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After traumatic brain injury, the brain extracellular matrix undergoes structural rearrangement due to changes in matrix composition, activation of proteases, and deposition of chondroitin sulfate proteoglycans by reactive astrocytes to produce the glial scar. These changes lead to a softening of the tissue, where the stiffness of the contusion core and peripheral pericontusional regions becomes softer than that of healthy tissue. Pioneering mechanotransduction studies have shown that soft substrates upregulate intermediate filament proteins in reactive astrocytes; however, many other aspects of astrocyte biology remain unclear. Here, we developed a platform for the culture of cortical astrocytes using polyacrylamide (PA) gels of varying stiffness (measured in Pascal; Pa) to mimic injury-related regions in order to investigate the effects of tissue stiffness on astrocyte reactivity and morphology. Our results show that substrate stiffness influences astrocyte phenotype; soft 300 Pa substrates led to increased GFAP immunoreactivity, proliferation, and complexity of processes. Intermediate 800 Pa substrates increased Aggrecan+, Brevican+, and Neurocan+ astrocytes. The stiffest 1 kPa substrates led to astrocytes with basal morphologies, similar to a physiological state. These results advance our understanding of astrocyte mechanotransduction processes and provide evidence of how substrates with engineered stiffness can mimic the injury microenvironment.

BACKGROUND: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. METHODS: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. RESULTS: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. CONCLUSIONS: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. We sought to elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb after treatment with serum or the α-adrenergic agonist phenylephrine. Depletion of Bmal1 decreased the expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by micrococcal nuclease-quantitative PCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting natriuretic peptide B transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented phenylephrine-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

BACKGROUND: Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. METHODS: We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. RESULTS: Plasma proteomics identified high protein abundance levels of B2M (β2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-β, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-β. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

AimAstrocytes respond to stressors by acquiring a reactive state characterized by changes in their morphology and function. Molecules underlying reactive astrogliosis, however, remain largely unknown. Given that several studies observed increase in the Amyloid Precursor Protein (APP) in reactive astrocytes, we here test whether APP plays a role in reactive astrogliosis.MethodsWe investigated whether APP instigates reactive astroglios by examining in vitro and in vivo the morphology and function of naive and APP-deficient astrocytes in response to APP and well-established stressors.ResultsOverexpression of APP in cultured astrocytes led to remodeling of the intermediate filament network, enhancement of cytokine production, and activation of cellular programs centered around the interferon (IFN) pathway, all signs of reactive astrogliosis. Conversely, APP deletion abrogated remodeling of the intermediate filament network and blunted expression of IFN-stimulated gene products in response to lipopolysaccharide. Following traumatic brain injury (TBI), mouse reactive astrocytes also exhibited an association between APP and IFN, while APP deletion curbed the increase in glial fibrillary acidic protein observed canonically in astrocytes in response to TBI.ConclusionsThe APP thus represents a candidate molecular inducer and regulator of reactive astrogliosis. This finding has implications for understanding pathophysiology of neurodegenerative and other diseases of the nervous system characterized by reactive astrogliosis and opens potential new therapeutic avenues targeting APP and its pathways to modulate reactive astrogliosis.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimers disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

Schizophrenia (SZ) is a serious mental illness and neuropsychiatric brain disorder with behavioral symptoms that include hallucinations, delusions, disorganized behavior, and cognitive impairment. Regulation of such behaviors requires utilization of neurotransmitters released to mediate cell-cell communication which are essential to brain functions in health and disease. We hypothesized that SZ may involve dysregulation of neurotransmitters secreted from neurons. To gain an understanding of human SZ, induced neurons (iNs) were derived from SZ patients and healthy control subjects to investigate peptide neurotransmitters, known as neuropeptides, which represent the major class of transmitters. The iNs were subjected to depolarization by high KCl in the culture medium and the secreted neuropeptides were identified and quantitated by nano-LC-MS/MS tandem mass spectrometry. Several neuropeptides were identified from schizophrenia patient-derived neurons, including chromogranin B (CHGB), neurotensin, and natriuretic peptide. Focusing on the main secreted CHGB neuropeptides, results revealed differences in SZ iNs compared to control iN neurons. Lower numbers of distinct CHGB peptides were found in the SZ secretion media compared to controls. Mapping of the peptides to the CHGB precursor revealed peptides unique to either SZ or control, and peptides common to both conditions. Also, the iNs secreted neuropeptides under both KCl and basal (no KCl) conditions. These findings are consistent with reports that chromogranin B levels are reduced in the cerebrospinal fluid and specific brain regions of SZ patients. These findings suggest that iNs derived from SZ patients can model the decreased CHGB neuropeptides observed in human SZ.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

IntroductionThe regeneration of pulp tissue is crucial for true regenerative endodontic treatment, which requires a reduction in osteogenic differentiation. Garcinol, a histone acetyltransferase inhibitor, is a natural regulator that is known to suppress the osteogenic differentiation of dental pulp stem cells. In this study, the inhibitory effect of garcinol on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) was evaluated using three-dimensional culture under in vitro and in vivo conditions.MethodshDPSCs were obtained from caries-free third molars and cultured with 10 μM garcinol for 7 days in an ultra-low attachment plate. The cell stemness and expression of osteogenic differentiation-related genes were analyzed using reverse transcription-polymerase chain reaction and single-cell analysis. A transplantation experiment was performed in mice to investigate whether garcinol-treated hDPSCs showed restrained osteogenic differentiation.ResultshDPSCs cultured in the U-shaped ultra-low attachment plate showed the highest expression of stemness-related genes. Garcinol-treated hDPSCs demonstrated downregulation of osteogenic differentiation, with lower expression of bone sialoprotein, which is related to bone formation, and higher expression of dentin sialophosphoprotein, which is related to dentin formation. However, the garcinol-treated hDPSCs did not show any alterations in their stemness. Consistent results were observed in the transplantation experiment in mice.ConclusionsGarcinol reduced the osteogenic differentiation of hDPSCs, which can contribute to true regenerative endodontic treatment.

