Expression Of Matrix-related Genes Research Articles

Potential of combined red and near-infrared photobiomodulation to mitigate pro-osteoclastic and inflammatory gene expression in human mandibular osteogenic cells

Appropriate regeneration of jawbone after dental or surgical procedures relies on the recruitment of osteoprogenitor cells able to differentiate into matrix-producing osteoblasts. In this context, photobiomodulation (PBM) has emerged as promising therapy to improve tissue regeneration and to facilitate wound healing processes. The aim of this study was to determine the effect of PBM on human osteoprogenitor cells isolated from mandibular trabecular bone.Bone marrow stromal cell cultures were established from 4 donors and induced toward osteogenic differentiation for 14 days in a standard osteogenic assay. Cells were irradiated with a combined red/near-infrared (NIR) laser following different schedules and expression of osteogenic, matrix-related, osteoclastogenic and inflammatory genes was analyzed by quantitative PCR.Gene expression analysis revealed no overall effects of PBM on osteogenic differentiation. However, a statistically significant reduction was observed in the transcripts of COL1A1 and MMP13, two important genes involved in the bone matrix homeostasis. Most important, PBM significantly downregulated the expression of RANKL, IL6 and IL1B, three genes that are involved in both osteoclastogenesis and inflammation.In conclusion, PBM with a red/NIR laser did not modulate the osteogenic phenotype of mandibular osteoprogenitors but markedly reduced their expression of matrix-related genes and their pro-osteoclastogenic and pro-inflammatory profile.

Loss of periostin function impairs ligament fibroblast activity and facilitates ROS-mediated cellular senescence.

Anterior cruciate ligament (ACL) injuries pose a significant challenge due to their limited healing potential, often resulting in premature arthritis. The factors and mechanisms contributing to this inadequate healing process remain elusive. During the acute phase of injury, ACL tissues express elevated periostin levels that decline over time. The functional significance of periostin in ligament biology remains understudied. In this study, we investigated the functional and mechanistic implications of periostin deficiency in ACL biology, utilizing ligament fibroblasts derived from patients and a murine model of ACL rupture. Our investigations unveiled that periostin knockdown compromised fibroblast growth characteristics, hindered the egress of progenitor cells from explants, and arrested cell-cycle progression, resulting in the accumulation of cells in the G0/G1 phase and moderate apoptosis. Concurrently, a significant reduction in the expression of cell-cycle and matrix-related genes was observed. Moreover, periostin deficiency triggered apoptosis through STAT3Y705/p38MAPK signaling and induced cellular senescence through increased production of reactive oxygen species (ROS). Mechanistically, inhibition of ROS production mitigated cell senescence in these cells. Notably, invivo data revealed that ACL in Postn-/- mice exhibited a higher tearing frequency than wild-type mice under equivalent loading conditions. Furthermore, injured ACL with silenced periostin expression, achieved through nanoparticle-siRNA complex delivery, displayed an elevated propensity for apoptosis and senescence compared to intact ACL in C57BL/6 mice. Together, our findings underscore the pivotal role of periostin in ACL health, injury, and potential for healing.

Effects of different combinations of mechanical loading intensity, duration, and frequency on the articular cartilage in mice

BackgroundUnderstanding how healthy articular cartilage responds to mechanical loading is critical. Moderate mechanical loading has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical loading is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical loading on healthy knee articular cartilage in vitro and in vivo.Methods and resultsIn this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages.ConclusionLow-intensity, low-frequency, and long-duration mechanical loading is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions.

