The corpus luteum is an endocrine gland that synthesizes the steroid hormone progesterone. luteinizing hormone (LH) is a key luteotropic hormone that stimulates ovulation, luteal development, progesterone biosynthesis, and maintenance of the corpus luteum. Luteotropic and luteolytic factors precisely regulate luteal structure and function; yet, despite recent scientific progress within the past few years, the exact mechanisms remain largely unknown. In the present review, we summarize the recent progress towards understanding cellular changes induced by LH in steroidogenic luteal cells. Herein, we will focus on the effects of LH on inter-organelle communication and steroid biosynthesis, and how LH regulates key protein kinases  responsible for controlling steroidogenesis and autophagy in luteal cells. The ovary is a highly dynamic organ that undergoes some of the most dramatic structural and functional changes of any adult tissue. Reproductive potential and reproductive life span are determined by the ovarian follicles, which are the major endocrine and reproductive compartments of the ovary. The follicles are comprised of three essential cells: theca cells, granulosa cells, and the oocyte. Together, these cells interact in a synergistic manner to secrete sex steroids and protein hormones that contribute to oocyte maturation for successful fertilization.Gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion from the hypothalamus, inducing the release of the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), from the anterior pituitary gland. FSH acts directly on granulosa cells of the developing ovarian follicle to stimulate growth of the follicle, secretion of estrogen and expression of the LH receptor (LHCGR) ,2,3. LH The corpus luteum is a transient endocrine gland that synthesizes peptide and steroid hormones and serves as the primary source of progesterone during estrous/menstrual cycles and early pregnancy ,11. ProgProgesterone plays a vital role in fertility. Expression and activation of the progesterone receptor (PGR) is required for ovulation . FurtherIn the present review, we summarize the recent progress in LH-triggered metabolic events in luteal cells. We review the contribution of various organelles and inter-organelle communication as a fundamental platform for the maintenance of basic luteal cell functions. The review also focuses on the role of LH on the activity of two main kinases  responsible for regulating metabolism, as well as metabolic events triggered downstream of 5′-AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (MTOR) in luteal cells. Due to the narrow focus of this review, the authors apologize for the inability to include all of the excellent contributions by other scientists studying the formation and regression of the corpus luteum.Luteinization is a complex process characterized with rapid proliferation of endothelial cells, and cessation of proliferation and hypertrophy of the steroidogenic cells. In studies using a rat model, ovulatory doses of LH inhibited proliferation of granulosa cells . IncreasIn domestic farm animals like the cows, sheep and pigs, the small and large luteal cells are highly steroidogenic and differ based on the size, morphology, and function ,56,57. IIntracellular organelles were once thought to function as individual units. However, recent progress in the understanding of membrane dynamics has revealed intracellular membrane compartments engage in extensive communication, either indirectly, or directly through membrane contacts, orchestrating dynamic communication between organelles that can be modulated to serve cellular requirements. Inter-organelle communication is fundamental for cell and tissue homeostasis as it regulates vital processes including calcium ions homeostasis, lipid and protein synthesis, metabolism, and cell death ,64,65,66Mitochondria play a diverse role in cellular physiology from cell survival and metabolism, to initiating cell death mechanisms by producing free radicals and releasing apoptotic proteins . These hThe dynamin family member DRP1 is a key mediator of outer mitochondrial fission. DRP1, a large dynamin GTPase, is recognized as the major player in mitochondrial fission in mammals , but theGenetic knockdown of DRP1 and MFF, but not fission protein FIS1, disrupts exogenous stimuli–induced mitochondrial fission and apoptosis in HeLa and MEF cells . RecentlMitochondrial dynamin like GTPase  is a mitochondrial dynamin related GTPase that is characterized as an inner mitochondria membrane fusion protein . OPA1 isS-OPA1) and long (l-OPA1) isoforms that are released into the intermembrane space or tethered to the inner mitochondria membrane, respectively [l-OPA1 contains a transmembrane domain and mediates mitochondrial fusion, while S-OPA1 takes on a soluble form and is involved in modulating mitochondrial energetics. Alterations to mitochondrial physiology, such as loss of mitochondrial membrane potential, ATP depletion or induction of apoptosis, leads to further proteolytically processing of l-OPA1 by OMA1 to generate the S-OPA1 isoform that is released into the intermembrane space. Regulation of OPA1 processing allows for alterations in the mitochondrial network through post-translational mechanisms that are more rapid than changes in gene expression [Constitutive proteolytic processing of OPA1 by YME1 like 1 ATPase (YME1L) and/or OMA1  generates short  homeostasis and regulating apoptosis; while the ER is involved in protein folding, lipid metabolism and Ca2+ homeostasis [The mitochondria and ER are dynamic organelles that play a vital role in maintaining cellular homeostasis. Mitochondria are responsible for synthesizing ATP, maintaining intracellular of calcium ion  and translocase of outer membrane 22 (TOM22) are both involved in the regulation of STAR activity at the outer mitochondria membrane. In steroidogenic cells, STAR interacts with VDAC2 prior to its translocation to the mitochondrial matrix [In steroidogenic cells, cholesterol is transported to the mitochondria by STAR, a transport protein that regulates cholesterol transfer within the mitochondria ,119. STAl matrix . This inl matrix . In the l matrix .Mitochondrial dynamics are dependent on interactions with microtubules and actin filaments, both components of the cellular cytoskeleton. Mitochondrial-cytoskeletal interactions have a well-established role in mitochondrial motility . Actin c2+ storage, protein synthesis and lipid metabolism [The ER is a large, dynamic organelle essential for maintaining cellular functions including Catabolism . ER servtabolism ,131,132.ER homeostasis is sustained by ER chaperone proteins, glucose-regulated protein 78 (GRP78), heat shock protein 90 kDa beta member 1 (GRP94), calreticulin (CRT) and protein disulfide isomerase (PDI). ER stress occurs when the capacity of the ER to fold proteins becomes saturated, often as a result of impair protein glycosylation or disulfide bond formation, or by overexpression/mutations in proteins entering the secretory pathway ,134. In Recent studies demonstrated connection between metabolic hormones such as Visceral-adipose-tissue derived serine protease inhibitor (Vaspin) and ER homeostasis. Vaspin, also known as Serpin A12, is an adipokine that plays important roles in glucose metabolism and inflammation. Vaspin is a member of the serine protease family and binds to its receptor 78-kDa GRP78 ER chaperone. Like adiponectin and leptin, the level of vaspin are affected by gender, with significantly higher expression in females . RecentlLipid droplets are unique organelles that serve as lipid reservoirs storing neutral lipids such as cholesterol esters and triglycerides ,143,144.Ovarian luteal tissue is easily distinguished from other tissues due to the abundant lipid content and cytoplasmic lipid droplet . Luteal O-acyltransferase, alpha beta hydrolase domain containing 5 and lower levels of adipose triglyceride lipase, and sterol O-acyltransferase proteins [Lipid droplets have a unique architecture enclosed by a phospholipid monolayer that is decorated by a specific set of proteins which forms the boundary between the aqueous cytosol and the hydrophobic inner lipid core. The PLIN family of lipid droplets coat proteins that are important for stabilizing lipid droplets structure and provide a platform for protein assembly on the lipid droplets surface. PLIN1 localizes to the surface of adipose tissue lipid droplets, however it is not clear whether PLIN1 binds at the surface or is embedded within the phospholipid monolayer. The activity of PLIN1 is regulated in a PKA-phosphorylation dependent manner, and its phosphorylation is known to rapidly initiate lipolysis. Adipocytes highly express PLIN1, a major lipid droplet binding protein, whereas PLIN1 is undetectable in lipid droplets of the bovine corpus luteum . Luteal proteins . The preproteins . Howeverproteins .N-terminal region of PLIN1 to initiate the hydrolysis of cholesterol esters. This mechanism is not prominent in bovine luteal cells that express little PLIN1 [Luteal lipid droplets are known to be regulated by luteal trophic hormones, such as LH, which mobilize lipid droplet cholesterol, and luteolytic hormones, such as PGF2α, which increase lipid droplet triglyceride content ,158,159.Although HSL has broad substrate specificity, the kinetics of hydrolysis of neutral lipids and cholesterol esters vary based on fatty acid size and degree of unsaturation. Structurally, fatty acids consist of a carboxylic acid cap, a long aliphatic carbon chain, and hydrogen atoms along the length of the chain. In steroidogenic tissue, HSL more readily mobilizes fatty acids by hydrolysis as their chain length decrease (between 12–24 carbons) and degree of unsaturation increases ,167. TheThe notion that lipid droplets and mitochondria interact is well-supported. Luteal mitochondria also interact with lipid droplets at specific contact sites ,59. ProtLysosomes are the terminal degradative compartment of endocytosis, phagocytosis and autophagy . LysosomLysosomes play a dual role in both lipid transport and lipid biogenesis and integration of these two processes may play important roles in progesterone biosynthesis and luteal function ,171. OneLysosomes have been implicated in multiple events in the ovary, such as follicular and oocyte atresia, ovulation, steroidogenesis, and luteal regression ,174,175.Changes in lysosomes number and size, as well as enzyme activity have been implicated in late luteal regression in the porcine and bovine ,177. LysThe maintenance of cell function requires utilization and metabolism of nutrients, which are an important source of proteins, lipids, amino acids, nucleotides, and energy. This is achieved through a network of well-synchronized cellular metabolic pathways that direct the utilization of various nutrients. Characterizing the principles underlying these cellular pathways can provide important insights into the understanding