Extensive Remodeling Research Articles

Dangers of under‐treatment and over‐treatment with inhaled corticosteroids in children with asthma

AbstractTwo children, both under the care of specialists for mild persistent asthma, flirted with mortality. One lost and one won the battle. A 16‐year‐old boy never received ICS therapy despite extensive airway inflammation and remodeling and died due to mismanagement of an asthma exacerbation. A 6‐year‐old girl developed iatrogenic Cushing's syndrome during 18 months of continuous treatment with high, FDA‐unapproved doses of both ICS and INCS and nearly died during an adrenal crisis. The role of ICS under‐treatment and over‐treatment and the possibility that recommendations in asthma guidelines and information in FDA package labels could have prevented both outcomes are explored.

Sex-specific regulation of the cardiac transcriptome by the protein phosphatase 2A regulatory subunit B55α

Protein phosphatase 2A (PP2A) regulatory subunit B55α has been implicated in the transcriptional regulation of cardiac growth and fibrosis by suppressing HDAC5/MEF2 signalling in cardiomyocytes. We created and characterised two mouse models with global or cardiomyocyte-specific disruption of the gene encoding B55α (Ppp2r2a) to conduct the first detailed exploration of B55α in the heart. Global homozygous B55α knockout mice died in utero, while heterozygous mice had thinner left ventricular walls at 12 months, an effect more pronounced in males. At 10–12 weeks of age, cardiomyocyte-specific B55α knockout mice displayed normal cardiac morphology with increased left ventricular collagen deposition, identifying B55α as a negative regulator of cardiac fibrosis. Gene expression analyses demonstrated extensive remodelling of the cardiac transcriptome in male but not female mice, revealing a sexually dimorphic role for B55α in cardiac transcriptional regulation. These findings provide a basis for future work investigating B55α in cardiac stress settings.

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with “epidrugs” is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Peripheral arterial pathology and osteoarthritis of the knee: US examination of arterial wall stiffness, thickness, and flow characteristics

BackgroundOsteoarthritis (OA) is a widespread chronic joint disorder characterized by the degeneration of articular cartilage, extensive bone remodeling, ligamentous fibrosis, and synovial inflammation impacting millions. Shared factors like phenotypic similarities, hypofibrinolysis, and inflammation constitute similarities in pathophysiology and clinical manifestations between OA and peripheral vascular disease (PVD). This study investigated peripheral arterial flow characteristics, vascular wall thickness, and stiffness in knee OA to clarify a potential association with early atherosclerosis. MethodsThe OA cohort consisted of 35 knees with a mean age of 69 years. The control cohort consisted of 58 knees with a mean age of 68 years. Subjects underwent arterial ultrasound scanning of the common femoral, superficial femoral, and popliteal arteries. Data measured included peak systolic volumetric flow, intima-media thickness, systolic and diastolic vessel diameter, and simultaneous EKG and flow curves. Structural and functional vascular data were quantified using the incremental Young's modulus, pulse wave velocity (PWV), and distensibility. ResultsSignificantly elevated arterial volumetric flow, measures of arterial stiffness, and intima-media wall thickness were observed in those with OA compared to those without. PWV as calculated by the Bramwell-Hill equation were found to be significantly greater in all three vessels of patients with OA. ConclusionsThis study supports the association between peripheral arterial pathology and knee OA, consistent with shared clinical and phenotypic traits. The observed characteristics of early vascular pathology suggest potential pathophysiologic linkages between OA and PVD. This foundational framework provides avenues for mechanistic studies exploring the relationship between these two disease processes.

