Cellular Components Research Articles

Network pharmacology and molecular docking approach to elucidate the mechanisms of safflower, phellodendron, scutellaria baicalensis, coptis, and gardenia in hand-foot syndrome.

Safflower, phellodendron, scutellaria baicalensis, coptis, and gardenia (SPSCG) are medicinal plants with a wide range of anti-inflammatory and antioxidant effects. However, the related mechanism of SPSCG against hand-foot syndrome (HFS) has yet to be revealed. To investigate the mechanisms of SPSCG in the treatment of HFS using the Network Pharmacology. Active ingredients and targets of SPSCG for HFS were screened by the Chinese Medicine Systems Pharmacology (TCMSP) and Swiss Target Prediction databases. Potential therapeutic targets were collected from the GeneCards and OMIM databases. Subsequently, protein-protein interactions (PPI), Gene Ontology (GO) annotations, and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to investigate the potential mechanism of the SPSCG in HFS. Then, molecular docking and molecular dynamics simulations were performed to predict the binding interactions between the active compound and the core target. Finally, vitro experiments were used to verify the repair effect of key ingredients of SPSCG on cell damage caused by 5-Fluorouracil. Quercetin, kaempferol, β-sitosterol, and stigmasterol were identified as the major active components of SPSCG. GO analysis showed a total of 1,127 biological processes, 42 terms cellular components, and 57 molecular functions. KEGG analysis showed that the MAPK, TNF, and IL-17 signaling pathways were significantly enriched. The PPI analysis discovered that EGFR, CASP3, AKT1, CCND1, and CTNNB1 shared the highest centrality among all target genes. The experimental results confirmed that these SPSCG active ingredients could treat HFS by reducing inflammation reaction and promoting cell damage repair. SPSCG may alleviate HFS by exerting antioxidative effects and suppressing inflammatory responses.

Understanding the Significance of Raffinose Family Oligosaccharides in Seed Physiology

Seed vigour and longevity are pivotal agronomic traits with profound implications for crop yield, food security, and the global economy. Seed longevity, often defined by the duration of seed viability, is crucial for effective gene bank management, influencing seed regeneration cycles and ensuring the long-term conservation of plant genetic resources. The inevitable process of seed deterioration, driven by a complex network of biochemical reactions, leads to altered metabolism and damage to critical cellular components such as membranes, DNA, mitochondria, proteins, and the antioxidative defence system. In recent research, Raffinose family oligosaccharides (RFOs) have emerged as key contributors to enhancing seed vigour and longevity. These sugars perform multiple functions in plants, including tolerance to abiotic and biotic stresses, regulation of seed germination, and maintenance of desiccation tolerance, all of which are essential for overall seed health. Studies suggest that RFOs significantly bolster seed vigour and longevity through mechanisms such as cytoplasmic vitrification, water replacement, and osmoprotection in dried seeds. These oligosaccharides are particularly abundant in the seeds of leguminous crops and are also found in roots and specialized storage organs. The glassy state formed by RFOs is vital for protecting cells from oxidative damage caused by reactive oxygen species (ROS), enhancing the stability of enzymes, and preventing deleterious conformational changes in proteins. Furthermore, delaying the degradation of RFOs has been shown to inhibit premature germination, underscoring their critical role in early seedling development and successful crop establishment.

