The FASEB Journal

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.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

ObjectiveInvestigate how Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) orchestrates a catabolic shift in chondrocytes during early osteoarthritis (OA). MethodsCartilage, osteochondral plugs and chondrocytes were collected from patients undergoing total hip arthroplasty or non-OA donors. SOX9 levels were assessed via immunoblotting or immunohistochemistry (IHC). Sox9 levels were also assessed by IHC in knee joints from wild-type (WT) and Camkk2-/- mice that underwent sham or destabilization of medial meniscus (DMM), with or without STO-609 (0.033 mg/kg) treatment. Co-immunoprecipitation followed by mass spectrometry was performed to identify CaMKK2 interacting proteins in chondrocytes. Kinase assays were analyzed by immunoblotting and phosphosites identified by mass spectrometry. Proteasome function was assessed in murine and human chondrocytes lacking or expressing kinase-active or kinase-inactive CaMKK2. ResultsInhibition or loss of CaMKK2 increased SOX9, whereas the expression of kinase-active, not inactive, CaMKK2 reduced Sox9 in human and mouse OA cartilage. Proteomic analysis of CaMKK2 immunoprecipitates revealed the presence of ubiquitin E3 ligase Ubr4 and the 19S proteasome regulatory particle (RP). CaMKK2 kinase activity was dispensable for its interactions with Ubr4, 19S RP, and Sox9-ubiquitin conjugates, and kinase-inactive CaMKK2 attenuated Sox9 degradation in chondrocytes. Further, CaMKK2 phosphorylated the 19S RP ATPase Psmc5 on Ser136, and an intact kinase increased proteasome activity in chondrocytes. ConclusionsOur findings identify CaMKK2 as a dual-function regulator of chondrocyte UPS with a scaffolding role to assemble UPSUbr4-19S RP around polyubiquitinated proteins such as Sox9, and a catalytic role to enhance proteasome function, potentially through Psmc5 phosphorylation, thereby linking chondrocyte inflammatory signaling to Sox9 degradation and cartilage degeneration.

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.

In eutherian mammals, blastocyst implantation is often associated with a quasi-inflammatory reaction in the endometrium, which is resolved with the establishment of the definitive placenta. This is understandable in the case of invasive placentation, since implantation entails a nidatory injury to the maternal tissue due to the invading blastocyst. Quasi-inflammatory processes have also been documented in pregnant pigs, even though the blastocyst only attaches to, rather than invades into, the endometrium of the uterus. In this study, we asked what processes in early porcine pregnancy lead to the resolution of attachment-associated inflammation. In generic wound healing the transition from a pro- to an anti-inflammatory state is caused by a corresponding transition from M1 to M2 polarized macrophages following efferocytosis by macrophages of apoptotic neutrophils. In order to determine whether this scenario applies to the pregnancy-related resolution of inflammation in the porcine uterus, we produced a series of bulk transcriptome samples spanning days (D) 13 to 25 of gestation. This time span corresponds to the transition from pre- to post-attachment stages of pregnancy. We found slower changes in the transcriptome between D20 and D25 than prior to D20, suggesting a turning point in pregnancy-related reprogramming. The turning point at D20 corresponds to the time of firm attachment of trophectoderm to uterine luminal epithelium and the cessation of IFNG signaling from the blastocyst. This transition coincides with increased expression of RNAs of genes implicated in resolution of inflammation and M2 polarization such as ARG1, MRC1/CD206, CD86, TGFb1 and IL10, as well as a significant increase in expression of HGPD, the enzyme that metabolizes prostaglandins. While immunoreactivity for ARG1 was found in putative macrophages in the sub-epithelial stratum compactum, other markers of M2 polarized macrophages were localized to non-immune cells: MRC1 was found on fibroblast-like stromal cells, CD86 on trophoblast cells, and IL10 in luminal and glandular epithelia. These results suggest that intrauterine immune regulation is decoupled from that of the rest of the body by engaging non-immune cell types as anti-inflammatory mediators during the peri-attachment period of pregnancy.

Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo. Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish (Danio rerio) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9-deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

Increased mechanical loading induces skeletal muscle growth and, at the ultrastructural level, promotes myofibrillogenesis and the radial growth of myofibrils. However, the mechanisms regulating these ultrastructural adaptations are not known. Here, we sought to determine whether the mechanistic target of rapamycin complex 1 (mTORC1) regulates these processes. To accomplish this, muscle-specific, tamoxifen-inducible raptor knockout (iRAmKO) mice were used to inhibit signaling through mTORC1, and growth was induced with a model of chronic mechanical overload (MOV). Using a next-generation fluorescence imaging pipeline for ultrastructural analyses, we found that mTORC1 is a critical regulator of the myofibrillogenesis and radial growth of myofibrils that occur in response to MOV. Together with other recent advances in the field, we propose a model in which mTORC1 acts as a gatekeeper that permits the retention, rather than the synthesis, of proteins that drive the ultrastructural adaptations.

