Now α2-ARs lurk on the horizon involving hypertension causality, renal denervation for hypertension, injury from falling in the elderly and prazosin's mechanism of action in anxiety states such as posttraumatic stress disorder (PTSD). Our goal here is to focus on this horizon and bring into clear view the role of α2-AR-mediated mechanisms in these seemingly unrelated conditions. Our narrative begins with an explanation of how experiments in isolated perfused kidneys led to the discovery of a sodium-retaining process, a fundamental mechanism of hypertension, mediated by α2-ARs. In this model system and in the setting of furosemide-induced sodium excretion, α2-AR activation inhibited adenylate cyclase, suppressed cAMP formation, and caused sodium retention. Further investigations led to the realization that renal α2-AR expression in hypertensive animals is elevated, thus supporting a key role for kidney α2-ARs in the pathophysiology of essential hypertension. Subsequent studies clarified the molecular pathways by which α2-ARs activate prohypertensive biochemical systems. While investigating the role of α1-adrenoceptors (α1-ARs) versus α2-ARs in renal sympathetic neurotransmission, we noted an astonishing result: in the kidney α1-ARs suppress the postjunctional expression of α2-ARs. Here, we describe how this finding relates to a broader understanding of the role of α2-ARs in diverse disease states. Because of the capacity for qualitative and quantitative monitoring of α2-AR-induced regulatory mechanisms in the kidney, we looked to the kidney and found enlightenment.It was indeed a Don Quixote-like pursuit of the mechanism of essential hypertension when we serendipitously discovered  Rana pipiens) skin. In these experiments, the skin of the rear thigh of Rana pipiens was draped over a 1.2 cm cylinder and light reflectance from a parallel light source was quantified by a photometer; then, MSH was applied. Microscopically, MSH treatment causes a rapid migration of tiny black granules from the dense core granules to completely blacken the skin within a few minutes. Epinephrine, via α-adrenoceptors, causes a reverse migration of the granules back into the dense core granules and a tremendous increase in the reflected light in several minutes. We found methyldopamine and methylnorepinephrine to be 30 to 100 times as potent as the naturally occurring dopamine and norepinephrine metabolites of DOPA in reversing MSH-induced darkening of frog skin [α2-ARs and the functional classification of α-adrenoceptors [α2-AR subtypes, referred to as α2A-AR, α2B-AR, and α2C-AR, have been cloned. Despite having only approximately 50% amino acid sequence identity, these three α2-AR isoforms have similar structures, pharmacology, and signaling mechanisms, yet differ in tissue distribution (see Aantaa and coworkers [While pursuing the hidden antihypertensive mechanism of methyldopa (Aldomet) , 2, we eoceptors , 5. Sincoworkers  for reviα2-ARs were discovered in normal kidneys [α2-ARs was also present in prazosin (selective α1-AR antagonist) pretreated normal rats [α2-ARs occupied the anatomic and functional domain unique to α1-ARs in membrane enclosed postjunctional sites . Acute reduction of systemic arterial pressure thus interferes with results of studies of these drugs in vivo, so in vitro perfusion techniques were essential to study these mechanisms. However, technical problems of kidney perfusion precluded use of these techniques as well.Renal excretory function is dependent on high oxygen tension in the perfusate and oncotic effects of albumin to prevent severe edema in isolated, perfused kidneys. Previous studies failed because bubbling of oxygen denatured and precipitated albumin which clogged arterioles. We overcame these technical problems using the reverse phase of pediatric dialysis coils for oxygenation of albumin/ficol (oncotic activity) perfusates which sustained rat kidney functioning for hours –13. For α1-AR antagonist prazosin; however, the α2-AR antagonist yohimbine had no effect. This is not surprising since α1-ARs are considered the primary postjunctional α-AR subtype mediating noradrenergic neurotransmission via a signaling mechanism that involves coupling to phospholipase C via Gq/11 with subsequent production of inositol trisphosphate, diacylglycerol, and calcium release from the endoplasmic or sarcoplasmic reticulum [α2-AR antagonist) infusion was required to block sympathetic nerve-induced sodium retention [α2-AR density in the kidney had doubled after three days of prazosin administration. Thus, α1-AR blockade induces synthesis of new α2-ARs which then occupy the otherwise exclusive postjunctional domain and function of α1-ARs . Notably, norepinephrine (NE) infusion did not cause sodium retention. Thus, a barrier encompasses the sympathetic nerve terminal/α1-AR linkage which impedes access of circulating NE to the innervated α1-ARs mediating sodium retention. From these observations, it appears that normally functioning α1-ARs are suppressive to α2-AR synthesis. In fact, in genetic hypertensive rats where excess α2-ARs exist, there is a defect in α1-AR signaling [ α2-ARs in genetic hypertension  mediates excess sodium retention as part of the fundamental cause of hypertension [α1-ARs suppress the expression of α2-ARs is unknown, and it is imperative to investigate this mechanism in future studies. We speculate that activation of the inositol trisphosphate/calcium/diacylglycerol axis by α1-ARs in the postjunctional cell leads to either reduced production of α2-ARs, decreased trafficking of α2-ARs to the postjunctional membrane, or increased degradation of α2-ARs.In this isolated perfused rat kidney model, we found that subpressor sympathetic nerve activation promoted sodium retention which was blocked by the eticulum . Howeverf α1-ARs . Inasmucignaling . It shourtension . Currentα2-AR-mediated signaling is illustrated further by the contrasting effects of α2-AR activation on sodium retention/excretion by kidneys under different hormonal influences. For example, vasopressin infusion causes water and sodium retention [α2-AR activation causes sodium excretion. Alternatively, in the presence of furosemide, adenylate cyclase is activated, sodium excretion is increased, and α2-AR activation induces sodium retention [α2-AR signaling is context dependent in many other tissues, for example, the brain. Most importantly, isolated kidney perfusion techniques were critical in discovery of increased synthesis and redistribution of α2-ARs which may explain diverse clinical phenomena as described below.The complexity of etention , and wheetention , 18. We α1-AR-mediated retention of sodium [β-adrenoceptor activation of the renin-aldosterone-sodium retaining mechanism [High blood pressure is arguably the most important public health problem in the world today. If the kidney has an impaired capacity to excrete sodium  and dietary sodium intake is excessive, an increase in arterial pressure is required to cause natriuresis to restore sodium balance . Thus, hf sodium , 23 and echanism –28.α1-AR and β-adrenoceptor blocking agents, when used alone, have very low efficacy during long-term usage in hypertension treatment. Why? For prazosin, the reason is that excess α2-ARs and new function thereof occur with blockade of α1-ARs [α2-ARs functionally for α1-ARs impairs the efficacy of prazosin. We suspect that similar excess and translocation of α2-ARs occur on blood vessels, especially on veins, the reservoir of blood needed to sustain cardiac output, and hence blood pressure, in the upright position.Thus, it is remarkable that f α1-ARs  for thref α1-ARs , 30 and β-adrenoceptor blockers when used alone is because the dominant controller of renin release is a pressor-volume mechanism [β-adrenoceptors. However, when the sympathetic nervous system is reflexly activated by peripheral acting antihypertensive drugs, β-adrenoceptor blockade of excess renin release is a powerful blood pressure lowering mechanism [The low antihypertensive efficacy of echanism  independechanism –28. Exteechanism , detailsechanism . Moreoveechanism . It is iechanism .α2-AR-specific mRNA is on the Y-chromosome and one or more are on somatic chromosome(s) [α2-ARs in causality of human hypertension. Incidentally, in order for increased α2-ARs to sustain a tonal retention of sodium, our model presumes an endogenous factor stimulatory to a renal adenylate cyclase pool/isoform that is linked to sodium excretion by the kidneys. Who will discover this remarkable substance?With regard to hypertension, sex is an important biological variable with premenopausal women having a lower blood pressure compared to age-matched men . Interesosome(s) , 36. Thiα2-ARs in hypertension is provided by studies in the spontaneously hypertensive rat (SHR), a genetic model of hypertension with excessive expression of renal α2-ARs. Despite normal plasma and kidney levels of angiotensin II, as well as normal expression of angiotensin II type 1 receptors (AT1Rs) in the kidney, this model of genetic hypertension is fully dependent on the renin-angiotensin system for development and maintenance of an elevated blood pressure [α2-ARs are Gi-coupled receptors that upon activation release large quantities of βγ G-protein subunits from the Gi ternary complex , and AT1Rs are Gq-coupled receptors that upon stimulation release αq G-protein subunits from the Gq ternary complex . As discussed by Selbie and Hill [βγ subunits bind to specific isoforms of phospholipase C (PLC), the ability of αq to activate the enzymatic activity of PLC is remarkably enhanced. PLC, in part via activation of protein kinase C (PKC), then promotes renal vasoconstriction. In SHR kidneys, but not kidneys from genetically normotensive rats, this signaling “cross-talk” or “coincident signaling” is engaged when α2-ARs are stimulated with the selective agonist UK 14,304 and AT1Rs are simultaneously activated with angiotensin II [α2-ARs in SHR kidneys provides an enlarged membrane-localized pool of βγ subunits which tethers the scaffolding protein receptor for activated C kinase 1 (RACK1) to the cell membrane [βγ subunits released from α2-ARs, αq subunits released from AT1Rs, PLC, and PKC [α2-AR/Gi-mediated release of βγ in this mechanism is highlighted by recent findings that Gi-coupled receptors are particularly effective in releasing active βγ subunits. In contrast, other G-proteins  tend to restrain the signaling capabilities of βγ subunits released from their corresponding αβγ complexes [α2-AR-mediated release of αi from the αiβγ ternary complex may also contribute to the enhanced renovascular response to angiotensin II in SHR kidneys  has shown promise as a treatment for resistant hypertension [α1-ARs to suppress the synthesis of α2-ARs. Thus, RDN likely would upregulate the expression