The present review aims to provide a critical appraisal of the sonographic diagnosis and follow-up and to evaluate the optimal clinical management of monochorionic twin pregnancies where one of the twins is complicated by selective fetal growth restriction (sFGR). The classification is based on the umbilical artery (UA) diastolic flow reflecting the outcome. If the sFGR twin has positive diastolic flow (Type I) then the prognosis is good, and it does not require close surveillance. Biweekly or weekly sonographic and Doppler surveillance and fetal monitoring are recommended strategies to detect unpredictable complications in type II and type III forms, which are defined by persistently absent/reverse end-diastolic flow (AREDF) or cyclically intermittent absent/reverse end-diastolic flow (iAREDF) in the umbilical waveforms, respectively. The latest forms are associated with an increased risk of unexpected fetal demise of the smaller twin and 10–20% risk of neurological injury in the larger twin in addition to the overall risk of prematurity. The clinical course can be affected by elective fetal therapy  or elective delivery in the presence of severe fetal deterioration. The prediction of the clinical outcome in complicated cases of type II and III sFGR cases remains elusive. Novel routines in fetal and placental scans in order to predict neurological impairments and unexpected fetal death to optimize the delivery time-point are needed. Approximately 20% of twin pregnancies are monochorionic (MC), constituting 1 out of 250 pregnancies, and 70–75% of them are monozygotic twins . Almost Generally, twinning gestation poses a higher risk of perinatal morbidity (iatrogenic preterm delivery) and mortality as compared to that of singletons ,10,11,12This review intends to provide a comprehensive critical description of the clinical management and diagnostic follow-up with sonography concerning the complications of MC twins with a focus on sFGR.Overall, one out of three MC twin pregnancies will develop specific complications by way of congenital defects and growth restriction based on placental insufficiency of one or both twins and nearly always present interfetal placental vascular connections encompassing twin-to-twin transfusion syndrome (TTTS), isolated discordant growth, twin anemia–polycythemia sequence (TAPS), twin reversed arterial perfusion (TRAP), or intrauterine demise ,20,21. TSelective fetal growth restriction (sFGR) is a condition that occurs in twin pregnancies when one of the fetuses is supplied with insufficient nutrient and oxygen content through the placenta to grow at a normal rate ,23. It iThe prenatal diagnosis of FGR is sometimes problematic and it is not possible to find all the cases. Plenty of EFW formulas are available, and some of them are more accurate in normal ranges; however, in the diagnosis of FGR the Hadlock B formula had the best correlation . On the The early forms appear in the midtrimester period (below 20 weeks of gestation) and affect 10–12% of MC twins , whereasThe pathological feature of the early and more serious form is the variation of the size, types, and number of anastomoses and the distance of the cord insertions, and, hence, the intensity of the placental intertwin blood transfusion varies extensively, presenting a wide range of unique clinical manifestations ,34. The The interfetal vascular anastomoses are randomly distributed between the MC twins, influencing the imbalanced placental blood flow between the fetuses and affecting the clinical course. The main feature of MC twins is the existence of placental anastomoses, which can be AA, veno-venous (VV), or arterio-venous (AV). Vascular placental connections result in bidirectional fetofetal transfusion, a sort of third circulatory system between two fetuses. AV anastomoses have unidirectional flow (from recipient to donor) and are vascular AV connections where a placental cotyledon is perfused by an artery from one fetus and drained to the other one. Blood transport through the intertwin AA and VV anastomoses in either direction, usually opposite to the AV anastomoses (from donor to recipient), may compensate for the imbalances in blood volume or pressure between the twins. AA anastomoses connect the two cords in the placenta, directing from one or two UAs. These types of anastomoses are usually thick, with a diameter of ≥2 mm that allows a larger amount of blood interchange. The smaller twin receives oxygenated blood from its co-twin via placental anastomoses to partially compensate for the placental insufficiency ,31,33,35The worse clinical evolution is characterized by large placental discordance with very few intertwin anastomoses . Aside fThe classification of the subtypes is based on the end-diastolic flow (EDF) in the UA of the growth-restricted fetus that corresponds well to three clinical expressions ,21. ThesThe most frequent subform is type I sFGR fetuses, with an incidence of 29–63.5% ,43 whichFor the most severe variant (type II) (incidence: 22.4–36.5% of the sFGR) ,43, a peThe type III subform  ,42 is acThe apparently benign clinical evolution provides a low risk of hypoxic damage to the FGR fetus and facilitates the gestation until 32 weeks, or in milder form to 34 weeks. The fetal deterioration is unpredictable, with scanning of the DV flow, umbilical vein Doppler, or biophysical profile changes, and elective delivery around 32 weeks is recommended with weekly follow-up if venous Doppler is normal ,21,34,44Generally, the most severe complications are attributed to the type II forms, but type III sFGR fetuses are also at a high risk of very preterm delivery, intrauterine fetal death of the smaller twin, and neurological sequelae of the twin with normal growth. The severity of the complications is highly related to the grade of growth restriction and its progress, and the gestational age at diagnosis. UA Doppler examination cannot be used as a prognosticator of imminent fetal death, but the manifestation of absent or reverse atrial flow in the DV or abnormal venous Doppler can be used for appropriate timing of delivery. Fetal therapy (laser therapy or selective fetocid) has been proposed for use only in severe cases diagnosed at the <24–26th weeks of gestation or at signs of imminent fetal demise before viability. Type II and III sFGR pregnancies treated with laser therapy or cord occlusion have a higher rate of perinatal mortality and premature birth but a lower rate of morbidity compared with those managed expectantly ,22. HoweThe clinical pattern of the growth-restricted twin differs prominently from that of singleton or in DC pregnancies where there are two entirely separated chorionic ’units’. sFGR fetuses in MC pregnancies show a remarkably longer latency between the onset of the alteration of the umbilical flow and delivery (10–14 weeks) compared with the 3–6 weeks reported in singletons with FGR with deteriorated umbilical blood flow ,39. The The umbilical Doppler ultrasound patterns of the smaller (recipient) twin are not always stable, and a considerable proportion of the sFGR changes its classification. The highest risk of fetal death is encountered in pregnancies with type I sFGR that evolves type II or III or type II subforms pass into type III subform ,54. NoneA slowly progressive intertwin discordance may contribute to the late sFGR of the compromised twin, while normal growth can be described for the healthy fetus. Late sFGR has always normal EDF and can be demonstrated solely in the third trimester (>28 weeks of gestation) and is not observed in the second trimester. The rate of late onset sFGR was reported to be 6.3% among MC pregnancies. A remarkably benign clinical outcome with a low risk of intrauterine fetal death (8%) is associated with this group. An underlying placental insufficiency results in a progressively developing weight discrepancy reaching a mean of 30%. The reported frequency of TAPS was 38%, which suggests the significance of the monitoring of peak systolic velocity (PSV) of the middle cerebral artery (MCA) and pulsatility index (PI) in DV for close timing of pregnancy in these cases . Long-tesFGR occurs in 10–15 percent of DC twins, but unlike MC pregnancies, DC twins have entirely separate fetal-placenta blood circulations, and thus the larger twin is unaffected ,57.A detailed description of other complications than sFGR in MC twins is beyond the scope of this review; however, a short interpretation of the MC-related unique complications is provided. The sFGR implicates separate pathomechanisms from TTTS ), TAPS ,60, TRAPTTTS is a serious condition that occurs in about 10–15% of monochorionic twin pregnancies usually before 26 weeks ,65. In mFetoscopic laser coagulation is the optimal therapy to considerably improve the co-twin prognosis by clogging abnormal anastomoses . CoagulaTAPS appears in 3–5% of MC twins, usually in the third trimester (mainly after 26 weeks) . PostnatsFGR and TAPS can be individually observed or can co-occur in MC twin pregnancy. The placental anastomoses of sIUGR with TAPS are typically small. sIUGR with TAPS had smaller differences in placental share and larger distances between umbilical cord insertions. Late sFGR, when the growth rate reduces gradually, could be accompanied by abnormal blood flow in the umbilical cord artery during the diastole. This allows the donor fetus to transfuse blood chronically to the recipient fetus through these anastomoses, leading to anemia in the donor and hypervolemia in the recipient fetus sIUGR with TAPS had smaller differences in placental share and larger distances between umbilical cord insertions .TAPS must be regularly screened by means of routine middle cerebral artery (MCA) Doppler ultrasound examination (MCA Vmax >1.5/<1 MoMs in the twins) since it has no other manifestations besides the DV PI measurement . If untrIn TRAP, a parabiotic twin has an absent or non-functioning cardiac system and receives blood from the normally developing twin, often referred to as the ‘pump twin’. Because one heart is pumping blood for both twins, the condition places an enormous demand on the heart, putting the pump twin at risk for cardiac failure. TRAP can develop as a complication of TTTS laser treatment of TTTS. In combined cases, the formation of sFGR is independent of TRAP development .AFD is usually defined as a difference in amniotic fluid volumes in a twin pregnancy. An AFD ≥ 4 cm cutoff is associated with a significantly increased risk of the development of TTTS (70%) ,78. Accurate diagnosis/identification of chorionicity and amnionicity is of paramount importance ,21,79. TThe lowest birth weight and gestation at birth can be reported in MC twins compared to singleton or DC pregnancies. Growth-restricted fetuses (birth weights below the 5th percentile) are more often observed in MCDA pregnancies (37.8%) than in MCMA pregnancies (33.3%) where at least one fetus is born alive, and only the former rate is significantly higher compared to DCDA twins 31.2%). However, both fetuses were discovered to be growth-restricted in most of the pregnancies in these cases %. Howeve. Twin feThe perinatal mortality of MCMA twins is between 30 and 40% . The oveThe lowest birthweight and gestation at birth can be observed in MC twins compared to DC twins . DetermiForemost, first and early second trimester sonography should be applied in aiding the determination of amnionicity/chorionicity ,82, majoComplicated cases where sFGR is combined with TTTS, which deteriorates the condition of the donor twin urgently, require immediate laser therapy, so the first step in the diagnostic algorithm is to assess TTTS. When routine sonography is performed, TTTS and sFGR should be assessed first, whereas TAPS and particularly AFD are arbitrary phenomena . PrimariGenerally, there is no evidence of optimal monitoring of MC pregnancies, but most studies recommend a sonographic follow-up at least biweekly/weekly, since TTTS can appear even days after an examination with conclusion of normal values ,21,97. TAccordingly, a simultaneous follow-up of the fetal heart monitoring (computer-task CTG) from viability (from 24th weeks of gestation) or later from 28th weeks) and onwards is often integrated into the twin monitoring system ,97,99. Hth weeks The surveillance intensity and approach vary in the studies with no obvious consensus ,102,103 It is of paramount importance that timely delivery is mainly guided by avoiding fetal loss in MC pregnancies . ElectivHowever, the timing process of delivery carries uncertainty and depends on many factors: flow parameters , the extent of the weight discordance and the growth restriction, the gestation at diagnosis, the available technical factors, and finally the parents’ preferences, keeping in mind that a substantial proportion of sFGR twins, particularly in type II or type III, have very low birthweight due to prematurity . MC twinning poses a greater perinatal risk compared to DC gestations ,109,110;The presence of one of the growth-restricted twins has a critical influence on the perinatal outcome ,11,12,17The duration of pregnancy is the longest for sFGR type I cases (between 33.0 and 36.0 weeks of gestation) compared to type II (between 27.6 and 32.4 weeks) and type III cases (between 28.3 and 33.8 weeks). Accordingly, the lowest intrauterine demise (0–4%) ,111 and Generally, type I is characterized by good perinatal outcome and is recommended to be managed expectantly. The adverse perinatal outcome in severe type II and III cases raises concerns on the necessity of further prognostic markers. Laser placental coagulation therapy is the suggested treatment for pregnancies complicated by sFGR with coexisting TTTS. However, the elimination of the acute interfetal transfusion imbalances by laser surgery in isolated sFGR to improve the perinatal outcome and to avoid the potential long-term neurological consequences for the cotwin is still to be addressed. The adequate prediction of the risk of transfusion imbalances may assist in better understanding the risk of unpredictable fetal death in sFGR fetuses.