Cholesterol elimination in mammals depends largely on the biliary secretion of cholesterol and its conversion to bile acids, followed by their fecal loss. Human studies suggest an association between blood vitamin D levels and blood cholesterol; however, the mechanistic impact of sustained elevation of 1,25(OH)2D3 (active vitamin D) on cholesterol flux remains unclear. Here, we used two complementary mouse models--a genetic model with chronically elevated plasma 1,25(OH)2D3 (-klotho KO mice) and a pharmacological model of repeated 1,25(OH)2D3 administration in wild-type mice--to define the mechanism by which 1.25(OH)2D3 regulates the hepatic-intestinal programs controlling cholesterol elimination. -klotho KO mice showed increased fecal excretion of both cholesterol and total bile acids. Hepatically, Sr-b1, Abcg5/Abcg8, Abca1, Cyp7a1, and Mrp2 transcriptions were increased, whereas Cyp27a1 and Bsep was unchanged. Duodenal Npc1l1 was reduced, and ileal Asbt showed a decreasing trend. In the administration model, fecal bile acid levels increased by day 3, consistent with the induction of hepatic Mrp2 expression from day 3. Bsep exhibited a biphasic change, enhanced at early phase and downregulated to basal levels later and Asbt was unchanged. Increased fecal cholesterol emerged later (day 15), accompanied by late-phase induction of Abcg5/Abcg8 and suppression of Npc1l1. Together, we propose that sustained elevation of 1.25(OH)2D3 is associated with coordinated hepatic and intestinal transcriptional remodeling that promotes cholesterol disposal, with an early increase in fecal bile acid loss preceding the enhanced fecal cholesterol excretion.

We investigated effects of three aerobic exercise interventions, varying in amount and intensity with durations of 8-9-months on small RNA (smRNA) expression and regulatory pathways in skeletal muscle and plasma from 120 participants. Using untargeted smRNA sequencing focused on miRNAs and piRNAs, adjusting for demographics and bodyweight, we identified 124 muscle smRNAs altered by exercise amount and 15 by intensity, and 47 plasma smRNAs altered by intensity and one by amount. These smRNAs were enriched in metabolic, transcriptional, translational, and cell cycle pathways. Exercise-induced changes in several smRNAs-six from muscle and five from plasma-and exercise-induced reduction in body weight, aligned with improvement in insulin sensitivity (p<0.05). These findings demonstrate tissue-specific regulation of smRNAs by exercise and identify potential candidates for exercise mimetics to modulate muscle insulin sensitivity.

BackgroundPolycystic kidney disease (PKD) is the leading genetic cause of renal failure, resulting in the accumulation of fluid filled cysts and gross enlargement of the kidney. Mutations in PKD1 or PKD2, which encode ciliary polycystin proteins, are the most common cause of PKD. These proteins function in a cilia-dependent cyst activation (CDCA) pathway-one that requires cilia for its pro-cystic function-yet the molecular driver(s) of this pathway are unknown. ARL13B is a regulatory GTPase enriched in cilia and links to renal cystogenesis. ARL13B possesses guanine nucleotide exchange factor (GEF) activity for ARL3, another ARL with links to cilia. MethodsWe used two distinct Arl13b mouse alleles to investigate whether ARL13B is a component of the CDCA pathway: Arl13bV358Aencodes for enzymatically normal ARL13B that is undetectable in cilia, and Arl13bR79Qencodes for cilia-localized ARL13B lacking a residue critical for its ARL3 GEF activity. We used these alleles in a Pkd1-deficient adult mouse model and investigated renal morphology (H&E and cystic index analysis), physiology (blood urea nitrogen measurements), renal fibrosis (picrosirius staining and -smooth muscle actin levels), renal injury (SOX9 immunofluorescent staining and quantification), and Wnt signaling ({beta}-catenin and cyclin D1 protein levels). ResultsWe found that loss of ciliary ARL13B or mutation of a single residue critical for its ARL3 GEF activity suppressed Pkd1-dependent cysts. We observed reductions in kidney size, cystic index, and blood urea nitrogen. We also observed suppression of renal fibrosis, renal injury, and {beta}-catenin and cyclin D1 protein levels. ConclusionsOur results identified a subcellular location and mechanism driving Pkd1-dependent renal cystogenesis. We demonstrated that expression of a critical residue for ARL13Bs GEF activity specifically in cilia is a key mechanism of the CDCA pathway driving renal cystogenesis. Key PointsO_LILoss of ciliary ARL13B suppressed renal cystogenesis in an adult mouse model of polycystic kidney disease (PKD) without ablating cilia C_LIO_LILoss of ciliary ARL13B or mutation of the residue critical for its GEF activity did not affect renal morphology or physiology in a PKD mouse model C_LIO_LIMutation of a residue critical for ARL13B activation of ARL3 suppressed cystic phenotypes in Pkd1-dependent cysts C_LI

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.

