A trial of awake prone position should be considered.•The decision of intubation and mechanical ventilation should not be considered based on a single isolated parameter, such as hypoxemia degree or the extent of pulmonary infiltrates in imaging tests. Silent hypoxemia is common in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this article, the possible pathophysiological mechanisms underlying respiratory symptoms have been reviewed, and the presence of hypoxemia without hypoxia is also discussed. The experience we have gained since the start of the Coronavirus disease 19 (COVID-19) pandemic has changed our point of view about which patients with respiratory involvement should be admitted to the intensive care unit/high-dependency unit for mechanical ventilation and monitoring. In patients with clinically well-tolerated mild to moderate hypoxemia (silent hypoxemia), regardless of the extent of pulmonary opacities found in radiological studies, the administration of supplemental oxygen therapy may increase the risk of endothelial damage. The risk of sudden respiratory arrest during emergency intubation, which could expose healthcare workers to infection, should be considered along with the risks of premature intubation. Criteria for intubation need to be revisited based on updated evidence showing that many patients with severe hypoxemia do not show increased work of breathing. This has implications in patient management and may explain in part reports of broad differences in outcomes among intubated patients. Coronavirus disease 19 (COVID-19) pandemic has proven to be a challenge in different areas of care for patients admitted to intensive care units. Application of oxygen therapies and respiratory support measures is challenging because COVID-19 differs from other viral respiratory infections . Given the lack of knowledge supported by scientific evidence, medical treatment has been administered based on expert opinions. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a peculiar form of hypoxemic respiratory failure that differs greatly from other types of lung involvement induced by bacterial respiratory infections, showing the presence of endotheliitis, thrombotic vascular events, and a delayed hyperinflammatory systemic reaction.Hypoxia is defined as a reduction in oxygen levels in tissues. It is not measured directly by a laboratory test but is instead characterized by lactate production due to the activation of anaerobic metabolism. Hypoxemia is defined as a drop in the arterial pressure of oxygen below 80 mmHg. There are five pulmonary mechanisms that result in hypoxemia including alveolar hypoventilation, right to left shunt, diffusion–perfusion abnormality, diffusion impairment and ventilation–perfusion mismatch, These usually manifest as shortness of breath. “Silent  hypoxemia” has been defined as a form of hypoxia that is well tolerated by early-stage patients with no sensation of dyspnea or increased respiratory work 2O) SARS-CoV-2 infection is a systemic complex disease with multiple organ involvement, and respiratory alterations may be caused by several factors, including the coexistence of hyperperfused and hypoperfused areas (the relative percentage of these areas differs according to the severity and stage of the disease). Hypoxemia is mainly caused by the presence of poorly aerated areas and a low ventilation/perfusion ratio or normally aerated/hyperperfused areas. Dual-energy computed tomography (CT) scans have demonstrated the presence of lung perfusion deficits that increase dead space. The dual-energy CT pattern characteristic of SARS-CoV-2 infection with perfusion abnormalities described recently indicates that we are observing a new clinical process The temporal evaluation of silent hypoxemia in patients with COVID-19 requires a supply of inhaled oxygen at low concentration and noninvasive respiratory support using supplemental high-flow oxygen therapy through a HFNC or continuous positive airway pressure (CPAP) systems, together with awake prone positioning, if possible. Other measures are more controversial; these include the use of noninvasive ventilation systems, which may spread the virus through aerosols, and artisanal systems consisting of facial masks with positive-pressure input, which are associated with the risk of severe respiratory acidosis in chronic respiratory patients in case these systems are abruptly withdrawn accidentally or voluntarily In other types of hypoxemic respiratory failure, such as those caused by bacteria The respiratory infection caused by SARS-CoV-2 exhibits different physio-pathological, clinical, and responses to respiratory support. A shunt effect could be suspected given the poor response to the increase in the fraction of inhaled oxygen with oxygenation. This would correspond to a greater extent with an effect derived from extensive lung involvement and an alteration in the relationship ventilation–perfusion ratio. Lung involvement is bilateral and patchy, with hyperventilated areas alternating with hypoventilated areas.2, resulting in hypercapnia. Recently, a series of clinical conditions that assist in decision-making regarding orotracheal intubation have been suggested, and these include signs of unsustainable respiratory work and the development of respiratory acidosis With respect to dead space, the clinical picture with COVID-19 could differ from that seen in ARDS secondary to pneumococcal pneumonia, where diffuse alveolar involvement is an impediment to the elimination of COIn many cases, absence of a significant impairment in pulmonary compliance and resistance, evidenced in ventilator mechanics and parameters, has been reported Finally, involvement of the microcirculation in the viral infection, resulting in compensatory pulmonary vasoconstriction, can lead to so-called hypoxic vasoconstriction ,28, withSevere hypoxemia  and hypoxia (decreased oxygen diffusion in tissues and cells) are concepts that are usually correlated (hypoxic hypoxemia), although hypoxia may be caused by other processes.2] in arterial blood) that does not lead to significant hypoxia is difficult to explain based on usual pathophysiological mechanisms It is common when assessing patients with COVID-19 to observe a dissociation between the absence of clinical manifestations and the detection of moderate/severe hypoxemia through arterial blood gas values. Together with the presence of diffuse opacities observed in radiological studies, these findings usually indicate the need for hospital admission. Hypoxemia  have difficulty diffusing oxygen into the blood but are still able to readily diffuse CO2 out of their blood. Silent hypoxemia as a physiologic phenomenon does not appear to be COVID-19-specific. Rather, it seems to follow established physiological principles.COA high oxygen concentration (and low nitrogen concentration) may induce toxicity, with individual susceptibility ,42. This2 values obtained from arterial samples can be influenced by some kind of physiological conditions. If we analyze the dissociation that also occurs between peripheral oxygen saturation and the degree of hypoxemia, oximetry-estimated saturation (SpO2) can differ from true arterial oxygen saturation, mainly at an SpO2 of 95% 2 values <40 mmHg (SpO2 of approximately 75%), and when oxygen extraction increases, normal oxygen consumption is maintained How can the phenomenon of nonhypoxic hypoxemia be explained? A question worth asking is whether the PaOAreas of uncertainty exist. For example, does the SARS-CoV-2 infection trigger an alteration in the oxygen transport by hemoglobin? Could it be related to hypoxic vasoconstriction phenomena? Does noninvasive ventilation ,46 inducGiven the great variability of clinical presentation, different clinical phenotypes associated with COVID-19 have been defined 2 monitoring. Difficulties in endotracheal intubation should be anticipated. A hallmark of COVID-19 is the rapid development of refractory hypoxemia with a poor response to oxygen supplementation, suggesting intrapulmonary shunting. This can overwhelm local resources, resulting in shortages of trained staff, oxygen supplies, and ventilators 2 is 93% (instead of 90%) and by a factor of 8.5 if the target SpO2 is 98%. Thus, the titration of oxygen concentration for achieving an SpO2 of 90% has been implemented for COVID-19 management in regions where oxygen shortages were documented.The two most important strategies to consider when deciding whether endotracheal intubation is required are respiratory observation and continuous SpO(1)2 remains above 80%, no hypoxia manifestations should be expected. Thus, maintaining SpO2 values lower than those usually indicated to start supplemental oxygen administration (93%) should be considered if they are clinically well tolerated.In patients with clinically well-tolerated mild to moderate hypoxemia (silent hypoxemia), regardless of the extent of pulmonary opacities, the administration of supplemental oxygen therapy is required. Provided that SpO(2)Patients with symptomatic moderate to severe hypoxemia could benefit from the administration of supplemental oxygen through reservoir masks or HFNCs, although no evidence of outcome improvement with CPAP systems has been reported (3)The decision of intubation and mechanical ventilation should not be made based on a single isolated parameter, such as hypoxemia severity or the extent of pulmonary opacities observed in imaging tests. If required, it should be performed by the most skilled available operator after preoxygenation and rapid-sequence induction of sedation using neuromuscular blockade with succinylcholine or rocuronium ,52. LateLastly, we schematically outline the following temporal sequence of measures for respiratory management depending on the increase in clinical severity:(1)Subjective patient sensation of physical exhaustion and sensation of nervousness or anxiety associated with difficulty to clear secretions and refractory hypoxemia.(2)2 above 85%.Appearance of severe tachypnea and its analytical translation represented by increasing respiratory alkalosis and the patient being unable to maintain SpO(3)2) despite supplemental oxygen therapy with reservoir mask, a HFNC, or CPAP systems.Deterioration of clinical status Encephalopathy with significant alteration of the level of consciousness, with development of hypercapnia or acidemia.(5)Progression to multiple organ failure/dysfunction.(6)Severe myocardial dysfunction.Thus, intubation should be indicated in conjunction with the degree of clinical involvement documented by the development of any of the following conditions:In summary, silent hypoxemia is common in patients with SARS-CoV-2 infection. The risk of sudden respiratory arrest during an emergency intubation, which could expose healthcare workers to infection, should be considered along with the risks of premature intubation. Careful monitoring of respiratory status to identify whether intubation is required is the main goal. Criteria for intubation need to be revisited based on updated evidence showing that many patients with severe hypoxemia do not experience increased work of breathing. This has some implications in patient management and may explain in part the broad differences in outcomes reported among intubated patients.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Most studies of the regulation of proteolysis by the ubiquitin proteasome system have focused on the control of ubiquitination. However, it is now clear that the activity of the 26S proteasome and rates of protein degradation in cells are also tightly regulated through proteasome phosphorylation. Here we demonstrate that agents that raise cGMP and activate cGMP-dependent protein kinase  stimulate proteasome activities and intracellular proteolysis without affecting autophagy. Furthermore, we showed that raising cGMP reduced the levels of the disease-causing mutant tau in a zebrafish model by increasing its degradation, and also decreased the associated morphological abnormalities. Thus, activating the proteasome via cGMP is a promising strategy to prevent the progression of neurodegenerative diseases. Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington’s disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases. In mammalian cells, the great majority of proteins are degraded by the ubiquitin proteasome system (UPS) . These p13cAMP and PKA were shown to increase the ability of purified 26S proteasomes to hydrolyze polyubiquitinated proteins, ATP, and small peptides via phosphorylation of Serine 14 in Rpn6, a subunit of the 19S regulatory complex , 15. ActcGMP is an intracellular second messenger that is synthesized by soluble guanylyl cyclases in response to nitric oxide  or by trAlthough the ability of these agents to alter protein turnover generally or to combat the progression of neurodegenerative disease has received little or no attention, in a mouse model of a cardiomyopathy caused by overexpression of mutant αβ crystallin, raising cGMP with sildenafil was shown to reduce cardiac hypertrophy . This trHere we have systematically investigated whether different pharmacological agents that raise cGMP influence 26S proteasome function and the degradation of different classes of cell proteins. These studies demonstrate that PKG stimulates multiple 26S proteasome activities by phosphorylation of a different proteasome component than is modified by PKA. Also, unlike cAMP, cGMP stimulates the degradation of the bulk of cell proteins, and not just short-lived proteins. Because of these large increases in protein degradation, we also studied whether raising cGMP increases protein ubiquitination and autophagy. A number of major neurodegenerative diseases are caused by the accumulation of misfolded or mutant proteins, and in many cases the build-up of such aggregation-prone proteins impairs proteasome function, which leads to further defects in protein homeostasis , 20, 21.To raise the intracellular levels of cGMP, we used two inhibitors of PDE5, tadalafil or sildenafil, and two stimulators of soluble guanylyl cyclases, BAY41-2272 or cinaciguat. Addition of tadalafil (100 nM) or BAY41-2272 (100 nM) to human neuroblastoma cells (SH-SY5Y) caused a rapid increase in the proteasomes’ chymotrypsin-like peptidase activity in cell lysates A. This iThis rapid stimulation and slow decline of peptidase activity occurred without any change in the levels of proteasome subunits B. In addTo verify that these increases in peptide hydrolysis were due to greater 26S proteasome activity, SH-SY5Y cells were treated for 30 min with tadalafil, BAY41-2272, or these two agents together, and the 26S proteasomes were then purified using the UBL domain as the affinity ligand D. SimilaThe physiological substrates of 26S proteasomes are ubiquitinated proteins, whose degradation is linked to ATP hydrolysis . The proSI Appendix, Fig. S1A). Although 26S proteasomes are abundant in both the nucleus and cytosol, PKG is localized primarily to the cytosol . Therefore, we tested whether activated PKG in cells stimulates the activities of proteasomes in both fractions. After treatment of SH-SY5Y cells with tadalafil or dimethyl sulfoxide (DMSO) for 30 min and hypotonic lysis, nuclei were separated from the cytosol by differential centrifugation and then 26S proteasomes were purified from each fraction by the UBL method. After tadalafil treatment, the cytosolic, but not the nuclear proteasomes, exhibited greater peptidase activity  with increasing concentrations of human PKG1α for 1 h, the levels of phosphorylated bands at 100 and 130 kDa in the proteasome preparations increased in a concentration-dependent manner F. There SI Appendix, Fig. S1C). However, the overexpression of PKG did increase the amount of phosphorylated proteins detected in the proteasome preparations above levels in particles from cells transfected with an empty vector. In addition, the overexpressed PKG copurified with the 26S proteasomes . Similar increases in the levels of phosphorylated proteins and the amount of PKG that copurified with proteasomes were seen after treatment of HEK293 cells with the soluble guanylyl cyclase stimulator cinaciguat .Prior work has demonstrated that phosphorylation of two sites on the 19S regulatory particle increases proteasomal activities: Rpn6-S14 by PKA , 15 and The subunit Rpt6 has also been reported to be phosphorylated by PKG  and on S3H-phenylalanine for 20 min to label short-lived proteins. After the 3H-phenylalanine was removed, fresh media containing large amounts of nonradioactive phenylalanine and cycloheximide were added to prevent reincorporation of the labeled amino acids released by proteolysis. The degradation of the labeled proteins was then measured by assaying the generation of radiolabeled amino acids .Raising the level in cells of cAMP with pharmacological agents or hormones enhances selectively the degradation of short-lived proteins , 15, whino acids . Adding radation A. A simiSI Appendix, Fig. S3A). Thus, cGMP and PKG enhance the degradation of these abnormal polypeptides.To determine whether raising cGMP also stimulates the degradation of damaged or misfolded proteins, which are short-lived, we induced the production of incomplete proteins by exposing SH-SY5Y cells for 1 h to puromycin, which is incorporated into newly synthesized proteins and causes premature termination of the polypeptides which are then rapidly hydrolyzed . DegradaSI Appendix, Fig. S3B).These increases in proteolysis presumably also reflect accelerated degradation of many short-lived regulatory proteins. Therefore, we measured by western blot the degradation in the presence of cycloheximide of p53, whose rapid ubiquitination and degradation have been extensively characterized . The bre3H-phenylalanine for 16 h, chased for 2 h with nonradiolabeled phenylalanine to allow the hydrolysis of labeled short-lived proteins, and then the degradation of the 3H-labeled long-lived proteins was measured . These rates of degradation of long-lived proteins were linear for at least 4 h and were surprisingly high. In fact, these rates were greater than those seen in such cells upon serum or nutrient deprivation  or by inhibition of PKG with Rp-8-Br-PET-cGMPs  . In these cells, as in other lines tested, cinaciguat stimulated the degradation of both short- and long-lived proteins . Surprisingly, tadalafil by itself did not increase protein degradation in these cells  or in HEK293 cells , probably due to a low level of basal cGMP synthesis. However, combining cinaciguat with tadalafil in BJ5A and HEK293 cells produced even larger increases in proteolysis . Treating BJ5A cells with carbachol, a pan muscarinic receptor agonist, stimulated the degradation of both short- and long-lived proteins , a well-characterized PKG substrate, and increased proteasomal peptidase activity , as also seen upon treatment of cells with agents that raise cGMP .To determine whether the increased levels of polyubiquitinated proteins were due to increased ubiquitination or decreased deubiquitination of cell proteins, we used cytosolic extracts from HEK293 cells from which nuclei and mitochondria were sequentially removed by differential centrifugation. Incubating these extracts with 1 µM cGMP for 30 min in the presence of the broad-spectrum phosphodiesterase inhibitor IBMX activated PKG, as shown by increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP)  and the hydrolysis of ubiquitin-amc . Thus, cGMP increases the levels of polyubiquitinated proteins by stimulating the ubiquitination of cell proteins more than it enhances their degradation by proteasomes.To evaluate whether cGMP also decreases the rate of deubiquitination of cell proteins, HEK293 cells were treated for 30 min with bortezomib to increase the levels of ubiquitinated proteins and cytosolic extracts were prepared as above, but in the presence of an inhibitor of the ubiquitin-activating enzyme instead of the inhibitors of deubiquitinases. Adding cGMP to these extracts did not change the rate of deubiquitination of cell proteins E. FurtheSI Appendix, Fig. S9C). Furthermore, exposure to these agents for only 1 d also increased proteasomal peptidase activity in the lysates of WT larvae  with 1 µM sildenafil or 3 µM rolipram did not alter the levels of assembled 26S proteasomes in the lysates as measured by native PAGE and western blot . In these lysates, unlike those of human cells . Treatment with 10 µM sildenafil or 30 µM rolipram, but not with the lower concentrations of either agent, increased the intensity of the upper band of the doublet and decreased the intensity of the lower band . This interesting effect, however, did not correlate with changes in activity because 26S proteasomes from larvae treated with 1 or 10 µM sildenafil or 3 or 30 µM rolipram showed similar increases in peptidase activity .At these concentrations, neither drug had any overt toxic effects on the larvae. There was also no change in the levels of proteasome subunits in the lysates of the larvae, and the treatments . Most of the zebrafish larvae expressing the A152T mutant tau in the nervous system exhibit an abnormal curvature of the dorsal spine . Furthermore, the degree of rod photoreceptor degeneration was determined by analyzing the ratio of the rod protein ZPR3 (rhodopsin) to the cone protein ZPR1 (arrestin) measured by western blot. Both sildenafil and rolipram increased this ratio compared to that seen in siblings that received only the vehicle , indicating that these agents prevented the degeneration phenotype. This effect was not due to decreased expression of the transgene, because these treatments did not change the levels of EGFP mRNA measured by qRT-PCR .Overexpression in the zebrafish retina, under the rhodopsin promoter, of human WT tau or the P301L mutant tau, which also causes frontotemporal dementia, leads to degeneration and death of the rod photoreceptors . To deteIn Huntington’s disease, mutant huntingtin containing an expanded polyglutamine repeat accumulates in protein aggregates within neurons. To test whether raising cAMP or cGMP could also reduce the levels of such aggregates, zebrafish larvae expressing exon 1 of human huntingtin with 71 glutamine repeats in rod photoreceptor cells (Rho::EGFP-HD71Q) were treated for 5 d with sildenafil or rolipram, and the number of microscopically visible aggregates in the retina was measured. Both agents reduced the number of huntingtin aggregates G. Thus, The regulation of protein breakdown by cGMP and PKG differs in several respects from other known mechanisms that control intracellular proteolysis. Pharmacological agents that inhibit PDE5 or stimulate soluble guanylate cyclase rapidly increase not only the activities of 26S proteasomes, but also the levels of ubiquitinated proteins and the total rate of intracellular protein degradation. Raising cGMP content even further by combining these treatments caused a further increase in the magnitude, rapidity, and duration of these responses. The increase in ubiquitin conjugates, proteasomal degradation, and overall proteolysis were all evident within 5 min after addition of these drugs to cultured cells and these changes occurred too rapidly to result from changes in gene transcription. It is noteworthy that these increases in proteasomal activity and ubiquitinated proteins were only observed in the cytosol, where PKG is localized, not the nucleus. The stimulation of proteasome activity and protein turnover by cGMP and PKG appears to be a general cellular response because similar changes were seen in HEK293, neuroblastoma, myotubes, and fibroblasts, as well as