Osteoinductive activity of photobiomodulation in an organotypic bone model

Photobiomodulation (PBM) is a technique that harnesses non-ionizing light at specific wavelengths, triggering the modulation of metabolic pathways, engendering favourable biological outcomes that reduce inflammation and foster enhanced tissue healing and regeneration. PBM holds significant promise for bone tissue applications due to its non-invasive nature and ability to stimulate cellular activity and vascularization within the healing framework. Notwithstanding, the impact of PBM on bone functionality remains largely undisclosed, particularly in the absence of influencing factors such as pathologies or regenerative therapies. This study aims to investigate the potential effects of PBM using red (660 nm) (RED) and near-infrared (808 nm) (NIR) wavelengths within an ex vivo bone culture system - the organotypic embryonic chicken femur model. A continuous irradiation mode was used, administering a total energy dose of 1.0 J, at an intensity of 100 mW for 10 s, which was repeated four times over the course of the 11-day culture period. The primary focus is on characterizing the expression of pivotal osteoblastic genes, the maturation and deposition of collagen, and the formation of bone mineral. Exposing femora to both RED and NIR wavelengths led to a notable increase in the expression of osteochondrogenic transcription factors (i.e., SOX9 and RUNX2), correlating with enhanced mineralization. Notably, NIR irradiation further elevated the expression of bone matrix-related genes and fostered enhanced deposition and maturation of fibrillar collagen. This study demonstrates that PBM has the potential to enhance osteogenic functionality within a translational organotypic bone culture system, with the NIR wavelength showing remarkable capabilities in augmenting the formation and maturation of the collagenous matrix.

ASH2L, Core Subunit of H3K4 Methylation Complex, Regulates Amelogenesis

Histone methylation assumes a crucial role in the intricate process of enamel development. Our study has illuminated the substantial prevalence of H3K4me3 distribution, spanning from the cap stage to the late bell stage of dental germs. In order to delve into the role of H3K4me3 modification in amelogenesis and unravel the underlying mechanisms, we performed a conditional knockout of Ash2l, a core subunit essential for the establishment of H3K4me3 within the dental epithelium of mice. The absence of Ash2l resulted in reduced H3K4me3 modification, subsequently leading to abnormal morphology of dental germ at the late bell stage. Notably, knockout of Ash2l resulted in a loss of polarity in ameloblasts and odontoblasts. The proliferation and apoptosis of the inner enamel epithelium cells underwent dysregulation. Moreover, there was a notable reduction in the expression of matrix-related genes, Amelx and Dspp, accompanied with impaired enamel and dentin formation. Cut&Tag-seq (cleavage under targets and tagmentation sequencing) analysis substantiated a reduction of H3K4me3 modification on Shh, Trp63, Sp6, and others in the dental epithelium of Ash2l knockout mice. Validation through real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence consistently affirmed the observed downregulation of Shh and Sp6 in the dental epithelium following Ash2l knockout. Intriguingly, the expression of Trp63 isomers, DNp63 and TAp63, was perturbed in Ash2l defect dental epithelium. Furthermore, the downstream target of TAp63, P21, exhibited aberrant expression within the cervical loop of mandibular first molars and incisors. Collectively, our findings suggest that ASH2L orchestrates the regulation of crucial amelogenesis-associated genes, such as Shh, Trp63, and others, by modulating H3K4me3 modification. Loss of ASH2L and H3K4me3 can lead to aberrant differentiation, proliferation, and apoptosis of the dental epithelium by affecting the expression of Shh, Trp63, and others genes, thereby contributing to the defects of amelogenesis.

Abstract 15180: Inhibition of Tgfβ Signaling Enables Durable Ventricular Pacing by TBX18 Gene Transfer

Introduction: Somatic gene transfer of an embryonic transcription factor, TBX18, could directly reprogram ventricular myocytes to induced pacemaker myocytes. We and others have previously reported that direct myocardial gene delivery of TBX18 created ventricular pacing in heart block animals in vivo. However, the de novo biological pacing was short-lived, diminishing its translational value. Hypothesis: Our preliminary RNAseq and mass spec data indicate pronounced Tgfβ signaling in TBX18-transduced cardiomyocytes. Tgfβ signaling induces fibrosis in multiple organs including the heart. We hypothesized that TBX18 triggers Tgfβ signaling and results in fibrosis, leading to loss of durable ventricular pacing. Methods: Adenoviral vectors expressing TBX18 or GFP was delivered to neonatal rat ventricular myocytes (NRVMs) in vitro or to the left ventricular apex of adult rats with complete atrioventricular block (CAVB). Results: In vitro experiments showed increased population of vimentin+ fibroblasts and alpha smooth muscle actin+ myofibroblasts in TBX18-NRVMs compared to GFP-NRVMs at 2 weeks after gene transfer. Single cell transcriptome data indicated that TBX18 triggered substantial changes in extracellular matrix-related gene expression and Tgfβ signaling. Quantitative RT-PCR and ELISA validated scRNA-seq data, indicating fibroblast activation. TBX18-NRVMs showed faster automaticity than that control did, but their automaticity began wavering after day 7. Treatment of TBX18-NRVMs with a Tgfβ signaling inhibitor, A83-01, mitigated fibroblast activation by TBX18 and preserved their automaticity. Direct delivery of TBX18 into ventricular myocardium resulted in focal fibrosis as well as expansion of vimentin+ fibroblasts at day 7. TBX18 gene transfer initiated ventricular pacing in a rat model of CAVB, but ventricular pacing diminished after one week. In contrast, treating TBX18-injected animals with A83-01 enabled sustained biological pacing beyond three weeks of after gene delivery, accompanied by reduced fibrosis in situ. Conclusions: Inhibition of Tgfβ signaling is sufficient to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

