The clockwise spiral of troughs marking the Martian north polar plateau forms one of the planet’s youngest megastructures. One popular hypothesis posits that the spiral pattern resulted as troughs underwent poleward migration. Here, we show that the troughs are extensively segmented into enclosed depressions (or cells). Many cell interiors display concentric layers that connect pole- and equator-facing slopes, demonstrating in-situ trough erosion. The segmentation patterns indicate a history of gradual trough growth transversely to katabatic wind directions, whereby increases in trough intersections generated their spiral arrangement. The erosional event recorded in the truncated strata and trough segmentation may have supplied up to ~25% of the volume of the mid-latitude icy mantles. Topographically subtle undulations transition into troughs and have distributions that mimic and extend the troughs’ spiraling pattern, indicating that they probably represent buried trough sections. The retention of the spiral pattern in surface and subsurface troughs is consistent with the megastructure’s stabilization before its partial burial. A previously suggested warm paleoclimatic spike indicates that the erosion could have occurred as recently as ~50 Ka. Hence, if the removed ice was redeposited to form the mid-latitude mantles, they could provide a valuable source of near-surface, clean ice for future human exploration. Planum Boreum (PB), the Martian north polar plateau, consists of a water–ice cap that features a network of troughs arranged in a clockwise spiral pattern2 Fig. A. The hi9. A potential solar cycle of only ~ 1.5 Ka could have also modulated the depositional episodicity7. These polar materials consist of two distinct units7: upper and lower layered deposits   linear depressions . Unlike the troughs, the undulations have surfaces that lack layered exposures10. On the other hand, the LLD consist of moderately high-albedo layers locally resolvable down to decimeter and meter thicknesses; possibly numbering in the hundreds to thousands in the thickest sequences that reach several hundred meters of relief11. Superposed over both the ULD and LLD is the youngest unit—a tens-of-cm-thick residual ice cap and local deposits of dark sedimentary material2.The NPLD consist of hundreds to thousands of layers formed by episodic ice accumulation due to cyclic climatic fluctuations with various periodicities on the order of tens and hundreds of thousands to millions of yearsly) Fig. . The ULDns, Fig. 10). Unli1 and that the trough surfaces might be at least ~ 3.6 ± 2.5 Ma7. This hypothesis implies that the NPLD-forming ice was stable over enormous geologic timescales, as appears to be regionally the case for the ice that forms PB’s basal unit1 and its surrounding zones of the Vastitas Borealis Formation (VBF), an ice-rich deposit that covers most of the planet’s northern plains12.Detailed mapping of the polar plateau’s stratigraphy combined with crater count statistics suggest that the NPLD could have started to accumulate during the Middle Amazonian (~ 1 Ga)7 and those inferred from paleoclimatic simulations5. The latter view suggests NPLD accumulation may have started ~ 4 Ma when a decrease in Mars’ mean obliquity to ~ 25° destabilized the mid-latitude ice reservoirs, resulting in climatic conditions that facilitated water vapor transport and widespread deposition to the north pole5. In accordance with this geologic context, an estimate is that the upper few hundred meters in the NPLD, including the materials exposed within the troughs, could have formed during the last ~ 370 Ka This is an order of magnitude younger than the ~ 3.6 ± 2.5 Ma age determined from crater counts7. Thus, NPLD accumulation times of millions to hundreds of millions of years all seem plausible, and previous models of NPLD accumulation all make assumptions about processes for which much uncertainty exists.However, there is a standing disagreement between the NPLD ages derived from geologic investigations15 and the later analysis of subsurface radar reflectors obtained by the Shallow Radar (SHARAD) sounder onboard the Mars Reconnaissance Orbiter19 suggest the troughs initiated about halfway through the NPLD accumulation and that they migrated poleward as further accumulation of the NPLD ensued. The hypothesized trough migration is generally explained by water–ice transfer from the troughs’ warmer equatorward-facing slopes to their colder poleward-facing slopes. The processes involved include solar insolation-driven sublimation of equatorward-facing ice, downslope transport of water vapor via katabatic winds, ice-vapor condensation onto the cold, poleward-facing slopes, and perhaps mechanical erosion and sedimentation of ice particles17. Simulations of the sublimation from the trough walls, combined with proposed NPLD accumulation rates recreate the trough-migration paths inferred to exist in the subsurface from radar-sounding data20. The trough migration process has also been invoked to explain the development of their spiral pattern as poleward migration rearranged systems of initially disorganized troughs21.Surface geologic investigations of Viking Orbiter dataIn this article, however, we show geologic evidence indicating that in-situ erosion led to the formation of the currently exposed troughs and undulations and that their current spiral arrangement resulted from their progressive growth and spatial integration.We used Esri’s ArcGIS software as the primary mapping and analytical tool for the spatial, topographic, and stratigraphic characterizations of troughs and undulations across the entire north polar plateau, Planum Boreum. Because this morphologic mapping required high-resolution images registered to an accurate digital elevation model (DEM), we used (~ 6 m/pixel) Mars Reconnaissance Orbiter Context Camera (CTX) and (~ 18 m/pixel) Thermal Emission Imaging System (THEMIS) VIS (visible light) image mosaics georeferenced to a Mars Orbiter Laser Altimeter (MOLA) DEM . We mapped the troughs’ inner segmentation into cells by topographically tracing the upper margins of the interior enclosed depressions that constitute the cells Fig. . To perfIn plan view, most NPLD troughs have simple elongate, quasi-elliptical outlines exhibiting topographic segmentation Fig. . Each seHere, we present the first comprehensive map of the NPLD trough cells Fig. A. Our maOur map shows that the concentrically layered outcrops within trough cells are common across the entire polar plateau Fig. –S8, comp22. Hence, the difference between these two trough types , which we equate to the amount of evacuated NPLD materials  