Maintenance Of Cell Morphology Research Articles

Significant alleviation of cadmium toxicity in Thalassiosira weissflogii through the combined effect of high silicon and zinc supplementation

Cadmium (Cd), a prevalent pollutant in near-shore seawater ecosystems, poses a significant threat to marine biota. Zinc (Zn) and Cd can have interchangeable physiological effects in marine plankton, and both can influence the synthesis of siliceous walls. Consequently, the concentrations of zinc and silicon may jointly impact the toxicity of Cd. This study endeavors to elucidate the combined effect of silicon (Si) and zinc (Zn) on the growth and physiological responses of the marine diatom species Thalassiosira weissflogii when subjected to Cd-induced stress conditions (190.7 μg L−1). Our results reveal statistically improvements (p<0.05) in biomass production, reductions (p<0.05) in superoxide dismutase (SOD) activity and extracellular secretion in T. weissflogii when high concentrations of Si (172 µmol L−1) and Zn (18.3 nmol L−1) were applied simultaneously. Additionally, examination of Cd bioconcentration factors (BCF) derived from Cd uptake kinetics and intracellular Cd content measurements underscores the ability of elevated Si and Zn concentrations to reduce intracellular Cd accumulation (p<0.05). The ability of Si and Zn to mitigate Cd toxicity was further evidenced by increased cellular biosilica content and maintenance of cell morphology, suggesting a protective role in preserving structural integrity and growth. Our findings underscore the synergistic benefits of Si and Zn in enhancing the resilience of T. weissflogii to Cd stress, providing valuable insights into the potential use of nutrient amendments as a strategy for mitigating heavy metal contamination in marine environments.

Diffusion model predicts the geometry of actin cytoskeleton from cell morphology.

Cells exhibit various morphological characteristics due to their physiological activities, and changes in cell morphology are inherently accompanied by the assembly and disassembly of the actin cytoskeleton. Stress fibers are a prominent component of the actin-based intracellular structure and are highly involved in numerous physiological processes, e.g., mechanotransduction and maintenance of cell morphology. Although it is widely accepted that variations in cell morphology interact with the distribution and localization of stress fibers, it remains unclear if there are underlying geometric principles between the cell morphology and actin cytoskeleton. Here, we present a machine learning system that uses the diffusion model to convert the cell shape to the distribution and alignment of stress fibers. By training with corresponding cell shape and stress fibers datasets, our system learns the conversion to generate the stress fiber images from its corresponding cell shape. The predicted stress fiber distribution agrees well with the experimental data. With this conversion relation, our system allows for performing virtual experiments that provide a visual map showing the probability of stress fiber distribution from the virtual cell shape. Our system potentially provides a powerful approach to seek further hidden geometric principles regarding how the configuration of subcellular structures is determined by the boundary of the cell structure; for example, we found that the stress fibers of cells with small aspect ratios tend to localize at the cell edge while cells with large aspect ratios have homogenous distributions.

Autism risk gene Cul3 alters neuronal morphology via caspase-3 activity in mouse hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.

HAI-1 is required for the novel role of FGFBP1 in maintenance of cell morphology and F-actin rearrangement in human keratinocytes.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

Adhesion and Colonization of the Probiotic Lactobacillus plantarum HC-2 in the Intestine of Litopenaeus Vannamei Are Associated With Bacterial Surface Proteins.