of renal α2-ARs in the renal sympathetic neuroeffector junction. Upregulated α2-ARs may express agonist-independent (constitutive or intrinsic) activity [α2-ARs sensitizes kidneys to the detrimental effects of angiotensin II and nitric oxide synthase inhibition.To the extent that rtension , 59. Howrtension . Why thertension . Howeveractivity  or couldactivity  revealedα1-ARs. In this context, postural hypotension can be severe during the first few days of prazosin treatment or reinitiation of treatment [α1-AR antagonists are used frequently to reduce urethral tone in prostatic obstruction in elderly men, in stress disorders and in treatment of hypertension. Fortunately, severe orthostasis is temporary, and we attribute reversal thereof to stimulation of newly expressed α2-ARs that occupy the otherwise exclusive postjunctional domain of α1-ARs. The exaggerated temporary orthostasis is due to two mechanisms: one is reduced retention of sodium and water by the kidney causing volume contraction in venous reservoirs; the second mechanism is due to a decrease in venous tone which occurs during the time interval before multiplication and assumption of α1-AR function by newly synthesized α2-ARs.Falling is a frequent cause of serious injury and death in the elderly and orthostatic hypotension often causes falling. With prazosin, blood pressure drops in the standing position because of blockade of postsynaptic reatment , 30. Praα-adrenoceptor junction and its protective barrier discovered in the isolated kidney perfusion model. In this regard, pioneering work by Solomon Langer [α2-ARs that are inhibitory to NE release. In pheochromocytoma, high levels of circulating NE have access to these presynaptic α2-ARs that are inhibitory to NE release. Thus, α2-AR-mediated inhibition of NE release in the standing position may also contribute to orthostatic hypotension in pheochromocytoma.Orthostatic hypotension is often severe in patients with excess circulating NE in pheochromocytoma, an apparent oxymoron. In the past this was attributed to volume contraction from venoconstriction which could, of course, be a contributing factor. However, this hypotension can also be explained with the model of the sympathetic nerve/n Langer  and Klaun Langer  demonstrα2-ARs mediate suppression of anxiety such that withdrawal of treatment with α2-AR agonists  precipitates anxiety and sleeplessness [α2-ARs in the kidney which assumes additional activity in sodium retention, a similar phenomenon may occur in the brain. Thus, we hypothesize that the efficacy of prazosin in PTSD is mediated at least partially by upregulation of α2-ARs. This is a testable hypothesis that if proven true would indicate a new avenue of research for effective PTSD treatments.Propranolol –69, prazlessness , 74. Sinα2-ARs were discovered along with their substitution for α1-ARs following prazosin administration, microdissection/microchemical techniques simultaneously pursued correlates of the physiological studies involving the adenylate cyclase inhibitory role of excess α2-ARs. Thus, the renal adenylate cyclase stimulatory mechanism of furosemide was discovered in which furosemide was shown to increase urinary cyclic AMP excretion by approximately 5-fold [+-K+-2Cl− symporter  of about 10 mV (lumen positive with respect to the interstitium). This lumen positive PD drives the reabsorption of Ca2+ (which being a cation is repulsed by a positive PD) via the paracellular pathway. Loop diuretics, such as furosemide, bind to and inhibit the Na+-K+-2Cl− symporter, which destroys the lumen positive PD and thereby reduces the driving force for Ca2+ reabsorption. This is why loop diuretics increase Ca2+ excretion leading to hypocalcemia [2+ levels stimulate parathyroid hormone (PTH) release, which mobilizes Ca2+ from bone leading to decreased bone density. However, the aforementioned mechanism does not rule out the possibility that furosemide increases Ca2+ mobilization from bone via an additional, and more direct, mechanism involving stimulation of adenylate cyclase in osteoblasts/osteoclasts, similar to PTH. Currently, huge doses of furosemide are used repeatedly in race horses to prevent lung hemorrhage. Via the aforementioned mechanisms, furosemide in race horses would be expected to absorb excess calcium from bone causing osteoporosis and predisposition to leg fracture, which may explain the large number of deaths in race horses due to leg fracture. Indeed, loop diuretics are also known to promote osteoporosis in humans [Normally, the operation of the Nacalcemia . Low sern humans , 77.α2-ARs? Inasmuch as α2-ARs inhibit the adenylate cyclase/cAMP pathway, one would predict that α2-AR agonists might reduce osteopenia/osteoporosis. However, as with the kidney, the effects of α2-AR signaling in bone also appear to be context dependent since the published data suggest that α2-AR antagonists, rather than agonists, protect against bone loss. For example, female mice with double knockout of α2A-ARs and α2C-ARs present a surprising phenotype characterized by (1) increased bone mass; (2) reduced bone resorption; (3) augmented bone formation; (4) increased and better connected and more plate-shaped trabeculae in the femur and vertebrae; (5) increased cortical thickness in the vertebrae; and (6) increased tibial and femoral strength [α2-AR agonist clonidine stimulates resorptive activity of osteoclasts in culture [α2A-AR gene are associated with osteoporosis and significantly increase α2A-AR mRNA levels in human bone samples by stabilizing mRNA [α2-AR antagonists for prevention and treatment of osteopenia/osteoporosis, including disease promoted by loop diuretics.How does loop diuretic-induced bone disease relate to strength . Moreove culture . Recent ing mRNA . Togetheα1-AR suppression of synthesis, redistribution, and function of α2-ARs that appears applicable to several clinical phenomena. These include the basic mechanism of hypertension, unexpectedly low efficacy of renal denervation for hypertension, severe injury from falling, especially in the elderly, and a mechanism of action for prazosin in stress disorders (PTSD). These kidney experiments also revealed the context-dependent effects of α2-ARs [α2-AR regulation in hypertension via retention of excess sodium appears very promising. Millions of people ingesting 20 times the real minimum daily requirement for sodium [α2-AR-sodium retaining mechanism could be paradigm shifting. It could prevent heart attacks and failure, strokes and aortic ruptures, many cases of renal failure and dementias, remarkable goals indeed. Coincidentally, the discovery of α2-ARs may lead to a possible treatment for osteopenia/osteoporosis, including disease promoted by loop diuretics, thus reducing the incidence of bone fractures in humans and animals. Although not discussed here, potential uses for α2-AR drugs keep expanding and include glaucoma, ocular hypertension, rosacea, preeclampsia, chronic pain, anesthesia, autonomic dysfunction, attention-deficit hyperactivity disorder, opioid withdrawal, diabetes, neuropsychiatric disorders, and migraines. We conclude that hypotheses concerning potential biologic mechanisms of α2-ARs are guideposts of major future and continuing discoveries. The discovery of α2-ARs, their altered regulation, and their potential roles reviewed herein are examples of such guideposts.Because of directly measurable effects of neuroregulatory control mechanisms in the kidney, we discovered a tonal mechanism of f α2-ARs , 81, whir sodium  have no


The akinetic/rigid (AR) motor subtype of Parkinson’s Disease is associated with increased rates of motor and cognitive decline. Cross-sectional studies examining the neural correlates of AR have found abnormalities in both subcortical and cortical networks involved in motor planning and execution relative to controls. To better understand how these cross-sectional findings are implicated in the unique decline associated with the AR subtype, we examined whether baseline AR symptoms are associated with longitudinal decline of these networks, in contrast to other motor symptoms such as tremor. Using whole brain multiple regression analyses we found that worse AR symptoms at baseline were associated with greater gray matter loss over four years in superior parietal and paracentral lobules and motor cortex. These regions also showed altered connectivity patterns with posterior parietal, premotor, pre-supplementary motor area and dorsolateral prefrontal regions in association with AR symptoms across subjects. Thus, AR symptoms are related to gray matter decline and aberrant functional connectivity in a network of frontal-parietal regions critical for motor planning and execution. These structural and functional abnormalities may therefore be implicated in the more aggressive course of decline associated with the AR relative to tremor-dominant subtype. Differential patterns of neuropathology and rates of decline across individuals with PD may therefore manifest as specific symptom profiles6. While recent cross-sectional functional and anatomical neuroimaging studies have found distinct correlates of AR symptoms, including motor and posterior parietal cortical regions as well as basal ganglia (see ref. 7 for review), the pattern of longitudinal changes involving these neural substrates and their association with initial AR symptoms remains largely unknown. The current study therefore examined longitudinal changes in gray matter regions associated with AR symptoms measured at a baseline time point. We also determined if such regions exhibit altered functional connectivity patterns in relation to AR symptoms, which may identify a link between early AR symptom severity and neuronal pathways associated with accelerated spread of PD pathology8.Motor symptoms in Parkinson’s Disease (PD) are heterogenous, with the akinetic/rigid (AR) motor subtype being associated with poorer prognosis and increased risk of dementia compared to the tremor subtype9. While both symptom types have been associated with alterations in motor cortex and basal ganglia12, critical distinctions between the two have also been proposed. Specifically, tremor symptoms have been linked to altered interactions between globus pallidus and the cerebellothalamic circuit14, while AR symptoms have been associated with altered anatomy and function of basal-ganglia motor loops, specifically in projections from putamen to globus pallidus, thalamus and ultimately motor cortex17. These overlapping yet distinct network alterations may offer insights into the unique longitudinal trajectory associated with each symptom dimension. Specifically, recent work has proposed that the initial decline of basal ganglia causes compensatory mechanisms within the cerebellothalamic network, resulting in tremor symptoms