Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common disorders that disrupt the quality of life of millions of people. These two chronic diseases cause damage to the joint cartilage and surrounding tissues of more than 220 million people worldwide. Sex-determining region Y-related (SRY) high-mobility group (HMG) box C, SOXC, is a superfamily of transcription factors that have been recently shown to be involved in various physiological and pathological processes. These include embryonic development, cell differentiation, fate determination, and autoimmune diseases, as well as carcinogenesis and tumor progression. The SOXC superfamily includes SOX4, SOX11, and SOX12, all have a similar DNA-binding domain, i.e., HMG. Herein, we summarize the current knowledge about the role of SOXC transcription factors during arthritis progression and their potential utilization as diagnostic biomarkers and therapeutic targets. The involved mechanistic processes and signaling molecules are discussed. SOX12 appears to have no role in arthritis, however SOX11 is dysregulated and promotes arthritic progression according to some studies but supports joint maintenance and protects cartilage and bone cells according to others. On the other hand, SOX4 upregulation during OA and RA was documented in almost all studies including preclinical and clinical models. Molecular details have indicated that SOX4 can autoregulate its own expression besides regulating the expression of SOX11, a characteristic associated with the transcription factors that protects their abundance and activity. From analyzing the currently available data, SOX4 seems to be a potential diagnostic biomarker and therapeutic target of arthritis. Osteoarthritis (OA) and rheumatoid arthritis (RA) are two forms of arthritis with a similar clinical phonotype, causing joint pain, stiffness, and probably disability, but occurring for different reasons and differing in pathophysiological mechanisms and in treatment strategies. These two common disorders affect more than 220 million people in the world. Osteoarthritis, the most common type, is a degenerative joint disease, characterized by the progressive deterioration of the articular cartilage or of the entire joint (the articular cartilage and the joint lining), the ligaments, and the subchondral bone. In addition, although inflammatory conditions can be associated with OA, basically, it is not an inflammatory disease. On the other hand, RA is a chronic systemic autoimmune inflammatory disorder characterized by the infiltration of inflammatory cells at the synovial lining resulting in hyperplasia and the destruction of cartilage and bone tissues (reviewed by Pap and Korb-Pap ). TypicaSOX) genes have been reported in the mammalian genomes, which encode eight groups of SOX TFs, named from A to H. However, during the past decade, studies have found links between the dysregulation of sex-determining region Y-related (SRY) high-mobility group (HMG) box C (SOXC) levels and the progression of arthritis disorders. SOXC is a superfamily of TFs that have been recently shown by several studies to be involved in the onset and pathogenesis of arthritis. Interestingly, the great majority of these studies have been conducted during the last 5 years [SOXC gene family encodes SOX4, SOX11, and SOX12 TFs that contain a conserved high mobility group (HMG)-box domain by which SOXC proteins are able to bind to minor grooves in the DNA. SOXC TF binding to DNA can lead to alterations in the chromatin architecture associated with changes in the downstream genes at both transcriptional and functional levels. At least in vitro, while the three have similar DNA-binding abilities, the mammalian SOXC unique features in the SOX11 DNA binding domain allow the more potent transactivation function of SOX11; however, SOX12 was found to be the weakest [To date, there is no cure for OA or RA, and the available therapeutic protocols are able to partially reduce the pain, relieve the symptoms, improve quality of live and increase survival. However, an understanding of the involved signaling biomolecules and their targets and inhibitors is advantageous in designing new therapeutic arthritis drug or drug combinations. A little over 30 years ago, the gene of the male sex-determining region of the Y chromosome (SRY) was discovered, which later facilitated the discovery of the whole family of essential regulatory gene-encoding transcription factors (TFs) that control cell fate in many processes ,3. To da 5 years ,15,16,17 weakest .In the current review, we aim to highlight SOX4 and SOX11 transcription factors as a diagnostic biomarker and therapeutic targets of arthritis diseases, and to clarify their role in regulating osteoarthritis and rheumatoid arthritis in relation to the involved downstream and upstream signaling molecules. An overview of the role of SOXC transcription factors in OA and RA has not been conducted before.SOXC genes, SOX4 and SOX11 are involved in synovial hyperplasia during arthritic disease through enhancing the survival of FLS, but do not affect the FLS proliferation rate [SOXC genes expression in the articular chondrocytes synovial lining significantly decreased synovitis and the erosion of the articular cartilage [At local inflammatory sites in humans, a specific type of CD4+ T cell was found to enhance ectopic lymphoid-like structure (ELS) formation through the production of the chemokine CXCL13 and other mediators . These eion rate . Furtherion rate . Moreoveion rate . In the artilage . Thus, tIn healthy tissues, FLSs populate at the synovial lining of the joints and maintain the synovial fluid homeostasis through producing cartilage-protecting proteins and enhancing joint lubrication. However, in arthritic inflammatory conditions, FLSs are epigenetically changed and transformed into a main source of catabolic enzymes and inflammatory cytokines that promote the degeneration of joints . In addiDevelopmental biology studies support the idea that SOX4 and SOX11 are essential in promoting articular and bone formation ,18. HoweThe progression of OA disorder was recently documented in several studies to be associated with upregulated levels of SOX4  As a critical mediator of the TNF-induced transformation of FLS, SOX4 interacts with RELA  to regulate the expression of TNF downstream genes and thus maintains the transformation of FLS and inflammatory pathology in arthritis ,23. (2) Targeting the SOXC proteins or the mechanisms that stabilize them during arthritis through the systemic delivery of inhibitors has been suggested as an option to be explored ,32,33. HThrough direct and indirect binding to SOX4 TF, several upstream factors and signaling molecules have been recently reported to target SOX4 during arthritis, such as ROS)/TGF, TNF, SMOC2, AGEs, Lnc PART1, and the miRNA, miR-31 . The proOn the other hand, the endogenous secreted modular calcium-binding protein 2 (SMOC2) has been recently recognized as a key molecule to control the aggressive behavior in FLSs during arthritis. Mechanistically, SMOC2 exerts this function via SOX4-mediating transcriptional regulation leading to increased levels of upregulated motor protein myosin1c (MYO1C) and hence causing cytoskeleton remodeling and enhancing synovial migration and invasion during RA .In human chondrocytes, the inflammation and degradation of the cartilage-specific proteoglycan core protein and the onset and development of OA was reported to be regulated by advanced glycation end products (AGEs). Mechanistically, in a dose-dependent manner, AGE-induced chondrocyte injury was found to upregulate the levels of both SOX4 and phosphorylated p38. In the meantime, treatment with the p38 inhibitor reduced AGE-induced SOX4 expression, indicating that the upregulation of SOX4 that resulted from AGE treatment is mediated by p38 . HoweverBesides the above-mentioned signaling molecules, preclinical studies have suggested natural anti-inflammatory and antioxidant biomolecules to target SOX4 and SOX4 signaling molecules and treat arthritis. In this regard, the classic nonsteroidal anti-inflammatory drug Feprazone was reported to have a promising effective role in treating arthritis. This has been proven mechanistically by the ability of Feprazone to reduce the degraded aggrecan in the human chondrocytes induced in TNF-α treated models, where it inhibited the loss of aggrecan via the suppression of the SOX-4/ADAMTS-5 signaling pathway . In addiSOX4 gene expression during inflammatory diseases [Based on recent data, SOX4 was found to autoregulate its own expression besides regulating its family-member SOX11. As recently interpretated by Jones and colleagues , the autdiseases . SOX4 podiseases . In thisSOX4 gene 3′ (UTR) SNP and osteoporosis susceptibility, three SNPs loci, rs79958549, rs139085828, and rs201335371, at the SOX4 gene were found to be significantly associated with the risk of osteoporosis [Osteoporosis, a metabolic bone disease affecting millions of the world’s population, is characterized by a lower bone mineral density, bone fragility, and a loss of bone mass. It commonly occurs during the postmenopausal period in women and in the elderly population due to the increase of bone remodeling and the imbalance between bone osteoclastic and osteoblastic activity. An impaired osteoblast associated with decreased bone formation in SOX4 +/− mice suggested an important role for SOX4 in the formation and the resorption of bone . SimilarAs a downstream target of TNF-α , SOX4 waOn the other hand, primary Sjögren’s syndrome (pSS) is an autoimmune condition in which a pathogenic type of T helper cells (CCR9+) is upregulated in patients and contributes to the immunopathology of pSS ,47. In sAlthough osteoarthritis and rheumatoid arthritis are two forms of arthritis occurring for different reasons, both appear to be regulated by similar signaling mechanisms. Both are chronic disorders; while OA is a degenerative disease, RA is an inflammatory one. Based on the currently available data, SOX12 seems not to be involved in arthritis disease progression, while SOX11 is dysregulated and enhances arthritic progression according to some studies, but is required for joint maintenance and protects cartilage and bone cells according to other studies. However, SOX4 appears to be a potential diagnostic biomarker and therapeutic target of arthritis. Subsequently, further studies are required to fully understand the mechanistic actions of SOXC proteins during arthritis progression and many knowledge gaps still need to be filled, which may help to find effective treatments for osteoarthritis and rheumatoid arthritis.


