERSORTx performs estimation based on the reference file, which is referred to as signature matrix, and allows users to create custom signature matrix to identify specific phenotypes. In the current study, we created two custom signature matrices using two single cell RNA sequencing data of hepatic cells and performed deconvolution for bulk microarray data of liver tissues including PSC and PBC patients.MethodsCustom signature matrix files were created using single-cell RNA sequencing data downloaded from GSE185477 and GSE115469. Custom signature matrices were validated for their deconvolution performance using validation data sets. Cell composition of each hepatic cell phenotype in the liver, which was identified in custom signature matrices, was calculated by CIBERSORTx and bulk RNA sequencing data of GSE159676. Deconvolution results were validated by analyzing marker expression for the cell phenotype in GSE159676 data.ResultsCIBERSORTx and custom signature matrices showed comprehensive performance in estimation of population of various hepatic cell phenotypes. We identified increased population of large cholangiocytes in PSC and PBC livers, which is in agreement with previous studies referred to as ductular reaction, supporting the effectiveness and reliability of deconvolution analysis in this study. Interestingly, we identified decreased population of small cholangiocytes, periportal hepatocytes, and interzonal hepatocytes in PSC and PBC liver tissues compared to healthy livers.DiscussionAlthough further studies are required to elucidate the roles of these hepatic cell phenotypes in cholestatic liver injury, our approach provides important implications that cell functions may differ depending on phenotypes, even in the same cell type during liver injury. Deconvolution analysis using CIBERSORTx could provide a novel approach for studies of specific hepatic cell phenotypes in liver diseases.

Ameliorated in vitro anti-cancer efficacy of methotrexate loaded zinc oxide nanoparticles in breast cancer cell lines MCF-7 & MDA-MB-231 and its acute toxicity study.

Traditional therapies often struggle with specificity and resistance in case of cancer treatments. It is therefore important to investigate new approaches for cancer treatment based on nanotechnology. Zinc oxide nanoparticles (ZnONPs) are known to exhibit anti-cancer properties by inducing oxidative stress, apoptosis, and cell cycle arrest. Methotrexate (MTX) known anti-folate shows specificity to folate receptors (FR) and interrupts healthy functioning of cells. This study proposes the use of previously characterized biocompatible Methotrexate loaded Zinc oxide nanoparticles (MTX-ZnONPs) as a dual action therapeutic strategy against breast cancer cell lines, MCF-7 (MTX-sensitive) and MDA-MB-231 (MTX-resistant). To elucidate the cytotoxicity mechanism of MTX-ZnONPs in depth In-vitro study was carried out. In vitro assays, including cell cycle analysis, apoptosis assay, and western blot analysis to study the protein expression were performed. Results of these assays, further supported the anti-cancer activity of MTX-ZnONPs showing apoptotic and necrotic activity in MCF-7 and MDA-MB-231 cell line respectively. In vivo acute oral toxicity study to identify the LD50 in animals revealed no signs of toxicity and mortality up to 550 mg/kg body weight of animal, significantly higher LD50 values than anticipated therapeutic levels and safety of the synthesized nanosystem. The study concludes that MTX-ZnONPs exhibit anti-cancer potential against breast cancer cells offering a promising strategy for overcoming resistance.

A novel prognostic signature based on smoking-associated genes for predicting prognosis and immune microenvironment in NSCLC smokers

BackgroundAs a highly heterogeneous tumor, non-small cell lung cancer (NSCLC) is famous for its high incidence and mortality worldwide. Smoking can cause genetic changes, which leading to the occurrence and progress of NSCLC. Nevertheless, the function of smoking-related genes in NSCLC needs more research.MethodsWe downloaded transcriptome data and clinicopathological parameters from Gene Expression Omnibus (GEO) databases, and screened smoking-related genes. Lasso regression were applied to establish the 7-gene signature. The associations between the 7-gene signature and immune microenvironment analysis, survival analysis, drug sensitivity analysis and enriched molecular pathways were studied. Ultimately, cell function experiments were conducted to research the function of FCGBP in NSCLC.ResultsThrough 7-gene signature, NSCLC samples were classified into high-risk group (HRG) and low-risk group (LRG). Significant difference in overall survival (OS) between HRG and LRG was found. Nomograms and ROC curves indicated that the 7-gene signature has a stable ability in predicting prognosis. Through the analysis of immune microenvironment, we found that LRG patients had better tumor immune activation. FCGBP showed the highest mutation frequency among the seven prognostic smoking related genes (LRRC31, HPGD, FCGBP, SPINK5, CYP24A1, S100P and FGG), and was notable down-regulated in NSCLC smokers compared with non-smoking NSCLC patients. The cell experiments confirmed that FCGBP knockdown promoting proliferation, migration, and invasion in NSCLC cells.ConclusionThis smoking-related prognostic signature represents a promising tool for assessing prognosis and tumor microenvironment in smokers with NSCLC. The role of FCGBP in NSCLC was found by cell experiments, which can be served as diagnostic biomarker and immunotherapy target for NSCLC.