and possibly a more benign form of disease progression20. In contrast, neural decline associated with AR symptoms may reflect pathways of initial PD progression, thus highlighting the importance of evaluating longitudinal neural patterns associated with early AR symptoms.AR symptoms are commonly defined as a composite of motor deficits classified as rigidity, bradykinesia and akinesia, while tremor symptoms are defined via items associated with postural and kinetic rhythmic involuntary movements as measured in the Movement Disorder Society Unified Parkinson’s Disease Rating Scale24, it has also been suggested that AR symptoms may not be entirely accounted for by dopamine depletion and its effects on basal ganglia-cortical interactions (see ref. 15 for review). In particular, AR symptoms have been linked to abnormalities in motor planning26, which is reliant on a frontal-parietal network, consisting of posterior parietal cortex, pre-supplementary motor area (SMA) and premotor cortex, which projects to motor cortex (for review see refs. 28). Several neuroimaging studies have reported associations between AR symptoms and alterations in connectivity30, activation, and volume33 of regions within this fronto-parietal network including pre-SMA, motor cortex, putamen, and parietal cortex. Overall, these findings suggest that the putamen, as well as frontal and parietal inputs to motor cortex may be closely related to AR symptom severity at discrete time points. However, to the best of our knowledge no studies have evaluated the longitudinal neural correlates of AR. This information would be critical for understanding the mechanisms by which individuals with greater AR symptoms are at increased risk for poorer outcome .While cross-sectional studies have consistently found a relationship between AR symptoms and reduced levels of putamen dopamine, activation and volume24 and a frontal-parietal cortical network33, we hypothesized that these regions would show increased gray matter decline for those with more severe AR symptoms at a baseline time point. We also examined whether regions that decline anatomically at a greater rate in association with early AR symptoms would have altered functional connectivity patterns associated with individual differences in AR symptoms as well. Such analyses may therefore reveal pathways by which disease spread occurs at a greater rate for patients with more severe AR symptoms.Therefore, we sought to examine the relationship between longitudinal gray matter changes and early measures of AR symptoms in contrast to tremor symptoms. Given the literature relating AR symptoms to altered anatomy and function of two distinct circuits with inputs to the motor cortex, specifically the putamenF (3819) = 70.21, p < 0.001) and tremor (F (3819) = 14.41 p < 0.001) domains increased significantly over time.We first confirmed that motor symptoms progressed over the four-year period in a subset of the PPMI sample by conducting a repeated measures analysis of variance on the 274 participants who had clinical and behavioral assessments available at all four time points . Additionally, a cluster in the motor cortex was prominent, though not meeting the cluster corrected significance threshold  . Surprisingly, no significant relationship between AR scores and putamen volume decline were found, even at a more lenient threshold (p < 0.005).To examine the extent to which baseline motor symptoms related to basal ganglia and cortical gray matter values over time, we estimated the impact of baseline AR and tremor symptom severity on net gray matter changes across the four time points  by applying multiple regression analyses on the voxel compression maps (VCM) while controlling for confounding factors (see “Methods”). In a sub-sample of 50 participants, individuals with more severe AR symptoms at baseline showed significantly greater net gray matter volume loss across the 4 years in the paracentral (PCL) and posterior superior parietal lobule (SPL) at the whole brain level 35.In contrast, baseline tremor scores did not show any significant relationship with longitudinal gray matter volume changes in the whole brain analyses. The correlations between tremor and gray matter loss in ROIs defined in the above AR analysis were also not statistically significant Fig. . CriticaWe further utilized multiple regression analyses to examine the across-subject relationship between motor symptoms and functional connectivity using a sub-sample of 87 participants with resting-state fMRI data. We specifically examined whether the regions with significant longitudinal gray matter loss would also show alterations in their functional connectivity in association with higher AR symptoms.p < 0.001 voxel-wise threshold, though at a reduced threshold (p < 0.005), there was an association between AR symptoms and stronger functional connectivity between PCL and the left premotor cortex and dorsolateral prefrontal cortex (dlPFC) , except for the dorsolateral prefrontal cluster which trended towards significance . However, the actual resting-state values extracted from this cluster did not significantly differ between the groups . Additionally, these functional connectivity patterns were not significantly associated with tremor symptom severity 35.Individual variations in AR symptoms were significantly associated with posterior superior parietal lobule (SPL) functional connectivity; this parietal region showed weaker functional connectivity with the anterior portion of the SPL in relation to more severe AR symptoms see Fig. . Greatersee Fig. . These asee Fig. , while tThe current study demonstrated that PD individuals with more severe AR symptoms exhibited greater gray matter volume reduction over four years and altered functional connectivity within a frontal-parietal network involved in motor planning and execution. Critically, these patterns of gray matter decline and altered functional connectivity were not found for the tremor subscores. The implications of these findings are discussed below.36. Specifically, we found longitudinal anatomical decline associated with baseline AR symptoms in superior parietal lobule (SPL), a region heavily implicated in motor planning and awareness, as well as paracentral lobule (PCL) and motor cortex, regions primarily involved in the execution of planned movements40. These findings corroborate studies that have found lower gray matter volume and altered activation in SPL and cortical motor areas in relation to AR symptoms33. Similar patterns of reduced gray matter in the SPL, PCL and motor areas have also been found in several studies that compare PD patients and healthy controls42. However, these latter studies did not distinguish between AR and tremor subtypes, suggesting that patterns of anatomical changes associated with PD categorically may well be related to distinct motor profiles within the disease. Our findings therefore further implicate frontal and parietal motor control regions in the unique longitudinal trajectory specifically associated with AR symptoms.Our findings support our initial hypothesis that baseline AR symptoms related to volumetric changes and altered functional connectivity within a frontal-parietal network30. Specifically, we found weaker connectivity between anterior and posterior portions of SPL in relation to worse AR symptoms. Functional parcellations of the SPL have found the anterior segments to be more strongly connected with sensory processing regions, while posterior segments are more strongly connected with regions involved in executive functions43. Accordingly, with the current findings, it is possible that AR symptoms are related to abnormal integration of these sensory and motor planning processes that occur within the SPL. Additionally, we found altered connectivity in relation to AR symptoms between regions responsible for motor output (motor cortex and PCL) and regions involved in motor planning, including premotor cortex, dorsolateral prefrontal cortex, and pre-SMA46. Similar to the altered SPL connectivity, altered connectivity between these motor regions in relation to AR suggests altered integration of motor planning with execution, though studies evaluating this with direct experimentation are needed to substantiate this claim.We also found altered functional connectivity of the SPL, PCL, and motor cortex in relation to AR symptoms, which is in line with several studies that have found functional alterations in motor cortex and parietal regions in relation to AR symptoms47, while activity of dorsolateral prefrontal cortex has been strongly implicated in working memory tasks and visuospatial information processing50. Additionally, impairments in inhibition and working memory in PD patients have been associated with alterations in these regions53, while abnormalities in these cognitive control systems may also play a role in mood disturbances in PD56. Our findings are therefore in line with previous studies that have found a strong relationship between AR symptoms and executive function impairments in PD patients (see ref. 57 for review), symptoms which may have common underlying neural correlates. Future studies should directly evaluate this possibility using longitudinal methods in order to define neural decline that may relate to both cognitive and motor domains.Furthermore, several of the regions which exhibited altered connectivity in relation to AR are also implicated in cognitive control processes. In particular, the connectivity between pre-SMA and motor cortex has been found to play a central role in response inhibition60, our results do highlight the potential role of networks beyond the basal-ganglia-cortical loop in AR symptoms. This is in line with a growing body of literature that has begun to examine the relationship of PD symptoms, such as tremor and memory decline, and dysfunction of systems beyond dopaminergic basal ganglia loops63. Future studies interested in differentiating the neural correlates of motor symptoms in PD, as well as how these neural correlates decline over time, should therefore expand their analyses to include systems beyond those associated with basal ganglia function, specifically frontal and parietal regions involved in motor planning and output.Finally, as both cortical and subcortical networks input to the motor cortex, a region we found to be associated with AR symptoms, we cannot rule out the relationship between decline of subcortical systems and initial AR symptoms. However, as we did not find significant AR-related anatomical decline in the putamen, a region that was expected to be directly affected by the progressive death of dopamine neurons associated with PD pathology64, with studies finding that functional network paths predict regions of increased anatomical