Crh) acting at cognate type 1 receptors  modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1–) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1– neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1– neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1– neurons, which may have important implications for the transduction of CRF signaling in the CeA.The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF,  Our novel identification of specific molecular, morphologic and functional properties to distinguishing CRF receptor type 1 (CRFCrh) plays an important role in emotional regulation via hypothalamic and amygdalar circuits under normal physiologic conditions (1) antagonists on mood disorders have been unsuccessful  expressing neurons are located in several brain regions including the central nucleus of the amygdala (CeA), which functions as the main output nucleus for amygdala functions  subset. Thus, we used a bacterial artificial chromosome (BAC) transgenic mouse line expressing the green fluorescent protein (GFP) under the Crhr1 promoter (CRF1:GFP) to readily identify neurons expressing CRF1  neurons display no ongoing tonic inhibition  co-expression patterns of CRF1 (via GFP reporter) with CBPs parvalbumin (PV), calretinin (CR), and calbindin (CB); (2) co-expression patterns of Crhr1 with Penk, Pdyn, Sst, Npy, Crh, and Prkcd; (3) dendritic spine morphology and density; and (4) basal and CRF-modulated glutamatergic transmission. We found that Penk and Sst have the highest percentage of co-expression with Crhr1 in both the CeM and CeL, and that Penk is enriched in CeM Crhr1+ neurons compared to their Crhr1– neighbors. We also show that CeM CRF1+ neurons exhibit an overall lower spine density and differential fractions of mature versus immature spines compared to CRF1– neurons. Consistent with a comparable density of mature spines in these two populations, we found no difference in spontaneous EPSCs (sEPSCs) or miniature EPSCs (mEPSCs). Importantly, acute CRF application increased overall CeM glutamatergic transmission and does not affect amplitude or kinetics of EPSCs in either population. These data provide important information about the neurobiology of CeA CRF1+ neurons that may have critical implications for their functional role under physiological and pathological conditions.Given the critical role of CRF and CRF1:GFP mice were generated on a mixed C57BL/6J x BALB/c background using BAC recombination  were anesthetized (isoflurane) and perfused with cold PBS followed by 4% paraformaldehyde (PFA). Brains were dissected and immersion fixed in PFA for 24 h at 4°C, cryoprotected in sterile 30% sucrose in PBS for 24–48 h at 4°C or until brains sank, flash frozen in pre-chilled isopentane on dry ice, and stored at –80°C. Free floating 35-µm brain sections were obtained using a cryostat and kept at 4°C in PBS containing 0.01% sodium azide.Mice , mouse anti-PV , mouse anti-CR , and mouse anti-CB . Next, sections were triple washed in PBS for 10 min at RT with gentle agitation followed by a 1-h secondary antibody incubation in PBS (in the dark). The following secondary antibodies were used: Alexa Fluor 488 goat anti-chicken  and Alexa Fluor 568 goat anti-mouse . Sections were then washed  and mounted in Vectashield .Sections were washed in PBS for 10 min at room temperature (RT) with gentle agitation and then blocked for 1 h at RT in blocking solution . Primary antibody was incubated at 4Sections were imaged on a Zeiss LSM 780 laser scanning confocal microscope . All microscope settings were kept the same within experiments during image acquisition. Analyst was blind to the identity of the red fluorescent signal (CBP) when performing cell counts, and analysis was performed manually in an unbiased manner at four anterior-posterior levels (equidistant sections located –1.00 through –1.70 mm from bregma). Data are presented as mean ± SEM.n = 3–4) were anesthetized (isoflurane) and perfused with cold PBS/Z-fix . Brains were dissected and immersion fixed in Z-fix for 24 h at 4°C, cryoprotected in sterile 30% sucrose in PBS for 24–48 h at 4°C or until brains sank, flash frozen in pre-chilled isopentane on dry ice, and stored at –80°C. Brains were then sliced on a cryostat in 20-µm-thick sections, mounted on SuperFrost Plus slides  and stored at –80°C.Mice  in RNase-free conditions. To perform the RNAscope in situ hybridization, a target retrieval pretreatment protocol was performed as outlined in the manual . Briefly, slides were submersed in target retrieval buffer  at 95–98°C for 10 min, immediately washed in distilled water, dehydrated with 100% ethanol (storage at –80°C if required), and lastly digested with Protease IV for 20 min at 40°C. Following this pretreatment the RNAScope Fluorescent Multiplex Reagent kit User Manual  was followed and slides were mounted with DAPI-containing Vectashield . A negative control  was run in tandem for each experiment. The probes used from ACD Biotechne were as follows: Crhr1 , Gad2 (439371-C3), Slc17a7 (416631-C2), Penk (318761), Pdyn (318771), Sst (404631), Npy (313321), Prkcd (441791), Crh (316091), and eGFP (400281).Slides were imaged on a Zeiss LSM 780 laser scanning confocal microscope . All microscope settings were kept the same within experiments during image acquisition. To perform quantification, ImageJ  was usedCrhr1+ nuclei expressing a marker of interest was determined by dividing the number of co-labeled nuclei by the total number of Crhr1+ nuclei. The percentage of Crhr1– nuclei co-expressing a marker of interest was determined by dividing the number of cells expressing the marker of interest but not Crhr1 by the total number of Crhr1– nuclei per image. Crhr1+ compared to Crhr1– patterns of co-expression was assessed and normalized to the Crhr1– cells.Next, the percentage of nuclei positive for one or both probes and the percentage of signal co-expression were calculated. Percentage of total nuclei was determined by dividing the total number of nuclei expressing that marker by the total number of DAPI positive nuclei per image. The percentage of t test was used to assess significance of the CeL versus CeM expression difference for each gene, as well as the Crhr1+ to Crhr1– percentage co-expression of each gene. Analysis and statistics were performed using R programming  were anesthetized (isoflurane) and perfused with cold PBS/4% PFA. Brains were extracted and immersion fixed in 4% PFA at 4°C for 2 h before being sectioned coronally into 100-μm slices on a vibrating microtome .Mice . The slices were triple washed with PBS for 10 min followed by secondary antibody incubation for 1 h at RT in blocking solution . The slices were triple washed in PBS and mounted on slides with Prolong Diamond .Sections were biolistically labeled with DiI -coated 1.1-μm tungsten particles delivered with a Bio-Rad gene gun and incubated in PBS overnight at 4°C before immunostaining. Slices were permeabilized in 0.01% Triton X-100 for 15 min at RT with gentle agitation and blocked for 30 min at RT with gentle agitation in blocking solution (10% NGS in 0.01% Triton X-100). Primary antibody incubation was performed overnight at 4°C in PBS  and CRF1– neurons . The experimenter was blind to cell type (CRF1+ vs CRF1–) when performing spine quantification. Data obtained in each mouse were averaged for each cell type, such that the number of mice was used as the n in statistical analyses. Spine densities and spine type proportions in CRF1– and CRF1+ neurons were compared using paired t tests. Data are presented as mean ± standard error. In all cases, p < 0.05 was the criterion for statistical significance.Slides were imaged for CeA on a Zeiss LSM 710 laser scanning confocal microscope . All microscope settings were kept the same within experiments during image acquisition. ImageJ  was usedn = 12) were briefly anesthetized with 3–5% isoflurane and decapitated. Brains were rapidly removed and placed in an ice-cold high-sucrose solution (pH 7.3–7.4) that contained: 206.0 mM sucrose, 2.5 mM KCl, 2.5 mM CaCl2, 7 mM MgCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, 5 mM glucose, and 5 mM HEPES. Brains were cut into coronal sections (300 µm) using a Leica 1200s vibratome (Leica Microsystems) and placed in oxygenated (95% O2/5% CO2) artificial CSF (aCSF) solution composed of the following: 130 mM NaCl, 3.5 mM KCl, 2 mM CaCl2, 1.25 mM