cluWe hypothesize that the following sequence of events links the troughs’ formational history to the spiral pattern’s emergence Fig. . Followi23 performed a wind field simulation over a significant portion of PB . We attribute the elongate cell shapes to erosion by wind vortices , vertex-to-midsection intersections produced widespread forking patterns  increases with distance from the pole. Possibly, the erosive power of wind vortices that developed within the pits and growing cells also increased near the plateau’s margins due to increases in slope and entrainment of fines. Consequently, while the sublimation pits’ initial distribution was probably random, their subsequent growth would have been greater closer to PB’s periphery, where the troughs are most developed and best integrated.4 km3 estimate. A consequence of this geologic scenario is that the spiral pattern must have also developed before ULD emplacement.We produced the first comprehensive map of the PB undulations. The map reveals that the undulations follow and extend the troughs’ clockwise spiraling pattern, particularly on upper plateau surfaces Fig. A. Locall7, and before ULD deposition ~ 10 Ka7. The age of the LLD trough strata remains uncertain, with estimates ranging from ~ 3.6 ± 2.5 Ma7 to ~ 370 Ka13. The younger age bracket for trough development (between ~ 370 Ka and ~ 10 Ka) suggests that trough development could represent evidence for one of the most recent episodes of climate change on Mars. There is additional evidence of potential warmer paleoclimates slightly more recent than ~ 370 Ka. For example, the migration of aeolian bedforms in the Meridiani region ~ 50–200 Ka28 is interpreted as an indicator of a significant increase in Mars’ atmospheric density and pressure. There are also possible aqueous debris flows from ∼ 190 Ka29 in Istok crater , a very young crater in Aonia Terra. Perhaps the water ice removal recorded in the uppermost sequences of the NPLD exposures along troughs’ walls and floors was coeval with these recent paleoclimate events.Our interpretation of the undulations as ULD-buried trough sections implies that the troughs developed largely after the accumulation of the LLD7, the geologic scenario arises that trough excavation might have been a cumulative process spanning numerous high-obliquity cycles. In this case, the larger troughs, mostly along the PB periphery, would have likely been exposed to more intense and reoccurring resurfacing during these cycles. Hence, they might be sites where alternating erosion and deposition more frequently took place during trough excavation, particularly in areas where NPLD sediments were remobilized and not completely removed from these larger troughs. Evidence of these intercalated inner-trough erosional depositional cycles is preserved as widespread unconformities mapped in some of these larger trough sets7.Conversely, if the LLD surfaces exposed within the troughs are ~ 3.6 ± 2.5 Ma3 during periods of high mean obliquity (> 35°) transported enormous water vapor volumes to lower latitudes8. Recondensing water vapor likely formed widespread lower latitude icy deposits, including the extensive Latitude Dependent Mantle (LDM)30, plains regions with evidence of excess ice34 and glaciers at mid-latitudes36. Moreover, this atmospheric and paleoclimatic cyclicity is thought to have also generated tropical mountain glaciers37. However, thus far, these atmospheric models face a significant handicap —  their implied large-scale NPLD erosional events, which led to the equatorward water vapor transport, have not been successfully reconciled with direct observational evidence in the exposed stratigraphy. The evidence to support these events was limited to the documentation of unconformities inferred from theoretical stratigraphic reconstructions3 and radar subsurface detections13.Three-dimensional Global Climate Models [GCMs] indicate that PB water ice sublimation4 km3 of water ice from PB , leads us to conclude that upwards of ~ 25% of the ice deposited at non-polar latitudes to form the LDM could have originated from the ice lost in the polar erosional event we propose here. This approximation does not include the potentially coeval retreat of a circum-polar ice sheet, which Tanaka7 reconstructed based on the mapping and characterization of numerous NPLD outliers, nor from a potential mass of eroded volatiles from the ULD-buried troughs that now form the undulations, as hypothesized above.Our proposed geologic scenario fills this gap by interpreting the entire spiral PB troughs as erosional landforms. We estimated that trough excavation could have removed ~ 4 × 10 PB Fig. . Compari36. Transient melting would have dissolved and increased within these materials the concentration of perchlorates, a widespread Martian surface compound and toxic to humans39.The assumption of the recent timeline  for the polar erosional event carries potentially significant implications for future human exploration. Following the proposed water vapor deposition at lower, non-polar latitudes, these young deposits would not have existed sufficiently long to experience large obliquity excursions. Hence, they would have remained wholly frozen during their entire post-depositional history, avoiding possible episodic interstitial thawing and refreezing such as that associated with wet-based glaciation3 of water ice were removed during trough growth, which is volumetrically equivalent to 16 billion Olympic pools. Drinking three liters a day would take 2283 years for a single person to drink an Olympic-sized pool.Hence, these young “clean” ice deposits could be an essential resource for future human explorers. We estimated that ~ 40,000 km40 or shallowly buried water ice within the upper meter of the Martian surface at the mid-latitudes42. Additionally, the deposits’ youthfulness implies that they likely formed widespread near-surface stratigraphy, enabling easy access. Such deposition may account for the relatively pure water ice beneath a centimeters-thick soil cover discovered by the Phoenix lander in the high latitudes of the northern plainsStratigraphy in trough walls reveals that layering is horizontal and occurs evenly, with uniform thicknesses, in the plateau wall all around the troughs. Hence, the bulk of the exposed stratigraphy consists of layers that were emplaced during the construction of PB.The mantles on pole-facing slopes Figs. , S8, S9 Trough, undulation, and layer geometries indicate in-situ erosion on the LLD surface, below the ULD. Troughs tend to be deeper, wider, and more connected toward the margins of PB, where some trough floors expose basal unit