BackgroundPhenome-wide association studies (PheWAS) can reveal novel associations between variants in drug-target genes and disease and, as such, can be used to predict new drug-indication pairs for repurposing drugs with a known mechanism of action. A platelet-derived growth factor receptor beta (PDGFR{beta}) PheWAS demonstrated that patients with a single nucleotide variant that reduces PDGFR{beta} expression exhibit a higher prevalence of chronic skin ulcers, skin grafts, and reconstructive surgeries. Recombinant human platelet derived growth factor BB (rhPDGF) is a therapeutic that binds to and activates PDGFR{beta} and has received FDA approval for multiple indications, including improving healing of lower extremity diabetic neuropathic ulcers, augmenting periodontal bone and soft tissue reconstruction, and stimulating orthopedic bone regeneration. Leveraging a drug-repurposing methodology informed by PheWAS, we hypothesize that rhPDGF will provide therapeutic benefit in the treatment of other complex wounds, like full-thickness surgical wounds of the head or neck that cannot heal by primary intention following skin cancer excision. MethodsThis prospective, double-blinded, single-site study aims to enroll 40 participants, randomized at a ratio of 1:1, comparing the efficacy of an advanced wound matrix saturated with rhPDGF or saline. Comparisons will be stratified by anatomical location (scalp/forehead versus face/neck) and maximum surgical defect dimensions (< 3cm versus > 3cm). The primary outcome of this study will evaluate the time in days to 81-100% granulation of the wound bed by expert clinical assessment of daily photographs. Secondary outcomes will assess the superiority of the rhPDGF-enhanced wound matrix relative to control with respect to wound granulation rate, epithelialization, complete wound healing, and patient reported outcomes (PROMs). DiscussionAlthough reconstructive techniques are available for healing complex head and neck wounds following skin cancer excision, these procedures are invasive, and older, frail patients are often suboptimal candidates. There remains a need for less invasive therapeutic approaches that reduce the healing time and mitigate the morbidity associated with chronic wounds. A PheWAS analysis identified complex wounds requiring reconstructive surgery as a novel drug-indication pair for repurposing rhPDGF. This protocol is designed to evaluate the efficacy of an rhPDGF-enhanced advanced wound matrix for healing complex head and neck wounds post skin cancer excision that cannot heal by primary intention. Clinical trial registrationThis trial is registered at ClinicalTrials.gov (NCT06634030).

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease (ILD) characterized by progressive fibrosis, irreversible loss of lung elasticity, and chronic respiratory failure, with a mean survival of 3-5 years. The disease is believed to result from repeated alveolar epithelial injury that sustains transforming growth factor-beta (TGF-{beta}) signaling, driving fibroblast-to-myofibroblast differentiation and excessive collagen deposition. Although current IPF models--including animal studies, 2D cultures, and basic 3D systems--have enhanced understanding of disease mechanisms, they inadequately replicate epithelial-fibroblast interactions, extracellular matrix (ECM) remodeling, and epithelial barrier dysfunction. To address this limitation, we engineered a 3D lung co-culture model that simulates the physiological epithelial-fibroblast crosstalk and ECM remodeling characteristic of IPF. Our model embeds fibroblasts within a collagen-hyaluronic acid matrix overlaid with an epithelial monolayer cultured at an air-liquid interface. Basolateral TGF-{beta} exposure generated a profibrotic microenvironment that weakened epithelial barrier integrity and drove myofibroblast differentiation marked by elevated -SMA and vimentin. Elevated pro-inflammatory cytokine secretion and increased collagen disorganization further demonstrated active fibrogenesis. Together, these features show that our model captures key early events in IPF pathogenesis and offers a versatile platform for next-generation lung-on-a-chip studies in fibrotic disease.

