Experimental Cell Research

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Iron absorption in the small intestine has classically been described by the duodenal DMT1/FPN1 pathway for inorganic non-heme iron, yet emerging evidence suggests that chemically distinct iron forms may use region-specific routes. Nicotianamine (NA), a plant-derived metal chelator, can form NA-iron (NA-Fe) complexes and has been proposed to support intestinal iron absorption through amino acid transporter pathways. However, direct comparisons of transepithelial transfer of inorganic iron and NA-Fe across defined small intestinal regions under controlled epithelial conditions remain limited. Here, we established region-specific 2D epithelial monolayers derived from duodenal and proximal jejunal crypt organoids from male ICR mice cultured on Transwell inserts. Transcriptomic profiling indicated partial retention of regional identity, and barrier integrity was confirmed by junctional marker localization, transepithelial electrical resistance, and low paracellular permeability. We then examined expression and polarized localization of candidate transporters for inorganic iron (Dmt1/Fpn1) and NA-Fe (Pat1/Lat2). Finally, we quantified transepithelial transport using apical loading of isotope-labeled iron (55Fe) or NA-55Fe and measured radioactivity appearing in the basolateral compartment as the primary readout of transepithelial flux. Basolateral appearance of inorganic 55Fe was comparable between duodenum- and proximal jejunum-derived monolayers, whereas NA-55Fe exhibited significantly greater basolateral appearance in proximal jejunum-derived monolayers. These findings demonstrate that organoid derived, region-specific monolayers provide a tractable epithelial platform to evaluate iron form-dependent, region-specific transepithelial transfer and to enable further mechanistic dissection of NA-Fe transport. NEW & NOTEWORTHYNon-heme iron absorption may depend on iron chemical form and intestinal region, but direct epithelial comparisons are scarce. We established duodenum and proximal jejunum derived murine intestinal organoid monolayers on Transwells and quantified transepithelial flux using isotope-labeled iron. Inorganic 55Fe showed no clear regional difference, whereas NA-55Fe displayed greater basolateral appearance in proximal jejunum-derived monolayers. This platform enables mechanistic studies of NA-iron complex transport.

