Changes In Compartmentalization Research Articles

Regulation of the mitochondrial integrated stress response and ATF4 by acute contractile activity in mouse muscle

In the context of energetically demanding conditions, such as exercise, skeletal muscle relies on mitochondria to support its metabolic requirements. Maintenance of mitochondria and their bioenergetic availability is thus critical to sustain the metabolic flexibility of muscle. The quality of the organelle network is highly regulated by the coordination of mitochondrial quality control (MQC) processes. One MQC pathway that has garnered recent interest is the integrated stress response (ISR), which is activated in response to a variety of stimuli. The transcription factor ATF4, denoted as the main effector of this response pathway, serves to ameliorate cellular stress by upregulating a plethora of cytoprotective genes, such as CHOP and ATF5. Current literature has shown that the ISR is activated upon mitochondrial stress/perturbations to restore optimal organelle function and health. However, the precise mechanism that constitutes the activation of ISR and thus the response of ATF4 following acute exercise-induced stress is unknown. Therefore, our objective is to determine the extent of ISR/ATF4 induction in vivo and to test whether this pathway is required for the maintenance of muscle function. To investigate this, a mouse in situ hindlimb protocol was utilized to acutely stimulate muscles at 0.25-1 tetani/per second for 9 mins, followed by a 1-hour recovery period. The kinases, CAMKIIα and JNK2, were robustly phosphorylated/activated 6-fold immediately following the protocol. The initial stage of ISR activation, reflected in the ratio of phosphorylated to total-eIF2α protein levels, was also increased in response to acute contractile activity, evident in the recovery phase. Downstream of ISR activation, the total protein expression of ATF4 remained unchanged, however, contractile activity induced an increase in the localization of ATF4 to the nucleus. Robust 2-fold increases in the mRNA expression of ATF4 and CHOP were also observed and enhanced following the recovery period. Changes in ATF4 mRNA appeared to be independent of transcriptional activation, as assessed using an ATF4 promoter-reporter plasmid. An in vitro cell free mRNA decay assay revealed an increase in ATF4 mRNA stability post-stimulation, possibly as a result of cellular compartmental changes in the RNA binding proteins, HuR and CUGBP1. RNA sequencing analyses also confirmed the observed increase in related ISR genes following an exhaustive bout of exercise. These data suggest that acute contractile activity is indeed suffcient to induce mitochondrial stress and activate the ISR, corresponding to the induction of ATF4. Future work will reveal the underlying mitochondrial signal responsible for activating this pathway, as well as the downstream consequences for the regulation of MQC processes in maintaining mitochondrial homeostasis. This work was supported by funds from the Canadian Institutes for Health Research (CIHR). Victoria C. Sanfrancesco is the recipient of the CIHR Frederick Banting and Charles Best Canada Graduate Scholarship-Master's (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.

Identifying quantitatively differential chromosomal compartmentalization changes and their biological significance from Hi-C data using DARIC

BackgroundChromosomal compartmentalization plays a critical role in maintaining proper transcriptional programs in cell differentiation and oncogenesis. However, currently the prevalent method for comparative analysis of compartmentalization landscapes between different cell types is limited to the qualitative switched compartments.ResultsTo identify genomic regions with quantitatively differential compartmentalization changes from genome-wide chromatin conformation data like Hi-C, we developed a computational framework named DARIC. DARIC includes three modules: compartmentalization quantification, normalization, and differential analysis. Comparing DARIC with the conventional compartment switching analysis reveals substantial regions characterized by quantitatively significant compartmentalization changes without switching. These changes are accompanied by changes in gene expression, chromatin accessibility, H3K27ac intensity, as well as the interactions with nuclear lamina proteins and nuclear positioning, highlighting the functional importance of such quantitative changes in gene regulation. We applied DARIC to dissect the quantitative compartmentalization changes during human cardiomyocyte differentiation and identified two distinct mechanisms for gene activation based on the association with compartmentalization changes. Using the quantitative compartmentalization measurement module from DARIC, we further dissected the compartment variability landscape in the human genome by analyzing a compendium of 32 Hi-C datasets from 4DN. We discovered an interesting correlation between compartmentalization variability and sub-compartments.ConclusionsDARIC is a useful tool for analyzing quantitative compartmentalization changes and mining novel biological insights from increasing Hi-C data. Our results demonstrate the functional significance of quantitative compartmentalization changes in gene regulation, and provide new insights into the relationship between compartmentalization variability and sub-compartments in the human genome.

Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs.

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semiquantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROX Deep Red, and Amplex Red for functional characterization of the spatiotemporal mode of ROS production, delivery, and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye loading and undisturbed root hair tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of nonelongating root hairs, but with contrasting reasons for this impairment, we found that: (i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, (ii) CellROX Deep Red labels ROS in mitochondria, (iii) Amplex Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, (iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks Amplex Red-reactive ROS in apoplast, and (v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROX Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free premature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.

Abstract 4716: Characterization of 3D genome in nasopharyngeal carcinoma (NPC)

Abstract Background: Epstein-Barr virus (EBV) can be detected in more than 90% of NPC patients in Hong Kong. It has been shown that EBV infection could hijack the human epigenome in B cell lymphoma and EBV+ gastric cancer, however, the role of EBV in epigenetic dysregulation remains largely unknown in NPC. Recently, our epigenomic landscape study demonstrated EBV was associated with changes of chromatin accessibility in NPC, which were largely involved in the binding sites of CTCF, a master regulator of chromosome organization. Therefore, we aim to further characterize the 3D chromatin structure in NPC. Methods: We used the Hi-C (Omni-C Seq) to profile the chromatin interactions in the EBV-positive (EBV+) NPC, EBV-negative (EBV-) NPC, and normal immortalized epithelial (NPE) cell lines. The Omni-C Seq data were integrated with the ATAC-Seq, RNA-Seq, and CUT&RUN-Seq data. The histone markers including H3K4me3, H3K27ac, and H3K27me3, were applied to classify the active and inactive status of the chromatins. The chromatin looping and topologically associated domains (TADs) were defined by the CTCF binding sites. Results: We observed a much larger compartmentalization size in EBV+ NPC with a consistent increase of active to inactive (A to B) compartments, compared to NPE and EBV- NPC. Pathway enrichment analysis showed that these changes potentially influenced immune response, cell differentiation and development, metabolism, ERBB pathway, and calcium signaling transduction. In addition to the changes in compartmentalization, we observed a reduction of CTCF binding signals at the TADs boundaries and the loss of chromatin looping at the 10kb resolution. Consistently, the aggregate peak analysis has shown a significant reduction of the CTCF anchor, indicating the abandonment of architectural CTCF in EBV+ NPC. More importantly, we found EBV tethering to the host chromatins. In the EBV genome, the tethering sites were often located at the regions associated with OriP, EBNA1, and BARTs. In the human genome, the tethering sites were dramatically enriched in the inactive to active (B to A) compartments, which were highly associated with the enhancer regions with H3K27ac. We identified a specific target gene CD74 with EBV tethering at the nearby super-enhancer region. Our results suggest the upregulation of CD74 could be controlled via remodulating the host 3D genome in EBV+ NPC. The spatial transcriptome analysis indicated that the expression of CD74 was highly correlated with HLA-DRA. The spatial analysis further showed that the NPC patients with upregulated HLA-DRA often had de novo distant metastasis after treatment (p=0.007). Conclusions: The EBV tethering to the host genome has a significant impact on host 3D genome organization, affecting chromatin accessibility and gene regulation, providing survival advantages of cancer cell growth and potentially contributing to immune suppression and metastasis. Citation Format: Dittman Lai-Shun Chung, Larry Ka-Yue Chow, Wei Dai. Characterization of 3D genome in nasopharyngeal carcinoma (NPC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4716.

Medial Varus Proximal Tibial Resection is Superior to Pie-Crusting of the Medial Collateral Ligament During Primary Total Knee Arthroplasty

