ADAMTS family members control mammalian development and disease, primarily through their function as proteases, by regulation of extracellular matrix composition. Until recently, ADAMTS6 was known as one of the orphan proteinases of the nineteen-member family with a relatively unknown expression pattern and function. Emerging focus on this enzyme has started to uncover these unknowns and revealed a vast importance and requirement of ADAMTS6 in cardiovascular and musculoskeletal development. In addition, ADAMTS6 has been linked to numerous disease settings including several types of cancer. This review summarizes the necessity of ADAMTS6 during development, its role in disease and requirement for essential prospective studies to fully realize its biological implications and potential for therapeutic intervention. Their rfication .O-fucosyltransferase-2 (POFUT2) is an enzyme that adds O-linked fucose to the TSRs, which are numerous in ADAMTS6, in the endoplasmic reticulum to promote trafficking of substrates that include ADAMTS proteases   . It was d below) .Adamts6 is expressed in numerous vertebrates in a variety of tissues. In zebrafish, it is enhanced 200-500-fold after 8 h post fertilization and expressed in all organs surveyed including high expression in the liver, kidney and skeletal muscle as assessed by quantitative real-time PCR (qRT-PCR)  as assayed by RT-PCR and northern blot analysis as well as adult chondrocytes (qRT-PCR) . Express tissues . In humadrocytes .It is also expressed in a variety of tumors from different origins and associated with other pathological situations as described in detail below. However, the role of ADAMTS6 in these and other yet undescribed tissues and disease settings are not fully uncovered. ADAMTS6 has long been considered an “orphan protein” with very little data on its expression profile, function, and substrate targets. Very recent studies have started to uncover these unknowns and provide a strong case for the necessity of ADAMTS6 in ECM maintenance during development and its implications during disease processes. The main processes that ADAMTS6 has been identified to participate in will be discussed in the following sections, spanning embryology through cancer and other disease settings.Adamts6 is expressed throughout the developing heart with strong staining noted in the outflow tract, valves and myocardium  identified ADAMTS6 as a promising, but not yet genome-wide significant, susceptible loci for osteosarcoma  14.5 to E18.5 in the hindlimb bones with the fibula being the most drastic example. Adamts6−/− growth plates are disorganized and have a reduction of key chondrogenic markers, including aggrecan, cartilage link protein and Sox9 with no change in chondrocyte proliferation or cell death , there were abundant microfibrils present, which points to a direct role of ADAMTS6 in microfibril formation or turnover. Therefore, these proteins were further explored as possible substrates. Direct binding of ADAMTS6 to fibrillin-1, fibrillin-2 and fibronectin was noted in Biacore assays and proteomic assays revealed that they were all indeed substrates through identification of specific cleavage sites  resulted in genetic reversal of limb abnormalities exhibited in Adamts6−/− mice with restoration of chondrocyte growth plate markers  which had additional musculoskeletal phenotypes compared to Adamts6−/− alone consisting of forelimb buckling in a similar manner to Adamts6−/− hindlimbs and subcutaneous hemorrhage, neither of which appear in Adamts10−/− mice  . Their rADAMTS10 . Overexp samples . In this−/− mice . It woulADAMTS6 was shown to be either upregulated or downregulated in the same cancer type based on microarrays, clinical samples, and in vitro assays. White the overall majority of studies show that increased expression of ADAMTS6 is pro-cancer and correlates with a negative outcome, there are a few exceptions noted.ADAMTS6, like many other ADAMTS family members, has a pro- or anti-tumor role in various types of cancer, spanning several organ systems. In some instances, ADAMTS6 expression is upregulated in breast carcinoma tissue samples with highest expression in myoepithelial cells  and associated with loss of H3K27me3 on its promoter  through regulation of anterior gradient 2 (AGR2), an endoplasmic reticulum protein family member . In thisogenesis . Importapromoter . Increasinvasion . TherefoADAMTS6 as one of the most differentially expressed genes in a survey of data sets of human colorectal cancer as compared to normal tissue  and RNA-seq data revealed that high survival . Consisth cancer . Gene seADAMTS6 was significantly downregulated in placental site trophoblastic tumor (PSTT), a rare type of gestational trophoblastic neoplasms through interrogation of microarray data and confirmed by qRT-PCR analysis ADAMTS6 RNA, a type of noncoding RNA, was downregulated after treatment of IL-1β   in several disease settings. database . It was (ADAMTS6 . A furth(ADAMTS6 . Bioinfoof IL-1β . circADA exon 24 . Overexpartilage . Knockdo pathway . Taken ta (ESCC) . ConversADAMTS6 was upregulated, as assessed by microarray and confirmed by qRT-PCR analysis, in kidney epithelial cells in an in vitro model of Fabry disease, an X-linked lysosomal disorder (ADAMTS6 is highly expressed in osteoarthritic cartilage and synovium as identified from qRT-PCR analysis (ADAMTS6 as a susceptible locus for inguinal hernia (Adamts6 expression, as determined by RNA-seq and qRT-PCR analysis, was noted in mouse connective tissue equivalent to human transversalis fascia, which when weaken or defective can lead to an inguinal hernia (ADAMTS6 associated with changes in central corneal thickness in the eye and may lead to disease if not properly maintained (Additional disease settings involving ADAMTS6 have been reported, but have limited data and require follow up studies. disorder . ADAMTS6analysis . Two GWAl hernia . Adamts6l hernia . And finintained .Adamts6−/− hindlimbs, as compared to control, with no change in RNA levels (Adamts6-deficient embryonic hindlimbs, another tissue and setting could uncover a yet unknown role and function (Like other family members, ADAMTS6 participates in ECM remodeling and thus targets will likely be found in this area. Evidence that fibrillin-1, fibrillin-2 and fibronectin were potential substrates was shown through increased immunofluorescence staining in A levels . They weA levels . In a seA levels . ADAMTS6A levels . The clefunction . Westernfunction .ADAMTS6 is expressed in numerous cell types and tissues during development and disease and its roles in these settings will not be fully realized until additional tissue-specific substrates are identified. Thus, an unbiased proteomic search in select ECM-enriched populations is necessary to uncover additional substrates. Once these cleavage sites are verified and shown to be critical, utilizing in vitro and in vivo means, only then can the role of ADAMTS6 in these settings be realized which is necessary to reveal new avenues of research towards therapeutic intervention.While these few ADAMTS6 substrates have been suggested and fibrillin-2 verified through cleavage site identification and genetic interaction, additional focus on substrate verification and identification is warranted. This review summarizes the known ADAMTS6 connections to development and diseases known currently. This will undoubtedly expand in the near future and beyond as this once “orphan protein” starts to be recognized and more studies are undertaken, such as whole genome sequencing of rare disorders in humans and preclinical models as well as proteomic screenings. ADAMTS6 participates in a myriad of processes including cardiac and musculoskeletal development as well as cancer and disease and interacts with many ECM molecules and signaling pathways as a result. Greater understanding of the ADAMTS6 regulatory network is vital to predict undesired outcomes when attempting to modify consequences of disease.Adamts6-deficent mice have made a considerable impact on the ECM field and have led to the discovery of its necessity in cardiovascular and musculoskeletal biology (Adamts6 in disease settings. Adamts6 is expressed in differing cells lineages and it is difficult at present to discern cell type specific functions. Therefore, a conditional genetic approach is warranted in future in vivo studies. The full picture of the role of ADAMTS6 can only begin to be realized once the substrates are identified and verified. This will allow for delineation of molecular pathway and mechanistic insight into its role in the ECM during tissue development, homeostasis, and disease settings. While a few substrates have been suggested and some verified, an unbiased, proteomics approach is warranted to discern the full landscape. Only then will it be readily suitable to generate targeted approaches for therapeutic applications. biology . HoweverAdamts6−/− musculoskeletal tissues during development (fbn1 postnatal expression as compared to fbn2, which is virtually absent starting in early postnatal tissues, is a result of abundance fibrillin-1 transcription masking a fibrillin-2 microfibril core (Fibrillin microfibrils are a necessary component of the ECM that provides mechanical stability to tissues and regulates several signaling pathways such as TGFβ and BMP . That ADelopment . Whetherril core . TherefoA further function of ADAMTS6 was uncovered in focal adhesion biology. Through a series of experiments utilizing a human retinal epithelial cell line, it was shown that ADAMTS6 negatively regulates focal adhesions . AdditioAdamts6-deficient embryonic myocardium had a reduction of Cx43 staining, a predominant myocardial gap junction protein (Adamts6+/− myocardium without a change in Gja1 mRNA levels, which suggested post-transcriptional regulation (ADAMTS6 was shown to have a role in gap junctions in ventricular myocardium.  protein . This wagulation . These fIn summary, ADAMTS6 is a major component of the ECM and regulates several biological processes during development and disease . Further