of cell function. Cellular metabolism is regulated by two main protein kinases i.e., AMPK and MTOR, which act in an antagonist manner. Both protein kinases are expressed in the granulosa and theca cells, oocytes and luteal cells ,182,183.AMPK is a trimeric protein composed of catalytic α subunit, scaffolding β subunit, and a regulatory γ subunit and is activated in response to reduced levels of ATP (elevated AMP/ATP ratio) . IntraceAMPK regulates diverse metabolic events including inhibition of lipolysis and synthesis of fatty acids by phosphorylating HSL and acetyl-coA carboxylase alpha (ACACA) . HSL intIn contrast, treatment with LH increases the phosphorylation of HSL at Ser563 (activation site), a site specific for PKA, and decreases HSL phosphorylation at Ser565 (inactivation site). Additionally, LH prominently reduces phosphorylation of AMPKα at Thr172 (activation site), while simultaneously increasing the phosphorylation of AMPKα at Ser485 (inactivation site) . These fMetformin, an oral medication commonly used to treat type 2 diabetes, is a known activator of AMPK. Metformin is thought to activate AMPK by the inhibition of complex I of the electron transport chain leading to reductions in ATP production and increased content of AMP. The cellular bioavailability of metformin is controlled by membrane transporter proteins belonging to the solute carrier family (SLC) and multidrug resistant transporters of the superfamily of ATP-binding cassette (ABC) transporters . In ovarThe MTOR is a conserved serine/threonine kinase, which forms two structurally and functionally distinct complexes i.e., MTORC1 containing MTOR, RPTOR (regulatory associated protein of MTOR complex 1 or raptor), MLST8 , and PRAS40 (proline-rich AKT substrate of 40 kDa); and MTORC2 consisting of MTOR, RICTOR (RPTOR independent companion of MTOR complex 2), mLST8, and mSIN1  ,193. MTOThe activation of MTORC1 is dependent on nutrients and growth factors . In the Autophagy, so called ‘self-eating’, is a process during which cells destroy damage or used organelles to recycle inter-cellular content and building blocks required to maintain basic cellular functions . AutophaAutophagy is inversely regulated by the metabolic regulators, AMPK and MTOR. Activation of AMPK regulates autophagy at different levels including inactivation of MTOR via phosphorylation of RPTOR (Ser772 or Ser792) or TSC1/2 (Thr1227 or Ser1345) as well as direct activation of proteins related to autophagy induction and autophagosomes formation such as Unc-51-like kinase 1 (ULK1 Ser555 and Ser317) or (Ser93 or Ser15) . In addiIn the ovary, autophagy has been associated with follicle development and atresia as well as luteal formation and regression ,218,219.Many independent studies demonstrate the occurrence of autophagy during luteal regression . In cowsRecent studies demonstrate that AMPK and MTOR1 differentially regulate autophagic signaling pathways in bovine luteal cells ,213. In The cyclical development and regression of ovarian follicles and corpora lutea in the ovary represent one of the most fascinating endocrine-controlled, developmental biology systems in the adult. Proper systemic and local controls of these processes ensure fertility and fetal development, or resumption of the ovarian cycle in the absence of pregnancy. To understand the mechanisms of action of hormones and other factors that control ovarian steroidogenesis and cell fate it is imperative to appreciate the contribution of various intracellular organelles and potential inter-organelle communication as a fundamental platform for regulation and maintenance of basic luteal cell functions.Understanding the intracellular mechanism of action of hormones and other factors requires knowledge of the contributions of two vital proteins kinases  to ovarian cellular metabolism, inter-organelle communication and steroidogenesis.LH is a key luteotropic hormone that stimulates ovulation, luteal development, progesterone biosynthesis and maintenance of the corpus luteum. A summary of cellular and metabolic changes induced by LH in steroidogenic luteal cells is shown at What is the repertoire of intracellular signaling events triggered by luteotropic and luteolytic agents? Where are these signals located in the cell and how do they affect cellular function?Can advanced cellular and organelle imaging techniques be used to improve our understanding of inter-organelle communication? Can this information be used to better understand cellular metabolism and control of steroidogenesis?Will analysis of the follicular and luteal proteome reveal actionable targets for improvement or control of fertility? Can we identify specific hormone-responsive protein kinases and their substrates in follicular and luteal cell types?How do hormones control post-translational changes other than phosphorylation  and what is their contribution to ovarian function?What important cellular metabolic changes occur during the follicular to luteal transition? Can these be targeted to rescue or terminate luteal function?What are the intracellular metabolic changes induced by luteotropic and luteolytic agents? Can metabolites be identified that can regulate steroidogenesis and cellular fate?Over the last several years, considerable new research by many laboratories has identified several key factors and signaling mechanisms that play crucial roles in the cyclical development and regression of ovarian follicles and corpora lutea. These studies have contributed to significant enhancement of the basic knowledge underlying the mechanisms of hormone action, aiding the development of novel clinical approaches to improve female fertility or manage infertility-associated disorders. However, many questions remain unanswered:


Haemophilus influenzae; those affected during nonpeak months or during non-respiratory syncytial virus (RSV) predominant months; those with wheezing at presentation; those with clinical characteristics such as atopic dermatitis or a family history of asthma in a first-degree relative; and those infants infected with RSV genotypes ON1 and BA, have the greatest likelihood of benefiting from albuterol. Presently, this patient profile could serve as the basis for rational albuterol administration in patients with viral bronchiolitis, at least on a therapeutic trial basis, and it could also be the starting point for future targeted randomized clinical trials (RCTs) on the use of albuterol among a subset of infants with bronchiolitis.Although there is increasing evidence showing that infants with viral bronchiolitis exhibit a high degree of heterogeneity, a core uncertainty shared by many clinicians is with regard to understanding which patients are most likely to benefit from bronchodilators such as albuterol. Based on our review, we concluded that older infants with rhinovirus (RV) bronchiolitis, especially those with a nasopharyngeal microbiome dominated by  Data from several studies have shown that affected patients exhibit a high degree of heterogeneity in their clinical presentation, immune responses, and molecular immune signatures, and show a high probability of distinct responses to different therapeutic options (phenotype-specific treatment strategies).7For decades, it has been a well-established position that bronchiolitis treatment is mostly supportive, focusing only on observation, hydration, and oxygen supplementation. Although prior evidence-based medicine (EBM) clinical practice guidelines (CPGs), such as the 2006 American Academy of Pediatrics (AAP) bronchiolitis CPG, recommended the use of bronchodilators on a trial basis,8 Just as it is inappropriate to use albuterol indiscriminately for all patients with the diagnosis of “viral bronchiolitis”, it would also be inappropriate to fail to administer it to patients who could benefit from it. Using albuterol based on specific patient characteristics or biomarkers, that is, the use of personalized precision medicine, instead of a “one-size-fits-all” treatment strategy could help contribute to the treatment of “viral bronchiolitis” in a more cost-effective way. Although the most appropriate way to reach the above-mentioned goal is to conduct randomized clinical trials (RCTs) using biomarkers to stratify patients most likely to respond to treatment with albuterol, on a provisional basis it could be of central importance to identify the patient profile that has the greatest likelihood of benefit from albuterol based on current knowledge of immune responses and molecular immune signatures.However, despite this novel and promising way of understanding the disease, a core uncertainty shared by many clinicians is with regard to understanding which patients are most likely to benefit in some way from each of the currently available medications .9 This is because despite the lack of RCTs demonstrating a beneficial effect of albuterol in certain phenotypes of viral bronchiolitis  up to now, there is a scientific rationale that supports this practice. Seumois et al.10 examined the correlation between expression of asthma-specific genes and lung physiological measures such as forced expiratory volume in 1s (FEV1), bronchodilator reversibility (BDR) following albuterol treatment, and methacholine challenge, and found a moderately good correlation  between BDR and transcriptional profiling of Th2 cells , suggesting that the molecular program in circulating Th2 cells may influence BDR. We, therefore, aimed to summarize and analyze the current evidence that could help identify possible phenotypes or subgroups of bronchodilator responders among infants with “viral bronchiolitis”.This patient profile could serve as a basis for rational albuterol administration in patients with “viral bronchiolitis”, at least on a therapeutic trial basis.11 Notably, Human rhinovirus (HRV)-infected infants display a different acquired immunological response compared to infants with RSV bronchiolitis, with a predominant Th-2 polarization, exhibiting significantly higher Th2 cell frequencies, significantly higher Th2 index ,12 and increased airway interferon lambda receptor 1 (IFNL1R) transcript levels.13 Additionally, in infants with viral bronchiolitis, HRV and RSV infections have different nasal airway microRNA profiles associated with a nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling. Hasegawa et al.14 demonstrated that infants with HRV have higher levels of NFκB induced type-2 cytokines (IL-10 and IL-13) compared to those with RSV infection. Along the same lines, there is evidence showing that RSV and RV bronchiolitis are associated with significantly different nasopharyngeal metabolomes and bacterial metagenomes. Stewart et al.15 demonstrated that RSV and RV are associated with different metabolic pathways and that the associated bacterial functional capacity is derived primarily from Streptococcus pneumoniae in RSV bronchiolitis and from Haemophilus influenzae in RV bronchiolitis. This evidence supports the