BackgroundMedial varus proximal tibial (MPT) resection or soft tissue releases (STRs) of the medial collateral ligament (MCL) in the form of pie-crusting can be performed to achieve a balanced knee in a varus deformity. Studies comparing the 2 modalities have not been addressed within the literature. Therefore, the aims of this study were to assess the following: (1) compartmental changes between the 2 methods and (2) changes in patient-reported outcome measurements. MethodsUsing our institution’s total joint arthroplasty registry, patients who underwent primary total knee arthroplasty from January 1, 2017, to December 31, 2019, were identified. The MPT resection and STR patients were 1:1 matched with baseline parameters yielding 196 patients. Outcomes of interest included: changes in compartmental pressures at 10, 45, and 90° degrees and change to the Short-Form 12, Western Ontario and McMaster Universities Osteoarthritis Index, and Forgotten Joint Scores (FJSs) at the 2-year follow-up period. A P value less than .05 was used as our threshold for statistical difference. ResultsThe MPT resection led to significant reductions in compartmental pressures at 10° [43 versus 19 pounds (lbs.), P < .0001], 45° (43 versus 27 lbs., P < .0001), and 90° degrees (27 versus 16 lbs., P < .0001) compared to STR. MPT resection also had significantly improved Short-Form 12 (47 versus 38, P < .0001), Western Ontario and McMaster Universities Osteoarthritis Index (9 versus 21, P < .0001), and Forgotten Joint Score (79 versus 68, P = .005). ConclusionBone modification was superior to pie-crusting of the MCL in achieving consistent pressure balancing and improved outcomes. The investigation can guide surgeons on the preferred method to achieve a well-balanced knee.

DcHiC detects differential compartments across multiple Hi-C datasets

The compartmental organization of mammalian genomes and its changes play important roles in distinct biological processes. Here, we introduce dcHiC, which utilizes a multivariate distance measure to identify significant changes in compartmentalization among multiple contact maps. Evaluating dcHiC on four collections of bulk and single-cell contact maps from in vitro mouse neural differentiation (n = 3), mouse hematopoiesis (n = 10), human LCLs (n = 20) and post-natal mouse brain development (n = 3 stages), we show its effectiveness and sensitivity in detecting biologically relevant changes, including those orthogonally validated. dcHiC reported regions with dynamically regulated genes associated with cell identity, along with correlated changes in chromatin states, subcompartments, replication timing and lamin association. With its efficient implementation, dcHiC enables high-resolution compartment analysis as well as standalone browser visualization, differential interaction identification and time-series clustering. dcHiC is an essential addition to the Hi-C analysis toolbox for the ever-growing number of bulk and single-cell contact maps. Available at: https://github.com/ay-lab/dcHiC.

Tibiofemoral knee osteoarthritis progresses symmetrically by knee compartment in the GOGO cohort

ObjectiveTo evaluate the degree of symmetry of knee osteoarthritis (OA) structural severity and progression of participants with a mean follow-up time of 3.8 years. DesignParticipants from the Genetics of Generalized Osteoarthritis (GOGO) study (n ​= ​705) were selected on the basis of radiographic evidence of OA in at least 1 knee, availability of radiographs at baseline and follow-up, and no history of prior knee injury or surgery. Incidence and progression of osteoarthritis were determined by radiographic Kellgren-Lawrence (KL) grade; compartmental OA progression was determined by change in joint space width of lateral and medial tibiofemoral compartments. Total OA progression was the sum of change in KL grade of both knees. ResultsCompared with left knees, right knees had more severe KL grades at baseline (p ​= ​0.0002) and follow-up (p ​= ​0.0004), McNemar's χ2 ​= ​34.16 and 26.08, respectively; however, both knees progressed similarly (p ​= ​0.121, McNemar's χ2 ​= ​10.09). Compartmental changes were symmetric across knees: medial r ​= ​0.287, p ​= ​0.0002; lateral r ​= ​0.593, p ​= ​0.0002. Change in joint space width in the medial compartment was negatively correlated with change in the lateral compartment of the same knee (left knees: r ​= ​−0.293, p ​= ​0.021; right knees: r ​= ​−0.195, p ​= ​0.0002). ConclusionsAlthough right knees tended to have more severe OA at both baseline and follow-up, radiographic progression did not differ by knee and compartmental progression correlated across knees. Given this trend in generalized OA, the risk of progression for both knees should be considered, even if only one knee has radiographic OA at baseline.

Dynamic Reconfiguration of Subcompartment Architectures in Artificial Cells.