The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear.To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling.Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice.Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated β1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor.Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH) is a disease characterized by occlusion of distal pulmonary arteries resulting in increased resistance to flow and culminating in heart failure. The appearance of fragmented elastic laminae in pulmonary arteries from patients with PAH suggested the presence of heightened elastase activity in the vessel wall that was identified as neutrophil elastase in human cells and in experimental animals with pulmonary hypertension.1) production and release of elastase, 2) generation of neutrophil extracellular traps, and 3) adhesion mediated by vinculin that directly contributes to the pathogenesis of PAH. This phenotype results from a cell-autonomous and non–cell-autonomous increase in two human endogenous retrovirus K family retroviral elements.We describe a novel phenotype of neutrophils related to an increase in Pulmonary arterial hypertension (PAH) is a disease characterized by occlusion of distal pulmonary arteries resulting in increased resistance to flow and culminating in heart failure. Current treatments improve quality of life and survival primarily by dilating pulmonary arteries, but these agents do not address the mechanism underlying the progressive pulmonary vascular pathology (reviewed in ). The apNeutrophils are major sources of NE; increased release of NE is observed in PAH neutrophils , and cirNE is critical for NET formation, and the enzyme remains proteolytically active when bound to NETs, thus contributing to tissue damage . ElevateTo understand their contribution to the pathogenesis of PAH, we isolated circulating neutrophils from patients with PAH versus healthy control subjects and first assessed NE production and release, NET formation, and adhesion and migration, including transendothelial migration. We performed proteomic and transcriptomic profiling to identify the mechanism underlying the abnormalities observed. Our results indicated a novel antiviral response in PAH neutrophils that explained the PAH neutrophil phenotype. The mechanism accounting for increased elastase was related to an elevation in the human endogenous retrovirus (HERV)-K envelope in neutrophils, and the mechanism explaining increased vinculin (VCL)-mediated adhesion could be attributed to elevated circulating concentrations of HERV-K deoxyuridine triphosphate diphosphatase (dUTPase). We showed that PAH neutrophil EVs contain elevated NE as well as the HERV-K envelope and that they induce pulmonary hypertension in mice, except when pretreated with elafin . Taken txpanded Methods section is available in the online supplement.An Esee Tables E1–E4 in the online supplement).Whole blood from patients with PAH was obtained from the Stanford PAH biobank, and blood from healthy volunteers was obtained from the Stanford Biobank and the Stanford Blood Center. Pulmonary arterial endothelial cells (PAECs) were obtained through the Pulmonary Hypertension Breakthrough Initiative  and were cultured in endothelial cell complete medium.NE activity was measured using EnzChek Elastase Assay Kit (Thermo Fisher Scientific).d-lysine–coated coverslips were treated with phorbol myristate acetate (PMA) with vehicle  or elafin for 60 minutes. Cells were washed with PBS and then stained with cell-impermeable SYTOX Green and for H3 citrullinated and NE as described in the Expanded Methods section of the online supplement.Neutrophils adherent to poly-Neutrophils adherent to fibronectin were treated with 100 ng/mL of IL-8, and cell migration was observed in real time using a confocal microscope.Transmigration and transendothelial migration assays were performed in a modified Boyden chamber using the Corning FluoroBlok 24-well plate.Neutrophils were incubated on fibronectin-coated slides or on a monolayer of PAECs at 37°C for 5 minutes, washed with PBS, then imaged under light microscopy.Neutrophils were stained for Alexa Fluor 700–conjugated integrin-β2 (ITGB2), and data were acquired using a Beckton Dickenson LSR II flow cytometer and analyzed using FlowJo.Adherent neutrophils were fixed with 4% paraformaldehyde and treated with HERV-K envelope antibody, followed by Alexa Fluor 488–conjugated secondary antibody and DAPI staining.Pooled EV samples were visualized using a JEOL JEM-1400 120-kV electron microscope.xpanded Methods section in the online supplement.See the EqPCR was performed using Powerup SYBR Green PCR Master Mix (Applied Biosystems).Primer sequences are listed in Table E7.HL-60 cells were transfected with HERV-K envelope vector or enhanced green fluorescent protein control vector (VectorBuilder) using HL-60 Cell Avalanche Transfection Reagent (EZ Biosystems).EVs were isolated from plasma using the EV Total Isolation Chip (ExoTIC). ExoTIC-harvested EVs were further purified using Dynabeads conjugated to CD66b/CEACAM-8 (carcinoembryonic antigen–related cell adhesion molecule 8).Proteomic analysis used the liquid chromatography system directly coupled inline with an Orbitrap Fusion Lumos Mass Spectrometer.Total RNA was prepared using the Takara Bio SMARTer Stranded Total RNA-Seq Kit version 2 Pico Input Mammalian kit and sequenced using the HiSeq 2500 sequencer.1) PBS vehicle, 2) pooled EVs of healthy donor controls, 3) pooled PAH EVs, or 4) pooled elafin-treated PAH EVs. Cardiac function, right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular morphometry were assessed. The first cohort consisted of the first three groups with n = 3 in each group, and the second cohort added the fourth group with n = 6 in each group.Adult male mice (8 wk of age) were administered tail vein injections twice weekly for 5 weeks of n is the number of control subject or patient cells studied. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test, or it was determined by two-sided unpaired t test. Data with a sample size n < 8 were analyzed using the Mann-Whitney nonparametric test. Samples with counts of 3 are confirmatory of other assays or represent technical replicates from cell lines or pooled samples. Pathway enrichment analysis was conducted using the Integrated Molecular Pathway-Level Analysis (IMPaLA) webtool.Bars represent the mean ± SEM, and RNA-sequencing (RNA-seq) data were deposited in the National Center for Biotechnology Information Sequence Read Archive with accession number PRJNA822411. Proteomic data are available via ProteomeXchange with identifier PXD023134. Code related to RNA-seq data analysis is available on the Benayoun lab GitHub website.xpanded Methods section of the online supplement. Demographics and other data related to the cohorts used in each of the experiments are found in Tables E1–E4 in the online supplement. Although we requested samples from patients with idiopathic PAH, a small proportion (3 of 68) were later reclassified as having drug- and toxin-related group 1 PAH  analysis of CD66b and CD16, as shown in Figure E1A and described in the Eup 1 PAH , and 5 oup 1 PAH . A compaup 1 PAH , and theElevated NE activity was normalized to neutrophil counts. These were also increased in this cohort of patients with PAH compared with control subjects , resultiIncreased NE production and NETosis suggested that other neutrophil functions could be altered or exaggerated in response to inflammatory stimuli. The functions considered were adhesion, migration, and transendothelial migration. We observed heightened adhesion to a fibronectin substrate in PAH versus control neutrophils  that we P value of 0.04 but an FDR of 11% , but 483 differentially regulated proteins were identified with a false discovery rate (FDR) of 10% . PathwayR of 11%  and woulDR of 6% . No signP = 0.03; FDR, 9%) and C-X-C motif chemokine receptor 2 (CXCR2)  . Using an FDR <5%, we identified 1,483 differentially expressed genes as displayed in the heat map . By usinPKR) was determined to be a central node , IFNAR1 (type I IFN receptor), and IFNGR2 (IFN-γ receptor 2) and a decrease in ARIH1 (ariadne RBR E3 ubiquitin protein ligase 1) are critical in mounting an effective antiviral immune response in neutrophils  was transfected with a HERV-K envelope vector or a control green fluorescent protein vector, and gene expression was analyzed by qPCR . Overexp NE mRNA  and prot NE mRNA . There wPKR or RIG-1 mRNA or in NE protein . The dose of dUTPase chosen was based on previous studies by our group showing the impact of dUTPase on cytokine release , monocytes can release HERV-K dUTPase, and circulating concentrations of this retroviral protein were increased in PAH versus control plasma samples . We adde release  and EndM release . We thenTransport of increased NE via neutrophil EVs was observed in bronchial secretions from patients with COPD, and these EVs caused COPD-like changes in mice . A CD66bNeutrophil EVs isolated from pooled PAH and control plasma were administered by tail vein injection twice per week for 5 weeks to adult male mice  accordinDespite many advances in our understanding of the sequelae of chronic inflammation related to PAH pathology, the role of neutrophils has remained elusive. Here, we provide evidence of neutrophil dysfunction in PAH that we link to an antiviral response that results in adverse remodeling of the pulmonary vasculature. PAH neutrophils produce an increase in NE, leading to a propensity to form NETs, and are characterized by increased adhesion and reduced transendothelial migration attributed to elevated concentrations of VCL , 36. AugOur previous studies attributed the increase of NE in PAH to its production by pulmonary arterial SMC in patients and experimental models . Here wePAH neutrophils are further characterized by increased adhesion and reduced migration that we relate to an increase in neutrophil VCL previously shown to be responsible for these abnormalities . FurtherA PAH neutrophil antiviral signature consistent with an IFN innate immune response  is relatOverexpression of HERV-K envelope in a neutrophil cell line was sufficient to induce the production of NE. Although the increase in NE likely represents the activation of an innate immune response, future studies will be of interest in delineating the molecular mechanism involved. HERVs can also mediate a chronic IFN response in association with the production of double-stranded RNA , 51, andInfection of monocyte-derived macrophages with HIV led to the upregulation of VCL, and, as a consequence, VCL negatively affected the propagation of the virus . SimilarPathologic EVs are recognized in many diseases, including autoimmunity , cancer