Eya3 and Eya4 are two Eya genes expressed in adult myogenic stem cells, where they may act as SIX cofactors. We analyzed muscle regeneration in single and compound Eya3 and satellite cell-specific Eya4 mutant mice. A kinetic analysis of muscle regeneration after Notexin injury of the Tibialis Anterior revealed no major phenotype at 4, 14, and 30 days after injury in terms of PAX7+ cell number and myofiber cross-sectional area in Eya3 mutants, while all parameters were decreased in Eya4 mutants and further worsened in Eya3/Eya4 double mutants, in which we also observed a modification of the myofiber phenotype at 30 days after injury. Satellite cells were cultured ex vivo and Eya4 deletion was induced by Ad-Cre-mediated recombination. While single Eya3 mutant cells showed normal proliferation and differentiation, double mutant cells exhibited normal proliferation but failed to fuse. Analysis of their transcriptome revealed that the expression of Myomixer, Follistatin, and Noggin was severely downregulated specifically in double mutant cells, explaining their fusion deficiency. To gain a better understanding of the involvement of Eya genes during embryonic development and the genesis of PAX7+ myogenic stem cells, we analyzed Eya1 / ;Eya2 / , Eya3 / , Eya4 / , and Eya3 / ;Eya4 / E18.5 mutant fetuses at the limb and craniofacial levels. In Eya1 / ;Eya2 / fetuses, we confirmed the absence of distal limb muscles and observed reduced craniofacial muscles. In Eya3 / ;Eya4 / fetuses, craniofacial myogenesis appeared preserved and PAX7+ myogenic stem cells were present. BackgroundThe Eyes absent (Eya) genes encode transcriptional co-activators and phosphatases that function within the PAX-SIX-EYA-DACH (PSED) regulatory network. In skeletal muscle, EYA proteins cooperate with SIX homeoproteins to control myogenic gene expression during both embryonic development and adult regeneration. While Eya1 and Eya2 are predominantly expressed in embryonic myogenic progenitors and Eya3 and Eya4 are the dominant paralogs in adult satellite cells (SC), the specific and redundant contributions of individual family members to myogenesis remain poorly characterized. MethodsWe analyzed compound Eya mutant mice during adult Tibialis anterior muscle regeneration and during embryogenesis. We complemented this analysis by performing ex vivo myogenic stem cell cultures from compound Eya mutants and examining their fusion capacity. ResultsAnalysis of muscle regeneration following Notexin injury revealed that Eya2 and Eya3 single mutants display no major regenerative deficit. In contrast, satellite cell-specific deletion of Eya4 (Eya4sc/sc) caused a transient impairment of early regeneration, with reduced numbers of smaller regenerating MYH3+ (embryonic myosin heavy chain) myofibers and a transient decrease in SC number at 4 days post-injury (dpi). Compound Eya3-/-;Eya4sc/scdouble mutants showed a more severe and persistent phenotype, with decreased myofiber cross-sectional area, reduced myonuclear accretion, accumulation of PAX7+ cells associated with regenerated myofibers, and altered fiber-type composition at 14 and 30 dpi. Ex vivo analysis of double mutant SCs revealed a specific and complete blockade of myogenic fusion without defects in proliferation or MYOD expression. Transcriptomic analysis identified severe downregulation of Myomixer, Noggin, and Follistatin in differentiating Eya3-/-;Eya4-/- SCs. Open-access SIX1 and SIX4 ChIP-seq publicly available data confirmed direct binding at the Myomixer, Noggin, and Follistatin loci, supporting a direct SIX-EYA transcriptional mechanism. In parallel, embryonic analysis demonstrated that Eya1-/-;Eya2-/-E18.5 fetuses lack distal limb musculature and display severe craniofacial muscle hypoplasia, while in Eya3-/-;Eya4-/-fetuses limb and craniofacial musculature developed with no detectable defects. ConclusionsThese results reveal distinct temporal requirements for EYA proteins in skeletal muscle: EYA1 and EYA2 are essential SIX cofactors for embryonic myogenic fate acquisition in hypaxial and craniofacial progenitors, while EYA3 and EYA4 act redundantly in adult satellite cells to enable myogenic fusion by maintaining BMP antagonist expression and Myomixer activation downstream of the SIX-EYA transcriptional complex.

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

Interleukin-6 (IL-6), produced by skeletal muscle and extramuscular tissues, regulates skeletal muscle function through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. However, the interaction between intrinsic (locally produced) IL-6 and extrinsic (circulating) IL-6 in skeletal muscle remains unclear. We investigated whether and how intrinsic expression of IL-6 in cultured primary human myoblasts influences their response to extrinsic stimulation with recombinant human IL-6 (rhIL-6). Using gene silencing, we found that suppression of intrinsic IL-6 enhanced rhIL-6-induced phosphorylation of STAT1 and STAT3. Silencing STAT3 also increased rhIL-6-induced STAT1 phosphorylation, but silencing STAT1 had no effect on STAT3 phosphorylation. Pretreatment of myoblasts with neutralising anti-IL-6 antibodies increased phosphorylation of STAT1 and STAT3 induced by 50 ng/mL rhIL-6, whereas pretreatment with 5 ng/mL rhIL-6 reduced this response. Despite increased JAK/STAT signalling, IL-6 silencing decreased glucose and oleic acid uptake and oxidation under both basal and rhIL-6-stimulated conditions. Collectively, our results imply that intrinsic IL-6 restrains activation of the JAK/STAT pathway by extrinsic IL-6, but acts synergistically with it to promote myoblast energy metabolism.