Clinical overlap between healthy athletes and athletes with arrhythmogenic right ventricular cardiomyopathy

Abstract Introduction The extensive physiological cardiac remodelling that results from the haemodynamic stress of endurance exercise shares many features with the pathological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC), giving rise to a significant diagnostic challenge for clinicians. Contemporary discriminators of disease have been derived from comparisons between healthy athletes and non-athletic ARVC patients. Differentiation between healthy and diseased athletes may be more difficult. Purpose To assess published clinical differentiators of physiological remodelling from ARVC in a cohort of endurance athletes. Methods This project utilises data from the Pro@heart consortium comprising elite endurance athletes that have received comprehensive cardiac phenotyping using cardiac magnetic resonance imaging, echocardiography, resting and ambulatory electrocardiography and cardiopulmonary exercise testing. Measures of cardiac structure and function with potential to differentiate health and disease were assessed in healthy endurance athletes without symptoms or suspicion of ARVC, endurance athletes diagnosed with ARVC (meeting ARVC task force criteria) and athletes with suspected sub-clinical ARVC (complex ventricular arrhythmias and ≥1 task force criteria). One way ANOVA assessed differences in continuous variables, while Chi Square with pair wise Z test and Bonferroni p value correction were used to test for differences in nominal data. Results Athletes with ARVC and athletes with suspected ARVC displayed more clinical indicators of ARVC compared to healthy athletes (table 1). While a greater proportion of athletes with ARVC and suspected ARVC met ≥1 and ≥3 indicators of pathology compared to healthy athletes, it was relatively common for healthy athletes to meet ≥1 criteria (53%) and ≥3 criteria (14%) (table 2). Comparison of mean values showed right ventricular ejection fraction (RVEF) and right ventricular fractional area change (RV FAC) were lower in athletes with or suspected to have ARVC compared to healthy athletes, however no significant differences in burden of ventricular premature beats (VPB) were noted (table 1). A high burden of VPBs, evidence of ventricular arrhythmia, evidence of late gadolinium enhancement (LGE), a low RVEF, and a reduced RV FAC were more prevalent in athletes with ARVC compared to healthy athletes (table 2). However, there was clinically relevant overlap in all measures between healthy and diseased athletes (table 2). Right ventricular to left ventricular volume ratio was a particularly poor discriminator of pathology (table 1 & 2). Conclusion Electrophysiological, structural and functional abnormalities are not uncommon in healthy athletes resulting in significant overlap with athletic ARVC patients. Future research is required to identify better clinical discriminators of disease, and to assess the clinical significance of abnormal measures in ostensibly healthy athletes

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B.

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4 , lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Rapid onset fibrotic remodeling and ventricular dysfunction induced by phenylephrine involve targeted reprogramming of myocyte and fibroblast transcriptomes.

Catecholamine dysregulation is a common feature of multiple acute and chronic cardiac conditions, including heart failure. To investigate the role of altered α-adrenergic stimulation on cardiac function, we developed a short-term exposure model, administering phenylephrine subcutaneously to mice for one week. Compared to vehicle-injected controls, phenylephrine-treated animals exhibited increased ejection fraction, decreased chamber size, diastolic dysfunction and ventricular hypertrophy in the absence of hypertension. Remarkably, these animals developed extensive fibrotic remodeling of the tissue that plateaued at 24 hours and myocyte hypertrophy localized to regions of fibrotic deposition after 3 days of treatment. Transcriptome analyses of purified myocyte and fibroblast populations from these hearts revealed an unexpected role for myocytes in the production of extracellular matrix. Comparison with other models of cardiac stress, including pressure overload hypertrophy and cytokine activation of fibroblasts, identified stimulus-specific transcriptional circuits associated with cardiac pathology. Given the rapid, robust fibrotic response that preceded myocyte hypertrophy, intercellular communication analyses were conducted to investigate fibroblast to myocyte signaling, identifying potential crosstalk between these cells. These studies thoroughly describe and phenotypically characterize a new model of short-term catecholamine stress and provide an atlas of transcriptional remodeling in myocytes and fibroblasts.

Novel therapeutic strategy for intractable keloids: Suppression of intracellular mechanotransduction and actin polymerisation via Rho-kinase pathway inhibition.