NaH2PO4, 24 mM NaHCO3, and 10 mM glucose. Slices were incubated in aCSF for 30 min at 37°C, followed by a minimum 30-min equilibration at RT (21–22°C) before use.Mice  optics and CCD camera . A 60× magnification water immersion objective (Olympus) was used for identifying and approaching neurons. To avoid photolytic damage, initial exposure to episcopic fluorescence illumination was brief (<2 s). We detected fluorescent neurons using the Lumen 300 LED system (Prior Scientific) and captured images using Luminera software (Lumenera Corp.). Whole-cell current-clamp and voltage-clamp recordings were obtained with patch pipettes  using a Multiclamp 700B amplifier (Molecular Devices), sampled at 20 kHz, low pass filtered at 10 kHz, digitized , and stored on a computer using pClamp 10 software (Molecular Devices). Series resistance was not compensated but monitored throughout the experiment, and cells with a series resistance >15 MΩ or a >20% change were excluded.2, 10 mM HEPES, 2 mM Na-ATP, and 0.2 mM Na-GTP. Drugs were dissolved in aCSF and applied by bath perfusion. To isolate sEPSC and mEPSC, recordings were performed in the presence of GABAA (30 µM bicuculline) and GABAB (1 µM CGP 55845A) receptor antagonists, and tetrodotoxin  was included for mEPSCs. CeA neurons were held at –60 mV for voltage-clamp recordings and maintained at –70 mV for current-clamp recordings.The intracellular solution used for voltage and current clamp recordings was composed of 145 mM potassium gluconate, 0.5 mM EGTA, 2 mM MgClWe purchased CGP 55845A (1 µM) and CRF (200 nM) from Tocris Bioscience, bicuculline methiodide (30 µM) and TTX (0.5 µM) from Sigma.t test or one-sample t test using Prism 6 (GraphPad Prism). In all cases, p < 0.05 was the criterion for statistical significance, and n represents the number of cells or mice as indicated.Frequency, amplitude, rise, and decay time of EPSCs were analyzed and visually confirmed using a semi-automated threshold based mini detection software . Average EPSC characteristics were determined during baseline and experimental drug conditions in 3 min bins containing a minimum of 60 events. All detected events were used for event frequency analysis, but superimposed events were eliminated for amplitude and kinetic analysis. Data are presented as mean ± standard error, and statistical significance was assessed using a two-tailed 1+ neurons  and vesicular glutamate transporter 1 (Slc17a7) were used as GABAergic and glutamatergic neuronal markers, respectively. In situ hybridization in both the CeL , Prkcd , Pdyn , and Sst ] except Penk, which was expressed at equivalent levels in CeM and CeL (–0.07-fold), and Npy , whose mRNA density was higher in CeM. Additionally, we observed that Crhr1 density was higher in the CeM compared to the CeL  neurons was lower than in CRF1– (16 spines/10 μm) neurons  = –3.3, p < 0.05; 1+  and CRF1– neurons  = 4.9, p < 0.05) while the proportion of thin spines was significantly lower (t(3) = –4.9, p < 0.05) in CRF1+ neurons compared to CRF1– neurons. There were no differences in the proportion of stubby spines (t(3) = 0.4, n.s.) and filopodia (t(3) = –1.0, n.s.) between the two cell types.Higher levels of GFP and  the CeL –4, as prthe CeL –. We therstaining A,B. Spin1+ and CRF1– CeM neurons. Before recording glutamatergic activity, whole-cell current clamp recordings with a step protocol consisting of hyperpolarizing to depolarizing current injections were obtained from each cell to determine cell type based on spiking characteristics. As previously described, CeA neurons are composed of three principal cell types: regular spiking, low threshold bursting, and late spiking neurons  = 1.3, n.s.) and amplitude (t(26) = 1.23, n.s.) in CRF1+  and CRF1– neurons  = 0.75, n.s.) and amplitude (t(26) = 0.70, n.s.) in CRF1+  and CRF1– neurons  in CRF neurons C–H. We o1+ and CRF1– populations to CRF, we tested the effect of CRF  on mEPSCs (1+ (t(10) = 2.78, p < 0.05) and CRF1– (t(8) = 2.68, p < 0.05) CeM neurons (1+: t(10) = 0.50, n.s.; CRF1–: t(8) = 1.12, n.s.; 1+: t(10) = 1.28, n.s.; CRF1–: t(8) = 0.95, n.s.; t(10) = 1.32, n.s.; CRF1–: t(8) = 0.85, n.s.; To assess differences in the functional responsivity of CRFn mEPSCs I–N. We f neurons J but did1+ neurons in the CeA of adult male mice. We found that CB is the predominant CBP expressed in the CeA but overall there is no specific enrichment or exclusion of CBPs in Crhr1+ neurons. Co-expression analysis using in situ hybridization revealed Crhr1 is co-expressed mostly with Penk and Sst and least with Npy. In the CeM, Penk is significantly enriched in CRF1+ neurons compared to CRF1– neurons. Morphologically, CRF1+ neurons have a lower spine density but a higher proportion of mushroom spines compared to CRF1– neurons. Accordingly, basal excitatory transmission between CRF1+ and CRF1– neurons are similar. Additionally, acute CRF application increases glutamatergic transmission in both CRF1+ and CRF1– neurons. The lack of enrichment or exclusion of any particular CBP or neuropeptide in this population contributes to the difficulty in identifying and studying this subpopulation in this heterogenous region.Here, we investigated the neurochemistry, morphology, and physiology of CRF1–expressing neurons project out to the dorsolateral BNST  are associated with increased synaptic strength, whereas immature spines (thin/filopodia) may sustain limited synaptic signaling  spines, resulting in a comparable density of functional spines in CRF1+ and CRF1– neurons.We characterized the dendritic morphology of CeA CRFignaling . Abnormaignaling . Previouignaling . Most neignaling . Here, w1 activity  and drives appropriate behavioral outputs. Previous work has demonstrated a major role of the CRF system, and particularly the CRF1 receptors, within the amygdala complex to influence cellular functions that produce maladaptive behavior in animal models. The amygdalar CRF1 system represents a common pathway for the convergence of stress, addiction, pain, depression, memory formation and anxiety-related signaling  under pathologic conditions, such as alcohol dependence and anxiety disorders. The multiplicity of neuronal subpopulations and the complexity of local microcircuits provide numerous targets for potential dysregulation by drugs of abuse  and stress, and their potential clinical relevance.Our new evidence on the CRF


Meanwhile, the FP showed lower α-amylase and higher α-glucosidase inhibitory activities, but the RP showed similar levels of α-amylase and α-glucosidase inhibitory activities. Taken together, these results suggested that fermented pepper extract using water should be expected to have potentially inhibitory effects against both hyperglycemia and hypertension.This study was investigated to evaluate the antioxidant activity, the angiotensin I-converting enzyme (ACE) inhibition effect, and the α-amylase and α-glucosidase inhibition activities of hot pepper water extracts both before and after their fermentation. The fermented pepper water extract (FP) showed significantly higher total phenol content, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition effect, metal chelating activity and ACE inhibition activity compared to the non-fermented raw pepper water extract (RP) ( Hypertension is a condition of oxidative stress, therefore a decline in the antioxidant system can be fatal in hypertension patients, which can cause atherosclerosis and other hypertension-induced organ injuries . SeveralMeanwhile, angiotensin I is activated by renin secreted from the kidney; and angiotensin I-converting enzyme (ACE) changes angiotensin I to angiotensin II . Angioteα-amylase and α-glucosidase are related to the hydrolysis of dietary carbohydrates and the absorbance of glucose from the small intestine to the blood, respectively ,14. ContBacillus licheniformis showed significantly decreased capsaicin content, and pungency was decreased also, and this microorganism can utilize capsaicin as an energy source [Pepper is currently being widely used for cooking in both traditionally and contemporary cuisine, and it contains a variety of phytochemicals such as capsaicin and ascorbate . Capsaicy source ,23,24.This study was performed to evaluate the antioxidant activity and the key enzyme inhibition effects related with hypertension and diabetes mellitus using fermented pepper, for the purpose of developing new functional food ingredients.The pepper was obtained from a local market . All reagents were obtained from Sigma-Aldrich .Bacillus licheniformis SK1230. It was then incubated for 11 days at 37 °C in a shaking incubator . After fermentation, the fermented pepper was freeze dried. For the raw pepper, the same amount of sterilized (inactive) Luria–Bertani (LB) broth  was added (to substitute for the presence of microorganism), then freeze dried. Powdered raw pepper and fermented pepper were stored at −20 °C before use and were used as samples in this experiment. According to previously reported capsaicin analysis method [p < 0.05).The pepper was blended and freeze dried. To prepare fermented pepper, 40 g of powdered pepper was mixed with 1 L of distilled water. After sterilization, the sample was inoculated with 5% of s method , the amo2B4O7 : 0.2 M H3BO3 = 135 : 165, pH 8.3) was stirred at 4 °C for 24 h in dark room. Then, it was centrifuged at 12,000 rpm for 30 min. The supernatant was used as the ACE solution [To prepare the ACE solution, 1 g of commercial rabbit lung acetone powder  in 10 mL buffer  reagent was prepared by mixing 1 g of DNS with 50 mL of distilled water. Then, 30 g of sodium potassium tartrate tetrahydrate was slowly added. 