ObjectiveLoss of skeletal muscle mass and performance is a hallmark of ageing. Mitochondrial function has been suggested as a critical determinant of skeletal muscle performance. However, mixed results have been reported regarding mitochondrial function in older individuals. Therefore, the primary objective of this systematic review is to determine whether 31P-MRS-derived {tau}PCr, reflecting mitochondrial oxidative capacity, is reduced in ageing skeletal muscle. MethodsA preregistered systematic literature review was performed using the databases MEDLINE, EMBASE, SPORTDiscus, and Cochrane Central Register of Controlled Trials (CENTRAL). Papers were included if they reported {tau}PCr as measured by 31P-MRS; and studied individuals over 65 years of age in combination with a younger control group. Differences between young and older groups were assessed using random effects meta-analysis. ResultsWe included 20 papers in total, of which 2 measured 2 muscles, 5 focused on the tibialis anterior (TA) muscle, 11 on the calf muscles, 5 on the quadriceps, and 1 on the flexor digitorum longus. No statistically-significant differences were found in {tau}PCr between older and younger adults for all muscles combined (Hedges g=0.11 (p=0.487). Inter-study heterogeneity was high ({tau}2=0.36, I2=72.49%, H2=3.64). Sub-analyses for the individual muscles showed longer {tau}PCr in the quadriceps (g=0.65, p<0.001) in older adults, but shorter {tau}PCr in the TA muscle (g=-0.64, p<0.001). For the calf muscles, no differences were detected between older and young individuals (g=0.20, p=0.377). ConclusionNo uniform age-related decline was found for {tau}PCr when comparing all studies together. Substantial heterogeneity was observed between the individual muscles, with {tau}PCr being prolonged in the upper leg muscles in older adults, but shortened in the tibialis anterior. This suggests more work using standardised settings and well-defined cohorts is needed.

Pluripotent stem cells derived from livestock species represent valuable systems for studying early mammalian development and for establishing renewable, well-defined cell sources; however, direct comparative characterization of distinct pluripotent stem cell platforms in sheep remains limited. In this study, we established and evaluated two ovine pluripotent stem cell types: reprogrammed induced pluripotent stem cells (siPSCs) and embryonic disc-derived stem cells (sEDSCs). Both siPSCs and sEDSCs exhibited core features of pluripotency, including compact colony morphology, alkaline phosphatase activity, expression of key pluripotency-associated markers, and maintenance of a normal ovine karyotype. Flow cytometry and quantitative RT-PCR analyses revealed broadly overlapping yet distinguishable pluripotency marker expression profiles between the two cell types. Functional pluripotency was confirmed by embryoid body formation and in vitro differentiation into derivatives of all three germ layers. To further assess lineage-specific differentiation competence and compare functional outputs relevant to mesodermal differentiation, both pluripotent stem cell types were directed towards the adipogenic lineage. While siPSCs and sEDSCs were each capable of adipogenic differentiation, differences in differentiation efficiency and marker expression were observed. Together, these findings demonstrate that ovine siPSCs and sEDSCs share core pluripotency characteristics while retaining distinct molecular and functional properties, providing a robust comparative framework for studies of ovine pluripotency, lineage specification, and stem cell biology.

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([&ge;]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

BackgroundSkeletal muscle represents around 40% of total human body weight and exhibits remarkable plasticity. It can hypertrophy, atrophy, or regenerate in response to changes in activity, nutrient availability, or injury. The main component of striated muscle, the myofiber, is a post-mitotic, multinucleated cell that contains the muscles contractile unit, the sarcomere. The myonuclei within these fibers are specialized and differ in terms of gene expression and localization. Adult muscles also contain various other cell types, including adult muscle stem cells (MuSCs), macrophages, fibro-adipogenic progenitors (FAPs), and endothelial cells. MuSCs are central to muscle plasticity, and are capable of activation, proliferation, differentiation, and fusion to form new myofibers during regeneration, or to fuse with existing myofibers during hypertrophy. Muscle hypertrophy and myofibers enlargement involve increased protein synthesis and reduced protein degradation, as well as myonuclear accretion following satellite cell activation. Multiple signaling pathways, such as the mTOR pathway and the RhoA/SRF mechanotransduction pathway, are involved in these processes. MethodsWe performed single-nucleus RNA sequencing (snRNA-seq) on plantaris muscles of adult mice, comparing samples 7 days after hypertrophy induction (overload, 7OV) to non-hypertrophied controls (Ctl). RNAscope experiments on isolated myofibers identified the heterogeneity of myonuclei along the myofiber. ResultsSnRNA-seq analysis revealed a previously unknown population of myonuclei (UM). UM-Ctl, which is present only in the Ctl condition, and UM-7OV, only in the 7OV condition. These myonuclei are localised at the tips of myofibres. Furthermore, we determined that UM-7OV are not newly fused myonuclei from activated satellite cells. Trajectory analyses suggest that UM-Ctl transition into UM-7OV during hypertrophy, returning to a near-basal homeostatic state after 21 days of overload (21OV). Gene expression analysis showed that UM-Ctl and UM-7OV have distinct gene expression profiles compared to other myonuclei and respond differently to hypertrophy. ConclusionOur findings suggest the existence of a specific population of myonuclei with unique localization and gene expression profiles, which play distinct roles at baseline and during hypertrophy. These results highlight the differential properties of myonuclei in the myofiber and their potential specific functions in muscle homeostasis and adaptation.