Keloid is a dermal fibrotic disorder characterised by excessive extracellular matrix production by fibroblasts. Despite the significance of mechanostimulation in fibrotic diseases, its association with keloid pathophysiology or treatment remains unexplored. We investigated the role of mechanical force in keloid formation and elucidated the significance of Rho-associated coiled-coil-containing kinase 1 (ROCK1) as a mechanoresponsive target for keloid treatment. Patient-derived keloid fibroblasts (KFs) were subjected to cyclic stretching ranging from 0 to 20% elongation using a cell-stretching system. We observed the inhibitory effects of the ROCK1 inhibitor Y27632 on KFs and keloid formation. Validation was performed using keloid xenograft severe combined immune-deficient (SCID) mouse model. ROCK1 was overexpressed in KFs isolated from patients. Cyclic stretching induced fibroblast proliferation and actin polymerisation by activating Rho/ROCK1 signalling. Treatment with Y27632 downregulated fibrotic markers, reduced the migration capacity of KFs, and induced extensive actin cytoskeleton remodelling. In keloid xenograft SCID mouse model, Y27632 effectively suppressed keloid formation, mitigating inflammation and fibrosis. The ROCK1 inhibitor Y27632 is a promising molecule for keloid treatment, exerting its effects through actin cytoskeleton remodelling and nuclear inhibition of fibrotic markers in keloid pathogenesis.

12489 Senolytics Dasatinib And Quercetin Alleviate Type 1 Diabetes-Related Cardiac Fibrosis In Male Akita Ins-/+Mice

Abstract Disclosure: N. Kadoumi: None. G. Zatarah: None. J. Stokar: None. P. Shivam: None. I. Gurt: None. R. Dresner-Pollak: None. People living with type 1 diabetes (T1D) have a markedly shorter life expectancy compared to non-diabetic age- and sex-matched controls primarily due to cardiovascular disease. Diabetic cardiomyopathy is characterized by structural and functional alterations that occur independently of hypertension or coronary artery leading to congestive heart failure. The pathogenesis of diabetic cardiomyopathy involves endothelial cell dysfunction, microangiopathy, mitochondrial dysfunction, and oxidative stress, resulting in inflammation, extensive remodeling, cardiomyocyte hypertrophy, interstitial fibrosis, and apoptosis. T1D is associated with accelerated aging. Cellular senescence is a hallmark of aging, it is characterized by cell cycle arrest, distinct morphological alterations and the release of molecules referred to as the senescence-associated secretory phenotype (SASP). Senescent cardiomyocytes secrete SASPs that promote activation of fibroblasts, hypertrophy of cardiac muscle cells, and dysfunction of cardiac endothelial cells. An increase in cardiac cellular senescence was demonstrated in mouse models of T1D. Senolytics are compounds that clear senescent cells. Senolytics were shown to improve multiple outcomes in age-associated conditions in mice and humans.We investigated the effects of senolytics Dasatinib (D) and Quercetin (Q) on cardiac fibrosis in the AKITA Ins-/+ mouse model of T1D.We first assessed if there is increased collagen type1 expression in the heart ventricles and perivascular in 3-month-old in AKITA Ins-/+ vs. WT male mice using Immunofluorescence. Increased perivascular and interstitial expression of collagen 1 was found in AKITA Ins-/+ compared to WT age-and sex-matched mice (0.0811±0.008177 µm2 vs. 0.0533± 0.007960 µm2 ,P=0.0476), (105.7±10.29 µm2 vs. 54.85±14.61 µm2, P=0.0476), respectively. Next, 3-month-old AKITA Ins-/+wmale mice were randomly assigned to treatment with Dasatinib (5 mg/kg) and Quercetin (50 mg/kg) or vehicle administered once monthly by oral gavage for a total of four months (n=20, 10 in each group). Heart samples were analyzed for gene expression, collagen 1 protein by western blotting and Immunofluorescence and analyzed using QuPath 0.5.0 Software. Interstitial and perivascular fibrosis was separately assessed in the right and left ventricles. Treatment with D+Q resulted in a 2-fold decrease in COL1A1 gene and Collagen 1 protein expression by RT-PCR and western blot analysis (P= 0.011 and 0.042, respectively) in AKITA Ins-/+ mice. Immunostaining analysis revealed reduced Collagen 1 deposition in both the left and right ventricles (105.7±10.29 µm2 vs. 43.53±16.31 µm2, P=0.0571), as well as in the perivascular area (0.0811±0.008177 µm2 vs. 0.052±0.006163 µm2, P=0.1143). Treatment with senolytics D+Q reduces the expression of Collagen I, a key factor in myocardial fibrosis, in adult male AKITA Ins-/+ mice. Presentation: 6/3/2024