Horizontal semicircular canal site pathology of benign paroxysmal positional vertigo demonstrating three types of nystagmi on positional test were studied. We have attempted to design a protocol for its diagnosis and treatment.320 patients of HSC-BPPV were subjected to two types of positional tests. Of these, patients with bilateral steady apogeotropic nysatgmus were treated with VAV modification of Semont’s maneuver. Patients with unsteady or changing apo/geotropic signs were converted into steady geotropic ones by repetitive positional tests; followed by barbecue maneuver with forced prolong positioning.Overall 88% of patients had a total recovery. 92% of patients with geotropic nystagmus showed no symptoms after second maneuveral sitting. 85% of patients with apogeotropic nystagmus recovered fully after third maneuveral sitting.Correct identification of subtypes of HSC-BPPV is based on provoked nystagmus by positional tests. After locating the site and side on the basis of nystagmic pattern, physician can apply the appropriate PRM.II a. Benign Positional Paroxysmal Vvertigo (BPPV) is a common cause of dizziness, particularly in people older than 60 years. Its incidence is estimated to be 6000/million of population in those whose illness persists longer than 30 days, although this is probably a gross estimate. BPPV is easily identifiable with bed side clinical test . All three semicircular canals  may be the source of BPPV, although the literature quotes the posterior semicircular canal as the most common source. Various theories exist regarding the pathogenesis and localization of BPPV. Calcium carbonate embedded in proteinaceous matrix on surface of utricle,Variants of BPPVIn recent time a specific attention is made to detect BPPV related to Horizontal Semicircular Canal (HSC). The HSC variant of BPPV (HSC-BPPV) is usually induced by Hallpike-Dix test and or by head rotation in plane of HSC with head hanging at 30°‒45°.Depending on the appearance of induced nystagmus; following variants of HSC-BPPV can be identified and classified into three pathological subgroups viz: 1) Most common type is bilateral geotropic nystagmus where Free Floating Particles (FFP) detached from utricular macula travelling in post arm of HSC; 2) Second common type, which shows bilateral unstable nystagmus (Changing from apogeotropic to geotropic after few Particle Re-Positioning Maneuvers (PRM). Pathophysiology may be related to the shift of FFP from anterior arm of HSC to posterior arm of HSC; 3) Third type, which can be evoked in form of bilateral stable or fixed apogeotropic nystagmus, can be interpreted as a cupulolithiasis.Of the above three types described, the third type shows most aggressive longer lasting symptoms often accompanied with neuro-vegetative phenomena .In spite of the listed signs and symptoms above, at present we lack in producing the model which can navigate to produce standard protocol for treatment of HSC-BPPV unlike that of PSC-BPPV: as Epley’s maneuver is not effective to cure HSC-BPPV.First undocumented attempt to remove FFP from HSC was done by Vannucci et al.9As the abundant literary articles have proved by describing various PRMs to be more effective and quicker in curing the BPPV pathology when compared with natural recoveryA healthy male 42 yrs. old approached us in February 2002 with history of recurrent episodic vertigo. The vertigo was often triggered with change in bed position from supine to left lateral and lasting for 2–3 min. The vertigo was lessened with lying on supine. He preferred to lie down on right lateral position during sleep. His systemic and ear, nose, throat, systemic and neurological examination was unremarkable. The bedside Hallpike positional test done repeatedly showed changing nystagmus (apogeotropic as well geotropic) pattern in horizontal axis. Patient was put on intermittent short therapy of vestibular suppressants and vestibular adaptive exercises for 3 months which failed to improve the vertigo. In May 2002 he met with an fatal road accident and died of severe chest compressive injury. His temporal bone autopsy specimen was available for HPE study which revealed the adherent “debris” to HSC cupula as well as few free particles in membranous canal portion .Figure 1This institutional study has been granted an exemption by ethical committee by document number IEC/SH/08/1995 which is in compliance with Helsinki declaration (1964) as well with (ICMR guidelines 1990). From May 1995 till October 2019, we examined 320 consecutive patients were diagnosed to be of purely HSC-BPPV origin on the basis of positional tests in a central city vertigo clinic. Duration of symptoms ranged from 15 to 770 days (average duration 65 days). All patients described brief episodic intense vertigo while changing the positions in bed as lateral to supine and vis-a-vis with the duration of intense phasic vertigo lasting between few seconds to 2 min. Pure Horizontal positional nystagmus was seen during Dix-Hallpike test in majority of the cases and in modified head rotational positional test , vide infra) , Fig. 4.Before performing specific physical therapy (PRM),1)For HSC cupulolithiasis VAV-Semont’s (modified II stage of Semont’s) maneuver was done. Patient sits upright, neck then extended to 45° for 10 s and then made to quickly lie down on the affected side followed by rotating the head by 45 ° to return to the starting downwards facing the earth and maintaining this position for 3–5 min and then bringing him back to upright position. Usually, patient reports relief from head heaviness . If not,2)For HSC canaloithiasis, Barbecue maneuver is performed at home or in wards with his headfirst from affected side to normal side followed by trunk rotating in each step by 90° in a sequence from healthy side to affected side . Each step is followed by 2 min pause, after attaining final lateral position on healthy side patient is asked to maintain FFP for 10 h.PRM was performed as: All the maneuvered subjects were asked to report the details of recovery on our media network after 48 h and at 10 days, 6-months and 2-years: those non-beneficiaries were asked to attend repeat physical therapy.Few non-responders to PRMs were put on intermittent vestibular suppressant and Vestibular Adaptation Exercises (VAE).Of 320 healthy individuals with female: male ratio of 3:2  reported short lasting episodic rotatory vertigo. Of these 63 subjects had neuro-vegetative symptoms. Vertigo induced from supine to lateral was present in 210 subjects and from sitting to lying down (vis-a-vis) in 55 subjects in their history, further 46 subjects reported the onset of vertigo with either change in posture; majority of these reported to be at ease, during episodic vertigo with eyes closed. There was some predilection of Cupulolithiasis occurring more in females compared to males with neuro-vegetative symptoms.Dix-Hallpike and VAV tests were applied randomly in 320 suspected HSC-BPPV patients; shows 117 cases of cupulolithiasis and 193 cases of canalolithiasis were inducible by Dix-Hallpike test, while 148 cases of cupulolithiasis and 165 cases of canalithiasis were inducible by VAV test . When coInitial observation was with reports of 130 cases with geotropic and 190 cases with apogeotropic forms of nystagmus after combination of both type of positional test. Final observation listed before applying PRM was as; canalolithiasis with geotropic nystagmus presentation were 190 and with apogeotropic nystagmus were 130 ; hence 6The side of lesion was labeled after closely co-relating the history with findings of positional tests .PRM was applied as: VAV-Semont’s maneuver in all 130 of cupulolithiatic cases (with assisted head shakes only in 20 cases) and Barbecue maneuver with FFP for 10 h in 190 Canalolithiatic cases. Complete resolution was noted in 77% (105/130) in cases of cupulolithiasis after 1st PRM and in 85% (114/130) after 3rd PRM. Canalolithiatic group showed complete resolution in 87% (165/190) after 1st PRM with FPP and 90% 174/196) after 2nd PRM with FPP.Benign paroxysmal positional vertigo represents 15%–22% of all otogenic dizziness.The challenges in managing HSC-BPPV are: 1) Detection of affected side; 2) Detection of sites; short arm of HSC vs. long arm of HSC; 3) Deciding the sequential steps to elicit the nystagmus; 4) Choosing the correct PRMs to yield optimum results.1)Side detection can be decided on basis of correct history taking and type of nystagmus which is bilateral horizontal beating with apo/geotropic with non/fatiguing . At a ti2)Site in HSC can be decided again on evoked apo/geotropic nystagmus and consistent/non consistent (refer hereafter as stable/unstable) . Stable apogeotropic feature indicates copular lesion while unstable apogeotropic3)We found following steps to be effective to evoke the nystagmus optimally. a) Cessation of the vestibular suppressants for 48 h. b) Correct history pertaining to the details of episodic vertigo  Performing Dix-Hallpike and VAV positional tests with 2–3 times repetition and recording with VNG. In case of un-stable apogeotropic nystagmus we tried to convert it into stable geotropic one ; by repeating VAV test or occasionally doing VAV-Semont's maneuver. This was possible due to bringing gravitational factor to produce effective elimination of FFP along the effective vector created nearing to gravity vector Vg = Ve,  in to acThese are met successfully as:4)Once these steps are done PRM is selected on basis of protocol .Figure 5Yun-Hoon ChoungPatients with stable apogeotropic (Cupulolithiasis) are treated with VAV-Semont’s PRM. If it fails, then head shakes is implemented followed by repeating of VAV-Semont’s PRM. Failing this, the patient is put on to short duration (2–4 days) of vestibular suppressants and VAE.Unstable apo/geotropic nystagmus is tried to be converted preferably in to stable geotropic following which barbecue PRMThe stable geotropic nystagmus showing subjects were treated with barbecue PRM and FPP for 10 h.Literature has expressed possible fear of FFP migration to other canals or sites. We haven’t come across any such incidental finding in 320 individuals.The strategic treatment reported in this series of HSC-BPPV has resulted complete resolution of vertigo over 88% as against 42% in control groupIn the last century, treatment with diagnosing ability looked bizarre. Diagnosing abilities in suspected BPPV has been greatly improved upon in last 2 decades. The Senior author witnesses the shifting paradigm in the management of BPPV in recent time; especially after considering maiden evidence of cupulolithiasis on HPE in a case. Hence, on the basis of fixed cupular particles or FFP one is able to locate the side and site of culprit attributed to a troublesome recurring peripheral vertigo. The key factor in the management of HSC-BPPV are well studied nystagmus pattern in co-relation to correctly expressed history and choosing the correct maneuver pertaining to final observation made of combined positional test . The proThe authors declare no conflicts of interest.