Background: Metabolic syndrome (MetS) is a precursor to the development of many diseases (atherosclerosis, diabetes, etc.). It is marked by disruptions in glucose and lipid metabolism, along with hypertension. Numerous types of risk factors contribute to the development of the MetS, inflammation and insulin resistance are present throughout the metabolic abnormalities. Chrysanthemum indicum L. is a traditional Chinese plant used for both tea and medicine, known for its high content of total flavonoids, which are important secondary metabolites. Our research led to the extraction of a Buddleoside-Rich Chrysanthemum indicum L. extract (BUDE) which has demonstrated anti-inflammatory properties. Nonetheless, the specific role and mechanism of BUDE in preventing MetS remain unclear. Methods: The study initially evaluated the role of BUDE in preventing MetS. Subsequently, it investigated the anti-inflammatory properties of BUDE in the liver and pancreas in response to unhealthy diets. It then examined the level of insulin resistance and pancreatic β-cell function induced by inflammation. Additionally, an lipopolysaccharide (LPS)-induced macrophage inflammation model was used to further investigate the ameliorative effects of BUDE in inflammation. Results: BUDE has hypotensive, hypoglycemic and hypolipidemic effects. It can also resolve the imbalance between macrophage subpopulations, impede the triggering of the NF-κB signaling pathway, reduce the secretion of inflammatory mediators, ameliorate insulin resistance, and safeguard organs such as the liver and pancreas from inflammatory damage. These effects collectively contribute to preventing the development of MetS. Discussion: BUDE has the ability to modulate macrophage-mediated inflammation, leading to improved insulin resistance. Additionally, it delivers antihypertensive, hypoglycemic, and hypolipidemic effects, offering a potential for preventing MetS. [ABSTRACT FROM AUTHOR]

The cGAS-STING pathway is a pivotal element of the innate immune system, recognizing cytosolic DNA to initiate the production of type I interferons and pro-inflammatory cytokines. This study investigates the alterations of the cGASSTING signaling components in the cortex and hippocampus of mice aged 24 and 108 weeks. In the cortex of old mice, an increase in the dsDNA sensor protein cGAS and its product 2030-cGAMP was observed, without corresponding activation of downstream signaling, suggesting an uncoupling of cGAS activity from STING activation. This phenomenon may be attributed to increased dsDNA concentrations in the EC neurons, potentially arising from nuclear DNA damage. Contrastingly, the hippocampus did not exhibit increased cGAS activity with aging, but there was a notable elevation in STING levels, particularly in microglia, neurons and astrocytes. This increase in STING did not correlate with enhanced IRF3 activation, indicating that brain inflammation induced by the cGAS-STING pathway may manifest extremely late in the aging process. Furthermore, we highlight the role of autophagy and its interplay with the cGAS-STING pathway, with evidence of autophagy dysfunction in aged hippocampal neurons leading to STING accumulation. These findings underscore the complexity of the cGASSTING pathway's involvement in brain aging, with regional variations in activity and potential implications for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Background: Odontogenic cysts and tumors develop from the dental follicle of asymptomatic impacted teeth. Odontogenic tissues express the epidermal growth factor receptor family (EGFR), which mediates cell proliferation, survival, and neoplastic differentiation. The present study aimed to compare the immunohistochemical expression of EGFR and human epidermal growth factor receptor 2 (HER2) in the dental follicle of impacted wisdom teeth with normal and abnormal radiographic size. Methods: In this analytical study, immunohistochemical staining of EGFR and HER2 was performed on 30 normal and 30 abnormal follicles of impacted third molars. Follicles with a width of <2.5 mm were considered normal, whereas those with a width of ≥2.5 mm were regarded as abnormal. The immunoreactive score (IRS) was used to report the expression levels of EGFR and HER2. The obtained data were analyzed using SPSS software. Age and sex were compared in normal and abnormal groups with independent t test and Chi square test, respectively. P<0.05 was considered statistically significant. Results: The EGFR and HER2 overall expression was high in all normal and abnormal follicles. The comparison of the percentage of stained cells and intensity of EGFR and HER2 staining in normal and abnormal follicles were not significantly different (P=0.73, P=0.63, P=0.95, respectively). Conclusion: Due to the high expression of EGFR and HER2 in normal and abnormal follicles, as well as the lack of significant differences in these two groups, the radiographic size of dental follicles might not indicate the potential capabilities of their cells, and more research in this field is recommended. [ABSTRACT FROM AUTHOR]

To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that the rapid recruitment of all 3 Drosophila calcium-responding and phospholipid-binding Annexin proteins (AnxB9, AnxB10, and AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair. [ABSTRACT FROM AUTHOR]

Simple Summary: Oral lichen planus (OLP) is a chronic inflammatory disease of autoimmune nature and unknown etiology which affects approximately 1% of the world's population. The most important feature of OLP is its behavior as an oral potentially malignant disorder (OPMD). The current study is the first systematic review and meta-analysis designed to evaluate the degree of existing scientific evidence on the cancer hallmarks proposed in 2011 by Hanahan and Weinberg, defined as the characteristics that cells must fulfill in order to be considered neoplastic cells in all types of tumors that affect humans. This systematic review and meta-analysis includes 110 studies which recruited 7064 cases of OLP, in which the expression of 104 molecular biomarkers were analyzed through an immunohistochemical technique. The earliest oncogenic molecular mechanisms that could justify the malignant transformation of this disease are analyzed in depth and critically discussed on the basis of evidence. We aimed to qualitatively and quantitatively analyze, through a systematic review and meta-analysis, the current evidence on the differential expression of the hallmarks of cancer in oral lichen planus (OLP) samples, in order to know the earliest molecular mechanisms that could be involved in the malignant transformation of this oral potentially malignant disorder. We searched MEDLINE/PubMed, Embase, Web of Science, and Scopus for studies published before November 2023. We evaluated the methodological quality of studies and carried out meta-analyses to fulfill our objectives. Inclusion criteria were met by 110 primary-level studies, with 7065 OLP samples, in which the expression of 104 biomarkers were analyzed through immunohistochemistry. Most OLP samples showed sustained cell proliferation signaling (65.48%, 95%CI = 51.87–78.02), anti-apoptotic pathways (55.93%, 95%CI = 35.99–75.0), genome instability (48.44%, 95%CI = 13.54–84.19), and tumor-promoting inflammation events (83.10%, 95%CI = 73.93–90.74). Concurrently, OLP samples also harbored tumor growth suppressor mechanisms (64.00%, 95%CI = 53.27–74.12). In conclusion, current evidence indicates that molecular mechanisms promoting hyperproliferative signaling, an antiapoptotic state with genomic instability, and an escape of epithelial cells from immune destruction, are developed in LP-affected oral mucosa. It is plausible that these events are due to the actions exerted by the chronic inflammatory infiltrate. Malignant transformation appears to be prevented by tumor suppressor genes, which showed consistent upregulation in OLP samples. [ABSTRACT FROM AUTHOR]