and Vastitas plains materials. Hence, their in-situ excavation might have been enhanced throughout the more steeply sloping areas of PB, probably due to increased insolation on these areas when compared to the flatter, and more central (hence higher latitude), parts of the polar plateau Fig. .The cells and troughs exhibit complex intersections from which their spiral distribution emerges, suggesting that the pattern is a late-stage consequence of the troughs’ erosional history. The troughs’ growth patterns reflect an overall growth trend in directions orthogonal or oblique to regional katabatic winds, suggesting a possible morphogenetic connection.The troughs largely ceased developing before ULD accumulation, given that apparently these materials completely filled some of their sections . Hence, Mars’ current and recent climate does not include the atmospheric conditions that led to trough formation and the development of the spiral pattern.We propose that future investigations regarding the origin and evolution of the NPLD troughs need to account for the following observations and related interpretations:44.Our geologic investigation reveals widespread segmentation of the north polar troughs, which most likely resulted during geologically recent in-situ erosion. The inferred growth trends are consistent with the erosion occurring preferentially orthogonally and obliquely to katabatic wind directions and suggest that the spiral pattern is a consequence of the troughs’ progressive spatial integration. Furthermore, the spiral pattern’s connection to landforms indicative of cell and trough intersections implies that there has been no appreciable repositioning of the trough axes subsequent to (and during) the emergence of the spiral arrangement. Our hypothesis explains that the troughs’ spiral trends developed over the plateau’s current topography. A similar in-situ erosional history was also likely responsible for the origin of the south polar spiral troughs1, the NPLD contain layers deposited during the last billion years, then formation of the troughs could have exposed a paleoclimatic record spanning about a fourth of the planet’s geologic history. If on the other hand, the NPLD formed during the last 4 Myrs3, the exposed layers likely comprise an extensive paleoclimatic record of the geologically recent evolution of the Martian paleoclimate.A non-migratory nature of the polar troughs implies that the strata that they expose on both their equator and pole-facing slopes can be linked to paleoclimatic conditions accompanying the construction of the Martian polar plateaus. If, as previously proposed19 show possible trough migration paths beneath some of the undulations. If the undulations present within these areas are indeed buried migratory troughs, a reasonable  prediction is that  their migration paths would (1) trace a shrinking spiral , or (2) the pathways that led to the emergence of the spiral pattern . However, due to the orbital inclination of the Mars Reconnaissance Orbiter, the innermost areas of PB lack observations, which makes these determinations challenging.SHARAD subsurface radar data characterizations in Smith and HoltThe interpretation that inclined radar discontinuities are trough migration paths is partly based on the observation that their uppermost zones terminate right beneath trough floors. However, it remains unclear whether other geologic scenarios could explain the radar-reconstructed stratigraphy. For example, some of these could be buried unconformities, which in the past surfaced at breaks-in-slope Fig. . TroughsMajor uncertainties and controversies that remain unresolved include:Supplementary Information


TGR5 is a G protein-coupled bile acid receptor that is increasingly recognized as a key regulator of glucose homeostasis. While the role of TGR5 signaling in immune cells, adipocytes and enteroendocrine L cells in metabolic regulation has been well described and extensively reviewed, the impact of TGR5-mediated effects on hepatic physiology and pathophysiology in metabolic regulation has received less attention. Recent studies suggest that TGR5 signaling contributes to improvements in hepatic insulin signaling and decreased hepatic inflammation, as well as metabolically beneficial improvements in bile acid profile. Additionally, TGR5 signaling has been associated with reduced hepatic steatosis and liver fibrosis, and improved liver function. Despite the beneficial effects of TGR5 signaling on metabolic health, TGR5-mediated gallstone formation and gallbladder filling complicate therapeutic targeting of TGR5 signaling. To this end, there is a growing need to identify cell type-specific effects of hepatic TGR5 signaling to begin to identify and target the downstream effectors of TGR5 signaling. Herein, we describe and integrate recent advances in our understanding of the impact of TGR5 signaling on liver physiology and how its effects on the liver integrate more broadly with whole body glucose regulation. TGR5 is a transmembrane G-protein coupled receptor (GPCR) for bile acids that is ubiquitously expressed in mouse and human tissues ,3,4,5,6.While the role of TGR5 signaling in immune cells, adipocytes, and enteroendocrine L cells has been well described and extensively reviewed, the impact of TGR5 on hepatic physiology and pathophysiology has received less attention. TGR5 is highly expressed in the non-parenchymal cell-types of the liver ,21,22,23TGR5 is a member of the rhodopsin-like family of GPCRs . TGR5 iss), whereby ligand binding to TGR5 results in the activation of adenylyl cyclase-cAMP-PKA signaling and the associated downstream effects, including the recruitment of cAMP response element-binding protein (CREB) to target genes [In 2002, Maryuama et al., discovered TGR5 as a novel GPCR that, in response to bile acids, enhanced cAMP production in human cell lines . Togetheet genes ,2. Howevet genes , Rho kinet genes , mammaliet genes , as wellet genes . TGR5 is ubiquitously expressed in rodent and human tissues and has a well-defined role in many regulatory functions that affect whole body metabolism ,13,15,20A growing body of literature suggests that TGR5 signaling plays an essential role in hepatic metabolic regulation. Our group recently identified a previously unknown role for hepatocyte TGR5 signaling in regulating whole body glucose homeostasis and insulin sensitivity; however, the underlying signaling pathway through which this occurs remains unresolved . Moreoves to increase intracellular cAMP levels, resulting in increased ERK1/2 signaling and promotion of proliferation -fed controls [−/−Tgr5 mice fed a HFD are more