CD73-Positive Cell Spheroid Transplantation Attenuates Colonic Atrophy

The incidence of inflammatory bowel diseases (IBD) is increasing worldwide. Mesenchymal stem/stromal cells (MSCs) have immunomodulatory functions and are a promising source for cell transplantation therapy for IBD. However, owing to their heterogeneous nature, their therapeutic efficacy in colitis is controversial and depends on the delivery route and form of transplanted cells. Cluster of differentiation (CD) 73 is widely expressed in MSCs and used to obtain a homogeneous MSC population. Herein, we determined the optimal method for MSC transplantation using CD73+ cells in a colitis model. mRNA sequencing analysis showed that CD73+ cells exhibited a downregulation of inflammatory gene expression and an upregulation of extracellular matrix-related gene expression. Furthermore, three-dimensional CD73+ cell spheroids showed enhanced engraftment at the injured site through the enteral route, facilitated extracellular matrix remodeling, and downregulated inflammatory gene expression in fibroblasts, leading to the attenuation of colonic atrophy. Therefore, the interaction between intestinal fibroblasts and exogenous MSCs via tissue remodeling is one mechanism that can be exploited for colitis prevention. Our results highlight that the transplantation of homogeneous cell populations with well-characterized properties is beneficial for IBD treatment.

Aging Fibroblasts Adversely Affect Extracellular Matrix Formation via the Senescent Humoral Factor Ependymin-Related Protein 1

Skin senescence is characterized by a decrease in extracellular matrix and the accumulation of senescent fibroblasts in the dermis, and their secretion of humoral factors. Ependymin-related protein 1 (EPDR1) is involved in abnormal fibroblast metabolism and collagen deposition, however, its relation to skin aging is unclear. We investigated whether and how EPDR1 is involved in age-related dermal deterioration. When young dermal fibroblasts and senescent cells were co-cultured in a semipermeable membrane separation system, the young fibroblasts showed decreased gene expression of collagen type I α1 chain (COL1A1) and elastin, and increased expression of matrix metalloproteinase (MMP)1 and MMP3. Senescence marker expression and EPDR1 production were increased in the culture medium of senescent cells. Treatment of young fibroblasts with recombinant EPDR1, enhanced matrix-related gene expression and suppressed COL1A1 expression, whereas EPDR1 knockdown had the opposite effects. EPDR1 gene and protein expression were increased in aged skin, compared to young skin. These results suggest that senescent cells affect nearby fibroblasts, in part through EPDR1 secretion, and exert negative effects on matrix production in the dermis. These results may lead to the discovery of potential candidate targets in the development of skin anti-aging therapies.