This second volume of a Research Topic devoted to the investigation of the control of urinary bladder in physiological and pathological conditions reiterates its relevance. As a subject with several “facets” , not surLamy et al. have shown that Angiotensin-(1–7) administrated intravenously or topically (in situ) onto the urinary bladder (UB) elicits an increase in the intravesical pressure. The authors also demonstrated that Mas receptors for Angiotensin-(1–7) and ACE-2, an enzyme required for Angiotensin-(1–7) synthesis, are expressed in the bladder. Therefore, they suggested that this peptide acts in the UB to increase the IP and can be also locally synthesized in the UB.The urine storage and voiding from the bladder are mediated by both central and peripheral mechanisms, nevertheless they still remain to be fully elucidated. Interestingly, Pang et al. presented in this topic shows previous functional imaging studies and combines them with brain regions involved in bladder control, demonstrating interactions between these regions, and brain networks, as well as changes in brain function in diseases affecting the urinary bladder. Pang et al. extend the working model proposed by The review of Phelps et al. aimed to identify the possible similarities in extracellular Ca2+ requirements between muscarinic, histamine, 5-hydroxytryptamine (5-HT), neurokinin-A (NKA), prostaglandin E2 (PGE2), and angiotensin II (ATII) receptors for mediating contractile activity of the urinary bladder (urothelium and lamina propria). Despite the finding that the specific requirement of Ca2+ on contractile responses varies depending on the receptor, Phelps et al. suggested that extracellular Ca2+ has a key role in mediating G protein-coupled receptor contractions of the urothelium and lamina propria.Several bladder diseases arise due to abnormal contractions , and PheAresta Branco et al. investigated possible mechanosensitive mechanisms of ATP hydrolysis in the LP at the anti-luminal side of nondistended (empty) or distended (full) murine (C57BL/6J) detrusor-free bladder model. The authors demonstrated that mechanosensitive degradation of ATP and ADP by membrane-bound and soluble nucleotidases in the LP reduces the availability of excitatory purines in the LP at the end of bladder filling. Hence, they suggested a possible safeguard mechanism to prevent overexcitability of the bladder, in which adequate proportions of excitatory and inhibitory purines in the bladder wall are determined by distention-associated purine release and purine metabolism.Two of the studies examined purinergic mechanisms that operate within the bladder. The first investigated the role of purine metabolism in purinergic mechanisms. Earlier studies have shown that adenosine 5′-triphosphate (ATP) released from the urothelium has a prominent role in bladder mechanotransduction . UrothelTaidi et al. investigated the role of P2X7R in acrolein-induced inflammatory damage in primary cultured porcine bladder urothelial cells. The authors demonstrated that acrolein induced a significant reduction in urothelial cell viability and barrier function, which was protected by the presence of P2X7R antagonist. Thereby, Taidi et al. suggested that P2X7R blockade may be a possible therapy in patients with bladder cystitis evoked by cyclophosphamide treatment.The second purinergic study examined the role of P2X7 receptors in the bladder. The purinergic P2X7 receptor (P2X7R) is expressed abundantly on the bladder urothelium and its role in inflammation and cell death has been increasingly recognized . It is wCarew et al. investigated the expression of myosin 5a (Myo5a), which is a motor protein that facilitates the directed motion of synaptic vesicles along actin fibers, in the regulation of excitatory neurotransmission in the bladder. The authors demonstrated that Myo5a is localized in cholinergic nerve fibers in the bladder and identified several Myo5a splice variants in the detrusor and suggested that the abundance of each is likely critical for efficient synaptic vesicle transport and neurotransmission in the bladder.The dysregulation in neurotransmission has been implicated in several lower urinary bladder conditions, however the mechanisms underlying the neurotransmitter release in the bladder still require elucidation. Oliveira et al. has shown that the treatment of mice with methylglyoxal (MGO), which is a compound generated during glycolysis and present in high levels in the plasma of patients with diabetes mellitus  production, which was markedly higher in the detrusor muscle than in the urothelium. They suggested that MGO accumulation increases AGE formation, which activates the RAGE-ROS signaling and consequent Rho kinase-induced muscle sensitization, which leads to detrusor overactivity.High glucose levels can induce changes in the urinary bladder. mellitus , inducesmellitus . OliveirYesilyurt et al., bladder weight and blood glucose from 16 studies were evaluated and concluded that the presence and extent of bladder enlargement varied markedly depending on the diabetes models. The authors also suggest that particularly in type 2 diabetes models, the bladder enlargement is primarily driven by glucose levels/glucosuria.Despite the urinary bladder is markedly enlarged in the streptozotocin-induced type 1 diabetes mellitus in rats, which may contribute to the frequent diabetic uropathy, very little is known about the bladder changes in type 2 diabetes models. Diabetic polyuria has been proposed as the pathophysiological mechanism behind bladder enlargement. In the review of Mansfield et al. discusses the findings of recent laboratory and clinical studies, providing the relationship between urinary tract infection, bladder inflammation, and the pathophysiology of OAB. The authors suggest that urinary tract infection may be an underappreciated contributor to the pathophysiology of some OAB patients who are resistant to standard treatments.Overactive bladder (OAB) has been accepted as an idiopathic disorder defined by urinary urgency, increased daytime urinary frequency and/or nocturia, with or without urinary incontinence. This clinical syndrome is characterized clinically by an absence of other organic diseases, including urinary tract infection. However, a growing body of evidence has shown that a significant proportion of OAB patients have active bladder infection. The review of Gao and Rodriguez highlights recent findings about stress-related animal models as water avoidance stress, social stress, early life stress, repeated variable stress, chronic variable stress, intermittent restraint stress and others, demonstrating that different types of chronic stress induce relatively distinguished changes at multiple levels of the micturition pathway.Urinary bladder function can also be affected by chronic psychological stress leading to an exacerbated lower urinary tract dysfunction as in OAB or interstitial cystitis-bladder pain syndrome , 2015b. Beaman et al. about a rare disease called urofacial syndrome (UFS), which is an autosomal recessive congenital disorder of the urinary bladder characterized by voiding dysfunction and a grimace upon smiling  are expressed in neural like cells with a migratory phenotype, postulated to be pelvic ganglia precursors. Thereby, Beaman et al. suggested that biallelic variants of LRIG2 should be also implicated in the rare UFS.This topic also brings a novelty showed by  smiling . Biallel smiling . BladderIn conclusion, this Research Topic encompasses a broad range of studies from basic science to clinical and certainly challenges researchers to further investigate unsolved questions. We trust that the valuable lessons learnt about the urinary bladder will be useful further for the development of novel therapeutic approaches upon the growing number of patients with bladder dysfunctions worldwide.