zebrafish larvae. However, cell types differ widely in their content of phosphodiesterases and guanylate cyclases and in the physiological factors that promote cGMP synthesis. Therefore, it remains to be determined to what extent in different tissues, hormones , signaling molecules , neurotransmitters , or drugs that raise cGMP also stimulate proteasome function and overall proteolysis by the UPS.Although both cGMP and cAMP enhance proteasome activity and the degradation of short-lived proteins, they probably do not stimulate the ubiquitination and degradation of the same short-lived proteins. cGMP and cAMP are generated in different physiological states and often have opposite effects on cell function, especially in the cardiovascular system. In hearts, epinephrine via cAMP increases cardiac output, while acetylcholine via M2 muscarinic receptors raises cGMP and decreases cardiac output. PKG’s ability to also stimulate the breakdown of the bulk of cell proteins clearly distinguishes its effects on protein degradation from those of PKA and must have important physiological consequences. For example, in hearts adrenergic agonists via cAMP induce hypertrophy , while aThe increases in overall degradation by the UPS with these pharmacological treatments were surprisingly large and even exceeded the increases in proteolysis reported in cells in various catabolic states, such as cachexia and muscle atrophy  or upon A variety of protein kinases have been reported to alter proteasome activities , althougIt has been claimed that increases in proteasome activity are associated with or even require greater formation of 26S proteasomes . HoweverBecause these PKG-induced changes in proteasome activities resemble those induced by PKA, it is unclear , 15 how It is noteworthy that after a rise in cGMP, the cell’s content of ubiquitinated proteins increased, despite the enhanced proteasomal destruction of ubiquitinated proteins and total proteolysis by the UPS. Increases in the levels of ubiquitin conjugates are often interpreted as evidence of proteasome inhibition. However, the rapid rise in ubiquitin conjugates with PKG activation implies that the increase in protein ubiquitination must have exceeded the increase in their degradation. A similar rise in ubiquitin conjugates also occurs in atrophying muscles and during starvation despite the activation of 26S proteasomes and the increased proteolysis by the UPS . Thus, iA large variety of neurodegenerative diseases result from the accumulation of misfolded, aggregation-prone proteins, which are generally found in intracellular inclusions conjugated with ubiquitin. This build-up of proteins likely represents a failure of the cell’s protein quality control machinery. In fact, there is growing evidence that such aggregates or microaggregates can impair the functioning of the 26S proteasomes , 20, 21,Surprisingly, raising cGMP with sildenafil had very similar effects in these zebrafish larvae as rolipram, not only in the tauopathy models but also in a Huntington’s disease model caused by the expression of a huntingtin exon 1 with a highly expanded polyQ sequence. Although PKA and PKG have very different physiological actions, their key shared property in these responses must be their capacity to enhance the degradation of short-lived misfolded proteins by the UPS. Remarkably, both sildenafil and rolipram, while enhancing proteasome activity and tau degradation, reduced neuronal death and the gross morphological abnormalities caused by overexpression of mutant tau in neurons. Furthermore, the treatments did not have any evident deleterious consequences, probably because they simply augment a physiological mechanism for protein quality control. While activation of PKG and PKA reduced the levels of the aggregated proteins, these structures are generally much larger than proteasomes. So, it remains unclear whether proteasomal digestion involves faster degradation of these misfolded proteins before they aggregate, or whether the proteasomes can extract ubiquitinated proteins from such aggregates for degradation. It also remains unclear whether these beneficial effects result solely through proteasome activation or through enhanced ubiquitination of the toxic proteins.Thus, agents that raise cGMP in the central nervous system represent a promising new strategy to combat Alzheimer’s, Huntington’s, and other neurodegenerative diseases. In humans, the PDE5 inhibitors and the soluble guanylyl cyclase stimulators used here poorly cross the blood brain barrier. However, brain-penetrant PDE5 inhibitors  and soluThe present findings and other’s prior observations on a mouse model of cardiomyopathy caused by an αB crystallin mutation , 44 demoSI Appendix.For details on general methods, reagents, antibodies, proteasome purifications, proteasome activity assays, measurements of intracellular protein degradation, cell-free assays, statistics, and zebrafish husbandry and experiments, please refer to All zebrafish procedures were performed in accordance with the UK Animals (Scientific Procedures) Act with appropriate Home Office Project and Personal animal licenses and with local Ethics Committee approval. Studies were performed in accordance with ARRIVE guidelines.All data in this report are available to all readers. The reagents used in this study, as well as further information, are available upon request from A.L.G.Supplementary File


Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization. Fertilization is regarded as one of the greatest feats of nature, beginning with a sperm combining with an oocyte, during which these two terminally differentiated germ cells are converted into a totipotent zygote  is an inheritable type of epigenetic mark that provides molecular memory to preserve the transcriptional order during mammalian early embryo development   embryos, the efficiency of embryonic development is much lower than that in normally fertilized embryos, owing to the multiple epigenetic barriers that impede SCNT-mediated reprogramming, including abnormally higher levels of DNA methylation in cloned embryos  method  facilitates the expression of ZGA-related genes in SCNT embryos but results in no improvement of the birth rate or ntESC establishment efficiency. Conversely, knockdown of Kdm6b (another H3K27me3 demethylase) promotes both ZGA and SCNT efficiency  leads to increased DNA methylation levels, followed by a significant H3K4me3 decrease in bivalent developmental genes  overexpression in embryos and Suv39h1/h2 (H3K9me3 methyltransferases) knockdown in donor cells can rescue the transcription of ZGA-related genes impeded by H3K9me3, which dramatically improves the developmental rate of blastocysts. Notably, subsequent experiments using an embryo biopsy system and single-cell transcriptome sequencing identified Kdm4b, another H3K9me3 demethylase, as a key factor in the 2-cell arrest of SCNT embryos  mice for all three histone H3K9 methyltransferases (SETDB1 and SUV39H1/H2) show a three-fold reduction in body weight and are much smaller in size compared to the control mice  loci and activates 2-cell/4-cell embryo genes by regulating the enhancer activity of MT2, associated with the increased deposition of H3K4me1, H3K27ac, and H3K14ac  complex retroelements are the most abundant class of retroelements in mammals  method revealed the dynamics of 3D genome architecture reprogramming in mouse early embryo development (Du et al., Dramatic higher-order chromatin structure reprogramming indicates the importance of epigenetic reorganization during mammalian early development. However, little is known about the molecular basis of the reprogramming of the 3D chromatin structure. The establishment of TADs depends on DNA replication rather than ZGA, which reveals the function of cell cycle transition in genome structure dynamics (Ke et al., Kdm4b partially ameliorates abnormal 3D chromatin structures, suggesting that H3K9me3 modification in donor cells is a barrier to chromatin structure reprogramming. This indicates a correlation between the organization of the 3D genome architecture and histone modifications.Moreover, a recent study by our group revealed spatiotemporally dynamic chromatin reorganization in SCNT embryos (Chen et al., CTCF is highly expressed at the ZGA stage of human embryos, which coincides with the time at which TADs are observed, indicating the critical role of CTCF in the establishment of the higher-order chromatin structure during early human embryo development. Intriguingly, the establishment of TADs in human embryos is ZGA dependent, which leads to the hypothesis that the correlation between TAD formation, DNA replication and gene activation differs among species or cell types (Ke et al., The dynamics of the 3D chromatin architecture during human embryogenesis have recently been revealed. Unlike mouse sperm, human sperm lack TADs and the expression of the chromatin regulator CTCF (Chen et al., During the development of pre-implantation embryos, ZGA occurs predominantly at the two-cell stage in mice and the eight-cell stage in humans, which involves global DNA demethylation, chromatin remodeling, spatial reorganization of the genome and substantial transcriptional changes. Recent discoveries have shed light on the potential regulatory mechanisms that drive subsequent biological events upon fertilization, but many such mechanisms remain to be investigated. Future work is needed to further elucidate the molecular details that underlie the interaction between epigenetic remodeling and the totipotency transition as well as cell fate decisions in early mammalian embryo development.Epigenetic reprogramming during early embryonic development is an exquisitely controlled process, involving both global re-establishment of most epigenetic marks and locus-specific regulation. In recent years, due partly to single-cell and low-cell-number epigenomic studies, our understanding of the epigenetic reprogramming landscape of pre-implantation development has improved considerably. However, how reprogramming is regulated at different genome loci remains unknown. Different transcription factors must play important roles in determining the locus-specific epigenetic transition pattern. The identification of these factors and the underlying mechanisms will improve our understanding of cell fate transitions and mammalian early development. Cell fate transitions are coordinated by the synergistic action of multiple types of epigenetic remodeling. Multiomics analysis is needed to clarify the fundamental principles underlying totipotency acquisition and cell fate decisions.in vitro culture (Ma et al., The mechanism of epigenetic remodeling seems to vary in different species; for example, TAD establishment depends on DNA replication in mouse embryos, while that in human embryos requires ZGA. The exploration of the epigenetic regulation mechanism of human early embryo development will continue to be an important area of research, the results of which will be beneficial for diagnosing and treating human infertility. It is worth mentioning that human embryos show high individual heterogeneity, including a high proportion of aneuploid embryos, which may to some extent conceal the detailed characteristics of the histone modifications and 3D chromatin structures that exist during human embryogenesis. Notably, remarkable progress has recently been made in mapping the molecular architecture of lineage specification during gastrulation and early organogenesis in mouse embryos (Peng et al., In summary, recent advances in sequencing technologies have given rise to research probing the regulatory network based on epigenome reprogramming during mammalian pre-implantation embryonic development, and further studies are urgently needed to reveal the underlying molecular mechanisms.