decline in PD patients6. While we did not directly test the disease spread hypothesis in our current work, our findings lend support for this hypothesis, and further suggest that AR symptoms are associated with distinct patterns of neural decline and disease spread along cortical networks integrating motor planning with execution. Additionally, future work aiming to evaluate disease spread in PD should take motor symptom subcategories into consideration. Evaluating disease spread via AR and tremor subtypes may ultimately provide critical information for disease prognosis and treatment planning, with future work ideally utilizing fully overlapping participants in both anatomical and functional analyses.Additionally, our work is the first, to the best of our knowledge, to demonstrate that AR symptoms have distinct patterns of anatomical decline that are associated with altered functional connectivity, in contrast to tremor symptoms. This relationship is critical as recent work in PD has substantiated the hypothesis that neurodegenerative disease pathology spreads along the brain’s connectomeOur study had several limitations that should be noted. Although the resting-state analysis was informed by the longitudinal gray matter findings, not all participants had data of both neuroimaging modalities available for analysis. While the differences in sample is an important limitation, there were no significant differences in the demographic variables or the extracted resting-state values between the group of participants included in both neuroimaging analyses and those included only in the resting-state analysis. Future studies though should ideally integrate multiple modalities in completely overlapping samples. Additionally, larger sample sizes would afford better detection of subtle longitudinal changes in structural and functional images. Our analysis did demonstrate that individual differences in baseline AR symptom severity related to gray matter decline in frontal and parietal regions involved in motor planning and execution, while functional connectivity in motor planning regions was altered in relation to AR symptoms as well. These regions therefore may play a critical role in the unique longitudinal trajectory taken by AR dominant PD patients. Such findings may inform future studies aiming to understand the longitudinal trajectory of AR dominant PD patients, and ultimately help in disease prognosis and planning for such patients.www.ppmi-info.org/data). PPMI—a public-private partnership—is funded by the Michael J. Fox Foundation for Parkinson’s Research and partners, including Abbvie, Avid Radiopharmaceuticals, Biogen, Bristol-Meyers Squibb, Covance, GE Healthcare, Genetech, GlaxoSmithKline, Eli Lilly, Lundbeck, Merck, Meso Scale Discovery, Pfizer, Piramal, Roche, and UCB. This multi-site study collected standardized behavioral, neuropsychological, biospecimen and neuroimaging data from a group of 424 newly diagnosed individuals with PD, and follow-up data up to four years  after initial assessments. More specific details of the PPMI study can be found in a report published previously65. For up-to-date information on the study, visit the project webpage (www.ppmi-info.org). All participants enrolled in the PPMI study within 24 months of their initial diagnosis. Each participating PPMI site received approval from an ethical standards committee on human experimentation before study initiation. Written informed consent for research was obtained from all individuals participating in the study.Behavioral and imaging data were obtained from the Parkinson’s Progression Markers Initiative (PPMI) database . To determine if motor impairments became more severe over time we evaluated motor domain scores in 274 patients who had behavioral data collected at all four time points. Baseline demographic information regarding these participants can be found in Table Each patient’s overall motor impairment was indexed using the total (summed) score of the Unified Parkinson’s Disease Rating Scale (UPDRS) part IIIt’s = 0.144 – 1.39, p’s = 0.16 − 0.88). The imaging data were acquired at seven different sites . All imaging (MRI and fMRI) data were acquired using Siemens Trio Tim 3 Tesla magnets . We only included individuals who were not reported by the original experimenters as having any problems during the scanning session . Individual participants’ anatomical and functional images were visually inspected to ensure that the whole brain was covered and were screened for artifacts. Data were obtained from participants with the imaging parameters specified in the PPMI documentation as the following: high resolution structural images (T1-weighted MP-RAGE) were acquired with repetition time (TR) = 2,300 ms, echo time (TE) = 2.98 ms, flip angle (FA) = 9°, matrix = 256 × 256, field of view (FOV) = 256 mm, voxel size 1 × 1 × 1.2 mm3, 176 sagittal slices with slice thickness = 1.2 mm. Subjects were instructed to rest quietly, to keep their eyes open and not to fall asleep. Anatomical scans were visually inspected for all time points, and no participants were removed due to motion or other artifacts. At the 48-month time point the mean levodopa equivalent dose (LED)68 was 544 mg/day (standard deviation = 260 mg/day) . We therefore used the scans of these 50 individuals  in the final longitudinal volumetric analysis, 47 of whom also were included in the longitudinal behavioral analysis. It is worthy to note that these 47 participants had comparable baseline demographics relative to the 227 behavioral participants not included in the anatomical analysis . As a result, a total of 87 individuals  were included in the final resting-state functional connectivity analysis, 36 of whom had sufficient anatomical scans available to be included in the longitudinal anatomical analysis. Critically, these 36 subjects had motor and demographic characteristics comparable to the 51 subjects that only had resting-state scans analyzed , thus allowing for informative interpretation to be made of results across the neuroimaging data types. The mean LED at time of scan was 188 mg/day (standard deviation = 211 mg/day). The participants on medication did not undergo a medication washout before scanning. Table Resting-state functional images were acquired from the baseline, 12, 24, and 48-month time points, which included 18, 55, 64, and 78 scans respectively, though only 62 and 55 scans out of the total 24 and 48-month time points had consistent scanning parameters as defined in the PPMI documentation (see below). As the goal of our study was to determine if anatomical regions defined in our longitudinal gray matter analysis had functional implications, we chose to select the first time point at which functional scans were available for each participant, so as to obtain the highest number of functional scans possible. We therefore analyzed 94 participants with resting-state scans from the first time point at which each individual was scanned with consistent scanning parameters. Data were obtained with the imaging parameters specified in the PPMI documentation as following: Echo-planar images acquired for 8.29 min (212 volumes) with TR = 2400 ms, TE = 25 ms, flip angle = 80°, matrix = 68 × 68, FOV = 222 ×222 mm, 40 slices (ascending with 0-mm gap), and voxel size 3.25 × 3.25 × 3.25 mmhttp://dbm.neuro.uni-jena.de/cat12/) and SPM 12. For each participant, T1 images from each of the four time points were first registered and bias-corrected using serial longitudinal registration in SPM 1269. In this process each time point is reoriented, spatially warped, and intensity biased corrected relative to the average T1 image, resulting in four jacobian difference maps representing the extent to which voxels from each time point image expand  or compress  in relation to the average T1 image. Individuals’ average T1 images were then segmented into gray and white matter and cerebral spinal fluid using CAT12.Structural MRI data were processed using the computational anatomy toolbox (CAT12 r930) (70). More specifically, for each subject, we constructed a within-subject linear model in which each jacobian determinant image was regressed against time in years. The resulting beta image was then multiplied by the gray matter segmentation of the average T1 image for each subject. The product of this multiplication thus represented the net change of gray matter from baseline to 48 months, and is known as a voxel compressions map (VCM)70. Additionally, the within-subject average gray and white matter segmentation images were used to create a group specific DARTEL template71. Deformation fields from this process related each subject’s average gray matter segmentation to the DARTEL template and were used to normalize VCMs to the DARTEL template. Normalized VCMs were then smoothed by 4-mm FWHM.Net gray matter change was computed in a fashion similar to that implemented in recent studies  of p < 0.05 at the cluster level to correct for Type I error74.A group level multiple regression model was conducted in which baseline indexes for AR and tremor symptoms were used to predict gray matter changes (VCM maps), while age, LED at the final time point, scanner site, gender, and total intracranial volume at baseline were included in these models to control for confounding factors. Previous studies have shown that the corrected voxel peak threshold of www.fil.ion.ucl.ac.uk/spm). For each individual, functional images were corrected for slice timing and then realigned to the first image to correct for head motion between scans, while structural image was coregistered to the mean functional image. The structural image was then segmented and normalized to a template based on a sample of individuals with PD75 using affine registration followed by nonlinear transformation77. The resulting parameters were then applied to all functional images. Finally, the functional images were spatially smoothed with a Gaussian kernel of 6 mm at full width at half maximum.Standard image preprocessing was performed using Statistical Parametric Mapping  and derivative of the root mean square variance over voxels (DVARS) as in our previous work87. Each functional volume that exceeded the head motion limit FD (t) > 0.5 mm or DVARS(t) > 0.5% were included as a regressor in the final time series88. Two out of the 94 (2.12 %) processed resting-state datasets had >1/3 of time points that exceeded the motion limit, while five subjects were removed due to artifacts. On average, 9.3% of data points exceeded the motion limit in the remaining 87 datasets. The number of scans exceeding motion limits did not significantly correlate with the UPDRS III total score, or the AR and tremor subscales .Additional preprocessing was applied to reduce spurious fMRI signal variances that were unlikely to reflect neuronal activityz scores using the Fisher’s z transformation90: z = 0.5 log (1 + r) / (1 − r). The resulting z maps from each individual were used in the second-level multiple regression analyses.ROIs with significantly steeper longitudinal gray matter decline in association with higher AR scores were used as seeds in our resting-state analyses. The fMRI signal time courses were averaged across all voxels within each seed region. For each resting-state fMRI dataset, we computed the correlation coefficient between the averaged time course of each seed region and the time course of each voxel in the rest of the whole brain. The resulting r values were converted to p < 0.001 at the voxel level and a false discovery rate (FDR) of p < 0.05 at the cluster level to correct for Type I error, in line with current reporting guidelines74. A summary table for all multiple regression analyses conducted can be found in Supplementary Table The main analysis consisted of a group level multiple regression model for each seed region to determine the relationship between individual motor scores at time of scan and seed-based functional connectivity differences across subjects. These models also included the confounding factors of age, gender, scanner site, and LED at time of scan, and time point at which the resting-state scan was obtained. Unless otherwise noted, we present results that satisfy both an uncorrected threshold of Further information on research design is available in the Supplemental Materialreporting summary