We have introduced a rectangular fin inside the cylinder; the fin temperature is at  It was reached the hybrid liquid slowdown in heat transmission rate was greater than the slowdown in single nano. Accordingly, they noticed the implementation of the integration of hybrid nanoparticles Cu-Al2O3 and Al2O3 nanoparticles that led to a huge rise in nanoparticles speed with an improved 2O3-CuO/water) mixture nano in hollow vulnerable by [2O3-water nanofluid in a square-shaped container. They showed the relationship between the average weight and high heat source. Al-Rashed et al. [Heat transmission is utilized in various areas of industry represented in solar and thermal energy complexes, thermal insulation and metal melting, petrochemicals and cooling electronic components, fiber insulation, etc. Given applications, there is a need for high thermal operation ,6,7,8,9.rable by . The overable by  examinedd et al.  propose d et al.  evaluated et al. . Seddeghd et al.  securitid et al.  analyzedd et al.  examinedd et al.  tested td et al.  entropy Wavy cavities are infrequently used, especially for hybrid nanofluids, even though a range of cavity forms were taken into consideration in the literature. In technical applications like heating systems, space thermal protection, storage of energy, electronic devices, reactor safety gear, solar collectors, etc., and the study of confined enclosures are crucial. Bridging the gap few studies were given by researchers. Hence, the current work focus on the space of studying flow, heat, and entropy aspects of the wavy enclosure using hybrid nanofluid has been evaluated a. The inHere, It is supposed to be a thermal balance between the fluid and nanoparticles and no slide between water and particles. In a realistic setting, the majority of nanofluids utilized to increase heat transmission consist of extremely small particles. The nanofluids can therefore be seen as acting comparable to a traditional homogeneous single-phase liquid . The theThe following can therefore be stated for continuity, impetus, and heat equations ,31.(1)∂After introducing the following dimensionless numbers, the dimensionless system of equations and the appropriate boundary constraints are .(5) with reducing the fluid flow of hybrid nanofluid of kind AlAs the supporting proof of the previous claim, 2O3-Cu/H2O hybrid fluid were portrayed in Fractional volume (2O3-Cu/H2O hybrid fluid over the enclosure. As the porosity allows more thermal efficient particles which drive more temperature in the enclosure meanwhile the entropy also gets elevated can be noted through the intensifying diagonally dominant contours.Significant impact of undulation on the fluidity in the enclosure can be viewed through the streamlined patterns in Entropy formation across the enclosure for the improving wavy nature of its outer wall for improving values of undulation constraint under magnetic interference can be viewed in Generally, in this enclosure structure, the Entropy formation can be noted in the diagonal portions of the lower heated wall and the upper cold wall along with the right-angled corner part for higher magnetic states. As the undulation gets improved, the entropy spots get increased in the trough portions of wavy walls. Entropy formation level seems to be higher for the higher-end undulations even for the minimal magnetic interference.2O3-Cu/H2O nano level fluid in this study. Fluidity, thermal transference, average Nusselt number (Special kind of enclosure with two differently sized and shaped cylinders with a porous middle portion enveloped by the wavy outer wall has been engaged with the hybrid nano of Al❖Even though the thermal transmission process was the hotspot of this study, the porous medium includes the fluidity brought under consideration. Vital sign of improved fluidity was commonly found through the extended flow contour towards the narrow path between two distinct walls. Such a trend can be found as increasing for improving amounts of parameters like Rayleigh (❖2O3-Cu/H2O can be significant enough due to its elevated thermal properties than that of usual fluid. A diagonally dominant trend by the inner hot smaller wall and outer wavy cooler wall sets the favorable situation for thermal distribution. It can be found for the parameters such as Darcy number (Thermal distribution with effecting crucial constraints involved in this work has been plotted as a contour and clear sketch graph. Impact of a hybrid nanofluid of Al❖Vital thermal transmitting rate has been traced in terms of the average Nusselt number (❖To address this issue entropy study has been carried out and parametrical outcomes were plotted as counterplots and graphs. Highest entropy-forming situations were found in higher amounts of ❖For the best possible results, it is crucial to take preventative measures to identify and minimize transferred energy loss while conducting tests to find an effective thermal transmitting fluid.In the future study, an extension of this study can be carried out by using different models of nanofluids with different shapes and different qualities of nanoparticles, using the following valuable studies in ,37,38,39


Dear Editor,-1, acidemia [venous pH <7.3 or bicarbonate <15 mEq L-1] and ketonaemia) is classified as an important perioperative complication. Moreover, an ever-evolving field of diabetology and the concomitant polypharmacy present additional challenges in the sound perioperative management of these metabolic disorders. This is epitomised by the resurgence of interest in euglycaemic DKA (EDKA). Diabetic ketoacidosis (DKA)  adverse-event reporting systems reveal that a sizeable percentage of the DKA patients (as high as 35% in certain scenarios) demonstrates blood glucose levels <200 mg dL4 The commonly used gliflozins include canagliflozin, dapagliflozin, and empagliflozin. Their clinical effects are primarily due to inhibition of sodium-glucose transport protein 2 resulting in reduced renal glucose reabsorption and glycosuria leading to euglycaemia.5 This class of agents came into the limelight due to their purported additional benefits like weight loss, reduction in incidence of heart failure independent of glycaemic control, improved renal outcomes, and hepatoprotective properties in non-alcoholic steatohepatitis.6The usual triggers associated with EDKA are infection, starvation, vomiting or diarrhoea, and cocaine overdose. Recent reports of heightened EDKA risk with the perioperative use of sodium-glucose cotransporter-2 (SGLT2) inhibitors or gliflozins has captivated attention, particularly with these agents being increasingly employed as second-line hypoglycaemics.4 outlined an accentuated risk of perioperative EDKA associated with gliflozins. The aforementioned in conjunction with other recent incriminating literature led to an updated FDA prescribing information in the march of 2020 with canagliflozin, dapagliflozin, and empagliflozin to be withheld for at least 3 days prior and ertugliflozin to be withheld at least 4 days prior to an elective surgery.7 The American Association of Clinical Endocrinologists and American College of Endocrinology also recommend that gliflozins be withheld preoperatively or in other situations with typical DKA triggers, like severe infection.8However, a prominent case series by Lau et alEuglycaemic DKA is a rare clinical entity wherein a high degree of caution must be exercised. With ever growing number of diabetics and frequent use of gliflozins, EDKA incidence is bound to increase. Fasting patients with history of gliflozin use should be evaluated for high anion gap metabolic acidosis and ketonaemia despite having controlled blood glucose to unmask an underlying EDKA, thereby reducing mortality and morbidity. This highlights the fact that the pharmacodynamic, pharmacokinetic, and safety profile of the prescribed hypoglycaemics warrants due consideration in the conduct of safe anaesthesia.


Bone is a complex tissue populated by a highly heterogeneous mix of cell types in different compartments. The endosteal compartment is a key site for bone remodelling and provides a supportive microenvironment to harbour haematopoietic and mesenchymal stem cells, as well as cancer cells that grow in bone. The purpose of this review is to summarize recent findings of studies in bone using single-cell RNA sequencing and emergent spatial RNA sequencing to describe different bone-resident cell types and their molecular programs.Single-cell RNA sequencing identified novel and transcriptionally distinct cell clusters within different bone cell lineages, including MSCs, osteoblasts, chondrocytes, fibroblasts, osteoclasts and cells of the vasculature. Spatial transcriptomics methods provide information on the localization of the different cell populations.Single-cell transcriptomics provided valuable insights into long-standing knowledge gaps in the cellular heterogeneity of bone-resident cells in unprecedented detail, paving the way for studies to further investigate the different cell populations and to develop cell-based therapies for bone diseases. Musculoskeletal disorders, including osteoporosis and cancers that grow in bone, represent a major burden of disease. The endosteal surface in the skeleton is a main site of bone remodelling, a tightly controlled process involving osteoclasts, which remove bone and osteoblasts, which lay down new bone. These cells are coordinated by osteocytes, which are the master cell regulators in the skeleton. Other cell types and cell states, such as cells of the vasculature and haematopoietic cells, are also likely to contribute to the control of the skeleton. Bone is also a main site for cancer metastasis, with up to 90% of patients with advanced prostate cancer, and 60–70% of breast cancer patients have bone involvement with advanced disease . The sucDespite the key role of the endosteal compartment in bone remodelling and disease, the specific identity of the cells that comprise the compartment remains unclear. Traditional tools of assessing the functions of cells within complex tissues rely largely