To improve efficacy of solid cancer treatment, efforts have shifted towards targeting both the cancer cells and the surrounding tumour tissue they grow in. The lysyl oxidase (LOX) family of enzymes underpin the fibrotic remodeling of the tumour microenvironment to promote both cancer growth, spread throughout the body and modulate response to therapies. This review examines how the lysyl oxidase family is involved in tumour development, how they can be targeted, and their potential as diagnostic and prognostic biomarkers in solid tumours.The lysyl oxidase (LOX) family of enzymes are a major driver in the biogenesis of desmoplastic matrix at the primary tumour and secondary metastatic sites. With the increasing interest in and development of anti-stromal therapies aimed at improving clinical outcomes of cancer patients, the Lox family has emerged as a potentially powerful clinical target. This review examines how lysyl oxidase family dysregulation in solid cancers contributes to disease progression and poor patient outcomes, as well as an evaluation of the preclinical landscape of LOX family targeting therapeutics. We also discuss the suitability of the LOX family as a diagnostic and/or prognostic marker in solid tumours. The extracellular matrix (ECM) provides a supporting scaffold for cells to differentiate, communicate and grow within and plays an important function in maintaining tissue homeostasis ,2. This Dysregulation of the deposition and composition of ECM components can both lead to and accelerate disease. For example, excessive deposition of collagens leads to fibrosis, characterized by a dense ECM. In solid cancers, the reprogramming of fibroblasts to cancer associated fibroblasts (CAFs) leads to desmoplasia, in which the ECM is remodeled by degradation of the existing, structured “normal” ECM and replaced by excessive, disordered “tumour” ECM that feeds into disease progression .Fibroblasts are the primary cell type involved in the production of matrix components such as fibrillar collagen. Collagens are typically heterotrimeric helical polymers  that form organized fibers comprised of bundled collagen fibrils  3,10]. ,10. 3,10One of the key enzymatically controlled stages of fibrillar collagen synthesis is the formation of stabilizing cross-links catalyzed by the lysyl oxidase (LOX) family of enzymes ,17. ThisStructurally, all LOX family members contain a highly conserved C-terminal copper binding domain consisting of three histidine residues which, when bound to copper, cause oxidation of Tyr355 and a conformational change of the active site to form a unique lysine tyrosylquinone (LTQ) cofactor. LOX and LOXL1 also both contain a cleavable, N-terminal pro-peptide domain cleaved by BMP1, whereas LOXL2-4 contain four repeating scavenger receptor cysteine-rich (SRCR) domains. In healthy tissue, the synthesis of the LOX family is tightly regulated to control the amount of active LOX family members present. The structure, genetic sequence and cross-linking activity of the LOX family has previously been extensively reviewed ,20,21,22The degree of cross-linking of collagens and elastin within the ECM varies across tissue types and is thought to be related to the tensile strength required for tissue function ,23. LOX In cancer, dysregulation of the deposition, degradation, and post translational modification of collagens significantly alters the structure and function of the ECM ,29. As aCancer associated fibroblasts (CAFs) are one of the most abundant types of stromal cell within the tumour microenvironment and are morphologically, epigenetically and metabolically distinct from their originating fibroblasts . They prLOX family members are upregulated in a number of different cancer types, and high levels of LOX family expression, as well as elevated deposition of fibrillar collagen in tumours, is associated with poor patient outcomes see  42,43,44,45. ChaPotent fibrogenic signaling effectors such as TGF-β1 have been shown to be regulators of LOX expression in diseases such as cancer ,80,81,82Expression of LOX family members typically causes stiffening of the ECM in cancer through increased catalytic activity, and this may result in further regulation of PI3K signaling though the mechano-activation of the FAK/Src pathway. Increases in LOX, LOXL2 and LOXL4 expression in the ECM of cancer is linked to an increase in focal adhesion kinase (FAK) and Src phosphorylation in colorectal adenocarcinoma ,89, gastAdditionally, LOX expression has been linked to MAPK signaling through p38-MAPK, ERK and JNK pathways in cardiac fibroblasts and breast cancer models ,83. In pLOX and LOXL1 are directly regulated at the post-translational level by BMP1 and ADAMTS proteinases, which are also responsible for cleaving collagen. Catalytically inactive LOX and LOXL1 are secreted as a pro-LOX into the extracellular space where BMP1 cleaves off the N-terminal pro-peptide, releasing the mature, active enzyme . Cancer After embryonic development, LOX and LOXL1 can be found across various tissues including the heart, kidneys, testes, and uterus, whereas LOXL2, LOXL3 and LOXL4 are less widely expressed. Overall, LOX family activity is thought to be restricted until needed for processes such as maintaining tissue homeostasis and acute wound healing . In tumoECM remodeling fosters cancer growth and enhances cells’ abilities to metastasize ,114,115.LOX family member involvement in shaping the ECM at both primary and secondary tumour sites see , has resSince irregular LOX family expression has been linked to poor prognosis , notablyGiven that current diagnostic and prognostic methods used in the clinic often require a tissue biopsy for histological analysis, the development of non-invasive or non-operative techniques would be favorable. LOX family members may have potential in this context since they are detectable in human bodily fluids such at the blood and urine. Some early preclinical success in this has demonstrated the detection of LOXL2 within both murine and human blood samples of breast cancer patients, where hydrogen peroxide release, a product of LOX family activity, was detected via the use of a gold-based electrochemical biosensor . In thisThe complexities of correlating LOX family expression and tumour staging are typified in prostate cancer, where high LOX expression has been correlated with advanced tumour stage; however, low LOX expression was associated with decreased overall survival . In otheIndeed, different LOX family members have been reported to act as tumour promoters or suppressors depending on the cancer type See . AdditioIn addition to CAF mediated fibrosis and the collagen cross-linking activity of the LOX family, the chemotherapy induced wound healing response which often occurs at both primary and secondary sites typically results in an increase in matrix deposition. As a result, this exacerbated change in ECM deposition and stiffness may contribute to the development of acquired chemoresistance, likely due to changes in cellular signaling. Evidence for this is supported in 2D models, where overexpression of LOX had no effect on cancer cell sensitivity to doxorubicin, compared to 3D models where the cells in a collagen matrix showed reduced sensitivity to doxorubicin following LOX overexpression . IncreasSurgery can result in an increase in LOX family expression as a result of a wound healing response at the site of surgery . Plasma Many tumours display increased chemoresistance in the presence of hypoxia, and the expression of LOX family members in some chemo-resistant tumours is upregulated in response to hypoxia ,45. HypoCombination treatment of chemo-resistant tumours with doxorubicin and inhibitors of LOX, or components of the pathways involved in LOX regulation such as FAK or Src, have also been shown to decrease tumour growth, suggesting that LOX and FAK/Src signalling play a role in chemoresistance . It is pThe well characterized beta-aminopropionitrile (β-APN) is a non-specific, irreversible pan-LOX family inhibitor which covalently binds to the active site of LOX family enzymes . It was The highly specific small molecule inhibitor of LOXL2, PAT-1251, which is based on a benzylamine with 2-substituted pyridine-4-ylmethanamines, was developed by PharmAkea . Although found to be well tolerated and successfully passing through a phase I clinical trial , to date no phase II clinical trials have been completed with PAT-1251. Pre-clinical studies in vivo of the aminomethiophene based LOX inhibitor CCT365623 showed inhibition caused delayed tumour development and reduced lung metastasis in mouse breast cancer models ,155,156.Another LOXL inhibitor to show early promise in cancer treatment was the anti-LOXL2 monoclonal mouse antibody AB0023, which was effective in reducing intratumoral collagen density in vivo in a murine model of pancreatic cancer . FollowiOther anti-LOXL2 antibodies have been developed in mice, such as GS341  which caA number of studies to date have examined the use of LOX family inhibitors in combination with current chemotherapeutic treatments simultaneously. Given that lysyl oxidase inhibitors reduce the fibrotic ECM and desmoplasia in cancer, it is possible that initial treatment with lysyl oxidase inhibitors prior to administering chemotherapeutics will improve patient outcomes by priming the cancer microenvironment.Downregulation of LOX family expression or activity through indirect mechanisms may similarly be beneficial in targeting the LOX family, especially if approaches can be administered in combination with chemotherapeutics . BMP1 isLOX family activity may additionally be targeted through limiting the copper available to the cells. Copper is critically required for correct folding of LOX family enzymes, and the catalytic activity of their active sites. The copper chelator ammonium tetrathiomolybdate (ATTM) has been shown to reduce bleomycin-induced pulmonary fibrosis in mice by blocking LOX family activity . FurtherD-penicillamine (D-pen) is another orally administered copper chelator, initially suggested to be a LOXL2 inhibitor. Previous work showed in an in vivo breast cancer model that treatment with D-pen significantly reduced the number of lung metastases formed in mice compared to control groups . Rather Taking a different approach, Mohankumar et al.  developeLOX family member expression can also be modified by targeting components in the pathways leading to their expression . For exaThe remodeling of the extracellular matrix in cancer plays an important role in controlling the progression of disease and influences cell growth, motility and survival. As such, combining conventional chemotherapeutic targeting of cancer cells in solid tumours with a stromal targeting agent is likely to improve patient outcomes by disrupting pro-tumourigenic ECM remodeling. The LOX family of enzymes are favorable targets for anti-stromal therapeutics due to their importance in cancer development and progression when compared to healthy state ECM. Each LOX family member is critically involved in matrix deposition, and subsequently matrix stiffening, directly influencing the invasive and proliferative abilities of cancer cells, as well as the generation of pre-metastatic niches that support metastatic colonization. Additionally, highly LOX family expressing tumours have increased LOX family levels detectable in plasma , and thuAlthough no inhibitors of the LOX family have currently been approved for routine clinical practice, preclinical and clinical trials underway are developing LOX family inhibitors which have to date shown high specificity and low toxicity. Novel classes of LOX family inhibitors have been designed to target either single, dual, or multiple LOX family members and these high degrees of specificity towards individual members of the LOX family offer significant promise. With this in mind, studies where LOX family inhibition appears to be correlated with poorer outcomes in early clinical trials should be further evaluated. Some LOX family members have been identified as tumour suppressive or tumour promoting in different cancer types and so investigating the individual LOX family member contributions to a specific cancer is essential to determine the most effective LOX family targeting combination. Additionally, further preclinical in vivo investigation would be valuable to examine when best to administer LOX family inhibitors in order to block tumour progression. Significant changes in the ECM of the tumour microenvironment during progression means that treatment regimens for early, late and advanced stage patients is likely to differ.Recent work has resolved the partial crystal structure of LOXL2 . Whilst Alternately, indirect inhibition of LOX family members through the downregulation or inhibition of enzymes or signaling pathways required for LOX family expression may prove to be viable with further preclinical investigation, however such methods often lack target specificity. Ultimately, efforts continue towards the development of a well-tolerated method of targeting LOX family activity in cancer, and further investigation into the feasibility of LOX family targeting in a range of cancers and the interplay between different family members in this process will be required in the future, particularly given the sometimes contradictory roles different members of the LOX family can play in tumorigenicity.With these barriers to anti-stromal targeting in mind, it is essential that treatment is complemented with a LOX family specific companion biomarker. Preclinical research has increased in utilizing LOX family members for the development of non-invasive methods of diagnosing and staging disease. Due to the secreted nature of the LOX family members, their detectable presence in the blood, and the well-established correlation between LOX family enzyme expression and prognosis in many solid tumours, this unique family offers promise as an inexpensive and non-invasive companion biomarker for highly stromal tumours. Although current non-invasive diagnostic measures remain in the preliminary stages of development, the LOX family holds huge potential as stromal targets in cancer.