Breast cancer (BC) is one of the most common malignancies and the leading cause of cancer-related mortality in women. Androgen receptor (AR) is frequently expressed in diverse BC subtypes. Accumulating evidence has revealed that AR might be a predictive or prognostic factor and a drug target in BC. AR expression and AR pathways differ in various BC subtypes, thereby resulting in controversial inferences on the predictive and prognostic value of AR. Herein, we summarized the roles of AR in different BC subtypes and AR-targeting therapies based on preclinical and clinical studies. Moreover, we highlighted the possible efficacy of a combination therapy via exploiting the AR-related mechanisms and the research on therapeutic resistance. Breast cancer (BC) represents the most common malignancy in women, and is one of the leading causes of cancer-related fatality  , 3, whilAndrogen receptor (AR), a member of the steroid receptor superfamily, is expressed in many human tissues, among which BC tissue has the third-highest expression of AR , 9. It hIn this review, we focused on the roles and prognostic significance of AR and AR-targeting therapies in different BC subtypes based on preclinical and clinical studies. We also summarized the possibility of combination treatment and research on therapeutic resistance.ClinicalTrials.gov was searched for ongoing clinical trials.We performed a comprehensive search of the PubMed database for articles written in English and published before January 2020 (mainly after 2010), using the following search terms, alone or in combination: “androgen receptor,” “androgen,” “breast cancer,” “prognosis,” “androgen receptor agonist or antagonist,” and “resistance.” In addition, bibliographies of relevant articles were searched for additional references. The website AR is a 919-amino-acid hormone-regulated transcription factor. The gene responsible for AR measures 180 kb in length and is located on the chromosome, Xq11-12 . AR compAR is situated in the cytoplasm, and without the ligand, it adheres to heat shock proteins (HSPs) . Once anIn ER-positive BC, AR has been demonstrated to interfere with ER-dependent transcription through competition with the ER for binding sites at the estrogen response elements (EREs) or through competition for the transcriptional co-modulators . Indeed,In HER2-positive BC, AR transcriptionally regulates the levels of WNT7B that leads to the transfer of β-catenin into the nucleus. In the nucleus, the AR/β-catenin complex identifies the modulatory regions of HER3 and raises its transcription under the cooperation of the transcription factor, FOXA1. HER3 and HER2 form a heterodimer that stimulates MYC gene expression and PI3K/AKT pathway, thereby resulting in cell proliferation and tumor growth  29). Th. Th29). It is known that the PI3K/AKT/mTOR pathway is involved in BC development . In TNBCERβ, a second form of estrogen receptor, is found in ~30% of TNBC cases . ERβ oveAdditionally, in TNBC, it was found that AR could adjust the cell behavior via modulating the interaction between microRNAs and lncRNAs. The AR negatively induced lncRNA (ARNILA) was reported to serve as a competitive endogenous RNA of miR-204 that promotes the expression of its target gene, Sox4, which boosts epithelial–mesenchymal transition (EMT), invasion, and metastasis of TNBC. Therefore, ARNILA is associated with poor progression in TNBC .AR can be detected in most BCs, and the frequency of its positivity differs between different BC subtypes and ethnicities , 37–42. AR is expressed in 70–95% of ER+ BCs , 37, 38.On the contrary, the AR/ER ratio has been considered to affect the prognosis and response to anti-estrogen endocrine therapy. Cochrane et al. revealed that ER+ BC patients with an AR/ER ratio > 2, had a 4-fold increased risk of failure with tamoxifen therapy .P = 0.039) and OS . Besides, patients treated with first-line trastuzumab, AR+ tumors had longer PFS  and 5-year OS rate  compared with AR-negative subjects  in those with AR+ subtypes than that in those with AR-negative subtypes (P = 0.011)  . Besides= 0.011) .However, in a meta-analysis of 27 studies, including 4,914 TNBC patients, AR expression was not related to DFS, OS, distant DFS, or recurrence-free survival . MoreoveA multi-institutional study of 1,407 TNBC patients from six international cohorts found that AR status presents population-specific patterns related to OS. AR positivity is a biomarker of favorable prognosis in the Nigerian and US cohorts, whereas it correlated with poor prognosis in the Indian, Norway, and Ireland cohorts, while being neutral in the UK cohort .To some extent, the prognostic discrepancy mentioned above may be owing to differences in sample sizes, the methodology of detection, the antibody used to test AR, the cut-off values used to define AR positivity, the ethnic composition of cohorts, adjuvant treatments, and follow-up time of studies , 64, 65.Natural and synthetic androgens have been used as a treatment approach in BC with AR expression , 66, 67;So far, androgens and AR agonists have been explored in ER+ BC. A retrospective analysis of ER+ MBC patients revealed that the clinical benefit rate (CBR) of fluoxymesterone was 43% after progressing to conventional hormonal therapy, independent of the AR expression level . EnobosaThe AR-directed antagonists are mostly used in advanced BC, especially TNBC . PreclinFurthermore, a large preclinical study showed that enzalutamide significantly reduced proliferation, migration, and invasion, while increasing apoptosis in 11 BC cell lines (three non-TN and eight TNBC); moreover, growth inhibition was dependent on the expression of AR, which is consistent with the findings of another research that involved four different non-LAR TNBC cell lines of the MSL, BL2, and mesenchymal-like subtypes demonstrating enzalutamide-mediated cell inhibition , 81. A pBeyond that, a randomized phase II study in locally recurrent (unresectable) or metastatic AR+ TNBC treated with darolutamide or capecitabine is currently recruiting patients NCT03383679)  (83). An679  (83)Aside from monotherapy approaches, the combination of AR-targeted therapy with other therapies as new potential strategies has gathered considerable interest. Previous studies have revealed that in AR+ TNBC, PIK3CA kinase mutations were more common and that targeting AR alone could potentially enhance tumor growth . A studyCyclin-dependent kinases (CDKs) are a family of protein kinases that play a crucial role in eukaryotic cell proliferation. CDK4 and CDK6, members of the CDK family, are activated by cyclin D and induce the transition from G1 phase to S phase in cell proliferation . PalbociIn the studies involving BC, little research has been done on the association between AR and immune response. In a previous study on prostate cancer, increased PD-L1/2-positive dendritic cells and PD-1-positive T cells were found in the blood of patients progressing to enzalutamide, compared to those naïve or responding to enzalutamide, suggesting that the combination of enzalutamide and anti-programmed death-1 therapy may improve the prognosis in patients with enzalutamide resistance . MeanwhiFurthermore, AR antagonists have been combined with conventional therapies, such as radiotherapy and chemotherapy, with agreeable results. For example, Barton et al. in a preclinical model, reported that the combination of enzalutamide with paclitaxel (simultaneous and subsequent) was more effective than paclitaxel alone in reducing tumor recurrence . There aWhile the results of AR-targeted therapies for BC are promising, some patients eventually show disease progression due to drug resistance. The underlying mechanisms of drug resistance in BC are still being explored, while those identified in prostate cancer may also apply to BC. In prostate cancer, the failure of AR-targeted therapy is related to AR gene amplification and mutation, interaction among coactivators, and overexpression of active AR splice variants, such as AR-V7 lacking the C-terminal LBD –96. UndeAnalysis of various splice variants revealed that 53.7% of primary BC samples had AR variant 7 (AR-V7) mutations . InteresMoreover, recent studies have shown that the aberrant activation of the EMT and Wnt/β-catenin pathway also contributes to enzalutamide resistance in prostate cancer –107.AR is a burgeoning and promising therapeutic target that potentially improves the survival outcome of BC patients. This review emphasizes the role of AR and AR-related mechanisms in various BC subtypes from bench to bedside. However, additional researches are needed to elucidate the significance of AR, in particular, identification of appropriate AR detection methods and novel markers of AR responsiveness to better select the patients who may benefit from AR-related treatments. Up till now, there is no clinical guideline concerning the methods to test AR positivity. Immunohistochemistry (IHC) is the cheapest method and is widely used in the definition of AR positivity in BC . The optFQ and JH: conception and design. MC, YY, and KX: write, review, and revision of the manuscript. LL: supervision. All authors contributed to the article and approved the submitted version.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.