insulin-resistant than +/+Tgr5 mice, as assessed by an insulin tolerance test [+/+Tgr5 mice, but not in whole body −/−Tgr5 mice [Olea europaea and a TGR5 agonist, decreased fasting plasma insulin concentrations and improved glucose tolerance in mice [−/−Tgr5 mice [Targeted disruption of TGR5 in mouse models of metabolic disease has identified a role for TGR5 signaling in the maintenance of whole body glucose homeostasis and insulin sensitivity. For instance, Thomas et al., have shown that mice with a gain-of-function of TGR5 have improved glucose tolerance, whereas whole body controls . Previounce test . Furthernce test ,28,29,48−/− mice . Similar in mice . Additio−/− mice . Until r−/− mice ,17,34,50HEP+/+Tgr5 mice, but not in Hep−/−Tgr5 mice. Furthermore, we found that this effect occurred independent of body weight and GLP-1 secretion [However, since the liver is central to whole body glucose regulation, this suggests that hepatic TGR5 signaling may be a critical contributor to the overall metabolic benefits of TGR5. Therefore, we administered Compound 18 to hepatocyte-specific TGR5 knockout mice to assess the role of hepatocyte TGR5 on glucose regulation . We founecretion . Therefo−/−Tgr5 mice exhibited more hydrophobic biliary, circulating, and hepatic bile acid pool compositions, as well as decreased muricholic acid (MCA)/CA ratios in both the plasma and liver, as compared to wild-type controls [−/−Tgr5 mice had an increased ratio of 12-α-hydroxylated bile acids to non-12-α-hydroxylated bile acids, which was specifically characterized by increased taurocholic acid (TCA) and decreased tauromuricholic acid (TMCA) [Alterations in bile acid homeostasis have been linked to the pathogenesis of insulin resistance and obesity ,53,54,55controls . Furtherd (TMCA) . Similard (TMCA) . The resd (TMCA) . INT-767Cyp7a1 expression was similar between whole body +/+Tgr5 and −/−Tgr5 mice, but that fasting induction of hepatic Cyp8b1 was attenuated in whole body −/−Tgr5 mice [Cyp7a1 and Cyp8b1 expression, whereas treatment with INT-777 only decreased hepatic Cyp8b1 expression [Cyp8b1 expression  in gallbladder epithelial cells. Upon activation of TGR5, the subsequent increase in cAMP resulted in insertion of ABST into the apical membrane, leading to enhanced uptake of bile acids from bile into biliary epithelial cells . Through−/−Tgr5 mice experience exacerbated inflammatory responses and hepatic fibrosis compared to wild-type controls [Chronic inflammation is increasingly recognized as a key driver of insulin resistance . Metabolcontrols . In linecontrols ,15,20. Mcontrols . Togethe+/+Tgr5 and −/−Tgr5 mice following RYGB [Bariatric surgery is currently the most effective treatment for obesity and is associated with high rates of T2DM remission ,68. Manying RYGB . Overall−/−Tgr5 mice exhibit increased liver lipid deposition compared with male wild-type mice. Furthermore, HFD-fed male whole body −/−Tgr5 mice exhibit increased liver lipid deposition compared with female whole body −/−Tgr5 mice, suggesting that the effect of TGR5 signaling on liver lipid deposition may be influenced by sex [Non-alcoholic fatty liver disease (NAFLD) is a common co-morbidity of obesity and develops due to an accumulation of lipid in the liver. NAFLD can progress to non-alcoholic steatohepatitis (NASH), characterized by inflammation, progressive fibrosis, and hepatocellular damage and can eventually progress to cirrhosis or hepatobiliary cancer. Loss of TGR5 signaling has been shown to promote liver lipid deposition in preclinical studies, suggesting that TGR5 signaling may protect against NAFLD. For example, HFD-fed male whole body d by sex .de novo lipogenesis, and promoted expression of anti-inflammatory genes and genes related to lipid uptake [ob/ob mice on a HFD supplemented with trans-fat, cholesterol, and fructose treated with INT-767 exhibited a greater decrease in liver parenchymal fibrosis and inflammatory infiltrates compared to mice treated with OCA [Studies investigating the effects of TGR5 agonists on NAFLD further demonstrate a role for TGR5 in protection against liver lipid deposition. Treatment of HFD-fed mice with INT-777 decreased plasma free fatty acids and liver steatosis, liver fibrosis and plasma AST and ALT levels . Similard uptake . In linewith OCA . Moreovewith OCA . Althoug−/−Tgr5 mice [−/−Tgr5 mouse models used, duration of HFD-feeding, and variation in diet composition in each study [Studies in mouse models of bariatric surgery report conflicting results on the role of TGR5 in the effect of bariatric surgery to reduce liver lipid deposition. Ding et al., reported that VSG decreased hepatic steatosis in wild-type but not in whole body −/− mice . In cont−/− mice . The disch study .Overall, these findings suggest that enhanced TGR5 signaling attenuates hepatic triglyceride accumulation and fibrosis, and improves liver function; however, the mechanisms through which this occurs remain unknown C. Furthe−/−Tgr5 mice relative to wild-type controls [−/−Tgr5 mice are protected against the formation of cholesterol crystals and gallstones when fed a lithogenic diet [Therapeutic targeting of TGR5 for the treatment of metabolic disease has been hindered by the wide range of effects associated with systemic TGR5 activation. In particular, a major side effect of TGR5 agonists is the inhibition of gallbladder emptying, leading to gallstone formation and cholestasis. TGR5 is highly expressed in the gallbladder and cholangiocytes . TGR5 sicontrols ,43. Howenic diet , which s−/−Tgr5 mice exhibit decreased cholangiocyte and hepatocyte proliferation and increased liver injury in response to cholestatic stress [TGR5 expression in the biliary tract is also crucial for protection against bile acid overload. For example, TGR5 signaling strengthens the biliary epithelial barrier resulting in decreased hepatocyte necrosis in the presence of obstructive cholestasis in mice . In contc stress . In addic stress . Comparac stress , suggestc stress . To thisIn light of the ubiquitous expression of TGR5 throughout the liver, hepatic TGR5 signaling is increasingly recognized as a key contributor to whole body metabolic regulation. Nevertheless, the wide range of effects associated with liver TGR5 signaling complicates therapeutic targeting of TGR5. Further delineation of the downstream mediators of TGR5 signaling in the liver is needed to enable rational development of compounds targeting mediators of the metabolically beneficial actions of TGR5 in the liver. Such a compound could be coupled with an GLP-1 receptor agonist to develop a highly effective multimodal therapy for the treatment of type 2 diabetes.