Secondary healing of fractured bones requires an application of an appropriate fixator. In general, steel or titanium devices are used mostly. However, in recent years, composite structures arise as an attractive alternative due to high strength to weight ratio and other advantages like, for example, radiolucency. According to Food and Drug Administration (FDA), the only unidirectionally reinforced composite allowed to be implanted in human bodies is carbon fiber (CF)‐reinforced poly‐ether‐ether‐ketone (PEEK). In this work, the healing process of long bone assembled with CF/PEEK plates with cross‐ and angle‐ply lay‐up configurations is studied in the framework of finite element method. The healing is simulated by making use of the mechanoregulation model basing on the Prendergast theory. Cells transformation is determined by the octahedral shear strain and interstitial fluid velocity. The process runs iteratively assuming single load cycle each day. The fracture is subjected to axial and transverse forces. In the computations, the Abaqus program is used. It is shown that the angle‐ply lamination scheme of CF/PEEK composite seems to provide better conditions for the transformation of the soft callus into the bone tissue. [ABSTRACT FROM AUTHOR]

Ewing sarcoma is an uncommon neoplasm considered in the differential diagnosis of tumors with "small round cell" morphology, but its occurrence in the gynecologic tract has only been sporadically documented. Herein, we describe the largest cohort of Ewing sarcoma localized to the female genital tract to date, and emphasize their clinicopathologic resemblance to more common gynecologic neoplasms. Ewing sarcoma (n= 21) was retrospectively identified from 5 institutions. The average patient age was 35 (range 6-61) years. Tumor sites included uterus (n= 8), cervix (n=4), vulva (n= 5), vagina (n= 1), broad ligament (n=1), inguinal area (n =1), and pelvis (n= 1). Nine of 18 cases in which slides were available for review demonstrated only classic round cell morphology, with the remainder showing a variable combination and prominence of variant ovoid/spindle or epithelioid appearance. Tumors showed diffuse membranous reactivity for CD99 (20/20) and were positive for NKX2.2 (8/8, diffuse) and cyclin D1 (7/7, of which 3/7 were patchy/multifocal and 4/7 were diffuse). They were negative for ER (0/6) and CD10 (0/6). Three cases were initially diagnosed as endometrial stromal sarcomas. EWSR1 rearrangement was confirmed in 20/21 by fluorescence in situ hybridization (n=15) and/or sequencing (n= 8). Of the eight tumors that underwent sequencing, 6 harbored FLI1, 1 ERG, and 1 FEV as the fusion partner. Of 11 patients with available follow-up, 5 died of disease, 1 developed lung metastases and 5 are alive with no evidence of disease. Ewing sarcoma of the gynecologic tract is a rare, aggressive entity that shares some morphologic and immunohistochemical features with other more common gynecologic neoplasms. In addition to the typical round cell appearance, variant spindled/ovoid to epithelioid morphology may also be observed and should prompt consideration of this entity with appropriate immunohistochemical and/or molecular studies. [ABSTRACT FROM AUTHOR]

Classic Hodgkin lymphoma (CHL) can arise in patients with low-grade B-cell lymphoma. The features of CHL arising in follicular lymphoma (FL) and its outcome are still unclear, mainly due to the very few cases reported. This study compares 17 patients with CHL and FL to 2 control groups: 1 of 26 patients with FL and a second of 60 patients older than 40 when diagnosed with CHL. Of the FL and CHL patients, 8 had simultaneous FL and CHL, while 9 had FL first, followed by CHL 4.7 years later on average. The age at the diagnosis of FL was 61 years for patients with synchronous FL and CHL and of 60 years for FL, followed by CHL at 65 years. Patients with FL only were, on average, 59 years old at presentation, while CHL patients were 61. FL was grade 1-2 in 75% of FL and CHL patients and 67% of FL first and CHL second patients, lower proportions than in the FL control group-92%. Epstein-Barr virus (EBV) was detected in a lower fraction (29%) of the FL and CHL group than in CHL-only controls (46%). BCL2 translocations were detected in 4 of the 7 cases with FL, but in positive cases, the rearrangement was also present in the CHL component, indicating a clonal relationship between FL and CHL. Patients with FL and CHL treated for CHL had an initial outcome more similar to FL than to CHL controls. [ABSTRACT FROM AUTHOR]