20 mL of 2 N NaOH was also added to the mixture, and distilled water was added until the mixture totaled 100 mL .g for 10 min, the supernatant was filtered using Whatman filter paper No. 2, and it was used for experimentation. The extract was prepared by means of a modified method published by Kwon et al. . Of the 2CO3 30 μL was added. After 1 h, absorbance was measured at 725 nm. Gallic acid was used to establish a standard curve, and results were expressed as mg of gallic acid equivalents per gram of dried sample weight.The total content of the phenol compounds was determined according to literature . Of the DPPH  radical scavenging activity by Blois  were perAnother antioxidant activity of extract was performed by metal chelating activity . Of the 2B4O7 : 0.2 M H3BO3 = 135 : 165, 0.4 M NaCl, pH 8.3) and an ACE solution of 80 μL was reacted for 30 min in a 37 °C water bath. Then, 200 μL 1 N HCl was added to stop the reaction. Ethylacetate (1.2 mL) was added, and the mixture was vortexed for 5 min. After centrifugation at 3000 rpm for 15 min, the supernatant of 0.8 mL was allowed to stand at 90 °C for removal of ethylacetate. After that, the residue was mixed with 2.4 mL of distilled water, and the absorbance was recorded at 228 nm . The blank contained HCl from the beginning to prevent the reaction of substrate and enzyme. Distilled water, instead of extract, was used for the control. The ACE inhibition activity was calculated as below:The ACE inhibition effect was performed by Chushman et al.’s method . An extrThe α-amylase inhibition effect was evaluated by a modification of the assay described by the Worthington Enzyme Manual . Of the The α-glucosidase inhibitory activity was measured by the modified method of Ranilla et al. . Of the t-test using SPSS 18.0 . The experiments were performed in triplicates. Significant difference was evaluated by p < 0.05.All results were stated as mean ± standard error of mean (SEM) and, analyzed by p < 0.05).Total phenol contents in extracts from raw and fermented peppers varied from 150 to 250 μg/mL , and it p < 0.05). Meanwhile, metal chelating activity was significantly higher than DPPH radical inhibition activity in both RP and FP (p < 0.05).Antioxidant activities were determined by both DPPH radical inhibition assay and metal chelating assay . The FP p < 0.05).The inhibition rate of angiotensin I-converting enzyme is exhibited in p < 0.05). Meanwhile, α-amylase and α-glucosidase inhibition activity in RP were similar each other (p > 0.05), but in FP, low α-amylase and high α-glucosidase inhibition activity were shown significantly (p < 0.05).α-amylase and α-glucosidase inhibition activity were seen in the RP and FP . Fermentp < 0.05). Generally, phenolics exist as conjugated forms together with sugars or other moieties. Glucosidase is related to the hydrolysis of glycosidic linkages, and it leads to the release of free phenolics [p < 0.05). This means that the FP had higher glucosidase activity than the RP. Therefore, it suggested that it resulted in the release of free phenolics with increasing glucosidase activity during fermentation of the pepper.Consistent with this study, Sim and Han  reportedhenolics . In thisp < 0.05). There were several ways to protect oxidation. Elimination of over produced free radicals which can react with biomolecules and cause chronic diseases [Meanwhile, antioxidant activity generally correlates with the amount of phenolic compounds. Vattem et al.  reporteddiseases  and heavp < 0.05). A relationship with higher ACE inhibition activity than before fermentation has been suggested. Higher ACE inhibition means lower conversion of angiotensin I to angiotensin II, a potent vasoconstrictor, and it seems to be a strategic treatment to protect against hypertension—which is a representative complication of diabetes [There have been several reports about ACE inhibition activities by fermented milk peptides , flavanodiabetes .p < 0.05). Martin and Montgomery [Ranilla et al.  recentlyntgomery  referencIn this study, we fermented pepper for 11 days with the intention of “capsaicin decrease” not “no capsaicin” in pepper for adjustable pungency. If the effect of fermented pepper is higher than that of raw hot pepper, it is thought to be resulted from the degradation of the ingredient of capsaicin or other fermentation by-product even if capsaicin remains in the fermented pepper. Indeed, the chromatogram of fermented pepper showed a greater area in some peaks compared to that of raw hot pepper (blue square in p < 0.05). Furthermore, lower α-amylase and higher α-glucosidase inhibition effects in FP could have inhibitory potential in diabetes. Therefore, it is suggested that water extract of fermented pepper might be appropriate for a functional prevention ingredient of hypertension and hyperglycemia as a natural resource.Fermented pepper extract using water was evaluated about antioxidant activity, ACE and enzymes relevant for hyperglycemia inhibition effects. Results showed significantly higher total phenol contents, DPPH radical inhibition activity, metal chelating activity, and ACE inhibition effect than those of RP (


From the early-2000s through to ~2012, we observe a slowdown in ice motion in response to increased surface melt, consistent with previous research. From 2013 to 2019 however, we observe an acceleration in ice motion coincident with atmospheric cooling and a ~15% reduction in mean surface melt production relative to 2003–2012. We find that ice velocity speed-up is greater in marginal areas, and is strongly correlated with ice thickness. We hypothesise that under thinner ice, increases in basal water pressure offset a larger proportion of the ice overburden pressure, leading to reduced effective pressure and thus greater acceleration when compared to thicker ice further inland. Our findings indicate that hydro-dynamic coupling provides the major control on changes in ice motion across the ablation zone of land terminating margins of the Greenland Ice Sheet over multi-annual timescales.The subglacial hydrological system critically controls ice motion at the margins of the Greenland Ice Sheet. However, over multi-annual timescales, the net impact of hydro-dynamic coupling on ice motion remains poorly understood. Here, we present annual ice velocities from 1992–2019 across a ~10,600 km Approximately 52% of this mass loss can be attributed to surface melt6, which increased in the late 2000s and early 2010s to levels unprecedented since at least 19007. Increases in surface melt have been driven by increasing air temperatures over Greenland since the mid-1980s8 and variability in cloud cover, both of which are forced by larger scale circulation patterns13. Increased cloud-cover warms the ice sheet interior through the trapping of longwave radiation15, whereas a reduction in summer cloud cover since 1995 has driven enhanced melt in the ablation zone through increasing the shortwave flux16. Moreover, the seasonal migration of the snowline causes the exposure of dark bare-ice, decreasing the albedo of the ice surface and reducing meltwater re-freezing, further driving surface