Abstract C036: Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma

Abstract PDAC growth is characterized by dependencies on both mutant KRAS signaling and autophagy. Our group and others previously demonstrated that inhibition of the RAF/MEK/ERK pathway in PDAC induces an increased dependence on autophagy, a metabolic nutrient scavenging process by which cellular components are recycled in times of nutrient stress. Combined inhibition of the ERK MAPK pathway and autophagy inhibited the growth of multiple preclinical models of PDAC. Based on these findings, combined ERK/MEK inhibition and hydroxychloroquine (HCQ) is currently under clinical evaluation (NCT03825289, NCT04386057). However, resistance to this combination has already been described. To identify sensitizers to CQ treatment, our lab performed a CRISPR-Cas9 mediated loss-of- function screen using an sgRNA library targeting genes associated with cancer signaling pathways. Surprisingly, we identified multiple autophagy related genes, indicating that concurrent inhibition of two nodes of the autophagy pathway may be a more effective method of inhibiting autophagy in PDAC. For example, we identified, PIK3C3, the gene that encodes for VPS34, a protein essential for the nucleation of autophagosomes, as a potential sensitizer to CQ treatment. Inhibition of VPS34 with the compound SAR405, resulted in both decreased autophagic flux and impaired proliferation. Additionally, VPS34 inhibition sensitized cells to CQ treatment, leading to further reduced proliferation. These results prompted us to hypothesize that inhibition of ULK1, a serine/threonine kinase critical for the initiation of autophagy, could also sensitize PDAC cells to CQ treatment. We found that ULK1 inhibition further reduced CQ-mediated anti-proliferative effects. Additionally, both ULK1 and VPS34 inhibitor treatment sensitized cells to PIKfyve inhibition with apilimod, a chemically and mechanistically distinct inhibitor of the termination phase of the autophagic pathway. Previous studies have indicated that autophagy inhibition alone is not a viable therapeutic avenue for the treatment of PDAC. Thus, we next determined whether vertical inhibition of the autophagy pathway improves the efficacy of combined inhibition of the RAS and autophagy pathway. We found that both VPS34 inhibition and ULK1 inhibition can decrease RAS inhibitor induced autophagic flux. Finally, vertical inhibition of the autophagic pathway via VPS34 inhibition and CQ sensitized PDAC cells to RAS inhibition to further reduce PDAC proliferation. Ongoing and future studies are aimed at elucidating the mechanism leading to decreased proliferation of combined anti-autophagy and RAS inhibitor therapies, as well as mechanistically understanding the effect of autophagy inhibition at multiple nodes on autophagic flux. Citation Format: Mallory K Roach, Jonathan M DeLiberty, Elyse G Schechter, Runying Yang, Noah L Pieper, Clint A Stalnecker, Kirsten L Bryant. Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C036.

Acute exposure to electronic cigarette components alters mRNA expression of pre-osteoblasts.

The use of traditional nicotine delivery products such as tobacco has long been linked to detrimental health effects. However, little work to date has focused on the emerging market of aerosolized nicotine delivery known as electronic nicotine delivery systems (ENDS) or electronic cigarettes, and their potential for new effects on human health. Challenges studying these devices include heterogeneity in the formulation of the common components of most available ENDS, including nicotine and a carrier (commonly composed of propylene glycol and vegetable glycerin, or PG/VG). In the present study, we report on experiments interrogating the effects of major identified components in e-cigarettes. Specifically, the potential concomitant effects of nicotine and common carrier ingredients in commercial "vape" products are explored invitro to inform the potential health effects on the craniofacial skeleton through novel vectors as compared to traditional tobacco products. MC3T3-E1 murine pre-osteoblast cells were cultured invitro with clinically relevant liquid concentrations of nicotine, propylene glycol (PG), vegetable glycerin (VG), Nicotine+PG/VG, and the vape liquid of a commercial product (Juul). Cells were treated acutely for 24 h and RNA-Seq was utilized to determine segregating alteration in mRNA signaling. Influential gene targets identified with sparse partial least squares discriminant analysis (sPLS-DA) implemented in mixOmics were assessed using the PANTHER Classification system for molecular functions, biological processes, cellular components, and pathways of effect. Additional endpoint functional analyses were used to confirm cell cycle changes. The initial excitatory concentration (EC50) studied defined a target concentration of carrier PG/VG liquid that altered the cell cycle of the calvarial cells. Initial sPLS-DA analysis demonstrated the segregation of nicotine and non-nicotine exposures utilized in our invitro modeling. Pathway analysis suggests a strong influence of nicotine exposures on cellular processes including metabolic processes and response to stimuli including autophagic flux. Further interrogation of the individual treatment conditions demonstrated segregation by treatment modality (Control, Nicotine, Carrier (PG+VG), Nicotine+PG/VG) along three dimensions best characterized by: latent variable 1 (PLSDA-1) showing strong segregation based on nicotine influence on cellular processes associated with cellular adhesion to collagen, osteoblast differentiation, and calcium binding and metabolism; latent variable 2 (PLSDA-2) showing strong segregation of influence based on PG+VG and Control influence on cell migration, survival, and cycle regulation; and latent variable 3 (PLSDA-3) showing strong segregation based on Nicotine and Control exposure influence on cell activity and growth and developmental processes. Further, gene co-expression network analysis implicates targets of the major pathway genes associated with bone growth and development, particularly craniofacial (FGF, Notch, TGFβ, WNT) and analysis of active subnetwork pathways found these additionally overrepresented in the Juul exposure relative to Nicotine+PG/VG. Finally, experimentation confirmed alterations in cell count, and increased evidence of cell stress (markers of autophagy), but no alteration in apoptosis. These data suggest concomitant treatment with Nicotine+PG/VG drives alterations in pre-osteoblast cell cycle signaling, specifically transcriptomic targets related to cell cycle and potentially cell stress. Although we suspected cell stress and well as cytotoxic effects of Nicotine+PG/VG, no great influence on apoptotic factors was observed. Further RNA-Seq analysis allowed for the direct interrogation of molecular targets of major pathways involved in bone and craniofacial development, each demonstrating segregation (altered signaling) due to e-cigarette-type exposure. These data have implications directed toward ENDS formulation as synergistic effects of Nicotine+PG/VG are evidenced here. Thus, future research will continue to interrogate how varied formulation of Nicotine+PG/VG affects overall cell functions in multiple vital systems.