concept that viral bronchiolitis should be considered to be a heterogeneous disease that involves a complex interplay among virus, microbiome, and host. Supporting this concept, Zhang et al.16 demonstrated that in infants <6 months of age during the first episode of severe RSV bronchiolitis, the relative abundance of Haemophilus, Moraxella, and Klebsiella was higher in infants who later developed recurrent wheezing by the age of 3 years than in those who did not. Also, in comparison to infants with RSV bronchiolitis, those infected with RV are more likely to be older, have a prior history of eczema, be treated with systemic corticosteroids,17 have a significantly shorter length of stay,18 and have an increased risk of subsequent development of childhood asthma.20 Additionally, it has been estimated that there is a 25% population-based increased risk of early childhood asthma following infant bronchiolitis occurring during RV-predominant months compared to asthma following infant bronchiolitis during RSV-predominant months.21 Interestingly, new concepts have recently emerged concerning a possible significant role of HRV in asthma inception. Recent genome-wide association studies (GWAS) have shown that the ORMDL3 locus in chromosome 17q, the most highly replicated GAWS finding for asthma to date, seems to exert its effects by increasing susceptibility to RV in early life.22 The hypothesis emerging from these data is that infants with RV-bronchiolitis, especially those with a nasopharyngeal microbiome dominated by H. influenzae, could be good candidates for administering or testing albuterol in an attempt to improve clinically important outcomes.Respiratory syncytial virus (RSV) and rhinovirus (RV) are the most important causative agents of viral bronchiolitis.23 after dividing infants according to hospitalization during the peak months and nonpeak months of the year (with RSV infections predominating during the peak months), found significant differences in terms of risk factors for respiratory diseases: infants hospitalized during the peak months had a lower family history of asthma, had more smoking mothers during pregnancy, had a slightly higher percentage of being breastfed, had a lower number of blood eosinophils, and had higher clinical severity scores. The authors hypothesized that infants hospitalized during the peak months of bronchiolitis epidemics and those hospitalized in nonpeak months might reflect two different populations of infants. The same group performed additional analyses aimed at testing the hypothesis that the balance of type-1/type-2 immune responses differs between these two populations of infants. They found that infants hospitalized during the nonpeak months had a significantly higher percentage of CD4 T cells producing IL-4, a slightly lower percentage of CD8 T cells producing IFNγ, and a significantly higher Th2 polarization than infants hospitalized during the peak months.Cangiano et al.,24 Although these findings could be explained by the predominance of RV infections that may occur during the non-RSV-predominant months, this seems not to be the case always, because, although in the 10 consecutive annual epidemics evaluated RSV was detected mostly during peak months, RV was equally distributed during the seasons.24 Although replication is needed, all these evidences collectively support the idea that albuterol could be used or tested in infants suffering from viral bronchiolitis during nonpeak months or during non-RSV-predominant months.The authors concluded that there are at least two different bronchiolitis phenotypes: previously healthy full-term infants, hospitalized with RSV bronchiolitis during the peak months, and infants with a possible genetic predisposition to atopy, hospitalized during the nonpeak months.25 analyzed data from two prospective, multi-center cohorts of children younger than 2 years old hospitalized with viral bronchiolitis. Severe bronchiolitis profiles were determined by latent class analysis, classifying children based on clinical factors and viral etiology. Among the four clinical profiles (phenotypes) identified, it is worth highlighting “Profile A”: patients characterized by a history of wheezing and eczema, wheezing at the emergency department (ED) presentation, and RV infection.Dumas et al.27 Interestingly, two independent studies aimed at a better understanding of predictors of prescription of albuterol among clinicians who attend infants with viral bronchiolitis have identified the age of patients as an independent predictor of prescription of albuterol, with the older patients being more likely to be prescribed this bronchodilator therapy.29Children in this profile were also more often boys, more often older (>6 months), and more often had a parental history of asthma. These results support the hypothesis that children in this profile present early signs of asthma and could be potential responders to albuterol. Additionally, older meta-analyses of the efficacy of bronchodilator therapy in viral bronchiolitis, which showed modest short-term improvements in some clinical features included older infants (greater than 1 year of age).Altogether, these results support the concept that the older the patient with viral bronchiolitis, the higher the probability of obtaining clinical benefit with albuterol use.30 Our group went further, evaluating nasal airway levels of type-2 cytokines (IL-13/IL-4). Children wheezing at first hospitalization and with at least one subsequent episode of asthma-like symptoms had higher nasal airway levels of type-2 cytokines (IL-13/IL-4), with no significant differences in other cytokines.31 Collectively, these data indicate that infants with wheezing at presentation have a higher probability for developing subsequent recurrent asthma-like symptoms with higher nasal airway levels of type-2 cytokines; therefore, suggesting a potential role of bronchodilators such as albuterol in treating their condition.Although infants requiring the first hospitalization due to viral bronchiolitis have a high risk of developing subsequent recurrent asthma-like symptoms, few studies have evaluated the utility of bedside clinical assessment  for predicting the risk of recurrence. Our group evaluated both individual risk factors and bedside clinical parameters for predicting recurrence after viral lower respiratory tract infection (LRTI) hospitalization in a cohort of young children. Wheezing and family history of asthma were identified as the only two independent predictors of recurrence after viral LTRI hospitalization in young children.6 there is increasing evidence showing that these recommendations have not yet had a major impact on all physicians’ behavior, not necessarily due to a lack of awareness of these guidelines.32 Specifically, there are reports showing rates of use of bronchodilators ranging between 18 and 90%, with substantial differences between countries and even between hospitals in the same country.34Despite the recommendations against the use of bronchodilators given in the more recent CPGs on viral bronchiolitis,35 reported that dexamethasone with salbutamol shortened the time for readiness for infirmary discharge during bronchiolitis episodes in patients with atopic dermatitis or a family history of asthma in a first-degree relative. Additionally, atopic dermatitis was identified as an independent predictor of inappropriate use of diagnostic tests and management of bronchiolitis, which in turn was mainly due to the use of inhaled or nebulized Beta-2 agonists.34 Interestingly, supporting this practice, there is evidence showing that atopic dermatitis aggravates allergic airways inflammation in patients with acute viral bronchiolitis.36Although clinicians may have decided to use albuterol and other bronchodilators intuitively, or perhaps even instinctively in patients with certain clinical characteristics, there is some evidence supporting this therapeutic decision. Specifically, Alansari et al.All this evidence collectively suggests that infants suffering from viral bronchiolitis with clinical characteristics such as atopic dermatitis or a family history of asthma in a first-degree relative could benefit from therapy with albuterol.37 studied the RSV genotype distribution, clinical presentation, and disease severity in 998 previously healthy term infants less than 1-year-old hospitalized for bronchiolitis over 12 epidemic seasons. Stratifying data according to genotypes NA1, ON1, and BA showed that when compared to infants infected with genotype NA1, those infected with genotype BA had less severe symptoms and more frequently had eosinophilia and familiar history of asthma. Similarly, although to a lesser extent, those with the ON1 genotype had more risk factors for asthma and atopy than infants infected with genotype NA1. Although future replication studies are required, these findings, therefore, imply that the less virulent RSV genotypes (ON1 and BA) preferentially cause bronchiolitis in infants with a possible genetic predisposition toward asthma and atopy. Accordingly, even though genotypification of RSV is not readily available for clinical use at this time, these findings suggest a potential role of bronchodilators such as albuterol in treating infants infected with RSV genotypes ON1 and BA.Midulla et al.Despite the recommendation against the use of albuterol in infants with viral bronchiolitis given in the main CPGs on viral bronchiolitis, there is increasing evidence showing that this recommendation is not being followed by a not inconsiderable percentage of pediatric care providers. Although at first glance this use of albuterol might appear inappropriate, there is increasing evidence showing that infants with viral bronchiolitis exhibit a high degree of heterogeneity in their clinical presentation, immune responses, and molecular immune signatures, as well as probably distinct responses to different therapeutic options such as albuterol. Identifying the patient profile that could benefit most from treatment with albuterol is essential for rational albuterol use in patients with viral bronchiolitis, at least on a therapeutic trial basis.H. influenzae; those affected during nonpeak months or non-RSV-predominant months; those with wheezing at presentation; those with clinical characteristics such as atopic dermatitis or a family history of asthma in a first-degree relative; and those infants infected with RSV genotypes ON1 and BA have the greatest likelihood of benefit from albuterol (Based on our review, we concluded that older infants with RV-bronchiolitis, especially those with a nasopharyngeal microbiome dominated by lbuterol .Presently, this patient profile could serve as a basis for rational albuterol administration in patients with viral bronchiolitis, at least on a therapeutic trial basis, and it could also be the starting point for future targeted RCTs on the use of albuterol among a subset of infants with bronchiolitis.


