Recent mouse model experiments support an instrumental role for senescent cells in age-related diseases and senescent cells may be causal to certain age-related pathologies. A strongly supported hypothesis is that extranuclear chromatin is recognized by the cyclic GMP–AMP synthase-stimulator of interferon genes pathway, which in turn leads to the induction of several inflammatory cytokines as part of the senescence-associated secretory phenotype. This sterile inflammation increases with chronological age and age-associated disease. More recently, several intracellular and extracellular metabolic changes have been described in senescent cells but it is not clear whether any of them have functional significance. In this review, we highlight the potential effect of dietary and age-related metabolites in the modulation of the senescent phenotype in addition to discussing how experimental conditions may influence senescent cell metabolism, especially that of energy regulation. Finally, as extracellular citrate accumulates following certain types of senescence, we focus on the recently reported role of extracellular citrate in aging and age-related pathologies. We propose that citrate may be an active component of the senescence-associated secretory phenotype and via its intake through the diet may even contribute to the cause of age-related disease. INK4A and p21WAF [WAF and p16INK4A, and transit into senescence [Classical cellular senescence was originally defined as an irreversible cell cycle arrest that was distinct from quiescence, terminal differentiation, and apoptosis. This form of senescence could occur following multiple rounds of cell division (replicative senescence ) or folld p21WAF  or EGR2 d p21WAF . In addinescence ,10, suggnescence ,12 but tnescence  for a reCIP1/WAF1 is an early consequence of telomere attrition and/or the induction of genotoxic stress, so some of the heterogeneity seen in senescent fibroblast populations could be due to the rate of progression of each cell towards senescence. Nevertheless, some of the cytokines of the SASP increase to detectable levels in the plasma of older humans and are proposed to be biomarkers of age-related conditions, such as frailty [The SASP is a collection of proteins secreted by a variety of senescent cell types and includes a large number of cytokines, chemokines and immunomodulatory molecules, growth factors, shed surface molecules, and survival factors together with promoters of angiogenesis, fibrosis, and tissue remodeling. The SASPs are to some extent cell type- and mechanism-specific and recent data using single-cell RNA sequencing indicates that the SASP may be temporally and highly heterogeneous in nature even within populations of the same cell type . The evo frailty . This st frailty  and is t frailty . However frailty  and it h frailty .WAF1/CIP1. These exposed DNA ends can be visualized by their association with numerous DNA damage recognition proteins such as ᵞH2.AX and 53BP1 [Replicative senescence occurs at the end of multiple rounds of replication known as the Hayflick limit  and is snd 53BP1 ,25,26.Telomere loss ,28 and tAdditionally, telomeres can contribute to senescence and aging in other ways. More recently it has been shown that telomeres are also especially sensitive to the induction of DNA double-strand breaks by oxidative damage due to their high G:C content and poor DNA repair capacity ,38 as a Terc or the catalytic subunit gene Tert is homozygously deleted and the telomeres allowed to shorten over four generations. Numerous age-related phenotypes ensue including premature greying of the hair, poor wound healing, increased cancer incidence, gut defects, infertility, shortened lifespan, decreased adipose tissue, and hair loss [WRN gene, which encodes the RecQ type DNA helicase for the unwinding of unusual DNA structures. WS has some common features with the Terc−/− mouse [TERT and TERC and hemizygous mutations in the telomerase enzyme component dyskerin (DKC) [Terc +/− mouse than the homozygous knockout mouse [DKC-deficient human fibroblasts have a very short replicative lifespan in vitro [The effects of dysfunctional telomeres on aging and mitochondrial function have mainly been studied in the telomerase-deficient mouse where either the telomerase RNA template gene air loss . Werner’/− mouse . The humin (DKC) . Howeverin (DKC) . Unsurprut mouse . Telomerut mouse  and DKC-in vitro . Howeverin vitro . Other lin vitro .In mice nullizygous for telomerase short telomeres give rise to mitochondrial dysfunction and increased mitochondrial ROS via p53 and peroxisome proliferator gamma co-activator 1 alpha  a phenoHowever, the above epidermal phenotypes are not so dramatic in human skin aging and recent data implicates aged melanocytes as the major senescent cell type in the human epidermis where telomere-associated DNA damage is induced in the keratinocytes by a paracrine mechanism . HoweverVery recently, dietary factors have emerged which may also impact the differences between DC and telomerase-deficient mice as well as offering a potential explanation for some in vitro findings on senescent cell metabolism. Of particular interest has been nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD+), molecules central to normal metabolic function and to the maintenance of a normal redox state within the cell. Decreased NAD+ levels have been implicated in senescence. NAD+ is an important cofactor to sustain glycolytic activity and is necessary for the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. While conversion of glyceraldehyde-3phosphate is accompanied by reduction of NAD+ to NADH further conversion of pyruvate to lactate is accompanied by NADH oxidation. Whether pyruvate is converted into lactate or enters the tricarboxylic acid (TCA) cycle is determined by the pyruvate dehydrogenase complex  but pyruvate can also act as a scavenger of reactive oxygen species such as hydrogen peroxide and contribute to antioxidant defense ,57.NAD+ has been shown to decline in several tissues with age while disruption in its biosynthesis has been implicated in age-related diseases. NR and NMN supplementation in the diet has been shown to be effective in restoring NAD+ levels and slowing down age-associated phenotypes . NAD+ inThe changes in senescent cell metabolism have been extensively reviewed recently  and so oIt has long been known that cancer cells shift their metabolism away from oxidative phosphorylation towards glycolysis even in the presence of atmospheric oxygen and this process is termed ‘aerobic glycolysis’ or the ‘Warburg Effect’ although this process can shift back towards oxidative phosphorylation in cancer stem cells reviewed in . NeverthRAS and BRAF can increase the flux of pyruvate into the TCA cycle and mediate the initiation of senescence by increasing the oxygen consumption rate and redox stress [RAS over-expression and proliferative exhaustion [PDHC (pyruvate-dehydrogenase complex) is the gatekeeping enzyme linking glycolysis to the TCA cycle. This step is regulated by reversible phosphorylation by PDK (pyruvate dehydrogenase kinase) enzymes (PDK1–4) that inhibit its activity. In contrast, dephosphorylation by PDP1 and PDP2 (pyruvate dehydrogenase phosphatase) stimulates PDHC activity. Oncogenes such as x stress . This isx stress . Furtherx stress . Convershaustion . Furtherhaustion . TherefoIn fully senescent fibroblasts cultured in the presence of FBS optimized for the culture of normal human cells, most glycolytic and pentose phosphate pathway (PPP) metabolites and enzymes and their transcripts increase and many TCA metabolites decline. In particular, the enzyme transcripts of the PDHC such as PDKs 1-4, and especially PDK4, are upregulated . In addiIn the two unbiased metabolic screens of senescent oral NHOF-1 oral and IMR90 embryo lung fibroblasts published so far, there are many similarities. Specifically, glucose together with glycolytic metabolites ,60 and pRecently, we introduced the concept of the reverse TCA cycle with respect to cancer cells  and hereAlthough no NADP+ data were available in primary oral fibroblasts or IMR90, all non-proliferating oral fibroblasts have low NADH and a high NAD+/NADH ratio and increased NADPH  arguing However, as emphasized above, dietary factors in both mice and humans together with cell culture conditions may influence phenotypes greatly and more work is required to test the above hypotheses.Whilst it is recognized that the above arguments are based on the only two unbiased metabolomic screens of senescent cells published so far, several changes are common to both. These include an increase in metabolites involved in glycolysis, the PPP and redox homeostasis, and the differences between the screens may stimulate the investigation into such variables and the ability of cells to deal with oxidative damage and the availability of dietary factors. Furthermore, many studies on senescent cells employ at the most two cell lines ,78 and fIn the oral fibroblast screen above we reported that several metabolites specifically accumulated or became depleted in the conditioned medium of senescent fibroblasts on a per cell basis . Whilst INK4A rise [In classical senescence induced by genotoxic stress the levels of total p53, p53 phosphorylation, and transcription factor activity increase  and thisK4A rise . This tyK4A rise . ClassicK4A rise  and a shK4A rise ,60,70.In the absence of pyruvate, another form of senescence involving mitochondrial dysfunction and the depletion of sirtuins (SIRTs) 3 and 5 can be detected termed mitochondrial dysfunction-induced senescence (MiDAS). However, SIRTs 3 and 5 transcripts do not decline in all fibroblast lines following replicative senescence . MiDAS iMiDAS is dependent on mitochondrial DNA and antagonized by NAD+ and its precursors such as NMN , which dThe role of p53 in metabolism  and fibrIn addition to the unbiased analysis of the internal senescent fibroblast metabolome ,70, we aC-mannosyl tryptophan (CMT) is thought to be epigenetically regulated and is associated with lung function, bone mineral density, and low birth weight in twins. Low birth weight in twins is linked to poorer health in later life and CMT may be an important indicator of human health span as well as chronological age . It has Citrate accumulated over 20-fold outside the senescent cells relative to the interior and relative to the controls and this is consistent with citrate being incompatible with glycolysis (see above) and requiring increased export in senescent cells. Citrate accumulates in IMR90, indicative of reduced activity of the ROS-sensitive mitochondrial aconitase ACO-2  but seneCitrate is an important metabolite as it is not only required for the TCA cycle. After export from the mitochondria or import from the extracellular environment, citrate can serve as a substrate for ATP citrate lyase to produce acetyl CoA (reviewed in ). In turThe specific accumulation of EC and a shift to glycolysis following senescence is not observed in all cell types such as mammary epithelial cells undergoing proliferative exhaustion in the absence of irreparable DNA damage . TherefoThe potential role of citrate in age-related pathologies is summarized in SLC13A5/INDY was originally identified as a regulator of lifespan in Caenorhabditis elegans lifespan [drosophila melanogaster [SLC13A5/INDY (mindy) in the liver reveals that mindy mediates type 2 diabetes and several characteristics of the age-related cardiometabolic syndrome including adiposity, non-alcoholic fatty liver, insulin resistance, and mitochondrial dysfunction [mindy is expressed in the adrenal medulla and mindy knockout mice show lowered levels of citrate, catecholamines, and significantly, heart