on imaging and functional assessment of genetic models based on single-cell markers to identify cell population of interest. However, there are various unappreciated differences in the cellular composition of bone cells in vitro and these issues potentially extend to in vivo studies using single markers to identify populations of interest, as many of the markers capture not one but multiple cell types . The useHere we review the insights scRNAseq has provided to our understanding of the endosteal bone compartment. We first describe the different methods of scRNAseq briefly and the cell types identified by these techniques in bone. We then report findings of more recent approaches involving spatially annotated transcriptomics, with limited application in bone so far but holds potential to provide even deeper insight into the cellular makeup of the endosteal bone compartment.A critical step in conducting a successful single-cell sequencing study of bone is generation of a single-cell suspension of skeletal cells while minimizing the impact on cell viability and transcriptional profile. Optimization of cell isolation procedures must aim to minimize the transcriptional impact of the isolation procedure at the same time that cell yield is maximized, as described in a recent protocol for bone scRNAseq .There are currently two main classes of methods to isolate single cells for RNA sequencing, which vary in the number of cells captured (high-throughput or low-throughput) and how the cells are selected for capture (targeted or untargeted) . The firStudies of scRNAseq in bone to date, which contain cells from the endosteal surface and bone marrow, mostly focused on the non-haematopoietic cell types of the murine bone, which provided valuable insights into the heterogeneity of early mesenchymal cells, osteogenic cells, fibroblasts and cells of the vasculature –15. The Lepr and Cxcl12 expression, which are characteristics of previously described Cxcl12-abundant reticular (CAR) cells [Cxcl12 and adipogenic markers such as Adipoq (adiponectin), as well as cells primed for osteogenic differentiation, with lower expression of Cxcl12 and high expression of osteogenic genes such as Sp7 (osterix) and Alpl . The adipo-primed cells were enriched for previously identified genes associated with human bone marrow mesenchymal stem cells [Osteogenic cells and adipocytes are believed to derive from mesenchymal stromal cells (MSCs). However, the identification of specific MSC populations remains challenging due to the limited number of specific markers available. scRNAseq studies identified multiple populations of MSC-like cells in bone with high levels of R) cells . Lepr+ cem cells , suggestAdipoq, Lpl, Apoe) and upregulates osteogenic genes  [To infer the hierarchical relationships of MSCs and osteogenic cell types, which include osteoblasts and chondrocytes, directionality of differentiation trajectory was assigned based on changes in transcriptional cell states using pseudotime analysis , 22. Wol2, Alpl) , 15.Mmp13, Postn, Alpl and Spp1, all markers of osteoblastic differentiation [Bglap2 , Col1a1, Sparc (osteonectin) and Ifitm5.The first committed osteoblastic cell state identified on the differentiation trajectory is a population of Runx2+ and Sp7+ cells with high expression levels of ntiation . HoweverSox9, Acan and Col2a1 and formed a continuum in the trajectory from pseudotime analysis [Ucma, a gene detected mostly in the “resting” zone of epiphyseal and vertebral cartilage [Ihh, Pth1r and Mef2c and terminally differentiated hypertrophic chondrocytes express high levels of Runx2, Ihh, Mef2c and Col10a1.Distinct clusters of chondrocytes were identified by Baryawno et al. due to the high number of non-haematopoietic cells sampled in the study. All identified chondrocytes express chondrocyte lineage genes analysis . The firartilage . Pre-hypFn1 (fibronectin-1), S100a4, Dcn (decorin) and Sema3c (semaphorin-3C) and lowly expressed the chondrocyte genes Sox9 and Acan, and were distinguishable from both MSCs and osteoblasts. A cluster expressed progenitor markers Cd34, Ly6a, Thy1 and Cd44, suggesting these fibroblasts are MSC-like [Scx (scleraxis) that regulate differentiation of tenocytes and ligamentocytes [Fibroblasts are cells of mesenchymal origin that are ubiquitous in the bone marrow and consist of phenotypically and functionally distinct subpopulations. Multiple fibroblast-like populations characterized by a high expression of extracellular matrix genes were identified, particularly in studies containing digested bones , 15. FibMSC-like . Additiontocytes . Howeverntocytes .Flt4, Tfpi, Stab2 and Vcam1 are highly specific to sinusoidal ECs and Cxcl12, Kitl, Gja4 and Vegfc to arterioles. Kusumbe et al. identified CD31highEMCNhigh type-H ECs in the transitioning zones of the metaphysis bridging arterioles with the sinusoids, which have been shown to play a role in osteogenesis [Emcn and Cd31 compared to sinusoids, suggesting that they might represent transitional H vessels [Cd34 and Ly6c1 but also express unique markers such as Cotl1 and Sox4.Bone is a highly vascularised organ with vertically aligned arteries carrying blood from the heart and smaller arterioles feeding into highly branched capillary beds . Capillaogenesis . By comb vessels . This poCspg4) as well as pericyte markers Acta2, Myh11 and Mcam [Lepr, in contrast to the high expression of Lepr in Lepr+ Cxcl12+ MSCs. This suggests that vascular SMCs and MSCs are clearly distinguishable and may represent distinct pools of progenitor cells.SMCs were found to highly express classical MSC markers Nestin and Ng2 , a classical DC marker. Deletion of Rank (receptor activator of nuclear factor-κB) specifically in CD11c-expressing cells inhibited osteoclast formation in vitro and in vivo. This finding supports previous reports of DCs differentiating into osteoclasts [Cited2  was identified to be a key molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Due to the in vitro nature of this study, contribution of other cell populations to osteoclastogenesis in the native bone marrow microenvironment is still unclear.Osteoclasts differentiate from precursors in the monocyte/macrophage lineage. The arrival and attachment of these precursors on bone surface are important steps in the initiation of osteoclast differentiation and bone remodelling. In vitro culture of bone marrow cells with recombinant RANKL and M-CSF can induce osteoclastogenesis, a model that has provided valuable insights into the molecular mechanisms of osteoclast differentiation. However, this culture system contains a highly heterogeneous cell population, which limits our understanding of the precise cell states and genes involved in different stages of osteoclast differentiation. A recent study by Tsukasaki et al. using scRNAseq on osteoclast culture of murine bone marrow cells treated with RANKL and M-CSF was able to overcome this limitation to reveal cell fate decision pathways during osteoclast differentiation in vitro at single-cell resolution . The auteoclasts , 29. TwoCtsk and Atp6v0d2 compared to osteoclasts, and expressed higher levels of surface markers including Axl, Cd11b, Ccr3, Vcam1, Cd74 and Cadm1 when compared to macrophages. The osteomorphs persisted after OPG-Fc withdrawal and exhibited hypersensitivity to RANKL, which drove them to form highly active osteoclasts. Osteoclast recycling therefore not only provides a new paradigm for understanding the behaviour of these cells in their in vivo physiological niche, but also explains the paradoxical acceleration of bone loss and vertebral fractures observed upon discontinuation of the anti-RANKL therapeutic denosumab [In an elegant study using dynamic real-time intra-vital imaging of live mice, McDonald et al. have shown that multinucleated osteoclasts underwent extensive fission to produce daughter cells, a novel cell state called osteomorphs, with the ability to fuse back with the parental network of osteoclasts, a process termed osteoclast recycling . Using senosumab –32 widelenosumab  and impoenosumab , 35.Changes within the bone niche during cancer growth and progression, as well as in stress conditions, were also investigated using scRNAseq. Baryawno et al. showed in a murine model of acute myeloid leukemia (AML) that cancer cell growth in bone led to a decrease in mature osteoblasts and a concurrent increase in preosteoblasts, with early MSC numbers remaining stable . This fiThe main limitation of scRNAseq approaches is the loss of spatial context of cells in the tissue being studied. To overcome this issue, spatially resolved transcriptomic imaging approaches have been developed, albeit with limited application currently in bone research. Incorporating laser capture microdissection and bulk RNAseq, Baccin et al. captured and sequenced cells from the arteriolar, sinusoidal and endosteal regions of the bone . Using tRecent scRNAseq studies have unravelled the complex heterogeneity of cell types present on the endosteal bone surface and bone marrow in unprecedented detail. This has provided valuable insights into long-standing knowledge gaps in the field, including the different cell states of osteogenic and osteoclastic cells, as well as the identities of MSCs. scRNAseq studies also provided evidence of the key role bone niche plays in disease states such as cancer, identifying potential targets amenable to therapeutic intervention.Despite these advances, there are still limitations in scRNAseq studies with key questions that remained unanswered. The unsupervised nature of clustering cells in scRNAseq analysis involves manual cluster annotation, which limits the reproducibility of the results. This is exemplified by conflicting differential hierarchies of many cell types between studies which led to distinct conclusions despite highly comparable data . Global In conclusion, scRNAseq is a promising tool for the charting of a comprehensive map of the cellular landscape in bone and their molecular processes, paving the way to the development of novel cell-based therapeutic targets for bone diseases.