The progressive appreciation that multiple types of RNAs regulate virtually all aspects of tissue function and the availability of effective tools to deliver RNAs in vivo now offers unprecedented possibilities for obtaining RNA-based therapeutics. For the heart, RNA therapies can be developed that stimulate endogenous repair after cardiac damage. Applications in this area include acute cardioprotection after ischemia or cancer chemotherapy, therapeutic angiogenesis to promote new blood vessel formation, regeneration to form new cardiac mass, and editing of mutations to cure inherited cardiac disease. While the potential of RNA therapeutics for all these conditions is exciting, the field is still in its infancy. A number of roadblocks need to be overcome for RNA therapies to become effective, in particular, related to the problem of delivering RNA medicines into the cells and targeting them specifically to the heart.  RNA therapies hold a great promise to treat several cardiac conditions, including stimulation of cardiac regeneration. This review details the main possible applications of RNA therapies and defines the technological and biological barriers that still need to be overcome for these therapies to become clinically effective..1The repair of damaged hearts remains a holy grail of clinical medicine. Advances in early revascularization after myocardial infarction, the development of devices sustaining the failing myocardium and better clinical management have significantly improved the outcome of heart failure. Still, no drug or treatment is available to protect the heart from damage or promote its regeneration. Neither are any effective treatments available to stimulate new cardiac blood vessel formation. All current medical therapies for myocardial infarction and heart failure essentially aim to sustain the function of the survived myocardium and are based on the combination of small molecules.2 these ncRNAs can be classified into one of three classes. The first class includes small RNAs, usually less than 50 nucleotides (nt), of which the most relevant ones are the small RNAs involved in the RNA interference (RNAi) pathway . A second class includes a heterogenous series of RNAs, typically between 50 and 500 nt, which include RNA polymerase III (Pol III) transcripts such as tRNAs or ribosomal 5S rRNAs, Y RNAs, small nuclear RNAs (snRNAs) and intron-derived small nucleolar RNAs (snoRNAs). A third class includes long noncoding RNAs (lncRNAs), which are more than 500 nt in length and are mostly generated by Pol II. These lncRNAs are estimated to be well in excess of 100 000 different molecules in humans.4lncRNAs also include circular RNAs (circRNAs) generated by backsplicing of coding and noncoding RNAs.5Over the last couple of decades, there has been a growing appreciation that a large set of noncoding RNAs (ncRNAs) regulate virtually all biological functions of the cells. According to a recent consensus statement,6 together with 2 mRNA-based COVID-19 vaccines.Together with protein-coding mRNAs, these ncRNA molecules participate in a very broad range of cellular processes, including in all cells of the cardiovascular system. All these processes are thus amenable to regulation by either exogenously administering RNA molecules or by targeting the endogenous RNAs using complementary small RNA segments. The RNA therapeutics field has expanded quicky over the last 5 years for several noncardiac conditions. To date (March 2023), 14 ncRNAs—9 antisense oligonucleotides (ASOs) and 5 short interfering RNAs (siRNAs) —have already reached clinical approval,To what extent and how quickly can we also envisage the development of a new class of RNA therapeutics for cardiac repair and regeneration, which could overcome the need for developing stem cell-based cell transplantation approaches? And what are the hurdles that still need to be overcome to develop such RNA therapies? Here, I will briefly discuss the main foreseeable applications in this area and highlight the barriers that still need to be overcome for effective clinical application.6 A third class of molecules includes aptamers, namely RNAs that bind a specific target, which can be either a biological or a small chemical molecule, by virtue of their 3D conformation7 Finally, a fourth class of ncRNAs includes a heterogenous group of small RNAs with different functions. Among these molecules, there are CRISPR RNAs to guide Cas molecules onto specific DNA targets and other cellular regulatory RNAs, such as Y RNAs.8The types of RNA therapeutics can essentially be divided into 2 categories according to their length (long and short RNAs), as these 2 kinds of molecules not only have different biological properties but also pose different challenges in terms of therapeutic administration. Long RNAs can either code for protein, acting as mRNAs, or not (lncRNAs). Short therapeutic RNAs are all noncoding and can in turn be classified into 4 different classes . The firApplications of RNA therapies for cardiac repair and regeneration can be foreseen in at least 4 areas. These include protection from acute myocardial damage (cardioprotection), induction of new blood vessel formation (angiogenesis), stimulation of cardiomyocyte proliferation (cardiac regeneration) and correction of cardiac inherited conditions (gene editing) . To date9 but also occurs more chronically in several other pathological conditions, ranging from disorders of cardiac overload (such as hypertensive cardiomyopathy or aortic stenosis) to cancer chemotherapy. As the formation of new cardiomyocytes does not occur at a clinically significant rate after birth, the goal of sparing cardiomyocytes from death is of paramount importance.Loss of cardiomyocytes accompanies virtually all conditions leading to heart failure. This can be dramatic during myocardial infarction, when the heart can lose as many as 25% of the 2-4 billion cells that are present in the left ventricle,10). Of note, the need for cardioprotection is not necessarily restricted to the time immediately following ischemia–reperfusion, as a vast number of cardiomyocytes can be saved in the first 2-3 days after the event in the so-called area at risk, which is significantly larger than the actual infart region.11Experimental evidence shows that several ncRNAs can exert an acute cardioprotective effect. Examples include antisense inhibition of miR-92a,12 miR-3413 or miR-124,14 administration of miR-19a/19b mimic,15 or inhibition of the cardiac-specific lncRNA ZFAS1.16Multiple studies in the last couple of decades have focused on the events that occur immediately after ischemia and reperfusion, showing that brief episodes of coronary occlusion/reperfusion preceding or following infarction  can reduce infarct size. Translation of these observations to the clinic, however, has failed so far (reviewed in ref.18:). MicroRNA-loaded EVs with a cardioprotective effect are also spontaneously released after myocardial infarction by platelets or different cells in the heart, in addition to being abundant in the circulation.19The potential of ncRNA therapies for acute cardioprotection is also indirectly supported by the observation that several cell types originally injected into the heart for their presumed regenerative properties do not lead to cardiac regeneration but still exert a transient cardioprotective activity through a paracrine effect, which also involves the production of ncRNA-rich extracellular vesicles  or circRNAs .A few proteins have also been described to exert acute and chronic cardioprotective activity. For example, recent work from my own laboratory has identified three novel cardioprotective proteins  from an AAV library of 1200 factors that were selected in vivo for cardioprotection in infarcted mice.The possible modalities for the delivery of small, ncRNA, or large RNA therapeutics to the heart for cardioprotection and the problems that still need to be overcome will be discussed later, as these are not significantly different from those encountered by the same types of molecules for other cardiac applications.23 some of the fibroblast growth factor (FGF) family members24 and the hepatocyte growth factor (HFG).25 All of these have been proven effective in driving angiogenesis in both small and large animal models.The induction of new blood vessel formation to counteract cardiac ischemia by gene transfer is one of the still unaccomplished goals of gene therapy. Angiogenesis is known to be controlled by a series of growth factors that act in concert to activate both endothelial and mural cells. For endothelial cells, these factors include, among several others, the VEGF family members with their different splicing isoforms,26 Based on the efficacy of a naked, modified mRNA (modRNA) coding for VEGF-A in mice27 and the results of preliminary safety experimentation, a phase II clinical trial was started based on the injection of this modRNA in the myocardium of patients receiving elective coronary artery bypass surgery.28 Earlier in the summer of 2022, however, this trial was removed from the company’s phase II development pipeline (https://www.astrazeneca.com/investor-relations/results-and-presentations.html). The problems that still need to be overcome for effective therapeutic angiogenesis will be discussed later.Taken together, these observations suggest that the delivery of the mRNA coding for these angiogenic factors, either alone or in combination, could induce therapeutic angiogenesis in ischemic hearts, as RNA gene transfer is more effective than plasmid transfection and less inflammatory than adenoviral vectors, which are the 2 modalities used by gene therapy clinical trials so far.30 Thus, an appealing possibility to induce cardiac regeneration is the stimulation of endogenous cardiomyocyte proliferation using gene or RNA transfer. An obvious problem, however, is how to identify the most effective therapeutic molecule.Cardiac regeneration in other species, including fish and amphibians, is not sustained by the expansion and differentiation of stem cells, but by the proliferation of already committed cardiomyocytes.31). The positive effectors of these pathways  are all activators of cardiomyocyte proliferation, and thus either these factors themselves or the regulatory pathways leading to their activation can in principle be leveraged for cardiac regeneration through mRNA or siRNA delivery of the activators and repressors respectively. Work from the Martin laboratory has already shown that the injection of an AAV vector expressing an shRNA against SAV1, a cofactor of the YAP inhibitory kinase MST1/2 (Hippo in Drosophila), leads to cardiomyocyte proliferation and cardiac regeneration in pigs.32 A similar application can be developed based on naked siRNA injection.At least 3 main signal transduction pathways sustain cardiomyocyte proliferation during embryonic, fetal and early neonatal life, and in principle can be stimulated by genetic manipulation. These are the Wnt/β-catenin, the Notch, and the Hippo pathways , miRNAs that also regulate the proliferation of cancer cells , along with individual miRNAs identified through systematic screenings . Other miRNAs are instead expressed in nonreplicating cardiomyocytes and have an inhibitory role . While the inhibition of these suppressive miRNAs using ASOs has a limited role in therapeutic cardiac regeneration, several laboratories have reported on the regenerative effect of the positive administration of pro-proliferative miRNAs. After myocardial infarction in mice, miR-199a or miR-590a,34 miR-294, a member of the miR-302 superfamily,35 and the miR-17 ~ 92 cluster member miR-19a/19b,15 in all cases expressed using AAV vectors, led to cardiac