Unlike the conventional mature neutrophils, immature neutrophils have been investigated for their regenerative properties; however, their limited availability necessitates alternative generation strategies. Here, we used a combination of dimethylsulfoxide (DMSO) and 1,25-dihydroxyvitamin D3 (D3) to differentiate myeloid leukemia (HL-60) cells into immature neutrophil-like cells. Differentiated cells exhibited reduced cell size, loss of uniformity, decreased nuclear-to-cytoplasmic ratio, band-shaped nuclei, increased proportion of CD11b+CD14+ cells (indicative of immature neutrophils), decreased proportion of CD11b+CD16+ cells (indicative of mature neutrophils), higher levels of arginase 1, TGF{beta}1 (markers of immature neutrophils), and no expression of CD16, MRC1 (markers of mature neutrophils and M2 macrophages, respectively). Proteomic analysis revealed enrichment of proteins associated with immature neutrophils and wound healing. Functionally, these cells supported limbal stem cell growth and wound closure in vitro, indicating relevance for corneal regeneration. Administration of these cells to ex-vivo and in-vivo alkali-injured corneas, resulted in significant effect on promotion of wound healing, with epithelial regeneration and decreased fibrotic markers, proving that such cells hold promise for clinical translation as a therapeutic tool for tissue repair.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

In this study, we evaluated the impact of different in vitro 3D culture modelling methods on the activity of doxorubicin (DOX) and 5-fluorouracil (5-FU) in human melanoma spheroids. Human melanoma A375 and IGR39 spheroids were generated using the hanging drop and non-adhesive surface methods. Spheroid growth dynamics were assessed by measuring changes in spheroid diameter. To compare the effects of anticancer drugs in spheroids of different sizes, spheroids of approximately 200 and 400 {micro}m were formed. Drug activity was evaluated based on spheroid growth and cell viability using the MTT assay. A375 spheroids formed using the non-adhesive surface method were more sensitive to DOX than spheroids formed using the hanging drop method. In smaller A375 spheroids, 10 {micro}M 5-FU reduced cell viability more effectively in spheroids formed using the hanging drop method. In contrast, IGR39 spheroids formed by the hanging drop method were more resistant than those formed on a non-adhesive surface. However, in IGR39 spheroids, the effects of DOX and 5-FU on growth and viability did not significantly differ between formation methods. In conclusion, A375 spheroid growth was not significantly influenced by the formation method, whereas IGR39 spheroid growth depended on the method used. A375 spheroids formed on non-adhesive surfaces were more sensitive to DOX, whereas 5-FU activity depended on drug concentration and spheroid size. In IGR39 spheroids, the effects of DOX and 5-FU on growth and viability were largely independent of the spheroid formation method. Based on these results, it can be concluded that the researchers should carefully select the spheroid formation method for their studies, as this may influence the results of the tested compounds effect on their size and viability.

During early development of the placenta, a subset of murine trophectoderm stem cells (TSCs) undergo endoreplication, an unusual form of cell division cycle that decouples DNA synthesis from cytokinesis, resulting in physiological polyploidy. Oscillations in CDK2 activity are essential for the orderly progression of the cell cycle to ensure replicated DNA is accurately partitioned into two daughter cells. However, it remains underexplored how the dynamics of CDK2 activity regulate endoreplication in the context of TSCs differentiation. To address this question, we leveraged the variability in cell fate decisions in an established in vitro system of TSCs differentiation that relies on removal of a growth factor, FGF4, to induce endoreplication. Using quantitative single-cell live confocal microscopy of a precise CDK2 biosensor, DHB-Venus, we identified at least three different outcomes upon FG4 removal: self-renewal, endoreplication, and migration. Our quantitative analyses showed high levels of Cdk2 activity in self-renewing cells whereas intermediate DHB-Venus turnover is linked to increased nuclear and cell size, indicating a shift to endoreplication. Importantly, we also characterize a third class of differentiating TSCs with migratory characteristics that correlate with low levels Cdk2 activity without a change in nuclear size. In sum, our results demonstrated a correlation between different fate outcomes and specific thresholds of CDK2 activity. Our findings show that TSCs can distinguish between different outcomes through modulating the central kinase of the cell cycle, CDK2, positioning it as a key regulator of early trophoblast differentiation. Summary StatementThis study investigates the oscillatory behavior of CDK2 activity during murine trophectoderm differentiation and its potential role in guiding cell fate decisions.