The rate of mass loss has nearly doubled from − 0.4 m w.e year−1 during 1,600 to 1978 to − 0.7 m w.e year−1 at present. In comparison Patagonia has lost just 11% of it’s LIA volume. Glacier ice in the Southern Alps has become restricted to higher elevations and to large debris-covered ablation tongues terminating in lakes. The accelerating rate of ice loss reflects regional-specific climate conditions and suggests that peak glacial meltwater production is imminent if not already passed, which has profound implications for water resources and riverine habitats.Rapid changes observed today in mountain glaciers need to be put into a longer-term context to understand global sea-level contributions, regional climate-glacier systems and local landscape evolution. In this study we determined volume changes for 400 mountain glaciers across the Southern Alps, New Zealand for three time periods; pre-industrial “Little Ice Age (LIA)” to 1978, 1978 to 2009 and 2009 to 2019. At least 60 km With decadal mean contributions of ~ 0.9 mm year−1 they account for 25% of total global sea level rise3. Furthermore, those contributions have accelerated recently5 and are expected to continue for many decades6. It is therefore crucial to understand not only the present day distribution of ice volume and contemporary rates of decline, but also to place modern rates of cryosphere and proglacial landscape change within a longer-term context. Reconstructing mountain glacier volumes is perhaps most obviously achieved by using moraines and trimlines that delimit former ice margin extent and ice thickness7.Mountain glaciers and ice caps produce global sea level rise contributions that over the last few decades have far exceeded those from the major ice sheets8 for the Patagonian ice fields, Carrivick et al.9 for several mountain glaciers on James Ross Island on the Antarctic Peninsula and that by Mark et al.10 for a small number of glaciers around Quelccaya ice cap in northern Peru. In a different approach using a parameterised mass balance gradient and an estimate of ice thickness applied along single glacier flow lines, Hoelzle et al.11 have calculated an ice volume loss across the Southern Alps of New Zealand of − 61% since the LIA. The paucity of distributed ice volume reconstructions, and hence the scant knowledge on the response of Southern Hemisphere glaciers to climate change since late pre-industrial times, is especially important to understand because there are inter-hemispheric differences in Holocene climate change (e.g.16). In addition, spatiotemporal heterogeneity for climate variability within the Southern Hemisphere is strong (e.g.18), and differences within regions can be amplified due to modes of variability that drive synoptic weather patterns23.Few studies have sought to quantify glacier volume changes in the Southern Hemisphere on a centennial-scale; i.e. since recession from nineteenth Century maximum ice limits, or the conclusion of pre-industrial times that is contemporaneous with the “Little Ice Age (LIA)” end. Some exceptions include the geomorphology-based studies by Glasser et al.24. This knowledge gap is important because those glaciers are highly responsive to climate change, especially air temperature changes25. Those short response times, coupled with high sediment availability, has produced exceptionally well-preserved sets of moraines, which delineate former ice margins (e.g.26). The most widespread and best-preserved moraines and trimlines across the Southern Alps represent the former extent of mountain glaciers between the fourteenth and nineteenth centuries, which is contemporaneous with the northern hemisphere LIA27. Details about the local expression of late pre-industrial era Southern Alps moraines that overlap with the classical definitions of northern European LIA are described in our Table SI There have been limited estimates of ice volume loss across the Southern Alps of New Zealand during the late Holocene25, and highly variable glacier responses to climate depending on terminus type , glacier hypsometry and glacier surface conditions29. In particular, the largest glaciers are presently stagnating and down-wasting (e.g.32) which distorts the apparent loss of ice if that estimate is based solely on areal extent. The aim of this study is therefore to quantify glacier volume loss since the “Little Ice Age” for the Southern Alps.Determination of modern rates of glacier geometry changes are complicated in New Zealand by limited direct and remotely sensed information, overall ice terminus advances during the 1980s and 1990s33 for the entire Southern Alps were obtained from the Randolph Glacier Inventory (RGI) via Global Land Ice Measurements from Space (GLIMS)34. We acknowledge the imperfections of this dataset but did not seek to subjectively modify it. Glacier outlines for year 2009 mapped by Sirguey and More35 for the Mt Cook region only were also obtained from GLIMS. In this study we derived glacier outlines for year 2019 using a land cover classification of 10 m resolution multi-spectral Sentinel-2 level-2A satellite imagery. The Sentinel-2 spectrally derived bands included the NDVI, NDSI, NDWI and the first principal component of an eigenvector analysis on a grey-level co-occurrence matrix analysis. The classification was conducted in Google Earth Engine using a Random Forest machine learning classifier (n = 50). Supplementary information (SI) has further detail on image pre-processing, image band calculation and the classification method.Glacier outlines for the year 1978 by Chinn36. First, crests of LIA-associated moraines37 were mapped . Subsequently, adjacent and surrounding valleys were examined within the hillshade of the DEM and in Planet47 imagery, mostly PlanetScope 3 m resolution https://www.planet.com/explorer/, and within orthorectified 1 m resolution aerial photographs48 for contiguous landform evidence.Glacier outlines for the LIA were primarily mapped using a hillshade