Renal cell carcinoma (RCC) refers to a group of tumors that develop from the epithelium of the kidney tubes, including clear cell RCC, papillary RCC, and chromophobe RCC. Most clear cell renal carcinomas have a large histologic subtype, genetic or epigenetic von Hippel-Lindau (VHL). A comprehensive analysis of the genetic modification genome suggested that chromosome 3p loss and chromosome gains 5q and 7 may be significant copy defects in the development of clear RCC. A more potent RCC may develop if chromosome 1p, 4, 9, 13q, or 14q is also lost. Renal carcinogenesis is not associated with chronic inflammation or histological changes. However, if regional hypermethylation of DNA in CpG C-type islands has already accumulated in cancer-free kidney tissue, it implies that the presence of malignant kidney lesions may also be detected by modified DNA methylation. Modification of DNA methylation in cancerous kidney tissue may advance kidney tissue to epigenetic mutations and genes, leading to more serious cancers and even determining a patient's outcome. The genetic and epigenetic profile provides accurate predictors for patients with kidney cancer. New genetic and epigenetic analysis technologies will help to speed up the identification of vital cells for kidney cancer prevention, diagnosis, and treatment. Cancer, as a genetic disorder, is associated with epigenetic abnormalities. It is clear that epigenetic disruption caused by the microenvironment is important in developing neoplasia . ChangesThe most common type of RCC is clear cell RCC (ccRCC), which accounts for 75% of all RCC cases . Recent  Emerging evidence suggests that modifying our body through exercise or a variety of foods such as ketogenic diets, low-carbohydrate diets, fasting, or exercise can alter the concentration of various metabolites, some of which can alter the function of proteins that cause epigenetic changes ,4. TheseDNA methylation may be the most studied epigenetic marker among the epigenetic components. DNA methylation is a type of post-genetic mutation that occurs in the cytosine sequence of 5'-C-phosphate-G-3' (CpG) dinucleotide, in which the methyl group S-adenylmethionine is exchanged with cytosine . AdditioThe main goal of this review is to provide an overview of the role of DNA methylation in the pathology of RCC. We will first discuss the relationship between epigenetics and DNA methylation before delving into recent developments in DNA methylation research in RCC and the role of DNA methylation in therapeutic approaches. Overall, these findings point to a novel method for identifying the gene for an epigenetic driver, the intended therapeutic target of a ccRCC treatment strategy, including self-medication.DNA methylation and histone modificationEpigenetics is the study of phenotypic mutations that do not involve DNA sequencing or just genes. It affects the function of genes by influencing their cellular and physiological phenotype expression . The varEpigenetic marker is very common in cancer cells representing both a central stage in the demethylation process and an important epigenetic marker in tumorigenesis . AlthougCancer epigenetics modificationsCancer epigenetics deals with mutations in the DNA of malignant cells and excludes mutations in DNA sequences . Loss ofDNA methylation in cancerThe methyl-CpG-binding domain (MBD) "epigenome readers" methyl-CpG binding protein 2 (MeCP2) and methyl-CpG-binding domain proteins 1-4 (MBD1-4) can detect DNA methylation . EpigeneAmong others, the hormone response ESR1, ESR2 in the p53 network , and HIC-1 promote tumor cell growth while increasing genetic instability and aggression . OncogenWhen a methyl group (CH3) is added to or removed from DNA, this is referred to as methylation. These mutations result in genetic mutations, which promote growth. overmethylation of DNA typically involves inserting a methyl group into a 5-carbon cytosine ring, yielding 5-methylcytosine . This reDNA methylation and cancer metastasisCancer metastasis involves stages of local invasion and proliferation. This is influenced by the oncogenic suppressive transcription factor (TFs) that regulate tumor microenvironment features . DNA metTumor-related genes and their role in renal carcinogenesisAlthough RCC classification is largely based on histology, the World Health Organization (WHO) classification has introduced genetic mutations as a sign of certain types of histological subtypes of RCC, for example, cell RCC is characterized by chromosome 3p loss and VHL gene dysfunction at 3p25.3 due to mutation or DNA methylation around the promoter region . This coType 1 papillary RCC develops in patients with genetic mutations who benefit from mesenchymal epithelial transition (MET) genetics. Transmembrane receptor tyrosine kinase is incorporated into MET's ligand, hepatocyte growth factor (HGF). MET activation of HGF causes tyrosine kinase activity, which facilitates several transduction cascades leading to many cellular processes such as mitogenesis and migration . HoweverThe excessive c-kit function (KIT) expression occurs in the chromophobe RCC, in contrast to the genetic modification of KIT: KIT is a type III receptor tyrosine kinase that participates in cell signaling . When KIRAS, v-RAF murine sarcoma viral oncogene B1 (BRAF) , TP53 7, retinobGenetic clustering of ccRCCsSince the genetic background of RCCs is not fully understood, array-comparative genomic hybridization (CGH) is being analyzed and modified using a customized bacterial artificial chromosome (BAC) array (MCG Whole Genome Array-4500) . The RCCClinical implications of DNA methylation as a marker of RCC diseaseClinically, most cases of RCC are less obvious and are now diagnosed as a result of the unintentional use of abdominal computed tomography (CT), ultrasound (US), and magnetic resonance tomography (MRT) for other medical reasons . Early d Hypermethylation of CpG islands is common in a variety of cancers, including kidney cancer, and is frequently associated with tumor-suppressor gene mutations and signatory mechanisms . During The Genomic Atlas Cancer Analysis (TCGA) confirmed a few well-known aspects of RCC while also expanding our understanding of many other factors, such as survival biomarkers . The finEpigenetic changes as targets for cancer therapyEpigenetic methods as a new treatment have aroused much interest in recent research over the past few decades. Epigenetic mutations can initiate disease and may predict clinical outcomes . Recent There have been reports that NOTCH method components are activated in kidney cell cancer and that components such as DLL4 may have therapeutic efficacy in pre-clinical models . HoweverThe recent revolution in DNA methylation revolutionized the traditional view of gene function and its alteration as the primary cause of cancer and its metastases. Recent epigenetic advances have revealed that genome packaging is just as important as the genome in regulating the fundamental cellular processes that cause cancer. A better understanding of these epigenetic changes in cancer will lead to better therapeutic modalities, which will improve patient morbidity and mortality. The combined approach of epigenetic therapy in addition to standard chemotherapy promises successful cancer treatment. We hope these additional therapeutic approaches may also help cancer stem cells that are unresponsive to standard chemotherapy and are more likely to develop early metastases. Understanding cancer stem cells and developing specific epigenetic drugs are essential to effectively reconstructing the abnormal cancer epigenome. Epigenetic modification patterns linked to cancer development and progression have the potential to be clinically useful. The development of DNA methylation markers may be useful for early cancer detection, cancer diagnosis, and cancer prognosis prediction. Recent epigenomic advances allow for high-precision mapping of methylation/acetylation status and miRNA levels in the genome, which can aid in the identification of biomarkers for various diseases. Understanding the molecular events that initiate and maintain epigenetic gene silencing could lead to the development of clinical cancer prevention and treatment strategies.


In this work, we exploited the in vitro priming model to reveal novel candidates for adult nerve regeneration. We performed a whole-tissue proteomics analysis of the in vitro primed dorsal root ganglia (DRGs) from adult SD rats and compared their molecular profile with that of the in vivo primed, and control DRGs. The proteomics data generated are available via ProteomeXchange with identifier PXD031927. From the follow-up analysis, Bioinformatics interventions, and literature curation, we identified several molecules that were differentially expressed in the primed DRGs with a potential to modulate adult nerve regrowth. We then validated the growth promoting roles of mesencephalic astrocyte-derived neurotrophic factor (MANF), one of the hits we identified, in adult rat sensory neurons. Overall, in this study, we explored two growth priming paradigm and shortlisted several candidates, and validated MANF, as potential targets for adult nerve regeneration. We also demonstrate that the in vitro priming model is a valid tool for adult nerve regeneration studies.Injuries to peripheral nerves are frequent, yet no drug therapies are available for effective nerve repair. The slow growth rate of axons and inadequate access to growth factors challenge natural repair of nerves. A better understanding of the molecules that can promote the rate of axon growth may reveal therapeutic opportunities. Molecular profiling of injured neurons at early intervals of injury, when regeneration is at the maximum, has been the gold standard for exploring growth promoters. A complementary  However, no pharmacological therapies are currently available for effective nerve repair because of the lack of knowledge of potent molecular targets. In this work, we performed whole-tissue proteomics studies using two growth priming models and revealed novel molecular candidates, including mesencephalic astrocyte-derived neurotrophic factor (MANF), for peripheral nerve regeneration. This work has not only generated a large list of potential molecular candidates for future research but also characterized a novel Peripheral nerve injuries affect millions of people worldwide . Severe in vitro model, known as in vitro priming model, has been recently developed to study neuron outgrowth  in adult rats  DRGs. We found differential expression of several growth regulatory molecules in the in vitro and in vivo primed DRGs compared with control, revealing potential molecular targets for nerve regeneration and repair. Through this approach, we found a previously unrecognized growth promoting role for the mesencephalic astrocyte-derived neurotrophic factor (MANF) in the peripheral nervous system (PNS), along with the detection of its specific receptor, neuroplastin, in PNS tissues.Several preclinical studies demonstrated that growth factors such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial-cell derived neurotrophic factor (GDNF) promote axon regeneration . Howeverutgrowth . This neult rats . HoweverIn vitro and in vivo priming of L4–L6 DRGs were done as described previously  + N2 supplement  + 100 ng/ml NGF  + Cytiva Hyclone antibiotic/antimycotic solution ] at 37°C and 5% CO2 conditions for 24 h. In vivo priming was done by performing axotomy in adult male SD rats. Briefly, a small incision was made at the mid-thigh level and muscle layers were separated using a fine scissor to expose sciatic nerve (SN). A blunt transection was then performed onto the SN for complete axotomy. The muscle layers and the skin were then sutured back, and the animals allowed to survive for 3 d before the isolation of DRGs. All animal protocols were approved by the Institutional Animal Research Ethics Board Committee.eviously . For theTotal proteins from DRGs were isolated using RIPA lysis buffer  containing Halt protease and phosphatase inhibitor cocktail . Protein concentration was determined by Bradford assay. Proteins were denatured with trifluoroethanol  and digested in-solution following a previously published protocol . BrieflyRattus norvegicus; UniProt May 2020 release date) nonredundant database using Spectrum Mill (Agilent Technologies) as the protein database search engine. Search parameters included a fragment mass error of 50 ppm, a parent mass error of 20 ppm, trypsin cleavage specificity, and carbamidomethyl as a fixed modification of cysteine. Carbamylated lysine, acetyl lysine, oxidized methionine, pyroglutamic acid, deamidated asparagine and phosphorylated serine, threonine, and tyrosine were used as variable modifications in various stages during the database search. In addition, semi-trypsin nonspecific C terminus and semi-trypsin nonspecific N terminus were