Functional, patient-derived 3D tri-culture models of the uterine wall in a microfluidic array.

Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues? We present a three-dimensional (3D) physiologically relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue. As a highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial-myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist. We employed microfluidic technology to characterize multiple miniaturized models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were assessed. Endometrial (n = 9) and myometrial biopsies (n = 4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5 × 5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterized by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both insulin-like growth factor binding protein 1 (IGFBP-1) and osteopontin secretion in response to hormone stimulation. When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of the protocol, epithelial cells localized to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate (cAMP) increased secretion of IGFBP-1, which indicates stromal decidualization, and enhanced epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes. Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may have influenced the myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having a hysterectomy, thus may not include the deeper basalis region, which may limit the physiological mimicry of the final models. Our novel approach to modelling the uterine wall in 3D captures all of the main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis. C.B. was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant. C.J.H. was supported by a Wellbeing of Women project grant (RG2137), SRI/Bayer and Wellcome Trust IFFS3. D.K.H. was supported by a Wellbeing of Women project grant (RG2137) and MRC clinical research training fellowship (MR/V007238/1). M.Z. is Director and Co-Founder of ScreenIn3D Limited. The other authors declare no conflict of interest. N/A.

Abstract A047: Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer

Abstract OBJECTIVE: KRAS-driven inflammatory signaling in pancreatic ductal adenocarcinoma (PDAC) promotes cancer-associated fibroblast (CAF) polarization and an immunosuppressive tumor microenvironment (iTME), driving therapeutic resistance. IL1α, a downstream product of KRAS signaling, activates the pro-inflammatory CAF (iCAF) phenotype and correlates with worse survival. Using patient and PDAC mouse model single-cell RNA sequencing (scRNAseq) data, we have shown that the IL1 Receptor (IL1R1) is highly enriched in the fibroblast compartment. However, the role of IL1R1 in modulating iCAFs and the iTME remains poorly understood. We hypothesize that CAF-specific disruption of IL1R1 signaling will reprogram cellular components of the PDAC TME to overcome the immunosuppressive milieu and therapeutic resistance. METHODS: The effects of IL1R1 inhibition on iCAF modulation in vivo were determined in Ptf1aCre/+, LSL-KrasG12D/+, Tgfbr2flox/flox (PKT) mice treated with IL1R1 antagonist. To determine if CAF-specific silencing of IL1R1 recapitulates the TME remodeling after IL1R1 inhibition, we generated a fibroblast-specific IL1R1KO mouse model Col1a2Cre/+;Il1r1flox/flox (CAF-Il1r1KO) and orthotopically injected KPC tumor cells for endpoint analysis. Flow cytometry was performed in primary tumors of CAF-Il1r1KO and littermate controls. Functional experiments evaluating myeloid-derived suppressor cell (MDSC) trafficking were performed using ex vivo bone marrow-derived cells (BMDCs) co-cultured with CAF/tumor-conditioned media (CM) with/without IL1R1 inhibition in a transwell system. MDSC contribution to iTME was assessed by co-culturing MDSCs from CAF-Il1r1KO tumors and splenic T cells from naïve C57BL/6 mice. To investigate the effects of IL1R1 deletion in overcoming therapeutic resistance, survival studies in CAF-Il1r1KO mice with chemotherapy were conducted. RESULTS: Treatment of PKT mice with IL1R1 antagonist diminished transcriptional levels of Il6 and Cxcl1, iCAF-associated cytokines, in the CAF compartment. Compared to littermate controls, orthotopic tumors in CAF-Il1r1KO showed significant reduction in tumor weights, increased CD8+ T cell infiltration, and overall reduction in polymorphonuclear (PMN) MDSC trafficking by flow cytometry. Moreover, migration of BMDCs was reduced when co-cultured with CAF/tumor CM and IL1R1 antagonist. In addition, co-culture of MDSCs from CAF-Il1r1KO mice with naïve T cells showed diminished inhibition of T cell functional activity. Chemotherapy treatment of orthotopic tumors in CAF-Il1r1KO mice showed significantly decreased tumor size and improved survival compared to tumors in littermate controls. CONCLUSION: Our findings illustrate a novel mechanism of CAF-specific disruption of IL1R1 signaling inhibiting trafficking of immunosuppressive MDSCs and increased infiltration of cytotoxic CD8+ T cells in the PDAC TME. These data pave the way for novel combinatorial treatment strategies targeting IL1R1 to overcome chemotherapeutic resistance through reprogramming of the stromal compartment in PDAC. Citation Format: Camille Acevedo, Samara Singh, Austin Dosch, Edmond W Box, Iago C Silva, Anna Bianchi, Siddharth Mehra, Haleh Amirian, Andrew Adams, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B Merchant. Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A047.