of an 8 m Digital Elevation Model (DEM)ped Fig. , and tho49, which compared favourably with the neoglacial ELA proposed by Porter50 for a transect between Mt Cook and Jollie Glacier  trend surface of the Equilibrium Line Altitude (ELA) for the LIA based on 22 cirque glaciers previously studied Fig. SI . By disc51, Table SI 52 and provides the uncertainty bounds of our mean rate.For mean volume change rates, a temporal datum during the LIA interval needed to be applied. It has been noted that the Southern Alps glaciers reached their greatest LIA extents at differing times . Volume uncertainty is far higher; about 20%, given the 3D surface interpolation as explained in the very similar study of glacier volume change since the LIA in NE Greenland53, and surface differencing methods, but is not dependent on the choice of DEM  and optical image (< 1 m) resolution used for digitising LIA glacier outlines, and subjective choices of the most prominent inner moraine and of trimlines. Nevertheless, in the vast majority of cases the geomorphological evidence is distinct, whilst digitising errors for smaller glaciers will have the largest relative effect in area measurement accuracy. For a typical valley glacier in the Southern Alps of 2 km54) was estimated using a perfect-plasticity centre-line model55 using the 1978 glacier outlines. That model was tested against dense bed topography data from five mountain glaciers around the world, including the few direct ice thickness measurements of any Southern Alps glaciers at Tasman Glacier . That model is well-suited to making rapid region-wide estimates of distributed ice thickness for mountain glaciers such as across southern South America56 and for the entire Antarctic Peninsula57. Year 2019 ice volume was estimated as the difference between our 2019 ice surface, which was derived in the same manner as for the LIA by interpolating between our 2019 outlines, and the ice-free DEM. More methodological detail is given in our Supplementary Information. A summary and depiction of our surface differencing is given in Fig. SI We also make a novel attempt to estimate the total volume of ice across the Southern Alps during the LIA by comparing our LIA surface elevations with those of an ice-free DEM Fig. SI . Our ice2 ± 104 km2. If we assume that the 440 km2 that we were unable to map has not changed since the LIA because it is mostly comprised of ice above the regional ELA, then combining that with the area that we have mapped produces a total glaciated LIA area of at least 1932 km2 ± 135 km2. Compared to the 1978 GLIMS inventory area of 1,463 km2 we find a loss of at least 24% in glacier ice areal extent since the LIA. For comparison, Chinn24 reported that New Zealand’s Southern Alps glaciers (based on 127 glaciers) had lost 38% of their length and 25% of their area since the LIA. Using newly generated glacier outlines for the year 2019  of glacier area in 2019 lies within the LIA ablation areas.Overall, we mapped LIA outlines of 400 Southern Alps glaciers. The vast majority of these represent the coalescence of many, now smaller, glaciers that have fragmented greatly since the nineteenth century. The total area we mapped as ice covered during the LIA was 1,492 kmThere has been major terminal recession of ice between 2009 to 2019; especially where ice is flowing and calving into proglacial lakes. The difference in terminus outlines between 2009 and 2019 far exceeds the 1978 to 2009 changes Fig. SI . One sig52), there has been major ice surface elevation lowering . This lost volume equates to a 41% to 62% of the total LIA volume, which was between 97 km3 and 145 km3. For comparison, Hoelzle et al.11 estimated a slightly larger volume of 170 km3 for the Southern Alps during the LIA.Based on the method used herein to calculate volume change of glaciers from a reconstructed LIA ice surface  was lost, or an additional 14% to 17% of the former LIA volume.The 2019 volume is estimated in this study to be between 33.7 km3 ± 0.9 km3. Godley has lost 3.49 km3 ± 0.7 km3, Murchison Glacier has lost 3.47 km3 ± 0.7km3, and Mueller has lost 2.27 km3 ± 0.4 km3. These four glaciers  together account for over one fifth of the total ice mass loss across the Southern Alps since the LIA.The largest elevation changes Fig. SI  and henc3 year−1 ± 0.1 km3 year−1 occurred if 1450 CE is taken as the LIA starting point, and from 1600 CE the rate is 0.18 km3 year−1 ± 0.1 km3 year−1. The near-end point of the LIA at 1800 CE yields a rate of volume loss of 0.38 km3 year−1 ± 0.2 km3 year−1  is in good agreement but slightly lower than the estimate of 61% loss by Hoelzle et al.8 reported 606 km3 lost from the North Patagonian ice field (NPI) and South Patagonian ice field (SPI) collectively, since the LIA. Taking the mid-point of the local LIA timespan in New Zealand of 1,600 CE, our calculated sea level rise contribution from the Southern Alps of 0.0007 mm year−1 is 62% of Glasser et al.’s 0.0018 mm year−1 for the NPI and 79% of their 0.0034 mm year−1 for the SPI. This slower centennial rate of mass loss in the Southern Alps compared to that of Patagonia is most easily explained by the far larger volume of ice in Patagonia than in the Southern Alps.In the Southern Hemisphere, Glasser et al.3 ± 1,191 km3, for the NPI and SPI combined56. Comparing this to the volume change of Glasser et al.8 suggests that only 11% of the LIA ice volume of Patagonia has been lost to date. In stark contrast, we find that across the Southern Alps the ice volume lost since the LIA equates to between 41 and 62% of the LIA total. This disproportionate ice loss could be most easily explained by the more northerly location of the Southern Alps in comparison to Patagonia, but probably is also a reflection that the smaller glaciers in New Zealand have faster response times.However, in terms of absolute volume change, it is instructive to note that a total remaining ice volume in southern South America of 5,561 km52. A mean summer