AR promoter in order to decrease AR expression. A newly discovered G4 compound that suppresses AR expression demonstrates selective killing of AR-expressing tumor cells, including CRPC lines. Our findings raise the significant possibility that G4-stabilizing drugs can be used to increase NCL transcriptional repressor activity to block AR expression in prostate cancer. Our studies contribute to a clearer understanding of the mechanisms that control AR expression, which could be exploited to overcome CRPC.The androgen receptor (AR) is a major driver of prostate cancer development and progression. Men who develop advanced prostate cancer often have long-term cancer control when treated with androgen-deprivation therapies (ADT). Still, their disease inevitably becomes resistant to ADT and progresses to castration-resistant prostate cancer (CRPC). ADT involves potent competitive AR antagonists and androgen synthesis inhibitors. Resistance to these types of treatments emerges, primarily through the maintenance of AR signaling by ligand-independent activation mechanisms. There is a need to find better ways to block AR to overcome CRPC. In the findings reported here, we demonstrate that the nuclear scaffold protein, nucleolin (NCL), suppresses the expression of AR. NCL binds to a G-rich region in the AR promoter that forms a G-quadruplex (G4) structure. Binding of NCL to this G4-element is required for NCL to suppress AR expression, specifically in AR-expressing tumor cells. Compounds that stabilize G4 structures require NCL to associate with the G4-element of the  Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men . AndrogeAR gene, AR gain of function mutations, induction of other signaling pathways that activate AR, and AR splice variants that display constitutive activity in the absence of ligand binding. Most CRPC cases have an increase in AR protein production  containing protease and phosphatase inhibitor cocktails (Millipore Sigma) for 5 minutes at 4° C and then cleared by centrifugation at 16,000 × g for 10 minutes at 4° C. All protein concentrations were determined using the bicinchoninic acid assay (Thermo Fisher Scientific).siRNA sequences against NCL, 5′-GGCAAAGCAUUGGUAGCAAtt-3′ (NCL1), and 5′-CGGUGAAAUUGAUGGAAAUtt-3′ (NCL2) were chemically synthesized and annealed by Ambion, Inc. BLAST analysis showed no homology of the siRNA sequences to any other sequences in the Human Genome Database. The siRNAs were transfected using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's directions. Scrambled siRNA used as a negative control was from Ambion.Total cell lysates were resolved by SDS-Tris PAGE and transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific) in Tris-glycine buffer containing 20% methanol. Proteins were detected by immunoblotting with an appropriate antibody overnight at 4° C . MembranAR proximal promoter sequence. Wild AR promoter reporter contains the DNA segment spanning -1075 to +304 of the AR promoter   quinoline as yellow solid. 1H NMR : δ 8.54 , 8.43 , 8.34 , 7.80 - 7.60 , 7.59 , 7.40 , 4.95 , 3.67 , 2.50 - 2.40 . MS (ESI): m/z 329.2 and 331.2 (M+H)+].To a stirred solution of 11-chloro-10H-indolo quinoline (8.0 g) in dry DMF (80 mL), 60% sodium hydride in mineral oil (4.3 g) was added at 0 ° C. The reaction mixture was stirred at the same temperature for 1 hour and 1,3-bromochloropropane (22.8 g) was added drop wise to the reaction mixture. The reaction mixture was poured into ice cold water and extracted with EtOAc (3 × 100 mL). The combined EtOAc layers were washed with water, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography using 2-4% EtOAc in hexane as an eluent to afford 6.0 g (57%) of 10-(3-chloropropyl)-11-chloro-These quindoline analogs were synthesized from 11-chloroquindoline, as shown in scheme 1. Structures, chemical nomenclature, and analytical data on tested compounds are shown in 3). After completion, the reaction mixture was poured on to ice-cold water and extracted with EtOAc (3 × 100 mL). The combined EtOAc layers were washed with water (3 × 200 mL) and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography using 2–4% MeOH in CHCl3 as eluent to afford 1.0 g (84%) of pure product as yellow color solid. This product was converted into dihydrochloride salt using methanolic HCl  at 0 ° C and then room temperature. Diethyl ether (50 mL) was added to the reaction mixture, and the resulting solids were collected by filtration. The product was recrystallized from a mixture of solvents  to yield 1.1 g of pure CV-GSA-02/30 as yellow color solid in 93% yield (A mixture of 10-(3-chloropropyl)-11-chloro-10H-indolo quinoline , sodium iodide  and N-(2-aminoethyl)-4-piperidinol  was stirred at 100 ° C for 24 h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3% yield .These compounds were synthesized using microwave irradiation at 130 ° C for 25 min. The reaction mixture was poured into water and extracted with dichloromethane (100 mL × 3). The combined organic layer was washed, dried, filtered, and concentrated. The resulting residue was purified on silica gel column to obtain pure compounds. Reactions details are discussed below.A mixture of 11-chloro-10-(3-chloropropyl)-10H-indolo quinoline , N1, N1-dimethylethane-1,2-diamine  and NaI  were reacted under microwave irradiation at 130 ° C for 25 min. After completion of the reaction, the reaction mixture was poured into water and extracted with dichloromethane (100 mL × 3). The combined organic layer was washed, dried, filtered, and concentrated. The resulting residue was purified on silicone gel column to give compound 25 mg as yellow solid .A mixture of 11-chloro-10-(3-chloropropyl)-10H-indolo quinoline , N1, N1-dimethylpropane-1,2-diamine  and NaI  were reacted to give compound 100 mg as yellow solid .A solution of 11-chloro-10-(3-chloropropyl)-10H-indolo quinolone , 2-(1-methylpiperidin-4-yl) ethan-1-amine  and NaI  in 2 mL DMF were reacted to obtain compound 52 mg as yellow solid .A solution of 11-chloro-10-(3-chloropropyl)-10H-indolo quinolone , 4-(2-aminoethyl) cyclohexan-1-ol  and NaI  in 2 mL DMF were reacted to obtain 50 mg of GSA1512 as yellow solid .AR G4 oligonucleotides -2,5-diphenyltetrazolium bromide (MTT) assay . Briefly


A global pandemic of a new highly contagious disease called COVID-19 resulting from coronavirus (severe acute respiratory syndrome (SARS)-Cov-2) infection was declared in February 2020. Though primarily transmitted through the respiratory system, other organ systems in the body can be affected. Twenty percent of those affected require hospitalization with mechanical ventilation in severe cases. About half of the disease survivors have residual functional deficits that require multidisciplinary specialist rehabilitation. The workforce to deliver the required rehabilitation input is beyond the capacity of existing community services. Strict medical follow-up guidelines to monitor these patients mandate scheduled reviews within 12 weeks post discharge. Due to the restricted timeframe for these events to occur, existing care pathway are unlikely to be able to meet the demand. An innovative integrated post-discharge care pathway to facilitate follow up by acute medical teams (respiratory and intensive care) and a specialist multidisciplinary rehabilitation team is hereby proposed. Such a pathway will enable the monitoring and provision of comprehensive medical assessments and multidisciplinary rehabilitation. This paper proposes that a model of tele-rehabilitation is integrated within the pathway by using digital communication technology to offer quick remote assessment and efficient therapy delivery to these patients. Tele-rehabilitation offers a quick and effective option to respond to the specialist rehabilitation needs of COVID-19 survivors following hospital discharge. In December 2019, a new highly contagious respiratory illness was observed to have developed in the Wuhan province of China. Over the following weeks, a rapid spread across the globe was observed as a result of what is now known to be infection with the severe acute respiratory syndrome (SARS)-CoV-2 virus, also called the coronavirus. The illness caused by the virus was labelled coronavirus disease or COVID-19 by the World Health Organization (WHO) on 11 February 2020 ,4. The tTwo Chinese nationals from the same family staying at a hotel in York were the first reported cases in the UK . SubsequIt is difficult to ascertain if premorbid fitness and wellbeing have a direct impact on the clinical course of the disease. It has been observed that lifestyle-related diseases such as obesity, insulin resistance and diabetes are linked to an increased risk of hospitalization and mortality. This has prompted suggestions that high levels of cardiorespiratory fitness might be protective to developing a severe case of COVID-19 . These lThe planned response by the health authorities to the pandemic largely focused on the prevention of infection by limiting spread and ensuring that the acute care services, including intensive care units (ICUs), were able to cope with the care of patients with the disease. There has been very minimal discussion on the possible need for specialist rehabilitation services as part of the planning of the national response to COVID-19. Understandably, the disease has had a massive impact on health care services. Routine clinical activities have been disrupted as part of the emergency preparation for the expected surge in hospital bed utilization at the peak of infection. The disruption of clinical activities has had a massive impact on specialist and community rehabilitation services. Many of the patients served by the specialist rehabilitation service fall within the vulnerable group, and some may require regular intervention procedures such as botulinum toxin injections to manage the residual deficits from their condition. Specialist rehabilitation services in England are classified on a three-tier structure, as defined by NHS England into Levels 1, 2, and 3 , which aPatients who recover from the infection, especially those that required ICU input, are likely to have on-going rehabilitation needs. In view of the novel nature of this disease, very little is known about the long-term residual deficits and the rehabilitation needs of this group of patients. While it is anticipated and expected that pulmonary rehabilitation (PR) will predominate during the acute episode with the need to improve oxygenation and facilitate the drainage of pulmonary secretions, the rehabilitation requirements following discharge is less known. It is assumed that those managed in the ICU may develop residual deficits such as critical illness neuropathy/myopathy and deconditioning commonly seen as part of critical care episodes.Evidence from previous coronavirus-related respiratory tract viral infection epidemics such as the SARS, which swept the world in 2002−2003, and middle east respiratory syndrome (MERS), which emerged in 2012, indicate the development of impairment of physical, mental, and social functioning brought about by respiratory compromise and deconditioning ,14,15. TThis paper proposes a model of a care pathway to mitigate against the impact on the rehabilitation services due to the response of the UK National Health Service in managing the COVID-19 crisis. The value of performing rehabilitation assessment and intervention for COVID-19 survivors alongside the mandatory medical follow-up requirements is elaborated. The issues of capacity and alternative ways of working within the rehabilitation services are explored, and tele-rehabilitation is proposed as a viable alternative to traditional face-to-face intervention. This paper advocates for a tele-rehabilitation pathway model based on the case of need. The proposal was influenced by the clinical experience of the authors, the clinical exigencies of this novel disease, and the local/regional service peculiarities.Rehabilitation is a key component of recovery following illnesses and major health interventions. It is an established clinical intervention in the