Apart from the lethal midline carcinoma (NUT carcinoma), NUTM1 translocation has also been reported in mesenchymal tumors, but is exceedingly rare. Here, we describe a series of 8 NUTM1-rearranged sarcomas to further characterize the clinicopathologic features of this emerging entity. This cohort included 2 males and 6 females with age ranging from 24 to 64 years (mean: 51 y; median: 56 y). Tumors occurred in the colon (2), abdomen (2), jejunum (1), esophagus (1), lung (1) and infraorbital region (1). At diagnosis, 6 patients presented with metastatic disease. Tumor size ranged from 1 to 10.5 cm (mean: 6 cm; median: 5.5 cm). Histologically, 4 tumors were composed of primitive small round cells to epithelioid cells intermixed with variable spindle cells, while 3 tumors consisted exclusively of small round cells to epithelioid cells and 1 tumor consisted predominantly of highgrade spindle cells. The neoplastic cells were arranged in solid sheets, nests, or intersecting fascicles. Mitotic activity ranged from 1 to 15/10 HPF (median: 5/10 HPF). Other features included rhabdoid phenotype (4/8), pronounced nuclear convolutions (2/8), prominent stromal hyalinization (2/8), focally myxoid stroma (1/8), foci of osteoclasts (1/8), and necrosis (1/8). By immunohistochemistry, all tumors showed diffuse and strong nuclear staining of NUT protein, with variable expression of pancytokeratin (AE1/AE3) (2/8), CK18 (1/8), CD99 (3/8), NKX2.2 (2/8), cyclin D1 (2/8), desmin (2/8), BCOR (2/8), S100 (1/8), TLE1 (1/8), and synaptophysin (1/8). Seven of 8 tumors demonstrated NUTM1 rearrangement by fluorescence in situ hybridization analysis. RNA-sequencing analysis identified MXD4::NUTM1 (3/7), MXI1::NUTM1 (3/7), and MGA::NUTM1 (1/7) fusions, respectively. DNA-based methylation profiling performed in 2 cases revealed distinct methylation cluster differing from those of NUT carcinoma and undifferentiated small round cell and spindle cell sarcomas. At follow-up (range: 4 to 24 mo), 1 patient experienced recurrence at 8.5 months, 4 patients were alive with metastatic disease (5, 10, 11, and 24 mo after diagnosis), 3 patients remained well with no signs of recurrence or metastasis (4, 6, and 12 mo after diagnosis). Our study further demonstrated that NUTM1-rearranged sarcoma had a broad range of clinicopathologic spectrum. NUT immunohistochemistry should be included in the diagnostic approach of monotonous undifferentiated small round, epithelioid to high-grade spindle cell malignancies that difficult to classify by conventional means. DNA-based methylation profiling might provide a promising tool in the epigenetic classification of undifferentiated sarcomas. [ABSTRACT FROM AUTHOR]

Cells exist in different phenotypes and can transition between them. A phenotype may be characterized by many different aspects. Here, we focus on the example of whether the cell is adhered or suspended and choose particular parameters related to the structure and mechanics of the actin cortex. The cortex is essential to cell mechanics, morphology, and function, such as for adhesion, migration, and division of animal cells. To predict and control cellular functions and prevent malfunctioning, it is necessary to understand the actin cortex. The structure of the cortex governs cell mechanics; however, the relationship between the architecture and mechanics of the cortex is not yet well enough understood to be able to predict one from the other. Therefore, we quantitatively measured structural and mechanical cortex parameters, including cortical thickness, cortex mesh size, actin bundling, and cortex stiffness. These measurements required developing a combination of measurement techniques in scanning electron, expansion, confocal, and atomic force microscopy. We found that the structure and mechanics of the cortex of cells in interphase are different depending on whether the cell is suspended or adhered. We deduced general correlations between structural and mechanical properties and show how these findings can be explained within the framework of semiflexible polymer network theory. We tested the model predictions by perturbing the properties of the actin within the cortex using compounds. Our work provides an important step toward predictions of cell mechanics from cortical structures and suggests how cortex remodeling between different phenotypes impacts the mechanical properties of cells. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is the predominant cause of dementia on a global scale, significantly impacting the health of the elderly population. The pathogenesis of AD is closely linked to neuroinflammation. The present study employs a bibliometric analysis to examine research pertaining to neuroinflammation and AD within the last decade, with the objective of providing a comprehensive overview of the current research profile, hotspots and trends. Methods: This research conducted a comprehensive review of publications within the Science Citation Index Expanded of the Web of Science Core Collection Database spanning the years 2014 to 2024. Bibliometric analyses were performed using VOSviewer (version 1.6.19) and CiteSpace (version 6.3.R1) software to visualize data on countries, institutions, authors, journals, keywords, and references. Results: A total of 3,833 publications on neuroinflammation and AD were included from January 2014 to January 2024. Publications were mainly from the United States and China. Zetterberg, Henrik emerged as the author with the highest publication output, while Edison, Paul was identified as the most cited author. The most productive journal was Journal of Alzheimers Disease, and the most co-cited was Journal of Neuroinflammation. Research hotspot focused on microglia, mouse models, oxidative stress, and amyloid-beta through keyword analysis. Additionally, keywords such as blood-brain barrier and tau protein exhibited prolonged citation bursts from 2022 to 2024. Conclusion: This study provides a comprehensive review of the last 10 years of research on neuroinflammation and AD, including the number and impact of research findings, research hotspots, and future trends. The quantity of publications in this field is increasing, mainly in the United States and China, and there is a need to further strengthen close cooperation with different countries and institutions worldwide. Presently, research hotspots are primarily concentrated on microglia, with a focus on inhibiting their pro-inflammatory responses and promoting their anti-inflammatory functions as a potential direction for future investigations. [ABSTRACT FROM AUTHOR]

Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance. [ABSTRACT FROM AUTHOR]