also used during the search to enhance protein identification. Validation of the Spectrum Mill search was performed at peptide and protein levels . Three samples each representing the control, in vitro primed and in vivo primed DRGs were used for the analysis. Differential expression of proteins in the in vitro and in vivo primed DRGs was determined relative to control based on a fold change (FC) of ≥2 in spectral intensities. Mass Profiler Professional  software was used for the statistical analysis (one-way ANOVA). A cutoff value of p < 0.05 and the Benjamini and Hochberg FDR set at <1% were used to obtain statistically significant results. Each of the significant proteins has more than two peptides identified. The mass spectrometry proteomics data are deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031927 (Tandem mass spectra were searched against the SwissProt rat (XD031927 .in vivo and in vitro samples. The STRING database and the Gene Ontology (GO) terms were used to retrieve the biological processes for all differentially expressed proteins (in vivo and in vitro samples compared with control), due their small number, we performed AmiGO2 gene ontology search for retrieving the biological functions  was supplemented to the culture for 48 h. The cultures were then fixed using 4% paraformaldehyde and stained with NF200  followed by incubation with the secondary antibody . The neurite outgrowth images were captured using the inverted fluorescence microscope (Zeiss Axio Observer) and the outgrowth parameters were measured using the WIS-Neuromath software .in silico digestion of MANF was performed. Database search of the acquired spectra, for the identification of MANF, was done as described above.We performed LC-MS/MS analysis in four individual sensory neuron cultures grown for 14 h to determine whether MANF is released by adult sensory neurons in culture. At first, total protein from culture supernatants was isolated using acetone precipitation protocol. Briefly, one part of the culture supernatant was added to 10 parts of acetone (1:10 ratio) and the mixture stored at −80°C overnight. The resulting supernatant was removed the next day, and 1:10 acetone was again added to the pellet and incubated at −80°C for another 1 h. The final supernatant was then removed and the pellet air dried. The sample pellet was dissolved in PBS before in-solution digestion, which was performed as described for the whole-tissue proteomics studies. Targeted MS analysis on tryptic peptides that resulted from n = 3) for each group, indicating reproducibility in the results  in the in vivo and in vitro primed DRGs are provided. Corrected p values and fold changes are also given. Potential functional roles of the proteins are also provided from the UniProt knowledgebase. Download Figure 1-1, XLS file.List of total proteins analyzed, and differentially expressed proteins  observed combinedly in the two models, 77 (∼29%) proteins showed a similar change (up or down) between the models, indicating that in vitro priming partially reproduces the molecular features that occur in in vivo primed DRGs.Fold change analysis revealed that 63 and 64 proteins were upregulated in the e models . Similarin vitro and in vivo primed DRGs , neural cell adhesion molecule 1 (Ncam1), and macrophage migration inhibitory factor (Mif) were previously shown to have growth regulatory roles in the PNS, while drebrin (Dbn1) and contactin-1 (Cntn1) were known to regulate CNS neurons , α-soluble NSF attachment protein (napa) and plasma membrane calcium-transporting ATPase 3 (Atp2b3) suggest that these proteins may not be critical for the communication of injury signals or transport of growth-related molecules across injured nerve tips and neuronal soma during regenerative reprogramming of adult neurons. Similarly, common downregulation of the actin modulators such as filamin-C and brefeldin A-inhibited guanine nucleotide-exchange protein 1 (Arfgef1) suggests that similar modes of cytoskeletal changes occur in neurons, and may be satellite glial cells, in the in vitro and in vivo primed DRGs. Although we found downregulation of a few growth promoters in the two models, additional molecular changes unique to these individual models may compensate the loss of these proteins for promoting outgrowth in the primed neurons. The unique protein changes observed in the two priming models are presented below.We found that 64 common proteins were downregulated in the two primed DRG models compared with control. The list of commonly downregulated proteins and their molecular functions identified from the UniProt knowledgebase are given in  neurons . Considein vivo primed DRGs with 50 and 54 proteins upregulated and downregulated, respectively, compared with control. The list of these proteins and their potential functions identified from the UniProt knowledgebase are given in Extended Data Fn1), signal transducer and activator of transcription 3 (Stat3) and E3 ubiquitin-protein ligase NEDD4, (Nedd4) are well known for their roles in PNS regeneration indicating the validity of our proteomics analysis (Map3k8), in this model suggests its potential role in promoting neurite extension. Similarly, the upregulation of the transport proteins such as dynamin-3 (Dnm3), twinfilin-1 (Twf1), cytoplasmic dynein 1 light intermediate chain 2 (Dync1li2), sodium/potassium-transporting ATPase subunit α−4 (Atp1a4), tubulin α−8 chain (Tuba8), and kinesin heavy chain isoform 5A (Kif5a) indicates their likely roles in communicating injury signals, and transporting growth promoting molecules across the injured nerve tips and neuronal soma, during the regenerative reprogramming of adult PNS neurons. Overall, our proteomics data substantiated that in vivo priming promotes key events such as cell metabolism, intracellular transport and trophic signaling cascades to facilitate neuron outgrowth and revealed additional molecular candidates for future exploration in adult nerve regeneration and repair models.Expression of 104 proteins was uniquely altered in analysis . The uprin vivo primed DRGs (Extended Data Tmod2), actin-related protein 2/3 complex subunit 2 (Arpc2), destrin (Dstn), and profilin-2 (Pfn2). The Tubulin polymerization-promoting protein family member 3 (Tppp3), which regulates tubulin dynamics, was also downregulated. We believe that this is because of in vivo priming favoring major cytoskeletal reorganization, a key prerequisite for neurite elongation. We found that both actin polymerizing (Arpc2) and depolymerizing (Dstn) proteins were downregulated indicating a balanced scenario. We also noted that the neurite outgrowth promoters, such as, caveolin-1 (Cav1) and elongation factor 2 (Eef2) were also downregulated after in vivo priming  and coronin-1B (Coro1b) were upregulated. Similarly, the transport proteins sodium/potassium-transporting ATPase subunit β−1 (Atp1b1), solute carrier family 2 facilitated glucose transporter member 1 (Slc2a1), and importin subunit β−1 (Kpnb1) were upregulated. Among the transcriptional regulators, prohibitin-2 (Phb2) and activated RNA polymerase II transcriptional coactivator p15 (Sub1) were upregulated. Transcriptional activation and actin-dependent cytoskeletal modifications are critical events for peripheral nerve regeneration. Similarly, molecular transport between Golgi and endoplasmic reticulum, and nuclear and cytoplasmic compartments is also critical for efficient nerve regrowth. We also found that the axon growth promoter integrin β−1 (Itgb1) and the dendritic spine maintenance protein cortactin-binding protein 2 (Cttnbp2) were upregulated in the DRGs after in vitro priming , protein RUFY3 (Rufy3) and neural cell adhesion molecule L1 (L1cam) were downregulated in the in vitro primed DRGs  in adult primary sensory neuron cultures and found that it promotes neurite outgrowth, especially in NF200high neurons, significantly , indicating that no significant release of MANF occurs in the culture conditions we employed.in vivo primed) DRGs and found that it is expressed in both NF200low and NF200high sensory neurons (high neurons and satellite glial cells have low intrinsic expression of MANF, while NF200low neurons express it at higher levels even after injury (low neurons may bind to neuroplastin receptors on NF200high neurons and glial cells and activate them for nerve regeneration. These possibilities warrant additional investigations of MANF-neuroplastin axis in the PNS.The knowledge about the specific receptor for MANF was unknown for a long time. However, a recent study by Yagi et al., demonstrated that MANF binds to neuroplastin receptor for mediating its anti-inflammatory actions . The exp neurons A. Import neurons B. We earr injury A. Collecin vitro and in vivo primed DRGs, we were able to shortlist molecular candidates that are likely modulators of adult axon growth. We believe that this approach is more robust in identifying potential candidates for adult nerve regeneration since the candidates were derived from two independent growth-priming models.Despite active research in the past several decades, no drug therapy is available for the effective repair of injured peripheral nerves. While nerve reconstruction surgeries may be helpful for short distance regeneration, a combined approach of surgery and drug therapy is highly desired for long-distance regeneration and better functional recovery . Here, blow sensory neurons, with very weak to no detectable endogenous levels in basal and injury activated glia. We also demonstrated the presence its receptor Neuroplastin, especially in NF200high neurons and injury activated glia, suggesting that exogenous MANF may modify cellular signaling in these cells by binding to neuroplastin. Substantiating this argument, we demonstrated that exogenous MANF promotes the outgrowth parameters of NF200high sensory neurons in culture. Efficient regeneration of large diameter NF200high axons is critical for peripheral nerve repair because of them being a major population that is heavily myelinated. Therefore, the growth promoting effect of MANF in this population is promising to consider it as a potential nerve repair agent. MANF has been previously shown to regulate glial cell signaling (in vivo cannot be ruled out, especially since its receptor is expressed in injury activated glia. Therefore, further validation of MANF in more rigorous peripheral nerve injury models in vivo is warranted. Overall, our proteomics exploration of primed DRGs identified previously unrecognized critical roles of MANF in the PNS.The priming of DRGs preconditions adult neurons for regeneration, and hence the commonly upregulated proteins in our two models are likely promoters of axon growth. However, the opposite may also be true, such that the upregulated proteins may be growth inhibitory. For example, preconditioning of injured neurons also induces expression of growth inhibitors . Hence, ignaling . Studiesignaling . Althougin vitro and in vivo priming. While their downregulation in the in vitro primed DRGs was not surprising, may be because of the lack of longer nerve connections to neuronal soma in this model requiring no major retrograde transport, such a downregulation in the in vivo DRGs was not expected as retrograde transport is critical for axotomy-driven nerve preconditioning (in vivo priming indicates that these proteins may be critically required for the long-distance retrograde transport in vivo. We also found the upregulation of the transport protein importin subunit α−5 in the in vivo primed DRGs although our focused analysis considering selected biological processes did not capture this protein. The common downregulation of the neurite outgrowth facilitators such as contactin-1, CDK5, NCAM-1, and peroxisome modulators in the primed DRGs were unexpected, but indicate the presence of upstream growth decelerating signals in these DRGs. Supporting this argument, several previous studies demonstrated that growth inhibitory signals are active past neuronal injury and during the early regenerative reprogramming of adult neurons (We found common downregulation of several transport proteins in the DRGs after itioning . However neurons .Overall, we identified 263 molecular candidates based on their differential expression in two growth priming models and tabulated their individual functions from the UniProt knowledgebase for further investigation. We also shortlisted most promising candidates among them based on their functional enrichments at the nerve growth-related biological processes. In addition, we identified a novel growth promoting role for MANF in the PNS along with the detection of its receptor neuroplastin in PNS tissues. This is a promising start and follow-up systematic screening of the shortlisted candidates in nerve regeneration models is warranted to explore the full potential of these candidates in improving adult nerve regeneration.