Endodontics is the branch of dentistry concerned with the morphology, physiology, and pathology of the human dental pulp and periradicular tissues. Human dental pulp is a highly dynamic tissue equipped with a network of resident immunocompetent cells that play major roles in the defense against pathogens and during tissue injury. However, the efficiency of these mechanisms during dental pulp inflammation (pulpitis) varies due to anatomical and physiological restrictions. Uncontrolled, excessive, or unresolved inflammation can lead to pulp tissue necrosis and subsequent bone infections called apical periodontitis. In most cases, pulpitis treatment consists of total pulp removal. Although this strategy has a good success rate, this treatment has some drawbacks . In a sizeable number of clinical situations, the decision to perform pulp extirpation and endodontic treatment is justifiable by the lack of therapeutic tools that could otherwise limit the immune/inflammatory process. In the past few decades, many studies have demonstrated that the resolution of acute inflammation is necessary to avoid the development of chronic inflammation and to promote repair or regeneration. This active process is orchestrated by Specialized Pro-resolving lipid Mediators (SPMs), including lipoxins, resolvins, protectins and maresins. Interestingly, SPMs do not have direct anti-inflammatory effects by inhibiting or directly blocking this process but can actively reduce neutrophil infiltration into inflamed tissues, enhance efferocytosis and bacterial phagocytosis by monocytes and macrophages and simultaneously inhibit inflammatory cytokine production. Experimental clinical application of SPMs has shown promising result in a wide range of inflammatory diseases, such as renal fibrosis, cerebral ischemia, marginal periodontitis, and cancer; the potential of SPMs in endodontic therapy has recently been explored. In this review, our objective was to analyze the involvement and potential use of SPMs in endodontic therapies with an emphasis on SPM delivery systems to effectively administer SPMs into the dental pulp space. The word endodontics comes from the old Greek “endo” meaning “inside” and “odont” for “tooth.” Endodontics is the branch of dentistry concerned with the morphology, physiology and pathology of the human dental pulp and periradicular tissues. Dental pulp is a connective tissue that can trigger an immune response when challenged by different stimuli. In vital teeth, injuries such as dental caries, trauma, operative procedures, and periodontal diseases can lead to pulp inflammation, which is termed pulpitis. Pulpitis represents one of the main emergencies in dental practice, is the most prevalent form of orofacial pain, and has been associated with the prescription and potential overuse of opioid analgesics . SimilarHow to best treat deep carious teeth with inflamed pulp is still controversial . For manAn alternative approach is to remove only the inflamed portion to maintain and preserve the healing potential of the pulp. The ultimate goal of this concept, so-called vital pulp therapy (VPT) is the preservation of pulp vitality and function. The benefits of VPT include the maintenance of defense mechanisms, pain perception as a warning system, healing and tissue regeneration, completion of root formation in young patients to strengthen thin dentin walls and prevention of long-term complications. Despite these advantages, VPT has not become standard practice among dentists or gained general acceptance in the dental community. VPT outcomes have been difficult to predict due to the variable inflammatory status of the remaining pulp tissue. Indeed, pulp inflammation often remains high, leading to pulp necrosis , 14–16. Thus, it is crucial to identify molecular and cellular agents that can attenuate immune/inflammatory events and promote the rapid return of tissue homeostasis. Blocking the immune response entirely with drugs such as anti-inflammatory or immunosuppressive agents is either ineffective, as the immune response contributes significantly to regeneration or has significant side effects . ExtensiBacteria are the main and initial cause of inflammation and pulp infection. Carious lesions, trauma, wear and cracks provide bacteria and their byproducts access to the pulp through the dentinal tubules. Odontoblasts are the first cells to contact bacteria and their toxins due to their peripheral location in dental pulp tissue. As other cells  play multiple roles, these immunocompetent cells initiate and orchestrate the oral immune response . OdontobThe low compliance of the pulp environment, the absence of collateral replacement due to terminal vascularization, and the diversity of the microflora at the sites of the carious lesions are responsible for a relative inefficiency of the immune response leading more easily than other tissues to necrosis . Innate Until recently, the importance of inflammation in pulpal healing has been underestimated and merely considered an undesirable side effect. New evidence suggests that inflammation is a prerequisite for tissue healing and pulp regeneration. Indeed, the initiated inflammatory response can lead to cell death and tissue destruction or toward wound healing and tissue regeneration. This dynamic balance is linked to the presence and concentration of pro- and anti-inflammatory mediators. A slowly progressing carious lesion can result in pulp healing that is no longer possible if inflammation progresses , 30. MorApical periodontitis (AP) is an inflammatory lesion of the apical and peri-radicular areas mainly caused by bacterial elements from an infected tooth. During pulpitis, bacteria aggregate into a biofilm that adheres to the canal wall. Biofilm development in the root canals is initiated just after the first invasion of the pulp chamber by oral bacteria following inflammatory breakdown of pulp tissue . This biBacteria exert their pathogenicity through direct and indirect mechanisms. Bacterial factors such as enzymes, exotoxins, peptidoglycan, lipoteichoic acid, and lipopolysaccharides are released into the periradicular tissue, leading to phagocytic influx and the production of proinflammatory mediators. Neutrophils are the first cells involved in the defense and progression of AP, and these cells cause the chemotaxis of monocytes and lymphocytes , 40. BonAdvances have been made in understanding the pathogenesis of AP, especially the identification of cells and mediators capable of modulating the immune system. The dynamic balance between the protective effect of inflammation and its destructive capabilities is an active field of investigation that includes immunotherapy –47, antiHistorically, the resolution of inflammation has been considered a passive process involving the dilution of chemokine gradients over time; thus, circulating leukocytes no longer sense gradients and are no longer recruited to the site of injury . HoweverThe resolution of inflammation is coordinated and regulated by a large number of mediators, including SPMs.  These mediators are derived from polyunsaturated fatty acids (PUFAs) and bind to G protein-coupled receptors (GPCRs)  Fig. 2)2). ApproArachidonic acid (AA) synthesis and its derivative products are key regulators involved in the host immune response. The oxygenation of AA initiates the synthesis of powerful bioactive compounds called eicosanoids. These metabolites include prostaglandins, leukotrienes, and lipoxins, which are lipoxygenase (LOX) interaction products. Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) were the first mediators identified that possess specific proresolving actions , 94. TheLipoxin has three synthetic routes that depend on the cellular environment and result in the formation of lipoxygenase eicosanoid products  Fig. 2)2). The fThe second route is linked to platelet-leukocyte interactions. This synthetic route takes place in the vascular system and involves 12-LOX, which is activated during peripheral blood platelet − leukocyte interactions .Interestingly, the third biosynthesis route is dependent on aspirin and leads to the generation of 15 epi-lipoxin A4, which is also known as aspirin-triggered lipoxin (ATL), and 15 epi-lipoxin B4. Aspirin induces the acetylation of cyclooxygenase-2 (COX-2), transforming AA into 15R-HETE. 15R-HETE is then transformed by leukocytic 5-LOX into 15-epi-LXA4 and 15-epi-LXB4 . The conDuring inflammatory reactions in vivo, the synthesis of lipid mediators occurs in two stages. In the initial acute phase of inflammation, the leukotriene B4 concentration increases with the infiltration of neutrophils. The accumulation of neutrophils also coincides with increasing prostaglandin E2 (PGE2) concentrations. This prostaglandin induces LOX activity, and the resultant lipoxins contribute to the resolution of inflammation , 103.Lipoxins play multiple roles in the inflammatory response, even at picogram-level concentrations . LipoxinResolvin (Rv) is an term for resolution-phase interaction products. These factors are natural exudate products defined by their potent bioactivities. These endogenous mediators are synthesized during the resolution phase of inflammation from ω-3 fatty acids, including eicosapentaenoic acid (EPA) to produce E-series resolvins  and docosahexaenoic acid (DHA) to produce D-series resolvins (RvD1–RvD6), following COX-2 acetylation or cytochrome p450 activity  RvD1–RvD6 protect against PMN-mediated reperfusion-induced organ injury . RvD2 prThe biosynthesis of protectins and maresins involves the formation of epoxide intermediates of DHA Fig.  90]. Wh. Wh90]. There is increasing evidence that SPMs play an important role in the reduction of pain , 121. SoSPMs have been studied by extensively and their potent effects on reducing inflammation have been documented, along with the development of a wide range of preclinical disease models using validated commercially available SPMs . Most ofA protective effect of RvE1 was shown in an animal model of pathogen-mediated inflammation . Histological analysis was performed at 24 and 72 h posttreatment and revealed that RvE1 reduced inflammation and induced milder pulp alterations  than the other treatments. These results also demonstrated the lack of benefit of directly applying corticosteroids to the pulp tissue . MoreoveNumerous studies support systemic oral administration of omega-3 PUFAs for their beneficial effects on inflammatory diseases, including rheumatoid arthritis, bowel diseases, and chronic periodontitis , 134. A This omega-3 regimen could also be used to treat AP because its pathological mechanisms of action involve comparable inflammatory and cellular mediators. Systemic oral administration of proresolving mediators (omega-3 PUFAs) decreased the expression of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-17 and increased production of the anti-inflammatory cytokine IL-10 in a rat model of AP . These rIn a rat model of apical periodontitis in immature rat teeth, RvE1 promoted root formation and reduced inflammation . RadiogrIn an animal model of tooth revascularization, RvD2 promoted the resolution of inflammation . RvD2 adSPMs are highly potent, efficacious endogenous ligands associated with positive outcomes in preclinical animal models. Additional studies are required to assess the safety and efficacy of SPMs as therapeutic agents in humans. The development of stable analogs and drug delivery systems is still in the research stage. SPMs are delicate in their physicochemical nature, require complex chemical synthesis and are prone to metabolic inactivation . ClinicaTo overcome these limitations, different pharmacological approaches are currently under investigation to effectively administer SPMs in the dental pulp space. Access to the dental pulp is generally easier than access to the periapical region. However, the root canal may be narrow with a small volume. Therefore, it is challenging to implant a preformed scaffold into the root canal that would seamlessly cover the entire space of the canal. This concern is one of the main issues that must be addressed when considering drug delivery systems to the dental pulp or the periapical region , 142. ThUnlike currently available anti-inflammatory drugs, such as anti–TNFα antibodies, SPMs can dampen excessive inflammation without compromising host defense. Furthermore, SPMs do not impair endogenous healing pathways but rather act locally to halt leucocyte recruitment and promote the resolution of the inflammatory response. The few studies that have explored the role of SPMs in endodontics have generated promising results regarding controlling pulp inflammation, promoting the resolution of apical inflammation, and reversing the bone tissue destruction caused by excessive neutrophil activity. Scientific and technical barriers remain due to the instability, complex and delicate physicochemical nature, and potential metabolic inactivation of SPMs. Hydrogel-based drug delivery systems represent a promising approach to overcoming the aforementioned obstacles, and these systems are currently being investigated for periodontal applications in particular. Although the tissues and treatments vary, pulpal and periapical diseases share many features; therefore, advances in SPM delivery systems for periodontal applications will greatly benefit both conditions. This would be an important milestone for modular hydrogel strategies and open a new way to design drug delivery systems for specific applications, such as the treatment of pulpitis and apical periodontitis.