TRNA lysidinylation is essential for the minimal translation system found in the apicoplast of Plasmodium falciparum.

For decades, researchers have sought to define minimal genomes to elucidate the fundamental principles of life and advance biotechnology. tRNAs, essential components of this machinery, decode mRNA codons into amino acids. The apicoplast of malaria parasites encodes 25 tRNA isotypes in its organellar genome - the lowest number found in known translation systems. Efficient translation in such minimal systems depends heavily on post-transcriptional tRNA modifications, especially at the wobble anticodon position. Lysidine modification at the wobble position (C34) of tRNACAU distinguishes between methionine (AUG) and isoleucine (AUA) codons, altering the amino acid delivered by this tRNA and ensuring accurate protein synthesis. Lysidine is formed by the enzyme tRNA isoleucine lysidine synthetase (TilS) and is nearly ubiquitous in bacteria and essential for cellular viability. We identified a TilS ortholog (PfTilS) located in the apicoplast of Plasmodium falciparum parasites. By complementing PfTilS with a bacterial ortholog, we demonstrated that the lysidinylation activity of PfTilS is critical for parasite survival and apicoplast maintenance, likely due to its impact on apicoplast protein translation. Our findings represent the first characterization of TilS in an endosymbiotic organelle, advancing eukaryotic organelle research and our understanding of minimal translational machinery. Due to the absence of lysidine modifications in humans, this research also exposes a potential vulnerability in malaria parasites that could be targeted by antimalarial strategies.

A Comparison of White and Yellow Seminal Plasma Phosphoproteomes Obtained from Turkey (Meleagris gallopavo) Semen.

Seminal plasma is rich in proteins originating from various male reproductive organs. The phosphorylation of these proteins can significantly impact sperm motility, capacitation, and acrosome reaction. Phosphoproteomics identifies, catalogues, and characterizes phosphorylated proteins. The phosphoproteomic profiling of seminal plasma offers valuable insights into the molecular mechanisms that influence semen quality and male fertility. Thus, the aim of this study was a phosphoproteomic analysis of white and yellow turkey seminal plasma. The experimental material consisted of 100 ejaculates from BIG-6 turkeys between 39 and 42 weeks of age. The collected white and yellow turkey seminal plasmas were analyzed for total protein content; the activity of selected enzymes, i.e., alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); and the content of reduced glutathione (GSH) and malondialdehyde (MDA). Phosphoproteins were isolated from white and yellow seminal fluids, and the resulting protein fractions were separated by SDS-PAGE and Western blotting. Phosphorylated residues were immunodetected, and the isolated phosphoproteins were identified (nano LC-MS/MS). Yellow seminal plasmas were characterized by higher levels of total protein, GSH, and MDA, as well as higher levels of ALP, ACP, and GPx activity. There were no significant differences in the activity of SOD and CAT. A total of 113 phosphoproteins were identified in turkey seminal fluids. The functional analysis demonstrated that these phosphoproteins were mainly involved in oocyte fertilization, organization and metabolism of the actin cytoskeleton, amplification of the intracellular signal transduction pathway, general regulation of transport, vesicular transport, proteome composition of individual cellular compartments, and the organization and localization of selected cellular components and macromolecules. Increased phosphorylation of the fractions containing proteins encoded by SPARC, PPIB, TRFE, QSOX1, PRDX1, PRDX6, and FASN genes in white plasmas and the proteins encoded by CKB, ORM2, APOA1, SSC5D, RAP1B, CDC42, FTH, and TTH genes in yellow plasmas was observed based on differences in the optical density of selected bands. The obtained results indicate that the phosphorylation profiles of turkey seminal plasma proteins vary depending on the type of ejaculate.

Biological Barriers for Drug Delivery and Development of Innovative Therapeutic Approaches in HIV, Pancreatic Cancer, and Hemophilia A/B.