melt and runoff17. Alongside changes in surface mass balance, roughly 48% of mass loss is due to increases in ice discharge through Greenland’s marine terminating outlet glaciers6. However, the dynamic response of the ice sheet to variability in surface mass balance and ocean conditions remains a large source of uncertainty in projecting future sea level rise18.The Greenland Ice Sheet (GrIS) has lost mass at an accelerating rate over the past two decades, with persistent mass loss observed since 199820. This is particularly prescient as the largely land-terminating margin in South West Greenland exhibits a strong and sustained negative mass balance22, and is projected to make a greater contribution to sea level rise with continued atmospheric warming and associated increases in surface runoff23.Land-terminating margins are isolated from processes acting at the ice/ocean boundary, and thus provide ideal study sites for investigating how the ice-sheet responds to atmospheric, and thus surface melt forcing27 where their impact on friction at the ice-bed interface is fundamentally important in controlling ice velocity29. Whilst initial research postulated that as these seasonal meltwaters drain to the base of the ice sheet, they would pressurise the basal hydrological system, reduce friction at the bed and so enhance glacier sliding30, other studies have argued that variability in meltwater input, rather than the volume itself, is more critical for driving ice acceleration32.Each summer, surface meltwaters drain from the ice-sheet surface to the bed via moulins and crevasses34. These inputs force a morphological switch from an inefficient, distributed subglacial drainage system to an efficient, channelised system when and where subglacial discharge is sufficiently turbulent to open subglacial channels36. This introduces a negative feedback whereby as the drainage system capacity increases in response to enhanced meltwater input, basal water pressures decrease as subglacial channels allow the efficient evacuation of subglacial water37, forcing a deceleration of the ice later in the melt season38.Both short-term and sustained increases in surface meltwater delivery to the glacier bed drive an increase in basal water pressure above the ice overburden pressure, reducing friction at the bed and so forcing a transient acceleration40. Multi-annual ice velocity slowdowns in southwest Greenland since the early-mid 2000s have been reported by numerous studies based on both GPS data43 and large-scale satellite-derived observations44. Ice velocities from GPS stations along a transect in Southwest Greenland, extending ~150 km inland from the margin between surface elevations of 340 m and 1850 m above sea level (a.s.l), show a 10% average slowdown from 1991–200742, coincident with increasing surface melt, and GPS data at North Lake show a slowdown of −0.9 ± 1.1 m yr−2 from 2006–201443. Over a much larger 8000 km2 region of Southwest Greenland, Tedstone et al.40 showed that ice velocity had decreased by 12% in 2007–14 compared to 1985–94, despite a 50% increase in surface meltwater production, with ice velocity decreasing by 1.5 m yr2 between 2002 and 2014. This long-term slowdown is attributed to the expansion of subglacial channels, both up-glacier and in their dimensions, as a result of the long-term increase in surface melt, enhancing the drainage of waters from the more extensive distributed component of the subglacial drainage system, thus reducing regional basal water pressures and so ice velocities40. The distributed component of the subglacial drainage system encompasses varying degrees of connectivity, and it is hypothesised that the reduction of basal water pressures in the weakly-connected areas of the drainage system specifically39 are critical to the observed slow-down as their recharge is slow (on the order of years39), resulting in widespread and extended depressurisation and so increased basal traction.More recently, research has focused on whether this hydro-dynamical coupling of ice flow at land-terminating margins results in a long-term trend in ice-motion in response to long-term increases in surface melt3, a period of relative stability in mass balance45 has been observed across Greenland, with 2017 having the lowest maximum surface melt extent since 199646. This stability is coincident with positive Arctic and North Atlantic Oscillations, promoting cyclonic conditions thereby reducing incoming solar radiation and enhancing precipitation46. Given that the proposed hydro-dynamic mechanism for a long-term velocity slowdown requires a continual increase in surface melt40, it would be expected that ice motion would respond to a sustained change in surface run-off and begin to stabilise or accelerate as surface melt decreases and the distributed subglacial hydrological system re-pressurises. Under reduced surface melt forcing, we would expect the up-glacier extent of efficient subglacial channels to decrease, allowing regions of the distributed drainage system that were, in previous years, drained by efficient channels to re-pressurise through the gradual recharge of meltwater via basal melting. Numerous GPS data show that this process occurs on a seasonal timescale, whereby following the deceleration of ice motion to a minimum in the late melt season, measured ice velocities show a gradual increase over the following winter48; this process has not however been observed to-date on a multi-annual timescale. This study therefore extends the West Greenland ice velocity time series, both spatially and temporally, in order to investigate how ice motion has responded to recent reductions in surface melt forcing, with the ultimate aim of improving our understanding of the mechanisms driving ice sheet motion.Since the record surface melt in 20122 in SW Greenland, extending ~300 km along the margin and ~50 km inland to an elevation of 1300 m. Our study region is considerably larger than that of Tedstone et al.41, extending a further ~120 km to the south. We apply feature tracking to 2665 pairs of Landsat scenes, separated by 352–384 days, over 14 path/row combinations. Subsequently, we derive robust ice velocity and uncertainty estimates50 for periods of 1, 2 and 4 years to construct a time series from 1992 to 2019, and assess the spatial distribution of velocity change between 1992–2003 and 2003–2012, and 2010–2012 and 2017–2019. Finally, we assess the implications of our results for future land-terminating ice-sheet motion in a warming climate in light of the dynamic response of the study region to the recent variability in meltwater forcing.Here we present observations of ice velocity from 1992–2019 across a predominantly land-terminating area of ~10,600 km2) of the region exhibiting reduced velocity in the latter period, and a mean regional slowdown of ~15.3%  of this second change map exhibiting increased velocity during the latter period  due to the hypsometry of our study region , an order of magnitude greater than previous work40 . The quality of the fit is further improved via break point analysis (see SI) which recognises three statistically distinct periods based on the pattern of velocity changes . A substantial slowdown of −1.64 ± 0.37 m yr−2 (p < 0.05), starting around 2003 (but which could range from 2002–2004 due to a gap in our data), occurs until 2012, after which the most recent period of ice motion exhibits a significant increasing trend .Over the time period from 1992 to 2019, the ice velocity anomaly time series reveals a mean slowdown of −0.72 ± 0.08 m yr.85 Fig. ). Betwee9 to calculate the mean surface melt production anomaly during the three distinct periods identified in our ice velocity anomaly time series; 1992–2003, 2003–2012, and 2012–2019  (~2.15 m yr−1 to ~2.77 m yr−1 (w.e.)) between 1992–2003 and 2003–2012. Following this period of sustained higher surface melt production, a ~15% decrease to 0.06 m yr−1 (~2.39 m yr−1) is observed for 2012–2019, when compared to the 2003–2012 mean. The period of ice velocity decrease therefore coincides with a period of enhanced ice surface melt, while rates of constant and accelerating ice velocity occur during periods of lower surface melt.We use the regional climate model MAR v3.10019 Fig. . Mean su40. More recent work however suggests that this may be a statistical construct43, whereby as more antecedent melt years