Phenotypic Alterations Research Articles

NOD1 deficiency ameliorates the progression of diabetic retinopathy by modulating bone marrow–retina crosstalk

BackgroundNucleotide-binding oligomerization domain-containing protein 1 (NOD1) plays a pivotal role in inducing metabolic inflammation in diabetes. Additionally, the NOD1 ligand disrupts the equilibrium of bone marrow-derived hematopoietic stem/progenitor cells, a process that has immense significance in the development of diabetic retinopathy (DR). We hypothesized that NOD1 depletion impedes the advancement of DR by resolving bone marrow dysfunction.MethodsWe generated NOD1−/−-Akita double-mutant mice and chimeric mice with hematopoietic-specific NOD1 depletion to study the role of NOD1 in the bone marrow–retina axis.ResultsElevated circulating NOD1 activators were observed in Akita mice after 6 months of diabetes. NOD1 depletion partially restored diabetes-induced structural changes and retinal electrical responses in NOD1−/−-Akita mice. Loss of NOD1 significantly ameliorated the progression of diabetic retinal vascular degeneration, as determined by acellular capillary quantification. The preventive effect of NOD1 depletion on DR is linked to bone marrow phenotype alterations, including a restored HSC pool and a shift in hematopoiesis toward myelopoiesis. We also generated chimeric mice with hematopoietic-specific NOD1 ablation, and the results further indicated that NOD1 had a protective effect against DR. Mechanistically, loss of hematopoietic NOD1 resulted in reduced bone marrow-derived macrophage infiltration and decreased CXCL1 and CXCL2 secretion within the retina, subsequently leading to diminished neutrophil chemoattraction and NETosis.ConclusionsThe results of our study unveil, for the first time, the critical role of NOD1 as a trigger for a hematopoietic imbalance toward myelopoiesis and local retinal inflammation, culminating in DR progression. Targeting NOD1 in bone marrow may be a potential strategy for the prevention and treatment of DR.

Functional and Transcriptome Analysis Reveal Specific Roles of Dimocarpus longan DlRan3A and DlRan3B in Root Hair Development, Reproductive Growth, and Stress Tolerance.

Ran GTPases play essential roles in plant growth and development. Our previous studies revealed the nuclear localization of DlRan3A and DlRan3B proteins and proposed their functional redundancy and distinction in Dimocarpus longan somatic embryogenesis, hormone, and abiotic stress responses. To further explore the possible roles of DlRan3A and DlRan3B, gene expression analysis by qPCR showed that their transcripts were both more abundant in the early embryo and pulp in longan. Heterologous expression of DlRan3A driven by its own previously cloned promoter led to stunted growth, increased root hair density, abnormal fruits, bigger seeds, and enhanced abiotic stress tolerance. Conversely, constitutive promoter CaMV 35S (35S)-driven expression of DlRan3A, 35S, or DlRan3B promoter-controlled expression of DlRan3B did not induce the alterations in growth phenotype, while they rendered different hypersensitivities to abiotic stresses. Based on the transcriptome profiling of longan Ran overexpression in tobacco plants, we propose new mechanisms of the Ran-mediated regulation of genes associated with cell wall biosynthesis and expansion. Also, the transgenic plants expressing DlRan3A or DlRan3B genes controlled by 35S or by their own promoter all exhibited altered mRNA levels of stress-related and transcription factor genes. Moreover, DlRan3A overexpressors were more tolerant to salinity, osmotic, and heat stresses, accompanied by upregulation of oxidation-related genes, possibly involving the Ran-RBOH-CIPK network. Analysis of a subset of selected genes from the Ran transcriptome identified possible cold stress-related roles of brassinosteroid (BR)-responsive genes. The marked presence of genes related to cell wall biosynthesis and expansion, hormone, and defense responses highlighted their close regulatory association with Ran.