A Novel Role for Biomechanical Forces in the Pathogenesis of Idiopathic Constipation

INTRODUCTION: Chronic constipation is a common gastrointestinal disorder, but an anatomic or physiologic cause is identified in only 5% of patients. Surgery to remove chronically dilated segments of intestine is offered as a last resort. Our goal is to understand how the intestinal microenvironment is affected by biomechanical forces such as chronic distention in idiopathic constipation. METHODS: To evaluate the effects of biomechanical force on the colon, we used 3 models: (1) colon from wildtype mice was cultured ex vivo for 24 hours with and without distention and extracellular matrix–related gene expression was assayed using reverse transcriptase-polymerase chain reaction; (2) an in vivo mouse model of enteric distention was assessed at 7 to 14 days by immunohistochemistry; and (3) human specimens from pediatric patients who underwent colon resections for chronic idiopathic constipation (n = 27) were histologically evaluated to clinically validate our findings (Figure).FigureRESULTS: Ex vivo and in vivo colon under tension demonstrated increased fibrosis compared with control. Tension resulted in significantly decreased expression of antifibrotic Gremlin-1 and increased expression of profibrotic interleukin-1β. Collagens and matrix metalloproteinases were downregulated while laminins were upregulated. Seventy-seven percent of patients with chronic idiopathic constipation displayed dilated and/or redundant colon on imaging. In surgical specimens, 26% had interstitial fibrosis, 33% had muscular hypertrophy, and 15% had myocyte vacuolization. These histologic findings were also seen in distended mouse colon. CONCLUSION: Chronic intestinal distention led to a profibrotic phenotype in all 3 models. We conclude that mechanically sensitive cells may be affected by chronic distention and contribute to intestinal dysfunction in idiopathic constipation.

TGF-β-Mediated Modulation of Cell-Cell Interactions in Postconfluent Maturing Corneal Endothelial Cells.

PurposeTransforming growth factor-beta (TGF-β) is known to influence many cell functions. In the corneal endothelium, TGF-β1 exerts contextual effects, promoting endothelial–mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-β isoforms participate in the formation of corneal endothelial intercellular junctions.MethodsCorneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-β isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-β2 were also studied. The beneficial effect of TGF-β2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor.ResultsThe results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-β isoforms were individually added during the maturation phase, and TGF-β2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-β2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points.ConclusionsThese results suggest that TGF-β2 can strengthen cell–cell and cell–substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

Initial assessment of gingival biotype as a potential source of variability in the migration, contraction and gene expression of fibroblasts

ObjectiveGingival biotype refers to the clinical classification of gingiva based on the thickness of the tissue, with thick gingival tissues more resistant to trauma and recession than the thin variant. However, to date there has never been an analysis of whether fibroblasts isolated from different biotypes possess inherent phenotypic differences. We hypothesized that gingival fibroblasts from thick and thin biotype would exhibit differences in migration, contraction and gene expression in vitro in the presence of either transforming growth factor beta one (TGF-β1) or tumor necrosis factor alpha (TNFα), two major cytokines involved in wound repair. DesignMigration was quantified using closure of scratch wound assays, contraction was assessed using attached and detached collagen lattices and extracellular matrix related gene expression using Taqman Realtime polymerase chain reaction. ResultsHuman gingival fibroblasts isolated from both biotypes showed similar rates of closure of scratch wounds, which was not influenced by the addition of TGF-β1 or TNFα. Fibroblasts from both biotypes contracted detached, but not attached, collagen gels to 50 % of their original weight although this contraction was not associated with incorporation of α-smooth muscle actin into stressfibres under any tested culture condition. Analysis of gene expression showed that POSTN, and ACTA2 mRNA levels did not significantly change, but CCN2 and COL1A2 mRNA levels were significantly higher in thick compared to thin fibroblasts in response to TGF-β1. ConclusionWhile supra-cellular factors influence the healing, esthetic outcomes and recession in thin gingival biotypes, differences in gingival fibroblast gene expression in response to growth factors may also play a role and warrants further investigation.

A bioinformatics approach to study the role of calcium phosphate properties in bone regeneration

Calcium phosphates (CaPs) are extensively used as biomaterials for bone repair and regeneration and are considered an interesting synthetic alternative for natural bone grafts. However, the bone regenerative potential of many CaP bone graft substitutes requires further improvements. The bioactivity of CaPs is regulated by a variety of properties, including their chemical composition and (surface) structural properties. In this study, we performed a comprehensive characterization of six CaP ceramics that had been previously tested in several in vitro and in vivo studies, allowing us to in silico distinguish osteoinductive and non-osteoinductive CaP ceramics. The numerical parameters obtained from this characterization were further correlated with osteogenesis-associated gene expression and pathways of osteoblastic MG63 cells cultured on these six CaP ceramics. The results showed, among others, the influence of both chemical and structural parameters on cytoskeleton- and extracellular matrix-related gene expression and osteogenesis-associated pathways such as MAPK and BMP. The bioinformatics approach presented here allows exploring the transcriptional landscape induced by CaP ceramics and gaining fundamental knowledge about cell-biomaterial interactions.