regeneration and restitution of cardiac function.Multiple evidence over the last 10 years, including the results of systematic screenings, has shown that several miRNAs regulate the rate of cardiomyocyte proliferation.36 In all these conditions, the possibility of delivering synthetic ncRNAs remains appealing. Available evidence already shows that, in mice, the single intramyocardial injection of miR-199a-3p or miR-590-3p,37 miR-19a/19b,15 miR302b/c,39 miR-19a/19b15 or miR-70840 mimics is effective for cardiac regeneration. A challenge remains how to formulate these mRNAs in clinically approvable preparations and prove their efficacy in larger animals.The permanent expression of pro-proliferative miRNAs from AAV vectors can be problematic for at least 2 reasons. First, expression of miRNA cannot be controlled over time and, second, both the desired miRNA strand and the miRNA produced from the complementary strand (the 5p and 3p miRNAs) can be generated within the transduced cells. This can lead to undesired effects over time, as recently proven by one of our experimentations in pigs using miR-199a.33) and the metabolic enzyme PKM2.41 Additionally, the transient activation of the 4 reprogramming factors Oct4, Sox2, Klf4, and c-Myc (OSKM), used to generate iPS cells, can partially dedifferentiate cardiomyocyte and stimulate their proliferation,42 while the administration of other transcription factors  can directly reprogram fibroblasts into cardiomyocytes.44Some extracellular and intracellular proteins were also proven to stimulate cardiomyocyte proliferation. These include a few growth factors , the extracellular matrix protein agrin, cell cycle regulators such as E2F family transcription factor, or cyclins A2, D1, and D2 . Besides the problems of delivery, the main issue for such applications is the relatively short half-life of long RNAs, which, in contrast to small ncRNAs that can persist for over 1 week in an active form,37 have a much shorter duration.45 In addition, activation of the innate immune response is usually significant using long RNAs, despite the chemical modifications that are introduced during in vitro transcription.45For all these positively acting proteins, the delivery of their in vitro transcribed, modified mRNAs to the heart could be conceived as a mean to stimulate regeneration. The L. Zangi laboratory has already provided evidence that the administration of a modified mRNA for PKM2 can induce regeneration after myocardial infarction in both mice46 and phospholamban47 cardiomyopathies, delivery of miR-669a as a gene modifier for sarcoglycan dysfunction48 or pre-trans-spliced RNA molecules that induce trans-splicing of lamin A/C49).Several inherited cardiac conditions, including structural and functional genetic defects of cardiomyocytes (cardiomyopathies and inherited arrhythmia syndromes) are, in principle, amenable to be treated using RNA therapies. Noncoding RNA-based treatments have already been tested in some of the dilated cardiomyopathy preclinical models. These include ASOs for exon skipping in titin50). A clinical trial for the treatment of transthyretin amyloidosis51 by knocking out the mutated gene in the liver has already generated interim positive results (American Heart Association Scientific Sessions 2022).The CRISPR/Cas9 gene editing technologies now offer exciting new possibilities for direct DNA modification. The current standard for delivering a gene editing suite  is by using AAV vectors. These vectors, however, are not devoid of problems. They have a limited cloning capacity, which prompts for the identification or generation of smaller endonucleases, require high viral titres which can lead to adverse events, and determine indefinite persistence of the endonuclease in the transduced cells, which is not desirable for reasons of toxicity and immunoreactivity. The possibility of delivering the endonuclease mRNA and guide RNA as in vitro transcribed molecules hold the potential to overcome all these limitations. Multiple preclinical studies point to the feasibility of administering these RNAs in the context of lipid nanoparticles  that blunt activation of the innate immune response, and the co-transcriptional capping with the 5ʹ end Cap1 structure, which further helps suppress immunogenicity.55Nucleic acids are charged, hydrophilic and sensitive to nucleases, which are all obstacles to their effective administration. For ASOs, the introduction of chemical modifications can increase stability and pharmacological properties.56 natural nucleic acids with a phosphodiester backbone require either conjugation with a ligand or delivery through a carrier molecule. The only ligand for ncRNAs that have progressed towards clinical application for targeting the liver is a trimer of Nacetylgalactosamine , which binds the cellular asialoglycoprotein receptor . No such ligand still exists for the heart.A major problem remains the cellular uptake of these molecules, given the negative charge of nucleic acids. While short, single-stranded ASOs with a phosphorothioate backbone can cross a biological membrane when delivered as naked molecules,58 Most RNA applications, however, take advantage of the possibility of delivering nucleic acids using formulations based on polymers, lipids, or other nanosized particles. A specific type of formulation is lipid nanoparticles (LNPs) obtained by the Stable Nucleic Acid Lipid Particle (SNALP) technology. This originally dates back to the late 1990s and has been progressively improved in the early 2000s60 to become very popular after the COVID-19 vaccines. SNALP LNPs are 60-120 nm diameter particles in which the RNA is surrounded by a lipid bilayer formed of 4 lipids, of which one is an ionisable lipid that binds the RNA in acidic conditions to then become neutral at physiological pH (reviewed in ref.61). Besides the 2 COVID-19 vaccines, the SNALP technology was instrumental for the first siRNA product to be approved in the clinic, patisiran for transthyretin amyloidosis in 2018,62 and the above-mentioned in vivo gene editing clinical trial for the same condition.51In vitro transcribed, modified mRNA was reported to enter cardiomyocytes as a naked molecule when administered by intramyocardial injection in a sucrose–citrate buffer.63 Cardiac uptake was significantly increased after ischemia/reperfusion, but transfection was mainly of noncardiomyocyte cells in the infarcted area.While SNALP LNPs continue to be very promising and additional delivery methods are constantly developed, it is becoming quite clear that most systemically administered, nucleic acid-containing nanoparticles are phagocytosed by mononuclear cells in the liver, spleen, lymph nodes, and bone marrow or end up in the liver, while only a small fraction is retained in the heart. A recent study showed that the systemic injection of LNPs containing the DLin-MC3-DMA ionisable lipid, the same as patisiran, transfected the heart with an efficiency that was several orders of magnitude lower than that of the liver.6 for specific references). Methods can also be developed to enrich for a desired miRNA into engineered EVs64 and to include, within the EV membrane, specific ligands to achieve tissue targeting.65Therefore, a holy grail in the field of nonviral, cardiac RNA delivery is to find ligands that would permit specific targeting of cardiomyocytes, similar to the above-mentioned GalNac sugar for uptake in hepatocytes. A few cardiac-specific peptides have been identified through phage display panning techniques, or are ligands for cardiac cell-specific receptors, or antibodies against cardiomyocyte or endothelial cell receptors . The site of injection in the left ventricle muscle can be identified preoperatively by imaging techniques and confirmed during the procedure by electromechanical mapping or imaging.While waiting for an effective targeting system to be developed, cardiac applications can leverage the possibility of administering molecules directly to the heart. .The intramyocardial injection can be performed through surgical access using a mini-thoracotomy followed by trans-epicardial delivery, or during coronary bypass surgery. In a significantly less invasive manner, an intramyocardial injection can be performed by trans-endocardial delivery from an intraventricular catheter inserted percutaneously to reach the cavity of the left ventricle. Several of these catheters have been tested and approved for human application  effect, by which nanosized particles can extravasate from the vasculature and accumulate in tissue thanks to the generation of large fenestrations between endothelial cells due to inflammation.68 The EPR effect fades after approximately 48 h and thus provides a time window that is sufficiently long for therapeutic administration.An even more straightforward and less invasive application is intracoronary administration, which is commonly performed for percutaneous coronary intervention . This type of administration has already been used for cell delivery in over 30 clinical studies.69 Second, in experimental models VEGF-A requires at least 2-3 weeks of continuous expression before a stable vasculature is generated.71 Instead, the VEGF-A modified mRNA has a terminal half-life of 28 h after intracardiac administration.72 Third, the adult heart is a poorly angiogenic environment, in which new blood vessel formation is suppressed.73 Taken together, these observations reduce the surprise that the transient delivery of VEGF-A is poorly effective and prompt a better understanding of the underlying biology of the cardiac endothelium for effective therapies to be eventually developed.The success of RNA therapies for the heart not only requires a series of technical problems to be solved but also that effective RNA molecules are identified. This is well exemplified by the lack of success of all the clinical studies performed so far for therapeutic angiogenesis, and in particular of those using VEGF-A. Our current information indicates that: First, angiogenesis requires at least 30 different factors to lead to the formation of functional neo-vessels, while the vasculature formed in response to VEGF-A alone is leaky and not functional.A similar need for better basic understanding is universal in virtually all fields of cardiac biology. Another example derives from familial cardiomyopathies and inherited defects of the cardiac rhythm, which pose formidable problems to gene treatments. The cDNAs of several of the genes causing these conditions are too large to fit into AAV vectors, hence the recessive forms cannot be easily addressed by replacement gene therapy. Then, several of the mutated genes, in particular those coding for ion channels or desmosomal proteins, have a dominant or codominant inheritance, which would be amenable to gene disruption using CRISPR/Cas9 followed by repair by the cellular Non-Homologous-End-Joining (NHEJ) machinery. However, allele discrimination is poor single-point mutations, as is often the case, can lead to allele insufficiency. In these instances, precise gene correction of the mutated allele would be ideal, but homology direct repair is inefficient in cardiomyocytes. A better understanding of how to induce the homologous recombination machinery in postmitotic cells is thus required for the development of effective RNA treatments.The possibility of developing RNA-based medicines for cardiac repair and regeneration remains exciting and has disruptive potential. However, the successful generation of such a new class of drugs depends on our capacity to overcome the current technical obstacles in delivering nucleic acids to the heart and in better identification of the most effective molecules and their targets.