The chemical constituents and cytoprotective potential of Cyathea podophylla, a Vietnamese fern, remain poorly investigated. This study aimed to isolate its compounds and evaluate their in vitro cytoprotective activity against 6-hydroxydopamine (6-OHDA)-induced toxicity in F11 cells. Compounds were chromatographically isolated and structurally characterized using NMR and HR-ESI-MS. Seven compounds were identified: five phenolics (trans-cinnamic acid, (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one, p-coumaric acid, 3,4-dihydroxybenzoic acid, 4-O-acetyl-caffeic acid), 5-hydroxymethylfurfural, and butyl-{beta}-D-fructofuranoside. Six of these are newly reported for the Cyathea genus. In MTT assays, butyl-{beta}-D-fructofuranoside exhibited the strongest cytoprotective effect (69.6% cell protection at 10 {micro}M, p < 0.001), followed by (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (39.2% at 10 {micro}M). The remaining compounds lacked significant activity. These findings expand the phytochemical profile of Cyathea podophylla and provide preliminary evidence of its cytoprotective properties against 6-OHDA-induced injury, warranting further mechanistic and in vivo validation.

Lung adenocarcinomas frequently harbour actionable oncogenic mutations that are vulnerable to treatment with targeted therapies. While responses to targeted therapies are often initially dramatic, relapse is almost inevitable and prevents durable responses in advanced-stage patients. Relapse is, in part, caused by drug tolerant persister cells (DTPs) which are able to survive treatment by entering a reversible, dormant state. Although long non-coding RNAs (lncRNAs) regulate processes thought to allow DTPs to survive and become stably resistant, the potential roles of lncRNAs in DTPs are largely unknown. In this study, we sought to investigate the expression of lncRNAs in in vitro DTP models of lung adenocarcinoma. We found that the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were enriched in DTPs and that knocking down MALAT1 enhanced the effect of targeted therapies in both EGFR- and KRAS-mutant DTP models. To better understand pathways that MALAT1 might regulate in DTPs, bulk RNA-sequencing was performed and several pathways that may contribute to the actions of MALAT1 in DTPs were identified. Overall, our work describes a role for the lncRNA MALAT1 in DTPs in NSCLC and suggests that MALAT1 may be a novel target for the prevention of drug tolerance and subsequent resistance to targeted therapy in NSCLC.

Osteocytes and adipocytes represent cells with disparate functions. Osteocytes regulate bone metabolism (remodeling) and bone homeostasis, while adipocytes regulate energy metabolism and energy storage. Here, we demonstrate that osteocyte phenotype consists of adipocytic features which are under control of peroxisome proliferator-activated receptor gamma (PPARG), a master regulator of adipocyte differentiation and function. Using a mouse model with osteocyte-specific deletion of PPARG (OT{gamma}KO) and osteocyte cellular model of MLO-Y4 cells edited with CRISPR/Cas9 for PPARG deficiency, we are demonstrating that under PPARG control osteocytes produce and secrete adiponectin (ADIPOQ), and they are equipped in adipocyte-specific mechanisms for lipid-storage and their metabolism. Under PPARG, osteocytes accumulate lipid droplets which correlate with their capability to cover up to 20% of energy requirements from fatty acids metabolism. Although osteocytes like osteoblasts mainly express perilipin 2 (Plin2), however similarly to adipocytes, lipid droplets accumulation is associated with expression of perilipin 1 (Plin1) under PPARG control. Similarly, lipids accumulation and metabolism involve adipocyte-specific activities including fatty acids binding protein 4 (Fabp4), hormone-specific lipase (Hsl) and adipocyte-specific triglyceride lipase (Atgl), which expression are under PPARG control. These studies provide a new understanding of osteocyte biology which include adipocyte-like endocrine and lipid metabolism features probably reflecting an adaptation to their unique localization and a need for a maintenance of functional fitness in these conditions. They deepen our comprehension of the crossroads of osteocyte and adipocyte function and underscore the therapeutic potential of targeting common molecular pathways in both cell types for managing metabolic disorders and skeletal diseases.