Surface proteins are a type of proteins expressed on the surface of bacteria that play an important role in cell wall synthesis, maintenance of cell morphology, and signaling with the host. Our previous study showed that the probiotic Lactobacillus plantarum HC-2 improved the growth performance and immune response of Litopenaeus vannamei. To further investigate the probiotic mechanism, we determined the automatic aggregation ability of the bacteria and surface hydrophobicity of HC-2 after being treated with 5 M of lithium chloride (LiCl) and observed the morphology and adhesion of the bacteria to HCT116 cells. The results showed that with the removal of the HC-2 surface protein, the auto-aggregation ability and surface hydrophobicity of HC-2 decreased, and the crude mucus layer coated on the bacterial surface gradually dissociated. The adhesion rate of HC-2 to HCT116 cells decreased from 98.1 to 20.9%. Moreover, a total of 201 unique proteins were identified from the mixture of the surface proteins by mass spectrometry (MS). Several proteins are involved in transcription and translation, biosynthetic or metabolic process, cell cycle or division, cell wall synthesis, and emergency response. Meanwhile, a quantitative real-time PCR qPCR_ showed that HC-2 was mainly colonized in the midgut of shrimp, and the colonization numbers were 15 times higher than that in the foregut, while the colonization rate in the hindgut was lower. The adhesion activity measurement showed that the adhesion level of HC-2 to crude intestinal mucus of L. vannamei was higher than that of bovine serum albumin (BSA) and collagen, and the adhesion capacity of the bacterial cells decreased with the extension of LiCl-treatment time. Finally, we identified the elongation factor Tu, Type I glyceraldehyde-3-phosphate dehydrogenase, small heat shock protein, and 30S ribosomal protein from the surface proteins, which may be the adhesion proteins of HC-2 colonization in the shrimp intestine. The above results indicate that surface proteins play an important role in maintaining the cell structure stability and cell adhesion. Surface proteomics analysis contributes to describing potential protein-mediated probiotic-host interactions. The identification of some interacting proteins in this work may be beneficial to further understand the adhesion/colonization mechanism and probiotic properties of L. plantarum HC-2 in the shrimp intestine.

Three-Dimensional Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Promotes Matrix Metallopeptidase 13 (MMP13) Expression in Type I Collagen Hydrogels.

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

Ionomic and Metabolomic Analyses Reveal Different Response Mechanisms to Saline-Alkali Stress Between Suaeda salsa Community and Puccinellia tenuiflora Community.

Soil salinization imposes severe stress to plants, inhibits plant growth, and severely limits agricultural productivity and land utilization. The response of a single plant to saline-alkali stress has been well investigated. However, the plant community that usually works as a group to defend against saline–alkali stress was neglected. To determine the functions of plant community, in our current work, Suaeda salsa (S. salsa) community and Puccinellia tenuiflora (P. tenuiflora) community, two communities that are widely distributed in Hulun Buir Grassland in Northeastern China, were selected as research objects. Ionomic and metabolomic were applied to compare the differences between S. salsa community and P. tenuiflora community from the aspects of ion transport and phenolic compound accumulation, respectively. Ionomic studies demonstrated that many macroelements, including potassium (K) and calcium (Ca), were highly accumulated in S. salsa community whereas microelement manganese (Mn) was highly accumulated in P. tenuiflora community. In S. salsa community, transportation of K to aboveground parts of plants helps to maintain high K+ and low Na+ concentrations whereas the accumulation of Ca triggers the salt overly sensitive (SOS)-Na+ system to efflux Na+. In P. tenuiflora community, enrichment of Mn in roots elevates the level of Mn-superoxide dismutase (SOD) and increases the resistance to saline–alkali stress. Metabolomic studies revealed the high levels of C6C1-compounds and C6C3C6-compounds in S. salsa community and also the high levels of C6C3-compounds in P. tenuiflora community. C6C1-compounds function as signaling molecules to defend against stress and may stimulate the accumulation of C6C3C6-compounds. C6C3-compounds contribute to the elimination of free radicals and the maintenance of cell morphology. Collectively, our findings determine the abundance of phenolic compounds and various elements in S. salsa community and P. tenuiflora community in Hulun Buir Grassland and we explored different responses of S. salsa community and P. tenuiflora community to cope with saline–alkali stress. Understanding of plant response strategies from the perspective of community teamwork may provide a feasible and novel way to transform salinization land.

Actin-Binding Proteins as Potential Biomarkers for Chronic Inflammation-Induced Cancer Diagnosis and Therapy.