Acoustically accelerated neural differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) and their derived products offer great promise for targeted therapies and drug screening, however, the hESC differentiation process of mature neurons is a lengthy process. To accelerate the neuron production, an acoustic stimulator producing surface acoustic waves (SAWs) is proposed and realized by clamping a flexible printed circuit board (PCB) directly onto a piezoelectric substrate. Neural differentiation of the hESCs is greatly accelerated after application of the acoustic stimulations. Acceleration mechanisms for neural differentiation have been explored by bulk RNA sequencing, quantitative polymerase chain reaction (qPCR) and immunostaining. The RNA sequencing results show changes of extracellular matrix-related and physiological activity-related gene expression in the low or medium SAW dose group and the high SAW dose group, respectively. The neural progenitor cell markers, including Pax6, Sox1, Sox2, Sox10 and Nkx2-1, are less expressed in the SAW dose groups compared with the control group by the qPCR. Other genes including Alk, Cenpf, Pcdh17, and Actn3 are also found to be regulated by the acoustic stimulation. Moreover, the immunostaining confirmed that more mature neuron marker Tuj1-positive cells, while less stem cell marker Sox2-positive cells, are presented in the SAW dose groups. These results indicate that the SAW stimulation accelerated neural differentiation process. The acoustic stimulator fabricated by using the PCB is a promising tool in regulation of stem cell differentiation process applied in cell therapy. Statement of significanceHuman embryonic stem cells (hESC) are used for investigating the complex mechanisms involved in the development of specialized biological cells and organs. Different types of hESCs derived cell products can be used for cell therapy procedures aiming to regenerate functional tissues in patients who suffer from various degenerative diseases. Accelerating the hESCs’ differentiation process can considerably benefit the clinical utilization of these cells. This study develops a highly effective acoustic stimulator working at ∼20 MHz to investigate what roles do acousto-mechanical stimuli play in the differentiation of hESCs. Our results show that acoustic dose alters the extracellular matrix and physiological activity-related gene expression, which indicates that the acoustic stimulation is an important tool for regulating the stem cells’ differentiation processes in cell therapy.

Sexual Dimorphism in the Extracellular and Pericellular Matrix of Articular Cartilage

ObjectiveWomen have a higher prevalence and burden of joint injuries and pathologies involving articular cartilage than men. Although knee injuries affecting young women are on the rise, most studies related to sexual dimorphism target postmenopausal women. We hypothesize that sexual dimorphism in cartilage structure and mechanics is present before menopause, which can contribute to sex disparities in cartilage pathologies.DesignBovine knee was used as a model to study healthy adult cartilage. We compared elastic moduli under compression, abundances of extracellular and pericellular matrix (PCM) proteins using proteomics, and PCM constituency with tissue immunofluorescence. The gene expression of matrix-related genes under basal, anabolic, and catabolic conditions was assessed by quantitative polymerase chain reaction (qPCR).ResultsThe equilibrium modulus was higher in male cartilage compared with female cartilage. Proteoglycans were not associated with this biomechanical dimorphism. Proteomic and pathway analyses of tissue showed dimorphic enriched pathways in extracellular matrix (ECM)-related proteins in which male cartilage was enriched in matrix interconnectors and crosslinkers that strengthen the ECM network. Moreover, male and female tissue differed in enriched PCM components. Females had more abundance of collagen type VI and decorin, suggesting different PCM mechanics. Furthermore, the activation of regenerative and catabolic function in chondrocytes triggered sex-dependent signatures in gene expression, indicating dimorphic genetic regulation that is dependent on stimulation.ConclusionsWe provide evidence for sexual dimorphism in cartilage before menopause. Some differences are intrinsic to chondrocytes’ gene expression defined by their XX versus XY chromosomal constituency.