Simple Summary: Plexiform neurofibromas in neurofibromatosis type 1 (pNF1), characterized by aggressive growth and local invasiveness, are comprised primarily of Schwann cell-derived tumor cells (Nf1−/−) and the tumor microenvironment (TME). Fibroblasts (Nf1+/−), the most abundant cell type in the TME, are known to significantly contribute to pNF1 formation, but their precise role in the subsequent tumor progression is poorly understood. Using our established three-dimensional (3D) culture models with patient-derived pNF1 cells and fibroblasts, we observed that the fibroblast-derived secretome stimulates the growth and local invasiveness of pNF1 spheroids. Depletion of small extracellular vesicles (sEVs) from the fibroblast secretome completely eliminated the paracrine effect on pNF1 spheroid growth. These results suggest that targeting paracrine interactions between pNF1 tumor cells and fibroblasts may offer a potential therapeutic approach for reducing pNF1 tumor progression. Plexiform neurofibromas (PNs) occur in about a half of neurofibromatosis type 1 (NF1) patients and have garnered significant research attention due to their capacity for growth and potential for malignant transformation. NF1 plexiform neurofibroma (pNF1) is a complex tumor composed of Schwann cell-derived tumor cells (Nf1−/−) and the tumor microenvironment (TME). Although it has been widely demonstrated that the TME is involved in the formation of neurofibromas, little is known about the effects of the TME on the subsequent progression of human pNF1. Elucidating the molecular interactions between tumor cells and the TME may provide new therapeutic targets to reduce the progression of pNF1. In the present study, we focused on the contributions of fibroblasts, the most abundant cell types in the TME, to the growth of pNF1. To simulate the TME, we used a three-dimensional (3D) coculture model of immortalized pNF1 tumor cells (Nf1−/−) and primary fibroblasts (Nf1+/−) derived from pNF1 patients. We performed live-cell imaging of 3D/4D (3D in real-time) cultures through confocal microscopy followed by 3D quantitative analyses using advanced imaging software. The growth of pNF1 spheroids in 3D cocultures with fibroblasts was significantly greater than that of pNF1 spheroids in 3D monocultures. An increase in the growth of pNF1 spheroids also occurred when they were cultured with conditioned media (CM) from fibroblasts. Moreover, fibroblast-derived CM increased the invasive outgrowth and further local invasion of pNF1 spheroids. Interestingly, when small extracellular vesicles (sEVs) were depleted from the fibroblast-derived CM, the stimulation of the growth of pNF1 spheroids was lost. Our results suggest that fibroblast-derived sEVs are a therapeutic target for reducing the growth of pNF1. [ABSTRACT FROM AUTHOR]

Introduction: Cultivated meat has received growing attention since claims were made that cultivated meat can be produced more ethically and sustainably than the current meat production. However, there are still major challenges in the development of cell lines, scaffolding, growth media, and bioprocess, which need to be overcome to reach industrial production levels. Numerous technological innovations have been proposed to overcome these challenges but they have rarely been evaluated with regard to their social sustainability. Consequently, it remains unclear if and how cultivated meat would contribute towards creating inclusive food systems. Methods: To bring more clarity, the study identifies different technological solutions that are used or developed for production of cultivated meat and identifies the positive and negative claims about the expected contributions of these technological solutions to social inclusion in food systems, using evidence from the literature review and 11 expert interviews. An innovation radar for cultivated meat is proposed to visualise the variety of technological innovations and the claims about their expected contributions to social sustainability. Results: The technologies in the areas cell line development, scaffolding, growth media, and bioprocess are expected to have an impact on inclusion in consumption of cultivated meat. Some cell innovations are expected to raise cognitive barriers due to complex technologies that might be difficult for the consumer to understand. Cultural barriers are expected to be raised by cell innovation entailing genetic engineering and medium innovation using FBS or animal components, which is considered to harm animals. Further, regulatory barriers are expected in the EU if genetic modification is used in the production process, which concerns the areas cells, media, and scaffolding. The innovations for scaffolds are expected to mainly lower cost and cultural barriers since most technologies are already used in the food industry. Bioprocess innovations promise to lower cost barriers, however it must be considered, that most of the collected data for innovations in the bioprocess domain are based on assumptions. Discussion: The study concludes that at this point in time, the most socially sustainable approach to cultivated meat production is not obvious. Under the current technological state of the art, it is not thinkable that production and consumption of cultivated meat could be socially inclusive. As it remains poorly understood if technologies for cultivated meat production could raise or lower barriers to inclusive consumption and production, further research is needed. [ABSTRACT FROM AUTHOR]

Introduction: The Vel– phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin. [ABSTRACT FROM AUTHOR]

In recent years, the scientific community has increasingly recognized the complex multi-scale competency architecture (MCA) of biology, comprising nested layers of active homeostatic agents, each forming the self-orchestrated substrate for the layer above, and, in turn, relying on the structural and functional plasticity of the layer(s) below. The question of how natural selection could give rise to this MCA has been the focus of intense research. Here, we instead investigate the effects of such decision-making competencies of MCA agential components on the process of evolution itself, using in silico neuroevolution experiments of simulated, minimal developmental biology. We specifically model the process of morphogenesis with neural cellular automata (NCAs) and utilize an evolutionary algorithm to optimize the corresponding model parameters with the objective of collectively self-assembling a two-dimensional spatial target pattern (reliable morphogenesis). Furthermore, we systematically vary the accuracy with which the uni-cellular agents of an NCA can regulate their cell states (simulating stochastic processes and noise during development). This allows us to continuously scale the agents' competency levels from a direct encoding scheme (no competency) to an MCA (with perfect reliability in cell decision executions). We demonstrate that an evolutionary process proceeds much more rapidly when evolving the functional parameters of an MCA compared to evolving the target pattern directly. Moreover, the evolved MCAs generalize well toward system parameter changes and even modified objective functions of the evolutionary process. Thus, the adaptive problem-solving competencies of the agential parts in our NCA-based in silico morphogenesis model strongly affect the evolutionary process, suggesting significant functional implications of the near-ubiquitous competency seen in living matter. [ABSTRACT FROM AUTHOR]

Congenital heart disease (CHD) is the most common birth defect, requiring invasive surgery often before a child's first birthday. Current materials used during CHD surgery lack the ability to grow, remodel, and regenerate. To solve those limitations, 3D bioprinting is an emerging tool with the capability to create tailored constructs based on patients' own imaging data with the ability to grow and remodel once implanted in children with CHD. It has the potential to integrate multiple bioinks with several cell types and biomolecules within 3D-bioprinted constructs that exhibit good structural fidelity, stability, and mechanical integrity. This review gives an overview of CHD and recent advancements in 3D bioprinting technologies with potential use in the treatment of CHD. Moreover, the selection of appropriate biomaterials based on their chemical, physical, and biological properties that are further manipulated to suit their application are also discussed. An introduction to bioink formulations composed of various biomaterials with emphasis on multiple cell types and biomolecules is briefly overviewed. Vasculogenesis and angiogenesis of prefabricated 3D-bioprinted structures and novel 4D printing technology are also summarized. Finally, we discuss several restrictions and our perspective on future directions in 3D bioprinting technologies in the treatment of CHD. [ABSTRACT FROM AUTHOR]