Metalloproteinases were first identified as collagen cleaving enzymes and are now appreciated to play important roles in a wide variety of biological processes. The aberrant activity and dysregulation of the metalloproteinase family are linked to numerous diseases including cardiovascular and pulmonary diseases, chronic wounds, cancer, fibrosis and arthritis. Osteoarthritis (OA) is the most prevalent age-related joint disorder that causes pain and disability, but there are no disease-modifying drugs available. The hallmark of OA is loss of articular cartilage and elevated activities of matrix-degrading metalloproteinases are responsible. These enzymes do not exist in isolation and their activity is tightly regulated by a number of processes, such as transcription, proteolytic activation, interaction with their inhibitors, cell surface and extracellular matrix molecules, and endocytic clearance from the extracellular milieu. Here, we describe the functions and roles of metalloproteinase family in OA pathogenesis. We highlight recent studies that have illustrated novel mechanisms regulating their extracellular activity and impairment of such regulations that lead to the development of OA. We also discuss how to stop or slow down the degenerative processes by targeting aberrant metalloproteinase activity, which may in future become therapeutic interventions for the disease. Matrix metalloproteinases (MMPs), adamalysins ), and ADAM with thrombospondin motifs (ADAMTSs) belong to the family of zinc endopeptidases collectively referred to as metzincins. The metzincin superfamily is distinguished by a highly conserved motif containing three histidines that bind to zinc at the catalytic site and a conserved methionine that sits beneath the active site .The human genome contains 23 MMP genes and most MMPs have several domains: a signal peptide, a propeptide, a catalytic metalloproteinase domain, a linker (hinge) peptide and a hemopexin domain . MMPs thThe ADAM family is conserved type I transmembrane metalloproteinases related to the MMPs and ADAMTSs with human genome containing 20 ADAMs. The proteolytically active ADAMs mainly function as ‘sheddases’, cleaving the juxta-membrane region of their trans-membrane substrates to release the soluble ectodomain of the substrate to the extracellular milieu . This acADAMTS proteinases degrade ECM components and are secreted multidomain metalloproteinases, consisting of a signal peptide, a pro-domain, a metalloproteinase domain, a disintegrin domain, a thrombospondin type I motif, a cysteine-rich domain, a spacer domain, and a second thrombospondin motif of variable numbers of repeats at the C-terminal region . The humMost, if not all, of the metalloproteinases are tightly regulated by a number of ways, such as transcriptional regulation, proteolytic activation and interaction with tissue inhibitors of metalloproteinases (TIMPs). Not surprisingly, aberrant activity due to dysregulation of these metalloproteinase family are linked to numerous diseases including cardiovascular diseases, chronic wounds, pulmonary diseases, cancer, fibrosis, rheumatoid arthritis (RA), and osteoarthritis (OA) , 9.OA is the most prevalent age-related joint disorder that causes pain and disability, but there is no disease-modifying intervention currently available, except surgery at end stage of the disease. The hallmark of OA is manifested by progressive degradation of articular cartilage ECM due to elevated activities of matrix-degrading metalloproteinases. In this review, we describe the functions and roles of metalloproteinase family in articular cartilage integrity and degradation. We highlight recent studies that have illustrated novel mechanisms regulating the extracellular activity of cartilage-degrading metalloproteinases and their inhibitors. We also discuss how to stop or slow down the degenerative processes in OA by targeting impairment of such regulations, which may become therapeutic interventions for the disease.341 ~ Phe342 bond (where ~ indicates the cleavage site) in the aggrecan inter-globular domain (IGD) TIMP-3) or Thr2Gly mutation interferes with the inhibition of MMPs by TIMP-3, but inhibitory activity for ADAMTS-4 and 5, and ADAM17 is retained [34 of the inhibitor with MMPs is lost.[-1A]TIMP-3 overexpressed in mouse cartilage led to a significant protection of the articular cartilage in a murine surgical model of OA [The engineering of TIMPs has been an attractive approach to develop inhibitors for a group of metalloproteinases. The crystal structures of the TIMP-MMP complexes , 122 havretained , 123. Thel of OA  and a nael of OA . The unuserine proteinase inhibitors). Serpins are a unique and ancient family of proteins with an elegant mode of inhibition. Target proteinases cleave an extended reactive-centre loop or ‘bait region’, after which the serpin undergoes a major conformational change, and renders a hyperstable and covalent proteinase:serpin complex [Serine proteinase activity is countered by a host of anti-proteinases, the largest family of which are the serpins . They are transcriptionally modulated presumably by increased factors that are normally endocytosed but elevated in the presence of LRP1 antagonists. Mantuano et al.[Both ADAM17 and MMP-14 are responsible for shedding LRP1 in human chondrocytes . Howeverno et al. have shoLRP1 endocytoses not only aggrecanases and collagenases but also a number of other secreted metalloproteinases either directly or through making a complex with TIMPs , 132. In26Ala/Lys45Ala and Lys45Ala/Lys110Ala) without altering inhibitory activity against metalloproteinases. These TIMP-3 mutants have a longer half-life in cartilage and inhibit cartilage degradation at lower concentrations and for longer than wild-type TIMP-3. This illustrates that targeting the TIMP-3 endocytosis pathway is a potential strategy for inhibiting cartilage loss in OA. As mentioned above, sulphated GAGs including heparin, heparan sulphate and pentosan polysulphate are also able to inhibit TIMP-3 binding to LRP1, increase extracellular levels of TIMP-3 and thus protect cartilage ECM from degradation [A canonical mode of ligand recognition by LRP1 underlies a central role for the cysteine-rich complement-type repeats (CRs). These cage-shaped CRs present a characteristic acidic pocket that forms salt bridges with a specific lysine on the ligand moiety, as it was shown for a number of LRP1 ligands , 136. Prradation , 96. Howradation  found thThe ligand-binding regions in LRP1 occur in four clusters (clusters I-IV) containing between 2 and 11 individual ligand-binding CRs. Most of the ligands for LRP1 for which the binding sites have been mapped interact with ligand-binding repeats in clusters II and IV . As mentOA is now widely accepted as a whole joint disease, and indeed all the joint structures including the adjacent bone surfaces, the synovial lining of the joint cavity, tendons, ligaments, and menisci are affected in OA. Although chondrocytes have been considered to be responsible for maintaining cartilage homeostasis by balancing synthesis and degradation of matrix molecules, it is still not clear that the loss of chondrocytes affects cartilage integrity or not . FurtherEndocytic processes, including LRP1-mediated endocytosis, represent important mechanisms for regulating metalloproteinase activity, modulating extracellular levels of the enzymes and their endogenous inhibitors. In addition to endocytic scavenger functions, LRP1 can act as a signalling receptor via interaction of its cytoplasmic domain with various scaffolding and signalling proteins. A study by the Gonias group indicates that LRP1 can initiate different signalling pathways in response to binding of particular ligands to its extracellular domain . This raDuring the development of OA, cartilage ECM is slowly and gradually degraded. A prolonged period where biological changes are continuously taking place in a whole joint is a major challenge presented by the disease. If the rate of ECM degradation is reduced by the agents that inhibit activity of cartilage-degrading metalloproteinases—activation of these enzymes or LRP1 shedding without affecting the synthesis of ECM—this can be of benefit. However, inhibition of specific or multiple proteinases may be only effective at specific stages in the disease process. Further insights into regulations of individual enzymes in the complex environment at multiple stages of the OA development may allow us to develop effective approaches to ameliorate global joint pathology.