The taxane family of compounds, including Taxol/paclitaxel and Taxotere/docetaxel, are surprisingly successful drugs used in combination or alone for the treatment of most major solid tumors, especially metastatic cancer. The drugs are commonly used in regimen with other agents (often platinum drugs) as frontline treatment, or used as a single agent in a dose dense regimen for recurrent cancer. The major side effects of taxanes are peripheral neuropathy, alopecia, and neutropenia, which are grave burden for patients and limit the full potential of the taxane drugs. Especially in the current treatment protocol for peripheral neuropathy, taxane dosage is reduced once the symptoms present, resulting in the loss of full or optimal cancer killing activity.Substantial efforts have been made to address the problem of cytotoxic side effects of taxanes, though strategies remain very limited. Following administration of the taxane compound by infusion, taxane binds to cellular microtubules and is sequestered within the cells for several days. Taxane stabilizes and interferes with microtubule function, leading to ultimate death of cancer cells, but also damages hair follicles, peripheral neurons, and hemopoietic stem cells. Currently, cryo-treatment is practiced to limit exposure and side effects of the drug during infusion, though the effectiveness is uncertain or limited.A recent laboratory finding may provide a new strategy to counter taxane cytotoxicity, that a brief exposure to low density ultrasound waves was sufficient to eliminate paclitaxel cytotoxicity cells in culture by transiently breaking microtubule filaments, which were then relocated to lysosomes for disposal. Thus, ultrasonic force to break rigid microtubules is an effective solution to counter taxane cytotoxicity. The discovery and concept of low intensity ultrasound as an antidote may have the potential to provide a practical strategy to counter paclitaxel-induced peripheral neuropathy and alopecia that resulted from chemotherapy.Taxanes are a class of important drugs used in chemotherapy to treat several major cancers. This article reviews a new laboratory discovery that ultrasound can be used as an antidote for the peripheral cytotoxicity of taxane drugs and discusses the potential development and application of low intensity ultrasound to prevent side effects in chemotherapeutic treatment of cancer patients. Paclitaxel (brand name Taxol) is a key drug in the current treatment of several major solid tumors, including ovarian cancer Although generally paclitaxel is highly effective with tolerable side effects, several key side effects include peripheral neuropathy, neutropenia, and alopecia Taxus brevifolia) Paclitaxel, the first Taxane class of compounds, is a surprisingly successful anti-cancer drug The traditional view, largely based on studies of paclitaxel on cancer cells in culture, is that paclitaxel binds and stabilizes microtubules, and the key consequences relevant to its efficacy is mitotic inhibition and ultimately apoptosis However, alternative opinions of a minority contend that paclitaxel also kills cancer cells by a non-mitotic mechanism Nevertheless, the observations are consistent that microtubules are the specific and relevant drug targets Fig. 1). This proposed mechanism offers a possible alternative explanation for the well-established dogma that paclitaxel targets mitosis in cancer therapy; rather, paclitaxel likely aims at the weakened nuclear envelope of malignant cells. The study provides a new realization that paclitaxel can induce the generation of micronuclei in cells at S-phase by a non-mitotic mechanism Generally, in the presence of paclitaxel and its interference on microtubule function, the formation of multiple micronuclei likely results from aberrant, multipolar mitosis Another observation is that a brief treatment of ovarian cancer cells with paclitaxel induced the formation of rare cells with enlarged and deformed nuclei, likely a result of suppression of mitosis but continuous genomic replication The pharmacokinetic properties of paclitaxel in human patients have been documented Tumor cells and normal tissues take up and sequester abundant drugs into cells during the infusion, at several hundreds of times the concentration found in the extracellular space Fig. 2). Although it seems to have not been emphasized, this unique property of intracellular paclitaxel retention is likely an important factor for the success of paclitaxel as an anti-cancer drug.In laboratory studies, paclitaxel was found to be concentrated several hundred-fold into cultured cells Microtubules are polymers of alpha- and beta-tubulin heterodimers, and play multiple roles in cellular functions Tubulins are relatively stable, and the tubulin protein is removed by proteasome- (but not lysosome-) mediated degradation Fig. 3). Both neutropenia and peripheral neuropathy are often the dose limiting factors in cancer treatment using taxanes The common side effects of taxane chemotherapy are neutropenia, peripheral neuropathy, and alopecia Neutropenia is the principal dose-limiting toxicity of paclitaxel with an early onset (around day 8), when neutrophil counts drop by day 8 to day 11, followed by rapid recovery on days 15 to 21 Another highly proliferative cell type is the matrix cells of the hair follicles. Hair follicles undergo cycles of growth (anagen), regression (catagen), and relative quiescence (telogen) throughout life Fig. 3). Extensive laboratory research using cell systems, nonmammalian animal models, and rodent models has been attempted and is ongoing to understand the complex mechanisms of paclitaxel in causing peripheral neuronal damage Sensory neuropathy, presenting as numbness and pain of feet and hands, is often the dose-limiting toxicity of the taxane agents Oncologists have invested substantial research effort to find strategies to counter the side effects and fully realize the power of taxanes in the treatment of a wide range of cancer types The underlying pathobiology of paclitaxel chemotherapy-induced alopecia remains poorly understood. Recently it was shown that paclitaxel induces massive mitotic defects and apoptosis in transit amplifying hair matrix keratinocytes and also severely damages epithelial stem/progenitor cells in the bulge and outer root sheath of human scalp hair follicles In vitro studies indicate that paclitaxel causes neurite retraction and neuron cell degeneration, leading to the idea that paclitaxel targeting of axon microtubules is the cause of neuronal retraction and peripheral neuropathy In addition to neutropenia, which is often transient and readily recoverable, severe peripheral neuropathy is accumulative and is often the dosage limiting factor in treatment using taxane drugs 2O2, and subsequent activation of MMP-13, are involved in neurite degeneration Many studies and various proposed mechanisms have been reported Despite their recognized importance and the extensive efforts devoted to the issues related to paclitaxel side effects, to date, few approaches are practical and available to counter taxane side effects in chemotherapy Currently, no satisfactory methods are available to reverse the side effects of paclitaxel, though cooling of hands and feet to limit drug exposure has been studied as a possible strategy to limit peripheral neuropathy of hands and feet The cooling needs to be maintained during the entire paclitaxel infusion period, generally 3 to 6 hours The new discovery that low intensity ultrasound is an antidote of paclitaxel cytotoxicity 2) and high frequency (such as 50 MHz) is used for diagnostic (imaging) purposes. High intensity  ultrasound that can deliver strong energy is used for surgery and disruption through heating and acoustic cavitation. The medical application of ultrasound with an intensity that is low yet sufficiently high to produce biological activity is known as ultrasound physiotherapy 2). The most commonly used devices produce ultrasound waves with frequencies either around 1-3 MHz or 20-150 kHz (known as long wavelength ultrasound). Similar effects by either 1-3 MHz or 45 kHz ultrasound waves on cells and tissues were reported in several studies Ultrasound technologies have extensive applications in medicine, either for diagnosis (sonogram) or therapy 2) alone, for up to 10 minutes, had no noticeable impact on cultured cells, it actually reversed the cytotoxicity of paclitaxel in cancer cells Fig. 4) Fig. ), but the ultrasound exposure completely eliminated paclitaxel cytotoxicity  Fig. 4).A serendipitous discovery is that ultrasound can eliminate paclitaxel cytotoxicity in cells. Although exposure to low intensity ultrasound  Fig. 4).Based on these results, a concept was developed that ultrasound reverses cytotoxicity by disrupting rigid microtubule filaments induced by paclitaxel treatment of proliferative cells . This may reflect the underlying issue in peripheral neuropathy, which is thought to be caused by the impact of paclitaxel on the dynamics of the axonal long microtubule structure, presumably altering the distribution and structure of the peripheral neuron network and the transmission of nerve stimuli Fig. ). The paclitaxel bound microtubule fragments undergo lysosomal degradation. Additionally, paclitaxel is rapidly (8-24 hours) eliminated in the environment by binding to and being sequestered by cellular microtubules (about 10-20 µM in cells) In chemotherapy, paclitaxel also causes stabilization and bundling of microtubules in peripheral neuronal cells, and consequently leads to retraction . The human scalp is a strong barrier for the penetration of low frequency ultrasound energy Fig. ). The bath device produces 45-150 kHz, 1-3 W/cm2 ultrasound waves with adjustable frequency and energy levels. The probe device produces adjustable 1-3 W/cm2 and 1-3 MHz ultrasound waves.Since ultrasound treatment is considered a safe procedure, potential clinical development is eminently feasible. Ultrasound devices suitable to apply low intensity shock waves to the skin surface and hair follicles may be designed, and water may be suitable to be used as an ultrasound-transmitting medium (2) over just a few hours (e.g. 4 to 10 hours) following chemotherapy . Possibly, a second ultrasound treatment may be applied 24 hours after chemotherapy to ensure complete localized elimination of paclitaxel cytotoxicity  . The optimal timing of ultrasound exposure to reverse paclitaxel activity may be the window between the time when free paclitaxel is depleted, and the time required for causing damage and harm by the paclitaxel-bound rigid microtubules . Thus, ultrasound treatment appears to disrupt the persistent paclitaxel-bound rigid microtubules and thereby eliminate the prolonged cytotoxicity of paclitaxel.Paclitaxel is administrated through IV over a 4- to 6-hour time course. Upon completion of drug infusion, blood paclitaxel rapidly declines, and the drug enters and accumulates within cells Newer microtubule stabilizing drugs and delivering platforms, such as Abraxanes, cabazitaxel, epothilones (ixabepilone), laulimalide, and discodermolide, etc., are under development and coming into clinical application The application of ultrasound to counter paclitaxel-induced peripheral neuropathy seems to be very feasible and practical. In fact, probe ultrasound has been used in clinical trials to treat paclitaxel-induced peripheral neuropathy Paclitaxel (and other taxane drugs) binds and stabilizes microtubules, while ultrasound breaks microtubules, thus acting as an antidote. The mechanism may be quite self-explanatory. Based on our recent discovery that low intensity ultrasound treatment can effectively and almost completely neutralize the cytotoxic effects of paclitaxel in various cell types, a strategy may be developed to counter the side effects of paclitaxel-based chemotherapy for cancer patients. The concept that a brief exposure of paclitaxel-treated cells to ultrasound is sufficient to break paclitaxel-induced rigid microtubules opens up the possibility to use ultrasound locally to eliminate cytotoxicity at only the desirable anatomic sites, without affecting paclitaxel activity towards neoplastic cells. In essence, ultrasound is able to remove the persistent activity of paclitaxel at intended areas after systematic drug infusion. The possibilities to use this paclitaxel antidote can be contemplated to prevent chemotherapy-induced alopecia, and peripheral neuropathy, which will ensure the full use of paclitaxel dosage in treatment and improve the quality of life for cancer patients.