LncRNA VELRP Modulates Pulmonary Arterial Smooth Muscle Cell Proliferation and Promotes Vascular Remodeling in Pulmonary Hypertension.

Pulmonary hypertension is a devastating vascular disorder characterized by extensive pulmonary vascular remodeling, ultimately leading to right ventricular failure and death. Activation of PDGF (platelet-derived growth factor) signaling promotes the hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs), thus contributing to the pulmonary vascular remodeling. However, the molecular mechanisms that govern hyperproliferation of PASMCs induced by PDGF remain largely unknown, including the contribution of long noncoding RNAs (lncRNAs). In this study, we aimed to identify a novel lncRNA regulated by PDGF implicated in PASMC proliferation in pulmonary vascular remodeling. RNA-sequencing analysis was conducted to identify a novel lncRNA named vessel-enriched lncRNA regulated by PDGF-BB (VELRP). Functional investigations of VELRP were performed using knockdown and overexpression strategies along with RNA sequencing. Validation of the function and potential mechanisms of VELRP were performed through Western blot, RNA immunoprecipitation, and chromatin immunoprecipitation assays. We identified a novel vessel-enriched lncRNA with an increased response to PDGF-BB stimulus. VELRP was identified as an evolutionarily conserved RNA molecules. Modulation of VELRP in PASMCs significantly altered cell proliferation. Mechanistically, VELRP enhances trimethylation of H3K4 by interacting with WDR5 (WD repeat-containing protein 5), leading to increased expression of CDK (cyclin-dependent kinase) 1, CDK2, and CDK4 and consequent hyperproliferation of PASMCs. The pathological relevance of VELRP upregulation in pulmonary artery was confirmed using rat pulmonary hypertension models in vivo, as well as in PASMCs from patients with idiopathic pulmonary arterial hypertension patients. Specific knockdown of VELRP in smooth muscle cells using adeno-associated virus type 9 SM22α (smooth muscle protein 22α) promoter-shRNA-mediated silencing of VELRP resulted in a significant decrease in right ventricular systolic pressure and vascular remodeling in rat pulmonary hypertension model. VELRP, as an lncRNA upregulated by PDGF-BB, mediates PASMC proliferation via WDR5/CDK signaling. In vivo studies demonstrate that targeted intervention of VELRP in smooth muscle cells can prevent the development of pulmonary hypertension.

Cellulite Treatment With Electrotherapeutic Combinations: Cryolipolysis, Microcurrents, LED And Ultrasound N