Drosophila. Our review outlines the major peptidergic hormonal pathways known in Drosophila and presents a set of schemes of hormonal axes and orchestrating peptidergic systems. The detailed organization of the larval and adult Drosophila neurosecretory systems displays only very basic similarities to those in other arthropods and vertebrates.Hormones regulate development, as well as many vital processes in the daily life of an animal. Many of these hormones are peptides that act at a higher hierarchical level in the animal with roles as organizers that globally orchestrate metabolism, physiology and behavior. Peptide hormones can act on multiple peripheral targets and simultaneously convey basal states, such as metabolic status and sleep-awake or arousal across many central neuronal circuits. Thereby, they coordinate responses to changing internal and external environments. The activity of neurosecretory cells is controlled either by (1) cell autonomous sensors, or (2) by other neurons that relay signals from sensors in peripheral tissues and (3) by feedback from target cells. Thus, a hormonal signaling axis commonly comprises several components. In mammals and other vertebrates, several hormonal axes are known, such as the hypothalamic-pituitary-gonad axis or the hypothalamic-pituitary-thyroid axis that regulate reproduction and metabolism, respectively. It has been proposed that the basic organization of such hormonal axes is evolutionarily old and that cellular homologs of the hypothalamic-pituitary system can be found for instance in insects. To obtain an appreciation of the similarities between insect and vertebrate neurosecretory axes, we review the organization of neurosecretory cell systems in The online version of this article (10.1007/s00441-020-03264-z) contains supplementary material, which is available to authorized users. Drosophila melanogaster  from sensory inputs, releasing factors, hormonal targets and feedback. These hormonal regulatory circuits described are commonly based on both anatomical and functional analyses but in some cases only anatomical data are available. In assembling this summary, we also came across several peptidergic systems that are not releasing neurohormones into the circulation but still playing roles in integrating behavior and/or physiology in a global fashion. One example is the SIFa neurons mentioned above. Thus, we summarize both circuits using bona fide peptide hormones and those where peptides act globally by paracrine (and/or synaptic) signaling within the central nervous system (CNS). We also present data on neurosecretory cell systems in the ventral nerve cord and enteroendocrine cells of the intestine.To get a better appreciation of the similarities between insect and vertebrate neurosecretory axes, we review here the organization of neurosecretory cells in Drosophila (and other insects) and presents a set of schemes of hormonal axes. We find that the Drosophila neurosecretory systems, although embryologically somewhat similar to those of vertebrates or annelid worms, are quite divergent in larvae and adults and display an organization that deviates from the pattern seen in the other phyla.Our review outlines the major peptidergic hormonal pathways known in Drosophila  released from brain neurosecretory cells  act not only as circulating hormones on peripheral targets but also signal to brain neurons, probably in a paracrine fashion  have been shown to be intrinsically nutrient sensing , also known as gnathal ganglia. Secretory cells in the CC produce the peptides adipokinetic hormone (AKH) and limostatin (Lst) and cells in the CA produce juvenile hormone (JH), a sesquiterpenoid  has 24 cells, of 3–4 types and the LNC group in one hemisphere has 12 cells (of 2–3 types). These cell types are based on content of peptides determined using immunohistochemical approaches, sometimes in combination with Gal4 expression. Recent advances in single-cell transcriptomics may alter these numbers of cell types in the near future, as we begin to identify subtypes within supposedly homogenous groups of cells  and it has been assumed that these cells function as a group, i.e., that regulatory factors act on all IPCs. However, it seems that there are three subtypes of IPCs with respect to nutrient sensing: (1) IPCs with autonomous glucose sensing, (2) IPCs without glucose sensing and 3) those that receive inputs from two glucose-sensing DLPs (designated CNs)  and they additionally receive regulatory signals from brain neurons, CC, the intestine and the fat body Fig.  Ahmad e. The braIn adults, the IPCs are central to the regulation of metabolism, appetite and food seeking as shown in Fig. Another pathway that involves IPCs regulates metabolism-sleep interactions Fig. . A pair Drosophila can enter adult reproductive diapause when environmental conditions are adverse   locomotor activity and feeding rhythms, (2) feeding and excretion and (3) sperm release from spermatheca. It is not clear whether these represent three different axes or if there is an interactive link between these functional pathways.Drosophila and relies on several sets of peptidergic neurons spread out in the brain and SEZ and is not exclusively associated with the LNC-MNC cell groups  producing ITP have recently been shown to play a central role in regulation of feeding, drinking and excretion , resulting in three thoracic and nine abdominal neuromeres. In most insects studied, there are bilateral neurosecretory cells in each neuromere or ganglion  Fig. . BursicoSeveral peptides are produced in efferent abdominal neurons that “innervate” the hindgut, or reproductive tract Fig. ; neuronsDrosophila, the larval and adult midgut is a source of 11 different peptides , which represent another source of circulating hormones. Mammalian gut EECs express glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), ghrelin and cholecystokinin (CCK), which regulate various processes including regulation of hormone secretion, gut motility, nutrient homeostasis and feeding . Other peptides vary extensively between species. This common expression of several peptide hormones suggests that some ancient hormone functions have been evolutionarily conserved related to the control of feeding, metabolism, growth, reproduction and water homeostasis.There are not many comprehensive studies of peptide complement in specific brain neurosecretory cells of insects other than In the VNC, there are also some conserved peptidergic neurosecretory cell systems. For instance, pairs of LK . Systematic comparative studies of neuropeptide and peptide hormone functions in insects with larger and reduced peptidomes are required to resolve this.In Gryllus bimaculatus, AstB (MIP) is a regulator , from epitracheal (Inka) cells increases JH biosynthesis  in the eyestalks but also to some extent the anterior cardiac plexus  of the stomatogastric nervous system and the pericardial organs (POs) associated with the thoracic ganglia , hypothalamus-pituitary-thyroid (HPT) and hypothalamus-pituitary-gonadal (HPG) axes  are produced by glandular cells in the anterior pituitary and released into circulation via the vasculatory system Figs. , 13, wheThe general functions of a few insect LNC-MNC peptides partly overlap with those of some HP peptides: (1) DILPs produced by MNCs regulate growth (in larvae/pupae); (2) ion/water homeostasis is regulated by ITP and DH44; (3) metabolism by CRZ, ITP, DILPs and DH44 (and AKH from CC); and (4) gonad maturation and reproduction by DILPs (aspects of reproduction by DH44 and CRZ). In mammals, these functions are served by (1) growth hormone, (2) vasopressin, (3) TSH  and (4) FSH and LH  associated with the abdominal ganglia in insects Fig.  and the Drosophila and provided some comparisons with other insects as well as vertebrates. The regulation of Drosophila development, behavior and physiology relies on both (1) peptide hormones from LNCs, MNCs and other neurosecretory cells; (2) regulatory peptidergic interneurons that are part of high level neuronal circuits; and (3) peptidergic (and other) interneurons that regulate release of peptide hormones (sometimes in addition to being part of circuits). Hormonal axes in Drosophila are morphologically less distinctly organized (cells are more distributed) than those of the different hypothalamus-pituitary axes of vertebrates. Nevertheless, we can outline hormonal axes in Drosophila that regulate metabolism, appetite and food intake, reproductive behavior and physiology, sleep and activity rhythms and water and ion balance (excretion), as well as different developmental processes.We have outlined neurosecretory and peptidergic systems in Drosophila peptidome is somewhat reduced compared to several other insect species, suggesting that some peptide hormone functions are missing or taken over by other peptides. It would therefore be of great interest to widen the studies of insect peptide hormone functions and to explore other non-model insects with larger peptidomes. There are several Drosophila peptides whose adult functions are poorly known  and for some it even remains to be determined whether they have hormonal roles or not . With the multitude of novel imaging techniques available


Polycystic ovary syndrome (PCOS) is a complex multigenic disorder and women with PCOS suffer from several comorbidities. Although, obesity is a known risk factor for PCOS, the incidence of lean women with PCOS is on the rise. A systematic and comparative study on lean and obese PCOS with respect to genes, pathways and comorbidity analysis has not been attempted so far. Analysis of differentially expressed genes (DEGs) across tissue types for lean and obese PCOS revealed that the majority of them were downregulated for lean and obese PCOS. Ovarian and endometrial tissues shared several commonly dysregulated genes, suggesting shared PCOS pathophysiology mechanisms exist across tissues. Several pathways for cellular homeostasis, such as inflammation and immune response, insulin signaling, steroidogenesis, hormonal and metabolic signaling, regulation of gonadotrophic hormone secretion, cell structure and signaling that are known to be affected in PCOS were found to be enriched in our gene expression analysis of lean and obese PCOS. The gene-disease network is denser for obese PCOS with a higher comorbidity score as compared to lean PCOS. The cause of the disease can be attributed to genetic and lifestyle factors While obesity is a known risk factor for PCOS, not all women with obesity develop PCOS and not all women with PCOS are obese Metabolic syndrome, which is a constellation of conditions such as hypertension, abdominal obesity, insulin resistance and hypercholesterolemia, is commonly seen in women with obesity and PCOS ADIPOQ), leptin (LEP) and resistin (RETN) by adipose tissues is one of the important contributory factors to insulin resistance, cardiovascular diseases and metabolic disorders ADIPOQ is downregulated while LEP and RETN are upregulated in obese conditions ADIPOQ have been found to be lower in obese PCOS as compared to lean PCOS, and levels of LEP gene have