The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like cells is obtained. We monitored transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. Unexpectedly, we find maintenance of undecided Nanog/Oct4-PouV/Klf4-positive pluripotent-like pan-ectodermal stem-cells spanning the entire ectoderm late in the neurulation process with ectodermal patterning completed only at the end of neurulation when pluripotency becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency is found at all axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond “ectodermal-capacity” in chick and mouse embryos. How the NC establishes this exceptionally high pluripotent-like stem cell state remains poorly understood.Pluripotent epiblast stem cells, with the potential to become every cell type in the body, become restricted to germ layer-specific fates during gastrulation. Morphogen gradients polarize the body axes leading to proper tissue patterning. Medial-lateral patterning of the ectoderm germ layer results in formation of the medial future central nervous system (CNS) domain flanked by the lateral non-neural ectoderm (NNE) domains that become the skin . The neu2. A more recent study used Wnt1Cre and Oct4 reporter mouse lines to conclude that rather than maintaining pluripotency, the high level of NC stemness is enabled by the unique ability of the NC to re-activate the pluripotency program during the neurulation process at late neural fold stages well after gastrulation is completed3, a hypothesis also supported by pseudo-time analysis in a scRNA sequencing study of whole frog embryos4, and ectopic expression experiments of Ventx2 and Oct25 (frog homologues of Nanog and Oct4) in the ectoderm outside the NC domain5. We recently used the previous version of the method we developed, single cell Multiplex Spatial Transcriptomics, scMST /Oct4 and Klf4 transcription factors7, which are core components of a gene module that drives pluripotency in embryonic stem cells10. We defined this domain as a transient NC stem cell niche7. Recent studies have since confirmed the expression of pluripotency genes in the NC also in mouse and frog embryos5. However, defining the time at which the NC acquire stemness is critical for determining whether it is maintained or reactivated. Understanding how NC forms is highly clinically relevant : ~30% of all birth defects comprise of neurocristopathies, and the demand for using NC cells for regerative purposes is increasing. Furthermore, understanding of whether climbing upward the Waddigton’s epigenetic landscape11 truly is part of embryogenis will provide important clarity to the comprehension of pluripotency regulation in normal as well as malignant tissue growth.Two contradicting hypotheses have been recently proposed to explain this intriguing conundrum. One model suggests the exceptionally high stemness of the NC is retained from blastula stage  mainly based on continuos expression of key NC genes that promote pluripotencys, scMST , to inveTo this end, we investigated the temporal changes in stemness genes to gain insights into how the developing ectoderm can acquire a domain with high, pluripotency-like features so late in development. Multiple high-resolution approaches, including scMST, single cell, and bulk RNA sequencing  were combined to monitor gene expression across a broad series of developmental stages for a comprehensive analysis at the midbrain level , as thisNanog, PouV/Oct4, Klf4 together with Lin28, and 2) neural stem cells (light blue) that have a predominantly CNS profile, but also co-expressed pluripotency genes and a low level of NC genes. We speculate that this unique gene expression profile may provide plasticity to the DNT as it goes through major reconstruction to form the roof plate after the NC has emigrated from the neural epithelium12. The NC stem cells also co-express neural, glial, pigment and mesenchymal genes suggesting that at this premigratory stage they remain unspecified regarding their future NC lineage. Importantly, the DNT also consists of committed cells, including NC cells that do not have a stem cell profile and only express NC genes  as well as pluripotency factors, were chosen for our analysis . Due to To understand how NC stemness arises between gastrulation and neurulation, we analyzed the ectoderm at three different developmental stages that encompass neural fold elevation . In sum, these results suggest that the expression of pluripotency genes is not abruptly lost after gastrulation, but rather is maintained in the ectoderm until late neurulation stages. Importantly, the spatial distribution of the subgroups was consistent in all analyzed parallel embryos as shown by mapping back the pseudo-colored cells into the respective original images . Our findings thereby suggest that the loss of stemness proceeds in concordance with a much slower sepration of the ectodermal domains than previously thought, consistent with recent scRNAseq and imaging reports of a gradual ectodermal patterning process that is completed only near the end of neurulation stage17.Next, we analysed embryos at the early neural fold stage (1 somite). Intriguingly, we detected evidence for even broader stemness across the entire ectoderm: while some committed cells were found at their anticipated locations in the future epidermis and CNS, the majority of the cells met the criteria of a stem cell as evaluated by co-expression of the pluripotency genes –f”. Simi19. Second, consistent with a known role in neural induction20, ectodermal Sox2 expression clustered with neural progenitor markers and therefore was considered a neural stem cell rather than a pluripotency marker. Thus, we focused on Nanog, PouV /Oct4, and Klf4 co-expression to monitor pluripotency due to their known roles in epiblast and embryonic stem cell pluripotency9. We reasoned that if stemness were to be maintained from blastula stage onwards, continuous co-expression of the pluripotency genes would be readily detected. We therefore selected cells with scMST z-scores above the mean for all three pluripotency genes simultaneously. The results show that at the end of gastrulation (HH5), the ectoderm is comprised of numerous cells expressing a pluripotent signature and that these cells are maintained broadly throughout the entire ectoderm until the neural fold stage. Spatial restriction of these stem cells to the dorsal neural tube was only detected at the end of neurulation , these data do not support the idea that pluripotency is reactivated. As a validation of the sample dissection, the expression of the early NPB marker Pax7 started to rise soon after gastrulation, and the expression of the NC specifier gene Sox10 became prominent at the late neural fold stage, as expected . The linear plots revealed a slight overall decrease in transcript numbers of the pluripotency genes during the neurulation process, which raised the question of whether the transcript levels are biologically relevant and translated into protein. Immunofluorescence confirmed that Nanog was readily detectable in the NC domain at the end of neurulation . Together, scMST and bulk RNAseq analyses show that pluripotency is not lost in the ectoderm upon gastrulation but is maintained in the entire ectodermal germ layer as it is patterned into its functional domains. The high stemness, as evaluated by co-expression of core pluripotency genes, is eventually restricted to the dorsal neural tube  at the end of neurulation.These results prompted us to hypothesize that the pluripotency-like NC stemness is maintained in the ectoderm past the gastrulation stage by the continued co-expression of pluripotency genes. To test this, we used bulk-RNAseq to analyze dissected cranial NPB/NC regions from embryos collected only õne hour apart from each other  from gastrula to end of neurulation stage . As expefindings . In contNanog, PouV/Oct4 and Klf4 were continuously expressed in the ectodermal cells during neurulation  and analyzed the difference in gene expression in the NPB domains at neural fold stage (HH7/HH8−) by bulk RNA sequencing . The sam1. To test this, we performed multichannel fluorescent in situ hybridization (FISH) on older embryos  to address whether the pluripotency genes are expressed in the NC at all axial levels including the trunk, which does not give rise to mesodermal-like derivatives. Indeed, we detected continuous co-expression of pluripotency genes throughout anterior-to-posterior ectoderm  mouse embryos show co-expression of Nanog, PouV/Oct4 and Klf4 throughout the entire ectoderm  a requirement for maintaining cellular plasticity during the gradual patterning of the ectoderm, 2) the mechanism by which the NC domain acquires its exceptionally high stemness enabling it to form derivatives beyond “ectodermal capacity”, and 3) the mechanism by which the dorsal neural tube maintains plasticity as it transitions from NC to roof plate and ultimately the radial glia  in the ependymala stage  and thus5. However, stem cell potential is not necessarily equivalent to the final selection of the cell types the stem cell ultimately produces. In other words, the full potential may not always be utilized. Although the NC does not contribute to ecto-mesenchymal derivatives in the trunk region of the commonly used animal models including chick, frog, mouse and zebrafish models 29, reports from turtle embryos show trunk NC-derived bone in the plastron30, and transplant studies of axolotl trunk NC have shown their contribution to the branchial arch cartilage31. These reports indicate that the high stem cell potential may be an ancestral feature of the NC, initially maintained at all axial levels but lost secondarily in different vertebrate embryo models, according to species-specific needs . Furthermore, our findings do not rule out the possibility of additional, individual respective roles for the pluripotency genes later in NC development, such as supporting ecto-mesenchymal lineages at emigrating and migratory stage, as recently reported5. Thus, pluripotency defined by co-expression of core pluripotency genes is not to be confused with their individual expression, such as Oct4 detected both in the maturing neural ectoderm and mesoderm19.Importantly, we detect expression of pluripotency genes at all axial levels suggesting that they are broadly required for NC formation and ectoderm patterning. Furthermore, knockdown of the respective pluripotency genes affected overlapping gene regulatory circuitries involved in proliferation and stemness associated functions. These findings do not align with recent reports that link pluripotency-like features only to the anterior part of the embryo because of the unique ability of the NC to form the craniofacial skeleton in the vertebrate head 34. We show a comcomitant gradual loss of the pluripotency signature and increase in fate commitment during the ectodermal patterning process, suggesting possible involvement of similar epigenetic changes. On the other hand, neural crest cells do posses an exceptionally high pluripotent-like stem cell potential as judged by their ability to form derivatives beyond the ectodermal capacity. Our multiple lines of investigation combined argue for the original role of the pluripotency genes in enhancing stemness throughout the ectodermal patterning and neural crest specification process, allowing speculation of a dual role for the pluripotency factors in the ectoderm in both maintaining stemness and assisting gradual fate commitment. Future studies are also needed to investigate whether the neural crest cells truly are pluripotent or something in between pluripotency and multipotency (Pleistopotent1).Recent discoveries propose a role of pluripotency genes in