rate and blood pressure [mindy is expressed in neurons and systemic deletion of the mindy gene significantly improves memory performance and motor coordination of mice. Tissue-specific knockout of mindy in the neurons resulted in improved memory whereas liver-specific deletion did not [mindy may have a role in other tissues affecting the last phenotype. The improved memory of mindy-KO mice was associated with increased hippocampal neurogenesis and dendritic spine formation in dentate granule cells, which are well-documented contributors to enhanced memory performance [lifespan  and latenogaster  by mechanogaster ,98,99. Sfunction . In addipressure . These s did not  and neitformance . This isformance . Drugs tformance  and in vformance . Furtherformance ,106.SLC13A5/INDY in humans leads to severe disease such as early infantile epileptic encephalopathy-25/developmental epileptic encephalopathy-25 [+, known to enhance citrate uptake through SLC13A5/INDY expressed in neurons [mindy) and human (SLC13A5/INDY) phenotypes could be due to the blood–brain barrier [mindy knockout in the nervous system argue against this [SLC13A5/INDY expression is strongly associated with obesity, insulin resistance, and fatty liver in humans [However, these exciting findings should be tempered with caution. Firstly, it should also be noted that human SLC13A5/INDY has a much lower affinity for citrate than its mouse counterpart ,108 and pathy-25 ,110 revipathy-25 ,108. Brapathy-25 . This inpathy-25 . Interes neurons , is used neurons . This isnst this . Moreovenst this  and so tn humans . These in humans  where cin humans . Prostatn humans  and chanSLC25A1 and pmCiC having alternative first exons [Citrate is also imported by the bidirectional citrate transporter pmCiC, which is a variant of SLC25A1 directed to the plasma membrane instead of the mitochondrial membrane. This is due to st exons . pmCiC ist exons . The addst exons . pmCiC ist exons  and citrst exons . Therefost exons .We have recently reviewed the role of citrate in the cancer environment ,69. In aANKH gene is also expressed in the prostate and has recently been shown to export citrate in addition to malate, succinate, and phosphate [Prostate epithelial cells secrete high levels of citrate which is thought to aid the energy requirements of sperm . The plahosphate . Our preThere is now compelling evidence that senescent cells are instrumental in a wide range of age-related human pathologies and there is evidence that senescent cells can cause these pathologies in mice. One of the proposed mechanisms is the induction of an array of proteins collectively referred to as the SASP. One of the most popular hypotheses supported by a good body of evidence is that the SASP is induced by inflammatory signals mediated by extranuclear chromatin, irreparable DNA double-strand breaks, and dysfunctional mitochondria. However, there is insufficient evidence to conclude that senescent cells or the SASP cause aging as opposed to age-related disease. Furthermore, there is evidence to suggest that caloric intake can determine the induction of senescence and the SASP. Citrate is such a source of calories, increases in the plasma of humans with chronological age ,84 and i


Profound variations in care intensity of nursing home (NH) residents with advanced dementia exist for NHs within and across hospital referral regions (HRRs). Little is known about how these levels of influence relate. Nationwide 2016-2017 Minimum DataSet was used to categorize NHs and HRRs into 4 levels of care intensity based on hospital transfer and tube-feeding rates among residents with advanced dementia: low intensity NH in low intensity HRR; high intensity NH in low intensity HRR; low intensity NH in high intensity HRR; and high intensity NH in high intensity HRR. We used multinomial logistic regression to identify NH characteristics associated with belonging to each of 4-levels of intensity as compared to low intensity NH in low intensity HRRs (reference). We found high intensity NHs in high intensity HRRs were more likely to be in an urbanized area, not have an dementia unit, have an NP/PA on staff, have a higher proportion of residents who were male, age <65, of Black race, and had pressure ulcers, and relatively fewer days on hospice. Whereas in low intensity HRRs, higher proportion of Black residents was the only characteristic associated with being a high intensity NH. These findings suggest potentially modifiable factors within high intensity HRRs that could be targeted to reduce burdensome care, including having a dementia unit, palliative care training for NP/PAs, or increased use of hospice care. This study underscores the critical need to better understand the role race plays in the intensity of care of NH residents with dementia.


The recent surge in research on cannabinoids may have been fueled by changes in legislation in several jurisdictions, and by approval for the use of cannabinoids for treatment of some chronic diseases. Endocannabinoids act largely, but not exclusively on cannabinoid receptors 1 and 2 (CBR1 and CBR2) which are expressed in the bladder mainly by the urothelium and the axons and endings of motor and sensory neurons. A growing body of evidence suggests that endocannabinoid system constitutively downregulates sensory bladder function during urine storage and micturition, under normal physiological conditions. Similarly, exogenous cannabinoid agonists have potent modulatory effects, as do inhibitors of endocannabinoid inactivation. Results suggest a high potential of cannabinoids to therapeutically ameliorate lower urinary tract symptoms in overactive bladder and painful bladder syndromes. At least part of this may be mediated via effects on sensory nerves, although actions on efferent nerves complicate interpretation. The sensory innervation of bladder is complex with at least eight classes identified. There is a large gap in our knowledge of the effects of endocannabinoids and synthetic agonists on different classes of bladder sensory neurons. Future studies are needed to reveal the action of selective cannabinoid receptor 2 agonists and/or peripherally restricted synthetic cannabinoid receptor 1 agonists on bladder sensory neurons in animal models of bladder diseases. There is significant potential for these novel therapeutics which are devoid of central nervous system psychotropic actions, and which may avoid many of the side effects of current treatments for overactive bladder and painful bladder syndromes. The endocannabinoid system (ECS) consists of several endocannabinoids and their G-protein coupled receptors (GPCRs) together with synthetizing and degradation enzymes which are present in nearly every bodily tissue including bladder and dorsal root ganglion (DRG) neurons . The twoCannabinoids have been recently demonstrated to suppress pain behavior in various rodent models of inflammatory pain, such as arthritis and inflammatory bowel disease . In the In the bladder, CBR1 and CBR2 are expressed in urothelial cells, detrusor muscle and nerve fibres . DespiteCystometry studies demonstrate that endocannabinoids can modulate micturition intervals and threshold pressure, both of which may reflect bladder afferent function. However, their role in healthy bladders is inconsistent, most likely due to differences in particular drug used , route of administration and/or species studied see . For exaSpinal sensory neurons innervating the bladder play a key role mediating both storage and micturition. They are responsible for bladder sensations, ranging from physiological sensation of filling and fullness through to lower urinary tract symptoms such as urgency and pain . The blaclasses of bladder afferents within five major types of afferents (muscular afferents are “in series tension receptors” which respond to bladder distension and contraction and have receptive fields in the detrusor (non-encoding muscular afferents”) and 2) wide dynamic range afferents (“encoding muscular afferents”), which continue to increase firing across the full range of distension including noxious one afferents are sensitive to both low amplitude stretches and to light mucosal stroking  mucosal low-responding afferents which respond weakly to mucosal stroking, but may be activated by hypertonic stimuli  stretch-sensitive mechanoreceptors have been distinguished in the bladder (High-threshold afferents probably represent a heterogenous group of mechanoreceptors. Some have endings in the detrusor (high threshold muscular afferents) (high-threshold “vascular afferents”—have receptive fields associated with blood vessels in the suburothelium (“silentafferents” or “mechano-insensitive afferents” (In many  bladder . They ar bladder . High-thferents)  while hiothelium , similarothelium . Most ofothelium . In addiferents” . During ferents” .in vivo studies in rats. This effect is abolished by CBR1 and CBR2 antagonism, rimonabant and SR144528 , the density of CBR1 on nerve fibres in the suburothelium and detrusor is increased . HoweverIn patients with PBS, the expression of CBR1 in suburothelial, but not detrusor nerve fibres is significantly increased . CBR2 mRAEA levels in rat, but not mouse bladder are increased during bladder inflammation . ImportaIt is important to note that endocannabinoids such as AEA do not only act via CBR1 and CBR2 but can also activate TRPV1 channels, which are pro-nociceptive in the bladder and other tissues . Indeed,Animal models suggest that CBR2 may be a potential target to ameliorate pain and bladder inflammation in cystitis. Selective CBR2 agonists  reduce the inflammation, pain behavior, and urinary frequency associated with cystitis . WhetherChemically related to AEA, palmitoylethanolamide (PEA) is a ligand for PPARα . PEA hasThus, these data demonstrated that both CBR1 and CBR2 agonists are effective in decreasing pain behaviour and bladder hypersensitivity in animal models of PBS. The exact mechanisms of these effects remain to be established. It is still unclear which specific class(es) of bladder afferents contribute the most to analgesic effect of endocannabinoids as well as exact role of immune, urothelial, and other cells within the bladder wall.Despite some discrepancies in the expression of CBR1/CBR2 and the effects of exogenous and endogenous cannabinoids on sensory neurons and micturition in naive bladders and in animal models of OAB and PBS, preclinical research has identified the significant potential of cannabinoids as novel treatments for common bladder disorders. CBR2 agonists and/or peripherally restricted synthetic CBR1 agonists may significantly ameliorate lower urinary tract signs/symptoms in animal models of OAB and PBS. They are likely to lack the CNS psychotropic actions and sides effects of current treatments. Use of FAAH and other inhibitors to elevate the level of endogenous endocannabinoids currently appears less attractive since the action of endocannabinoids is complicated by off-target effects on ligand and voltage-gated channels, low efficacy, and possible side effects. This review has highlighted the paucity of basic research studies evaluating the direct effects of cannabinoids on the bladder sensory neurons. At least eight classes of bladder afferents, with very different roles in sensory signaling have been identified to date. To design better strategies for the treatment of lower urinary tract symptoms, further basic research studies are needed to elucidate the mechanisms of direct effects of endocannabinoids and their synthetic agonists on different classes of bladder afferents. This will help establish the precise mechanisms of their analgesic and anti-inflammatory actions in models of bladder diseases.