been reported to be lower in lean PCOS as compared to obese PCOS RETN were found to be similarly upregulated in obese and lean PCOS cases as compared to controls Altered secretions of adipokines such as adiponectin (SHBG) plays a major role in metabolic clearance of free androgens and other hydroxysteroid ligands to the target tissues and liver. Lower serum levels of SHBG in PCOS leads to elevated levels of circulating androgens Although PCOS and obesity are characterized by increased androgen production, the bioavailable androgen levels are normal in obese non-PCOS cases as compared to PCOS, due to its high clearance rate The factors associated with PCOS such as anovulation, insulin resistance and altered steroidogenesis are known to increase the risk of cancers in females with PCOS The information currently available for lean PCOS is scarce as most of the reported literature is based on patients managed in hospital or fertility clinics, which is known to better represent obese PCOS meta-analysis approach, where each study containing cases and controls was normalized and analyzed individually to identify differentially expressed genes and enriched pathways and then these results were compared across studies. Information available in literature was used to validate some of the resulting observations. The study has helped to generate novel mechanistic hypotheses for lean and obese phenotypes of PCOS and also to validate existing observations such as higher comorbidity in women who are obese and suffer from PCOS as compared to lean PCOS Here, we identify differentially expressed genes, enriched pathways and associated comorbidities for lean and obese PCOS, based on systematically reviewed and analyzed lean and obese PCOS data from the Gene Expression Omnibus (GEO) 2The workflow adopted in this study is illustrated in 2.1meta-analysis (The Gene Expression Omnibus (GEO) database was searched on 30th September 2019 to retrieve human microarray gene expression studies on PCOS using the query terms (((“polycystic ovary syndrome”) AND “Homo sapiens”[Organism]) AND gse[Filter]) AND “Expression profiling by array” [Filter]. 26 datasets were identified using this query, which were further manually curated for excluding studies that involved lncRNA, drug-treated samples, cell line studies and non-BMI matched samples. Eight GEO datasets  qualified for the analysis . The wom2.2meta-analysis approach, where each case-control study was separately analyzed from raw data to the differentially expressed gene stage, then these DEG lists were compared across studies affyoligobiomaRtlimmaThe CEL files were retrieved for the selected GEO datasets and each dataset was analyzed following a 2.3The DEGs obtained from each dataset were compared to detect commonly dysregulated genes in PCOS across diverse sample types and GEO platforms. For tissue-based analysis, the aforementioned list of DEGs were clustered based on their tissue source to identify commonly dysregulated genes across the tissue types. The array expression datasets were grouped based on their source, into four tissue types, namely ovarian, endometrial, adipose and skeletal. The ovarian group included metaphase II oocyte, cumulus cells and lutein granulosa cells. The endometrial group included cell types of epithelial, endothelial, stromal fibroblasts and mesenchymal stem cells. The adipose group had subcutaneous adipose tissue and omental adipose tissue. The skeletal group had skeletal muscle tissue.2.4Pathway enrichment analysis was performed using the Gene Set Enrichment Analysis (GSEA) method. The Java desktop application of GSEA (v. 3.0) developed by Broad Institute was used to identify statistically enriched gene sets and pathways in each of the datasets The GSEA output and normalized expression data were used to perform Enrichment analysis. The Enrichment Map Analysis Pipeline 2.5The KEGG disease database (Release 88.2) was used to obtain information for human diseases and its associated genes. The diseases were categorized as per International Classification of Diseases 11th Revision (ICD-11). The KEGG gene and disease IDs were used for mapping genes with the highest level of ICD11 classification. The DEGs were further mapped to this data to obtain the gene-disease association score (GDS) for PCOS. For each ICD-11 category, GDS was calculated as belowGene-disease association was further used to construct gene-disease networks for lean and obese PCOS.2.6GSVAcomplexheatmapThe R packages 33.1ETV3, GABPB1, ELF3, GABPA, ELF1, ELF4 and SRF) genes were identified to encode transcription factors using the iRegulon Cytoscape app A total of 5014 (unique = 4224) statistically significant DEGs were identified by analyzing the eight GEO datasets individually . Of these, 123 (unique = 96) genes were upregulated and 4891 (unique = 4101) genes were downregulated (Supplementary Table S3). Regardless of tissue type and phenotype, the majority of the genes were downregulated in PCOS. Endometrial cells of obese PCOS and cumulus cells of lean PCOS displayed the highest number of dysregulated genes . Seven  were found to be commonly dysregulated when analysis was restricted to two tissues (cumulus cells and subcutaneous adipose tissue) using GPL570 platform for lean PCOS (Supplementary Table S5).Array analysis of lean and obese PCOS, revealed that there were no commonly dysregulated genes across the platforms and tissue sources. Seven genes  3.3GPX7, SERPINI1, TMEM256, SVIP, MAT2A and SRGN) were commonly dysregulated in ovarian, endometrial and adipose tissues. Apart from these six genes, ovarian and endometrial tissues shared 181 (4.3%); ovarian and adipose tissues shared 21 (0.5%); adipose and endometrial tissues shared 45 (1.1%); endometrial and skeletal tissues shared 5 (0.1%) commonly dysregulated genes  revealed that there were 86 pathway gene sets (6.7%) common for lean and obese PCOS, 1031 pathway gene sets (80.8%) unique to lean PCOS and 159 pathway gene sets (12.5%) unique to obese PCOS cases  Cell-motility and immune response; (ii) FAK-related; (iii) ERBB1 and PDGFRB signaling; and (iv) Mitochondrial gene expression. Pathway gene sets involved in ‘Cell-motility and Immune response’, ‘FAK-related’ and ‘ERBB1 and PDGFRB signaling’ were downregulated in lean PCOS and upregulated in obese PCOS. Pathway gene sets involved in ‘Mitochondrial gene expression’ were upregulated in lean PCOS and downregulated in obese PCOS .Table 2D3.5CFH and CYP1B1 which are well studied for its role in eye diseases FN1, ACTN4 and TRPC6 mapped to the pathological condition of the glomerulus. It is noteworthy that DEGs identified from GEO datasets of obese PCOS and endometrial tissues displayed maximum GDS . The maximum number of DEGs were mapped to developmental anomalies (222 genes), followed by disorders of the metabolic (192 genes) and nervous system (127 genes). It is interesting to note that 37 DEGs mapped to disorders of the visual system (Supplementary Table S8). This list included genes such as imum GDS .Fig. 5GD3.5.1To visualize the DEGs-disease mapping as a map to support hypothesis generation, we constructed gene-disease networks for lean and obese PCOS. Although the disease categories that mapped to DEGs of lean and obese PCOS were similar, the number of DEGs associated with the diseases was much higher in obese PCOS as compared to lean PCOS (3.5.2GSVA was performed to assess the association of the dysregulated genes identified in ovarian tissue samples of lean and obese PCOS (GPL570 platform only) to reproductive and endocrine diseases. The genes associated with the disease category of pregnancy, childbirth or the puerperium were upregulated in the cumulus cell and metaphase-II oocytes derived from obese PCOS as compared to non-PCOS samples. Conversely, these genes were downregulated in cumulus cell derived from lean PCOS as compared to non-PCOS samples. The difference in the gene expression pattern in cumulus cells obtained from lean women and women with obesity and PCOS suggests that the etiology of PCOS may be different in the lean and obese PCOS . The genes associated with these reproductive and endocrine diseases were traced to the GSEA pathway enrichment plots to identify the pathways associated with these genes and compare the enrichment status of these pathways across lean and obese PCOS (Supplementary Tables S10 and S11).4PCOS has a multigenic etiology and women with PCOS are at risk of multiple comorbidities that include obesity, cardiovascular diseases and insulin-resistant diabetes.meta-analysis. A list of enriched pathways, associated DEGs and literature evidence for the association of these pathways with PCOS can be seen in Supplementary Table S12. Several research groups are working on potent insulin sensitizers such as inositol and its stereoisomers Insulin resistance, leading to hyperinsulinemia and type-2 diabetes, is frequently observed in women with PCOS While there are numerous high throughput studies that aim to delineate the genes and pathways that are dysregulated in obese PCOS and PCOS in general, the studies on lean PCOS are scarce and studies that compare obese and lean PCOS are far scarcer meta-analysis of gene expression studies and pathways for lean and obese PCOS from diverse human cell types/tissue sources and array platforms. The salient observations from the meta-analysis are discussed below.1)The pathophysiology of lean and obese PCOS seems to be differentIn the present study, we have attempted to address the above two aspects by collating information from literature and performing a 2)PCOS is caused mainly by downregulation rather than upregulation of associated genesAlthough the majority of DEGs associated with PCOS seems to be downregulated across the lean and obese PCOS phenotypes; the expression profile of genes from similar cell types seems to be different between lean and obese PCOS . Moreover, we did not find any commonly dysregulated genes between lean and obese PCOS datasets (Supplementary Table S5). The genetic contributors of lean and obese PCOS may therefore be different. However, additional research would be required to substantiate this hypothesis. The pathway analysis also reveals that the etiology of lean and obese PCOS is different .2)PCOS i3)Ovarian and endometrial tissues share several commonly dysregulated genesAnalysis of DEGs across tissue types for lean and obese PCOS revealed that the majority of them were downregulated . GSVA an.4)Pathway analysis reveals that cellular homeostasis is disrupted in lean and obese PCOSOvary is considered as the most important target organ of PCOS 5)The gene-disease network is denser for obese PCOS as compared to lean PCOSPathway analysis revealed that several fundamental pathways responsible for cell proliferation and survival such as those involved in gene expression, DNA recombination, cell cycle, cell structure and signaling are perturbed in lean and obese PCOS . A strikGene-disease networks mapped for dysregulated genes of obese PCOS are denser as compared to lean PCOS . This da4.1CYP1B1 in PCOS is well documented in literature, meta-analysis, which may have led to