participating the transition fom pluripotency to committed lineages by co-binding to lineage factors that redirect their binding to new enhancer sites, leading to regulatory interference from other factors inhibiting them to activate downstream targets, and eventually a gradual repression of the pluropotency gene transcription10. Perhaps this morphological difference has enabled the entire ectoderm with the unique feature of maintaining pluripotency-like characteristics after gastrulation to ensure a faithful ectodermal patterning process, leading to formation of the NC in vertebrates.Finally, our unexpected finding of sustained, broadly distributed ectodermal co-expression of pluripotency genes long after gastrulation has ceased challenges the dogmatic viewpoints of gastrulation in which pluripotency is lost during the formation of the three germ layers. It is worth noting that the formation of ectoderm is very different from endoderm and mesoderm, which both ingress from the pluripotent epiblast (primitive ectoderm) and lose pluripotency in the migration process, while less is known about the detailed molecular signatures that induce the commitment of definitive ectoderm to form from the remaining epiblast layer, which continues to be maintained as an epithelial sheetFertilized chicken eggs were obtained from the University of Connecticut (UCON) poultry farm  and Lassilan Tila , incubated at 37°C to reach the desired stage according to Hamburger and Hamilton (HH). See Wild-type C57BL/6J mice were obtained from the Jackson Laboratory. For timed matings, noon on the day a copulation plug was detected was considered to be 0.5 days postcoitus. Embryos were collected in a sex-unbiased manner. All mice were maintained under specific pathogen-free conditions at the NIH animal care facility. Mice were cared for, and all experiments were approved by the Administrative Panel on Laboratory Care, and the Institutional Animal Care and Use Committee(IACUC) of NIH. Mice were maintained on a normal chow diet.7, which was originally designed to analyze a single field of view at a single developmental time point. To govern analysis of multiple fields of views per sample and at multiple developmental stages, scripts that enable comparison of gene expression across all samples were added. Detailed instructions and codes are available in Github (https://github.com/KerosuoLab/Pajanoja_2023).The scMST pipeline was modified from our previously published technique Spatial Genomic Analysis 35) via a 4 nt long linker sequence resulting in a 60 nt-long probe. Expression of five genes was captured on each round using a spinning disc microscope with six channels. At the end of each round, signal was removed by using DNase I, followed by another round of hybridization by using probes to five new genes applied on the same sample. All hybridization and imaging steps were done in hybridization chambers  that were mounted on the coverslips. As the last step of the imaging rounds, cell borders were captured by using immunolabelling, which is critical for the machine learning algorithm-based 3D cell segmentation and quantitative dot counting of individual RNA transcripts within each single cell to work.In brief, multiplexing is based on serial single molecule fluorescent in situ hybridization (smFISH) rounds on cryosections. For each gene of interest, up to 24 short DNA probes (minimum 13 probes) that target the coding sequence of the same mRNA were used. 3’ end of each probe was linked with a HCR initiator sequence  was replaced with K-means clustering, an unsupervised machine learning algorithm that clusters dots based on their signal intensity, providing a more robust approach . Third, the Matlab code was further optimized for samples with multiple fields of views. Samples were z-scored across all three fields of views, therefore providing a more accurate and consisting level of “high” or “low” expression of a given gene within each cross section. Stage by stage analyses  was used to determine cells with overlapping pluripotency gene expression  nitric acid for 25 min, followed by 200nM NaOH for 15 min to make glass surface more conductive for silane binding. Silanization of the slides was performed with 1% (w/v) Triethoxysilylbutraldehyde (TESBA) made in ethyl alcohol for 5 min at room temperature. The slides were rinsed with 100% ethanol twice and once with distilled water, and heat-dried in an oven at 64 °C for 4h or overnight. Next, the slides were treated with (0.1 mg/ml) Poly-l-lysine (Sigma) in PBS for 20 min at room temperature, followed by three rinses with PBS. The coverslips were then air-dried and kept at 4 °C in an airtight storage for no longer than 4 weeks.The following changes were made to the sample preparation as compared to our previously published protocol 7 and 20 μm thick transverse cryosections were cut from the midbrain level onto silane coated coverslips.For scMST, samples were collected on Whatman filter papers, fixed in 4% PFA overnight, and washed 3 times with PBS-0.2% triton, dehydrated in ethanol and stored in −80°C. The embryos we incubated through a sucrose gradient  and stored at −80 °C. Replicates consisted of pooled tissue from at least five to seven embryos. Four replicates were collected per stage. For HH5 and HH6 replicates, rectangular patches lateral to the primitive streak were collected according to midbrain neural crest fate mapping as described 13. For premigratory stages beyond HH6, neural plate borders or neural fold apices corresponding to the midbrain neural crest domain were excised. Beyond 7ss, dorsal neural tubes containing neural crest from the midline to migratory front were collected.For bulk RNAseq experiments, embryos were collected on Whatman filter papers in PBS and midbrain level neural plate border and specified neural crest cells, respectively, from left and right sides were manually microdissected using tungsten needles, immediately placed into RNA Lysis Buffer  from the desired stage by using micro scissors, which were pooled from three to four embryos per sample and dissociated with a multi tissue dissociation kit  at 37°C for 20 min with intermittent pipetting to achieve a single cell suspension.A cocktail of E-cahderin (BD Transduction 610181), B-catenin (Abcam ab6391) and N-cadherin  primary antibodies were used to label the cell membranes. Secondary antibodies were AlexaFluor647 for E-cahderin and AlexaFluor488 for B-catenin and N-cadherin.Imaging was performed using an Andor Dragonfly 200 spinning disk confocal system coupled to a Zeiss AxioObserver . Coverslips were placed onto sealed coverglass rectangular chamber  in order to minimize errors for aligning images in each imaging round. A 63X apochromat water immersion objective (NA 1.27) was used for imaging cross sections. An Andor integrated laser engine provided the excitation light using 405 nm (100 mW), 488 nm (150 mW), 561 nm (100 mW), 594 nm (100 mW), 640 nm (140 mW), and 730 nm (30 mW) laser lines, and using suitable emission wavelengths for each fluorophore , respectively. A Photometrics Prime 95B CMOS  camera was used in 12-bit mode with 3X gain (13X 13 μm pixels). Exposure times and relative laser intensity was varied based on sample/fluorophore brightness (~ 500 ms and ~40% for each fluorophore). A Z piezo stage  allowed rapid imaging in Z. Images were collected every 0.5 μm over a 25 μm distance. All components were controlled by Micro-manager version 1.4.22 using the Andor driver and was programed by ADD. DAPI staining was imaged during the first round in addition to the five sets of probes (405 nm). For the final round of immunostaining, Alexa647 (640 nm) for E-cadherin with 500 ms with 20% laser power, and Alexa488 (473 nm) for B-catenin and N cadherin cocktail was used with 400 ms 20% laser power. Each cross section was captured using 3 fields of frames . Embryos were then dissected to isolate the dorsal neural folds, which were immediately placed into lysis buffer. RNA was extracted from the tissue using the RNAqueous-4PCR Total RNA Isolation Kit (AM1914) according to kit directions. Extracted RNA was then ethanol precipitated and resuspended in nuclease free water before storing at −80°C.Translation blocking morpholinos were designed to target the 5’ UTR in close proximity of the ATG for the respective genes: Nanog (CATGGTCGGGACGACACCTCCAG), PouV (CCGAGAGCTGCCTCCATGCTA), and Klf4 (CCGTCGTCCCGCCGAGGAGAGT). A control morpholino was designed to account for non-specific effects from electroporation (CCTCTTACCTCAGTTACAATTTATA). Morpholinos were conjugated on the 3’ end with carboxyfluorescein. Morpholinos were diluted to a 1.0 mM concentration and electroporated together with an empty pCIG vector as carrier DNA (1 μg/μL). Injections were two-sided, targeting the ectoderm in HH stage 4 chicken embryos, with control morpholino on the contralateral side. Chicken embryos were collected on Whatman filter papers and electroporated using 5.3V and 5 pulses (50mA/100mA), as previously described 40. Samples were pooled as shown in Figure S3d, and for 42 resource tool was used to find Gene Onthology (GO) terms using up-regulated genes (LFC > 0.75). Fold enrichments were obtained using statistical overrepresentation test, and p-values were calculated using Fisher’s Exact test and adjusted using the Bejamini-Hochberg false discovery rate (FDR) method for multiple test correction. For linear plots in Bulk RNA-seq was performed by the NIDCD/NIDCR Genomics and Computational Biology Core (GCBC). Libraries were created using the Nextera XT method. Samples collected from 12 different developmental stages were run on an Illumina NextSeq 2000 configured for 55 PE reads. The counts were generated by mapping via STAR aligner (v2.5.2a). Reads are 55×55bp in length, and samples contain between 43.7and 87.5 million uniquely mapped reads to gene regions. Counts were filtered to remove all genes which did not have more than 5 reads in at least 1 of the samples. Differential expression analysis was carried out using DESeq2 43 function as follows: Remove Unwanted Variation with replicate Samples (RUVs) was used to remove the batch effects by using factor analysis on the control samples. Additionally, one thousand in-silico control genes, identified as those with the lowest coefficient of variance among all samples, were passed to the function. Transformation of the data using RUVs revealed a PCA1/2 plot showing clear gene expression signatures for each KD group and control group. After normalization, differential gene expression (DGE) analysis was performed using DeSeq2, where each KD group was compared to all control samples. This revealed many genes, which showed a statistically significant change (p-adj <0.05) in expression levels between the KD groups and control, most of which were unique to only one KD group. Up and downregulated genes for each morpholino list were used for the overrepresentation test via PANTHER  44. Fold enrichments were obtained using statistical overrepresentation test, and p-values were calculated using Fisher’s Exact test and adjusted using the Bejamini-Hochberg false discovery rate (FDR) method for multiple test correction.Gene counts were produced by the NIDCD/NIDCR Genomics and Computational Biology Core using the STAR aligner in standard mode. The reads were mapped against a current ENCODE chicken release and the mapping parameters were derived from the GENCODE project. Counts were filtered to remove all genes that did not have more than 5 reads in at least 1 sample. Then counts were normalized by library size and analyzed with principal component analysis (PCA). A PCA1/PCA2 graph revealed an apparent batch effect preventing meaningful interpretation of the read counts, which was removed by