Notch signalling is an evolutionarily highly conserved signalling mechanism governing differentiation and regulating homeostasis in many tissues. In this review, we discuss recent advances in our understanding of the roles that Notch signalling plays in the vasculature. We describe how Notch signalling regulates different steps during the genesis and remodelling of blood vessels (vasculogenesis and angiogenesis), including critical roles in assigning arterial and venous identities to the emerging blood vessels and regulation of their branching. We then proceed to discuss how experimental perturbation of Notch signalling in the vasculature later in development affects vascular homeostasis. In this review, we also describe how dysregulated Notch signalling, as a consequence of direct mutations of genes in the Notch pathway or aberrant Notch signalling output, contributes to various types of vascular disease, including CADASIL, Snedden syndrome and pulmonary arterial hypertension. Finally, we point out some of the current knowledge gaps and identify remaining challenges in understanding the role of Notch in the vasculature, which need to be addressed to pave the way for Notch-based therapies to cure or ameliorate vascular disease. From the discoveries by William Harvey in the 1600s of the circulatory vascular system and its two major branches (the systemic and pulmonary circulation), we have been intrigued by the complexity of the vasculature, and there has been a continuous quest to unravel the mechanisms that generate and maintain our blood vessels. Progress was first made at the anatomical level, leading to a detailed characterization of the vasculature down to its finest capillary branches [. 2The Notch signalling pathway operates in most, if not all, multicellular species, and controls cell differentiation through cell–cell communication. The molecular mechanism for transmitting the Notch signal from the cell surface to the nucleus has been extensively reviewed –4 and wi. 3a). In parallel with the initial formation of a vasculature in the embryo proper, extraembryonic vasculogenesis occurs in the yolk sac, where the vasculature is built from ECs derived from haemangioblasts, which also give rise to haematopoietic cells [a).The vascular system originates through a process called vasculogenesis, which is characterized by de novo formation of blood vessels early in embryogenesis. Derived from mesodermal cells, endothelial progenitor cells (angioblasts) form a primitive vascular network (a plexus) starting around embryonic day (E) 7.5 in the mouse embryo. The early coalesced endothelial cells (ECs) become coated with mural cells (see below) and undergo a subsequent remodelling and differentiation into arteries, veins and capillaries  figure a. In paric cells . The haeb).Vasculogenesis is followed by a process called angiogenesis, which is defined as the generation of new blood vessels from the basic vascular network laid down during vasculogenesis. An important aspect of angiogenesis is the sprouting of new vessels from pre-existing ones. In the early vasculature, sprouting is executed by so-called tip cells, which take the lead in growing the new vessels and which are followed by stalk cells that form the lumen of the new vessel  figure b.a). The heart is formed from two principal founding cell populations: the first (primary) heart field, which gives rise to both atria and the left ventricle, and the second heart field, which generates the right ventricle and the outflow tract. The early heart is a three-layered structure, composed of an inner endocardium, a myocardium in the middle and an outer epicardium. Branching from the aorta, the coronary arteries wrap around the heart and provide the heart with blood circulation (figure 3a).A central component of the vascular system is the heart, which is the first functional organ to form in the embryo . Cardiogfigure 3b). The uptake of fluid into the lymphatic vessels is conducted via permeable junctions in the lymphatic ECs [In addition to the blood-transporting vasculature, the lymphatic vasculature drains plasma and proteins that are extravasated from the interstitium b. The upatic ECs , and theAt the cellular level, the vasculature contains ECs that form the inner layer, which is surrounded by mural cells: vascular smooth muscle cells (VSMCs) and pericytes. VSMCs cover larger vessels, i.e. elastic and muscular arteries, arterioles and veins, while pericytes cover the thinnest vessels, i.e. the capillaries and venules . The denIn addition to serving as a conduit system for nutrients, oxygen and waste products, the vasculature plays an important role in maintaining blood pressure through the regulation of vascular tone, which is achieved by a balance between vasoconstrictor and vasodilator signals. The vascular endothelium is able to sense haemodynamic changes and to respond via the release of vasoconstrictors and vasodilators . VSMCs r. 4. 4.1Notch signalling is important from the onset of vasculogenesis, as witnessed by early embryonic vascular phenotypes in mice where genes in the Notch pathway have been genetically ablated. Thus, targeting of Hey1 and Hes1 , Notch1 Notch gene targeting experiments furthermore revealed a role for Notch signalling in arterial versus venous fate specification: loss of Notch signalling led to reduced arterial endothelial differentiation at the expense of enhanced venous endothelial differentiation, and considerable progress has been made in decoding the underpinning molecular principles. Arterialization is mediated by Notch signalling acting epistatically over the Eph/ephrin signalling pathway, which is an important regulator of vascular cell fate specification . SpecifiAlthough the initial steps of arterial versus venous differentiation occur prior to differences in blood flow between arteries and veins have been established, it has been extensively discussed whether vascular flow (haemodynamics) is important for Notch signalling and that a higher pressure at the arterial side may contribute to arterial specification. Shear stress indeed upregulates vascular Notch signalling ,33, suggLymphatic vessels are not formed through vasculogenesis followed by angiogenesis. Instead, lymphatic ECs are generated from venous ECs during embryogenesis . This tr. 4.2b). Notch signalling and VEGF are important arbiters of the tip/stalk specification process. Sprouting is initiated by elevated VEGF levels (b), which activate the ERK pathway and upregulate the expression of Dll4 in the prospective tip cells [cis-inhibitory for Notch receptors in the tip cells, which reduces Notch signalling, promoting a higher level of ERK signalling in the tip cells [trans, i.e. on the tip cells [Drosophila melanogaster or Caenorhabditis elegans. During lateral inhibition, high levels of Notch ligand expression in one cell in a relatively homogeneous cell population activate Notch signalling in the neighbouring cells, forcing them to not take on the same cell fate as the cell with high ligand expression but opt for an alternative differentiation fate; for a review see [During angiogenesis, new blood vessels sprout from the vessels in the existing initial plexus, a process mediated by tip and stalk cells in the growing vasculature b. Notch F levels b, which ip cells . Integriip cells . The eleip cells ,43. Dll4ip cells ,45. Jaggip cells ; for a rip cells . A role ip cells . The cheip cells . In miceip cells . Notch sip cells . The funview see .−/− background resulted in effects in the vasculature of long bones but not in the intestine or retina [−/− background or targeting of ADAM10 (which executes S2 cleavage of the Notch receptor) led to enlarged subepicardial vessels and loss of mural cell ensheathment [While the role of Notch signalling in the tip/stalk segregation process appears to be general, the consequences of perturbing endothelial Notch signalling differ between different organs , which is caused by NOTCH3 mutations (see below) [The initial endothelial vasculature needs to be coated with mural cells (VSMCs and pericytes), and both platelet-derived growth factor (PDGF) and Notch signalling are important for mural cell coating. The PDGF signalling pathway plays an important role in the differentiation, proliferation and recruitment of mural cells to the endothelium. The PDGFB ligand is produced and secreted from the endothelium, promotes mural cell differentiation from mesenchymal progenitor cells and serves as an attractant for mural cell investment on the blood vessels ,60. Murae below) –65. In ze below) .−/− mice [−/− mice (see below for details). In subsequent studies, it was observed that Notch signalling executes a VSMC function, in part via regulation of PDGF signalling: Notch3 promotes PDGFRB expression and PDGFRB mRNA levels were reduced in the Notch3−/− mice [L1519P) elevated PDGFRB expression in infantile myofibromatosis, a non-metastatic cancer formed in bone, skin and muscle [Little is known about the role of Notch signalling at the earliest steps of VSMC differentiation from mesenchymal progenitor cells, but Notch3 is critically important for VSMC maintenance, as VSMCs are progressively lost at later postnatal stages in Notch3−/− mice ,67. In a−/− mice , a shift−/− mice . The disd muscle . Progresd muscle .. 5In addition to pivotal roles in the development of the vasculature, vascular Notch signalling is important at later stages, and it is increasingly realized that vascular Notch signalling plays a role as a regulator of homeostasis in the postnatal vasculature. As discussed above, ablation of CSL or Notch1 in postnatal ECs leads to enlarged vessels, dilated sinusoids and disorganized vascular architecture in the liver, accompanied by reduced levels of ephrin B2 . Analysilo monocytes) [per se but also has dramatic effects on other cell types and important physiological processes in various organs. To molecularly decode such ‘beyond vasculature' effects of vascular Notch perturbation is important, not least as a basis for future therapy development, as the effects relate to processes that are central from a disease perspective, such as metabolism and immune regulation.Perturbed vascular Notch signalling can also have ‘spillover’ consequences for the homeostasis of surrounding organs and non-vascular cell types. Notably, Dll4 expression on sinusoidal ECs plays a role in the differentiation of Kupffer cells (resident liver macrophages) . Ablationocytes) . Postnatnocytes) . Fischernocytes)  providednocytes) . In a sunocytes) , indicatin vitro studies corroborate that Notch signalling promotes the synthetic phenotype [in vivo, is warranted, in particular as one report identified a VSMC switch towards a synthetic phenotype in coronary vessels from Notch3−/− mice [−/− mice showed aberrant vascular tone regulation in distal resistance arteries (such as the brain and tail arteries), but not in large arteries (such as carotid arteries) [−/− mice were largely insensitive to vasoconstrictors and vasodilators, suggesting that Notch3 is critically required for the adaptive vasoactive response [As discussed in the previous section, under pathological or stress situations, VSMCs may switch from a contractile to a synthetic phenotype, and several lines of evidence support the notion that Notch signalling is an important regulator of the balance between the contractile and synthetic phenotypes  . Similarresponse .