Artificial cells are minimal structures constructed from biomolecular building blocks designed to mimic cellular processes, behaviors, and architectures. One near-ubiquitous feature of cellular life is the spatial organization of internal content. We know from biology that organization of content (including in membrane-bound organelles) is linked to cellular functions and that this feature is dynamic: the presence, location, and degree of compartmentalization changes over time. Vesicle-based artificial cells, however, are not currently able to mimic this fundamental cellular property. Here, we describe an artificial cell design strategy that addresses this technological bottleneck. We create a series of artificial cell architectures which possess multicompartment assemblies localized either on the inner or on the outer surface of the artificial cell membrane. Exploiting liquid–liquid phase separation, we can also engineer spatially segregated regions of condensed subcompartments attached to the cell surface, aligning with coexisting membrane domains. These structures can sense changes in environmental conditions and respond by reversibly transitioning from condensed multicompartment layers on the membrane surface to a dispersed state in the cell lumen, mimicking the dynamic compartmentalization found in biological cells. Likewise, we engineer exosome-like subcompartments that can be released to the environment. We can achieve this by using two types of triggers: chemical (addition of salts) and mechanical (by pulling membrane tethers using optical traps). These approaches allow us to control the compartmentalization state of artificial cells on population and single-cell levels.

Calcium cycling as a mediator of thermogenic metabolism in adipose tissue.

Canonical non-shivering thermogenesis (NST) in brown and beige fat relies on uncoupling protein 1 (UCP1)-mediated heat generation, although alternative mechanisms of NST have been identified, including sarcoplasmic reticulum (SR)-calcium cycling. Intracellular calcium is a crucial cell signaling molecule for which compartmentalization is tightly regulated, and the sarco-endoplasmic calcium ATPase (SERCA) actively pumps calcium from the cytosol into the SR. In this review, we discuss the capacity of SERCA-mediated calcium cycling as a significant mediator of thermogenesis in both brown and beige adipocytes. Here, we suggest two primary mechanisms of SR calcium mediated thermogenesis. The first mechanism is through direct uncoupling of the ATPase and calcium pump activity of SERCA, resulting in the energy of ATP catalysis being expended as heat in the absence of calcium transport. Regulins, a class of SR membrane proteins, act to decrease the calcium affinity of SERCA and uncouple the calcium transport function from ATPase activity, but remain largely unexplored in adipose tissue thermogenesis. A second mechanism is through futile cycling of SR calcium whereby SERCA-mediated SR calcium influx is equally offset by SR calcium efflux, resulting in ATP consumption without a net change in calcium compartmentalization. A fuller understanding of the functional and mechanistic role of calcium cycling as a mediator of adipose tissue thermogenesis and how manipulation of these pathways can be harnessed for therapeutic gain remains unexplored. Significance Statement Enhancing thermogenic metabolism in brown or beige adipose tissue may be of broad therapeutic utility to reduce obesity and metabolic syndrome. Canonical BAT-mediated thermogenesis occurs via uncoupling protein 1 (UCP1). However, UCP1-independent pathways of thermogenesis, such as sarcoplasmic (SR) calcium cycling, have also been identified, but the regulatory mechanisms and functional significance of these pathways remain largely unexplored. Thus, this mini-review discusses the state of the field with regard to calcium cycling as a thermogenic mediator in adipose tissue.

Pentad: a tool for distance-dependent analysis of Hi-C interactions within and between chromatin compartments

BackgroundUnderstanding the role of various factors in 3D genome organization is essential to determine their impact on shaping large-scale chromatin units such as euchromatin (A) and heterochromatin (B) compartments. At this level, chromatin compaction is extensively modulated when transcription and epigenetic profiles change upon cell differentiation and response to various external impacts. However, detailed analysis of chromatin contact patterns within and between compartments is complicated because of a lack of suitable computational methods.ResultsWe developed a tool, Pentad, to perform calculation, visualisation and quantitative analysis of the average chromatin compartment from the Hi-C matrices in cis, trans, and specified genomic distances. As we demonstrated by applying Pentad to publicly available Hi-C datasets, it helps to reliably detect redistribution of contact frequency in the chromatin compartments and assess alterations in the compartment strength.ConclusionsPentad is a simple tool for the analysis of changes in chromatin compartmentalization in various biological conditions. Pentad is freely available at https://github.com/magnitov/pentad.

Impact of Follicular Menstrual Phase on Body Composition Measures and Resting Metabolism.