Actin-binding proteins (ABPs), by interacting with actin, regulate the polymerization, depolymerization, bundling, and cross-linking of actin filaments, directly or indirectly, thereby mediating the maintenance of cell morphology, cell movement, and many other biological functions. Consequently, these functions of ABPs help regulate cancer cell invasion and metastasis when cancer occurs. In recent years, a variety of ABPs have been found to be abnormally expressed in various cancers, indicating that the detection and interventions of unusual ABP expression to alter this are available for the treatment of cancer. The early stages of most cancer development involve long-term chronic inflammation or repeated stimulation. This is the case for breast cancer, gastric cancer, lung cancer, prostate cancer, liver cancer, esophageal cancer, pancreatic cancer, melanoma, and colorectal cancer. This article discusses the relationship between chronic inflammation and the above-mentioned cancers, emphatically introduces relevant research on the abnormal expression of ABPs in chronic inflammatory diseases, and reviews research on the expression of different ABPs in the above-mentioned cancers. Furthermore, there is a close relationship between ABP-induced inflammation and cancer. In simple terms, abnormal expression of ABPs contributes to the chronic inflammation developing into cancer. Finally, we provide our viewpoint regarding these unusual ABPs serving as potential biomarkers for chronic inflammation-induced cancer diagnosis and therapy, and interventions to reverse the abnormal expression of ABPs represent a potential approach to preventing or treating the corresponding cancers.

Tailoring the growth and proliferation of human dermal fibroblasts by DNA-based polymer films for skin regeneration.

The use of DNA as a functional biomaterial for therapeutic, diagnostic, and drug delivery applications has been prominent in recent years, but its use as a scaffold for tissue regeneration is still limited. This study aimed to evaluate the biocompatibility and interaction of DNA-based polymeric films (DNA-PFs) with primary human fibroblasts (PHF) for regenerative medicine and wound healing purposes. The morphological characterization of the films was performed by scanning electron microscopy, SEM-energy-dispersive X-ray spectroscopy, and atomic force microscopy analysis. Cell viability, cell cycle kinetics, oxidative stress, and migration studies were carried out at 48 and 72 hr of incubation and compared to control cells. Cell adhesion was impaired in the first 24 hr, DNA-PFs with higher concentrations of DNA (1.0 and 2.0g/L) this effect was not seen in DNA-PFs (0.5g/L), explained by the difference in topography and roughness of DNA-PFs, but it was overcome after 48 hr of incubation. PHF seeded on DNA films showed higher proliferation and migration rates than the control after 48 hr of incubation, with the maintenance of cell morphology and lower cytotoxicity and oxidative stress during the evaluation time. Therefore, these results indicate that DNA-PFs are highly biocompatible and provide a suitable microenvironment for dermal fibroblasts to maintain their activity, helping build new and more complex biomaterials suitable for future tissue repair applications.

PHOTOBIOMODULATION INCREASES THE VIABILITY AND PROLIFERATION OF HUMAN PERIODONTAL LIGAMENT STEM CELLS CULTURED ON THE SURFACE OF POLYLATIC ACID FILMS

<h3>Backgorund</h3> Polylactic acid (PLA) is a biomaterial with diverse biomedical applications and has been a promising scaffold in tissue engineering mainly due to its biocompatibility, easy manipulation and low cost. Photobiomodulation using low-level laser irradiation has been shown to be a powerful tool to promote in vitro biostimulation in several cell types. The aim of this study was to evaluate the effectiveness of photobiomodulation on the viability and proliferation of human periodontal ligament stem cells (hPDLSC) cultured on PLA scaffolds. <h3>Methods</h3> PLA films were produced by solvent casting method and the surface topography was evaluated by scanning electronic microscopy (SEM) and atomic force microscopy (AFM). hPDLSC were isolated, characterized and cultured on the PLA films. Two groups were evaluated: Control - non irradiated; and Laser - irradiated with diode laser (InGaAIP) with wavelength of 660 nm, power of 30 mW, and a single dose of 1 J/cm² with radiation emitted continuously. Cell viability analyzes were performed 24 and 48 hours after irradiation using the the Alamar Blue biochemical assay and Live/Dead assay. Cell cycle events were assessed by flow cytometry and cell-biomaterial morphological interaction was evaluated by SEM. <h3>Results</h3> The films produced showed a flat and regular surface, with the occasional presence of small pores and an average roughness of 59.381 nm. The results of Alamar Blue assay showed a greater cell metabolic activity in irradiated group compared to control at 24 (<i>p</i><0.05) and 48 h (<i>p</i><0.001), which was confirmed in the Live/Dead assay by a higher density of viable cells in the Laser group. In the analysis of the cell cycle, the Laser group showed an increase of cells in the G2/M phase compared to the Control group (<i>p</i> <0.001). SEM images showed a higher cell density in the irradiated group, with maintenance of cell morphology and projections. <h3>Conclusion</h3> Taken together, the findings of this study demonstrated that photobiomodulation has the ability to increase the viability and proliferation of periodontal stem cells cultured on PLA scaffolds, which may contribute to the development of new studies using this protocol in periodontal tissue engineering.