Osteoarthritis (OA) is the most prevalent degenerative joint disease and the main cause of pain and disability in elderly people. OA currently represents a significant social health problem, since it affects 250 million individuals worldwide, mainly adults aged over 65. Although OA is a multifactorial disease, depending on both genetic and environmental factors, it is reported that joint degeneration has a higher prevalence in former athletes. Repetitive impact and loading, joint overuse and recurrent injuries followed by a rapid return to the sport might explain athletes’ predisposition to joint articular degeneration. In recent years, however, big efforts have been made to improve the prevention and management of sports injuries and to speed up the athletes’ return-to-sport. Biophysics is the study of biological processes and systems using physics-based methods or based on physical principles. Clinical biophysics has recently evolved as a medical branch that investigates the relationship between the human body and non-ionizing physical energy. A physical stimulus triggers a biological response by regulating specific intracellular pathways, thus acting as a drug. Preclinical and clinical trials have shown positive effects of biophysical stimulation on articular cartilage, subchondral bone and synovia. This review aims to assess the role of pulsed electromagnetic fields (PEMFs) and extracorporeal shockwave therapy (ESWT) in the prevention and treatment of joint degeneration in athletes. Athletes, due to the physical demands necessary for chasing sporting results, put significant stress on their joints and it is usual for them to suffer from articular cartilage defects. Chondral defects are linked to discomfort and physical weakness, the limiting of athletic activity and have been implicated as a potential risk factor in the development of early-onset osteoarthritis (OA).However, cartilage pathology is not always symptomatic: more than half of the asymptomatic athletes have a full-thickness defect .Chondral injuries are often present in sports subjects; the incidence rate in the knee is 36% compared to 16% in the general population .Concomitant ligament instability, misalignment, and previous injury may facilitate chondral lesions.Chondral damage can lead to an excessive load on the subchondral bone and therefore to bone edema, which can manifest itself with painful symptoms and limit sports participation.Over time, bone edema can resolve if adequately treated or can evolve towards bone necrosis , which, however, can also be secondary to vascular pathologies, or towards arthritic evolution.Non-operative strategies aim to improve effective hyaline cartilage regeneration by delivering growth factors or reducing inflammation .Many conservative therapies are available, such as chondroprotective drugs and nonsteroidal anti-inflammatory drugs (NSAID), Hyaluronic-acid or Platelet Rich Plasma (PRP) or staminal cells’ injection or biophysical stimulation.Biophysical stimulation is a non-invasive therapy currently employed in orthopaedics and traumatology practice to enhance the reparative abilities of the musculoskeletal system. Biophysical stimulation refers to the application of physical energy to a biological system to increase and facilitate tissue regeneration and anabolic activity .Biophysical stimulations act mainly at the level of the cell membrane. It plays a fundamental role in recognizing and transferring the physical stimulus to the various intracellular signalling pathways.Several types of non-invasive electrical stimulation devices have received US FDA approval for orthopaedic application and are classified into: electrical energy applied directly to the tissue by adhesive electrodes , ultrasound energy (low-intensity pulsed ultrasound system LIPUS) and electromagnetic energy applied by coils  or extracorporeal shock wave therapy (ESWT) or Low-Level Laser Therapy (LLLT) .Pulsed electromagnetic fields and extracorporeal shockwave therapy have strong evidence in the literature, so this narrative review aims to assess the role of PEMFs and ESWT in the prevention and treatment of joint degeneration in athletes.Several preclinical and clinical trials of PEMFs have shown positive effects of biophysical stimulation on articular cartilage, subchondral bone and synovia. After initial studies performed on animal cartilage cells, such as bovine or equine or guinea pigs’ cells ,7,8, stu2A and A3AR on the cell membranes of osteoblasts, chondrocytes and synoviocytes [The transmembrane signal recognition processes of PEMF were reported for the first time by Varani et al. . They diviocytes , and inhviocytes . IL-1β iviocytes .PEMF stimulation increased chondrocytes’ proliferation in patients without OA , and incFurthermore, some authors evaluated how PEMFs could influence the replication of chondrocytes cultured from subjects with OA. Stolfa and colleagues conducted three experiments with different PEMF signal parameters and different concentrations of chondrocytes and showed that this type of PEMF stimulated the metabolic activity of chondrocytes but there were no significant effects on cell proliferation. These results were not achieved in all experiments ; SchmidtThe sound frequency of PEMF is also debated; frequencies of 37 and 75 Hz were able to preserve the structural parameters of both cartilage and bone in the advanced phase of knee osteoarthritis. However, PEMF stimulation at 75 Hz compared to 37 Hz significantly improved cartilage preservation .The combined effect of PEMF and bone marrow concentrate (BMC) in the healing of osteochondral defects treated with a scaffold has been assessed in animal models by different authors. Both cellular and cartilage matrix parameters improved with the addition of PEMF stimulation compared to using the scaffold alone—the combination with BMC also facilitated osteochondral regeneration .i) a postsurgical treatment to quickly control local inflammation of the joint, and, over the long term, to maintain the mechanical and biological properties of the cartilage or engineered tissue, which can be used after arthroplasty to attenuate inflammatory processes involving periarticular tissues and reduce the chances of developing chronic pain or functional limitations [ii) a conservative treatment to limit the progression of the osteoarthritis degenerative process or the development of bone edema, or in association with surgery for risk fractures, delayed union and non-unions.In clinical practice, biophysical stimulation can be used proactively as: Damage to articular cartilage is increasingly identified as a source of joint limitation and reduced athletic performance in athletes, whether isolated or in conjunction with ligament or meniscal or tendon tears ; therefoFew authors have evaluated the role of PEMFs in chondral and osteochondral damage in athletes; for example, van Bergen and colleagues in a double-blind, randomized controlled trial of 68 young and athletic patients evaluated the effectiveness of PEMFs used for sixty days in the management of osteochondral ankle lesions after arthroscopy, considering the simple technology and ease of use and for the high potential to provide a safe and effective adjunct treatment option for talus osteochondral defects .In the literature, in studies that evaluate the PEMFs’ efficacy in patients with osteochondral lesions, it is not specified whether the sample under examination is from athletes .Initially, in 2009, Vavken et al. in a meta-analysis evaluated the positive effects of PEMF associated with some conservative therapies on the quality of life in patients with osteoarthritis of the knee .Later, Gobbi and colleagues also evaluated their use in the treatment of early osteoarthritis (Kellgren Lawrence < 2) and age < 60 for 2 years; the results were mixed as they showed an improvement in pain symptoms and KOOS and Tegner scores after one year of treatment and a worsening, instead, at two years . The autThe same author in a prospective level IV study enrolling 22 patients with a mean age of 48.4 years and with early OA, found at 1 year follow up a statistical improvement of KOOS, EQ-5D, Tegner score and IKDC after PEMF treatment for 45 days .Satisfactory results were also highlighted by Iammarrone and colleagues despite the small sample examined from young patients with patellofemoral pain syndrome (PFPS) .Marchegiani Muccioli et al., in a study on 28 patients with spontaneous osteonecrosis of the knee (SONK), assessed the clinical MRI effectiveness of PEMF therapy performed 6 h daily for 90 days. At 6-month follow up, a clinical improvement and a reduction of the SONK area were detected with MRI .PEMF therapy, with the same treatment protocol in 31 patients with focal knee chondral tears who were undergoing arthroscopy with chondroabrasion and/or perforations, involved a reduction from 75% to 26% with the use of NSAIDs, a higher KOOS at 90 days and a large number of patients that returned to normal daily sports activity at 3 years follow up .Collarile et al., in a prospective comparative study recruiting thirty patients, affected by grade III and IV International Cartilage Repair Society chondral lesions of the Knee treated with matrix-assisted autologous chondrocyte implantation (MACI), reported the patients who randomly received postoperative stimulation with PEMFs had a better clinical outcome both in the short- and long-term follow-up . These fSimilar results have been also reported by Cadossi et al., in a prospective comparative study recruiting thirty patients with grade III and IV Outerbridge osteochondral lesions of the talus (OLT) managed with a collagen scaffold seeded with bone marrow-derived cells (BMDCs), showed the patients who randomly received postoperative biophysical stimulation with PEMFs revealed a better clinical outcome – assessed using the American Orthopaedic Foot and Ankle Society (AOFAS) score; Visual Analog Scale (VAS) and Short Form-36 (SF-36)- at 12-months after surgery . TherefoBenazzo and colleagues also showed a reduction in the use of NSAIDs and faster functional recovery compared to the control group in patients who, after cruciate reconstruction, had been treated with a pulsed magnetic field; however, there was no statistical improvement in IKDC and SF-36 [Some authors, such as Gremion et al. and Ozgüçlü et al., found that a different pulsed signal therapy improved the clinical state of treated patients but there was no significant statistical difference to other conservative treatments such as physiotherapy and therapeutic ultrasound ,38.Nelson et al. and Bagnato et al., in a double-blind pilot clinical study with respectively 34 and 60 patients with OA treated with PEMF, showed that the VAS pain score decreased versus baseline and a reduction in the intake of NSAIDs ,40. Bagnp = 0.032) and a significant reduction in disability in daily activities according to the WOMAC score, compared to the placebo group [Wuschech, after a twice a day treatment for 5 min over a period of 18 days in patients with OA, found a significant reduction in stiffness  has been routinely utilized to treat soft tissue and bone-related musculoskeletal diseases and has been manifested clinically to be effective in plantar fasciitis , lateral6 mL/mm2 .The exact mechanism by which cells recognize an acoustic wave, converting it into biological responses, is currently largely unknown. According to the mechano-transduction theory, shockwaves