Biological barriers remain a major obstacle for the development of innovative therapeutics. Depending on a disease's pathophysiology, the involved tissues, cell populations, and cellular components, drugs often have to overcome several biological barriers to reach their target cells and become effective in a specific cellular compartment. Human biological barriers are incredibly diverse and include multiple layers of protection and obstruction. Importantly, biological barriers are not only found at the organ/tissue level, but also include cellular structures such as the outer plasma membrane, the endolysosomal machinery, and the nuclear envelope. Nowadays, clinicians have access to a broad arsenal of therapeutics ranging from chemically synthesized small molecules, biologicals including recombinant proteins (such as monoclonal antibodies and hormones), nucleic-acid-based therapeutics, and antibody-drug conjugates (ADCs), to modern viral-vector-mediated gene therapy. In the past decade, the therapeutic landscape has been changing rapidly, giving rise to a multitude of innovative therapy approaches. In 2018, the FDA approval of patisiran paved the way for small interfering RNAs (siRNAs) to become a novel class of nucleic-acid-based therapeutics, which-upon effective drug delivery to their target cells-allow to elegantly regulate the post-transcriptional gene expression. The recent approvals of valoctocogene roxaparvovec and etranacogene dezaparvovec for the treatment of hemophilia A and B, respectively, mark the breakthrough of viral-vector-based gene therapy as a new tool to cure disease. A multitude of highly innovative medicines and drug delivery methods including mRNA-based cancer vaccines and exosome-targeted therapy is on the verge of entering the market and changing the treatment landscape for a broad range of conditions. In this review, we provide insights into three different disease entities, which are clinically, scientifically, and socioeconomically impactful and have given rise to many technological advancements: acquired immunodeficiency syndrome (AIDS) as a predominant infectious disease, pancreatic carcinoma as one of the most lethal solid cancers, and hemophilia A/B as a hereditary genetic disorder. Our primary objective is to highlight the overarching principles of biological barriers that can be identified across different disease areas. Our second goal is to showcase which therapeutic approaches designed to cross disease-specific biological barriers have been promising in effectively treating disease. In this context, we will exemplify how the right selection of the drug category and delivery vehicle, mode of administration, and therapeutic target(s) can help overcome various biological barriers to prevent, treat, and cure disease.

Preparation, physico-biochemical characterization, and proteomic analysis of highly transparent corneal extracellular matrices for lamellar keratoplasty and tissue-engineered cornea construction

Corneal opacity and deformation, which often require corneal transplantation for treatment, are among the leading causes of monocular blindness. To restore corneal clarity and integrity, there is a need for an artificial stroma that not only matches the transparency of donated human cornea but also effectively integrates to the corneal tissue. In this study, a transparent decellularized cornea was successfully developed using the high hydrostatic pressure method with processing conditions optimized for corneal decellularization. Biochemical analyses demonstrated the effective removal of cellular components from the transparent decellularized corneas, while preserving collagen and glycosaminoglycans. Proteome analysis also revealed that core matrisome and matrisome-associated proteins remained following decellularization, similar to the composition observed in untreated corneas. The light transmittance of the transparent decellularized corneas was 86.4 ± 1.5 % in the visible region, comparable to that of donated human corneas. No complications, such as angiogenesis, were observed following interlamellar corneal transplantation in rabbits. The grafts were almost imperceptible immediately following surgery and achieved complete transparency within a few days, becoming indistinguishable even under a microscope. The transparent decellularized cornea presented here has promising potential as a material for application in lamellar keratoplasty.

The yeast Dothiora sorbi IOJ-3 naturally produced various filamentous sectors with distinct abilities by undergoing DNA demethylation

Some fungi have demonstrated the ability to adapt rapidly to changing environments by exhibiting morphological plasticity, a trait influenced by species and environmental factors. Here, an anamorphic yeast strain IOJ-3 exhibited unique sectorization characteristics, naturally producing diverse filamentous sectors when cultivated on potato dextrose agar (PDA) medium or natural culture medium for durations exceeding 13 days. The strain IOJ-3 and its filamentous sectors were identified as Dothiora sorbi. The morphology of the sectors was consistent and heritable. The life cycle of strain IOJ-3 was investigated through microscopic observation, emphasizing the development of conidiogenous cells as a crucial stage, from which filamentous sectors originate. Some physiological characteristics of IOJ-3 and filamentous sectors are compared, and strain IOJ-3 has a higher antibiotic tolerance than two filamentous sectors, IOJ-3a expands faster on the culture medium, and IOJ-3b can penetrate cellophane. A transcriptomic analysis was conducted to investigate the differentially expressed genes between the yeast form IOJ-3 and its two filamentous sectors, revealing a total of 594 genes that exhibited consistent differential expression relative to IOJ-3, including 44 silencing genes in IOJ-3 that were activated. Gene Ontology analysis indicated that these differentially expressed genes were primarily associated with the cellular component category. Furthermore, adding 5-Azacytidine accelerated filamentous sectorization and increased the proportion of filamentous cells of strain IOJ-3 in PD liquid media, suggesting that the filamentous sectorization observed in strain IOJ-3 is linked to processes of DNA demethylation. In conclusion, this study sheds light on the biological characteristics of D. sorbi regarding morphological transitions and provides substantial direction for exploring genes related to fungal filamentous development.