interesting observations.An important limitation of the study is that the analysis and conclusions presented here are dependent on the gene expression data on lean and obese PCOS existing in the public domain and the consistency maintained across the datasets for phenotype annotations. The study would have benefited with higher number of tissue and BMI-matched samples than the present availability in the GEO database. A second limitation, specifically for the comorbidity analysis, is the lack of a comprehensive and updated gene-disease association databases available for researchers. For example, although the association of of 5Tissue-based comparative analysis of the DEGs, pathway networks and GDS revealed that endometrium and ovary are important target organs of PCOS. The analysis offers evidence as to why women with obesity and PCOS are at higher risk of suffering from comorbidities as compared to lean PCOS. While the unifying mechanisms of obesity, metabolic-related disorders and PCOS are still unclear https://github.com/bic-nirrh/pcos-metaanalysis.The R codes used for the meta-analysis are freely available at in silico analysis; GDB was involved in study plan and writing the manuscript.S-IT was involved in study plan, data analysis and writing the manuscript; UG and SB were involved in literature review and manuscript writing; UG and KP was involved in Susan Idicula-Thomas: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition. Ulka Gawde: Methodology, Formal analysis, Writing - review & editing. Sameeksha Bhaye: Investigation, Writing - review & editing. Khushal Pokar: Formal analysis. Gary D. Bader: Conceptualization, Methodology, Writing - review & editing, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Paneth cells (PCs) are located at the bottom of small intestinal crypts and play an important role in maintaining the stability of the intestinal tract. Previous studies reported on how PCs shape the intestinal microbiota or the response to the immune system. Recent studies have determined that PCs play an important role in the regulation of the homeostasis of intestinal epithelial cells. PCs can regulate the function and homeostasis of intestinal stem cells through several mechanisms. On the one hand, under pathological conditions, PCs can be dedifferentiated into stem cells to promote the repair of intestinal tissues. On the other hand, PCs can regulate stem cell proliferation by secreting a variety of hormones (such as wnt3a) or metabolic intermediates. In addition, we summarise key signalling pathways that affect PC differentiation and mutual effect with intestinal stem cells. In this review, we introduce the diverse functions of PCs in the intestine. The intestinal epithelium, a single layer of columnar cells, lines the luminal surface of the intestinal mucosa and is regenerated throughout adult life. The intestinal epithelium is one of the most rapidly proliferating epithelia in mammals and plays an essential role in maintaining the balance between homeostasis and pathological condition . In the In the normal physiological state, the Wnt signalling cascade is key pathway that regulates the proliferation and differentiation of intestinal stem cells (ISC) –5. Lgr5 Secretory lineages are derived from a common progenitor that emerges to occupy the +5-cell position above the stem cell niches . A recenAs part of the niche, PCs represent a population of morphologically distinct and functionally specialised intestinal epithelial cells , which hIntestinal stem cells are a source of rapid renewal of the intestinal epithelium by giving rise to all type intestinal epithelial lineages , 8. StudIrradiation animal models are a very widely used vector for intestinal inflammation damage. The mechanism involved in acute intestinal radiation is supported by evidence; that is, it mainly involves extensive activation of tumour necrosis factor alpha (TNFα) cascades, activation of tight junction signalling, aryl hydrocarbon receptor (AhR)-mediated apoptosis, activation of cell cycle signalling pathways and activation of the coagulation system , 16. DexIntestinal stem cells are located in the intestinal crypts, where PCs are also located. Recent studies focused on defining the identity of intestinal stem cells and their interaction with their PC niche. Confocal cross-sectioning of the intestine crypt bottom showed an almost geometrical distribution of PCs and Lgr5 stem cells, leading to maximised heterotypic contact area (Paneth–stem cell) and minimised homotypic contact area . This feIn in vitro experimental research, PCs and Lgr5+ cells were sorted from untreated mice; their co-culture generated more organoids compared with Lgr5+ cells only . In DSS-In recent years, many studies have confirmed that PCs can regulate ISCs through various signalling pathways and growth factors. PCs itself could provide multiple secreted protein such as Wnt3, EGF and Notch ligand Dll4 and Dll1, which are crucial for stem-cell support , 11, 21 However, another study reported a transgenic mouse model , which made the PCs completely removed possible. Three months after tamoxifen administration, Lgr5+ cells manifest intact long-term self-renewal in the gut epithelium in the absence of PCs. The levels of Wnt target transcripts, including genes associated with cell proliferation, such as CD44, Myc and Ccnd1, were unaffected in Lgr5+ stem cells. However, the Ki67 labelling index and the BrdU expression increased in Atoh1−/− mice ; this phPCs that are terminally differentiated exit the cell cycle and do not express CBCs or proliferative markers during normal homeostasis in vivo , 33. ThiNotch and Wnt pathways play an important role in the plasticity of PCs. In animal and in vitro experiments, the Notch signalling pathway plays a key role in obtaining the stem characteristics of PCs. In vitro experiments, radiated PCs acquire stem cells features; this process leads to elevated expression of Notch pathway gene signatures, such as Hes1, Dtx4 and Adam17 compared with that under homeostatic conditions , 36. AnoIn the normal state, β-catenin is always nuclear positive in small intestinal PCs. β-catenin is a canonical target gene of the WNT signalling pathway, and it enters the nucleus and activates WNT signal. The positive nuclear expression of β-catenin in PCs probably indicates that PCs are sensitive to changes in Wnt signal within the crypt compartment. Acute loss of Wnt signalling results in depletion of PCs, whereas broad activation of canonical Wnt signalling results in the increase in the number of PCs , 39. HowIn addition to PCs, other differentiated intestinal epithelial cells, such as enterocyte cells, enteroendocrine cells (EECs) and other types of cells, can obtain stem features under certain conditions and promote intestinal injury repair. Enterocytes could acquire regeneration-associated genes by dedifferentiating into stem cells and Paneth-like cells when Lgr5+ stem cells are depleted . A subseThe lamina propria located below the intestinal epithelium of the intestine contains abundant mesenchymal cells, macrophages, immune cells and collagen. This area also plays an important role in repair of intestinal damage.Stromal cells surround the base of crypts and mainly include mesenchymal cells, fibroblasts, endothelial cells, neural cells and inflammatory cells scattered in between them; stromal cells have an important regulatory effect on intestinal epithelium. Mesenchymal cells and related collagen are necessary for regeneration of the intestinal epithelium. The matrix-mediated integrin signalling via YAP/TAZ mechano-transduction promotes intestine regeneration in collagen type I; this process is accompanied by Wnt activation, and the same phenomenon was observed in human ulcerative colitis . ISLR haInflammatory cells such as T cells and macrophages, by produced cytokines, influence intestine regeneration. CD4+ and CD8+ T cells suppress human colon organoid formation; one of the mechanisms is T cells derived from IFNγ through JAK1/STAT1 activation directly target ISCs and induce apoptosis . IFNγ alCytokines, such as IL-4, IL-22, IL-6, IL-33 and tumour necrosis factor (TNF), are also involved in maintaining the function of the intestinal epithelium. IL-4 is a cytokine that is involved in T cell differentiation and regulation of immunoglobulin production . In vitrThe regulation of intestinal epithelium is a comprehensive and complex process. In addition to the intestinal epithelium, sub-epithelium tissues and cytokines also provide support for the steady state of the intestinal epithelium. The activation of the Wnt pathway is necessary for ISCs to promote intestinal damage repair Fig. .Cell metabolism has been implicated in stem cell maintenance and differentiation in adult stem cell populations . Recent The mechanistic target of rapamycin (mTOR) is an important nutrition sensing element. It could regulate intestinal epithelial cells and ISCs during injury conditions , 72. ThePCs can affect the proliferation and differentiation of ISCs through different metabolic pathways. Under conditions of caloric restriction in mice, the numbers of PCs and Lgr5+ CBCs increased in vivo. In ex vivo study, co-culture with PCs from caloric restriction mice with intestine stem cell (ISCs from untreated or treated mice) gave rise to more and larger secondary organoid bodies than used normal PCs. The mechanism through which PCs augment stem cell function in response to calorie restriction involves the reduction of mTORC1 signalling in the cytoplasm. Under caloric restriction, downregulation of mTORC1 enhances the release of the paracrine effector bone stromal antigen 1 (Bst-1), which converts NAD+ to cyclic ADP ribose (cADPR) and promotes CBC proliferation  Fig. 2)2). FurthIn other studies, PCs affect the function of stem cells through other metabolic pathways. Study elaborated that Lgr5+ CBCs and PCs from the small intestine of mice displayed different metabolic programs. Compare with PCs, Lgr5+ CBCs showed higher mitochondrial activity. In Lgr5+ CBCs, the onset of differentiation relies on mitochondrial oxidative phosphorylation (OXPHOS), through which the number of crypts per organoid and the number of PCs increase. Treatment with glycolysis inhibitors in PCs strongly reduce their niche-supporting function and organoid formation. Addition of exogenous lactate, an end product of glycolysis, can strongly enhance the ability of Lgr5+ CBCs to form organoids . This exIn conclusion, Paneth cells serve as sensors for nutritional status affects the functions of Lgr5+ cells not only through growth factor signalling but also by providing them with a metabolic fuel source.Ageing is an inevitable biological event and is usually accompanied by decreased function, including diminished self-renewal ability of stem cells . This prThese data demonstrate that Notum produced by PCs attenuates the regenerative capacity of ageing intestinal epithelium in vivo by reducing Wnt activity in stem cells. PCs in contrast can promote the function recovery of ageing ISCs when fasting by metabolic-related pathways.Alterations in the number and function of