The occurrence, inheritance, and segregation of complex genomic structural variation in synthetic Brassica napus

“Synthetic” allopolyploids recreated by interspecific hybridization play an important role in providing novel genomic variation for crop improvement. Such synthetic allopolyploids often undergo rapid genomic structural variation (SV). However, how such SV arises, is inherited and fixed, and how it affects important traits, has rarely been comprehensively and quantitively studied in advanced generation synthetic lines. A better understanding of these processes will aid breeders in knowing how to best utilize synthetic allopolyploids in breeding programs. Here, we analyzed three genetic mapping populations (735 DH lines) derived from crosses between advanced synthetic and conventional Brassica napus (rapeseed) lines, using whole-genome sequencing to determine genome composition. We observed high tolerance of large structural variants, particularly toward the telomeres, and preferential selection for balanced homoeologous exchanges (duplication/deletion events between the A and C genomes resulting in retention of gene/chromosome dosage between homoeologous chromosome pairs), including stable events involving whole chromosomes (“pseudoeuploidy”). Given the experimental design (all three populations shared a common parent), we were able to observe that parental SV was regularly inherited, showed genetic hitchhiking effects on segregation, and was one of the major factors inducing adjacent novel and larger SV. Surprisingly, novel SV occurred at low frequencies with no significant impacts on observed fertility and yield-related traits in the advanced generation synthetic lines. However, incorporating genome-wide SV in linkage mapping explained significantly more genetic variance for traits. Our results provide a framework for detecting and understanding the occurrence and inheritance of genomic SV in breeding programs, and support the use of synthetic parents as an important source of novel trait variation.

Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Dynamic Effect of β-Lactam Antibiotic Inactivation Due to the Inter- and Intraspecies Interaction of Drug-Resistant Microbes.

The presence of β-lactamase positive microorganisms imparts a pharmacological effect on a variety of organisms that can impact drug efficacy by influencing the function or composition of bacteria. Although studies to assess dynamic intra- and interspecies communication with bacterial communities exist, the efficacy of drug treatment and quantitative assessment of multiorganism response is not well understood due to the lack of technological advances that can be used to study coculture interactions in a dynamic format. In this study, we investigate how β-lactamase positive microorganisms can neutralize the effect of β-lactam antibiotics in a dynamic format at the inter- and intraspecies level using microbial bead technology. Three interactive models for the biological compartmentalization of organisms were demonstrated to evaluate the effect of β-lactam antibiotics on coculture systems. Our model at the intraspecies level attempts to mimic the biofilm matrix more closely as a community-level feature of microorganisms, which acknowledges the impact of nondrug-resistant species in shaping the dynamic response. In particular, the results of intraspecies studies are highly supportive of the biofilm mode of bacterial growth, which can provide structural support and protect the bacteria from an assault on host or environmental factors. Our findings also indicate that β-lactamase positive bacteria can neutralize the cytotoxic effect of β-lactam antibiotics at the interspecies level when cocultured with cancer cells. Results were validated using β-lactamase positive bacteria isolated from environmental niches, which can trigger phenotypical alteration of β-lactams when cocultured with other organisms. Our compartmentalization strategy acts as an independent ecosystem and provides a new avenue for multiscale studies to assess intra- and interspecies interactions.

Anti-obesity effect of butyrate links to modulation of gut microbiome and epigenetic regulation of muscular circadian clock