are included in the average value, R2 tends further towards 1 , indicating that there is no significant relationship between annual ice velocity and annual runoff, consistent with earlier work48.Previous work has argued that there is a significant relationship between antecedent surface melt production and ice velocity2, and from 1992–1998, the South West Greenland land-terminating sector thinned by ~0.02–0.4 m yr−1 52. A period of stability characterised the late 1990s and early to mid-2000s52, followed by rapid thinning (~ 1–1.5 m yr−1). Whilst some datasets assume a constant thinning of a similar magnitude through to 201453, recent work points to a maximum thinning rate in 2012, after which thinning has continued at a more moderate rate54, albeit with differences in thinning trends between drainage basins55. Prior modelling work in our study region40 indicates that changes in driving stress due to thinning could only explain 17–33% of the slowdown signal beyond 10 km from the ice-margin, and none of the signal beyond 50 km. This modelling work applied 20 m of thinning over 30 years, equating to a thinning rate of −0.66 m yr−1. Outside of the period 2007–2011, there is no evidence for thinning rates greater than this during our study period56, and we thus do not believe that thinning rates could be responsible for more slowdown than already reported in Tedstone et al.40. Moreover, continued thinning from 2012 would be expected to continue to reduce ice motion, which contrasts with the acceleration in ice velocity observed during this period. As such, these findings imply that most of the ice velocity signal is controlled by processes operating at the ice-bed interface.Changes in ice thickness and surface gradient can contribute to changes in ice motion through their associated impacts on driving stress. Thinning at the margins of the Greenland Ice Sheet has been observed since the early 1990s57. Our results provide support for a previously postulated mechanism40, whereby under a sustained multi-annual increase in surface meltwater production, the subglacial drainage system is characterised by both a gradual increase in the extent of channelisation, as well as the time during which these channels remain open and are thus able to evacuate water from the surrounding distributed drainage system58. These conditions promote the drainage of waters from weakly-connected39 regions of the distributed drainage system, thereby reducing basal water pressure and associated basal lubrication and hence ice velocity over longer  timescales as effective pressures increase. Whilst it is hypothesised that increasing runoff to the bed will increase sediment pore water pressure, resulting in reduced sediment shear strength, increased sediment deformation and thus enhanced ice flow59, such behaviour contrasts with the observations reported here and elsewhere44. Consequently, our results suggest either that extensive layers of subglacial till are not ubiquitous across our study region or if they are present, the tills have not deformed pervasively in response to the sustained period of enhanced meltwater input.The subglacial hydrological system exerts a critical control on ice motion at land-terminating margins of the Greenland Ice Sheet46. Concurrent with decreasing meltwater runoff, we observe an acceleration in ice velocity. During the period of sustained high melt, the drainage of basal waters from a larger area of the distributed drainage system causes a gradual depressurisation of the background water pressure with an associated increase in effective pressure. Reduced surface meltwater production would result in seasonally less extensive and smaller subglacial channels and consequently these channels will undergo faster creep closure32. This results in a reduced time-period over which the main subglacial channels can evacuate water from the surrounding weakly connected, hydraulically distributed regions. As water pressure is no longer being systematically reduced, the background water pressure can increase through a gradual re-pressurisation of the subglacial hydrological system, as meltwater can recharge from basal melting without being evacuated by efficient subglacial channels.Surface melt production in our study region peaks in 2012, decreasing thereafter , consistent with trends observed across the ice-sheetN) is calculated as N = Pi − Pw, where Pi refers to the ice overburden pressure and Pw to the basal water pressure60. Repressurising the subglacial hydrological system causes basal water pressure (Pw) to increase, thus reducing effective pressure and causing ice to accelerate. As effective pressure is a function of ice thickness, for the same increase in basal water pressure we would anticipate acceleration to be greater where ice is thinner. Although we expect channel closure to occur earlier and faster under thicker ice (due to higher ice overburden pressure)50, modelling studies suggest that once channel pressure falls to 90% of overburden pressure, there is less than 2 days difference in closure time for channels with a cross-sectional area of 10 m2 compared to <1 m2 50. Moreover, it has been suggested that maximum channel closure rates are similar at both marginal and interior sites49. Consequently, we argue that any variation in repressurisation due to channel closure rates will only have a small impact upon effective pressures when compared to variability in ice thickness (which ranges from 0–1400 m across our study area). As a result, we hypothesise that the observed acceleration is greater for marginal regions of thinner ice due to the impact of basal meltwater recharge on water pressure in these areas offsetting a larger proportion of the ice overburden pressure  due to increasing noise in our dataset further inland. However, GPS observations reveal reduced velocities to at least 80 km inland (at ~1500 m a.s.l) in the years of record surface melt in 201048 and 201237 when compared to 2009.The inland limit and spatial extent of efficient channel formation is subject to considerable debate44, differences between studies exist regarding the magnitude of slowdown, and the proposed mechanism(s) driving this dynamic change. For example, whilst GPS data show a slowdown of −0.9 ± 1.1 m yr−2 at North Lake from 2006–201443, satellite radar data from 2000/01 to 2016/17 reveal no significant trend 44. At a regional scale, this radar data suggests a weaker slowdown trend than that reported either here, or by Tedstone et al.40 , across the same study area, with little in the way of significant changes between 2001/01 and 2016/17 across much of the southwest margin44. Methodological differences between the studies may affect the conclusions drawn as this study and that of Tedstone et al.40 applied feature tracking to image pairs either side of an annual baseline , whereas Joughin et al.44 composed mosaics of winter velocities, comprised of data sampled between September and May. Regardless, this raises the possibility that the previously observed slowdown40 may be related to some local process, rather than regional-scale surface melt forcing44, for example water piracy as a result of dynamic thinning on Jakobshavn Isbrae has been suggested as a driver of slowdown in the north of the West Greenland land-terminating sector44. However, by extending the study area ~120 km to the south, and by increasing the number of common pixels used in our time series by an order of magnitude compared to that of Tedstone et al.40, we extend confidence in the observed slowdown of ice motion from the early-mid 2000s. Moreover, by calculating an ice velocity anomaly72, we remove the influence of any biases between sensors from the observed trends, further increasing confidence that this sector of the ice sheet underwent significant deceleration during this period.Whilst multi-annual ice velocity slowdowns in southwest Greenland since the early-mid 2000s are observed here and in a number of other studies73. However, whilst current models can reproduce observed dynamics of Greenland’s land-terminating margins over days/weeks50, on a seasonal timescale74, and across several years75, there is a need to consider the longer, decadal and multi-decadal response of ice motion to surface meltwater forcing in order to better reproduce observed dynamics and project future change. On a decadal scale, recent modelling