DATA DRIVEN AND CELL SPECIFIC DETERMINATION OF NUCLEI-ASSOCIATED ACTIN STRUCTURE

In the specialized field of cellular mechanosignaling, the intricate relationship between the cytoskeleton and nucleus is crucial for determining the behavior and fate of mesenchymal stem cells (MSCs). Existing analytical methods for filamentous actin fibers (F-actin) suffer from issues of accuracy and reproducibility. To address this, our research introduces a significant advancement in image analysis by utilizing two deep-learning segmentation Convolutional Neural Networks (CNNs) based on U-Net architecture. This algorithm precisely quantifies F-actin and offers reproducible data from 3D confocal microscopy images. Our CNNs automatically extract the 3D shape of both the nucleus and individual actin fibers, thereby enabling the geometric reconstruction of these critical cellular components. We applied this methodology to MSCs exposed to low-intensity vibration, and hypothesized that mechanical stimuli will induce perinuclear actin remodeling. Confocal Z-stack images of MSCs were captured using a 40x oil lens and a Zeiss LSM 900 microscope. Nucleus and F-actin structures were stained with Hoechst 33342 and Phalloidin, respectively. Nucleus-based images were employed to mask and isolate actin fibers within the nuclear vicinity. A specialized deep-learning segmentation model was engineered within the PyTorch framework. For model training, a selective 2% of nuclei and F-actin images were annotated. Specifically, three layers from each of six representative nuclei, among approximately 500 cross-sectional layers, were meticulously selected; a similar approach was employed for actin. The model underwent 200 epochs of training. Post-training, the deep-learning model was employed to process all collected cross-sectional images of F-actin and nuclei. A subsequent analytical step involved the scrutiny of intersecting segmented fibers between consecutive layers for geometrically accurate actin fiber reconstruction. We analyzed 102 control and 110 vibration-treated mesenchymal stem cells (MSCs) by utilizing our deep-learning-based reconstruction algorithm on confocal Z-stack images. Our data revealed an 8% decrease in nuclear height from 5.73 µm to 5.28 µm (p < 0.001) upon application of low-intensity vibration. In contrast, the two groups observed no significant difference in nuclear volume. Accompanying these changes at the nuclear level, the following changes in the F-actin cytoskeleton were observed upon LIV application. The total number of actin filaments per cell increased from 117 to 150. The average volume of individual actin fibers rose from 0.40 µm³ to 0.43 µm³. Notably, the volume of fibers at the apical nuclear surface increased from 0.35 µm³ to 0.46 µm³. While the average length of actin fibers in the apical nuclear surface remained largely unchanged due to the addition of smaller fibers in LIV groups, the LIV group had a larger number of fibers that were larger than 15µm.

Demonstrating the staining ability of <i>Zingiber officinale</i> and <i>Curcuma longa</i> as replacement to Eosin Y in EA50

Nicholas Papanicolaou developed the Pap stain, a combination of three major stains used for cervical smears. Eosin-Azure (EA50), one of these stains, includes Eosin Y, Light Green SF, and Bismarck brown. This study explores the use of Curcuma longa extract as a substitute for Eosin Y in EA50. Cervical smears from premenopausal women were collected and stained using both the standard Papanicolaou stain and a modified version where Eosin Y was replaced with Curcuma longa extract. The modified stain's effectiveness was assessed based on the quality of staining of superficial cells. The modified stain (T.A- extract) successfully stained and highlighted superficial cells, while the Zingiber officinale extract (Z.A) performed poorly in staining the nuclei and cytoplasm. The staining ability of the Curcuma longa extract was found to be 100% comparable to the traditional Papanicolaou stain. Staining aims to clearly demonstrate cellular components for diagnostic purposes, and many existing stains are long-established but require modifications for better application. Using local alternatives can enhance availability and affordability. Curcuma longa is readily available and, based on our findings, can effectively replace Eosin Y in Eosin-Azure stains. Further research into the dye components of herbal products and their staining capabilities could lead to cost-effective, locally sourced alternatives to imported stains.