The role of the muscle circadian clock in regulating oxidative metabolism exerts a significant influence on whole-body energy metabolism; however, research on the connection between the muscle circadian clock and obesity is limited. Moreover, there is a lack of studies demonstrating the regulatory effects of dietary butyrate on muscle circadian clock and the resulting antiobesity effects. This study aimed to investigate the impacts of dietary butyrate on metabolic and microbiome alterations and muscle circadian clock in a diet-induced obesity model. Male Sprague–Dawley rats were fed a high-fat diet with or without butyrate. Gut microbiota and serum metabolome were analyzed, and molecular changes were examined using tissues and a cell line. Further correlation analysis was performed on butyrate-induced results. Butyrate supplementation reduced weight gain, even with increased food intake. Gut microbiome analysis revealed an increased abundance of Firmicutes in butyrate group. Serum metabolite profile in butyrate group exhibited reduced amino acid and increased fatty acid content. Muscle circadian clock genes were upregulated, resulting in increased transcription of fatty acid oxidation-related genes. In myoblast cells, butyrate also enhanced pan-histone acetylation via histone deacetylase inhibition, particularly modulating acetylation at the promoter of circadian clock genes. Correlation analysis revealed potential links between Firmicutes phylum, including certain genera within it, and butyrate-induced molecular changes in muscle as well as phenotypic alterations. The butyrate-driven effects on diet-induced obesity were associated with alterations in gut microbiota and a muscle-specific increase in histone acetylation, leading to the transcriptional activation of circadian clock genes and their controlled genes.

Differences in the Impact of Various Types of Exercise on Irisin Levels: A Systematic Review and Meta-Analysis.

Irisin, a myokine that is responsive to exercise, induces significant changes in subcutaneous adipose tissue. By promoting the browning of white fat tissue, it enhances energy expenditure, thereby addressing overweight and obesity. This systematic review and meta-analysis aimed to compare the effects of different types of physical exercises on irisin levels in overweight and obese adults. Specifically, the review focused on studies involving obese or overweight individuals who participated in exercise training for a minimum of 8 weeks, with measured and reported changes in serum irisin levels compared to a control group. Data were collected from four databases (Google Scholar, ISI Web of Science Core Collection, PubMed, and Scopus). The risk of bias was assessed using the Begg and Egger tests, and the results were synthesized. Initial searches identified 560 titles, out of which only seven met the criteria for inclusion in the systematic review. Statistical analysis demonstrated a significant increase in serum irisin concentration (SMD = 0.957, P = 0.005) among obese and overweight individuals who engaged in exercise, compared to the passive control group. High-intensity interval training (HIIT) (SMD = 1.229, P < 0.001) had a more pronounced effect on increasing serum irisin levels than other exercise protocols. Furthermore, the effectiveness of exercise varied based on the participants' weight status (significant changes for overweight individuals; P < 0.001 and insignificant changes for obese individuals; P = 0.1), age (significant changes for those under 40 years old; P < 0.001 and insignificant changes for those over 40 years old; P = 0.322), and gender (significant changes for men; P < 0.001 and insignificant changes for women; P = 0.285). Consequently, exercise can elevate serum irisin levels, leading to alterations in adipose tissue phenotype and thermogenesis, ultimately contributing to weight reduction in obese and overweight individuals.

Enhancing seed and cob characteristics in reciprocal crosses between purple waxy corn and sweet corn through plant growth-promoting rhizobacteria application

Maize is one of the major cereal crops. Different high yielding as well as hybrid varieties have been growing for higher maize production. Various fertilizers have also been used to increase the maize yield. But yield target is not achievable always and the fertilizers had detrimental effects on soil properties also. For sustainable maize production, we conducted a combined study of reciprocal cross and application of plant growth-promoting rhizobacteria (PGPR). In this study, we examine the traits of seeds and cobs resulting from reciprocal crossings between Purple Waxy Corn (PWC) and Sweet Corn (SC) that were subjected to treatment with PGPR. The outcomes of reciprocal crossings on the traits of seeds and cobs exhibited a distinctiveness that diverged from that of their respective progenitors. The seeds and cobs from reciprocal crosses exhibited novel variation in their characteristics. If the male parent is PWC, the F1 seeds exhibit alterations in both color and type on a single cob, so modifying the traits of the seeds in a direction aligns with those of the female parent. If the male parent is SC (♂), then F1 exhibits a phenotypic alteration in seed color, nugget color and seed type on a single cob which represents a distinctive trait variation between the two parents. An interaction between the application of PGPR, both in terms of timing and concentration and the kind of corn, was seen to affect the seed width, seed thickness and sugar content. The results of present study will be helpful for sustainable maize production.

HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.

Macrophages regulate healing-associated fibroblasts in diabetic wound

BackgroundRecovery from a foot ulcer is compromised in a diabetic status, due to the impaired tissue microenvironment that consists of altered inflammation, angiogenesis and fibrosis. Phenotypic alterations in both macrophages and fibroblasts have been detected in the diabetic wound. Recently, a fibroblast subpopulation that expresses high matrix metalloproteinase 1 (MMP1), MMP3, MMP11 and Chitinase-3-Like Protein 1 (CHI3L1) was associated with a successful diabetic wound healing. However, it is not known whether these healing-associated fibroblasts are regulated by macrophages.Methods and ResultsWe used bioinformatic tools to analyze selected public databases on normal and diabetic skin from patients, and identified genes significantly altered in diabetes. In a mouse model for diabetic wound healing, we detected not only a loss of the spatiotemporal changes in interleukin 1β (IL1β), IL6, IL10 and vascular endothelial growth factor A (VEGF-A) in wound macrophages, but also a compromised expression of MMP1, MMP3, MMP11, CHI3L1 and VEGF-A in healing-associated wound fibroblasts in a diabetic status. Co-culture with diabetic macrophages significantly reduced the expression of MMP1, MMP3, MMP11, CHI3L1 and VEGF-A in fibroblasts from non-diabetic wound. Co-culture with non-diabetic macrophages or diabetic macrophages supplied with IL6 significantly increased the expression of MMP1, MMP3, MMP11, CHI3L1 and VEGF-A in fibroblasts from diabetic wound. Moreover, macrophage-specific expression of IL6 significantly improved wound healing and angiogenesis in diabetic mice.ConclusionsMacrophages may induce the activation of wound-healing-associated fibroblasts, while the defective macrophages in diabetes may be corrected with IL6 treatment as a promising therapy for diabetic foot disease.

Yeast-derived nanocarriers enable binary reprogramming macrophages for cancer immunotherapy

Tumor-associated macrophages (TAMs) play an important role in promoting tumor growth and metastasis, thus reprogramming TAMs to modulate the tumor immune microenvironment is a promising tumor treatment stratagem. Here, we present novel yeast-derived nanocarriers (YNCs) as IPI-549 delivery system for binary reprogramming of TAMs to induce anticancer immunity. The nanocarriers were fabricated from yeast cell walls with immune-oncology agent IPI-549 loading and coated with a shielding polymer for prolonged blood circulation. After intravenous injection, these YNCs could accumulate at the tumor site and be phagocytosed by TAMs via the Dectin-1 pathway to induce macrophage polarization towards M1 phenotype. In combination with IPI-549, the YNC system has efficiently inhibited tumor growth and modulated the tumor immune microenvironment through macrophage phenotypic alteration and increase of T cell-mediated antitumor immunity, which was demonstrated in a 4T1 orthotopic breast cancer mouse model. All these features indicate the promising values of our YNC delivery system in clinical antitumor applications.

Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system.

The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.IMPORTANCENakaseomyces (formerly Candida) glabrata is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in N. glabrata. We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed invivo and exvivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction exvivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

Apigenin accelerates wound healing in diabetic mice by promoting macrophage M2-type polarization via increasing miR-21 expression.

The alteration of inflammatory phenotype by macrophage polarization plays an important role in diabetic wound repair. Apigenin has been reported to be anti-inflammatory and promote tissue repair; however, whether it regulates macrophage polarization to participate in diabetic wound repair remains to be investigated. We found that apigenin promoted miR-21 expression in LPS-stimulated RAW264.7 cells, inhibited cellular M1-type factor TNF-α and IL-1β secretion and increased M2-type factor IL-10 and TGF-β secretion, and accelerated macrophage conversion from M1 type to M2 type, whereas this protective effect of apigenin was counteracted by a miR-21 inhibitor. Moreover, we established a macrophage-HUVECs cell in vitro co-culture system and found that apigenin accelerated the migration, proliferation, and VEGF secretion of HUVECs by promoting macrophage miR-21 expression. Further, mechanistic studies revealed that this was mediated by the TLR4/Myd88/NF-κB axis. In in vivo study, diabetic mice had significantly delayed wound healing compared to non-diabetic mice, accelerated wound healing in apigenin-treated diabetic mice, and decreased M1-type macrophages and increased M2-type macrophages in wound tissues.