Simple Summary: The paper describes the statistical analysis of the response of gastric cancer cells and normal cells to broadband terahertz radiation up to 4 THz, both with and without the use of nanostructured contrast agents. A statistical analysis was conducted towards multiple pairwise comparisons in order to identify the narrow THz frequency ranges where the contrast is enhanced and allows a clear identification of tumor cells in the presence of contrast agents. The paper describes the statistical analysis of the response of gastric cancer cells and normal cells to broadband terahertz radiation up to 4 THz, both with and without the use of nanostructured contrast agents. The THz spectroscopy analysis was comparatively performed under the ATR procedure and transmission measurement procedure. The statistical analysis was conducted towards multiple pairwise comparisons, including a support medium (without cells) versus a support medium with nanoparticles, normal cells versus normal cells with nanoparticles, and, respectively, tumor cells versus tumor cells with nanoparticles. When generally comparing the ATR procedure and transmission measurement procedure for a broader frequency domain, the differentiation between normal and tumor cells in the presence of contrast agents is superior when using the ATR procedure. THz contrast enhancement by using contrast agents derived from MRI-related contrast agents leads to only limited benefits and only for narrow THz frequency ranges, a disadvantage for THz medical imaging. [ABSTRACT FROM AUTHOR]

Objective Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder. The understanding of the changes in adaptive immune cells and the crosstalk between innate and adaptive immune systems in AOSD is limited. This study aimed to examine the peripheral immune cell composition and inflammatory protein levels in AOSD patients. Methods Twenty-nine active AOSD patients were enrolled. Flow cytometry was used to analyse the cell populations in peripheral blood. Antibody chips were utilized to detect the protein expression profile in serum. Results In active AOSD patients, there was an increase in the percentage of classical and non-classical monocytes among peripheral blood mononuclear cells. The proportion of natural killer (NK) cells decreased, with an increase in CD56dim NK1 cells and a decrease in CD56bright NK2 cells compared with healthy controls (HCs). The percentage of naïve central memory T cells was decreased, while the percentage of effector and effector memory T cells was increased among adaptive lymphocytes. The proportion of naïve B and memory B cells was decreased, while plasma cells were increased in AOSD patients, indicating activation of the adaptive immune system. Additionally, the serum levels of 40 proteins were elevated in AOSD patients, primarily involved in cytokine–cytokine receptor interaction, inflammatory response and regulation of mitogen-activated protein kinase cascade. Conclusion Our findings showed the activation of the innate and adaptive immune system in AOSD. The protein–protein interaction analysis suggested potential communication between innate and adaptive cell subsets. These findings provide new insights into the pathogenesis of the disease and the development of targeted therapies. [ABSTRACT FROM AUTHOR]

Objective: Systemic lupus erythematosus (SLE), an autoimmune disease with incompletely understood etiology, has a strong genetic component. Although genome‐wide association studies (GWASs) have revealed multiple SLE susceptibility loci and associated single‐nucleotide polymorphisms (SNPs), the precise causal variants, target genes, cell types, tissues, and mechanisms of action remain largely unknown. Methods: Here, we report a comprehensive post‐GWAS analysis using extensive bioinformatics, molecular modeling, and integrative functional genomic and epigenomic analyses to optimize fine‐mapping. We compile and cross‐reference immune cell–specific expression quantitative trait loci (cis– and trans–expression quantitative trait loci) with promoter capture high‐throughput capture chromatin conformation (PCHi‐C), allele‐specific chromatin accessibility, and massively parallel reporter assay data to define predisposing variants and target genes. We experimentally validate a predicted locus using CRISPR/Cas9 genome editing, quantitative polymerase chain reaction, and Western blot. Results: Anchoring on 452 index SNPs, we selected 9,931 high linkage disequilibrium (r2 > 0.8) SNPs and defined 182 independent non‐human leukocyte antigen (HLA) SLE loci. The 3,746 SNPs from 143 loci were identified as regulating 564 unique genes. Target genes are enriched in lupus‐related tissues and associated with other autoimmune diseases. Of these, 329 SNPs (106 loci) showed significant allele‐specific chromatin accessibility and/or enhancer activity, indicating regulatory potential. Using CRISPR/Cas9, we validated reference SNP identifier 57668933 (rs57668933) as a functional variant regulating multiple targets, including SLE‐risk gene ELF1 in B cells. Conclusion: We demonstrate and validate post‐GWAS strategies for using multidimensional data to prioritize likely causal variants with cognate gene targets underlying SLE pathogenesis. Our results provide a catalog of significantly SLE‐associated SNPs and loci, target genes, and likely biochemical mechanisms to guide experimental characterization. [ABSTRACT FROM AUTHOR]

Objective Hydroxychloroquine (HCQ) has immunomodulatory, antithrombotic, cardiovascular, antimicrobial, and antineoplastic effects. In this study, we aimed to investigate the antiapoptotic and immunomodulator effects of intraperitoneal HCQ on hypoxic–ischemic (HI) injury in newborn rats. Study Design Wistar albino rats, 7 to 10 days old, were randomly divided into three groups: hypoxic–ischemic encephalopathy (HIE) group, HIE treated with HCQ group, and Sham group. Left common carotid artery ligation and hypoxia model were performed in HIE and HCQ groups. The HCQ group was treated with 80 mg/kg intraperitoneal HCQ every 24 hours for 3 days, while Sham and HIE groups were given physiological saline. After 72 hours, rats were decapitated and brain tissues were stained with hematoxylin and eosin, TUNEL, and IL-1β for histopathological grading and neuronal cell injury. Results Neuronal apoptosis was statistically lower in all neuroanatomical areas in the HCQ group compared with the HIE group. IL-1β-stained areas were similar in both HCQ and HIE groups but significantly higher compared with the Sham group. Histopathological grading scores were found to be lower in the HCQ group on the left parietal cortex and hippocampus region. Conclusion In this study, we have shown for the first time that HCQ treatment decreased apoptosis in HI newborn rat model in both hemispheres. HCQ may be a promising adjuvant therapy in neonatal HIE. Key Points HCQ decreased neuronal apoptosis in the ischemic penumbra of the rat brain. HCQ attenuates hypoxia–ischemia-induced brain injury in neonatal rats. HCQ has no anti-inflammatory effect on HI injury. [ABSTRACT FROM AUTHOR]