Transduced Olfactory Mucosa Cells Expressing Nerve Growth Factor for the Therapy of Experimental Spinal Cord Cysts.

A new gene-cell construct expressing nerve growth factor (NGF) has been developed. After obtaining engineered adenovectors Ad5-RGD-CAG-NGF and Ad5-RGD-CAG-EGFP, transduction efficiency and transgene expression were studied and multiplicity of infection was determined. The efficacy of transduced human olfactory ensheathing cells expressing NGF in restoring motor activity in rats has been shown in a limited period of time. Improved rat hindlimb mobility and cyst size reduction after gene-cell construct transplantation were more likely due to the cellular component of the construct.

Polyphenol-Mediated Modulation of Non-Coding RNAs: A New Therapeutic Approach for Hypertension - A Review.

Hypertension (HTN) is a leading risk factor for cardiovascular diseases (CVDs) and a major contributor to global morbidity and mortality. Conventional pharmacological treatments have been effective but are often accompanied by side effects and do not address all pathological aspects of the disease. Recent advances in molecular biology have identified non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), as key regulators in the pathogenesis of hypertension. These ncRNAs influence various cellular processes, such as gene expression, vascular tone, and inflammation, making them promising targets for therapeutic intervention. This review explores the potential of polyphenols, a diverse group of phytochemicals with potent antioxidant and anti-inflammatory properties, in modulating ncRNA expression and function. We discuss how polyphenols, such as epigallocatechin-3-gallate (EGCG), resveratrol, curcumin, and quercetin impact the regulation of ncRNAs, particularly focusing on their roles in reducing oxidative stress, improving endothelial function, and ameliorating vascular remodeling associated with hypertension. The review synthesizes current evidence from both in vitro and in vivo studies, highlighting significant findings and the mechanisms by which polyphenols exert their effects on ncRNA-mediated pathways. Moreover, we address the challenges of translating these findings into clinical applications, including issues related to bioavailability, dosing, and the complex interactions of polyphenols with other cellular components. Future directions for research are suggested, with an emphasis on the need for comprehensive clinical trials to establish the efficacy of polyphenol-based therapies targeting ncRNAs in hypertension management. By targeting ncRNAs, polyphenols offer a novel therapeutic strategy that could enhance the treatment landscape for hypertension and potentially other cardiovascular conditions.

Antifungal potential, mechanism of action, and toxicity of 1,4-naphthoquinone derivatives.

The rising prevalence of fungal infections and challenges such as adverse effects and resistance against existing antifungal agents have driven the exploration of new antifungal substances. We specifically investigated naphthoquinones, known for their broad biological activities and promising antifungal capabilities. It specifically examined the effects of a particular naphthoquinone on the cellular components of Candida albicans ATCC 60193. The study also assessed cytotoxicity in MRC-5 cells, Artemia salina, and the seeds of tomatoes and arugula. Among four tested naphthoquinones, 2,3-DBNQ (2,3-dibromonaphthalene-1,4-dione) was identified as highly effective, showing potent antifungal activity at concentrations between 1.56 and 6.25 μg mL-1. However, its cytotoxicity in MRC-5 cells (IC50 = 15.44 µM), complete mortality in A. salina at 50 μg mL-1, and significant seed germination inhibition suggest limitations for its clinical use. The findings indicate that primary antifungal mechanism of 2,3-DBNQ might involve disrupting fungal membrane permeability, which leads to increased nucleotide leakage. This insight underscores the need for further research to enhance the selectivity and safety of naphthoquinones for potential therapeutic applications.

Spatial resolved transcriptomics reveals distinct cross-talk between cancer cells and tumor-associated macrophages in intrahepatic cholangiocarcinoma

BackgroundMultiple studies have shown that tumor-associated macrophages (TAMs) promote cancer initiation and progression. However, the reprogramming of macrophages in the tumor microenvironment (TME) and the cross-talk between TAMs and malignant subclones in intrahepatic cholangiocarcinoma (iCCA) has not been fully characterized, especially in a spatially resolved manner. Deciphering the spatial architecture of variable tissue cellular components in iCCA could contribute to the positional context of gene expression containing information pathological changes and cellular variability.MethodsHere, we applied spatial transcriptomics (ST) and digital spatial profiler (DSP) technologies with tumor sections from patients with iCCA.ResultsThe results reveal that spatial inter- and intra-tumor heterogeneities feature iCCA malignancy, and tumor subclones are mainly driven by physical proximity. Tumor cells with TME components shaped the intra-sectional heterogenetic spatial architecture. Macrophages are the most infiltrated TME component in iCCA. The protein trefoil factor 3 (TFF3) secreted by the malignant subclone can induce macrophages to reprogram to a tumor-promoting state, which in turn contributes to an immune-suppressive environment and boosts tumor progression.ConclusionsIn conclusion, our description of the iCCA ecosystem in a spatially resolved manner provides novel insights into the spatial features and the immune suppressive landscapes of TME for iCCA.