induce the cellular mechano-transduction process, through which cells convert the shockwave mechanical signals into biochemical responses; mechanical stimulation of the cell membrane induces a conformational change of membrane proteins including integrins and ion channels. The activation of pathways, such as MAPKs and PI3K-Akt-eNOS, influences the transcription and expression of the genome ,50,51. AHuman osteoblasts exposed to shock waves show a dose-dependent increase in differentiation and growth secondary to the increased expression of the Transforming Growth Factor β1 (TGF-β1), which plays a fundamental role in osteogenesis and osteoblastic lineage differentiation ,54,55; sSimilar results were also found in rabbit models with osteochondral defects after ESWT showed improvements in the macroscopic characteristics of hyaline cartilage . The appThe role of subchondral bone throughout the early stages of OA showed that subchondral bone alteration might be a therapeutic focus in OA therapy ,65. WangESWT improved the healing of avascular tears by promoting meniscal cell proliferation and the upregulation of cartilage-repairing factors, such as CCN2, SOX9, aggrecan, and Col2a1, resulting in enhanced synthesis of a cartilage-specific extracellular matrix . Liu et The efficacy of ESWT in human and animal models with OA has also been demonstrated in several clinical trials in which improvements were observed in functional outcomes and pain relief with a reduction of VAS and WOMAC scores ,70,71,72The beneficial effect on OA pain could be explained by nerve fibre responses to ESWT treatment. Ohtori et al. showed that ESWT caused nerve fibre degeneration and reduced the expression of calcitonin gene-related peptide (CGRP) in dorsal root ganglia (DGR) neurons. The analgesic effect and the functional ability enhancement may be time-limited because of nerve regeneration that occurs in fibres 14 days after ESWT ,75. The Extracorporeal shock waves, used routinely for various musculoskeletal diseases, represent a valid therapeutic option for the treatment of the early stages of OA, resulting in an improvement in functional scores and pain; however, the benefits appear to be limited in time.Biophysical therapies with PEMF or ESWT can act to improve the symptoms and function of joints, such as the knee in patients with non-advanced OA or those who have suffered a trauma that has led to cartilage damage or subchondral edema. This can be very useful in athletes for an early return to sport and, above all, for preventing this damage from causing an arthritic evolution of the joint. However, in the literature, few studies use exclusively sportsmen or athletes as a sample to study. Particularly concerning the treatment with ESWT, studies that evaluate the effectiveness of the treatment are mainly on animal models while studies on human models focus on musculotendinous pathology.Further high-quality studies on athletes are needed to draw stronger conclusions.


Pulmonary hypertension (PH) is a condition characterised by an abnormal elevation of pulmonary artery pressure caused by an increased pulmonary vascular resistance, frequently leading to right ventricular failure and reduced survival. Marked sexual dimorphism is observed in patients with pulmonary arterial hypertension, a form of pulmonary hypertension with a particularly severe clinical course. The incidence in females is 2–4 times greater than in males, although the disease is less severe in females. We review the contribution of the sex chromosomes to this sex dimorphism highlighting the impact of proteins, microRNAs and long non-coding RNAs encoded on the X and Y chromosomes. These genes are centrally involved in the cellular pathways that cause increased pulmonary vascular resistance including the production of reactive oxygen species, altered metabolism, apoptosis, inflammation, vasoconstriction and vascular remodelling. The interaction with genetic mutations on autosomal genes that cause heritable pulmonary arterial hypertension such as bone morphogenetic protein 2 (BMPR2) are examined. The mechanisms that can lead to differences in the expression of genes located on the X chromosomes between females and males are also reviewed. A better understanding of the mechanisms of sex dimorphism in this disease will contribute to the development of more effective therapies for both women and men. Pulmonary hypertension (PH) is a condition characterised by an abnormal elevation of pulmonary artery pressure >20 mm Hg) and increased pulmonary vascular resistance, that frequently leads to right heart (RV) failure and death . Increas0 mm Hg aMarked sexual dimorphism is observed in patients with pulmonary arterial hypertension with the major disease registries reporting female to male ratios that are typically greater than 2:1 and range up to 4:1 . The femCuriously, despite being much more common in females, the prognosis in females with PAH is better than that in males . This obMore recently, it has become clear that the sex hormones cannot completely explain the sexual dimorphism of PAH and in this review we focus on the role of the sex chromosomes in producing this dimorphism, highlighting particularly the impact of genes encoded on the sex chromosomes on oxidant pathways.The potential roles of oestrogens and other sex hormones in producing the sex dimorphism observed in pulmonary hypertension have been extensively investigated and it is clear that they make important contributions to the differences between males and females ,13. For While oestrogen actions can account for improved right ventricular function in females with PAH, this mechanism clearly cannot account for the higher incidence of PAH in females. Additionally, there are other clinical observations that are not compatible with oestrogens as a driver of the higher incidence in females. For example, loss of ovarian oestrogen production due to early menopause leads to a subsequent increased incidence of PAH when compared to women with a menopause at a normal age . In cohoData from studies in animal models of pulmonary hypertension are also difficult to reconcile with the less severe PAH observed in female patients. For example, endogenous oestrogen production increased pulmonary arterial pressure and pulmonary vascular remodelling in female mice and rats with hypoxia or Sugen-hypoxia induced pulmonary hypertension ,24,25. IFinally, cell culture experiments show differences in responses between male and female cells in the absence sex hormones. This in vitro approach facilitates investigation of the acute  effects of sex hormones on cells although long lasting  effects established in the cells prior to isolation might persist . Male ceTaken together, these results show that ovarian oestrogen production cannot be the sole mechanism that causes the different PAH phenotype seen in males and females. A further important difference between males and females is the different compliment of sex chromosomes that each receives. Recently, considerable evidence has accumulated that genes encoded on the X and Y chromosomes also contribute to the sexual dimorphism observed in pulmonary hypertension. −) and instead an Sry transgene has been inserted into an autosome (Sry+). Breeding of an XY−(Sry+) male mouse with a standard XX female mouse results in four different combinations of gonads and sex: male XY−(Sry+), male XX(Sry+), female XX, female XY−. To remove the effects of sex hormones during adulthood and thus examine the separate effects of the sex chromosomes, Umar and colleagues removed the gonads from mice bred using the Four Core Genotypes model and 30 days later exposed them to hypoxia to induce PH [Umar and colleagues have used the Four Core Genotypes mouse model to examine the effects of the Y chromosome in the development of hypoxic pulmonary hypertension . The Founduce PH . Mice winduce PH . Mating nduce PH . It was nduce PH . Umar annduce PH . Recent nduce PH ,48.The Four Core Genotypes model can be used to examine the effects of genes encoded on the Y chromosome on the development of PH, with the exception of the Sry gene. To assess a potential role for SRY in protecting against PAH in males, Yan and colleagues explored a link between SRY and BMPR2 and found that SRY expression stimulated BMPR2 expression in human dermal fibroblasts and HEK293 cells . This fiOverall, there is now considerable evidence that the Y chromosome has an important role in producing the sex bias in hypoxia-induced pulmonary hypertension in mice although the mechanisms by which it acts are not fully explored. However, the contribution of genes located on the Y chromosome to sex dimorphism in humans has not yet been specifically investigated and therefore it is not clear how such genes could contribute to a reduced incidence of pulmonary hypertension in males. It is possible that when another stimulus that causes pulmonary hypertension is present  the products of these genes may act to reduce pulmonary vascular resistance and thus prevent clinically significant disease in circumstances that might otherwise have caused pulmonary hypertension. However, extensive further work will be needed to provide a more complete understanding of the role of the Y chromosome in human disease.The X chromosome contains around 2600 genes (NCBI gene) and differences in the expression of these genes are seen in females and males due to a number of different mechanisms  Figure . All malThe second mechanism by which sex chromosomes can lead to differences between males and females is mosaicism. In females one of the two X chromosomes in each cell is randomly inactivated . Hence, As a result, approximately 50% of cells in a female will express one polymorphism while the other 50% will express a different polymorphism. In contrast all cells in males will express a single  polymorphism. The mosaic pattern of expression in the female provides a buffer against the effect of detrimental imprinting or a disadvantageous allele inherited from any one parent. Thus, detrimental mutations and polymorphisms in genes located on the X chromosome are usually manifested in males more profoundly than in females due to female mosaicism . A further mechanism that can modulate the effects of mosaicism is secondary skewing . In a feWe have identified 25 genes located on the X chromosome that have been implicated in the pathogenesis of PH based on evidence of altered expression and activity in clinical samples from patients with pulmonary hypertension, evidence from animal models of pulmonary hypertension or both. Some of the genes are rare X-linked variants that cause pulmonary hypertension in humans as part of complex syndromes. These genes could contribute to sexual dimorphism by one of the mechanisms outlined above and they are listed in Interestingly, a substantial number of proteins encoded on the X chromosome are linked to pathways that alter ROS production and oxidant stress in the lung. Monoamine oxidases (MAOs) are mitochondrial enzymes involved in production of hydrogen peroxide via the catabolism of monoamines such as catecholamines and serotonin (5-HT). There are two isoforms of the enzyme MAOA and MAOB encoded on the short arm of the X chromosome at closely related loci and both are expressed in the pulmonary vasculature ,68. In t2•−) by transferring electron from NADPH to O2 and are also encoded on the X chromosome (NADPH oxidases 1 and 2 (NOX1 and NOX2) are membrane enzymes that