management of patients with various clinical conditions and has been demonstrated to be effective in optimizing clinical outcomes. Most of the evidence for the effectiveness of rehabilitation has been derived from organized stroke care . FactorsEvidence published by Chinese rehabilitation specialists identified the need for PR for survivors of the coronavirus infection. Pulmonary rehabilitation is recommended to improve lung function, exercise tolerance, and reduce fatigue post-COVID-19, particularly for those who required hospitalization . PulmonaIt is not uncommon for patients to develop a plethora of deficits as a consequence of the deconditioning that often accompany a stay in the ICU. With the COVID-19 situation, current efforts to limit the spread of the viral infection have necessitated the restriction of movement in the community. This movement restriction has the unintended consequence of preventing access to traditional physical face-to-face PR classes. Alternative means and methods to deliver PR and physical reconditioning programs are required. Additionally, these patients do develop significant psychological deficits that also require therapy input and support. Zhao et al.  made theTraditional  PR is beneficial to patients with chronic respiratory disease largely through the improvement in symptoms and functional capacity. This is attributed to improved muscle function rather than direct effects on lung function. It focuses on fitness and strength exercise, breathing exercises, postural drainage positions, patient education, and relaxation training. Pulmonary rehabilitation has been demonstrated to minimize the symptoms of patients with chronic obstructive airway disease (COPD). It can decrease the impact of disability and improve the quality of life . AdditioAssessing rehabilitation needs can easily and readily be done remotely through various assessment tools; however, the on-going restriction in terms of movement and the reduced capacity of community rehabilitation services due to limited manpower to actually deliver the required therapy in the community have the effect that the rehabilitation needs, even when identified, might not be fulfilled. Alternative ways of working will have to be explored if the rehabilitation needs of these patients are to be met with a reduced workforce. Tele-rehabilitation programs using appropriate technology have the potential to help deliver therapy in situations such as this. It has been demonstrated to be effective in PR of patients with COPD . It can Based on the documented evidence from previous outbreaks of similar respiratory virus epidemics, many of the discharged patients treated for COVID-19 are likely to have residual deficits affecting physical, psychological, and respiratory function. The evaluation and assessment of these needs will be required to appropriately plan for intervention. In view of the novel nature of this disease, it is anticipated that the clinical expertise to evaluate, assess, and monitor all discharged COVID-19 patients within this group for residual deficits will be limited within community therapy services. Furthermore, the resources to deliver the required intervention within the community will be limited. Though hospital-based services might have the clinical expertise to deliver the required input, the perennial issue of resource limitation makes it impractical for them to deliver the input. However, with tele-rehabilitation, discharged patients in the community can still access this expertise from hospital-based services. There are experienced therapy staff deployed from hands-on patient facing roles  during the peak of the pandemic. This group of staff can provide a readily accessible pool of experienced therapists to provide remote assessment and therapy. Tele-rehabilitation offers a means to deliver remotely supervised therapy by experienced clinicians to patients within their homes or community dwellings. Tele-rehabilitation is the application of telecommunication technology for supporting rehabilitation services , i.e., tThe use of remote rehabilitation methods is not new. Countries with isolated communities have had to utilize technology in order to offer equitable access to services and to provide more efficient use of staff time and reduce costs . In the Kairy et al. , in a syThis emerging field of rehabilitation has the advantage that the therapy can be delivered wherever is most convenient for the patient through the use of modern digital communication. The clinician is able to prescribe an array of rehabilitation programs and monitor adherence in the performance of the tasks. Feedback information, as well as an assessment of how well the patient is performing, can be provided—both in real time and following a review of data. Portability and versatility are two key advantages of tele-rehabilitation over the traditional modes of community therapy delivery. Tele-rehabilitation programs make use of natural body movements with minimal or no constraints from the use of peripheral equipment. This has the advantages that activities can take place in virtually any setting and the exercise program can be designed around the home environment. The use of visualization screens to provide instructions and as means of providing visual feedback makes it engaging and stimulating. Another advantage is that patients can participate in a tele-rehabilitation program regardless of discharge destination. Patients discharged to residential care facilities can also participate as long as they can be supported to follow the programs.The benefits to healthcare staff include a reduction in therapy space accommodation pressures because tele-rehabilitation only requires a small workspace footprint. Two therapists, one to monitor the screen to ensure patient safety and the other to demonstrate the exercises, could effectively conduct remotely supervised group therapy sessions. Overall, it requires fewer staff than in face-to-face groups, and the attendant reduction in footfalls within the therapy center further helps with maintaining social distancing rules within the workplace. Virtual waiting rooms can be created and monitored remotely by an administrator who has control over a number of clinics, and it is therefore more cost effective.There are potential drawbacks to tele-rehabilitation. Many of the tele-rehabilitation products on offer have little or no evidence of proven clinical efficacy or effectiveness. Their safety profile rather than clinical efficacy seems to be the main factor guiding their commercial availability. This can potentially restrict patient participation due to lack of access to the chosen technology or platform. Novel digital products and apps can be bewildering for some patients, though using a basic telephone facility as a minimum tool for the screening assessment makes the implementation of remote support and assessment feasible for most services. It is generally assumed nowadays that most people have access to a telephone, though internet access is not universal. A report published by the UK Office of National Statistics in 2019 suggested that only 7% of households across Great Britain do not have internet access. The proportion for the 65 years and older age group is higher, with 24% of them not using the internet in the last three months prior to the survey . A telepTele-rehabilitation offers a means to deliver the required therapy input while also maintaining the need for social distancing, which has been implemented as part of the raft of measures to slow the spread of the SARS-Cov-2 virus infection in communities. Tele-rehabilitation facilitates the continuation of a rehabilitation program within a patient’s home, which is often the preferred location by most patients for therapy input . Tele-reHome tele-rehabilitation systems are cost effective, especially when primarily used to monitor or evaluate patients during corrective therapy. The loss of physical human contact (face-to-face interaction) with the doctor or therapist is a potential drawback, but, as noted previously, the risk of cross-infection with such a highly contagious infective agent like the SARS-Cov-2 virus mitigates against this disadvantage. Potential factors that might prevent individual patients from accessing tele-rehabilitation program include cognitive impairment, severe pulmonary hypertension, unstable cardiovascular disease, uncontrolled seizure disorder, poor balance or vestibular control, and sensory and communication problems. For these patient cohorts, more traditional approaches enable closer risk management, but, for the majority, tele-rehabilitation offers a means to deliver supervised therapy by experienced clinicians to the patients within their home or community dwelling. The British Thoracic Society (BTS) guidelines  on PR caThe COVID-19 pandemic has had a massive impact on the UK National Health Service. Routine care pathways were suspended as part of the efforts geared towards ensuring that the healthcare system was not overwhelmed ,44. RehaIn order to ensure that post-discharged COVID-19 patients were appropriately monitored and provided with the required input, a group of clinicians with clinical expertise spanning across intensive care, respiratory medicine, infectious disease, rehabilitation medicine, and therapy disciplines based at the Hull University Teaching Hospital NHS Trust developed the proposed pathway . The iniDischarged COVID-19 patients will be managed along two streams based on whether they had intensive care input with respiratory support: mechanical ventilation, CPAP (continuous positive airway pressure) or high flow nasal oxygen, or not. Electronic coding will enable patients who had a hospital admission where they tested positive for COVID-19 to be identified to a pathway administrator. The codes also identify which patients require intensive respiratory support  from those who are able to remain on a ward (stream 2). The pathway administrator will receive weekly updates and then book patients into the appropriate assessment clinics. The pathway will incorporate two assessment points at four-to-six weeks and 12 weeks where clinicians make contact remotely with the patients . The fou® [Bespoke interventions tailored to individual circumstances will be provided based on the assessment . The tel® , an NHS Due to the nature of the on-going coronavirus pandemic and its impact on healthcare utilization, it is anticipated that many patients with COVID-19 discharged from hospitals will have residual rehabilitation needs. The evaluation and assessment of these needs will be required to appropriately plan for intervention. It is also anticipated that the resources to deliver the intervention in the community will be limited, thereby necessitating considerations for novel intervention delivery techniques.Patients should be offered the opportunity to participate in tele-rehabilitation programs in addition to any other input provided by community services. Tele-rehabilitation would be helpful for majority of patients who require input such as PR, psychological support, and nutrition advice. Patients unable to participate in a tele-rehabilitation program and those requiring highly specialist input for impairments such as balance deficits would require face-to-face assessment, and appropriate infection risk management will be implemented to support thisReferrals to services to support with dysphagia, nutrition, balance, cognitive difficulties, psychological support, or other neuromuscular sequelae should still be made as required. Participating in a tele-rehabilitation helps to avoid a gap in service provision following discharge from hospital. Service evaluation should be an integral component of such programs.We therefore propose that that in exploring the recovery trajectories of COVID-19 patients following discharge from hospital, supervised multidisciplinary tele-rehabilitation programs should be an integral component of the follow-up pathway because they provide an effective modality of assessing and managing residual deficits.