atmospheric circulation pattern for 1450 to 1800 CE was derived from quantitative temperature estimates based on the ELAs of 22 glaciers being lower than present, implicating increased frequency of trough synoptic types during the LIA at the expense of blocking types19. This glacier-based finding supports prior work that suggested a similar pattern existed for the LIA mean climate state, based on qualitative speleothem temperature and palaeo-water balance interpretations paired with quantitative tree ring temperature reconstructions52. Regional ‘trough’ circulation patterns during the LIA were nested within larger hemispheric-scale geopotential and Sea Surface Temperature (SST) anomalies  arising from interactions of multiple modes of variability, e.g. El Nino-Southern Oscillation (ENSO), Southern Annular Mode (SAM), Pacific South American (PSA) mode and Zonal Wave 3, which produced cooler regional LIA summer temperatures. More broadly, summer hemispheric-scale spatial field patterns48 hint at an important ENSO/SAM contribution, where El Nino Modoki combined with negative SAM, Zonal Wave (ZW) 3–458 and the PSA59 to promote regional-scale atmospheric flow that reduced surface air temperatures. The latter two modes of variability, along with the ENSO/SAM dynamic, are recognised as producing heterogeneous surface climate patterns for New Zealand and Patagonia simultaneously, and hence, could have contributed to very different timings and expressions for surface climate and LIA glacier activity at centennial timescales in those regions.Previous work synthesised multi-proxy data to infer regional atmospheric circulation patterns as a driver for pre-industrial New Zealand glacial advances32 estimated that Southern Alps ice volume decreased from 54.5 km3 in 1976 to 46.1 km3 by 2008. Our volume estimate for the year 1978 is in excellent agreement: we also calculate 54 km3 (43.2–64.8 km3). From this amount, a mean rate of 0.3 km3 year−1 volume loss from the mid-1970s to the mid-2000s is double that of our mid-scenario centennial rate (0.18 km3 year−1) of ice volume loss since the LIA  and extra-tropical circulation, including SAM and hemispheric-scale PSA and ZW3 wave patterns, was noted. Mackintosh et al.25 also concluded that by 2011 the situation had clearly reversed, and significant warm episodes were contributing to the rapid decline of Southern Alps ice volume.Recent work32) and as we have also found for the centennial time frame, approximately three-quarters of the total Southern Alps ice volume loss has occurred from the (~ 10) largest glaciers, which have a long (protracted) response time. Thus, whilst rapid retreat and glacier length decreases and area loss can be reported for the vast majority of New Zealand’s glaciers, the most salient points in terms of volume and mass and meltwater generation are that (i) committed losses (cf.60) for future decades are volumetrically large as a proportion of how much ice remains, and (ii) the overall percentage of ice loss relative to an initial state will likely diminish. Our analysis of ice volume in the Southern Alps remaining since the LIA therefore supports the suggestion61 that glaciers in climatically-sensitive regions are close to (or even past) peak glacial meltwater contributions to downstream hydrological systems. Nevertheless, many questions still remain about how meltwater contributions in glacierised catchments will contribute to rivers, and how it can help buffer extreme seasonal climate conditions.Furthermore, as highlighted  rapid-response glaciers progressively becoming restricted to high-elevation land close to the ELA, (ii) topographic shading and topographically-assisted mass inputs via avalanching and wind-blown snow becoming progressively more important proportionally as glacier area becomes restricted beneath steep headwalls, as found for European Alps glaciers63, and (iii) large valley floor glacier ablation tongues are stagnating beneath increasingly extensive, thick debris covers, but are continuing to lose mass fast because they are at low elevation and are influenced by proglacial lake interactions—including negative feedbacks associated with water contact ablation and tabular calving in front of the local grounding line.Overall, we suggest the Southern Alps rate of ice volume loss has recently accelerated. The glaciers there are now mostly characterised by; (i) small  topography across Southern Alps using the 1978 glacier outlines, and year 2019 glacier outlines, area and volume estimates. These spatially-distributed data complement a recent reconstruction of the last glacial maximum ice extent across the Southern Alps using similar methodsWe have determined an acceleration in the rate of volume loss and a progressively more negative mean annual balance from the LIA  to recent decades Fig. . This ac65, sedimentology and net sandur or outwash plain elevation changes (e.g.66). In particular, proglacial lake development affects glacier evolution, interrupts the flux of meltwater and sediments down valley68 and can control longer-term landscape evolution69. Our findings suggest that the timing of peak glacial meltwater production will soon be passed across the Southern Alps, if it has not been already, but we note that the associated spatio-temporal yields of proglacial sediment, minerals and nutrients have yet to be examined.Across the Southern Alps, former LIA ablation areas are now almost completely ice-free but the exceptions are the very large near-stagnant debris-covered ablation tongues that sit on valley floors at relatively low elevation. These large ablation tongues often terminate in proglacial lakes, which can exacerbate ice loss. The remaining ice at high-elevation is increasingly fragmented, thinning and disconnected from ablation areas thereby exposing increasingly large nunataks. These local glaciological controls determine the exceptionally dynamic proglacial geomorphologySupplementary information 1.Dataset S1.Dataset S2.