The Modulatory Effect of Adipose-Derived Stem Cells on Endometrial Polyp Fibroblasts

Endometrial polyps (EPs) are benign overgrowths of the endometrium, with the potential to cause severe complications, ranging from discomfort to inflammation and infertility. Dysfunction of endometrial fibroblasts may be a critical component leading to the development of polyps. Although surgical intervention is the common remedy for severe cases, it comes with drawbacks, including infection, bleeding, and risk of damage to the cervix and adjacent tissues. Adipose-derived mesenchymal stromal cells (ASCs) are at the focus of modern medicine, as key modulators of tissue homeostasis, inflammation, and tissue repair, rendering them prime candidate agents for tissue regeneration and cell-based therapies. In this study, EPs were isolated from patients admitted to the OB/GYN department at the Galilee Medical Center and extracted fibroblasts (endometrial polyp fibroblasts, EPFs) were isolated and characterized. ASCs were isolated from healthy patients. The effect of EPF- and ASC-conditioned media (CM) on polyp-derived fibroblasts was evaluated, in both 2D and 3D assays, as well as on the expression of matrix-related gene expression. Herein, EPFs exposed to ASC-CM exhibited reduced migration, invasion, contraction of hydrogels, and extracellular matrix deposition, compared with those exposed to EPF-CM. Altogether, this study suggests that ASCs may have a modulating effect on fibroblasts involved in forming EPs and may serve as the basis for conservative treatment strategies aimed at treating severe cases of EPs.

Effects of Collagen-Tripeptide and Galacto-oligosaccharide Mixture on Skin Photoaging Inhibition in UVB-exposed Hairless Mice.

Collagen-tripeptide (CTP) and galacto-oligosaccharide (GOS), which improve collagen homeostasis and barrier function in the skin, are widely used in the food industry to improve wrinkle-related parameters and skin health. In this study, the photoprotective effect of CTP/GOS mixtures (3:1, 1:1, and 1:3) in ultraviolet (UV) B-irradiated hairless mice was examined. Skin parameter analysis, histological approaches, molecular biology techniques and HPLC analysis were applied to investigate the photoaging protective effect, signaling pathways and changes in the microbiota. Oral administration of CTP/GOS mixtures ameliorated photoaged physical parameters and serum levels of pro-inflammatory cytokines compared to UV-irradiated control group. Administration of the 1:3 mixture showed significant changes in the extracellular matrix-related gene expression compared to other mixture groups. The cecal short-chain fatty acid (SCFA) content showed a significant increase in the CTP/GOS mixed group with a higher GOS content than the control group. In the 16S rRNA-based analysis of cecal microbiota, the relative abundance ratio of the Akkermansia genus belonging to the Verrucomicrobia phylum was higher in CTP and GOS mixture-administered groups than in the UV-irradiated control group. Taken together, CTP/GOS mixtures showed a synergistic effect on photoprotective activity through changes in the gene expression, cytokine levels and intestinal microbiota composition.

Inducible factors and interaction of pulmonary fibrosis induced by prenatal dexamethasone exposure in offspring rats

This study aimed to investigate the correlation between prenatal dexamethasone exposure (PDE) and susceptibility to pulmonary fibrosis in offspring. Healthy female Wistar rats were given dexamethasone (0.2 mg/kg.d) or an equal volume of normal saline subcutaneously from 9 to 20 days after conception. Some of their female offspring underwent ovariectomy (OV) at 22 weeks after birth. All animals were euthanized at 28 weeks after birth. The morphological changes related to pulmonary fibrosis and extracellular matrix-related gene expression were detected, and Two-way ANOVA analyzed the interaction between PDE and OV. The results showed that adult offspring rats in FD group (female rats with PDE treatment) had early pulmonary fibrosis changes, such as pulmonary interstitial thickening, and increased expression of type IV collagen (COL4), α -smooth muscle actin (α-SMA) and fibronectin (FN) in lung tissues compared with those in FC group (female rats with saline treatment). In addition, adult offspring rats in FDO group (female rats with PDE and OV treatment) showed signs of pulmonary fibrosis, including apparent extracellular matrix deposition, increased lung injury scores (P＜0.01, P＜0.05), and extracellular matrix related gene expression (P＜0.01, P＜0.05), compared with rats in FDS (female rats with PDE treatment alone) or rats in FCO group (female rats with OV treatment alone). Moreover, PDE and OV had an interactive effect on the development of pulmonary fibrosis in female adult offspring. This study first reported the correlation between PDE and susceptibility to pulmonary fibrosis in female offspring rats, as well as the synergistic effect of PDE and OV in this pathological event, which provided a basis for further understanding of the pathogenesis of fetal originated pulmonary fibrosis.