Adhesive and cohesive fracture of blood clots: Experiments and modeling

Blood clots represent living materials composed of a polymer network and an abundance of cells. They might fracture within the bulk material of the clot (cohesive fracture), at the interface between the clot and the surrounding tissue (adhesive fracture), or through a combination of both modes (hybrid fracture). The clot fracture within vascular systems and injury sites could lead to life-threatening conditions. Despite the significance, understanding and modeling the fracture behaviors of blood clots, including their dependence on mechanical loading and cellular components, remain in a nascent stage. In this study, we employ an integrated experimental-computational approach to comprehensively investigate the fracture behaviors of bovine blood clots. We explore various mechanical factors, substrates, and cellular components such as red blood cells (RBCs) and platelets. Our findings reveal that among various tissue substrates, blood clots exhibit the highest interfacial adhesion energy with muscle, and the lowest to the inner arterial lining, consistent with their biological function. Both interfacial adhesion energy and bulk fracture energy are rate-dependent, although they exhibit different dependencies. Also, RBCs and platelets have different effects on clot fracture. An increase in RBC content tends to toughen both adhesion and fracture of blood clots. However, an increase in platelet content enhances interfacial adhesion energy but lowers the bulk fracture energy. The platelet content also governs the shift from adhesive fracture to hybrid fracture. To model clot fracture, we developed two finite element models incorporating a coupled cohesive-zone and Mullins-effect approach to simulate pure shear fracture and peeling of blood clots. These models, validated through experimental data, elucidate the interplay between intrinsic fracture toughness, interfacial strength, and bulk energy dissipation during clot fracture. This study significantly advances our understanding of clot mechanics, providing valuable insights into the mechanics of similar living materials and the management of clot-related disorders such as hemorrhage and thrombosis.

Niabella digestorum sp. nov., a High Cell-Surface Hydrophobic Bacterium Isolated from Waste Digestion System.

A high cell-surface hydrophobic bacterium, strain A18T, was isolated from a waste digestion system in Chaozhou, China. Cells of strain A18T were Gram-stain-positive, aerobic, non-spore-forming, non-motile, and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene showed that strain A18T shared less than 94.2% sequence similarity to all validated species in the family Chitinophagaceae, and formed a distinct lineage close to genera Niabella and Terrimonas in the neighbor-joining tree, indicating that strain A18T is a novel species. Genome-based phylogenetic analyses revealed that strain A18T is affiliated to the genus Niabella. The cellular components, including iso-C15:0 and iso-C15:1 G as the major fatty acids, menaquinone-7 as the respiratory quinone and a DNA G + C content of 40.54% supported strain A18T as a member of the genus Niabella. However, the physiological and biochemical properties, such as enzyme activities, carbon source utilization and C18:0 3-OH as another major fatty acids, distinguished strain A18T from its close related species. Therefore, the name Niabella digestorum sp. nov. is proposed for this novel species. The type strain is A18T (= GDMCC 1.3242T = KCTC 92386T).

Characterizing the nonmonotonic behavior of mutual information along biochemical reaction cascades.

Cells sense environmental signals and transmit information intracellularly through changes in the abundance of molecular components. Such molecular abundances can be measured in single cells and exhibit significant heterogeneity in clonal populations even in identical environments. Experimentally observed joint probability distributions can then be used to quantify the covariability and mutual information between molecular abundances along signaling cascades. However, because stationary state abundances along stochastic biochemical reaction cascades are not conditionally independent, their mutual information is not constrained by the data-processing inequality. Here, we report the conditions under which the mutual information between stationary state abundances increases along a cascade of biochemical reactions. This nonmonotonic behavior can be intuitively understood in terms of noise propagation and time-averaging stochastic fluctuations that are short-lived compared to an extrinsic signal. Our results reemphasize that mutual information measurements of stationary state distributions of cellular components may be of limited utility for characterizing cellular signaling processes because they do not measure information transfer.