A single session of physical exercise can acutely reduce experimental pain—a phenomenon known as exercise-induced hypoalgesia (EIH). There is inconsistency in the literature as to (1) whether EIH differentially impact the perception of different types of nociceptive stimuli  and (2) whether exercise selectively modulates pain at exercising body parts, or also involves non-exercising body parts.We aim to characterize the effects of a single session of aerobic exercise on the sensitivity to stimuli activating skin versus muscle nociceptors, within or outside exercising body parts.We will recruit 40 healthy male participants aged 18-30 years. First, we will familiarize them with the sensory assessments and conduct a maximal cycling test. Then, we will assess, in two sessions, the sensitivity to blunt pressure, mechanical pinprick, contact heat and cold, and auditory stimuli before and after a cycling exercise versus a control session. Sensory testing will be performed over the rectus femoris muscle of the dominant leg  and over the flexor muscles of the non-dominant forearm (remote site). EIH will be quantified as the absolute and relative changes in sensitivity to the sensory stimuli before and after exercise.We hypothesize that EIH will be greater (1) in the exercise than in the control session, (2) at the local site compared with the remote site if local changes in nociceptive sensitivity contribute to EIH, and (3) when blunt pressure stimuli are used if EIH involves specific changes in sensitivity of muscle nociceptors.


Over the years, several studies have revealed an important link between thyroid disorders and gallstone disease. According to these studies, hypothyroidism and hyperthyroidism are associated with cholesterol gallstone disease. This association between thyroid hormone disorders and cholesterol gallstone disease is due to the importance of thyroid hormones on cholesterol synthesis, bile functioning and content, and gallbladder motility. Several genes and receptors have been found on the thyroid gland, liver, and gallbladder to verify this association. These genes affect thyroid hormone secretion, lipid metabolism, and bile secretion. Defects in these various gene expression and protein functions lead to bile duct diseases. Other causes that lead to cholesterol gallstone disease are supersaturation of the bile with cholesterol and impaired gallbladder motility, which leads to bile stasis. This article has discussed these factors in detail while highlighting the association between thyroid hormones and cholesterol gallstone disease.  The correlation between thyroid disorders and gallstone disease has been the subject of extensive investigation across several generations. In particular, various hypotheses have been proposed to explore the potential relationship between thyroid disorders and the occurrence of gallstones. The prevalence rate of gallstone disease in general populations ranges from 10% to 15%, indicating its wide occurrence and impact on a significant portion of the population [A study on the association of thyroid hormone deficiency and chronic gallstone disease suggests that the increased hydrophobic character of biliary atresias (BAs) due to the diminished expression of hepatic detoxification enzymes promotes cholesterol crystal precipitation . AnotherGallstone formation involves multiple factors, such as impaired bile flow, increased cholesterol saturation, and altered gallbladder motility. The pathogenesis of thyroid dysfunction in cholesterol gallstone formation varies between hyperthyroidism and hypothyroidism. Hyperthyroidism induces cholesterol gallstones by overexpressing hepatic nuclear receptor genes involved in cholesterol metabolism, while hypothyroidism promotes cholesterol gallstones through increased cholesterol biosynthesis .The association between cholesterol gallstones and thyroid disorders is influenced by several factors, including the overexpression of hepatic nuclear receptor genes  in hyperthyroidism and the promotion of cholesterol biosynthesis in hypothyroidism, both contributing to the development of gallstones in the biliary system . In thisBile formation and compositionBile is a body fluid produced by the liver that is essential for the excretion of cholesterol and toxins and also helps digest lipids from the small intestine . Bile coSex differences between the association of gallstone and thyroid disorders The relationship between gallstone disorders (GSD) and thyroid disorders associated with sex or gender is observed. In one of the studies, thyroid function parameters, total triiodothyronine (TT3), total thyroxine (TT4), and thyroid-stimulating hormone (TSH) were measured along with abdominal ultrasound for GSD diagnosis in euthyroid subjects. There was an inverse association between TT3/TT4 ratio and GSD in men. TT4 was an independent risk factor for GSD. These parameters were not significant in women. They also speculated that in women, the effect of thyroid function on GSD might be overshadowed by that of estrogen as it is a risk factor for GSD. It was concluded that low levels of TT3/TT4 ratio and high levels of TT4 were significantly and independently associated with GSD in euthyroid male subjects. There was no significant relationship observed in female subjects .In another study, hypothyroid and euthyroid mice were studied with respect to the claudin-1 expression in the liver. Claudin-1 plays a role in bile secretion. In hypothyroid patients, bile excretion is decreased, which leads to an increased prevalence of GSD. It was found that in hypothyroid females, there was elevated claudin-1 expression, while reduced claudin-1 expression was found in hypothyroid males compared to the euthyroid animals .Disturbing lipid homeostasisThe regulation of lipid metabolism is primarily governed by the thyroid hormone receptor (TR) and liver X-receptors (LXR) . CholestRegulation by liver x-receptor/retinoid x-receptor pathwayThe LXR/farnesoid X Receptor (FXR)/Takeda-G-protein-receptor-5 (TGR5) system is critically involved in maintaining the balance of cholesterol and lipid levels within the body ,16. LXR Although we have identified a considerable number of genes regulated by LXR, our understanding of genes suppressed by LXR is currently limited . We are Integration of cholesterol and triglyceride homeostasis by LXRsEarlier research has demonstrated that LXRs function as detectors of sterols and restrict the accumulation of cholesterol by reducing its absorption while enhancing its reverse transport and breakdown . SimultaRegulation by thyroid hormone and thyroid hormone receptorsThyroid hormones are vital for maintaining proper body composition and balancing lipid levels within the body . A recenElevated levels of cholesterol are observed in individuals with hypothyroidism and those with thyroid hormone resistance -30. WhenCYP7A1, also known as cholesterol 7α-hydroxylase, serves as the key enzyme that determines the rate of cholesterol conversion to bile acids and plays a central role in regulating cholesterol homeostasis ,36. It hInduction of human cholesterol 7α-hydroxylase (CYP7a1)The hepatic pathway responsible for bile acid synthesis is a highly coordinated metabolic process that plays a vital role in the absorption of dietary lipids and the regulation of serum cholesterol levels -43. CYP7Thyromimetic effects on reverse cholesterol transport (RCT)Thyromimetics are artificially created compounds that mimic the actions of thyroid hormones. These compounds are specifically designed to have a higher affinity for TRβ, primarily TRβ1, which plays significant roles in the liver for reducing serum cholesterol. This preference is in contrast to TRα1, which is associated with adverse effects of hyperthyroidism on the heart, muscles, and bones. Furthermore, thyromimetics are engineered to accumulate selectively in the liver, which is the primary site for regulating cholesterol metabolism . ReverseImpact on canalicular transportersThe composition of biliary lipids in vivo is determined largely at the level of the hepatocyte canalicular membrane . Bile foThyroid hormones, on the other hand, are known to control essential functions in growth, differentiation, and metabolism . Most ofGallbladder motilityOne of the major causes of GSD is due to gallbladder motility dysfunction. This leads to bile stasis, either due to intrinsic defects in the gallbladder or due to impaired cholecystokinin (CCK) release . CCK incImpaired bile flow and viscosityThe rate of bile salt secretion is a major determinant of bile flow, and the secretion of biliary cholesterol and phospholipids is closely related to that of bile salts . The stuA positive linear relationship between bilirubin UGT activity and the ratio of bilirubin di- to monoconjugates present in bile or formed by incubating liver homogenates in vitro at low concentrations of bilirubin (10 to 15 pM) was discovered, indicating that the conjugation activity in the liver primarily determines the makeup of bile pigment. Unconjugated bilirubin (UCB) and bilirubin monoconjugates (BMC) excretion rates reduced significantly and simultaneously in hypothyroidism, although bilirubin diconjugate (BDC) production decreased less noticeably. However, UCB and BMC excretion increased significantly, and simultaneously in hyperthyroidism, the increase in BDC production was far less prominent. Bile flow was found to be reduced by 54% in hypothyroidism. However, in the current study, hyperthyroid animals showed no alterations in bile flow . Using qSphincter of Oddi dysfunction The sphincter of Oddi (SO) may be the cause of the link between gallstone disease and thyroid dysfunction. The sphincter of Oddi (SO) motility regulates the flow of bile . By virtPrevious studies have shown that patients with CBD stones have a higher prevalence of being diagnosed with hypothyroidism than patients with gallbladder stones or age-, sex-, and hospital-admission-adjusted controls . A studyResearchers also come to the conclusion that T4 actively encourages relaxation in human SO that expresses TR beta1 and beta2. A transcriptional mechanism is used to carry out this function, which needs the synthesis of fresh mRNA and proteins and ultimately causes the activation of K+ channels .Rapid weight loss due to hyperthyroidism Weight loss can contribute to the occurrence of gallstones due to a potential mechanism in which bile with high cholesterol levels accumulates in the gallbladder and forms crystals . When obIt is widely recognized that thyroid dysfunction affects the composition and transportation of lipoproteins . In hypeImpact on enterohepatic circulation and detoxificationAccording to research, an estimated 19% of cholecystectomy patients suffered from hyperthyroidism and 17% from hypothyroidism . The intThe process of enteric digestion brings bile acids and lipids to the ileum to be actively transported with sodium to be reabsorbed. The hepatic portion of digestion occurs in the liver sinusoid through sodium transporters . In the Studies have shown that patients affected with gallstones have modified bile acid formation, containing elevated levels of hydrophobic bile acids and a decreased number of hydrophilic bile acids . This arMoreover, another aspect to consider is the importance and role of circulation in the kidneys on cholesterol reabsorption. Hypothyroidism results in decreased renal plasma flow, decreased glomerular filtration rate, and impaired urine concentration and dilation . ThyroidFurthermore, thyroid diseases, such as hypothyroidism, have been linked to alterations in bile metabolism. With reduced hepatic uptake and clearance of bile acids, a proper environment for the formation of CGD is ensured due to the increased number of circulating bile