PLOS Biology identifies a pathway that synergizes protein synthesis and degradation with remodeling of the microRNA (miRNA)-induced silencing complex (miRISC) to mediate synaptic scaling.Synaptic scaling allows neurons to adjust synaptic strength in response to chronic alterations in neuronal activity. A new study in  This Primer explores the implications of a new study in PLOS Biology which reveals how homeostatic synaptic plasticity requires coordinated and inter-dependent protein synthesis and degradation, as well as remodeling of the miRNA-induced silencing complex. Activity-dependent synaptic plasticity is thought to be a cellular mechanism for learning and memory. However, if left unchecked, persistent changes in synaptic activity would lead to destabilization of neuronal circuits and function. Homeostatic plasticity mechanisms, such as synaptic scaling, operate at a slower timescale to provide homeostatic control of synaptic activity and maintain neuronal firing rate within a dynamic range . SynaptiA receptor antagonist bicuculline, to induce hyperactivity and consequent synaptic downscaling, led to an enrichment of translation regulators, components of the proteasome, and the miRISC-associated E3 ligase Trim32 in polysomes, but to a depletion of MOV10, a helicase, and RNA-binding protein. This evidence suggests that chronic neuronal hyperactivity impacts on the polysome association of the proteasome and translation machineries, as well as elements of the miRISC.Srinivasan and colleagues describe that concomitant inhibition of the proteasome and translation blocks synaptic downscaling and the decrease in surface AMPA receptors triggered upon prolonged neuronal hyperactivity, suggesting that the 2 processes cooperate to induce synaptic downscaling. Indeed, components of the proteasomal machinery and of the microRNA (miRNA)-induced silencing complex (miRISC) were found to cosediment with translation initiation factors in hippocampal polysomes. This association depends on translating transcripts, since it is disrupted in polysomes isolated from RNAse-treated lysates. Interestingly, prolonged treatment of hippocampal neurons with the GABATo understand mechanistically how synaptic downscaling depends on proteomic remodeling that involves both protein translation and degradation, the authors focused on the opposite changes in the expression levels and polysome association of 2 of the miRISC components, Trim32 and MOV10, triggered by chronic neuronal hyperactivity. They found that Trim32 increased expression depends on mTORC-regulated Trim32 translation, which is necessary for proteasome-dependent MOV10 down-regulation. They further showed that Trim32 is the E3 ligase responsible for degradation of MOV10 mediated by the ubiquitin-proteasome pathway.A critical question addressed by Srinivasan and colleagues is whether the Trim32-MOV10-miRISC axis is sufficient to drive synaptic scaling. The approach used to tackle this issue was 2-fold: (i) MOV10 depletion using RNA interference was found to recapitulate decreased surface AMPA receptor levels and reduced amplitude of AMPA receptor–mediated miniature excitatory postsynaptic currents (mEPSCs), which occluded any further synaptic downscaling induced by prolonged hyperactivity; and (ii) Trim32 knockdown blocked downscaling of mEPSCs triggered by chronic bicuculline exposure. Together, these pieces of evidence support the involvement of the Trim32-MOV10 pathway in synaptic downscaling. Finally, the authors inquired into how this pathway could lead to the removal of surface AMPA receptors to bring about synaptic downscaling. Loss of MOV10 enhanced the expression of the immediate early gene Arc/Arg3.1, which is known to mediate homeostatic synaptic scaling of AMPA receptors via the activation of AMPA receptor endocytosis . In addiThe study by Srinivasan and colleagues is noteworthy as it supports the compelling idea that proteome dynamics mediating synaptic downscaling requires translation-dependent protein degradation and miRISC remodeling and as it finds that Arc is one of the downstream targets in this pathway, whose expression is regulated at the posttranscriptional level . It is n


COVID-19 is a disease caused by a new coronavirus SARS-CoV-2, primarily impacting the respiratory system. COVID-19 can result in mild illness or serious disease leading to critical illness and requires admission to ICU due to respiratory failure. There is intense discussion around potential factors predisposing to and protecting from COVID-19. The immune response and the abnormal respiratory function with a focus on respiratory function testing in COVID-19 patients will be at the center of this Perspective article of the Frontiers in Physiology Series on “The Tribute of Physiology for the Understanding of COVID-19 Disease.” We will discuss current advances and provide future directions and present also our perspective in this field. COVID-19 is a disease caused by a new coronavirus SARS-CoV-2, primarily impacting the respiratory system. COVID-19 can result in mild illness or serious disease leading to critical illness and requires admission to ICU due to respiratory failure.A major unresolved conundrum is the large spectrum of clinical presentations of patients with COVID-19, ranging from asymptomatic infections or symptomatic mild infections with fever, headache or mild respiratory symptoms (like cough or sore throat) and malaise in 80–85% of patients to flu-like illness and viral pneumonia. Within the “pneumonia phenotype” we also have a large clinical and pathophysiological spectrum that extends from only minor opacification with near normal chest radiographs and mild hypoxemia . Some of these patients develop an acute respiratory failure with severe hypoxemia of quick progression to a phenotype presenting with greater hypoxemia and higher respiratory rates  to severe diseases manifestations. Seriously ill patients develop severe hypoxemia requiring mechanical ventilation. Their CT scans document edema in the lower lobes, Angio-CTs detect multiple ground-glass opacities often showing micro-embolic lesions and lung ultrasonography that are consistent with interstitial injury with B lines (white lung). This latter phenotype is compatible with an organizing pneumonia with hypoxic vasoconstriction associated with severe hypoxemia . The last phenotype, less common than the previous one, represents an advanced stage with associated acute lung injury requiring mechanical ventilation . A subseIncreasing evidence suggests that these diverse clinical phenotypes might be explained by the immunological failure to control and restrict SARS-CoV2 infection of the lung. Failure and skewing of the adaptive immune system, promiscuous infection of epithelial (pneumocytes), endothelial as well as immune cells, coagulation defects and uncontrolled neutrophilic activation potentially govern the impact of COVID-19 on respiratory function and clinical phenotypes . An incrin situ hybridization as well as immunohistochemistry profiling (SARS-CoV-2 infection primarily targets the respiratory tract. For the virus to effectively enter the host cell it requires the membrane expression of angiotensin-converting enzyme-2 (ACE2) together with cofactors, such as transmembrane serine protease 2 (TMPRSS2), and furin . A plethrofiling . Despite+ T cell responses is essential for fast clearing of infected cells and maintaining long-term suppression of viral infections via immunological memory  that serve to initiate, amplify, and/or sustain host inflammatory responses . In partl memory . This tyl memory .1. The infected cells of the respiratory epithelium thus fail to launch a robust IFN-I response to SARS-CoV2 but at the same produce exuberant inflammatory cytokines which disrupts a balanced anti-viral innate and adaptive immunity  as suggested previously as a general feature of Coronoviruses  function and increased Th17 differentiation as observed in severely ill COVID-19 patients  capacity for carbon monoxide (CO)  reflects the capacity of CO transfer from the environment to the pulmonary capillary blood and represents the most clinically practical standard methodology to assess the gas exchange in the lung. In this review we will use the term TLCO. KCO, the transfer or diffusion coefficient is the rate constant for CO uptake from alveolar gas and is impacted mostly by the thickness and area of the alveolar capillary membrane, the volume of blood circulating in pulmonary capillaries coupling ventilated alveoli and the concentration and properties of hemoglobin in the alveolar capillaries blood ,B. KCO aA low TLCO is not exclusively determined by reduced VA , and resThe second most common abnormality in COVID-19 survivors is a restrictive ventilatory defect. A restrictive ventilatory defect is defined by a pathologically decreased total lung capacity. If caused by parenchymal lung disease, restrictive ventilator defect is accompanied by reduced gas transfer, which may be marked clinically by desaturation after exercise or even at rest (see the above paragraph).Total lung capacity is the greatest volume of gas in the lungs achieved after maximal voluntary inspiration. It depends on the static balance between the outward forces generated by inspiratory muscles during a maximal inspiratory effort and the inward elastic forces of the chest wall and lung. It is the lung that normally contributes the most to the elastic recoil forces of the respiratory system at total lung capacity. At total lung capacity, these two sets of forces are equal and opposite in sign. The decrease in total lung capacity usually reflects the reduced lung volumes either because of an alteration in lung parenchyma or because of a disease of the pleura, chest wall, or neuromuscular apparatus that may affect the pressure-generating capacity of the inspiratory muscles or the compliance of the lung or the compliance of the chest wall. Interstitial lung anomalies as such those observed in some forms of COVID-19  may resuRespiratory function testing has been performed in COVID-19 survivors at the time of hospital discharge and weeks after hospital discharge. This seems an important issue when dealing with COVID-19 survivors as these respiratory function testing anomalies may have a huge impact on the management, independency and quality of life of these patients as well as on the healthcare systems.1) and forced vital capacity (FVC) were lower compared to lower limit of normality values, while FEV1/FVC was higher compared to the upper limit of normality values. These results obtained in a very small sample size should be taken with caution as measure of Total Lung Capacity, preferably with plethysmography, was not included and the diagnosis of restrictive pattern was made exclusively on the reduced FVC, which is questionable and not acceptable . This trend of the gradual decrease in level of TLCO among patients was identical with the varying degree of severity. Of note, in 50% of the TLCO -impaired patients, the TLCO corrected for alveolar volume (TLCO/VA) was still within the normal range, which might indicate that TLCO decrease was more than the TLCO/VA in recovered subjects. In addition, the value of TLC as% of predicted in severe pneumonia cases was much less than that of pneumonia or mild illness, suggesting higher impairment of lung volume in severe cases. No significant difference among the discharged survivors with different severity in regard to other ventilatory defects  was observed.In the Mo’ study 110 patients with COVID-19 infection were enrolled, which included 24 cases of mild illness, 67 cases of pneumonia and 19 cases of severe pneumonia . SpiromeThese two studies, strongly suggest that respiratory function needs to be carefully investigated in COVID-19 patients, as it was already done for other atypical pneumonia, such as severe influenza A (H1N1) pneumonia . This is1, FEV1/FVC ratio, TLC, and TLCO values less than 80% of predicted values, respectively, whereas 49 and 23% of patients presented with maximum static inspiratory and expiratory pressure  values less than 80% of the corresponding predicted values. Compared with non-severe cases (n = 40), severe patients (n = 17) showed higher incidence of TLCO impairment , and significantly lower percentage of predicted TLC. Of note, only 11% of patients showed obstructive and 12% restrictive ventilatory defects  diffusion capacity and perhaps with persistent dyspnea but with no other associated anomalies in chest or CT scan imaging. Techniques capable of measuring more specifically the alveolar-capillary membrane, such as measurement of TLCO including inhaled gas mixtures containing two or three different oxygen fractions or combined TLCO and diffusing capacities of the lung for nitric oxide (DLNO) measurements, are welcome to shed light on the precise mechanisms of reduced TLCO in COVID-19 survivors particularly in distinguishing between interstitial and pulmonary capillary anomalies.