predicts increased ice-motion and thus ice flux into the ablation zone under enhanced summer melt within 44 km of the ice-margin76 . These predictions contrast with the results presented both here and from other GPS and satellite-derived observations44. Further work is therefore required to investigate what aspects of the model set-up or framework cause the modelled ice-motion to accelerate on a multi-annual timescale in response to increasing surface runoff, in contrast to numerous observations reported in this and other studies. Whilst we do observe reduced slowdown as ice thickness increases, our results and those of others show that ice motion within the ablation area has slowed as a result of increased surface runoff, and we do not observe any appreciable or spatially extensive increases in ice velocity as a result of an extended period of high surface melt. We therefore argue that in order to better represent ice dynamical processes and project future changes in coupled hydro-dynamic models, the models must better utilise the available data from field and satellite observations both in model set-up and for assessing model performance, and there is a need for changes in the numeric representation of processes, including ice-hydrology coupling.A variety of models exist to simulate the subglacial drainage system and its impact on ice dynamics in response to increasing meltwater fluxes in a warming climate72. Further, we see no evidence of speed-up at the elevations studied under a warming climate, irrespective of the bed conditions. Future work is required to improve observations of change at higher elevations, and at other land-terminating sectors of the ice sheet, in order to assess the extent to which these processes operate on an ice sheet scale.Our findings demonstrate that ice at surface elevations below 1300 m (a.s.l.) in the South West land-terminating region of the GrIS underwent deceleration during the mid to late-2000s and early-2010s with subsequent acceleration following sustained reductions in surface melt production, with this acceleration proportionally greater in areas of thinner ice. We hypothesise that this recent acceleration is driven by increases in basal water pressure offsetting a larger proportion of ice overburden pressure, and thus causing a greater reduction in effective pressure under thinner ice. This behaviour has not been observed previously on a multi-annual timescale. Whilst we have demonstrated that changes affecting the subglacial hydrological system are the most likely driver of ice velocity change in our study region, we are unable to conclusively state whether antecedent melt production is the predominant factor controlling ice motion, or whether some threshold in the amount of melt is required in order to drive a change in velocity. However, it is clear from our results that the period of ice deceleration occurred during a period of sustained high melt production, and ice motion accelerated subsequently as melt production decreased to consistently lower values from 2013 onwards. Moreover, we observed no indication of an ongoing slowdown once surface melt production exhibited a sustained decrease. Thus, the behaviour observed supports a process-based understanding of the links between hydrology and ice dynamics in this land-terminating sector of the Greenland ice sheet. The results displayed in this study show that the observed trends in ice velocity are not statistical artefacts or the result of biases between sensors78, with the latter potentially driving a positive feedback as a result of its influence upon fjord water circulation and thus submarine melt80. This positive feedback contrasts with our findings at land-terminating margins, indicating that at an ice-sheet scale, the relationship between hydrology and ice dynamics is complex and spatially variable. Since the extent to which surface melt processes impact on tidewater glacier dynamics remains uncertain, analysis of existing large-scale, multi-decadal datasets5 at marine-terminating margins should be undertaken with respect to surface melt change, in order to better project the future response of the Greenland ice sheet to changing atmospheric and oceanic conditions.Furthermore, while the results presented here relate specifically to land-terminating margins, it has been argued that the dynamics of tidewater glaciers are a product of both oceanic and atmospheric forcing81 to all Landsat pairs for the 14 path/row combinations that intersect our study region . We enhance the images by applying a principal component analysis to these bands, and subsequently, a high-pass gaussian filter is used to enhance surface features such as crevasses and reduce the impact of temporally stable features relating to the basal topography81. We use a reference window of 80 pixels and set the search window based upon prior velocity estimates83. Following Tedstone et al.40, we apply a median coregistration to the output velocity fields in order to remove errors associated with georeferencing.We apply optical feature tracking81. The final velocity field for each time period is composed of the median value of all velocity fields within the time period at each pixel. To calculate uncertainty at each pixel, we compute a 1σ confidence interval for each component of the velocity field in the form:et al.40, we discard pixels with σ > 60 myr−1 from the subsequent analyses. In addition, we mask out the tidewater glaciers to the very north of the study region, limit our analysis to pixels below 1000 m above sea-level, and retain only the velocity fields with an area coverage above 9250 km2. The median velocity is subsequently calculated across the 71703 pixels common to all the remaining merged velocity fields , identified by calculating the value beyond which the median absolute deviation of velocities over stable ground becomes asymptoticlds Fig.  in order72. To mitigate this effect, we follow the velocity anomaly approach of Dehecq et al.72. First, we calculate a mean of all velocity pairs covering the period 1992–2019 described as V0. The velocity anomaly is defined as the value of the difference vector Vt − V0 projected on the mean velocity vector:Recent work indicates that velocity magnitude has a biased mean, with this bias increasing with the standard deviation of the velocity components (and so with noise), causing an artificial negative velocity trend72. The resultant velocity anomaly fields are displayed in Fig. The result of this approach is to centre the noise distribution symmetrically around zero Fig.  such thaTo assess the long-term trends in our time series, we first compute a simple linear regression through the data, and then test whether the data can be divided into three statistically different segments Figures –S10 usin1 is the first period (i.e. 1992–2003) and c2 is the second period (i.e. 2003–2012). We remove pixels with uncertainty greater than 60 m yr−1. We also filter by Velocity Vector Coherence (VVC)81, which follows the form:We construct change maps displaying the percentage ice velocity change between the following periods; 1992–2003 and 2003–2012, 2010–2012 and 2017–2019. For each pair, median ice velocity for each period was calculated through fusing all constituent velocity fields via a spatio-temporal median, and uncertainty of the percentage change was calculated through a linear combination of the uncertainties of each period:7, forced by NCEP-NCARv1 from 1992 to 2019. We limit our analyses of SMB below an ice surface elevation upper limit of 1600 m (a.s.l.). Little lake drainage occurs above this elevation86 and it has been argued that this is an approximate maximum elevation where crevasses, and thus moulins, are likely to form87. Whilst surface meltwater can runoff from elevations above this, surficial drainage is less likely to occur in high elevation regions due to the shallower surface slope86. Consequently, below this threshold elevation, we can be confident that surface meltwater drains to the ice-bed interface, and so influences ice motion. Regardless, melt at all elevations has increased from the 1958–1987 average in the period 1988–201387, with the difference between the means of the period being positive at p < 0.05 from 400–2600 m (a.s.l.).We obtain surface mass balance (SMB) data from the MAR v3.10 regional climate modelSupplementary Information.