acids . AdditioFurther association of accumulation of bile acids and hepatocyte detoxification leads to CGD. The hepatocytes convert potentially toxic substances through detoxification . ConjugaImpact on nuclear receptor-mediated Lith gene expressionThe fundamental cause of CGD is a disturbed equilibrium between cholesterol, bile salts, and phospholipids . GeneticThe ABC transport proteins, which are expressed at the canalicular membrane and include ABCB4, the transporter for phosphatidylcholine , ABCB11,Nuclear receptors: Nuclear receptors (NRs) such as metabolite- and hormone-sensing transcription factors bind to their DNA target sites as a monomer , homodimer , or heterodimer (FXR and LXR) before forming heterodimers with the retinoid X receptor (RXR) to change gene expression in response to dietary or endocrine signals . Bile acLiver X receptor: The genes involved in sterol, bile acid, and lipid homeostasis are controlled by the LXRs, which include LXRα and LXRβ ,83. AddiFarnesoid X receptor: FXRs induce the expression of ABCB11 ,93. ABCBEstrogen receptor: ESR1 and ESR2 are two closely related classical homodimeric nuclear receptors that estrogens modulate to carry out their biological actions -98. As aImpact of thyroid hormone on nuclear-receptor mediated Lith gene (TR) expressionTRs come in two different isoforms, TR-α and TR-β (Nr1a1 and Nr1a2) respectively ,103. TheThe mouse CYP7A1 gene promoter exhibits interactions between TR and LXR, indicating cross-talk between the receptors . At the T3 activates the gene promoter, as evidenced by the up-regulation of mouse LXR-mRNA and protein expression in the liver. Recent research has demonstrated that the human LXR-promoter is regulated by LXR α . T3 alsoDespite the claim that TR does not appear to alter the lipid metabolic cascade , severalImpact of SREBPs in relation to thyroid hormone receptors on gallstone diseaseTranscription factors include SREBPs. SREBP-1c predominantly but not exclusively increases the transcription of genes needed for fatty acid production . In the At 28 loci, 32 gallstone disease association signals with genome-wide significance were identified using the additive model. Out of them, 11 are known variations, while 21 are novel variations. Through a recessive mode of inheritance, one of the novel common variations, rs708686 upstream of FUT6, dramatically increases its association with gallstone disease -122.In summary, the largest gallstone disease (GWAS) to date discovered 21 novel gallstone disease variants. The connections found specifically highlight the intestinal compartment of enterohepatic circulation in the pathophysiology of gallstone disease and emphasize the importance of sequence variants in genes involved in cholesterol regulation. We conclude that sequence variations influencing the quantity of cholesterol secreted into bile or the ratio of cholesterol to bile acids will likely result in the formation of gallstones.The interplay of the various underlying mechanisms involved in the association between thyroid hormones and gallstone disease was explored in this article. The thyroid hormone's effect on nuclear factors and cholesterol transport proteins is key in cholesterol homeostasis and the development of gallstone disease. Hypothyroidism may cause common bile duct stones by causing a delay in biliary tract emptying and lack of pro-relaxing action on the sphincter of Oddi, while hyperthyroidism, on the other hand, leads to increased cholesterol turnover and consequently causes crystallization of cholesterol-rich bile and formation of debris within the gallbladder. Many studies are still ongoing to fully understand the complex interaction that brings about these associations.


The ever-growing research on lymphatic biology has clearly identified lymphatic vessels as key players that maintain human health through their functional roles in tissue fluid homeostasis, immunosurveillance, lipid metabolism and inflammation. It is therefore not surprising that the list of human diseases associated with lymphatic malfunctions has grown larger, including issues beyond lymphedema, a pathology traditionally associated with lymphatic drainage insufficiency. Thus, the discovery of factors and pathways that can promote optimal lymphatic functions may offer new therapeutic options. Accumulating evidence indicates that aside from biochemical factors, biomechanical signals also regulate lymphatic vessel expansion and functions postnatally. Here, we review how mechanical forces induced by fluid shear stress affect the behavior and functions of lymphatic vessels and the mechanisms lymphatic vessels employ to sense and transduce these mechanical cues into biological signals. The advances in molecular, cellular, genetic, and imaging approaches in the past decade have led to the identification of specific lymphatic endothelial cell (LEC) markers, allowing the distinction between blood and lymphatic vessels and identification of the mechanisms controlling the development and stabilization of lymphatic vessels and their functions. Together, these discoveries have changed our perception of the lymphatic vasculature; namely, it is no longer believed to be a secondary vascular system but rather a system that is vital for human well-being and health. Indeed, the lymphatic system, with its extensive plexus of vessels and interconnected lymph nodes, plays essential roles in tissue homeostasis by clearing waste, in immunosurveillance by controlling immune cell trafficking and responses, and in organ morphogenesis by coordinating tissue regeneration , 2. MoreHere, we provide a brief overview of conventional and newly discovered biological functions of lymphatic vessels and their implications in health and diseases, as this topic has been well described in recent reviews , 2.Chy mice, a model of dermal lymphatic insufficiency, and our study . A. A144]. irection . Notablyirection . In an iirection  showed tInterestingly, the dynamic changes in LEC morphology observed in vitro in response to LSS have also been reported to alter lymphatic barrier function through a mechanism dependent upon Rac1-mediated actin dynamics . In line2, as determined in vitro based on cell alignment and NF-kB translocation, which is a well-studied response associated with vessel stabilization and suppression of inflammatory pathways [2, which is in the range of shear stress values observed in the rat mesenteric lymphatic collecting vessels [Consistent with the low and high levels of shear stress experienced by lymphatic and blood vasculature, respectively, the set point of BECs ranged between 10 and 20 dyne/cmpathways . On In c vessels . The hig vessels .Further supporting the involvement of flow-induced mechanical forces in maintaining the physiological functions of lymphatics, a study using in vitro and in vivo models revealed that transmural flow enhances lymphatic permeability, transport of macromolecules and DC migration into lymphatics  Fig. 2A2A. This 2 shear stress has been shown to trigger the release of ATP from LECs, which results in an increase in eNOS via activation of the purinergic P2X/2Y receptor and enhanced Ca2+ signaling [The effects of FFS on lymphatic function are not limited to initial lymphatics and also apply to collecting lymphatic vessels Fig. . The conignaling . Thus, sAlthough the response to FSS itself in LECs can be sufficient to maintain the complex structure of the vessel necessary for lymphatic functions, LECs may also integrate biochemical and fluid shear stress to do so Fig. . There iAltogether, these findings indicate that synergistic effects between biomechanical and chemical cues control lymphatic biology and functions, and more studies will be needed to uncover novel synergies between FSS and unexplored biochemical factors, such as chemokines and cytokines.Mechanotransduction is the process by which cells translate mechanical information into biological responses. The mechanotransduction of several shear stress responses in ECs, such as cell alignment, has been shown to require VEGFR2. However, the study of Bayens et al.  on diffe2 LSS, while they were more cuboidal under 4 dynes/cm2 ¼ Hz OSS and that these alterations in LEC alignment and cytoskeleton reorganization were triggered by the transcription factors Forkhead box protein C2 (FOXC2) and PROX1 [Several groups have focused their efforts on identifying flow-induced transcription factors mediating FSS responses in lymphatic vessels. An in vitro study showed that LECs become elongated and aligned under 4 dynes/cmnd PROX1  , a target gene of GATA2, as a key player in shear stress-dependent polarization of LECs  Fig. 2B2B. ThougAs reviewed above, FSS is an essential regulator of the stabilization of mature lymphatics, adult lymphangiogenesis and biological functions of lymphatics. To date, two potential shear stress-sensing mechanisms have been identified in lymphatics. The complex involving mechanosensory receptors at the cell‒cell junctions on the LEC surface including PECAM, VE- cadherin, VEGFR2 and VEGFR3 has been proposed as a mechanosensing mechanism of shear stress in lymphatics Fig. . Such meThe involvement of PECAM-1 in lymphatic mechanosensing was revealed in mice with PECAM deficiency, which led to defective lymphatic remodeling, affecting processes such as valve formation . NotablyThe molecular sensor that senses flow-induced mechanical force in LECs and activates ORAI1-mediated calcium entry has remained unknown until the recent follow-up study by Choi et al.  which idConsidering the vital functions that the lymphatic vessels play and the increasing list of their contributions to human diseases, it is imperative to improve our understanding of how these vessels sense and respond to environmental changes encompassing not only biochemical but also mechanical signals. As described in this review, recent research on biomechanical signals further supports the concept that lymphatic vessels are not “inert tubes” but rather highly plastic vasculature structures capable of responding to biomechanical changes and adapting their properties, such as permeability and contractibility, to changes in their surrounding microenvironment. However, many questions remain unresolved, including how biomechanical forces contribute to controlling lymphangiogenesis in pathological conditions such as cancer and metabolic and inflammatory diseases. However, identifying the specific role of biomechanical signals distinct from that of biochemical signals might be challenging because numerous downstream molecules are shared by both signals. It would also be interesting to investigate whether alterations in mechanosensing and/or mechanotransduction in lymphatics are associated with diseases that exhibit altered fluid flow. To this end, new tools may have to be developed to allow the visualization and accurate measurement of lymph flow. Although evidence supports the control of lymphatic functions by FSS, it is likely that lymphatic vessels are exposed to more than one biomechanical cue at a given time, particularly in a pathological setting, where the matrix composition or stiffness is expected to change. Thus, we need to understand how lymphatic vessels integrate multiple biomechanical signals and respond to them to avoid potential deleterious effects.More research is needed to further understand mechanotransduction and mechanosensing in lymphatic vessel during homeostasis and pathological conditions, and the results of such research may lead to the discovery of new promising therapeutic approaches. As an example, targeting the mechanical force sensor, Piezo-1 has shown encouraging preclinical results for the treatment of secondary or primary lymphedema by promoting the growth of functional lymphatic vessels.