EGCG-S Impacts Oxidative Stress and Infection of Enterovirus 69 in Lung Cells

Enteroviruses are responsible for emerging diseases which cause diverse symptoms and may result in neurological complications. An antiviral with multiple mechanisms of action can help prevent enterovirus mediated disease despite differences in the pathogenesis between enteroviruses, including the recently identified enterovirus 69 (EV-69) for which pathogenesis is not well understood. This study investigated the efficacy of epigallocatechin-3-gallate stearate (EGCG-S), a modified form of the antioxidant green tea catechin epigallocatechin-3-gallate (EGCG), in inhibiting EV-69 infection of lung fibroblast cells in vitro. Treatment with EGCG-S resulted in moderate protection from EV-69 mediated cytotoxicity as demonstrated by increased metabolic activity as well as maintenance of cell morphology and mitochondrial function. These effects were correlated with reduced hydrogen peroxide production in infected cells following EGCG-S treatment with concentrations less than 100 μM, suggesting a role for inhibition of EV-69 mediated oxidative stress. This study provides insight into characteristics of EV-69 infection as well as the efficacy of EGCG-S mediated inhibition of EV-69 infection.

Recombinant transgelin-like protein 1 from Mytilus shell induces formation of CaCO3 polymorphic crystals in vitro.

Transgelin is an actin cross-linking/gelling protein of the calponin family, which is associated with actin stress fibres, cell motility, adhesion and the maintenance of cell morphology. Transgelin-like proteins (TLPs) have also been identified as shell matrix proteins (SMPs) in several mollusc species; however, the functions of TLPs in biomineralization remain unknown. Transgelin-like protein 1 (TLP-1) was previously identified from the shell of Mytilus coruscus as a novel 19 kDa SMP with a calponin homology (CH) domain. To understand the role of TLP-1 in shell formation, the expression level and localization of the TLP-1 gene in biomineralization-related tissues were determined in this study. Furthermore, recombinant TLP-1 was expressed in a prokaryotic expression system with codon optimization, and an anti-rTLP-1 antibody was prepared based on the expressed recombinant TLP-1 (rTLP-1) protein. In vitro, rTLP-1 induced the formation of CaCO3 polymorphic crystals with distinct morphologies and inhibited crystallization rate and crystal interactions. Immunohistochemical, immunofluorescence, and pull-down analyses using the anti-rTLP-1 antibody revealed the specific locations of TLP-1 in biomineralization-related tissues and shell myostracum layer, and suggested the existence of a possible TLP-1 interaction network in the shell matrix. Our results are beneficial for understanding the functions of TLP-1, particularly through its CH domain, during shell mineralization.