ObjectivesMechanical cues are essential for maintaining cartilage function, yet how they integrate with molecular pathways dysregulated in osteoarthritis (OA) remains poorly defined in human tissue. Canonical Wnt signalling influences cartilage biology and cell-matrix interactions, but its role in integrin-dependent mechanoregulation in human cartilage is not fully understood. This study aimed to determine how Wnt activation affects chondrocyte responses to physiological mechanical loading, with a focus on 5{beta}1integrin and cytoskeletal organisation. MethodsHuman cartilage explants from non-OA and OA donors were subjected to short-term physiological cyclic compression. Canonical Wnt signalling was activated with CHIR99021, and integrin-mediated adhesion was modulated using the 5{beta}1 blocking peptide ATN-161 during loading. Chondrocyte responses were assessed by analysing mechanoresponsive and matrix-related gene expression, 5{beta}1 complex formation via proximity ligation assay and actin cytoskeletal organisation by confocal microscopy. ResultsOA chondrocytes exhibited a distinct integrin profile, characterised by increased ITGA5 and ITGB1 but reduced ITGA10 expression. In non-OA cartilage, canonical Wnt activation increased ITGB1 expression and 5{beta}1 integrin complex formation, while mechanical loading further enhanced ITGA5 and ITGB1 transcription under Wnt-activated conditions. Under control conditions, loading induced mechanoresponsive and anabolic gene expression in non-OA cartilage; these responses were attenuated following Wnt-activation and partially restored by 5{beta}1 blockade. Mechanical loading induced F-actin reorganization toward a more cortical distribution across cartilage zones, irrespective of disease status or treatment. Wnt activation did not result in distinct cytoskeletal phenotypes under load, and load-induced actin remodelling was comparable between groups. ConclusionThese findings identify 5{beta}1integrin as a key mediator linking canonical Wnt signalling to altered chondrocyte mechanoresponsiveness in human cartilage. While mechanical loading consistently induced cortical F-actin reorganization, Wnt-associated changes in load responsiveness arose primarily from integrin-dependent mechanisms rather than major alterations in actin organization. This study highlights the complexity of cartilage mechanoregulation and identifies integrin-mediated signaling as important contributors to canonical Wnt-driven alterations in load responsiveness relevant to OA.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

Collagens are key components of the extracellular matrix (ECM) that play a crucial role in maintaining structure, strength, and function of the lungs. Fibrillar collagens are crosslinked by enzymes such as lysyl oxidases and transglutaminases and organized into networks by proteoglycans and glycoproteins. Collagens are the main load-bearing components and along with elastin may impart a non-linear strain hardening behavior to the lung. In disease, collagen crosslinking and organization can be disrupted, possibly due to abnormal levels of enzymes or ECM components. Few studies have examined collagen crosslinking and organization in healthy and diseased human lungs. In this study, alterations in collagen crosslinking and organization were investigated in human lung control, fibrotic and chronic obstructive pulmonary disease (COPD) tissue sections. Ultra-performance liquid chromatography and second harmonic generation microscopy measured pyridinoline crosslinks and the distribution of mature and immature collagens within the decellularized scaffolds, respectively. Fibrotic scaffolds had higher total collagen but less crosslinking per mole of collagen compared with COPD donors. Image analysis by second harmonic generation microscopy showed mature collagens populated airway or blood vessel walls in all three groups and in the parenchyma of fibrotic scaffolds. Immature collagens, on the other hand, were mainly localized to parenchymal regions in control and COPD scaffolds, with fewer immature collagens in fibrotic parenchyma. Additionally, quantification of the mature to immature collagen ratio in defined regions of control and diseased scaffolds showed increased organized collagen in fibrotic tissue. Our study shows that collagen crosslinking and organization are disrupted in fibrotic and COPD lungs and these changes may be compartment specific and can contribute to aberrant mechanical properties of diseased lungs. Our findings highlight that along with total collagen content, collagen crosslinking and organization are equally important while investigating collagen-mediated pathological changes in lung tissue. These changes may have implications for developing ECM-based therapeutics for patients with lung diseases.

Ankle clonus is a sustained, involuntary, rhythmic muscle contraction frequently observed in humans with spinal cord injury (SCI). Although its pathophysiology remains incompletely understood, converging evidence suggests a role for brainstem systems in its generation. Following SCI, brainstem neuromodulatory inputs partially compensate for the loss of descending motor pathways by regulating motoneuron excitability during involuntary contractions, suggesting their involvement in the generation of clonus. To test this hypothesis, motoneuron excitability in response to Ia synaptic input was quantified using the soleus H reflex and maximal motor response (H/M ratio), and brainstem involvement was probed using the long lasting component of the cutaneous reflex (LLR) in the tibialis anterior and soleus muscles, as well as the StartReact response-an involuntary release of a movement triggered by a startling stimulus thought to engage the reticulospinal tract. We studied individuals with chronic SCI, both with and without ankle clonus, using standardized clinical tests across two days. Participants with clonus showed elevated H/M ratios, indicating increased motoneuron excitability, whereas those without clonus exhibited lower values than controls. Additionally, individuals with clonus exhibited longer LLR duration and greater LLR magnitude in both muscles, along with shorter reaction times to startle stimuli, consistent with enhanced monoaminergic and reticulospinal contributions. Notably, LLR duration was positively correlated with both StartReact response and H/M ratio. Together, these findings support a role for descending brainstem systems-particularly monoaminergic and reticulospinal pathways-in the maintenance of clonus in chronic SCI.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

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.