produce superoxide (Oromosome . NOX1 pr−/− deficient mice [−/y) leads to the spontaneous development of increased pulmonary vascular resistance and PH in mice in the absence of any precipitating cause [When NOX1 expression and activity in monocrotaline-induced pulmonary hypertension in rats was inhibited, pulmonary vascular resistance was reduced and pulmonary hypertension attenuated . Furtherent mice . While ang cause . This suThere is also evidence of NOX2 involvement in the pathogenesis of pulmonary hypertension. NOX2 protein is elevated in endothelial progenitor cells in hypoxia-induced PH in rats . Hypoxic2O2 to H2O [−/− model of pulmonary arterial hypertension, a model in which female mice develop more severe pulmonary hypertension [Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the conversion of G6P to 6-phosphogluconolactone, the first step on the pentose phosphate pathway, with the formation of NADPH from NADP. The pentose phosphate pathway supplies NADPH to glutathione reductase for the regeneration of glutathione (GSH) from reduced glutathione (GSSG). This GSH is used by glutathione peroxidase for the reduction of H2 to H2O . However2 to H2O . Thus, a2 to H2O . This “J2 to H2O . G6PD ex2 to H2O . Joshi artension . Interesrtension . The rea−/− mice develop pulmonary hypertension with increased oxidative stress and impaired endothelial function [ATPase copper transporting alpha (ATP7A) encodes a transmembrane Cu transporter, an essential cofactor for several enzymes that catalyse electron transfer, including cytochrome c oxidase, lysyl oxidase and superoxide dismutases ,122. Extfunction . Loss offunction . Restorafunction . Taken tfunction ,126. N-acetylglucosamine transferase producing two different isoforms, nucleocytosolic and mitochondrial, that catalyse the addition of N-acetyl-d-glucosamine to proteins and thus alter protein function [OGT encodes O-linked function . OGT delfunction ,129. Howfunction . Taken tA number of X encoded proteins regulate apoptosis which is of interest in the context of PAH where resistance to apoptosis is a prominent feature in the remodelled vasculature ,131. ApoHistone deacetylase 6 (HDAC6) is upregulated in the remodelled pulmonary vessel of patients with PAH . In contX-linked inhibitor of apoptosis (XIAP) is another regulator of apoptosis that is encoded on the X chromosome. Its expression is elevated in persistent pulmonary hypertension of the new born (PPHN) as a result of increased mitochondrial ROS production secondary to reduction of SOD2 activity. XIAP inhibits apoptosis by binding to and inactivating caspase activity . Of noteInflammation is an important pathogenic mechanism in the development of pulmonary hypertension of differing aetiologies ,136. The−/− mice  chronically infected with schistosomiasis develop worse pulmonary hypertension and pulmonary vascular remodelling whereas IL13RA1−/− mice have reduced remodelling [Two further genes on the X-chromosome, IL13RA1 and IL13RA2, play a role in the inflammatory processes activated in the hypertensive pulmonary circulation of some types of pulmonary hypertension. These genes encode, respectively, the low affinity interleukin 13 receptor subunit alpha 1 and the high affinity interleukin 13 receptor subunit alpha 2 ,89,90. Iodelling ,90.Pulmonary vascular remodelling and vasoconstriction are central processes in the development of pulmonary hypertension and six proteins involved in these processes are encoded on the X chromosome . Apelin Genes located on the X chromosome encode a number of transcription factors and non-coding RNAs that alter gene expression and contribute to the development of pulmonary hypertension . ImportaMethyl-CpG binding protein 2 (MECP2) protein is encoded on the X-chromosome and binds to methylated DNA promoter regions thus altering expression of specific genes, causing repression of some (15% of genes) and activation of other (85% of genes) . DuplicaIn addition to the regulation of gene effects by regulation of gene expression, gene effects are also regulated by post-transcriptional mechanisms. miRNAs regulate expression of more than 30% of human genes by translation inhibition or by targeting mRNAs for degradation ,151. MicLong non-coding RNAs can alter gene effects by multiple mechanisms including modulation of transcription, mRNA splicing, mRNA stability and mRNA translation . While tWhile many of the genes located on the X chromosome that we have discussed  have beeA particular example of interaction between X-linked genes and other stimuli causing pulmonary hypertension is found when autosomal mutations cause pulmonary hypertension in a sex dimorphic pattern. BMPR2 mutations, the first identified and most common of the heritable mutations, lead to PAH development in approximately 20% of those who inherit a mutation suggesting that a second pro-hypertensive stimulus, a “second hit”, is required to produce disease. Furthermore, even though BMPR2 is an autosomal gene, females with the mutations more commonly develop pulmonary hypertension than males ,51. Two The sex chromosomes play a potentially important role in the sex dimorphism observed in pulmonary hypertension. The Y chromosome is protective in hypoxia-induced pulmonary hypertension but the specific genes and mechanisms that are responsible for this have not yet been identified. There are also a large range of candidate genes located on the X chromosome that could contribute to sex dimorphism in pulmonary hypertension. Hence, genes located on the sex chromosomes are promising targets for further research to enhance our understanding of the differing disease mechanisms in males and females and to develop more effective, sex specific therapeutic interventions.


The sarcomeric myofilament proteins are essential players in the contraction of striated muscles. The troponin complex in the actin thin filaments plays a central role in the Ca2+-regulation of muscle contraction and relaxation. Among the three subunits of troponin, the Ca2+-binding subunit troponin C (TnC) is a member of the calmodulin super family whereas troponin I  and troponin T  are striated muscle-specific regulatory proteins. Muscle type-specific isoforms of troponin subunits are expressed in fast and slow twitch fibers and are regulated during development and aging, and in adaptation to exercise or disuse. TnT also evolved with various alternative splice forms as an added capacity of muscle functional diversity. Mutations of troponin subunits cause myopathies. Owing to their physiological and pathological importance, troponin variants can be used as specific markers to define muscle quality. In this focused review, we will explore the use of troponin variants as markers for the fiber contents, developmental and differentiation states, contractile functions, and physiological or pathophysiological adaptations of skeletal muscle. As protein structure defines function, profile of troponin variants illustrates how changes at the myofilament level confer functional qualities at the fiber level. Moreover, understanding of the role of troponin modifications and mutants in determining muscle contractility in age-related decline of muscle function and in myopathies informs an approach to improve human health.Ca Skeletal muscle contraction is initiated by an influx of Caarcomere .MYH7, MYH2, and MYH1 genes, respectively, as well as embryonic and neonatal isoforms encoded by MYH3 and MYH8 genes , type 2A (oxidative fast), and type 2B (glycolytic fast) fibers in humans . Based oH8 genes . Commonlyh4 gene . Heterogyh4 gene .Muscles playing a role in body posture are composed of more slow type I fatigue resistant fibers whereas muscles important for movement are composed of a higher percentage of fast type II fibers. Many muscles perform both roles and are heterogenic with regard to fiber type . Muscles2+-regulation is essential for muscle contraction and relaxation , the inhibitory subunit, and troponin T (TnT), the Tm-binding subunit. The troponin subunits are encoded by separate genes that have evolved into muscle fiber-type-specific isoforms. Whereas TnC has only two isoforms, one in fast skeletal muscle and the other in cardiac and slow skeletal muscles, TnI and TnT were evolved from a TnI-like ancestor and each has three isoforms for cardiac, slow skeletal, and fast skeletal muscles  and fast skeletal (TNNC2) muscles , fast skeletal (TNNI2), and cardiac (TNNI3) muscles  mice showed significantly greater variability in Cacontrols . The Sixcontrols .Sox6 gene is another important player in fast skeletal muscle differentiation as Sox6 KO mice show early postnatal lethality coinciding with the formation of secondary myotubes. Sox6 KO results in a decrease in fast skeletal muscle TnI and an increase in slow TnC and slow TnI . Further2+ sensitivity of the muscle fibers  while fast TnT and slow TnT are detectable around 12days p.c., coinciding with the decrease in cardiac TnT . Cardiace fibers . This wal muscle .Troponin I shows significant slow and fast isoform variations during development depending on the species and the muscle group studied. In the biceps femoris of pigs, fast TnI expression was lower in fetus and increased with age while slow TnI expression decreased, corresponding to a decrease in slow MHC I and an increase in fast MHC IIb . StudiesFast and slow TnI are co-expressed in embryonic muscles, and fast TnI is present from the very beginning of myogenesis. The expression of fast TnI is parallel with fast-twitch MHC IIx in adult mouse muscles . There iThough studies have indicated that changes in TnC during cardiac development confer unique functions , less isBy the mid-1970s, it had become apparent that fast skeletal muscle TnT had different isoforms in chicken leg and breast muscles . Monoclo2+ sensitivity of tension development, with higher molecular weight isoforms displaying greater sensitivity , a lethal recessive nemaline myopathy affecting approximately 1 in 500 births in the Amish communities in Pennsylvania and Ohio  encompass a group of autosomal dominant inheritance disorders leading to skeletal muscle weakness, wasting, and hyperexcitability, as well as insulin resistance and cardiac dysfunction, affecting approximately 1 in 8,000–20,000 people. The disorder can be traced back to the expansion of CTG repeats in the 3'-untranslated region of the DMPK gene, which encodes for a kinase that is a known regulator of muscle function and may be implicated in many different pathways. A number of genes involved in muscle functions are affected, and among these are ilaments . Differeiagnosis . The spliagnosis .2+-sensitive force generation, consistent with the role of fast TnT in modulating myofilament Ca2+-sensitivity  is a rare autosomal dominant disorder affecting joints and reducing function of the distal portions of the limbs without any associated muscle weakness. The disorder is delineated into the more severe form DA1, DA2A (Freeman-Sheldon syndrome), and DA2B . Depending on the classification, patients typically present with variable contractures affecting major joints including the hands and feet, while patients with DA2A and DA2B in addition are characterized by facial anomalies including deep-set eyes and small mouth, short stature, and scoliosis. Patients with DA show increased variability of muscle fiber size, largely confined to type II fibers. In some patients with DA, an in-frame internal deletion in ogenesis , and TNNh fibers .via alternative