Mitigating Milk-Associated Bacteria through Inducing Zinc Ions Antibiofilm Activity.

Dairy products are a sector heavily impacted by food loss, often due to bacterial contaminations. A major source of contamination is associated with the formation of biofilms by bacterial species adopted to proliferate in milk production environment and onto the surfaces of milk processing equipment. Bacterial cells within the biofilm are characterized by increased resistance to unfavorable environmental conditions and antimicrobial agents. Members of the Bacillus genus are the most commonly found spoilage microorganisms in the dairy environment. It appears that physiological behavior of these species is somehow depended on the availability of bivalent cations in the environment. One of the important cations that may affect the bacterial physiology as well as survivability are Zn2+ ions. Thus, the aim of this study was to examine the antimicrobial effect of Zn2+ ions, intending to elucidate the potential of a zinc-based antibacterial treatment suitable for the dairy industry. The antimicrobial effect of different doses of ZnCl2 was assessed microscopically. In addition, expression of biofilm related genes was evaluated using RT-PCR. Analysis of survival rates following heat treatment was conducted in order to exemplify a possible applicative use of Zn2+ ions. Addition of zinc efficiently inhibited biofilm formation by B. subtilis and further disrupted the biofilm bundles. Expression of matrix related genes was found to be notably downregulated. Microscopic evaluation showed that cell elongation was withheld when cells were grown in the presence of zinc. Finally, B. cereus and B. subtilis cells were more susceptible to heat treatment after being exposed to Zn2+ ions. It is believed that an anti-biofilm activity, expressed in downregulation of genes involved in construction of the extracellular matrix, would account for the higher sensitivity of bacteria during heat pasteurization. Consequently, we suggest that Zn2+ ions can be of used as an effective antimicrobial treatment in various applications in the dairy industry, targeting both biofilms and vegetative bacterial cells.

Deletion of NFKB1 enhances canonical NF-\u03baB signaling and increases macrophage and myofibroblast content during tendon healing

Flexor tendon injuries heal with excessive scar tissue that limits range of motion and increases incidence of re-rupture. The molecular mechanisms that govern tendon healing are not well defined. Both the canonical nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways have been implicated in tendon healing. The gene NFKB1 (proteins p105/p50) is involved in both NF-κB and MAPK signaling cascades. In the present study, we tested the hypothesis that global NFKB1 deletion would increase activation of both NF-κB and MAPK through loss of signaling repressors, resulting in increased matrix deposition and altered biomechanical properties. As hypothesized, NFKB1 deletion increased activation of both NF-κB and MAPK signaling. While gliding function was not affected, NFKB1 deletion resulted in tendons that were significantly stiffer and trending towards increased strength by four weeks post-repair. NFKB1 deletion resulted in increased collagen deposition, increase macrophage recruitment, and increased presence of myofibroblasts. Furthermore, NFKB1 deletion increased expression of matrix-related genes (Col1a1, Col3a1), macrophage-associated genes (Adgre1, Ccl2), myofibroblast markers (Acta2), and general inflammation (Tnf). Taken together, these data suggest that increased activation of NF-κB and MAPK via NFKB1 deletion enhance macrophage and myofibroblast content at the repair, driving increased collagen deposition and biomechanical properties.