PCs are associated with maintaining intestinal homeostasis. Thus, any mechanism that affects PC development or function could further change the intestinal homeostasis. In mice, PCs in the intestine differentiate around postnatal day 14 . In humaThe Wnt signalling pathway is vital to promote PC development and is also important for maintaining the undifferentiated state of intestinal crypt progenitor cells. Administration of Wnt3a increases the number of PCs in intestinal organoids  Besides the Wnt signalling pathway, many other growth factors affect the development of PCs]. The ErbB3 neuregulin (NRG) receptor is expressed in most epithelial tissues . In inteThe Notch signalling pathway plays a significant role in controlling the cell fate of intestinal epithelial progenitor cells into absorptive and secretory lineages. The inhibition of the Notch pathway by either chemical inhibition or knockout of associated genes  can increase the secretory phenotype of cells, mainly goblet cells , 108–110In addition to above signalling pathways, metabolism-related growth factors determined regulate the fate of PCs. Ketone bodies can mediate the proliferation ability of ISCs; 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) is the gene encoding the rate-limiting step for ketogenesis when the deletion of Hmgcs2 leads to pronounced expansion of PCs , 114. TuInflammatory factors are also involved in the regulation of PCs. For example, the deletion of Toll-like pattern recognition receptor (TLRs)9 could lead to downregulation of the number of PCs by blocking the release of interleukin17A (IL-17A) . The IL-We have discussed many growth factors and inflammatory factors that can mediate the development of PCs in different angles. The occurrence and differentiation of PCs is greatly affected by the Wnt signalling pathway but is minimally influenced by the Notch signalling pathway. In addition to traditional pathways, metabolic and inflammatory processes also affect the fate of PCs to a certain extent.PCs are a group of specialised epithelial cells of the small intestine and contain multiple secretory granules filled with antimicrobial peptides, which are essential for control of microbial growth and maintaining intestinal integrity. Previous studies and reviews focused on how PCs shape the intestinal microbiota or response to the immune system –126. ThiPCs are located at the bottom of intestinal crypts. As a terminally differentiated cell, the function of ISCs are partially regulated by paracrine-specific secreted proteins  or metabolic regulation under the conditions of intestinal homeostasis, including ageing and caloric restriction. The regulation of ISCs is mainly achieved by regulating the Wnt signalling pathway, but the metabolic regulation process, which promotes stem cell function by providing metabolic substrates. In the pathological state, the function of ISCs is enhanced, and PCs can acquire stem features to repair the intestinal mucosal epithelium. The strong plasticity of PCs is mainly achieved by reactivating the Notch signalling pathway.In the whole intestine, various cells of the intestinal lamina propria and cytokines play an important role in the steady state of ISCs and the regulation of PCs. As mesenchymal cells, fibroblasts can promote the function of ISCs by activating the Wnt signalling pathway or secreting Wnt and EGF; this process can partially overlap with the function of PCs. Similar to stromal cells, cytokines  also promote the proliferation of ISCs by activating Wnt.The development of PCs is also regulated by a variety of growth factors and cytokines especially under pathological conditions; the differentiation of precursor stem cells into PCs is greatly affected. PCs play an important role in the function of ISCs, in the ways that regulate PCs, which also means a certain effect on ISCs. The main factor that effects the differentiation of PCs is the Wnt signalling pathway.In summary, PCs can be regarded as the guardians of intestinal crypt function and have a huge regulatory effect on ISCs under pathological and physiological conditions. The interaction network between PCs and stromal cells and between PCs and differentiated intestinal epithelial cells remains unclear.


Already for centuries, humankind is driven to understand the physiological and pathological mechanisms that occur in our brains. Today, we know that ion channels play an essential role in the regulation of neural processes and control many functions of the central nervous system. Ion channels present a diverse group of membrane-spanning proteins that allow ions to penetrate the insulating cell membrane upon opening of their channel pores. This regulated ion permeation results in different electrical and chemical signals that are necessary to maintain physiological excitatory and inhibitory processes in the brain. Therefore, it is no surprise that disturbances in the functions of cerebral ion channels can result in a plethora of neurological disorders, which present a tremendous health care burden for our current society. The identification of ion channel-related brain disorders also fuel the research into the roles of ion channel proteins in various brain states. In the last decade, mounting evidence has been collected that indicates a pivotal role for transient receptor potential (TRP) ion channels in the development and various physiological functions of the central nervous system. For instance, TRP channels modulate neurite growth, synaptic plasticity and integration, and are required for neuronal survival. Moreover, TRP channels are involved in numerous neurological disorders. TRPM3 belongs to the melastatin subfamily of TRP channels and represents a non-selective cation channel that can be activated by several different stimuli, including the neurosteroid pregnenolone sulfate, osmotic pressures and heat. The channel is best known as a peripheral nociceptive ion channel that participates in heat sensation. However, recent research identifies TRPM3 as an emerging new player in the brain. In this review, we summarize the available data regarding the roles of TRPM3 in the brain, and correlate these data with the neuropathological processes in which this ion channel may be involved. The brain forms the control center of our body and is responsible for processing tremendous amounts of data to monitor and regulate our bodily functions at day and night. Such a precise control needs to be fast and accurate and requires highly sophisticated information processing. Our body accomplishes this task via neuronal cells, which form a complex connectome within the nervous system Purves, . Within Among the hundreds of ion channels expressed in the human brain, Transient Receptor Potential (TRP) channels form a diverse group of poly-modally activated cation channels and they are generally considered as molecular sensors of external and internal stimuli Clapham, . In totacanonical (TRPC) channel subfamily presents probably the most-studied TRP channel subfamily in the brain , which is of importance in descending modulatory pain pathways and seems to play an extended role in other brain functions, by enhancing glutamatergic neurotransmission  channel subfamily represents an especially heterogeneous group within the TRP superfamily and includes eight members in total (TRPM1-8). Most of them are non-selective calcium-permeable cation channels. As an exception, TRPM4 and TRPM5 are calcium-impermeable channels activated by intracellular calcium. As such, all TRPM family members can evoke calcium increases in cells, either by conducting calcium ions through their pores or by regulating the membrane potential, thereby activating/modulating calcium channels or transporters   in a total of nine patients , which was identified to be essential for the channel formation. Isoforms lacking ICFR, like TRPM3α7, do not form functional ion channels, probably due to a disturbed tetrameric channel complex formation and a decreased plasma membrane expression. Therefore, when co-expressed with the functional TRPM3α2, TRPM3α7 acts as a dominant negative regulator of the channel activity  P3 was found to be the most effective P2, and S100A protein  subunit, and amino acids on both the TRPM3 peptide and the Gβ1-proteins that mediate the interaction were identified. Interestingly, the interacting residues in Gβ1 only partially overlap with those involved in the inhibitory interaction with G-protein-coupled inward rectifier K+ (GIRK) channels , nor arachidonic acid, anandamide, linoleic acid, linolenic acid, and diacylglycerol analogs affected the channel , the Hv proton channel, which even does not possess a classical pore domain  and heart diseases (Mixed Arrhythmias and Dilated Cardiomyopathy). Some mutations in the voltage sensor of Kv7.2 and Kv7.3 subunits also result in the development of non-canonical pore currents. These currents can contribute to the hyperexcitability of neurons and are associated with benign familial neonatal seizures. Recently, two mutations in TRPM3 were found in patients with developmental and epileptic encephalopathies (DEE) , which belongs to the Interestingly, primidone and volatile anesthetics inhibited both the classical and the non-canonical pore mediated currents, which suggests that these inhibitors do not act as classical pore-blockers but rather inhibit a more general conformational change in the proteins . From all eight patients, seven carried an identical point mutation in the linker region between transmembrane segments 4 and 5. This mutation resulted in the substitution of a valine with a methionine (VM mutation)  . The remutation)   . More suutation)  who repoutation)  AlthoughTRPM3 gene were linked to several neurological disorders in human patients  pharmacological targeting or genetic alterations in animal models would be of immense value for our future quest to explore the role(s) of TRPM3 in the brain. Considering the broad hormonal regulation profile of TRPM3 (as discussed in section TRPM3 Is a Steroid Regulated Channel), it would be of further scientific value to apply behavioral tests to animals of different gender and different developmental stages. Such experiments will not only provide insights into the physiological roles of TRPM3 in the brain but could also illuminate the mechanisms of disease in selected TRPM3 animal disease models.Obviously, more research is required to investigate TRPM3 expression, regulation and function in different brain regions and cell types, and to validate its role in brain (patho)physiology. Nevertheless, given our current knowledge about the molecular and biophysical properties of TRPM3 and its recent genetic links to brain pathologies, it is tempting to imagine TRPM3 as an attractive potential new target for future drug interventions in neurological diseases such as epilepsy or autism spectrum disorders.KH and BIT drafted, corrected, and wrote this article. BIT was responsible for the funding acquisition. All listed authors qualify for authorship and all authors qualifying for authorship are listed above.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.