This study aimed to identify the effects of early follicular (EF) and midfollicular (MF) menstrual phases on body composition, resting metabolic rate (RMR), and respiratory quotient (RQ) assessment accuracy to identify an optimal testing period. Body composition was obtained from a four-compartment (4C) criterion model (fat mass (FM), fat-free mass, body fat percent, and dual-energy x-ray absorptiometry (DXA; FM, lean mass (LM), trunk FM, and trunk LM) in 19 eumenorrheic females (mean ± SD: age, 21.3 ± 3.1 yr, body mass index, 23.6 ± 1.8 kg·m-2). RMR (kcal·d-1) and RQ (a.u.) were measured via indirect calorimetry for 25 min. Body composition, RMR, and RQ were measured during the EF and MF phases. Dependent-samples t-tests were used to compare outcomes between EF and MF. 4C outcomes were similar between phases (P > 0.05). During EF, the following 4C components were significantly greater (P < 0.05): body volume (mean difference (MD) ± SD, 0.70 ± 1.05 L), extracellular fluid (MD ± SD, 0.27 ± 0.51 L), and body mass (MD ± SD, 0.56 ± 0.80 kg). DXA-measured LM, body fat percent, trunk LM, and trunk FM were similar (P > 0.05); however, DXA FM was significantly greater during EF (MD ± SD, 0.29 ± 0.40 kg; P = 0.005), yet within measurement error of the device. Although RMR was not significantly different between phases (MD ± SD, 6.0 ± 190.93 kcal·d-1; P > 0.05), RQ was significantly higher during EF (mean ± SD, 0.03 ± 0.06 a.u.; P = 0.029) compared with MF. Body composition from 4C and DXA do not seem to be affected beyond measurement error as a result of compartmental changes from the menstrual cycle. During MF, women oxidized more fat as demonstrated by a lower RQ. Researchers should aim to be more inclusive and schedule testing for females within 11-12 d from the onset of menstruation.

Abstract 2033: Rearrangement of the 3D genome structure in prostate cancer is a potential mechanism for disease exacerbation and classifies cancers according to progression

Abstract Prostate cancer (PCa) aggressiveness and propensity to metastasize can be influenced by gene expression patterns, genomic aberrations, and overall cellular and nuclear morphology. The 3D folding of chromosomes into loops, domains, and territories as they are packaged into the nucleus plays a key role in all of these processes, affecting proper gene regulation, DNA replication and repair, and nuclear physical properties. However, while nuclear atypia has long been used as a marker of PCa progression, little is known about what changes in 3D genome interactions occur or how these may influence cancer-promoting gene expression profiles or the nuclear malleability necessary for cancer cells to metastasize. Thus, we set out to characterize the role of the 3D genome changes in PCa, analyzing changes in the 3D genome structure of cell lines that model the continuum of disease. Using chromosome conformation capture (Hi-C) we have conducted a systematic comparison of the 3D genome structure of a normal epithelium model cell line (RWPE) to that of adenocarcinoma (LNCaP), bone metastatic (VCaP, PC3, 22RV1, LNCaP-C42B, MDaPCa2a and MDAPCa2b), and atypical metastasis (Brain - DU145) lines. Our study has shown that the open/closed chromatin compartmentalization identity (A vs B compartment) changes with progression. We have characterized a cohort of 316 genes whose compartmentalization changes from the B to the A compartment, suggesting that those genes become poised for transcription. These include androgen receptor, WNT5A and CDK14, as well as genes located in proximity to TMPRSS2, such as BACE2. The positive correlation between switches of compartmentalization identity and transcription activation has been confirmed by microarray. Of these B/A switch genes, 256 are grouped in 48 proximal clusters. We have also identified 84 genes whose compartments switch from the A to B compartment, suggesting a genomic rearrangement that could result in silencing. Such is the case of certain cadherins, annexins and mediators of inflammation. Of these, 64 are grouped in 16 clusters. Finally, we have observed that compartmentalization changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation; one of the main drivers of prostate cancer, present in 40% - 80% of prostate cancer patients. Interestingly, changes in gene transcription are also accompanied by changes in the structure, size, and boundaries of the topologically associating domains (TADs) surrounding the loci of interest, suggesting that 3D genome structure plays a role in the activation of alternate pathways for proliferation and survival. Citation Format: Rebeca San Martin, Priyojit Das, Renata Dos Reis Marques, Rachel P. McCord. Rearrangement of the 3D genome structure in prostate cancer is a potential mechanism for disease exacerbation and classifies cancers according to progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2033.