using the RUVseq (remove unwanted variation) Single cell sequencing was performed at FIMM Single-Cell Analytics unit supported by HiLIFE and Biocenter Finland. Single cell gene expression profiles were studied using 10x Genomics Chromium Single Cell 3’RNAseq platform. The Chromium Single Cell 3’RNAseq run and library preparations were done using the Chromium Next GEM Single Cell 3’ Gene Expression version 3.1 Dual Index chemistry. The Sample libraries were sequenced on Illumina NovaSeq 6000 system using read lengths: 28bp (Read 1), 10bp (i7 Index), 10bp (i5 Index) and 90bp (Read 2). Fastq files were generated by using the 10x Cell Ranger (v7.0.1) count pipeline of Cell Ranger as well as by performing alignment, filtering and UMI counting. Finally, Fastq files were mapped to chicken genome using a custom GRCg6a Gallus gallus genome annotation file that was constructed by extending selected 3’UTRs in order to account for incorrect annotations.45 in R. SoupX 46 and DoubletFinder 47 packages were used to filter out ambient RNA and doublets, respectively. Cleaned samples were merged and named based on their original file names. Following the standard Seurat workflow, artifact cells were further excluded by removing any cells that expressed more than 4000 genes and high mitochondrial content (>0.4%). Gene expression matrices were normalized and scaled using the NormalizeData and ScaleData functions. During the scaling, Cell Cycle Difference scores, which was calculated by difference of S from G2/M-phase specific gene scores via ‘CellCycleScoring’ function, were regressed out. Then, principal component analysis linear dimension reduction was conducted using using the ‘ElbowPlot’ function . Clustered cells were visualized via UMAP plot and manually annotated based on differentially expressed genes in each cluster . The clusters that were identified as “Ectoderm” were extracted and re-clustered using the ‘subset’ function and given colors based on their individual stages. Bar plots were used to demonstrate expression levels of selected genes. Each bar represents cell counts that express the gene of interest individually, and the values were normalized by total cell counts in each developmental stage or germ layer, respectively. Module scores were calculated based on individual list of genes we have used, using the AddModuleScore function in Seurat. For in depth analysis, cells in the ectoderm UMAP were labeled based on module score as “Pan-ectoderm” (Pan-ecto score >0) vs “Others” (Pan-ecto score <0). Differentially expressed genes between Pan-ectoderm vs Others were calculated via stage by stage comparison . Ratios of unspliced vs spliced RNA were calculated via scVelo dynamical modelling pipeline 22 in Python. For each sample, Loom files containing spliced and unspliced RNA expression matrices were created using the Velocyto.py command line tool. Ectoderm subset of HH5, 1 somite, 4 somite and 7 somite stages were selected for the analysis.Data cleaning, normalization and scaling was performed as follows: downstream analysis was carried out using Seurat package (v3.0.1) ™ Rapid Antibody Isotyping Kit, Thermofisher).Custom Nanog and FoxD3 Monoclonal antibodies  were commercially generated against full-length recombinant chicken proteins. Forty parental lines were received and tested by immunoblot and immunohistochemistry using chicken lysates and fixed embryos . Select parental lines were clonally expanded and monoclonal hybridoma isotypes were confirmed (Piercewww.molecularinstruments.com) for chicken Nanog (NM_001146142.1), PouV (NM_001309372.1), Klf4 (XM_004949369.3), Pax7 (NM_205357.1), and Sox10 (NM_204792.1). In situ HCR v3.0 with split-initiator probes was done according to whole-mount chicken protocol 48 with hybridization overnight at 37°C and amplification overnight at room temperature. Prior to hybridization, later stage embryos (HH12-HH14) were permeabilized with 10 μg/mL proteinase K for 2.5 minutes. Following amplification, embryos were washed with 5xSSCT, stained with DAPI, and post-fixed with 4% PFA for 15 minutes at room temperature. Embryos were mounted using ProLong Gold Antifade Mountant and imaged using an Andor Dragonfly spinning disk confocal microscope. For sections, embryos were embedded in gelatin and sectioned at 20 μm.Chicken (HH6- HH15) and mouse embryos (E7.75 – E8.5) were fixed in 4% paraformaldehyde, washed with PBS/0.2% Tween (PBST), dehydrated in methanol, and stored at −20 °C. All experiments involving animals were approved by the NIDCR Institutional Animal Care and Use Committee. HCR split-initiator probe sets were purchased from Molecular Instruments (


Hypertension is one of the leading causes of premature death in humans and exhibits a complex aetiology including environmental and genetic factors. Mutations within the glucocorticoid receptor (GR) can cause glucocorticoid resistance, which is characterized by several clinical features like hypercortisolism, hypokalaemia, adrenal hyperplasia and hypertension. Altered glucocorticoid receptor signalling further affects sodium and potassium homeostasis as well as blood pressure regulation and cell proliferation and differentiation that influence organ development and function. In salt-sensitive hypertension, excessive renal salt transport and sympathetic nervous system stimulation may occur simultaneously, and, thus, both the mineralocorticoid receptor (MR) and the GR-signalling may be implicated or even act interdependently. This review focuses on identified GR mutations in human primary generalized glucocorticoid resistance (PGGR) patients and their related clinical phenotype with specific emphasis on adrenal gland hyperplasia and hypertension. We compare these findings to mouse and rat mutants harbouring genetically engineered mutations to further dissect the cause and/or the consequence of clinical features which are common or different. Hypertension is a multifactorial disease and affects one-third of the human adult population . It has Glucocorticoids are ligands of the glucocorticoid receptor (GR), which is one of the most studied transcriptional regulatory factors (TRFs) involved in homeostasis and physiological regulation . GlucocoIn human, there are still questions that need to be addressed, (i) if and which kind of GR mutations are causative for the development of GR-mediated hypertension, and (ii) which are the main predisposing factors for this disease. Moreover, it was not clear until recently if some of these clinical features might be recapitulated in animal models to help dissect the underlying molecular mechanism. The aim of this review is to give an overview of GR pathological mutations found in humans and on studies of GR mutations done in experimental animals. We will discuss the prevalence of hypertension and adrenal hyperplasia in patients and animals carrying GR mutations and discuss possible mechanisms.NR3C1) gene, located on the short arm of chromosome 5 (5q31Y32) [The primary structure of the human glucocorticoid receptor is known since 1985 , which i5q31Y32) , encodes5q31Y32) . The two5q31Y32) . In 19905q31Y32)  demonstr5q31Y32) .The 5′ part of the sequence encodes the N-terminus domain  of the receptor Fig. . AF1 is NR3C1 gene mutations have been described in human  and zona glomerulosa mainly under the influence of ACTH and angiotensin II. At the age of 6 years, the zona reticularis will appear. Thus, GR may play a direct or indirect role in the adrenarche process. It could explain the adrenal gland hyperplasia diagnosed in a patient harbouring the E198X mutation only at the age of 12 and not at 3 years when the mutation has been found [Nine of these 14 GR mutations presented an elevated plasmatic level of the adrenocorticotropic hormone (ACTH)  is the first and most common cause of secondary hypertension  characteoduction , 77. Patoduction .It is important to note that PA might also be associated with hypercortisolism or subclinical Cushing’s syndrome. Cortisol-producing tumours were associated with aberrant cAMP-protein kinase A signalling. Furthermore, germline mutations in the tumour suppressor gene armadillo repeat containing 5 (ARMC5) were the underlying cause for primary bilateral macronodular adrenal hyperplasia (PBMAH) (see for review ). IndeedIn summary, due to the heterogeneity of the data collected, we cannot conclude that GR mutations are causative for PA. There is certainly additional need for evaluation of the renin-aldosterone ratio and the plasma renin activity (PRA). To further understand the underlying mechanisms of glucocorticoid resistance, adrenal gland hyperplasia and hypertension, animal models are useful to further dissect the development of the symptoms under normal and challenging conditions. In the following section, we will discuss the findings in transgenic rodents exhibiting GR mutations Table .Table 2O+/−) [+/− mice were lacking the GR-dependent regulation of the hypothalamus-pituitary axis (HPA) established during foetal development [geo reporter gene  concentrations and slightly hyperplastic adrenal glands  Table 22. Higher+/−) . Further+/−) . Therebyelopment . In modeelopment , 55. MicRβgeo/+, ) displayRβgeo/+, . A high  [+/em2, ) when ke [+/em2, . During  [+/em2, . While,  [+/em2, . FurtherTie−Cre) knockout mice were analyzed [To better understand the role of the vasculature in the generation and maintenance of glucocorticoid-mediated hypertension, vascular smooth muscle-specific  exhibited mildly increased baseline BP, but similar hypertensive response to dexamethasone [Persistent increased circulating glucocorticoids were proposed to contribute to nocturnal hypertension and induced a non-dipping blood pressure profile in mice with global reduction of GR abundance . BP furtsorption . Furthersorption . To furtPax8/LC1 . Overallethasone . Interesethasone . Salt loethasone .Glucocorticoid signalling regulates a wide range of systems in vertebrate organisms, and their responsiveness to glucocorticoids differs largely between individuals. Apart from the commonly used rodent models, the zebrafish has been proposed as an excellent in vivo model to study glucocorticoid resistance . As a noNR3C1 mutations exhibit adrenal hyperplasia, this clinical feature may not be a prerequisite to develop hypertension. Nevertheless, not all patients with adrenal hyperplasia were analysed for GR mutations. Environmental factors as varying daily salt intake might play an additional important role here. In the contrary, there is a strong correlation between NR3C1 gene mutations, salt-sensitive hypertension and high salt intake. Increased adrenocorticotropic steroids are known to promote renal sodium reabsorption and implicate both GR and mineralocorticoid receptor (MR) pathways that contribute to the pathogenesis of hypertension. We thus cannot exclude compensatory mechanisms and/or glucocorticoid-mediated mineralocorticoid activation. To dissect the underlying mechanisms, animal models are required that can be challenged and, hence, the development of hypertension can be followed over time. The use of inhibitors as sEH enzyme inhibitors in models with acute kidney injury (AKI), diabetic nephrology (DN), chronic kidney diseases (CKD), hypertension and other renal dysfunctions may present a potential therapy although this needs further experimental studies. More complete and standardized phenotyping of human subjects as well as adapted animal models will further help to dissect the underlying molecular mechanisms of adrenal gland hyperplasia and salt-sensitive hypertension.Glucocorticoid signalling stimulates several pathways concurrently and converges on common mechanisms. Although a relatively high number of human subjects suffering from glucocorticoid resistance and carrying