splicing of TnT to alter the thin filament regulation. The increase in low molecular splice forms of slow TnT in CMT type 1 muscle may have the effect of increasing force production as the less acidic slow TnT splice form confers increased Ca2+-activated tension (Charcot–Marie–Tooth disease (CMT) is a hereditary disorder affecting the peripheral nerves. CMT type 1 is classified by peripheral axon demyelination, resulting in reduced motor nerve conductive velocity, while CMT type 2 is classified by axonal degeneration. Patients with both CMT type 1 and type 2 experience weakening of muscles in the periphery limbs, including weakness and muscle atrophy of the feet and hands . In biop tension .TNNT3 was found in oculopharyngeal muscular dystrophy, resulting in changes in Ca2+ sensitivity  missense mutations led to scoliosis and respiratory weakness at birth among members of two families studied, though the condition appears to improve over time and suggests a compensatory effect of cardiac/slow TnC with increased reliance on slow twitch fibers with age. Unlike mutations in slow TnT, the reported TnC mutations do not appear to cause nemaline rods or dramatically alter the myofibrillar structure and solely act through decreasing Ca2+-sensitive force generation of the fibers (There are recently reported cases of TnC causing skeletal muscle myopathies. Inherited fast TnC (e fibers .In contrast, some myopathies, such as TP3 myopathy, acute quadriplegic myopathy, and neurogenic muscle atrophy, show changes in troponin subunits corresponding to atrophy of slow and/or fast fibers but appear to display no unique or abnormal loss or alteration of troponin .in vitro without the need of biopsies and open the possibility to induce disease-related proteins to study function (Muscle regeneration, either through grafts, stem cells, or satellite cells, has been a key goal for improving human health. As technologies develop, tissue engineering will continue to be a key approach in researchers’ toolbox, though difficulties with innervation, vascularization, and inflammatory reactions remain . Engineefunction . Myofilafunction .2+-regulation is a key determinant of skeletal muscle function, and troponin is the crucial regulator of Ca2+-dependent contraction and relaxation. While the expression pattern of MHC is complex and often involves co-expression of multiple isoforms within the same fiber, most fibers express only one isoform of TnI, TnT, and TnC. As fast and slow skeletal fibers differ markedly in their sensitivity to Ca2+, troponin plays a key role in defining fiber-specific functions. Thus, troponin isoforms may present a specific marker for classification of fast and slow skeletal fiber types as well as evaluation of developmental and differentiation states.Myofilament Ca2+-handling ability and specific force generation of skeletal muscles. A clear understanding of protein structure–function relationship informs how changes at the myofilament level confer functions at the fiber level, and the troponin subunit proteins present attractive markers for understanding muscle fiber-type-specific functions, growth and regeneration, age-related decline, myopathies, and compensatory adaptations.The importance of delineating fiber types can be seen in a number of muscle wasting and weakness disorders such as ANM and DM, where troponin mutations or dysfunctional aberrant splicing acts with precision on one specific fiber type. With data from models of age-related muscle decline, disuse, or exercise training, we are presented with a means to understand changes in troponin isoform expression for use in positively impacting human health by improving the CaJ-PJ conceived the topic of research. MR drafted the manuscript. MR and J-PJ edited and revised the writing. All authors contributed to the article and approved the submitted version.This work was supported in part by grants from the National Institutes of Health HL127691 and HL138007 to J-PJ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The localized surface plasmon resonance of metallic nanoparticles has attracted much attention owing to its unique characteristics, including the enhancement of signals in sensors and photothermal effects. In particular, hollow gold nanostructures are highly promising for practical applications, with significant advantages being found in their material properties and structures: 1) the interaction between the outer surface plasmon mode and inner cavity mode leads to a greater resonance, allowing it to absorb near-infrared light, which can readily penetrate tissue; 2) it has anti-corrosiveness and good biocompatibility, which makes it suitable for biomedical applications; 3) it shows a reduced net density and large surface area, allowing the possibility of nanocarriers for drug delivery. In this review, we present information on the classification, characteristics, and synthetic methods of hollow gold nanostructures; discuss the recent advances in hollow gold nanostructures in biomedical applications, including biosensing, bioimaging, photothermal therapy, and drug delivery; and report on the existing challenges and prospects for hollow gold nanostructures. Owing to recent advances in the fabrication and analysis of metal nanoparticles, localized surface plasmon resonance (LSPR) has been extensively investigated and used in a broad range of fields. LSPR is the strong oscillation of the electron cloud of metal nanoparticles induced by incident light . This phAmong the aforementioned gold nanostructures, hollow gold nanostructures, which have hollow cavities inside them, have been intensively investigated owing to their unique advantages over pre-existing solid gold nanostructures  . Herein,Similar to conventional gold nanostructures, hollow gold nanostructures are promising materials for biomedical applications, owing to their chemical stability, biocompatibility, and ease of surface modification. However, the most apparent difference between solid and hollow gold nanostructures is the existence of an inner cavity. The inner cavity provides additional functionalities to hollow gold nanostructures, such as anticancer drug loading and interaction between the inner and outer surfaces; thus, these structures can be utilized as a new strategy for chemo–photothermal therapy or light-responsive drug release systems. Hollow gold nanostructures can be synthesized with various sizes and morphologies  depending on several factors, including the synthetic protocol, reaction temperature, and reaction atmosphere . Althoug2B nanoparticles and the role of environmental oxygen for galvanic exchange, which gives two-fold tunability of hollow gold nanospheres. They synthesized two types of hollow gold nanospheres with different diameters and shell thicknesses: 1) identical outer diameters with different aspect ratios (the ratio of outer diameter to shell thickness), providing LSPR peak tunability  nability . Specifi+), which has a higher reduction potential. While the solid metal with the lower potential is oxidized and transformed into a metal ion (from A to A+), the metal ion with the higher potential is reduced and transformed into a solid metal (from B+ to B) at the contact surface. Through these processes, hollow nanostructures can be successfully synthesized with several characteristics: 1) tunable elemental compositions  further galvanic replacement by HAuCl4 (path [I] in 3)3 and NH4OH  easiositions , 3) closositions , 4) shorositions , and 5) ositions . For holositions . Liang eThe hollow structures were confirmed using transmission electron microscopy (TEM). In addition to nanospheres . They reAlthough galvanic replacement is a major synthetic method for hollow gold nanostructures, there are some drawbacks, such as 1) fragmentation or aggregation due to excessive galvanic replacement processes  and 2) t12C6C12Br2), played an important role as a soft template. C12C6C12Br2 is induced to self-assemble into various forms of vesicles, such as capsule-like or tube-like aggregates, depending on its concentration. Moreover, it provides structural stability during reduction because of its strong aggregation tendency. Recently, inert and relatively biocompatible gold nanostructures have been adopted for various biomedical applications owing to their attractive chemical and physical properties . Gold nain vivo environments owing to their high mechanical flexibility and stability; they can be used as suitable platforms for a variety of purposes, such as labeling with various ligands, biomolecules, and functionalization because of their atomically flat surfaces -based biosensor is one of the most powerful spectroscopic tools for biomedical field with high sensitivity and specific fingerprints and fast data collection speeds . A hollovia the thiol group. The fabricated biosensor detected HA proteins in phosphate-buffered saline (limit of detection (LOD): 1 pM) and diluted chicken serum (LOD: 1 pM) and confirmed that the LSPR peak increased as the concentration increased from 1 pM to 100 nM  and S100-b proteins in the blood was developed . The acto 100 nM .PTT is an effective cancer treatment method using nanoparticles that utilize a mechanism in which light is absorbed by nanoparticles and converted into heat within tumor tissues. When the local temperature in tumor tissue reaches 42–47°C, tumor cells—owing to their heat sensitivity being higher than that of normal cells—quickly undergo necrosis . This trGold nanostructures can be used to treat obesity. Their easy facile modification, superior stability, nontoxicity and biocompatibility make this possible. In addition, the properties of absorbing visible and near-infrared light to convert to thermal energy lead to effective photothermal treatment. One of the advantages of hollow gold nanostructure is that it is easy to control LSPR, which can be adjusted in size of nanostructure from the visible light field to the near-infrared field for cancer photothermal therapy. via anti-Spa, PDA was coated onto the surface and the nanostructure was activated by near-infrared radiation to convert photon energy into thermal energy, increasing the photothermal effect and degenerating the PDA coating to release the antibiotics  into polydopamine (PDA)-coated gold nanocages  Meeker . To induibiotics . Figure Hollow gold nanostructures have high photothermal conversion effects and excellent biocompatibility, can adjust the LSPR band in near-infrared (NIR) regions, and are suitable for drug delivery. The irradiation of gold nanoparticles with NIR lasers can accelerate the release of cargo by generated hyperthermia to the target location and ablate the tumor cells with favorable PTT .Cancer cell membrane-coated gold nanocages were fabricated by loading the anticancer drug Doxorubicin (DOX) into the inner core of gold nanocages and then coating with the cancer cell membrane derived from 4T1 breast cancer cells. A sulforhodamine B assay to determine the antiproliferative effect of gold nanocages showed that the strongest antiproliferative effect was on tumor cells cultured with cancer cell membrane-coated gold nanocages, resulting from the accelerated release of DOX from gold nanocages by laser-induced high heat and induced phototoxicity .  