Ideal implant placement may reduce surgical complications, such as nerve injury and lingual cortical plate perforation, and minimize the likelihood of functional and prosthetic compromises. Guided implant surgery [GIS] has been used as the means to achieveideal implant placement. GIS refers to the process of digital planning, custom-guide fabrication, and implant placement using the custom guide and an implant system–specific guided surgery kit. GIS includes numerous additional steps beyond the initial prostheticdiagnosis, treatment planning, and fabrication of surgical guides. Substantial errors can occur at each of these individual steps and can accumulate, significantly impacting the final accuracy of the process with potentially disastrous deviations from properimplant placement. Pertinent overall strategies to reduce or eliminate these risks can be summarized as follows: complete understanding of the possible risks is fundamental; knowledge of the systems and tools used is essential; consistent verification of bothdiagnostic and surgical procedures after each step is crucial; proper training and surgical experience are critical. This review article summarizes information on the accuracy and efficacy of GIS, provides insight on the potential risks and problems associatedwith each procedural step, and offers clinically relevant recommendations to minimize or eliminate these risks. The placement of osseointegrated dental implants is nowadays a common procedure in periodontal clinical practice. Implant therapy is driven by the patients' restorative needs and steered by the esthetic and functional demands of each case; concurrently,implant therapy can be limited by anatomic constraints. Therefore, correct implant positioning is critical if an esthetically and functionally acceptable restoration that can be maintained through proper oral hygiene is to be achieved. Furthermore, implantplacement must respect the various critical anatomic elements often present in the vicinity of a restoratively dictated implant site. Consequently, during diagnosis and treatment planning, the implant surgeon must pay close attention to both restorative andanatomic restrictions while selecting an alveolar bone site of adequate quality and quantity to ensure both appropriate and safe implant placement. Implant therapy diagnosis and treatment planning during the early years of osseointegration was based on clinicalexamination, study casts, and radiographs . The inherent limitations of these two-dimensional radiographic techniques were overcome with the adoption of digital computed tomography, and, subsequently, the more widespread use of dentalcone beam computed tomography that has allowed the detailed and precise three-dimensional evaluation of osseous topography . Guided [1] Patient scanning or initiator prosthetic diagnosis for the placement of the implant in the desired position .[2] Conversion of the digital imaging files and importation of the digital images into the software program.[3] Completion of virtual implant treatment planning.[4] Integration of a virtual model of the teeth with the cone beam computed tomography model [derived from an intraoral surface scan or an extraoral scan of stone casts .[5] Selection and fabrication of surgical guide based on the computed tomography model.[6] Implant placement using the fabricated surgical guide.[1] Involvement of all dental care providers from beginning, thus ensuring comprehensive diagnosis and treatment planning and better outcomes.[2] Serious potential complications of implant surgery that can be minimized by guided implant surgery- such as injury to the critical anatomical structures like sinus, nerves, vessels and teeth -15.[3] Allows flapless implant placement , which r[4] Better maintenance of the soft tissue profiles -Gingival margin position, interdental papilla.[5] From practice management point- guided implant surgery offers faster and more accessible record keeping and filing.[6] Avoidance of flap elevation and sutures results in less postoperative pain, edema, and bleeding, and immediate resumption of oral hygiene procedures -26.[7] Evidence suggests that putting an implant through the gingiva has no effect on osseointegration, bone levels, or aesthetic outcomes, as long as the soft tissue punch employed is not greater than the implant diameter -28.[8] When flap elevation and sutures are avoided, postoperative pain, edema, and bleeding are reduced, and oral hygiene practices can be resumed right away . In cont[1] Increased preoperative treatment planning time and additional cost of guide fabrication for each case.[2] Initial cost for the purchase of adequate computer hardware and software programs and special instructions and drills are expensive .[3] Additional training and familiarization with software will take a bit of time.[4] The routine and correct use of guided implant surgery demands a change in philosophy regarding implant placement and necessitates a learning curve and expenses related to training and familiarization with the softwareand the tools provided ,8; such In totally edentulous patients, the successful use of guided implant surgery combined with the simultaneous delivery of a prefabricated prosthesis for the rapid replacement of missing teeth has been described.The use of guided implant surgery in partially edentulous patients has also been evaluated; out of 250 implants (102 patients), 58% were placed flapless, one planned implant had to be changed to a shorter one, and in four posterior cases the limitedinterocclusal distance presented challenges during drilling; in eight cases, implant placement had to be delayed (guides could not be used) because of required bone augmentation, and for nine of the implants the final angle differed from the planned one,without any resulting clinical consequences. When guided implant surgery with bone supported guides (n = 16) or mucosa supported guides  was directly compared with the conventional implant placement protocol (n = 21), flapless procedureslasted significantly less time and resulted in less postoperative pain, analgesic consumption, and instances of trismus and of patient reported hemorrhage -34.HowevThe available randomized controlled trials suggest that guided implant surgery results in greater accuracy, less post operative pain or pain of shorter duration, less swelling, and less surgery time (guided implant surgery system dependent), but at higherfinancial cost; there were no differences between guided implant surgery and conventional surgery in terms of implant success/failure or clinical parameters such as marginal bone loss at 1 year .[1] Poor image quality and dimensional positional accuracies in the cone beam computerized tomography -38.[2] Improper or inadequate virtual implant positioning in treatment planning software.[3] Improper fit of the surgical guide or the surgical guide fracture .[1] Use of occlusal bite index to stabilize lower jaw and prosthesis- to improve image quality .[2] Knowledge of limitations and accuracy level of specific software - to correct the virtual implant positioning .[3] Use appropriate fixation such as three mini screws to stabilize mucosa or bone-supported guides along with special attention when using bone-supported guides -45.When using guided implant surgery the most important inaccuracy is in the vertical dimension (osteotomy depth), with inaccuracy in mesio-distal or bucco-lingual direction being clearly less. This is possibly due to the presence of debris in the implantcavity so that the implant cannot reach its final position, the resilience of the mucosal tissues in mucosa-supported guides, the setting of the gray values during segmentation, the blockage of the implant holders in the sleeves or by the crestal bone,and the deformation of the guide during surgery -47.Guided implant surgery can be an accurate and clinically advantageous procedure when implant therapy is called for. However, substantial errors can occur at each individual step and can accumulate, significantly impacting the final accuracy of the processwith potentially disastrous deviations from ideal implant placement. It is possible to eliminate or reduce these risks, provided that complete understanding of the guided implant surgery process, thorough and careful surgical technique, advanced comprehensivetraining, and adequate case preparation are always in place.