In certain industrial processes, ice aggregations on surfaces can occur under almost vacuum conditions and at very low to cryogenic temperatures due to residual water molecules. This aggregation can affect the performance of the process and it is therefore of interest to monitor such surfaces. In this paper, we present a capacitive ice measurement system capable to operate in vacuum and temperatures of about  Ice formation can be present in many applications and can adversely affect the performance of a system. Examples are heat exchangers in cooling systems, ice aggregation on wind turbines or overhead power-lines or even icing on surfaces of aircraft. Capacitive sensors have been considered a reliable method for ice detection in such applications ,2,3,4, aThe permittivity of ice varies according to frequency and temperature , as wellIn , the comT is the temperature. The value of the high frequency permittivity varied between 3.093 ± 0.003 at 2 K to 3.15 ± 0.02 at 200 K.Ice can be formed in many different ways, hexagonal ice, or ice lh, is the most common form of ice which is formed under normal pressure conditions on Earth. In , the higThe high frequency permittivity of crystalline ice was more recently addressed in ,14, the According to , the expf, the resultant permittivity could be given by the model in Equation  simulations were made to determine an appropriate length for the electrodes. The electrodes were assumed to be formed by the two inner layers of an in-vacuum coaxial cable consisting of an inner conductor layer and an outer insulating layer, where the conductor has 0.61 mm of diameter. The geometry used for simulations consisted of three electrodes placed at a fixed distance as shown in In It is also wished that the capacitance value stays within the operation range to be used in the proposed hardware and that the values vary according to the resolution of our hardware. This is taken into consideration when choosing the appropriate electrode length.Based on the discussion in the previous sections, a frequency of 250 kHz was chosen for the capacitive sensor system which is expected to be significantly above the cut-off frequency and thus in a range where the permittivity is less temperature dependent. In the proposed system compare , the senThe capacitive sensor was created using in-vacuum coaxial cables and the ground shield was the equipment where it was fixed. These electrodes were connected to an AD7142, a Capacitance-to-Digital Converter (CDC) with a resolution better than 1 fF, that transmitted the values via I2C to a nRF51822 micro-controller, which sent the samples of the two connected electrodes to a base-station via a wireless connection using the 2.4 GHz band with a fixed transmission rate.The base station was implemented using a Beaglebone Black Wireless (BBBW), which is a low-cost development platform. The software has been implemented in python for the base-station functionality, where the stability and reliability is ensured by a software supervisor, which checks continuously if the main script is still running and which restarts the system otherwise.The base-station functionality comprises the communication with the sensor where measured samples are being transmitted continuously. The sensor node is connected via wireless and not via cable to enable the sensor node to be mounted more flexibly if the need arises. On the base-station all received samples are supplied with time-stamps and stored in a buffer and in files in the local memory. For monitoring, supervision and cloud backup of the system, an Universal Serial Bus (USB) 4G modem is used.For this test, the sensor and the BBBW were mounted in the same closed package as the there was enough space for both systems. To monitor the hardware as well and record the influence of the temperature of the electronics on the measurement, an additional temperature and humidity sensor was added and connected via I2C directly to the BBBW. The setup is displayed in in The sensitivity needs to be compared to cross-sensitivities, in particular towards temperature. This applies to both, the sensor electronics and the sensor front end. The cross-sensitivity towards temperature variations is studied in the climate chamber and results are shown in The electronic circuitry will be placed outside of the vacuum chamber, in a temperature-controlled environment. Therefore, the temperature variation in this environment is quite low. Furthermore, the sensor front end will be kept at fairly constant temperature inside the chamber and this cross-sensitivity can be considered of low relevance.In order to simulate ice accumulation on the sensor surface in the laboratory, a test setup according to Results are shown in For the long-term experiments inside the equipment in the field, the electrodes were fixed as shown in The temperatures inside the equipment were around The signals show some cross-sensitivity to the process probably due to some temperature increase inside the equipment and the variations of the industrial process itself, which cause the smaller peaks in the signal. This is possible to see when comparing the capacitance measurements to the process temperature plot.To further show the long-term stability of the measurement system, The study shows that capacitive ice measurement at the given conditions is feasible. The sensor front end and the measurement hardware worked continuously during the time of the experiment, short term data losses occurred due to non hardware-related issues such as an unplugged power connection in the field. Consequently, a long-term operation of such a system under the given environmental conditions is also feasible.Even though the feasibility of the measurement system could be demonstrated, the threshold levels for the ice layer thickness have not been found as the degree of ice that occurred in the experiment was lower than expected. A change of the sensor front end may be required to optimally adjust the sensor for the required thickness. When the ice gets removed during periods of higher temperatures, water drops may remain on the sensor surface that lead to a change of the offset signal. Consequently, the offsets should be corrected every time the process restarts. As only three such events occurred during the test, the significance of the results should be enhanced by a continuation of the investigations.


This study aimed to isolate some of the currently circulating infectious bronchitis virus (IBV) strains from some broiler chicken farms in Al-Hasa and to do some molecular characteristics of these strains.We collected 300 tissue specimens, including the trachea, bronchi, lungs, and kidneys from some four commercial chicken farms showing respiratory manifestations. We tested these tissue specimens by the real-time polymerase chain reaction (RT-PCR) and gel-based PCR. We selected some PCR positive samples for isolation in the embryonated chicken eggs (ECE). We sequenced some PCR-positive samples and conducted phylogenetic analysis based on the obtained sequences.Our molecular surveillance revealed that 31.6% of the tested specimens were IBV positive by PCR. We selected some positive specimens showing low Ct values by the qRT-PCR for virus isolation by the ECE. The infected eggs showed hemorrhage, dwarfing, and death in some cases after three passages in the ECE. We sequenced some of the positive PCR specimens and used the obtained sequences to draw the phylogenetic tree based on the partial IBV-ORF-1a, N, and S1 gene sequences. The phylogenetic trees based on the IBV-N and S1 gene sequences showed that the circulating IBV strains in Al-Hasa during 2016 was showing a high degree of identity to some strains from Taiwan and Italy. Meanwhile, the grouping of these strains based on the IBV-S1 sequences revealed that the currently circulating IBV strains in Al-Hasa belonged to Gr.I.7 along with strains from Taiwan.Our results confirmed the continuous circulation of the IBV among the chicken population in Al-Hasa despite the intensive application of vaccines against this virus. Nidovirales, family Coronaviridae, and genus Gammacoronavirus [Escherichia coli and Mycoplasma gallisepticum) always exaggerate the viral pathogenesis and auscultate the mortality rates among the infected chicken population [Infectious bronchitis virus (IBV) is a highly contagious respiratory viral disease of chickens of all ages. The IBV belongs to the order onavirus ,2. The Ionavirus . This stonavirus . Based oonavirus . The IBVonavirus . The IBVonavirus . Secondapulation . The IBVpulation .Efforts to control spreading of the IBV infections through vaccination resulted in some variable outcomes. Many IBV strains and serotypes have emerged since its discovery >80 years ago; meanwhile, the misuse of the IBV vaccines complicates the evolution and emergence of new IBV strains ,10. ManyThe IBV infection in chicken was first reported in Saudi Arabia in 1984 in field samples by the real-time polymerase chain reaction (RT-PCR) using N primers . AnotherSeveral IBV variants and genotypes are currently circulating in the Middle East, Asia, and North Africa