Atlas of Cell Repertoire Within Neointimal Lesions Is Metabolically Altered in Hypertensive Rats.

High blood pressure has been suggested to accelerate vascular injury-induced neointimal formation and progression. However, little is known about the intricate relationships between vascular injury and hypertension in the context of arterial remodeling. Single-cell RNA-sequencing analysis was used to depict the cell atlas of carotid arteries of Wistar Kyoto rats and spontaneously hypertensive rats with or without balloon injury. We found that hypertension significantly aggravated balloon injury-induced arterial stenosis. A total of 36 202 cells from carotid arteries with or without balloon injury were included in single-cell RNA-sequencing analysis. Cell composition analysis showed that vascular injury and hypertension independently induced distinct aortic cell phenotypic alterations including immune cells, endothelial cells (ECs), and smooth muscle cells. Specifically, our data showed that injury and hypertension-induced specific EC phenotypic alterations, and revealed a transition from functional ECs to hypermetabolic, and eventually dysfunctional ECs in hypertensive rats upon balloon injury. Importantly, our data also showed that vascular injury and hypertension-induced different smooth muscle cell phenotypic alterations, characterized by deferential expression of synthetic signatures. Interestingly, pathway analysis showed that dysregulated metabolic pathways were a common feature in monocytes/macrophages, ECs, and smooth muscle cells in response to injury and hypertension. Functionally, we demonstrate that inhibition of mitochondrial respiration significantly ameliorated injury-induced neointimal formation in spontaneously hypertensive rats. This study provides the cell landscape changes of the main aortic cell phenotypic alterations in response to injury and hypertension. Our findings suggest that targeting cellular mitochondrial respiration could be a novel therapeutic for patients with hypertension undergoing vascular angioplasty.

Modulating TRPV4 Channel Activity in Pro-Inflammatory Macrophages within the 3D Tissue Analog.

Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(-)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson's trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(-) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

Extracellular Vesicle Clicking on Osteoimplants Through Biomimetic Molecular Adhesion Enables Immune‐Enhanced Osseointegration in Diabetics

AbstractExtracellular vesicles (EVs) derived from differentiating induced stem cells maintain their original stemness and differentiating trends and possess strong immunoregulatory capability. Biomaterials equipped with EVs are very promising in regenerative medicine. However, surface EV‐decoration on osteoimplants remains a challenge, due to the complexity of traditional molecular conjugations and the fragility of EVs. Here, mussel‐like molecular adhesion is combined with bioorthogonal click conjugation to introduce EVs on titanium (Ti) implants. The biomimetic adhesion and clickable molecular linkage allow mild and stable tethering of pre‐osteogenic mesenchymal stem cell (MSC)‐derived EVs on Ti implants. EV‐decorated implants exhibit significantly enhanced osseointegration on the bone‐implant surface under diabetic conditions, promoting increased expression of osteogenic genes. Modified surfaces impelled phenotypic alterations in macrophage polarization via multi‐pathway regulation, decreasing proinflammatory M1 macrophage formation, which can lead to the promotion of surface osteogenesis. On Ti rods implanted in a diabetic rat model, EV coating inhibited M1macrophages around the prosthesis, resulting in satisfactory long‐term osseointegration. This study offers a new perspective to represent a simple and effective means for surface EV decoration, providing an osteoimmunomodulatory effect to enhance the diabetic osseointegration of implants.