From hair colour to diagnosis

Hair pigmentation contributes extremely to the visual appearance and human-to-human communication, thus exerting enormous sexual and social impact. Follicular melanogenesis depends on genetic, biochemical and physical phenomena as well as proper epithelial-melanocytic interaction. Shades of grey, yellow, brown, red, and black produced by melanin define the exact determination of hair colour. Versatile clinical conditions depend on genetic melanogenetic changes, pigment transfer to bulb keratinocytes defects and impaired signal transduction pathways. Herein, an update of recent scientific advances on follicular melanogenesis and its pathological-driven compartmental changes reflecting specific disease phenotypes, are presented.

Knee joint distraction results in MRI cartilage thickness increase up to 10 years after treatment.

ObjectivesKnee joint distraction (KJD) has been shown to result in long-term clinical improvement and short-term cartilage restoration in young OA patients. The objective of the current study was to evaluate MRI cartilage thickness up to 10 years after KJD treatment, using a 3D surface-based approach.MethodsTwenty end-stage knee OA patients were treated with KJD. MRI scans (1.5 T) were performed before and at 1, 2, 5, 7, and 10 years after treatment. Tibia and femur cartilage segmentation and registration to a canonical surface were performed semi-automatically. Statistical parametric mapping with linear mixed models was used to analyse whole-joint changes. The influence of baseline patient characteristics was analysed with statistical parametric mapping using linear regression. Relevant weight-bearing parts of the femur were selected to obtain the average cartilage thickness in the femur and tibia of the most- (MAC) and least-affected compartment. These compartmental changes over time were analysed using repeated measures ANOVA; missing data was imputed. In all cases, P <0.05 was considered statistically significant.ResultsOne and 2 years post-treatment, cartilage in the MAC weight-bearing region was significantly thicker than pre-treatment, gradually thinning after 5 years, but still increased at 10 years post-treatment. Long-term results showed that areas in the least-affected compartment were significantly thicker than pre-treatment. Male sex and more severe OA at baseline somewhat predicted shorter-term benefit (P >0.05). Compartmental analyses showed significant short- and long-term thickness increase in the tibia and femur MAC (all P <0.05). ConclusionKJD results in significant short- and long-term cartilage regeneration, up to 10 years post-treatment.Trial registrationNetherlands Trial Register, https://www.trialregister.nl, NL419.

Taxa de prenhez e tamanho da ninhada de cadelas Buldogue Inglês inseminadas com sêmen fresco

The present study aimed to evaluate the estrus cycle of Bulldog bitches using vaginal cytology, and to inseminate these with fresh semen, leading to the determination of pregnancy rate and the size of the litter. Sixteen (16) English Bulldog breed bitches were used, all with good reproductive history and proved fertility, age ranging from 1.5 to 6 years. Associated with the proestrus detection (external signs: vulva edema and a blood-stained discharge) was performed the vaginal cytology in order to determine the estrus moment, as well was observed the female behavior of male acceptance. The artificial inseminations (AI) started when the vaginal cytology showed 90% of superficial cells, then repeated every 48h until the vaginal cytology change to diestrus (increasing the nucleated cell counting) and/or the bitch stop to accept the dog mating. To perform the semen collection were used the digital manipulation, all male breeders undergo to a previous seminal examination being select for use only ejaculates with more than 70% of total motility, vigor 3 and, at least, 200x106 of spermatozoa. The AI was performed using all the collected ejaculate. The gestation confirmation was made by transabdominal ultrasonography between 25 and 30 days after the last AI, the ultrasonographic evaluation after that was carried out periodically in order to determine the best moment to execute the c-section. The vaginal cytology evaluation, associated with the compartmental changes in the bitch was efficient to detect the estrus onset in 100% of the bitches. Get pregnant 14 of the 16 bitches inseminated (87.5%). The litters had a mean number of 5.6 puppies, of those 53.16% (42/79) were male and 46.84% (37/79) were female. The AI with fresh semen associated with vaginal cytology provided good fertility results and size of litters compatible in English Bulldog under these experimental conditions.

Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains.