The Effect of Glutathione on Itraconazole‐Associated Hepatocyte Toxicity in an In Vitro Canine Model

BackgroundItraconazole (ITZ) is used to treat many fungal infections in dogs. However, therapy is associated with dose‐dependent increases in liver enzymes in up to 42% of dogs. Glutathione (GSH) precursors are commonly used to treat ITZ‐associated hepatotoxicity, although no studies have investigated the efficacy of this practice.ObjectiveTo identify the effect of GSH on cell survival in an in vitro model of ITZ‐associated hepatotoxicity using canine primary hepatocytes.MethodsUsing a sandwich culture technique, pooled canine primary hepatocytes were incubated for 24 hours with 10‐fold dilutions of GSH (0 to 500 μM). Following GSH pre‐incubation, the cells were exposed to 5‐fold dilutions of ITZ (0 to 50 μM). Cell viability was determined using the neutral red assay after 4 and 24 hours of ITZ exposure (n=3). The effect of ITZ concentration, time, and GSH concentration on cytotoxicity was assessed using multivariate linear regression. Hepatocyte phenotype was assessed by measurement of albumin production using an enzyme‐linked immunosorbent assay (ELISA) kit for canine albumin and morphological assessment via phase contrast microscopy.ResultsCanine hepatocyte cytotoxicity significantly increased with ITZ concentration (p&lt;0.001) and time (p=0.004). Whereas, hepatocyte cytotoxicity significantly decreased with GSH treatment (p&lt;0.001). Maintenance of hepatocyte phenotype was confirmed by continued production of albumin and maintenance of cell morphology over time.ConclusionsThe in vitro model demonstrates dose‐ and time‐dependent ITZ cytotoxicity. Pre‐treating with GSH provides a protective effect against cell death in this model. These results suggest that GSH precursors may have a role in the management or prevention ITZ‐associated hepatotoxicity in dogs. Clinical trials are needed to evaluate their utility for this adverse drug reaction.

Transgelin Silencing Induces Different Processes in Different Breast Cancer Cell Lines.

Transgelin is a protein reported to be a marker of several cancers. However, previous studies have shown both up- and down-regulation of transgelin in tumors when compared with non-tumor tissues and the mechanisms whereby transgelin may affect the development of cancer remain largely unknown. Transgelin is especially abundant in smooth muscle cells and is associated with actin stress fibers. These contractile structures participate in cell motility, adhesion, and the maintenance of cell morphology. Here, the role of transgelin in breast cancer is focused on. Initially, the effects of transgelin on cell migration of the breast cancer cell lines, BT 549 and PMC 42, is studied. Interestingly, transgelin silencing increased the migration of PMC 42 cells, but decreased the migration of BT 549 cells. To clarify these contradictory results, the changes in protein abundances after transgelin silencing in these two cell lines are analyzed using quantitative proteomics. The results confirmed the role of transgelin in the migration of BT 549 cells and suggest the involvement of transgelin in apoptosis and small molecule biochemistry in PMC 42 cells. The context-dependent function of transgelin reflects the different molecular backgrounds of these cell lines, which differ in karyotypes, mutation statuses, and proteome profiles.

Progress in brewer's yeast cell wall stress response

Yeast cell wall plays an important role in the establishment and maintenance of cell morphology upon the cell wall stress. The cell wall of yeast consists of β-glucans, mannoproteins and chitin. The composition and structure remodel due to cell wall stress. Brewer's yeast cell wall exhibits stress response during long-term acclimation in order to adapt to environmental changes. This paper reviews the composition and structure of yeast cell wall and the molecular mechanisms of cell wall remodeling and signal pathway regulation.

Defining housekeeping genes suitable for RNA-seq analysis of the human allograft kidney biopsy tissue