A new triterpenoid saponin (1), along with five known compounds (2–6), was isolated from Bupleurum marginatum Wall. ex DC, of which compounds 2–4 were obtained for the first time from this plant. The structures were confirmed by the analysis of 1D, 2D NMR, and HR-ESIMS data, and comparison with previous spectral data. Anti-liver fibrotic activities of the isolates were determined as proliferation inhibition of LPS-induced activation of HSC-T6 in vitro. [ABSTRACT FROM AUTHOR]

Background: No other effects of atropine other than as an antagonist of muscarinic acetylcholine receptor (mAChR) have been found. Methods: In this study, human kidney epithelial cells were treated with different physiological regulators. Results: Subsequently, it was found that atropine could significantly induce autophagy as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and increased p62/SQSTM1 indicated incomplete autophagy. In addition, atropine induced autophagosome levels in a dose-dependent manner within a certain concentration range in human kidney epithelial cells. In atropine-treated mouse skeletal muscle cells containing nicotinic acetylcholine receptors and rat cardiac muscle cells containing mAchR, atropine induced autophagy in mouse skeletal muscle cells but not in rat cardiac muscle cells. Furthermore, atropine did not induce autophagy in tissue cells containing mAchR in vivo but did in tissue cells not containing mAchR. Conclusion: This study expands the application and understanding of atropine's action mechanism in the field of medicine. [ABSTRACT FROM AUTHOR]

• Suspended particulate matter induces the accumulation of ROS, which plays an essential role in oxidative stress. • Suspended particulate matter produces IL-8 and TGF-β1, which develops inflammation and fibrosis. • ROS, IL-8 and TGF-β1 interact together to induces SMAD3-dependent pathway. • Exposure to suspended particulate matter promotes epithelial-to-mesenchymal transition via TGF-β1-mediated ROS/IL-8/SMAD3 axis in A549 cells. Epidemiological evidence presents that dust storms are related to respiratory diseases, such as pulmonary fibrosis (PF). However, the precise underlying mechanisms of SPM-elicited adverse effects still need to be investigated. Epithelial-mesenchymal transition (EMT) process is a characteristic of PF. We discussed whether suspended particulate matter (SPM) is involved in EMT induction via transforming growth factor-β1 (TGF-β1). In this study, a detailed elemental analysis (55 elements), particle size, and morphology were determined. To investigate the toxicity of SPM, an MTT test was performed to detect cell viability. Next, A549 cells were exposed to selected concentrations of SPM (20 and 40 µg/mL) for single and repeated exposures. The DCFH-DA assay showed that exposure to SPM could produce reactive oxygen species (ROS). The ELISA assay demonstrated increased levels of interleukin-8 (IL-8) and TGF-β1 in the supernatant. Western blot was used to detect the expression of proteins associated with EMT and the SMAD3-dependent pathway. Results of western blot demonstrated that E-cadherin was reduced, whereas p-SMAD3, vimentin, and α-smooth muscle actin were elevated. Our findings indicated that SPM triggered EMT by induction of oxidative stress, inflammation, and the TGF-β1/SMAD3 pathway activation. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) protects the brain by actively allowing the entry of ions and nutrients while limiting the passage of from toxins and pathogens. A healthy BBB has low permeability and high selectivity to maintain normal brain functions. Increased BBB permeability can result from neurological diseases and traumatic injuries. Modern engineering technologies such as microfluidics and fabrication techniques have advanced the development of BBB models to simulate the basic functions of BBB. However, the intrinsic BBB properties are difficult to replicate. Existing in vitro BBB models demonstrate inconsistent BBB permeability and selectivity due to variations in microfluidic design, cell types and arrangement, expression of tight junction (TJ) proteins, and use of shear stress. Specifically, microfluidic designs have flow channels of different sizes, complexity, topology, and modular structure. Different cell types are selected to mimic various physiological conditions. These factors make it challenging to compare results obtained using different experimental setups. This paper highlights key factors that play important roles in influencing microfluidic models and discusses how these factors contribute to permeability and selectivity of the BBB models. [ABSTRACT FROM AUTHOR]

The article focuses on the high risk of inflammatory sensory and motor neuropathies associated with Brentuximab, an antibody-drug conjugate used in lymphoma treatment. Topics include the characteristics, risk factors, and management of Brentuximab-induced neuropathy, highlighting its inflammatory nature and distinct clinical features.

Aims: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes‐related conditions. We have identified a novel population of stromal cells within islets of Langerhans – islet stellate cells (ISCs) – which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. Methods: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell‐surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co‐culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. Results: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co‐culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co‐culture with MSCs. Conclusions: ISCs are a specific sub‐type of islet‐derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes. [ABSTRACT FROM AUTHOR]

Simple Summary: Spatial characteristics, including cell morphology and spatial distribution patterns, hold potential prognostic value in melanoma. This study builds on existing research by exploring these spatial factors through a computational pipeline, including both univariate and multivariate Cox proportional hazards models, supplemented by rigorous cross-validation techniques. Our findings reveal that spatial and morphological features can potentially enhance the prognostication of melanoma, supporting their integration into standard prognostic models. We also discuss potential limitations and outline directions for future research. In this study, the prognostic value of cellular morphology and spatial configurations in melanoma has been examined, aiming to complement traditional prognostic indicators like mitotic activity and tumor thickness. Through a computational pipeline using machine learning and deep learning methods, we quantified nuclei sizes within different spatial regions and analyzed their prognostic significance using univariate and multivariate Cox models. Nuclei sizes in the invasive band demonstrated a significant hazard ratio (HR) of 1.1 (95% CI: 1.03, 1.18). Similarly, the nuclei sizes of tumor cells and Ki67 S100 co-positive cells in the invasive band achieved HRs of 1.07 (95% CI: 1.02, 1.13) and 1.09 (95% CI: 1.04, 1.16), respectively. Our findings reveal that nuclei sizes, particularly in the invasive band, are potentially prognostic factors. Correlation analyses further demonstrated a meaningful relationship between cellular morphology and tumor progression, notably showing that nuclei size within the invasive band correlates substantially with tumor thickness. These results suggest the potential of integrating spatial and morphological analyses into melanoma prognostication. [ABSTRACT FROM AUTHOR]