reportedPAT, also referred to as optoacoustic tomography or photoacoustic imaging, is a powerful nonionizing optical imaging modality that integrates the advantages of both optical radiation and ultrasonic detection . This imThese bioimaging applications can be combined with therapeutic applications, such as drug delivery and PTT, to simultaneously perform treatment and diagnosis to enable precision medicine that can be tailored to each patient . NanoscaHollow gold nanostructures have drawn attention for their biomedical applications because of their unique physical and optical properties, such as facile LSPR tunability, drug loading capacity, and enhanced photothermal activity, compared to those of conventional solid gold nanoparticles. In this review, we summarized the recent research trends in hollow gold nanostructures, including synthetic methods, physical/optical characteristics, and recent advances in biomedical applications. Various forms of hollow gold nanostructures can be synthesized by either hard- or soft-templating methods, but the majority of synthesis is based on galvanic replacement. While the LSPR peaks of solid gold nanoparticles depend solely on the overall size of the particles, those of hollow gold nanostructures are greatly influenced not only by the size, but also by the thickness of the shell. Therefore, the LSPR peaks can be easily adjusted while maintaining the overall size of the particles, which provides additional tunability of the SERS signal or photothermal activity. The voids in the interior also provide additional functionality to hollow gold nanostructures by being utilized as nanoscale storage for impregnating functional materials , which is not possible in solid gold nanoparticles. Based on these characteristics, various hollow gold nanostructures have been widely used in several biomedical applications, including biosensors, photothermal therapy, and biomedical imaging. It should be noted that there have been many studies aimed at the synergistic effects of simultaneously utilizing the various functionalities of hollow gold nanostructures, such as combined chemo–photothermal therapy, light-responsive drug release, and plasmonic nanostructure-based bioimaging.Despite this progress, there are still many issues: 1) off- and on-target accumulation of hollow gold nanostructures and 2) preventing the aggregation and disassembly of hollow gold nanostructures. Large-sized hollow gold nanostructures (larger than 200 nm) can lead undesirable accumulation in the reticuloendothelial system ; on the other hand, very small-sized one cannot provide sufficient on-target accumulation of them due to the short circulation time. Even if the size of these structures is suitable for selective accumulation, the aggregation and disassembly of hollow gold nanostructures in human body can decrease the LSPR intensity. Therefore, researchers should consider 1) appropriate size of nanostructures for sufficient on-target accumulation and prevention of off-target side effect of them; 2) antibody conjugation which enhance the capability of target adhesion; and 3) the stability of hollow gold nanostructures in human body to provide good LSPR properties. We strongly believe that the above-mentioned problems will be overcome by further research using advanced synthetic methods or surface modifications in the future; then, hollow gold nanostructures will become increasingly important in biomedical applications because it is clear that these unique structures have many advantages compared to conventional nanoparticles. We hope that this review will help readers to comprehend the recent advances in hollow gold nanostructures and will provide great inspiration for researchers.


In the late 1980s, Paul Primakoff and colleagues showed that fertilization could be blocked in an in vitro sperm–egg fusion assay by inoculating them in the presence of a disintegrin and metalloprotease (ADAM)-specific antibody. This elegant experiment revealed the adhesive properties of these single-transmembrane spanning proteins and laid the foundation for several groundbreaking discoveries in the field of ADAM biology in the following years. While several ADAM proteins presumably function as adhesion molecules, some members of this family are catalytically active and regulate diverse biological processes, including neuronal differentiation, muscular tissue formation, inflammation and immune responses [In this Special Issue, we survey the recent advances in this field, adding formerly unknown insight into the aspects of ADAM biology, activity and substrate specificity. We have collected a series of research reports and review articles from prominent authors in the field, which explore the functional role of a variety of catalytically active ADAM proteases and their substrates, ranging from ADAMs 9, 10, 12, 15 and 17 to components of their substrate network.As it became more and more evident in the early 2000s that ADAM proteases serve as important molecular switches in various signaling circuits on the cell surface and in the extracellular space, the search for their substrates was rapidly expanding [Within this framework, we aimed to decipher the nature of the proteases that are responsible for the proteolytic release (ectodomain shedding) of a family of fibroblast growth factor receptors (FGFRs) from the cell surface. These receptor tyrosine kinases have been implicated in various cellular processes, such as embryonic development and wound healing, but have also been associated with enhanced tumor survival, angiogenesis and metastases . InteresBesides identifying the proteases involved in the release of these cell surface receptors, the functional consequences of this ADAM-mediated ectodomain shedding should be evaluated as well. In this regard, Anne Hanneken and colleagues examined the biological properties of FGFR1 ectodomain shedding and how this cleavage event is regulated. The authors not only found that FGFR1 is constitutively cleaved but that this released soluble form also inhibits FGF2-induced proliferation and competes with membrane-bound FGFR for ligand binding. Furthermore, the authors showed that the underlying shedding mechanism of FGFR1 shedding is regulated by two distinct signaling pathways . In summWhile we continue to discover novel substrates for these proteases, other ADAM substrates have been characterized in greater detail in recent years. Some of the presumably most extensively studied and best characterized ADAM substrates are the Notch receptors . Notch sADAM10 has also captured scientific interest because of its pivotal role in the non-amyloidogenic processing of the amyloid-β precursor protein in neurons, inhibiting the production of β-amyloid peptide in Alzheimer’s disease (AD). An interesting study, conducted by Izabela Vatanabe and colleagues, determined the activity of this protease in the plasma and cerebrospinal fluids of patients with amnestic mild cognitive impairment and mild AD . ConsideMany other chronic conditions, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), as well as various types of cancers, have also been linked to aberrant ADAM expression and activity . Often, Of note, a thorough analysis of additional substrates cleaved by ADAMs 10 and 17 and their activities in subcellular sites and multiprotein complexes is presented in the review article by Francesca Tosetti and colleagues [In addition, several studies have linked elevated levels of ADAM17 and connective tissue growth factor (CTGF) with the development of idiopathic pulmonary fibrosis. However, the underlying signaling cascades responsible for this increased expression in this malignancy remain poorly defined. Shu-Ching Ou and colleagues point to novel evidence regarding the impact of transforming growth factor-β (TGF-β)-mediated signaling pathways on the induced expression of CTGF in lung epithelial cells, uncovering the importance of ADAM17 as well as of ERK, RSK1 and C/EBPβ, which are well known for their functional role in epithelial–mesenchymal transition (EMT) . InteresAlthough ADAMs have been consistently associated with the development of several chronic diseases, they can also contribute to the detrimental effects in acute pathologies. In this context, ADAM17 has been shown to play a critical role in the pathogenesis of sepsis, an acute condition caused by a dysregulated response to infection. With a mortality rate of nearly 50%, there is an urgent need for the identification of novel therapeutic avenues for severe sepsis treatment. Towards this direction, Hemant Mishra and colleagues examined the effects of an ADAM17 function blocking monoclonal antibody in the pathogenesis of sepsis . In an iBesides modulating disease-relevant processes, ADAM proteins also regulate important physiological functions such as cell–cell interactions and the activation of various signaling pathways, as well as cell proliferation and differentiation . Anke SeIn addition, previous studies have shown an essential role for ADAM17 in differentiation of hypertrophic chondrocytes during endochondral ossification. In this process, growing cartilage is gradually replaced by bone to form the skeletal system. Renpeng Fang and colleagues point to novel evidence regarding the impact of iRhoms 1 and 2 in endochondral ossification, uncovering the importance of these inactive rhomboids in the regulation of bone growth by controlling the maturation of ADAM17 during differentiation of hypertrophic chondrocytes . ParticuAn excellent analysis of the signaling processes controlled by iRhoms and their regulatory role in immune responses to infections is presented in the review article by Mazin Al-Salihi and Philipp Lang . In addiWhile iRhoms 1 and 2 specifically interact with ADAM17, the tissue inhibitor of metalloprotease 3 (TIMP3) forms complexes with either matrix metalloproteases (MMPs) or ADAM proteins. Decreased levels of TIMP3 have been associated with aneurysms, cardiac inflammation and atherosclerosis, suggesting the potential utility of targeted TIMP3 delivery to inhibit aberrant metalloprotease activity in these diseases. Towards this direction, Anna Carreca and colleagues developed a high-resolution mass spectrometry-based approach to evaluate changes induced by stable overexpression of TIMP3 in the cell surface proteome [Similarly to iRhoms 1 and 2, a subgroup of tetraspanins, TspanC8, function as ADAM10 interaction partners and support its regulation and maturation. The role of this tetraspanin family in substrate recognition and their therapeutic potential are further discussed in a comprehensive and informative review article by Neale Harrison and Michael Tomlinson in this Special Issue . Though several studies herein have focused on the biology of ADAMs 10 and 17 specifically, little attention has been given to the variety of physiological functions and pathophysiological conditions that are influenced by other catalytically active ADAM proteins. An interesting article, written by Cheng-Wei Chou and colleagues, effectively summarizes the existing literature regarding the role of ADAM9 in developmental processes as well as in inflammation and degenerative diseases . MoreoveIn conclusion, the scientific discoveries presented in this Special Issue should inform future research and intervention initiatives and draw closer attention to the clinical importance of these ubiquitous molecular scissors, laying the foundation for treatment strategies to target ADAM-dependent signaling pathways that cause sustained inflammation and an increased risk for cancer as well as therapies to treat chronic diseases such as Alzheimer’s, cardiovascular disorders and cancer.