such as Iraq, Egypt, Libya, Iran, and Jordan . FurtherThis study aimed for isolation and molecular characterization of the IBV circulating strains of the IBV in Al-Hasa in the eastern region of Saudi Arabia. This region is one of the major hubs for intensive poultry production in Saudi Arabia.http://www.kfsh.med.sa/KFSH_WebSite/usersuploadedfiles%5CNCBE%20Regulations%20ENGLISH.pdf). The Animal Ethics Committee of the King Faisal’s University approved this protocol.We conducted this study according to the King Faisal’s University Animal Ethics protocols and the National Committee of Bio-Ethics, King Abdul-Aziz City of Science and Technology, Royal Decree No. M/59  and proved to be IBV-negative antibodies by ELISA. The inoculum was prepared as follows; IBV-suspected tissue suspensions were centrifuged for 10 min at 5000 rpm. Antibiotic mixture was added (penicillin and streptomycin) to each tissue suspension. We used 100 µl per each tissue suspension to inoculate through allantoic route into five embryonated eggs . The inoThe total viral RNAs were extracted from tissue using the QIAamp Viral RNA Mini Kit  was processed in parallel to each batch of specimens as described above.The RT-PCR was performed using the IBV RT commercial kits  . The reactions were conducted as per the kit’s instructions with some minor modifications. Briefly, each reaction consisted of 2×RT-PCR master mix  in a total reaction volume of 20 µl. Meanwhile, we used 5 µl of nuclease-free water as negative control. Furthermore, a positive control reaction was carried out in parallel to each run. Duplicate reactions were done per each specimen. The cycling parameters were 55°C for 10 min, then 95°C for 8 min, followed by 50 cycles at 95°C for 10 s, and 60°C for 1 min.We designed some primers to amplify the partial IBV-N gene. We designed the IBV-N primers based on the IBV-N (accession no: KT762154). The nucleotide sequences of the used IBV-N gene are IBV-NF (5’-CGCTGGAGAATTTCCTCTTG-3’) and the IBV-NR (5’-CTAGTCCCTAGCAGCCATGC-3’). Meanwhile, we used the following primers, IBV-S1F (5’-ACCGGCTGATGGATGGCAT-3’) and the IBV-S1 (5’-TTGCTTACAACCACCCTGTAG-3’), to amplify the partial IBV-S1 gene.The RT-PCR reactions were conducted in a 25 µl reaction volumes using the one-step RT-PCR kit . The reactions were conducted as per the manufacturer’s instructions. The reaction mixture consists of 10 µl of 5×reaction buffer, 2 µl of each primer, 2 µl of enzyme mixture, 2 µl of dNTP mixture (400 µM of each dNTP), 3 µl of the RNA template, and 4 µl of RNase-free water. Reverse transcription was started at 50°C for 30 min; initial Taq polymerase activation was performed at 95°C for 15 min, along with 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and then extensions at 70°C for 1 and 10 min.For visualization of the amplified PCR products, electrophoresis of 10 µl of each PCR products on 1% agarose gel was performed. This was done under the ultraviolet light and photographed by the Bio-Rad gel documentation system. 10 µl of per each PCR product was mixed with 3 µl of the loading buffer . A 100-bp, ready-to-load DNA-marker ladder was loaded in a separate, parallel lane.We processed a confirmed PCR-ECE-P3-IBV isolate for sequencing by the Illumina next-generation sequencing approach. We used this approach to decode the partial-length genome sequences of this IBV isolate as previously described .We constructed the phylogenetic tree based on the obtained partial IBV-ORF1-a, IBV-N, and S1 gene sequences. We constructed the tree using the multiple alignments of these sequences with other IBV sequences retrieved from the GenBank done using the Mega-7 package, and phylogenetic analysis was done by the maximum likelihood method using the best-fit model determination and at least 500 bootstrap replicates as previously described . MeanwhiWe applied the non-probability sampling strategy for our specimen collection with incidental assignment approach as previously described .The IBV infection in chickens produces a wide range of clinical syndromes. Some birds showed respiratory manifestation in terms of coughing, sneezing, and nasal discharges. Necropsy examination of some IBV-infected birds revealed serous, catarrhal, or caseous exudate in the tracheal, nasal passage, sinus, and bronchi congestion . The edeWe conducted molecular surveillance to check the prevalence of IBV across the Al-Hasa from October 2015 to April 2016. We investigated four IBV outbreaks across the Al-Hasa province by the RT-PCR . We testWe already tested different tissue specimens, including the trachea, lungs, and kidneys, from some suspected IBV-infected birds. Our results are showing that 11 tracheal tissue specimens of 20 were positive, while 6 lung specimens of 20 were positive; meanwhile, only two specimens from the kidneys were positive .We processed 15 specimens  after pooling from three IBV outbreaks. We did the propagation for three passages. The IBV isolation was successful only in case of two specimens. The first specimen was collected from an RT-PCR-IBV-positive chicken trachea collected from farm no. 7. The second specimen was collected from positive IBV-RT-PCR lungs collected from chicken farm no. 5 area . HoweverWe used three tissue suspensions  from three specimens representing three different IBV outbreaks in some chicken farms from Al-Hasa region. We followed up these three inoculums on the ECE for three subsequent passages. The IBV-infected tracheal specimen caused death and induced some pathological changes in two embryos from the inoculated embryos in the three passages. However, one of them showed no pathological changes or death at the end of the experiment. Meanwhile, the IBV-infected lung tissue specimens showed that only one embryo was affected per each passage.We used commercial available RT-PCR kits to confirm the identity of some suspected IBV-infected chicken tissues, as well as some fluids from the embryonated egg passages. We carried out the RT-PCR amplification for three representative specimens from the ECE passages P-1-3. Meanwhile, we already included six original specimens from the IBV-suspected outbreaks from different farms, including some pooled tissue specimens . Furthermore, we already included the IBV vaccine as a positive control of the reaction in addition to the kits positive control in the reactions. In addition to a negative control used a non-DNA template. Our results show that 6/9 (66.67%) of the three tested specimens representing embryonated eggs passages 1-3 were positive. Three specimens from selected IBV specimens were positive, as shown in Based on the reported IBV-ORF1a sequences, these strains were closely related to IBV-L1148 strains . The phyBoth the pairwise distance  and the The geographical distribution of the suspected outbreaks represents different regions across the Al-Hasa. Some chicken farms were selected around the Al-Hasa region as targets for our study. Some of these farms were reporting IBV outbreaks based on the obvious clinical signs and post-mortem lesions, while birds in some others were apparently healthy. The clinical examination of the affected birds showed depression, ruffled feathers, loss of the body weight, respiratory rales, nasal discharge, and lachrymal discharge (data not shown). Necropsy examination of some affected birds revealed congested trachea, caseous and bloody plugs at the tracheal bifurcations , congestWe conducted an IBV molecular surveillance among some selected chicken farms in Al-Hasa region by the RT-PCR technique using the conserved IBV-N primers. We tested 100 specimens from layers, and broiler non-vaccinated chicken’s flocks represented four suspected IBV outbreaks across the Al-Hasa. These tissue specimens were collected from various organs of the affected birds such as the trachea, lungs, and kidneys. We did pooling five organs per each tube considered as one specimen. We found that 19/60 specimens (31.67%) were positive . Our resWe used the generated sequences of three independent genes  in the IBV genome to do the phylogenetic analysis of the circulating strains in Al-Hasa region during 2016. We found great overlapping and consistency of the phylogenetic analysis based on the IBV-S1 and N genes. The reported sequences were closely related to other IBV strains reported in Taiwan, Italy, and China Figures--5. This Our results confirmed recent reports about the circulation of different IBV strains in the kingdom and the eastern region in specific ,29. ThisIBV continues to pose a great risk to the poultry industry in the Al-Hasa, Saudi Arabia. There is continuous circulation of the IBV between the broiler and layer chicken in this area. The currently circulating strains of the IBV belong to the Gr.I-17 along with other genotypes from Taiwan, China, and Italy.MGH designed and performed the experiments, oversee the entire work, and performed data analysis and interpretation. MuAA conducted the fieldwork, performed some laboratory experiments, and drafted the manuscript. MoAA helped in the data analysis and reading of the manuscript. All authors read and approved the final version of the manuscript.