BackgroundRNA-seq is poised to play a major role in the management of kidney transplant patients. Rigorous definition of housekeeping genes (HKG) is essential for further progress in this field. Using single genes or a limited set HKG is inherently problematic since their expression might be altered by specific diseases in the patients being studied.MethodsTo generate a HKG set specific for kidney transplantation, we performed RNA-sequencing from renal allograft biopsies collected in a variety of clinical settings. Various normalization methods were applied to identify transcripts that had a coefficient of variation of expression that was below the 2nd percentile across all samples, and the corresponding genes were designated as housekeeping genes. Comparison with transcriptomic data from the Gene Expression Omnibus (GEO) database, pathway analysis and molecular biological functions were utilized to validate the housekeeping genes set.ResultsWe have developed a bioinformatics solution to this problem by using nine different normalization methods to derive large HKG gene sets from a RNA-seq data set of 47,611 transcripts derived from 30 biopsies. These biopsies were collected in a variety of clinical settings, including normal function, acute rejection, interstitial nephritis, interstitial fibrosis/tubular atrophy and polyomavirus nephropathy. Transcripts with coefficient of variation below the 2nd percentile were designated as HKG, and validated by showing their virtual absence in diseased allograft derived transcriptomic data sets available in the GEO. Pathway analysis indicated a role for these genes in maintenance of cell morphology, pyrimidine metabolism, and intracellular protein signaling.ConclusionsUtilization of these objectively defined HKG data sets will guard against errors resulting from focusing on individual genes like 18S RNA, actin & tubulin, which do not maintain constant expression across the known spectrum of renal allograft pathology.

Gene expression patterns in synchronized islet populations

ABSTRACTIn vivo levels of insulin are oscillatory with a period of ~5–10 minutes, indicating that the islets of Langerhans within the pancreas are synchronized. While the synchronizing factors are still under investigation, one result of this behavior is expected to be coordinated and oscillatory intracellular factors, such as intracellular Ca2+ levels, throughout the islet population. In other cell types, oscillatory intracellular signals, like intracellular Ca2+, have been shown to affect specific gene expression. To test how the gene expression landscape may differ between a synchronized islet population with its reproducible intracellular oscillations and an unsynchronized islet population with heterogeneous oscillations, gene set enrichment analysis (GSEA) was used to compare an islet population that had been synchronized using a glucose wave with a 5-min period, and an unsynchronized islet population. In the population exposed to the glucose wave, 58/62 islets showed synchronization as evidenced by coordinated intracellular Ca2+ oscillations with an average oscillation period of 5.1 min, while in the unsynchronized population 29/62 islets showed slow oscillations with an average period of 5.2 min. The synchronized islets also had a significantly smaller drift of their oscillation period during the experiment as compared to the unsynchronized population. GSEA indicated that the synchronized population had reduced expression of gene sets related to protein translation, protein turnover, energy expenditure, and insulin synthesis, while those that were related to maintenance of cell morphology were increased.

Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR.

Ras-related C3 botulinum toxin substrate 1 (Rac1) plays critical roles in the maintenance of cell morphology by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Rac1 P29S mutant is known to strongly promote oncogenesis by facilitating its intrinsic GDP dissociation and thereby increasing the level of the GTP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the oncogenic P29S mutant. We demonstrate that the conformational landscape is markedly altered in the mutant, and the preexisting equilibrium is shifted toward the conformation with reduced affinity for Mg2+ , a cofactor that is critical for maintaining stable GDP binding. Our results suggest that the alternation of the preexisting conformational equilibrium of proteins is one of the fundamental mechanisms underlying their oncogenic activities.

Molecular mechanism of crolibulin in complex with tubulin provides a rationale for drug design

Microtubules (MTs) is one of the most important proteins in eukaryotic cells and plays a key role in the maintenance of cell morphology and cell division. The discovery and development of small molecule drugs targeting MTs has always been an important direction of anti-cancer research. Nowadays 4-Aryl-4H-chromenes have emerged as potent microtubule-targeting agents (MTAs) for various cancers. Crolibulin, a derivative of 4-Aryl-4H-chromenes, which has been progressed to Phase I/II clinical testing's for anaplastic thyroid cancer with the National Cancer Institute. However, the design and development of 4-Aryl-4H-chromenes family drugs have been hindered for a long time by the lack of structural information of the tubulin-agent complex. Here we report a 2.5 Å crystal structure of tubulin complexed with crolibulin. This complex structure reveals the interactions between crolibulin and tubulin, helps explain the results of the structure-activity-relationship (SAR) studies and provides a solid structural basis for the design and development of new 4-Aryl-4H-chromenes derivatives as MTAs.

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod‐shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin‐like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton‐controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.