•More particularly, two hypotheses on reduced TLCO could be tested in COVID-19 survivors: (1) a reduced TLCO with normal TLCO/VA may be in favor of definitive alveolar loss/destruction, with no optimistic perspectives of recovering; (2) a reduced TLCO with diminished TLCO/VA may be in favor of alveolar lesions  that are still evolving, with potential and optimistic perspective of some recovering, at least partial. We should therefore follow-up COVID-19 survivors to see whether they are able to recover from their DLCO anomalies.•A long-lasting follow-up in terms of respiratory function testing is proposed for COVID-19 survivors as results from literature are conflicting as to whether these patients may fully recover or even develop pulmonary sequelae.This combined perspective on basic immunological responses and physiological abnormalities might foster a better understanding of the disease course and may also shape future stratification of patients and treatment options.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cell therapy, particularly mesenchymal stem/stromal (MSC) therapy, has been investigated for a wide variety of disease indications, particularly those with inflammatory pathologies. However, recently it has become evident that the MSC is far from a panacea. In this review we will look at current and future strategies that might overcome limitations in efficacy. Many of these take their inspiration from stem cell niche and the mechanism of MSC action in response to the injury microenvironment, or from previous gene therapy work which can now benefit from the added longevity and targeting ability of a live cell vector. We will also explore the nascent field of extracellular vesicle therapy and how we are already seeing enhancement protocols for this exciting new drug. These enhanced MSCs will lead the way in more difficult to treat diseases and restore potency where donors or manufacturing practicalities lead to diminished MSC effect. It is almost impossible to catalogue all of the therapeutic investigations conducted and ongoing using stem cells. However, the excitement in this burgeoning field has been somewhat dampened by some less than stellar clinical trial results and persistent variability of effect due to production practicalities and correlative, rather than causative, potency assays. During these studies an immense amount of data has been generated regarding stem cell biology and possible mechanism of action in diseases, including the physiological niche where various stem cells are sourced, cell–cell contact-dependent mechanisms and a rich secretome containing small molecules, proteins, organelles and even full membrane bound bodies. Indeed, much of this data has been accumulated regardless of whether the overall subsequent clinical trials themselves were successful. These have prompted a multitude of strategies that could putatively be included in the cell manufacturing process to improve outcomes in patients. In this review we look at the more important of these strategies to give the reader an insight into the next generation of stem cell and stem cell-derived therapies.Often, in vivo delivery of therapeutic agents is hampered by obstacles such as their short half-life, poor solubility index, clearance shortly after administration, as well as a low targeting ability and potential toxicity toward healthy tissues. Therefore, many pharmacological, natural, and biological-based therapeutics require transport, protection, and direction toward the target site in vivo to improve their therapeutic index.. MSCs exhibit intrinsic therapeutic abilities in a range of injuries and diseases  was shown to induce enhanced osteogenic differentiation via induction of osteogenic markers RUNX family transcription factor (RUNX)-2 & alkaline phosphatase (ALP) and mineralisation of extracellular matrix. Mechanistically, CCPA were shown to activate wingless-related integration site (Wnt) signalling pathways, including the activation of dishevelled protein (DSh) while inhibiting glycogen synthase kinase 3b. Inhibition of dishevelled (Dsh) or Wnt signalling blockade interfered with CCPA mediated benefits from MSCs [MSCs are known to express adenosine receptors  . UtilisiExtracellular vesicles have been proposed as a viable replacement for MSC therapy with the additional benefit of being easier to produce and store. Preclinical studies have reported the potential of EVs as a therapeutic in liver  and cardThere is an abundance of evidence to suggest that the composition of EVs in the MSC secretome is largely influenced by the environment to which the MSCs are exposed , 121. SiPriming with inflammatory cytokines such as IL-1β, TNF-α or IFN-γ was shown to initiate the production of immune-modulatory factors including granulocyte colony-stimulating factor (G-CSF), factor H, and galectins , 134. RaLess is known about the capacity of priming to alter the EV content specifically, yet there is evidence to suggest that inflammatory preconditioning could help engineer specific MSC EVs. Inflammatory priming with TGF-β and IFN-γ cytokines promoted EVs to differentiate mononuclear cells into T-regs , while oPreclinical studies have reported the potential of EVs as a therapeutic in liver  and cardIt has become increasingly clear over recent years that, while stem cell therapy holds immense promise, the path to licensed medical options in the clinic remains difficult (see Table


Although ubiquitination is widely assumed to be the only regulated step in the ubiquitin–proteasome pathway, recent studies have demonstrated several important mechanisms that regulate the activities of the 26S proteasome. Most proteasomes in cells are inactive but, upon binding a ubiquitinated substrate, become activated by a two-step mechanism requiring an association of the ubiquitin chain with Usp14 and then a loosely folded protein domain with the ATPases. The initial activation step is signaled by Usp14’s UBL domain, and many UBL-domain-containing proteins  also activate the proteasome. ZFAND5 is a distinct type of activator that binds ubiquitin conjugates and the proteasome and stimulates proteolysis during muscle atrophy. The proteasome’s activities are also regulated through subunit phosphorylation. Agents that raise cAMP and activate PKA stimulate within minutes Rpn6 phosphorylation and enhance the selective degradation of short-lived proteins. Likewise, hormones, fasting, and exercise, which raise cAMP, activate proteasomes and proteolysis in target tissues. Agents that raise cGMP and activate PKG also stimulate 26S activities but modify different subunit(s) and stimulate also the degradation of long-lived cell proteins. Both kinases enhance the selective degradation of aggregation-prone proteins that cause neurodegenerative diseases. These new mechanisms regulating proteolysis thus have clear physiological importance and therapeutic potential. For many years, our lab has focused on furthering our understanding of the 26S proteasome, both because of its key role in intracellular proteolysis and because of its intriguing molecular mechanisms. The efficient degradation of cell proteins by the ubiquitin–proteasome system (UPS) depends on its capacity to selectively bind ubiquitinated proteins, to disassemble ubiquitin chains, to unfold a large variety of proteins and translocate them into the 20S core proteasome, and to release small peptide products. All of these steps have to be precisely timed and integrated to ensure the rapid clearance of misfolded and regulatory proteins, as well as the slower hydrolysis of more stable cell constituents. Despite the dramatic recent advances in our knowledge of these processes obtained by cyro-electron microscopy of the 26S complex and through biochemical and biophysical studies of its multistep mechanism, many key features of the 26S proteasome’s multistep mechanism are not yet fully understood. Further knowledge about these integrated mechanisms is not only of basic biochemical interest, but also should illuminate our understanding of proteotoxic disease in light of the growing evidence in many neurodegenerative disease models that proteasome function is impaired due to the accumulation of aggregation-prone proteins. The resulting defects in protein degradation most likely accelerate the further accumulation of misfolded proteins, which must interfere with normal cell function.This article reviews a number of important new insights about how the 26S proteasome’s activities are regulated that have emerged from our lab’s recent studies. A key technical development enabling many of these findings was our developing a one-step affinity purification method for gentle isolation of 26S proteasomes (the UBL-UIM method) . This apSurprisingly, this initial activation of the proteasome was not triggered by the interaction of the ubiquitinated substrate with one of the known receptors for ubiquitin chains , but instead by its binding to the proteasome-associated DUb, Usp14 ,3, whichThe further dissection of Usp14’s activation mechanism by Hyoung Tae Kim led to the important finding that this activation upon substrate binding was mediated by Usp14’s UBL domain, whose movement upon substrate binding to Usp14’s active site seemed to cause activation . In factWe have also studied in depth and describe below a very different type of proteasome activator, ZFAND5, a Zn finger protein, whose effects on proteasome activity raise intriguing mechanistic questions and are of clear biological importance . ZFAND5 Finally, we have described a very different, but physiologically very important, type of 26S proteasome activation, through phosphorylation of a proteasome subunit. A number of protein kinases have been reported to modify the 26S proteasome and even to alter its activity ,13, but It is of obvious interest to understand how the phosphorylation of different proteasome components by DYRK2, PKA, or PKG can stimulate the proteasome’s enzymatic activities and to learn how the resulting proteasome activation influences the degradation of different cell proteins. Proteasome activation by cAMP or cGMP leads to an enhanced capacity of the cell to degrade many short-lived cell proteins, including the clearance of various aggregation-prone proteins that are associated with human disease ,16,18. TDuring our efforts to understand how substrate de-ubiquitination on the proteasome may be linked to the ATP-dependent proteolytic steps, Andreas Peth found that the binding of a ubiquitin conjugate to the proteasomal deubiquitinating enzymes, Usp14/Ubp6 or Uch37/UchL5, allosterically activates the proteasome’s degradative mechanism ,3. This These two structural requirements for ATPase activation, a ubiquitin chain and a loose polypeptide domain, do not have to be covalently linked as they are in a ubiquitinated protein, because, on the proteasome, they interact with different parts of the 19S complex, the ubiquitin chain with Usp14 