−/− mouse, while there was a considerable loss of VSMCs [−/− mice [The role of Notch for pericyte maintenance during vascular homeostasis is currently more enigmatic. One report observed that pericyte numbers were relatively unaffected in the Notch3of VSMCs . By cont−/− mice . Similar−/− mice ,90. More. 6N3G73A promoted aggregation [in vitro [Considering the important role of Notch signalling in vascular development and homeostasis, it is logical that dysfunctional Notch signalling has been observed in a number of vascular diseases . Mutatioregation , but, inin vitro .Figure A1604T and NOTCH3L1515P) have been identified in patients with cerebral small vessel disease and white matter lesions [A1604T mutation correlated with migraine and white matter lesions in a family, although of a different kind from that which is typical for CADASIL, and was hypomorphic with regard to signalling [L1515P mutation, in contrast, resulted in hyperactive Notch signalling and correlated with white matter hyperintensities and headache [L1519P mutation (located four amino acid residues away from the NOTCH3L1515P mutation) instead presented with infantile myofibromatosis (see above), although the NOTCH3L1519P receptor, like the NOTCH3L1515P receptor, produced hyperactive Notch signalling [One may also ask whether cysteine-sparing NOTCH3 mutations, irrespective of their ability to generate GOM or not, can give rise to neurological or vascular problems simply by altering the signalling output. Two cysteine-sparing NOTCH3 missense mutations with aberrant signalling output  in patients with childhood PAH [Pulmonary arterial hypertension (PAH) has been linked to dysfunctional Notch signalling  is a disease in which the ductus arteriosus fails to close after birth. The VSMC-specific elimination of Jagged1  or the cN-acetylglucosamine transferase gene (EOGT), which encodes an enzyme that post-translationally modifies Notch receptors [In addition to CADASIL, PAH and cardiovascular diseases, the list of diseases with a vascular component and potential links to Notch dysregulation keeps growing . Gene fueceptors . Patienteceptors .. 7With the increasing number of diseases linked to aberrant Notch signalling, there is an obvious need to develop Notch-based therapies, to activate or dampen Notch signalling, depending on the disease situation at hand. Notch therapy development has been vigorously pursued for many years, and although interesting and promising preclinical data are mounting, there are still no Notch-specific therapies in regular clinical use ,141. In L1515P and NOTCH3L1519P mutations, which cause cerebral small vessel disease and infantile myofibromatosis, respectively, generate mutated hyperactive receptors, which, however, do not reach the cell surface [In addition to Notch inhibitors, there will also be a need for agents that can enhance Notch signalling, for use in situations where endogenous Notch signalling is hypoactive. As discussed above, some CADASIL mutations may fall in this category, and an activating Notch3 antibody was indeed used in a CADASIL mouse model to restore some of the observed phenotypes . It shou surface ,102; thu surface , and one surface  and used. 8Notch signalling plays a central role in both the development and homeostasis of the vasculature and regulates ECs as well as mural cells. The effects of Notch signalling range from balancing sprouting and branching of the vasculature via regulation of the tip/stalk balance in ECs to control arterial versus venous differentiation of mural cells and the balance between contractile and synthetic phenotypes in homeostasis, stress and disease. The number of vascular diseases that are caused or influenced by dysregulated Notch signalling is increasing and warrants efforts of developing Notch-based therapies. Despite recent progress, there are, however, several outstanding questions that remain to be answered, including how Notch produces appropriate downstream molecular outputs in the different cell types, given the relatively simple architecture of the pathway. Progress in this area is anticipated, not least because of rapid technological improvements and scalability of single-cell transcriptomics combined with increasingly precise transgenic models in both mouse and zebrafish. Detailed transcriptomic maps are beginning to be produced of the vasculature in different organs ,152–154,


Rising income inequality is strongly linked to health disparities, particularly in regions where uneven distribution of wealth and income has long been a concern. Despite emerging evidence of COVID-19-related health inequalities for adults, limited evidence is available for children and their parents. This study aimed to explore subtypes of families of preschoolers living in the disadvantaged neighborhoods of Hong Kong based on patterns of family hardship and to compare their patterns of parenting behavior, lifestyle practices, and wellbeing during the COVID-19 pandemic. Data were collected from 1338 preschoolers and their parents during March to June 2020. Latent class analysis was performed based on 11 socioeconomic and disease indicators. Multivariate logistic regressions were used to examine associations between identified classes and variables of interest during the COVID-19 pandemic. Four classes of family hardship were identified. Class 1 (45.7%) had the lowest disease and financial burden. Class 2 (14.0%) had the highest financial burden. Class 3 (5.9%) had the highest disease burden. Class 4 (34.5%) had low family income but did not receive government welfare assistance. Class 1 (low hardship) had lower risks of child maltreatment and adjustment problems than Class 2 (poverty) and Class 3 . However, children in Class 1 (low hardship) had higher odds of suffering psychological aggression and poorer physical wellbeing than those in Class 4 (low income), even after adjusting for child age and gender. The findings emphasize the need to adopt flexible intervention strategies in the time of large disease outbreak to address diverse problems and concerns among socially disadvantaged families. Family hardship is a multidimensional concept describing material conditions, social position, and economic position that are below the minimally acceptable level ,2. FamilIn terms of approaches to capture the heterogeneity in the family hardship profile, multidimensional approaches that analyze different indicators to determine the most appropriate set of group memberships have several advantages over unidimensional approaches. For example, one multidimensional approach is to first assign a weighting factor to each indicator based upon either expert opinions or through principal component analysis and then aggregate the indicators as a composite socioeconomic status (SES) index . HoweverSince December 2019, the coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on communities and people across the world. A growing body of research has demonstrated the direct effect of transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on health and wellbeing . The panHong Kong has a high Gini coefficient  of 0.539 in 2016 , denselyThis study surveyed families of preschoolers living in two districts (Sham Shui Po and Yuen Long) of Hong Kong during March to June 2020  when face-to-face classes were suspended . We chosSpecifically, we identified 32 under-resourced kindergartens from a total of 137 kindergartens in both districts using a grading checklist that evaluated (1) teacher qualification(s) and teaching experience; (2) teacher/student ratio; (3) school campus total area, classroom size, outdoor space, play areas; (4) teaching facilities, including the reading corner, books, toys, and number of available electronic devices; (5) school fee; (6) socio-economic background of students reflected by the percentage of families (i) receiving government social-security assistance, (ii) receiving a school fee remission, and (iii) having parents with a low education background; and (7) additional school support, including subsidies, financial, social service, and training. The overall percentage of preschool students receiving a school free remission  in Hong Kong was 14% at the time of this study, whereas the percentage for our selected kindergartens was 53.1% (ranging from 22.7% to 72.6%). We subsequently invited principals and teachers of 32 kindergartens in these two districts to assist in subject recruitment, and they all accepted our invitation. We offered parents the option to complete the survey either online or through a paper and pen format at home. All the study measures were administered in Traditional Chinese. Upon obtaining school consent, the teachers were instructed to provide either the e-survey link or paper questionnaires to each student depending on their parent’s preference. The e-survey database automatically recorded each response in a safe and secure manner, whereas parent-completed paper questionnaires were collected by schools and subsequently sent to the research office for further processing. According to the information provided by the schools, 5705 parents were invited, and 1338 (20.0%) returned the completed questionnaires. The responding parents were usually the mother (98%), and all were competent at reading and comprehending our questionnaires. This study was approved by the ethics committee of the Institutional Review Board of the Hong Kong University/Hospital Authority Hong Kong West Cluster (Reference UW 20–177). Informed consent was obtained from the parents of all participating families.Based on evidence from the previous literature and their relevance to the local context , we inclChild maltreatment episodes in the preceding three months were reported by the parents using the Parent-Child Conflict Tactics Scale (CTS-PC) . The CTSChild psychosocial problems during school closure were measured using the parent version of the Strength and Difficulties Questionnaire (SDQ) for 2- to 4-year-olds , which hParenting stress during school closure was self-reported by the parent using the 17-item Parental Stress Scale (PSS) , which