Many adults with phenylketonuria (PKU) rely on medical nutrition therapy  diet with protein substitutes/medical foods) to maintain blood Phe concentrations within recommended ranges and prevent PKU-associated comorbidities. Despite disease detection through newborn screening and introduction of MNT as early as birth, adherence to MNT often deteriorates from childhood onwards, complicating the assessment of its effectiveness in the long term. Via a modified Delphi process, consensus (≥70% agreement) was sought on 19 statements among an international, multidisciplinary 13-member expert panel. After three iterative voting rounds, the panel achieved consensus on 17 statements related to the limitations of the long-term effectiveness of MNT (7), the burden of long-term reliance on MNT (4), and its potential long-term detrimental health effects (6). According to the expert panel, the effectiveness of MNT is limited in the long term, is associated with a high treatment burden, and demonstrates that adults with PKU are often unable to achieve metabolic control through dietary management alone, creating an unmet need in the adult PKU population. PAH) gene and is identified in medically advanced countries via newborn screening detection of elevated blood phenylalanine (Phe) concentrations [4), the natural cofactor of the PAH enzyme, can be offered to a portion of the PKU population expressing residual PAH enzymatic activity [®) can be an option for adults with uncontrolled blood Phe (>600 µmol/L) in the United States (US) or for those >16 years of age in Europe [Phenylketonuria (PKU) results from biallelic pathogenic variants in the phenylalanine hydroxylase  ,11. The Due to the paucity of evidence on the long-term outcomes of adults with adequately controlled PKU, expert opinions were collected through a modified Delphi process, leading to the development of a set of consensus statements that aim to shed light on the long-term effectiveness of MNT and improve the understanding of the impact of MNT on the health outcomes of adults with PKU.Limitations of long-term effectiveness of MNT (7 statements);Burden of long-term reliance on MNT (4 statements);Potential detrimental health effects related to long-term reliance on MNT (8 statements).A modified Delphi process was applied to gain consensus from an international, multidisciplinary 13-member panel of experts with extensive experience in the management of adults with PKU . The panThrough an iterative process of individual voting rounds on a secure online platform, each of these statements was assessed by the expert panel along with providing feedback when not accepting a given statement. The gathered feedback was used to revise the statements without consensus (<70% agreement), which were voted on again in two subsequent rounds. All statements included the option “can’t judge” in case the expert lacked experience to vote on a specific statement. If the option “can’t judge” was chosen, the answer was excluded from the agreement calculations.Statement #1ConsensusDue to a combination of factors, the sustainability of MNT worsens from early adolescence onwards to an extent that strictly following the Phe-restricted diet becomes difficult for most patients, limiting the effectiveness of MNT to control blood Phe concentrations in the long term.100%Statement #2ConsensusFor adults with PKU on MNT, the ability to maintain blood Phe concentrations ≤360 µmol/L throughout life is limited to a minority of patients having a milder disease phenotype, significant discipline in the setting of adequate resources and continued access to care, or to temporary circumstances of intensive medical involvement and support, such as pregnancy.100%Studies in both Europe and the US have demonstrated that achievement of guideline-recommended target ranges starts to worsen during early adolescence and declines further in adulthood ,19,20,21Statement #3ConsensusThe majority of adults with PKU are unable to reach and sustain physiological blood Phe concentrations by strictly adhering to MNT, including a medically prescribed Phe-restricted diet.100%Statement #4ConsensusCurrent treatment targets, albeit evidence-based, allow blood Phe concentrations that are above the physiological range due to the lack of data on the outcomes of adults with PKU with physiological blood Phe concentrations (30–120 µmol/L).100%It has been reported in the literature that patients adherent to MNT do not always succeed in achieving metabolic control ,31. ContStatement #5ConsensusThe current treatment targets may not fully prevent the risk for developing PKU-associated comorbidities, including difficulties in neurocognitive functioning.100%Statement #6ConsensusAdults with PKU are prone to develop deficits in executive functioning, impeding their ability to manage the complexities of their diet, including intake of low Phe foods and frequent administration of protein substitutes/medical foods.77%Recent retrospective observational studies as well as expert opinions have led to the conclusion that it is nearly impossible to achieve physiological blood Phe concentrations (30–120 µmol/L) on MNT, even in adults who strictly follow their dietary restrictions ,11,31. IStatement #7ConsensusDue to the sustainability challenges related to MNT, adults with PKU should be offered pharmacological treatments to lower blood Phe levels when available.85%While currently available and emerging pharmacological treatment options  may allow patients to achieve and sustain physiological blood Phe concentrations , the sigAccording to a recent survey, about 70% of adult patients are dissatisfied with treatment plans that still require protein restriction . For somStatement #8ConsensusIn some countries, the availability and reimbursement of prescribed special low-protein foods and protein substitutes/medical foods are limited, resulting in a high financial and time burden that can cause worry and stress in the adult PKU population.100%Statement #9ConsensusIn adults with PKU, poor metabolic control can contribute to mental health and psychosocial issues. These issues can be exacerbated by the burden of MNT, especially in countries with limited access to protein substitutes/medical foods and inadequate dietetic support.100%Statement #10ConsensusElevated blood Phe concentrations increase the risk for the development of neuropsychological comorbidities, such as anxiety and depression. These can be exacerbated in patients with attempted but unsuccessful adherence to MNT, who often experience feelings of guilt and decreased emotional well-being.85%Limited adherence of adult patients to MNT is not always by choice. In many regions, reimbursement of special manufactured low-protein foods and protein substitutes/medical foods remains an issue, especially for the adult PKU population ,78,79,80There is consensus that patients with PKU should avoid elevations in blood Phe throughout life to preserve neurotransmitter production and myelin synthesis ,2,3. AltStatement #11ConsensusDespite struggling with long-term adherence to MNT and/or having poor metabolic control, some adults paradoxically have a good self-reported QoL due to becoming accustomed to living with PKU and its management.85%Studies have shown that in particular, patients with PKU whose attempts to strictly follow the dietary restrictions are unsuccessful perceive their treatment burden to be high, which, for some, can cause guilt and embarrassment ,47,81,88According to the general population, the Phe-restricted diet and PKU-related symptoms are both associated with disutilities  depending on the degree of protein restriction and symptom severity . UnaffecStatement #12ConsensusThe synthetic nature of the Phe-restricted diet can result in micronutrient deficiencies, especially in patients who discontinue protein substitutes/medical foods but continue to restrict their protein intake.92%Statement #13ConsensusRecent evidence suggests that MNT alters the gut microbiome of adults with PKU, requiring further research to determine its impact on health outcomes.77%Depending on the disease severity, adults with PKU should reduce the intake of natural protein and instead consume synthetic protein substitutes/medical foods (Phe-free amino acids or casein glycomacropeptide-based protein substitutes) supplemented with minerals, vitamins and essential fatty acids to provide sufficient nutrients ,5. To coStatement #14ConsensusAlthough future studies are needed, MNT can increase the risk for disordered eating behaviours due to the restrictive nature, limited food choices and constant focus on diet, especially in patients with low Phe tolerance.100%In recent years, interest in studying the gut microbiome of patients with metabolic disorders such as PKU has grown ,123,124.Statement #15ConsensusPatients with PKU may be at an increased risk of becoming overweight and should be monitored for metabolic comorbidities.100%Abnormal or irregular eating behaviours are inherently related to the restrictive nature of MNT ,125,126.Statement #161Consensus Adults with PKU are at risk for reduced bone mineral density. The aetiology is multifactorial and may be related to an increase in osteoclastogenesis in response to elevated blood Phe concentrations and/or inadequate intake of nutrients present in protein substitutes/medical foods, particularly in non-adherent patients and those with attempted but unsuccessful adherence.100%1 One advisor voted “can’t judge”.Although a recent systematic review and meta-analysis did not find an association between obesity and adherence to MNT in the overall PKU population, a subset of patients with a severe disease phenotype were reported to have a higher body mass index (BMI) than healthy controls . In agreStatement #17ConsensusFor some adults with PKU, protein substitutes/medical foods can cause gastrointestinal discomfort, especially when mitigation strategies are not followed.100%Osteopenia is another important yet controversial health concern in PKU. Similar to studies of obesity, conflicting results have been published on alterations in bone mineral density (BMD) due to variability among the included study participants and assessments used for measuring bone health. A recent systematic review concluded that BMD is reduced in the PKU compared with the general population despite BMD being in the normal range for most patients . In PKU,Comorbidity-claims-based studies reported a significantly increased use of gastrointestinal agents in the adult PKU population compared with matched controls ,17. ThisDespite the availability of pharmacological treatments, MNT remains the mainstay of treatment for many adults with PKU . TheoretDue to the lack of published evidence, there remains uncertainty on the safety of current guideline-recommended target ranges. Although childhood Phe remains the most important determinant of early and late neurocognitive outcomes, the limited effectiveness of MNT in the long term can still increase the risk for developing deficits in neurocognitive functioning in adulthood ,148,149.It has been suggested that both elevated blood Phe concentrations and severe protein restriction together with the intake of synthetic protein substitutes/medical foods may also compromise a patient’s physical health . Many soIn addition to physical health manifestations, the US retrospective claims-based study specifically evaluated the prevalence of neuropsychiatric comorbidities in adults with PKU, out of which many occurred more frequently in the PKU population than the general population . It is wThere remains much uncertainty on the long-term outcomes of adults with PKU as well as a general lack of evidence to support the safety of guideline-recommended target ranges in adulthood. Therefore, some of the statements, especially those that describe the limited long-term effectiveness of MNT, are solely based on the opinion of the expert panel . Because most of the experts are practicing in countries where newborn screening programmes for PKU have been implemented for more than 50 years, the statements focus on early-diagnosed and -treated adults with PKU. Nevertheless, similar opinions may be true for late-diagnosed patients, who are generally even more vulnerable to the long-term detrimental effects of elevated blood Phe. In addition, the current findings cannot be extrapolated to the paediatric PKU population, who may have different unmet needs.There remains an unmet treatment need in the adult PKU population that was reflected through 17 statements that describe the limitations of MNT with regards to the limited long-term effectiveness of MNT, the burden of long-term reliance on MNT and the potential detrimental health effects related to long-term reliance on MNT. Globally, MNT is the mainstay of PKU treatment. However, due to the burden of MNT in the long term, many adult patients are unable to follow it adequately and therefore may be at risk for detrimental health effects that can be related to the lack of metabolic control and/or micronutrient deficiencies. Future research with existing, emerging and more effective treatments should focus on the identification and assessment of patient-reported outcomes to clearly differentiate the impact of blood Phe from the burden of treatment, evaluate whether alternative treatments can address the unmet needs of the adult PKU population and determine if patients would benefit from achieving physiological Phe.