The final stage of liver development is the production of hepatocytes and cholangiocytes  from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc‐RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes.Differentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT‐PCR and RACE, we cloned a novel lncRNA  that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc‐RHL concurrently with differentiation, we show that lnc‐RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage.Here, we report the discovery of lnc‐RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc‐RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc‐RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes.Deficiency for lnc‐RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc‐RHL is a key regulator of bipotency in HepaRG cells. HepaRG cells serve as a useful model of hepatic differentiation. A novel lncRNA (lnc‐RHL) is induced when bipotent hepatic progenitor cells are induced to differentiate. Using inducible knockdown approaches, we show that lnc‐RHL regulates the final stage of liver development in which hepatocytes and cholangiocytes are formed from bipotent progenitor cells. Deficiency for lnc‐RHL alters the balance of hepatocytes and cholangiocytes over the course of differentiation. During gastrulation, mesendoderm arises, from which definitive endoderm is specified through the combined action of Nodal and BMP signalling. Subsequently, the embryonic liver bud arises from the ventral region of the anterior foregut after exposure to inductive signals from adjacent cardiac mesoderm, and within the liver bud, a population of bipotent hepatic progenitor cells  is specified. HPCs are able to differentiate into either hepatocytes or cholangiocytes .The investigation of developmental mechanisms leading to terminally differentiated hepatocytes has been hampered by deficiencies in the culture of primary hepatocytes, which rapidly lose their metabolic activity during cell culture. Though key liver developmental pathways have also been informed by studies of differentiating pluripotent stem cells, these fail to yield fully mature hepatocytes with full metabolic activity in vitro. However, a cell line called HepaRGLong non‐coding RNAs (lncRNAs) are a set of highly diverse transcripts present in all mammalian cells that participate in a wide range of molecular and cellular regulatory mechanisms.Recent advances show that lncRNAs are intimately involved in key aspects of normal liver function, development, regeneration and disorders of the liver. The lncRNA‐LALR1 (lncRNA associated with liver regeneration 1) increases hepatocyte proliferation after partial hepatectomy by triggering responses in the Wnt/β‐catenin pathway leading to cyclin D1 expression.APOA1 gene within the 11q23.3 apolipoprotein gene cluster.We were intrigued by the recent discovery of a human lncRNA called APOA1‐AS, an antisense lncRNA that regulates the neighbouring 22.16) were plated in a T25 collagen‐coated flask in HepaRG growth medium for 14 days. Differentiation of HepaRG cells was induced by adding HepaRG differentiation media and growth media (in a 1:1 ratio) from days 15 to 20, then pure HepaRG differentiation medium until day 33. Medium was changed every 2‐3 days. HepG2 cells were cultured in Dulbecco's modified eagle medium (DMEM) containing 1gm/L glucose and 10% foetal bovine serum (FBS) . All cell lines were maintained at 37°C in humidified incubators with 5% CO2.Frozen liver biopsies were procured from the Liver Tissue Cell Distribution System, University of Minnesota. Two media were used for HepaRG culture: HepaRG growth medium was prepared by adding HepaRG growth Supplement  to 500 ml of William's E medium (Thermo Fisher Scientific). HepaRG differentiation medium was prepared by adding HepaRG Differentiation Supplement  to 500 ml of William's E medium. Both were supplemented with 2 mM Glutamax and antibiotics . For the expansion phase of HepaRG culture, HepaRG cells (1.5 × 102.2RACE PCR was performed using a Clontech SMARTer RACE 5’/3’ Kit (cat. # 634 858). Gene‐specific primers Table  were obt2.3Packaged lentivirus containing 3 shRNAs designed to target lnc‐RHL (based on our complete lnc‐RHL sequence) was procured from transOMIC Inc, and expressed from a pZIP‐inducible lentiviral vector as well as a non‐targeting scrambled control shRNA. Stable lnc‐RHL shRNA and scrambled control HepaRG cell lines were generated by transducing the lentiviral particles per the manufacture's protocol. Briefly, HepaRG cells were seeded to 50% confluency and cultured for 24 hours in HepaRG growth medium before transduction in a 6‐well plate. HepaRG cells were transduced with the lentiviral shRNA in a mixture of polybrene 8.5 μg/mL  in complete HepaRG growth medium for 24 hours. Medium was then changed to HepaRG growth medium for an additional 48 hours. Growth medium was supplemented with puromycin (2.5µg/ml) (Sigma‐Aldrich) for all subsequent culture. All inducible shRNA knockdown experiments were performed using HepaRG cells of passage 17‐20. Induction of shRNA inserts was performed by adding doxycycline (1.5µg/ml). Fresh media with doxycycline was replaced every 24 hours since the half‐life of doxycycline in media is 24 hours.2.4Taq 2X master mix (M0270L) purchased from New England Biolabs, using primers described in Table RT‐PCR for lnc‐RHL was performed using 2.5‐Δ∆CT method and normalized to β‐actin mRNA expression.Total RNA was isolated from HepaRG cells using the NucleoSpin RNA Plus Kit according to the manufacturer's protocol (Macherey‐Nagel). RNA samples were treated with RNase free DNase I (cat. #18047019—Thermo Fisher Scientific). Total RNA was then used for first‐strand cDNA synthesis by an iScript cDNA synthesis kit . Primers Table  were des2.6P‐value less than .01, and three asterisks represent a P‐value less than .001.All data were analysed by GraphPad Prism software . A one‐way ANOVA was performed followed by a post hoc Tukey‐Kramer test with multiple comparisons to determine significant gene expression changes. One asterisk represents a p‐value less than 0.05, two asterisks represent a 33.1APOA1 gene within the 11q23.3 apolipoprotein gene cluster.regulator of hepatic lineages). The annotated length of RNAseq reads was 333 bases, flanked at the 5’ end by APOA4 and at the 3’ end by APOA5 genes  to precisely ascertain the structure of lnc‐RHL. First, we amplified lnc‐RHL–specific cDNA using primers designed to anneal to lnc‐RHL within the annotated 333‐base sequence and universal RACE primers to yield 5’ and 3’ RACE products. Initial 5’ and 3’ RACE products yielded a faint smear of cDNA (not shown), so a second RACE amplification was performed using nested lnc‐RHL–specific primers and the same universal RACE primers. This strategy yielded crisp 5’ (~300 bp) and 3’ (~650 bp) RACE products Figure . We clonGAPDH and APOA1). We also subjected the lnc‐RHL sequence to analysis by other programs including NCBI ORF finder, Coding Potential Assessment Tool (CPAT)lnc‐RHL gene similar to a CR1 transposable element, subfamily class L3, but this is located on the opposite, untranscribed strand of the lnc‐RHL gene. We also determined a predicted secondary structure of lnc‐RHL using the folding algorithm Vienna  and found two very short ORFs of 30 and 29 codons Figure . Predicta Figure A. This pa Figure B.3.2Bipotent HepaRG cells differentiate into both hepatocytes and cholangiocytes using an established differentiation protocol Figure . SimilarWe also assessed levels of lnc‐RHL and relevant marker gene expression before and after differentiation using qRT‐PCR Figure . lnc‐RHL3.3We next investigated whether genetic deficiency of lnc‐RHL might have consequences for expression of linked genes (similar to APOA1‐AS), or possibly, the ability of HepaRG cells to differentiate. To do this, we expressed lnc‐RHL–specific shRNAs from an inducible lentiviral vector to allow the protracted knockdown of lnc‐RHL during the entire course of differentiation or after differentiation was complete. We transduced undifferentiated HepaRG cells with a set of three lentivirus preparations  each harbouring a doxycycline (Dox)‐inducible shRNA predicted to target lnc‐RHL Figure . In piloAll transduced HepaRG cells expressed GFP upon Dox induction, and GFP expression was not detected in the absence of Dox Figure . In the We next assessed the impact of lnc‐RHL deficiency upon the expression of relevant mRNAs including hepatocyte markers, 11q23.3 mRNAs and cholangiocyte‐related mRNAs Figure . First, The reduced production of hepatocytes in differentiating HepaRG cultures made deficient for lnc‐RHL could be due to a defect in differentiation itself, or cell death of hepatocytes induced by lnc‐RHL deficiency. We therefore assessed if lnc‐RHL deficiency might induce apoptosis by performing a flow cytometry assay using Annexin‐V detection Figure . We foun4bona fide lncRNA: (1) lnc‐RHL contains only two very short ORFs  was shown to regulate the expression of adjacent The authors declare no conflicts in this study.B. Prabhakar and T. Rasmussen designed research; B. Prabhakar, SW Lee, A. Bochanis and W. He performed experiments. J. Manautou and T. Rasmussen edited the report for intellectual content. B. Prabhakar and T. Rasmussen wrote the report, and all authors approved the final report.Supplementary MaterialClick here for additional data file.