Signaling Responses to Stroma-Derived Soluble Factors Are Associated with Outcome and with Expression of Microenvironment-Related Genes in Pediatric AML

Pediatric AML has a relapse rate approaching 40%, indicating that resistance to chemotherapy remains a critical problem. We are focused on identifying and overcoming environment-mediated mechanisms of resistance. We previously reported that the sensitivity of G-CSF- and IL-6-induced STAT3 signaling was significantly associated with outcome in pediatric AML patients. In this follow-up study, we used conditioned medium (CM) from HS5 stromal cells as a more physiological stimulus to evaluate the capacity of primary AML cells to activate signaling pathways.We studied 111 diagnostic bone marrow samples from patients who enrolled on Children's Oncology Group (COG) AML treatment studies, AAML03P1 or AAML0531, and provided informed consent to bank bone marrow. The same chemotherapy backbone was used in both trials. All samples had at least 70% viability after thawing. Thawed cells were divided into aliquots for stimulation with 50% CM, 10 ng/ml G-CSF, or 5 ng/ml IL-6. Unstimulated cells were used for isotype controls and for determination of basal signaling levels. Following 15 min stimulation, cells were fixed and processed for FACS analysis of CD45, pY-STAT3, pY-STAT5, pERK1/2, and pAKT. Responses were expressed as the fold change in mean fluorescence intensity for stimulated cells over unstimulated cells (ΔMFI).The ΔMFIs for pY-STAT3 and pY-STAT5 varied between 0.1 and 31.8. The ΔMFIs for pERK1/2 and pAKT rarely exceeded 2. Samples with a robust response to one stimulus generally responded robustly to the others. The Pearson correlation coefficient (r) for CM-induced pY-STAT3 v. G-CSF-induced pY-STAT3 was 0.8511 (p<0.00001). The CM-induced pY-STAT5 and G-CSF-induced pY-STAT5 responses were significantly but less strongly correlated (r=0.2765; p=0.0043). Cut point analyses identified response thresholds that distinguished patients with higher EFS from those with lower EFS. We found that higher CM-induced pY-STAT5 was significantly associated with an inferior EFS (Figure 1), with HR 2.06 for those with ΔMFI > 1.96 (p=0.034). Previously we found that higher inducible pY-STAT3 was associated with significantly better EFS (Redell, et al, Blood, 2013; Long, et al, Oncotarget, 2017). In this study, no cut point for CM-induced pY-STAT3 ΔMFI distinguished patients with good or poor EFS. Cytogenetics and FLT3/ITD were not significantly different for groups above and below the ΔMFI cut points. The finding that robust STAT5 signaling is associated with inferior EFS suggests that a factor that signals primarily through STAT5 and not STAT3 (e.g. GM-CSF, IL-3, FL) could contribute to treatment resistance and relapse.Interestingly, most of the samples that failed to activate STAT3/5 pathways in response to G-CSF also failed to respond to the cocktail of factors in CM, suggesting a generalized signaling dysfunction. To investigate intrinsic gene expression differences, we leveraged existing RNA-seq data for the samples for which we generated signaling responses. We obtained RNA-seq data for 27 samples from the NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. RNA-seq data for an additional 45 samples were provided through collaboration with the University of Washington. Of these 72 samples, 29 samples were resistant to CM-induced pY-STAT3 (ΔMFI <2, CM-R) and 43 samples were sensitive (ΔMFI > 2, CM-S). Raw counts were normalized and analyzed by EdgeR with GLM algorithm and blocking to control for batch effects from the two datasets. We found that 219 features were significantly differentially expressed (DE; FDR <0.05), with 192 being expressed more highly in the CM-R group. Gene set enrichment analysis identified the “matrisome” gene set, including genes encoding growth factors and matrix proteins, as being significantly enriched in the CM-R group. For example, genes encoding G-CSF, GM-CSF, WNT7B, and integrin B3 were upregulated in CM-R samples. Additionally, a number of non-HOX homeobox genes, including DLX2, DLX3 and MSX2, were increased in CM-R samples. There was no difference in the mean %blasts for the samples in the CM-R and CM-S groups, arguing against the increased expression of matrix-related genes being due to a higher proportion of non-blast cells in the CM-R samples. Our integration of gene expression with inducible STAT3/5 responses will yield novel insights into extrinsic survival signaling and mechanisms of dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.