The dominance of the granulomatous infiltrate and the often sparse lymphocytic infiltrate frequently with minimal cytological atypia are features that distract from the correct diagnosis, even when raised by the clinician. We describe the clinical and histopathologic characteristics of 3 cases of granulomatous cutaneous T-cell lymphoma, illustrate the close clinical and pathologic relationship between GMF and GSS and emphasize the diagnostic difficulties that the granulomatous infiltrate can present. Furthermore, we demonstrate, for the first time, considerable elastolysis in a significant proportion of classical (Alibert-Bazin) MF lesions and therefore postulate that the differences observed between GMF and GSS are one of degree and secondary to their anatomic location rather than reflecting meaningful separate entities.Granulomatous cutaneous T-cell lymphoma includes mycosis fungoides with significant granulomatous inflammation (GMF) and granulomatous slack skin (GSS), listed in the WHO classification as a subtype of mycosis fungoides (MFs). Epidermotropism is only observed in half of the cases and cytological atypia is often minimal.3 Thus, many such cases are misdiagnosed as granulomatous dermatitis, with subsequent diagnostic delay.6 GMF lesions clinically present as classic (Alibert-Bazin) MF.7 In contrast, granulomatous slack skin (GSS) is clinically distinctive, with erythematous patches and plaques, often poikilodermatous that develop into pendulous folds of lax skin occurring at intertriginous regions.8 Despite this distinct clinical presentation, it has a similar histology to GMF; a granulomatous proliferation with variably heavy admixed T lymphocytes. Nevertheless, numerous multinucleated foreign body giant cells and loss of elastic fibers are hallmarks of GSS; however, elastophagocytosis is only directly observed in a minority.3 Although elastic tissue loss is also observed in GMF, it is reported to be more extensive in GSS lesions.9Granulomatous mycosis fungoide (GMF) is a rare subtype of cutaneous T-cell lymphoma (CTCL) occurring in approximately 6% of all MF cases.In this study, we present 3 cases of granulomatous CTCL, which illustrate the histologic overlap between GMF and GSS, and also demonstrate often considerable dermal elastic tissue loss in classic (Alibert-Bazin) MF.+ CD45RA + T-cell phenotype with loss of CD7. In the first biopsy, CD68 confirmed the dominant macrophage component  MF were analyzed with elastic van Gieson histochemistry for assessment of dermal elastic tissue. None originated from the axilla or groin. Elastic van Gieson staining confirmed substantial loss of dermal elastic tissue in lesional samples in 16 patients, without the clinical formation of lax skin folds Figs.  and 9. O10 but uncommonly observed in primary cutaneous lymphomas.6 Partly in view of their rarity, the clinicopathologic features of granulomatous CTCLs are often not recognized. Furthermore, in GMF and GSS, the dermal infiltrate may be dominated by granulomas rather than lymphocytes, often with a lack of epidermotropism and little cytological atypia of the lymphocytes, features that do not readily suggest a diagnosis of cutaneous T-cell lymphoma and often leading pathologists to favor inflammatory and infectious conditions. Moreover, in cases that present clinically with skin laxity or folds, the clinician may not propose lymphoma as a candidate diagnosis.Granuloma formation is well-documented in systemic lymphomas,The 3 cases presented herein highlight the similarities between GMF and GSS. In cases 2 and 3, there were clinical areas of skin laxity or folds, 1 of which (case 3) had more prevalent areas of patches of MF. Case 2 was dominated by multinucleate—foreign body type—giant cells, whereas case 3 had a predominantly lymphocytic dermal population and scattered admixed giant cells, with some foci of sparse epidermotropism; it was this case that had the only observable elastophagocytosis. Case 1 presented with clinical features of MF, but the diagnosis was delayed histologically because of the dominance of epithelioid granulomata and/or a more diffuse population of individual macrophages, some multinucleate, with admixed lymphocytes having little atypia. Indeed, lymphocyte cytological atypia was mild in all cases. Case 2 was notable for the striking dominance of foreign-body type giant cells and very few lymphocytes, perhaps an indication of a late stage in the disease process, supported by this patient having the longest prediagnosis clinical period.3 In particular, no specific histologic feature was uniquely associated with GMF or GSS. The mechanism for bulky pendulous lesions in GSS is believed to be secondary to a reduction in dermal elastic tissue and the presence of multinucleate giant cells a response to this. Interestingly, however, elastophagocytosis was directly observed in only 1 of our cases, despite multiple biopsies and numerous granulomata, and was also observed only rarely in the EORTC series. The apparent close relationship between GSS and GMF raised the question of elastic tissue abnormalities as a feature of MF generally. Our finding that most biopsies from Alibert-Bazin MF lesions also have significant absence of elastic fibers underlines this feature as a common finding in these diseases, and simultaneously suggests that the granuloma formation itself is unlikely to have a pathogenic role. It can be postulated, therefore, that the GMF and GSS differ from classical MF in the extent of this process, and GMF and GSS depending on whether the marked and widespread elastolysis affects gravity-sensitive sites. The basis for this presumed elastolysis is unclear. Metalloproteinases are enzymes that play a central role in tissue remodeling including the digestion of elastic and collagenous tissues. Although much work has been performed characterizing the process in the invasion and metastasis of carcinomas,11 there are as yet no mechanistic studies in cutaneous lymphoma. Because most patients with MF, GMF, and GSS do not succumb to advanced and metastatic disease, the fact that dissolution of elastic tissue seems a widespread observation, implies that elastolysis is not an indicator of aggressive disease and imminent dissemination.In 2008, the EORTC published the results of a workshop analyzing 15 cases of GMF and 2 of GSS and made some of these observations.12 Unlike GSS, the lesions do not affect the flexural areas and elastophagocytosis is rarely observed. Poikilodermatous MF is characterized by wrinkled skin lesions with a “cigarette paper”-like appearance along with atrophy, reticulate pigmentation, telangiectasia, and scaling. Histologically, a perivascular lymphohistiocytic infiltrate is present in the dermis with evidence of ectatic vessels and pigmentary incontinence. Unlike granulomatous CTCL, epidermotropism is often observed and a loss of elastic fibers in the superficial dermis is documented.13 Cutis-laxa-like MF, which is characterized by large areas of wrinkled skin on nonflexural sites is histologically comprised of an interstitial infiltrate of atypical lymphocytes and histiocytes within collagen bundles. Unlike in granulomatous CTCL, the cerebriform lymphocytes exhibit epidermotropism with focal loss and fragmentation of elastic fibers in the absence of elastophagocytosis.14 Finally, the clinical observation of skin wrinkling in patches of classical MF is commonplace  MF, could clinically result in wrinkling or laxity of the skin. However, because there are no significant reproducible histologic differences between GMF and GSS, we propose that the distinction between the GMF and GSS mirrors the degree of elastolysis, response to the elastic fragmentation, and—most significantly—the anatomic location of the disease, rather than these representing distinct clinicopathologic entities.


Heterogeneous fibre types exist in skeletal muscle as a continuum from slow- to fast-twitch to facilitate specialized function. Type I (oxidative fibres) present a slow-twitch phenotype, characterized by high oxygen capacity and increased fatigue resistance. In contrast, type IIa (fast oxidative glycolytic phenotype) and type IIx (fast glycolytic) present faster twitch speeds and contractile force a faster time to fatigue and a recued reliance on oxidative energy metabolism.et al. in 3Function used femoral artery transection of the lower limb in  mice to induce hypoxia in vivo and anoxia ex vivo to challenge different muscles  to hypoxia and anoxia insults. Following this process, the authors tested the functional consequence of oxygen availability by measuring muscle force production and metabolic changes during anoxia exposure (3 h). After a hypoxic insult, the authors observed higher fibrotic damage in EDL and SOL compared with FDB skeletal muscle, suggesting that muscle location might alter functional capacity in the absence of oxygen.It has been proposed that the location of the muscle  to the site of hypoxic insult might dictate the functional and necrosis response to reduced oxygen availability. To test this hypothesis, a recent study published by Amorese et al.3 showed that during anoxia insult in the presence of potassium cyanide (KCN), an inhibitor of mitochondrial respiration, the FDB skeletal muscle maintained force production compared to the EDL, despite lower mitochondrial content, respiration and membrane potential. Thus, the authors elegantly demonstrated that the FDB response to oxygen availability differed to other muscles in the lower leg, a response that did not seem to rely on altered mitochondrial function.Unlike other skeletal muscles located in the distal region, the FDB skeletal muscle presented preserved contractile function during anoxic conditions compared with EDL and SOL suggesting that this unique phenotype might be attributed at least in part by oxygen tension or energetic supply (glycolysis). To test this directly, Amorese Given the KCN results, the authors speculated that the attenuated force and energy production of the FDB may be attributed to an upregulation of glycolytic metabolism. To test this hypothesis, the authors incubated FDB muscle with the glycolysis inhibitor bromopyruvate (BrP) for 3 h. All three skeletal muscles  treated with BrP displayed a force reduction at a similar timepoint, as well as similar glycogen depletion in anoxia exposure, confirming that FDB does not depend on glycolysis more than other muscles. To further delineate the unique phenotype of the FDB skeletal muscle, Amorese and colleagues compared the proteome of the three skeletal muscles  using tandem mass tag (TMT)-labeled mass spectrometry and analyzed differential expression patterns of over 2000 proteins across skeletal muscles. This approach identified that the number of glycolytic proteins normalized to the total number of identified proteins was significantly lower in the FDB compared to EDL but not SOL.Considering the deleterious effect of the glycolytic inhibitor on the force production of the FDB skeletal muscle, the authors examined the abundance of the transmembrane glucose transporter protein type 1 (GLUT1), a rate limiting step in glucose metabolism. The results showed that GLUT1 protein content in FDB skeletal muscle was 1.9-fold higher than EDL and 2.1-fold higher than SOL. Additionally, the authors investigated whether GLUT1 plays a decisive role in fatigue resistance in FDB utilizing skeletal muscle-specific GLUT1 knockout mice (GLUT1 KO). The deletion of GLUT1 in the FDB skeletal muscle resulted in a marked loss of force production following anoxia exposure whereas this response was preserved in wild-type mice. Furthermore, force production in FDB skeletal muscle was significantly different between genotypes after 100 min of anoxia exposure whereas no genotype effect was found under oxygenated conditions.4 Protecting skeletal muscle from hypoxia is a critical need to improve tissue survival during states of acute hypoxia or during muscle wasting. Thus, understanding the potential mechanisms by which FDB can maintain function in the absence of oxygen may provide important new information as to how and why hypoxia and anoxia are detrimental to muscle function in clinical scenarios.5The work from Amorese and colleagues is a wonderful example of how skeletal muscle has evolved to deal with specific environmental stresses to meet energetic demands. This remarkable plasticity to adapt to environmental, physiological, and pathological conditions exhibited by skeletal muscle is conferred by changes in muscle contractile phenotype and metabolic shift.6 are common features of sedentary behavior resulting from hospitalization, limb immobilization, or bedrest. Systemic hypoxia, has been identified as contributing to poor musculoskeletal states in multiple clinical populations, including chronic obstructive pulmonary disease (COPD)7 and heart failure.8 Amorese and colleagues have identified a unique phenotype of the FDB muscle and its ability to maintain its function in the complete absence of oxygen. Thus, studies such as this, where different muscles are examined in response to fundamental metabolic/environmental stressors may identify novel biology to explain why muscles can or cannot adapt to alterations in demand. Certainly, these results support the inclusion of numerous tissue analysis in rodent metabolic studies factoring in the relevance of anatomical properties of muscle in specific disease pathologies. To support this notion, recent work from Naruse et al. (2023) have reported that muscle-specific atrophy is varied in human skeletal muscle in response to aging.9 Furthermore, the trajectory of muscle loss was variable when comparing across muscle groups , providing useful information for development of strategies to prevent muscle loss. Indeed, measuring mechanistic responses in both atrophy-prone and atrophy-resistant muscles, has recently been proposed as a valuable approach to identifying novel pathways in disuse atrophy models, which may provide novel targets for therapeutic intervention.10 Thus, rather than considering divergent responses as a biological anomaly, perhaps studying muscle such as the FDB can provide valuable information into the unique adaptive potential of skeletal muscle in health and disease.Disuse atrophy, characterized as a loss of muscle mass, reduced muscle strength, and metabolic dysfunction,