Cellulite is a multifactorial condition present in 80% to 90% of post-pubertal women and is one of the most intolerable aesthetic imperfections. It is a morphological alteration of the integumentary tissue, directly interfering with self-esteem. There are several theories about the pathophysiology of cellulite, and several different therapeutic regimens have been developed, from topical treatments to mechanical or electrotherapybased devices. In this brief review, we summarize the scientific scenario to determine the clinical evidence regarding the safety and efficacy of cellulite treatment options. Clinical protocols and the author's experience using a combination of external procedures are also discussed. Therapies such as ultrasound and shock waves have shown improvements in cellulite and a good safety profile, but the combination therapy red LED, microcurrent, plate cryolipolysis and 40 kHz ultrasound have demonstrated the most beneficial results in cellulite reduction. Although there is a lack of scientific evidence for treatments that improve cellulite, future emerging options and their combination may pave the way to eradicating this aesthetic concern, Objective: The objective of this work was to review the literature on cellulite and the main electrotherapeutic resources used, to demonstrate the effectiveness of the technique that associates red LED before the cryolipolysis technique in a mobile handle and association with microcurrents and finishing with low frequency ultrasound in the licus system in the treatment of cellulite, reducing edema, inflammation and decreasing adipose tissue in addition to toning the dermis with the presentation of clinical cases. Materials and Methods: In this study, we performed a comprehensive review of all scientific articles available and indexed in PubMed and Web of Science in the last 20 years about this technology and its effects on cellulite. We discussed the scientific evidence available in clinical studies and analyzed the effects and possible mechanisms of action of LED, cryolipolysis, microcurrents and the use of 40 kHz ultrasound in cellulite. The treatments were performed with LED at 4 joulles per cm2, cryolipolysis technique with mobile plate and associated microcurrents, with intervals for reperfusion. We used 4 series of 5 minutes of freezing and in the intervals 3 minutes for return of circulation, that is, reperfusion without injury, and finished with 40 kHz ultrasound at 0.3 watts/cm2 for 2 minutes per cm2. We demonstrated the result after 4 sessions. Conclusion: In the author's opinion, a combined approach where combined approaches are used strategically and in a staggered manner to produce synergistic results has the best clinical outcome. For example, the use of red LEDs prior to the combination of microcurrents simultaneously with cryolipolysis and finishing with 40 kHz ultrasound in a weekly LICUS system can result in improvements in severe cellulite in approximately 2 months. More large-scale studies, particularly regarding combined approaches, need to be conducted to evaluate the long-term results of cellulite therapies in terms of safety, efficacy, and patient satisfaction. improve the appearance of cellulite, sometimes to a satisfactory degree, but never eradicate cellulite, as this involves extensive remodeling of the tissue. potential to be effective in lysing subdermal collagen, such as that seen in the dermal septa (the underlying cause of cellulite). References Despite multiple therapeutic approaches attempting to treat cellulite, no procedure has proven to be successful in the long term, requiring weekly or biweekly maintenance of treatment

Palaeohistology of Macrospondylus bollensis (Crocodylomorpha: Thalattosuchia: Teleosauroidea) from the Posidonienschiefer Formation (Toarcian) of Germany, with insights into life history and ecology.

The Posidonienschiefer Formation of southern Germany has yielded an array of incredible fossil vertebrates. One of the best represented clades therein is Teleosauroidea, a successful thalattosuchian crocodylomorph group that dominated the coastlines. The most abundant teleosauroid, Macrospondylus bollensis, is known from a wide range of body sizes, making it an ideal taxon for histological and ontogenetic investigations. Previous studies examining thalattosuchian histology provide a basic understanding of bone microstructure in teleosauroids, but lack the taxonomic, stratigraphic, and ontogenetic control required to understand growth and palaeobiology within a species. Here, we examine the bone microstructure of three femora and one tibia from three different-sized M. bollensis individuals. We also perform bone compactness analyses to evaluate for ontogenetic and ecological variation. Our results suggests that (1) the smallest specimen was a young, skeletally immature individual with well-vascularized-parallel-fibered bone and limited remodeling in the midshaft periosteal cortex; (2) the intermediate specimen was skeletally immature at death, with vascularized parallel-fibered bone tissue interrupted by at least 10 LAGs, but no clear external fundamental system (EFS), and rather extensive inner cortical bone remodeling; and (3) the largest specimen was skeletally mature, with parallel-fibered bone tissue interrupted by numerous LAGs, a well-developed EFS, and extensive remodeling in the deep cortex. Macrospondylus bollensis grew relatively regularly until reaching adult size, and global bone compactness values fall within the range reported for modern crocodylians. The lifestyle inference models used suggest that M. bollensis was well adapted for an aquatic environment but also retained some ability to move on land. Finally, both larger specimens display a peculiar, localized area of disorganized bone tissue interpreted as pathological.

MRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

Emergence and stability of endoplasmic reticulum network streaming in plant cells

The endoplasmic reticulum (ER) network is highly complex and highly dynamic in its geometry, and undergoes extensive remodeling and bulk flow. It is known that the ER dynamics are driven by actin–myosin dependent processes. ER motion through the cytoplasm will cause forces on the cytoplasm that will induce flow. However, ER will also clearly be passively transported by the bulk cytoplasmic streaming. We take the complex ER network structure into account and propose a positive-feedback mechanism among myosin-like motors, actin alignment, ER network dynamics for the emergence of ER flow. Using this model, we demonstrate that ER streaming may be an emergent feature of this three-way interaction and that the persistent-point density may be a key driver of the emergence of ER streaming.

Secondary Envelopment of Human Cytomegalovirus Is a Fast Process Utilizing the Endocytic Compartment as a Major Membrane Source.

Secondary envelopment of the human cytomegalovirus (HCMV) is a critical but not well-understood process that takes place at the cytoplasmic viral assembly complex (cVAC) where nucleocapsids acquire their envelope by budding into cellular membranes containing viral glycoproteins. Previous studies presented controversial results regarding the composition of the viral envelope, suggesting trans-Golgi and endosomal origins, as well as intersections with the exosomal and endocytic pathways. Here, we investigated the role of endocytic membranes for the secondary envelopment of HCMV by using wheat germ agglutinin (WGA) pulse labeling to label glycoproteins at the plasma membrane and to follow their trafficking during HCMV infection by light microscopy and transmission electron microscopy (TEM). WGA labeled different membrane compartments within the cVAC, including early endosomes, multivesicular bodies, trans-Golgi, and recycling endosomes. Furthermore, TEM analysis showed that almost 90% of capsids undergoing secondary envelopment and 50% of enveloped capsids were WGA-positive within 90 min. Our data reveal extensive remodeling of the endocytic compartment in the late stage of HCMV infection, where the endocytic compartment provides an optimized environment for virion morphogenesis and serves as the primary membrane source for secondary envelopment. Furthermore, we show that secondary envelopment is a rapid process in which endocytosed membranes are transported from the plasma membrane to the cVAC within minutes to be utilized by capsids for envelopment.

Transcriptional re-programming of liver-resident iNKT cells into T-regulatory type-1-like liver iNKT cells involves extensive gene de-methylation.

Unlike conventional CD4+ T cells, which are phenotypically and functionally plastic, invariant NKT (iNKT) cells generally exist in a terminally differentiated state. Naïve CD4+ T cells can acquire alternative epigenetic states in response to different cues, but it remains unclear whether peripheral iNKT cells are epigenetically stable or malleable. Repetitive encounters of liver-resident iNKT cells (LiNKTs) with alpha-galactosylceramide (αGalCer)/CD1d-coated nanoparticles (NPs) can trigger their differentiation into a LiNKT cell subset expressing a T regulatory type 1 (TR1)-like (LiNKTR1) transcriptional signature. Here we dissect the epigenetic underpinnings of the LiNKT-LiNKTR1 conversion as compared to those underlying the peptide-major histocompatibility complex (pMHC)-NP-induced T-follicular helper (TFH)-to-TR1 transdifferentiation process. We show that gene upregulation during the LINKT-to-LiNKTR1 cell conversion is associated with demethylation of gene bodies, inter-genic regions, promoters and distal gene regulatory elements, in the absence of major changes in chromatin exposure or deposition of expression-promoting histone marks. In contrast, the naïve CD4+ T cell-to-TFH differentiation process involves extensive remodeling of the chromatin and the acquisition of a broad repertoire of epigenetic modifications that are then largely inherited by TFH cell-derived TR1 cell progeny. These observations indicate that LiNKT cells are epigenetically malleable and particularly susceptible to gene de-methylation.