and the polypeptide with the ATPase ring. This two-step activation mechanism also accounts for the important findings by Matouschek and colleagues that proteasomal degradation of a ubiquitinated substrate requires both substrate ubiquitination and the presence of an unfolded domain in it ,21.The finding that Usp14/Ubp6 is a central regulator of 26S function see  has beenThe finding that Usp14 functions in proteasome activation was quite surprising because Usp14/Ubp6 had been shown by Finley’s group to inhibit 26S proteasome activity both allosterically . Thus, UOur finding that Usp14 can function both as an inhibitor and an activator of proteolysis appeared contradictory and initially was quite perplexing. The simplest hypothesis to resolve these apparently conflicting roles of Usp14/Ubp6 would be that Usp14 normally inhibits proteolysis allosterically, but when it binds a ubiquitin chain on a potential substrate, Usp14 allosterically stimulates the proteasome’s degradative activities  2,3,4].,3,4.2,3,Indeed, in the absence of a substrate, and independently of its role as a DUb, Usp14 was shown by Hyoung Tae Kim to inhibit multiple proteasomal processes , this inUsp14KO proteasomes also have a higher basal ATPase activity than WT 26S, and surprisingly, these proteasomes do not require ubiquitin chain binding to stimulate ATP hydrolysis. By itself, an unfolded protein can stimulate ATP hydrolysis maximally . In thisNotably, Usp14KO 26S, unlike the WT, can degrade efficiently certain unstructured proteins without ubiquitination, which probably is a result of their faster translocation into the 20S particle and greater ATP hydrolysis. Moreover, 26S proteasomes lacking Usp14 are not just an informative laboratory construct, but also must be of physiological importance since Usp14 is present normally on only a minor fraction of cell proteasomes and exists freely in the cytosol. Interestingly, Chueh-Ling Kuo in our lab has shown that the presence of ubiquitinated proteins promotes the binding of free Usp14 to the proteasome, from which it dissociates when the ubiquitinated substrates are hydrolyzed . Thus, tThe suppression of proteasomal ATP hydrolysis by Usp14 may also be important in preventing ubiquitin-independent proteolysis in vivo. These findings together provide a clear rationale for these bidirectional effects of Usp14 on proteasome function. In the absence of a ubiquitin-conjugated substrate, Usp14 can help to prevent wasteful ATP consumption and the non-selective degradation of non-ubiquitinated proteins by the proteasome .Because of the importance of Usp14’s UBL domain for its association with the proteasome and for the resulting stimulation of Usp14’s deubiquitinating activity , we hypoWhen Usp14’s catalytic domain binds a ubiquitin chain, Usp14 can sterically inhibit Rpn11’s deubiquitinating activity. The finding that the UBL domain alone stimulates Rpn11’s activity against ubiquitin chains suggests an unexpected coordination between the actions of these two proteasomal DUbs. Furthermore, with casein, a protein with an unfolded domain, present, the UBL domain alone was found to stimulate ATP hydrolysis by the proteasomes. Because the UBL, by itself, alters the activity of the ATPases in a similar manner as does the binding of a ubiquitin chain or ubiquitin aldehyde to Usp14 , it is v5-DHFR and the ubiquitinated Sic1). Additionally, the isolated UBL domains derived from Ubiquilin1 and Rad23B also activated proteasomes, suggesting that this is a general property of such domains. Moreover, when a UBL domain was expressed in HeLa cells as the fusion EGFP-UBL, there was a clear enhancement of overall cellular proteolysis that was accompanied by a fall in the cellular level of ubiquitin conjugates ..17].Moreover, like forskolin, these hormones promoted the selective degradation in the hepatocytes of short-lived proteins, which include misfolded and regulatory proteins, but not the bulk of cell proteins. In additional experiments, proteasome activities and Rpn6 phosphorylation were also shown to increase similarly in working perfused rat hearts following epinephrine treatment, and also in leg muscle biopsies from exercising human volunteers, and in electrically stimulated rat hindlimb muscles . FurtherThese observations in diverse cells and tissues clearly demonstrated that proteasome activation and enhanced degradation of short-lived proteins are physiological responses to cAMP and not just pharmacological artifacts. These findings also confirmed our earlier conclusion that Rpn6-S14 is a bona fide PKA target  and thatProteasomal activity and the ability to degrade short-lived cell proteins thus appear to rise together with the increased glycogenolysis and triglyceride breakdown that are induced in the liver, muscle, and heart by epinephrine via cAMP during the fight-or-flight response and in the liver by glucagon in fasting. In tissues of fasted mice, even up to 2 days, when degradation rates are very high, proteasomal activities increased without any changes in 26S content. There has been appreciable controversy about how starvation affects proteasome content in cultured mammalian cells. Proteasome levels have been reported to rise very rapidly upon nutrient deprivation , to fallThis increased proteasome activity in mouse muscles and liver was clearly evident by 12 h after food was removed from fed animals and thus represents a rather rapid metabolic response to food deprivation. This timing suggests that a similar enhancement of proteolysis should also occur in humans in these tissues after an overnight fast . Incidentally, this rapid response long precedes the FoxO-mediated induction of ubiquitin ligases and autophagy genes that leads to muscle wasting, especially the breakdown of myofibrils, which is evident in rat and mouse muscles at 1–2 days after food deprivation . MoreoveBecause of these exciting discoveries about cAMP and PKA and especially their ability to enhance the cell’s capacity to degrade disease-associated, mutant proteins, we decided to investigate whether other signal transduction systems had similar effects and focused on cGMP and protein kinase G (PKG) . While cThe ability of cGMP and PKG to alter protein turnover generally or to combat the progression of neurodegenerative disease had not been studied systematically. In a mouse model of a cardiomyopathy caused by overexpression of mutant αβ crystallin, Ranek et al.  showed tExtensive studies in our lab by Jordan VerPlank demonstrated that cGMP and PKG also stimulate multiple proteasome activities and cellular proteolysis by the UPS without any effect on autophagy .The experiments implicating cGMP and PKG in proteasome regulation  resembleAlthough the 26S proteasomes activated by treatments that raise cGMP behaved similarly to those from cells where cAMP levels rose, the effects on cAMP and cGMP on cellular protein degradation were quite different. Experiments with radiolabeled amino acids showed that raising cGMP, like raising cAMP, stimulated the degradation of short-lived proteins . HoweverNaturally, we also tested if raising cGMP, like raising cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases . Our colTo further test if agents that raise cGMP may help to combat the progression of other neurological diseases, in collaboration with Laura Feltri and Lawrence Wrabetz  in still unpublished studies, we studied a mouse model of Charcot Marie Tooth 1B, a demyelinating neuropathy caused by the expression of mutant myelin protein zero (S63del). In the sciatic nerve of the S63del mouse, 26S proteasome activities are reduced, ubiquitinated proteins accumulate, and the Unfolded Protein Response (UPR) is activated. Treatment of the mice with sildenafil for 5 days restored proteasome function and reduced levels of polyubiquitinated proteins and markers of the UPR. Remarkably, myelin thickness, decreased amyelinated axons, and improved nerve conduction were observed. Thus, through its ability to stimulate proteasome activity and intracellular protein homeostasis, pharmacological agents that raise cGMP have the potential to combat diverse neurodegenerative and other proteotoxic diseases.Together, these studies have led to an important, new insight into the functioning of the UPS and proteostasis. It was widely assumed that the cell’s capacity for degradation is fixed. However, these studies with cAMP clearly demonstrate that a variety of hormones that activate adenylate cyclases, but have diverse physiological roles, all stimulate proteasome activity and the selective degradation of short-lived cell proteins. Moreover, these responses are surprisingly rapid. After vasopressin addition to renal epithelial cells, proteasome activity and Rpn6-S14 phosphorylation rose maximally within 5 min and returned to control levels by 60 min . SimilarThe physiological importance of this new, dynamic regulation of proteasome function remains unclear. Presumably, enhanced degradation of misfolded and damaged proteins helps cells to maintain proteostasis during stress. In addition, the accelerated breakdown of preexistent regulatory proteins induced by cAMP or cGMP can facilitate changes in cell protein composition upon transitions to new physiological conditions . Activation of proteolysis by cAMP and cGMP was observed in many cell types and may occur in nearly all mammalian cells. However, the specific consequences of this new mechanism for regulating proteolysis in different cells remain to be determined.Although the rate-limiting, highly selective step in proteolysis by the UPS is normally substrate ubiquitination, the finding of proteasome activation implies that the degradation of many proteins can also be regulated at the subsequent step, ubiquitin conjugate degradation by the proteasome, and that the half-lives of many proteins can change rapidly with alterations in the proteasome’s phosphorylation state. This ability of PKA, DYRK2, and PKG to enhance rates of proteolysis by increasing proteasome activity represents a new mode of regulation of protein breakdown ,13. WhilThese two mechanisms to regulate degradation by the UPS do not necessarily function independently. In fact, in addition to enhancing proteasomal activity, cAMP and cGMP stimulate within minutes the ubiquitination of some cell proteins ,16, suggThe present studies also strongly suggest that fasting, exercise, or other conditions that raise cAMP or cGMP levels might also be beneficial in promoting the clearance of potentially toxic proteins and stimulating protein degradation in diseases in which proteasome function is impaired ,69,70,84The rapid enhancement of the cell’s capacity to degrade short-lived proteins during exercise is of particular physiological interest. Such proteins represent only a minor fraction of cell constituents; therefore, even their complete hydrolysis and metabolism of the constituent amino acids cannot provide a significant source of energy to a fasting or exercising organism. Perhaps, after exercise, the enhanced capacity to degrade misfolded proteins enables the muscles to eliminate proteins damaged mechanically by the repeated contractions or by free radicals generated by mitochondrial metabolism. Another attractive possibility would be that the PKA-accelerated degradation of short-lived regulatory proteins with exercise, early in fasting, or in various hormonal responses facilitates adaptive changes in the cell’s protein composition. As these cells adapt to new physiological conditions, it would seem advantageous to degrade certain preexistent regulatory proteins or critical enzymes that are deleterious under the new conditions. In such cells, cAMP and cGMP also stimulate via CREB the expression of new proteins which are more appropriate for these new conditions. More rapid elimination of some preexistent regulatory proteins could synergize with this enhancement of new gene expression to promote the cellular adaptation to the new physiological states.