ObjectiveAnterior cruciate ligament (ACL) tears increase the risk for developing posttraumatic osteoarthritis (PTOA). Females have greater risk for both. However, studies defining sex-specific protein responses in human cartilage after ACL injury are lacking. We hypothesize that articular cartilages response to an injurious environment differs depending on sex. DesignWe compared the proteomic profiles of normal cartilage with injured cartilage harvested from the intercondylar area during ACL surgery. Sex-specific injury effects were estimated through contrasts between Injured Male and Normal Male and between Injured Female and Normal Female. Pathway enrichment analysis was done using gene ontology (GO) and compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the Matrisome AnalyzeR. ResultsFrom the 2,188 proteins identified, males and females shared 1,121 upregulated and 23 downregulated proteins in injured compared to normal cartilage. Analysis of ECM proteins and enriched pathways revealed mostly similar male and female responses to an injurious environment, with evidence of early cartilage remodeling in both sexes. Nevertheless, more than 240 proteins were affected specifically by sex, and significant sex differences were found in inflammation, ECM-related, and metabolic pathways. Males were enriched mostly in "ECM-receptor interaction", while females were enriched in "Citrate cycle (TCA cycle)", "Fatty acid degradation", and "Fatty acid metabolism" pathways. ConclusionArticular cartilage shows signs of remodeling soon after ACL injury, even when only exposed to an injurious environment rather than being physically impacted. Sex differences were observed in inflammation, metabolic pathways, and ECM synthesis.

IntroductionCancer progression is driven not only by tumor cells but also by interactions between the extracellular matrix (ECM), stromal cells, and immune cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major drivers of ECM remodeling, assembling ECM with aberrant organization. Extra domain A fibronectin (EDA-FN), a cellular FN containing an extra type III domain, is upregulated in the TME. EDA-FN regulates cellular behavior and has been associated with poor patient prognosis. Macrophages are among the most abundant immune cells within the TME, where they contribute to TME remodeling and inflammation to promote cancer cell invasion and metastasis. However, how tumor-associated matrix-specific cues regulate macrophage behavior remains largely understudied. PurposeHere, we developed a fibroblast-derived matrix platform that captures the structural imprint of tumor-associated EDA-enriched matrices and investigated how matrix-specific cues regulate macrophage behavior in the absence of ongoing soluble factor cues. MethodHuman mammary fibroblasts (HMFs) preconditioned in incubated low-serum media (lNC, or control) and MDA-MB231 metastatic breast cancer cell-conditioned media (mTCM) were cultured on polyacrylamide gels of 2 kPa and 20 kPa, respectively, followed by decellularization. Matrix organization, including fiber alignment, width, and intrafibrillar spacing, was quantified from confocal images. Decellularized EDA-FN-enriched matrices were subsequently reseeded with macrophages to assess macrophage morphology, phenotype, and matrix interactions. ResultsThe combined effects of tumor-derived soluble factors and pathological stiffness induced a CAF-like phenotype in HMFs, accompanied by cytoskeletal reorganization and microarchitectural alterations of EDA-FN-enriched matrices. Tumor-associated matrices exhibited increased alignment, narrower fiber width, and enlarged intrafibrillar spacing compared to control matrices. These aberrant, tumor-associated matrix-derived features were associated with altered macrophage behavior, including heterogeneous morphology, enhanced localized EDA-FN matrix loss beneath the cell body, and a hybrid phenotype with a shift toward a CD206-dominant profile. ConclusionsThese findings demonstrate the feasibility of obtaining EDA-FN-enriched matrices to isolate matrix-specific cues for investigating macrophage-ECM interactions. Furthermore, this platform can be leveraged to identify matrix-targeting therapeutic approaches for modulating macrophage function within the TME.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.