Simple Summary: Scientists are studying proteins like kinesin spindle protein and Aurora B, crucial for cell division and potential targets in cancer treatment. Drugs aimed at these proteins show promise in lab tests for killing cancer cells, but in clinical trials alone, they are not always effective, possibly due to varied cancer cell responses. To enhance their efficacy, researchers are exploring combinations with other cell-killing drugs. Our study focused on Navitoclax, an inducer of cancer cell death, tested alongside Ispinesib and Barasertib, targeting kinesin spindle protein and Aurora B, respectively. Together, these drugs induced significant cancer cell death, mainly through apoptosis. Moreover, imaging techniques revealing their combined effects suggest that combining these drugs could be a potent cancer treatment strategy, warranting further investigation in clinical trials. Many proteins regulating mitosis have emerged as targets for cancer therapy, including the kinesin spindle protein (KSP) and Aurora kinase B (AurB). KSP is crucial for proper spindle pole separation during mitosis, while AurB plays roles in chromosome segregation and cytokinesis. Agents targeting KSP and AurB selectively affect dividing cells and have shown significant activity in vitro. However, these drugs, despite advancing to clinical trials, often yield unsatisfactory outcomes as monotherapy, likely due to variable responses driven by cyclin B degradation and apoptosis signal accumulation networks. Accumulated data suggest that combining emerging antimitotics with various cytostatic drugs can enhance tumor-killing effects compared to monotherapy. Here, we investigated the impact of inhibiting anti-apoptotic signals with the BH3-mimetic Navitoclax in oral cancer cells treated with the selective KSP inhibitor, Ispinesib, or AurB inhibitor, Barasertib, aiming to potentiate cell death. The combination of BH3-mimetics with both KSP and AurB inhibitors synergistically induced substantial cell death, primarily through apoptosis. A mechanistic analysis underlying this synergistic activity, undertaken by live-cell imaging, is presented. Our data underscore the importance of combining BH3-mimetics with antimitotics in clinical trials to maximize their effectiveness. [ABSTRACT FROM AUTHOR]

Reversible protonic ceramic electrochemical cells (R-PCECs) demonstrate great feasibility for efficient energy storage and conversion. One critical challenge for the development of R-PCECs is the design of novel air electrodes with the characteristics of high catalytic activity and acceptable durability. Here, we report a donor doping of Hf into the B-site of a cobalt-based double perovskite with a nominal formula of PrBa0.8Ca0.2Co1.9Hf0.1O5+δ (PBCCHf0.1), which is naturally reconfigured to a double perovskite PrBa0.8-xCa0.2Co1.9Hf0.1-xO5+δ (PBCCHf0.1-x) backbone and nano-sized BaHfO3 (BHO) on the surface of PBCCHf0.1-x. The air electrode demonstrates enhanced catalytic activity and durability (a stable polarization resistance of 0.269 Ω cm² for ~100 h at 600 °C), due likely to the fast surface exchange process and bulk diffusion process. When employed as an air electrode of R-PCECs, a cell with PBCCHf0.1 air electrode demonstrates encouraging performances in modes of the fuel cell (FC) and electrolysis (EL) at 600 °C: a peak power density of 0.998 W cm-2 and a current density of -1.613 A cm-2 at 1.3 V (with acceptable Faradaic efficiencies). More importantly, the single-cell with PBCCHf0.1 air electrode demonstrates good cycling stability, switching back and forth from FC mode to EL mode ±0.5 A cm-2 for 200 h and 50 cycles. [ABSTRACT FROM AUTHOR]

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Toxoplasma gondiis propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasites ability to efficiently invade and regulate components of the hosts infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasmas ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasites diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogens tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cells response to limit infection.

Mast cells (MCs) are tissue-resident immune cells of a hematopoietic origin that play vital roles in innate and adaptive immunity. Human MCs can be isolated and differentiated from various tissue sources, including cord blood, when supplemented with cytokines such as stem cell factor, interleukin 3, and interleukin 6. Our current research study has shown significant differences in the marker expressions of human cord blood-derived mast cells (hCBMCs) based on donor dependency and the type of medium used for culturing and differentiation. These findings are particularly relevant given the challenges of obtaining specialty media influencing MC phenotypic marker expressions. We found that hCBMCs cultured in StemSpanTM-XF medium had a moderate expression of mast/stem cell growth factor receptor Kit (c-KIT) (mRNA and protein), low expressions of FcεRI (mRNA) and TLR2 (mRNA and protein) but had high levels of MRGPRX2 (mRNA and protein) expressions. In contrast, hCBMCs cultured in Stem Line II medium expressed FcεRI and TLR2 (mRNA and protein) with higher c-KIT but had lower MRGPRX2 expressions compared to the hCBMCs cultured in the StemSpanTM-XF medium. These results suggest that it is crucial to consider both donor dependency and the medium when investigating MC functions and that further research is needed to fully understand the impact of these factors on the hCBMC marker expressions.

Bone repair via endochondral ossification is a complex process for the critical size reparation of bone defects. Tissue engineering strategies are being developed as alternative treatments to autografts or allografts. Most approaches to bone regeneration involve the use of calcium composites. However, exploring calcium-free alternatives in endochondral bone repair has emerged as a promising way to contribute to bone healing. By analyzing researches from the last ten years, this review identifies the potential benefits of such alternatives compared to traditional calcium-based approaches. Understanding the impact of calcium-free alternatives on endochondral bone repair can have profound implications for orthopedic and regenerative medicine. This review evaluates the efficacy of calcium-free alternatives in endochondral bone repair through in vivo trials. The findings may guide future research to develop innovative strategies to improve endochondral bone repair without relying on calcium. Exploring alternative approaches may lead to the discovery of novel therapies that improve bone healing outcomes.