mChild physical wellbeing during school closure was reported by the parent using the physical functioning scale of the Pediatric Quality of Life Inventory™ 4.0 generic core scales (PedsQL™) , which hThe parent was asked to report the weekly frequency of recreational and learning activities with their children during school closure using the Chinese Parent-Child Interaction Scale (CPCIS), which was developed in Hong Kong and demonstrated good psychometric properties among local preschoolers . The CPCChildren’s lifestyle practices were measured in three aspects, namely exercise, sleep, and screen use (for learning or gaming purpose). The parent answered the question, “how long (in hours) did your child perform [the activity of interest] during a weekday/weekend?” The average amount of daily time spent on each activity was calculated by averaging the parent proxy-reported amount of time spent on the activity of interest during weekends and weekdays using the weighted average formula ([2 × weekend + 5 × weekday] ÷ 7).Child sex  and age were reported by the parent. We included these variables as covariates in the regression models.Descriptive statistics for family hardship indicators, parenting and lifestyle practices, demographic characteristics, and wellbeing scores were reported as mean and standard deviation for continuous variables and frequencies and percentages for dichotomous variables upon checking the distribution of data. Missing values on each variable of interest ranged from 0.7% to 60%. In order to minimize the bias attributable to missing information, missing data were imputed at the item level by means of multiple imputation using the MICE package in R . All anamaxiter) through which the estimation algorithm cycled was 3000. To automate the search for the global—rather than the local—maximum of the log-likelihood function, the number of repetitions (nrep) was set to 30. Multinominal logistic regressions were conducted to generate an effect estimate, odd ratio (OR), for the association of membership in different latent classes with child maltreatment variables, whereas generalized linear regressions were used to generate an effect estimate, unstandardized regression coefficient (β), for the association of class membership with lifestyle practices and wellbeing scores. Analyses were adjusted for possible confounding effects of child age and gender. Statistical significance was set at a 2-sided p < 0.05. LCA was conducted using the R package “poLCA”, which estimates model parameters with maximum likelihood techniques using expectation-maximization and Newton–Raphson algorithms . The optThe total sample comprised 1338 preschoolers, with 790 (59%) completing the paper survey and 548 (41%) completing the e-survey . The meaFive latent class models were estimated, and their fit indices are presented in p = 0.013), faced higher parenting stress , displayed more externalizing  and internalizing problems , and enjoyed fewer parent-child learning  and recreational activities . On the other hand, compared to Class 1 children, Class 3 children were more likely to have psychological regression experiences , showed more externalizing problems , and spent less time exercising . Lastly, compared to Class 1 children, Class 4 children were less likely to have psychological aggression experiences , faced higher parenting stress , had better physical wellbeing  and longer daily sleep duration , and enjoyed fewer recreational activities .Results of regression analyses  indicateThis study demonstrated the feasibility of using sociodemographic and disease characteristics of parents and children as a basis for the classification of family hardship subtypes. We identified four distinct and interpretable subtypes of family hardship based on 11 socioeconomic and disease indicators. The identified family classes were labeled as low hardship, poverty, poor health, and low income, respectively. The results highlight the severity of wealth and income disparities among socially disadvantaged families in Hong Kong. In addition, there were notable differences in parenting behavior, lifestyle practices, and wellbeing across these family classes. This investigation extends previous findings by showing that the poorest of the poor are most vulnerable to the effects of disease outbreaks and disasters . low hardship class had lower risks of being neglected than those in the poverty class, and lower risks of being psychologically aggressed than those in the poor health class. However, when compared to those in the low income class, children in the low hardship class had higher odds of being psychologically aggressed, even after adjusting for child age and gender. A possible explanation is that when compared to the low income class, parents in the low hardship class may spend more time with children at home during the pandemic, thereby increasing the likelihood of parent-child conflicts. In this study, although all children were living in disadvantaged neighborhoods, the low hardship and low income classes differed in the aspects of family income and parental education level. A previous study has posited that the pandemic not only exacerbates pre-existing social inequalities but also creates new forms of disparities [poverty and low income classes have a relatively high probability of having low family income, but the poverty class, by comparison, has a more disadvantaged profile characterized by high probabilities of having government welfare assistance, single parent, and less educated/sick father. It is possible that such a profile, rather than low income per se, accounts for the higher risk of child neglect in the poverty class than the low hardship class. Previous studies have shown that family poverty and parental depressive symptoms were associated with child neglect and psychological abuse ,39. Echoparities . Buildinpoverty class experienced higher levels of children’s externalizing and internalizing problems and greater parenting stress compared to those in the low hardship class, suggesting that adjustment difficulties could be among the factors associated with the increased occurrence of child neglect in poor families under this pandemic. On the other hand, parents in both poor health and low hardship classes had similar stress levels, but children in the poor health class exhibited significantly higher levels of externalizing problems, suggesting that children’s behavioral issues could also be a possible reason for the occurrence of psychological aggression in families with a high disease burden under the COVID-19 pandemic. As shown in previous research [Consistent with previous findings about the association between poverty and poor health ,42, thisresearch , parentslow income class than the low hardship class, children in the low hardship class were more likely than those in the low income class to suffer psychological aggression. This could be because our data captured parenting stress but no other types of stress. For example, the better-off families may experience decreases in income or net profit during the pandemic [low hardship class were found to have poorer physical well-being than those in the low income class, suggesting that children in better-off families are also vulnerable to the physical health impact of the pandemic.Interestingly, although parenting stress was higher in the pandemic , which, poverty and low income classes and lower levels of parent-child learning activities in the poverty class relative to the low hardship class are consistent with the current literature regarding social deprivation that could result from physical distancing measures [low income class relative to those in the low hardship class suggests that children in better-off families could have a higher risk of inadequate sleep during school closure. We also observed shorter daily exercise duration among children in the poor health class compared to those in the low hardship class, indicating that families with a high disease burden need more guidance or motivational resources to engage young children in physical activities during the pandemic. Previous research has reported that people with mental health problems or disabilities are more affected by the pandemic [In addition, we explored the activity patterns and lifestyle practices of children in different family hardship classes. The findings of lower levels of parent-child recreational activities in both the measures  and provpandemic . Public This study has several limitations that should be considered when interpreting the results. First, all data were based on parent self-report or proxy-report questionnaires, and some indictors had a relatively large proportion of missing values. Although multiple imputation was used to fill in the missing values, the uncertainty of parameter estimation cannot be ruled out. Future research should ascertain the associations observed in this study using multiple data sources, such as multiple informant reports or electronic health records, to enhance data availability and reliability. Second, it is important to note that although families were recruited from disadvantaged neighborhoods, they were not representative of the whole Hong Kong population. Therefore, caution is needed when generalizing our results to other populations. Third, due to the cross-sectional design, we have no pre-pandemic data on the variables of interest, and thus we cannot conclude whether the observed variations in parenting and lifestyle practices and wellbeing between family hardship classes emerged following the pandemic. Fourth, our LCA model focused on socioeconomic and disease characteristics. Other factors, such as academic and job pressures, social support, and access to public services and facilities, could be explored in future studies. poverty) showed higher odds of child neglect, demonstrated more parental and child adjustment problems, and had fewer parent-child activities than Class 1 (low hardship). Class 3  demonstrated similar patterns except lifestyle practices with children having a shorter daily exercise duration relative to Class 1. Class 4 (low family income) had lower odds of psychological aggression towards children but higher parenting stress and fewer parent-child recreational activities, although their children had higher levels of physical wellbeing and slept more than those in Class 1. The findings suggest a need to adopt different intervention strategies to mitigate the COVID-19-related health inequalities among those in poverty and other families in general. The present study identified four classes of family hardship and highlighted their associations with parenting behavior, lifestyle practices, and wellbeing during the COVID-19 pandemic. Class 2 (