Respiratory performance in Duchenne muscular dystrophy: Clinical manifestations and lessons from animal models.

Duchenne muscular dystrophy (DMD) is a fatal genetic neuromuscular disease. Lack of dystrophin in skeletal muscles leads to intrinsic weakness, injury, subsequent degeneration and fibrosis, decreasing contractile function. Dystropathology eventually presents in all inspiratory and expiratory muscles of breathing, severely curtailing their critical function. In people with DMD, premature death is caused by respiratory or cardiac failure. There is an urgent need to develop therapies that improve quality of life and extend life expectancy in DMD. Surprisingly, there is a dearth of information on respiratory control in animal models of DMD, and respiratory outcome measures are often limited or absent in clinical trials. Characterization of respiratory performance in murine and canine models has revealed extensive remodelling of the diaphragm, the major muscle of inspiration. However, significant compensation by extradiaphragmatic muscles of breathing is evident in early disease, contributing to preservation of peak respiratory system performance. Loss of compensation afforded by accessory muscles in advanced disease is ultimately associated with compromised respiratory performance. A new and potentially more translatable murine model of DMD, the D2.mdx mouse, has recently been developed. Respiratory performance in D2.mdx mice is yet to be characterized fully. However, based on histopathological features, D2.mdx mice might serve as useful preclinical models, facilitating the testing of new therapeutics that rescue respiratory function. This review summarizes the pathophysiological mechanisms associated with DMD both in humans and in animal models, with a focus on breathing. We consider the translational value of each model to human DMD and highlight the urgent need for comprehensive characterization of breathing in representative preclinical models to better inform human trials.

Revisiting secondary mitral regurgitation threshold severity: insights and lessons from the RESHAPE-HF2 trial.

This article revisits the severity threshold for secondary mitral regurgitation (MR), focusing on insights and lessons from the RESHAPE-HF2 trial. It aims to challenge the traditional effective regurgitant orifice area (EROA) threshold of ≥0.40 cm2 used for intervention, suggesting that earlier intervention may benefit patients with lower EROA. It also explores how transcatheter edge-to-edge repair (TEER) improves outcomes in patients with secondary MR and assesses the impact of left ventricular (LV) remodeling on treatment success. The RESHAPE-HF2 trial evaluated the use of TEER in patients with moderate-to-severe secondary MR, comparing outcomes in those with an EROA ≥0.2 cm2 and no extensive LV remodeling. TEER significantly reduced heart failure hospitalizations and improved quality of life in these patients. This supports the notion that patients with less severe MR, who still show symptoms despite optimal medical therapy, may benefit from earlier intervention. Comparisons with COAPT and MITRA-FR trials underscored the importance of selecting patients based on MR severity relative to LV dilatation. The RESHAPE-HF2 trial highlights the need to reconsider the current EROA threshold for secondary MR intervention. TEER has shown to be beneficial even in patients with lower MR severity, suggesting that earlier intervention could improve outcomes. A more dynamic and integrated approach, considering both MR severity and LV remodeling, is essential for optimizing patient selection and treatment success.

Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition.

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein α-catenin. Using correlative cryo-fluorescence microscopy and cryo-electron tomography, we identify F-actin featuring domains of nanoscale oscillating curvature at cytoskeleton-adhesion interfaces enriched in zyxin, a marker of actin-myosin generated traction forces. We next introduce a reconstitution system for visualizing F-actin in the presence of myosin forces with cryo-electron microscopy, which reveals morphologically similar superhelical F-actin spirals. In simulations, transient forces mimicking tugging and release of filaments by motors produce spirals, supporting a mechanistic link to myosin's ATPase mechanochemical cycle. Three-dimensional reconstruction of spirals uncovers extensive asymmetric remodeling of F-actin's helical lattice. This is recognized by α-catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by a mechanosensitive protein, providing a direct structural glimpse at active force transduction through the cytoskeleton.