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

Fruit setting rewires central metabolism via gibberellin cascades

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Alignment changes after open-wedge high tibial osteotomy result in offloading in the patellofemoral joint: a SPECT/CT analysis.

The patellofemoral (PF) joint may be adversely affected by medial open-wedge high tibial osteotomy (OWHTO). This study aimed to evaluate the PF compartmental changes using combined single-photon emission computed tomography (SPECT) and conventional computed tomography (CT) after OWHTO to provide clinical guidance regarding the PF joint pressure and force. Patients with medial osteoarthritis and varus malalignment > 5° were treated using OWHTO. Patients with a minimum 2-year follow-up were included in the study. The patellar positions were evaluated based on the radiographic parameters. The changes in chondral lesions during second-look arthroscopic examination were evaluated, and the PF joint arthritis grade was recorded on patellar Merchant radiographs using Kellgren-Lawrence classification. The PF compartmental changes according to SPECT/CT analysis after OWHTO were evaluated in all patients. The scintigraphic uptake was graded on four scales. Patients were divided into improved and unimproved groups according to the PF compartmental grade using the SPECT/CT uptake grading system. At a mean follow-up period of 47.0months (range 25-74months), the mean mechanical femorotibial angle changed significantly from varus 6.3° (range 5-12°) to valgus 2.6° (range 0-8°); p < 0.001) postoperatively. The radiological parameters presenting patellar positions, including the tibial slope, patellar convergence angle, and lateral tilt angle, did not change significantly between the preoperative values and the 2-year follow-up values. The mean patellar height significantly decreased (0.07 ± 0.14, p = 0.001 according to the Blackburn-Peel index and 0.32 ± 0.23, p < 0.001 using the modified Insall-Salvati ratio). The average tibial tubercle to trochlear groove (TT-TG) distance significantly decreased from 14.1 to 12.2mm (p < 0.001). The Q angle also significantly decreased from 9.8o to 7.7o (p = 0.008). Chondral lesions of the patella and trochlear groove revealed significant deterioration; at 2years after OWHTO, the arthritic grades of the PF joints worsened significantly, as determined by radiography (p = 0.007). Scintigraphic uptake in the PF joint was significantly lower (from 2 to 1) at 2years postoperatively compared to that immediately after the index operation (p < 0.001). Only 4 of 56 (7.1%) patients showed increased uptake. Comparison between the improved and unimproved groups according to scintigraphic uptake changes revealed that the changes in the cartilage status on the patellar undersurface and TT-TG distance were the most significant predictive factors of increased scintigraphic uptake in the PF joint after OWHTO. Alignment correction by OWHTO result in PF compartment offloading and should be considered when identifying the surgical indications for OWHTO. Therapeutic, Level IV.

Inflammation-related plasma and CSF biomarkers for multiple sclerosis

Effective biomarkers for multiple sclerosis diagnosis, assessment of prognosis, and treatment responses, in particular those measurable in blood, are largely lacking. We have investigated a broad set of protein biomarkers in cerebrospinal fluid (CSF) and plasma using a highly sensitive proteomic immunoassay. Cases from two independent cohorts were compared with healthy controls and patients with other neurological diseases. We identified and replicated 10 cerebrospinal fluid proteins including IL-12B, CD5, MIP-1a, and CXCL9 which had a combined diagnostic efficacy similar to immunoglobulin G (IgG) index and neurofilament light chain (area under the curve [AUC] = 0.95). Two plasma proteins, OSM and HGF, were also associated with multiple sclerosis in comparison to healthy controls. Sensitivity and specificity of combined CSF and plasma markers for multiple sclerosis were 85.7% and 73.5%, respectively. In the discovery cohort, eotaxin-1 (CCL11) was associated with disease duration particularly in patients who had secondary progressive disease (P CSF < 4 × 10-5, P plasma < 4 × 10-5), and plasma CCL20 was associated with disease severity (P = 4 × 10-5), although both require further validation. Treatment with natalizumab and fingolimod showed different compartmental changes in protein levels of CSF and peripheral blood, respectively, including many disease-associated markers (e.g., IL12B, CD5) showing potential application for both diagnosing disease and monitoring treatment efficacy. We report a number of multiple sclerosis biomarkers in CSF and plasma for early disease detection and potential indicators for disease activity. Of particular importance is the set of markers discovered in blood, where validated biomarkers are lacking.