DNA helicases have emerged as a prominent class of nucleic acid-metabolizing enzymes that play important roles in genome maintenance and cellular homeostasis. DNA helicases not only play essential roles in replication, DNA repair, and recombination, but they also influence gene expression and chromosome structure and act to resolve dynamic DNA structures that pose a source of genomic instability. Although the basic mechanisms of the unwinding of structured nucleic acids by DNA helicases share some features of commonality, research shows that there are specialized DNA-unwinding mechanisms that can be regulated by assembly states, post-translational modifications, and protein interactions.With the discovery of genetic disorders linked to helicase gene mutations and various cancers associated with helicase defects, it has become more important than ever to describe and characterize the molecular and cellular functions and pathways of DNA helicases to gain new understanding and move toward translational approaches in medicine.Genes entitled DNA Helicases: Mechanisms, Biological Pathways, and Disease Relevance is dedicated to a compilation of articles on DNA helicases from leading experts in the field. This Genes Special Issue provides diverse and informative perspectives on a spectrum of DNA helicase proteins and teaches lessons about their molecular and genetic functions and, more broadly, their roles in genome homeostasis.A Special Issue of History of DNA Helicases, an article by Brosh and Matson, sets the table for the Genes Special Issue by providing an in-depth and comprehensive assessment of how the study of helicases has progressed since their first discovery nearly 45 years ago [ears ago . InnovatE. coli [Genes Special Issue.So much of our knowledge of how helicases operate has been gleaned from seminal biochemical and genetic studies with bacterial proteins. Bianco addresses, in his review article, an in-depth discussion of the complex interactive and helicase-centric processes that occur at stalled replication forks in E. coli . DependiSaccharomyces cerevisiae and Saccharomyces pombe [One of the more prominent families of DNA helicases in terms of disease relevance is the RecQ grouping. Gupta and Schmidt provide an excellent launchpad for considering the important roles of RecQ helicases by providing a comprehensive assessment of the RecQ helicases in the model genetic organisms es pombe . With suAlthough there have been extensive studies of the three human RECQ helicases encoded by genes in which biallelic mutations are linked to diseases characterized by chromosomal instability, premature aging, and cancer, there is much less known about the two remaining human RECQ helicases: RECQ(L)1 and RECQ(L)5. Therefore, I was highly pleased when Drs. Sharma  and JansComing from a more global perspective, Huselid and Bunting take on the difficult but rewarding task of discussing, in their review article, how DNA helicases play diverse and complex roles in the regulation of homologous recombination, an important means for cells to use to heal lethal double-stranded breaks that arise from genotoxic insults or broken replication forks in a manner that suppresses their deleterious effects on chromosomal stability . Both prIn addition to DNA repair pathways, certain specialized DNA helicases are important for their ability to resolve unusually structured nucleic acids that deviate from the conventional DNA double helix and potentially interfere with normal cellular DNA replication or transcription. Lansdorp and van Wietmarschen provide an insightful review article on how the Fe–S cluster helicases FANCJ (mutated in Fanconi anemia) and RTEL1 (mutated in Dyskeratosis congenita), as well as the RECQ helicase BLM (mutated in Bloom’s syndrome), act upon a unique four-stranded DNA structure known as a G-quadruplex (G4) to regulate gene expression and maintain genomic stability .Genes Special Issue on DNA helicases describing FANCJ’s novel interaction with a cellular translesion polymerase and associated factor (PCNA) to synthesize past G4 obstacles [My own lab has taken a particular interest in the functions of FANCJ in nucleic acid metabolism and found that the helicase plays a vital role in smoothing out G4 DNA structures to allow efficient replication in human cells ,9,10. Cobstacles . With thAnother family of DNA helicases with profound roles in genome maintenance is represented by the PIF1 helicases. Muellner and Schmidt provide a critical analysis and summary of the PIF1 family of DNA helicases in yeast, providing an excellent foray into understanding their complex genetic and molecular roles . By theiGenes Special Issue on DNA helicases, the Bochman lab contributed a research article describing their findings from a molecular-genetic analysis of the S. cerevisiae Pif1 [In this iae Pif1 . AlthougA conserved eukaryotic helicase of considerable interest, but less well studied than PIF1 family members, is DNA helicase B (HELB), the topic of a review article by the Byrd lab . HELB isWith the beneficial articles on eukaryotic nuclear DNA helicases, I was pleased to have the Falkenberg lab contribute a review on the mammalian replicative mitochondrial DNA helicase TWINKLE and other human mitochondrial DNA helicases . Given tHistory of DNA Helicases [As communicated in our review, elicases , the fieelicases .Genes Special Issue represents a very comprehensive and significant source of information with testable models and emerging hypotheses to advance the field and bring a new cadre of scientists into the fold of helicase and nucleic acid metabolism research.Overall, this collection of articles on DNA helicases provided in the


Several studies have demonstrated that thromboembolic events increased in patients with coronavirus infection, usually occurring in elderly patients with severe illness, associated with comorbid diseases such as diabetes and hypertension. Portal vein thrombosis (PVT) is a rare venous thromboembolic disease occurring typically in patients with an underlying disease such as decompensated cirrhosis with or without hepatocellular carcinoma (HCC).  To evaluate incidence of occurrence of acute PVT in cirrhotic patients infected with 2019 coronavirus disease (COVID-19).  This cross-sectional, observational study involved 70 patients of the liver cirrhosis: (group A) 28 patients with liver cirrhosis infected with COVID-19, and 42 patients with liver cirrhosis as the control group matched for age and sex (group B). All patients were subjected to thorough medical history, routine investigations , imaging in the form of abdominal and Doppler ultrasonography to assess the presence of acute PVT, serum ferritin, D-dimer, C-reactive protein, and PCR of COVID-19 for group A only. p < 0.05).  There was a significant difference between the two groups regarding Doppler ultrasound findings as 3 of the patients in group A had PVT (10.7%), 2 of them had HCC diagnosed by triphasic CT abdomen, and only 1 patient in group B had PVT (2.3%) ( In cirrhotic patients infected with COVID-19, portal vein thrombosis may be a potential complication even in the absence of hepatocellular carcinoma; further prospective studies with longer follow-up may be needed. A new severe acute respiratory syndrome coronavirus began in Wuhan in December 2019 and then rapidly spread throughout China and became worldwide [Portal vein thrombosis is a rare venous thromboembolic disease typically occurring in patients with an underlying disease such as decompensated liver cirrhosis with or without hepatocellular carcinoma, systemic lupus erythematous, pancreatitis, and other hypercoagulable conditions , 4. SeveThe diagnosis of PVT in patients with cirrhosis is usually made during routine ultrasound examination in asymptomatic patients or following a new event of hepatic decompensation . SplanchThis cross-sectional, observational control study involved 70 patients of liver cirrhosis with or without decompensation divided into two groups: group A (28 patients with liver cirrhosis infected with COVID-19), and 42 patients with liver cirrhosis only as the control group matched for age and sex (group B). The patients with liver cirrhosis were selected randomly from the outpatient clinic of the Tropical Medicine department, Minia University Hospital, during the period from January 2021 to March 2021. Patients were collected according to the following inclusion criteria: cirrhotic patients presented with acute onset of fever, any upper respiratory symptoms, history of fatigue, acute abdominal pain, and/or history of diarrhea and vomiting; patients were excluded from the study if they had any history of PVT or any other thromboembolic events before the symptoms of the COVID-19 has appeared. Informed consent was obtained from all subjects (for both participation and publication of that work). The local ethics committee for human subject research reviewed and approved the study protocol and consent forms; all procedures performed in the study were in accordance with the ethical standards of the national research committee.History taking including the personal history, present illness of acute onset of fever, any upper respiratory symptoms, history of fatigue, acute abdominal pain, history of diarrhea and vomiting, and history stressing on history of DMBasic investigations including complete blood count (CBC), liver and renal function tests, and INRImaging in the form of abdominal and Doppler ultrasonography to assess the presence of PVTSerum ferritin, D-dimer, C-reactive protein, LDH, and PCR of COVID-19 for group A patients onlyAll patients were subjected as follows:Z test between the two groups. Statistical significance was defined as p values less than 0.05.The collected data were inserted, tabulated, and statistically anatomized using software version 24 of Statistical Package for Social Sciences program (SPSS). Quantitative data were expressed as mean + standard deviation (SD), while qualitative data were expressed as proportions. Comparisons between groups for qualitative data were analyzed by test of proportion of the Demographic and laboratory characters of the studied cases are given in p value (˂0.05). The three COVID-19 patients were diabetic, two of them had HCC diagnosed by triphasic CT abdomen. Many case reports described the occurrence of acute portal vein thrombosis as a consequence of COVID-19 infection in noncirrhotic patients, and the condition was associated with ground glass opacities in CT chest [Portal vein thrombosis is considered the commonest thrombotic complication in patients with hepatocellular carcinoma on top of liver cirrhosis . PVT is CT chest –24. OtheCT chest , 25, 26.