Platinum-based drugs induce phenotypic alterations in nucleoli and Cajal bodies in prostate cancer cells

PurposePlatinum-based drugs are cytotoxic drugs commonly used in cancer treatment. They cause DNA damage, effects of which on chromatin and cellular responses are relatively well described. Yet, the nuclear stress responses related to RNA processing are incompletely known and may be relevant for the heterogeneity with which cancer cells respond to these drugs. Here, we determine the type and extent of nuclear stress responses of prostate cancer cells to clinically relevant platinum drugs.MethodsWe study nucleolar and Cajal body (CB) responses to cisplatin, carboplatin, and oxaliplatin with immunofluorescence-based methods in prostate cancer cells. We utilize organelle-specific markers NPM, Fibrillarin, Coilin, and SMN1, and study CB-regulatory proteins FUS and TDP-43 using siRNA-mediated downregulation.ResultsDifferent types of prostate cancer cells have different sensitivities to platinum drugs. With equally cytotoxic doses, cisplatin, and oxaliplatin induce prominent nucleolar and CB stress responses while the nuclear stress phenotypes to carboplatin are milder. We find that Coilin is a stress-specific marker for platinum drug response heterogeneity. We also find that CB-associated, stress-responsive RNA binding proteins FUS and TDP-43 control Coilin and CB biology in prostate cancer cells and, further, that TDP-43 is associated with stress-responsive CBs in prostate cancer cells.ConclusionOur findings provide insight into the heterologous responses of prostate cancer cells to different platinum drug treatments and indicate Coilin and TDP-43 as stress mediators in the varied outcomes. These results help understand cancer drug responses at a cellular level and have implications in tackling heterogeneity in cancer treatment outcomes.

Cucumber Auxin Response Factor CsARF10a Regulates Leaf Morphogenesis and Parthenocarpic Fruit Set in Tomato

Auxin response factors (ARFs) are pivotal transcription factors involved in many aspects of auxin-dependent developmental processes. While functions of ARFs have been extensively studied in Arabidopsis, their distinct role in cucumber remains unclear. In this study, a cucumber auxin response factor homolog, CsARF10a, was cloned and overexpressed in tomato plants. RT-qPCR analysis indicated that the expression abundance of CsARF10a was significantly decreased in cucumber leaves and female flowers, and the expression level of CsARF10a was relatively low in pollinated fruits and hormone-treated fruits compared with that in unpollinated fruits. Moreover, the overexpression of CsARF10a in tomato resulted in multiple phenotypic changes, including a wider leaf blade, delayed fruit ripening, and parthenocarpic fruit set in CsARF10a-OE lines. Taken together, our research shed light on the regulatory importance of CsARF10a in regulating various phenotype alterations and laid a solid foundation for further functional studies.

Protein Restriction in Metabolic Health: Lessons from Rodent Models.

Consumption of protein-rich diets and supplements has been increasingly advocated by individuals seeking to optimize metabolic health and mitigate the effects of aging. Protein intake is postulated to support muscle mass retention and enhance longevity, underscoring its perceived benefits in age-related metabolic regulation. However, emerging evidence presents a paradox; while moderate protein consumption contributes to health maintenance, an excessive intake is associated with an elevated risk of chronic diseases, notably obesity and diabetes. Furthermore, recent studies suggest that reducing the ratio of protein intake to macronutrients improves metabolic parameters and extends lifespan. The aim of this study is to review the current evidence concerning the metabolic effects of protein-restricted diets and their potential mechanisms. Utilizing rodent models, investigations have revealed that protein-restricted diets exert a notable influence over food intake and energy consumption, ultimately leading to body weight loss, depending on the degree of dietary protein restriction. These phenotypic alterations are primarily mediated by the FGF21 signaling pathway, whose activation is likely regulated by ATF4 and the circadian clock. The evidence suggests that protein-restricted diets as an alternative approach to calorie-restricted regimes, particularly in overweight or obese adults. However, more research is needed to determine the optimal level of restriction, duration, and long-term effects of such interventions.