Physiological saline (0.9% NaCl) and deionized water were frozen in a laboratory chest freezer and impedance was monitored throughout freezing and thawing. The resistive and reactive components of electrical impedance were measured for these samples during freezing and thawing (heating) within a temperature range between 20 °C and −48 °C. The impedance of saline solution and de-ionized water increases sharply at the freezing point, similar to what is known for, e.g., complex tissues, including meat. Yet, only the saline solution impedance shows another sharp increment at a temperature between −30 and −20 °C. Changes of the electric properties after solidification suggest that the latter is linked to transformations of the ice lattice structure. We conclude that the electrical properties might serve as sensitive indicators of these phase changes. In the 1950s to 1980s much research had been dedicated to advancing the understanding of the low frequency properties of ice and snow, and to exploiting these properties for glaciological purposes ,3. The e3O+ and OH−, and the Bjerrum or valence defects. Petrenko [Jaccard  and PetrPetrenko  reportedPetrenko . Ionic dPetrenko .Furthermore, the electrical properties of ice are very sensitive to small concentrations of certain impurities that can be incorporated in the hydrogen-bonded network to generate protonic point defects . Many reIn this paper, we use impedance spectroscopy to measure the impedance of different types of ice in the laboratory. The goal of this experiment is to gain insight into the electrical properties of the physiological saline and deionized water during the freezing and melting process. Physiological salt solution (0.9% NaCl solution) resembles key features of intra- and extracellular fluids of biological materials, such as muscle tissue. Impedance measurements are of a high interest because they can provide relevant information for meat characterization. Meat is an aggregation of cells in about 60–80 % of fluid. Two thirds of fresh meat fluid is inside the cell and one third is in the extracellular space. Both intracellular and extracellular fluids are electrolytes containing free ions able to transport electrical charge. However, not much is known about impedance in frozen meat and the electrolytes it contains.Electrical impedance will be used to identify factors that influence the ice formation of saline solution and de-ionized water and that make impact on either the quality of frozen biological materials or their electrical properties. Moreover, this experiment will allow to link electrical impedance measurements of ice with freezing rate and temperature.In these fundamental laboratory studies on ice crystals, the electric impedance of ice was measured using a Zürich Instruments MFIA impedance analyzer. The frequency range was from 10 Hz to 1 MHz and the applied voltage was 600 mV rms. A tetrapolar electrode setup was used. The stainless steel needle electrodes had diameters of 2 mm and a length of 12 mm with 18 mm distance between the middle pick-up electrodes. Electrode pins were inserted into the upper surface of the tested solutions. 0.9% physiological saline solution and deionized water was used as samples for impedance measurements as a function of temperature.3 (width × depth × height). The sample, as well as the freezer chamber temperature, was measured using calibrated T-type thermocouples, which were connected to Lutron TM-947SD four-channel thermometer . Temperature at the center of the sample was obtained by inserting a T-type thermocouple at a pre-determined location. Surrounding air temperature was measured close to the cup. The measurement was carried out continuously under freezing before and after solidification and under subsequent heating.The sample holder was made of a plastic cup. Freezing was carried out in a commercial chest freezer  that was set to a temperature of −50±2 °C. The internal dimensions of the freezing chamber were 116 × 60 × 85 cmThe chest freezer was set to the required temperature 12 hours before the experiment.The impedance analyser was placed close to the chest freezer for subsequent measurements.Sample and electrode – connected with the impedance analyser cables – were moved into the freezer.The temperature sensors were placed inside and outside the sample (in the freezing chamber). The temperatures was recorded in 1 second intervals.Then the impedance was measured in defined temperature intervals until the sample's centre temperature was close to the freezing chamber temperature. At this point we left the sample in the freezer for 24 hours. To subsequently perform the impedance measurements during thawing (defrosting) samples were transferred to an incubator (20 °C). Impedance was measured until the sample's centre temperature reached 15 °C.We grew a 500 ml sample of physiological saline ice and monitored its impedance throughout its growth. We prepared experiments based on the following procedure:The conducted research is not related to either human or animal use.The impedance of de-ionized water was measured during freezing as well as heating (melting). The impedance measurements were conducted in dynamical conditions of temperature and phase transformation, so the curves can be a little distorted. A typical temperature dependence of impedance measurement result for different selected frequencies, is shown in Similarly, typical temperature dependency curves for electrical impedance data of selected frequencies for 0.9% physiologic saline from −30 to 20 °C, are shown in As the figure demonstrates, the saline electrical impedance increases with temperature decrement. Starting from 20 °C, the impedance increase very slowly as the temperature falls down to the freezing point.When the temperature falls below the freezing points, the impedance keeps increasing. This result is in contradiction with the result reported by Sauerheber , which sIn two different temperature ranges, between −2 and 2 °C, and from −30 to −20 °C, the impedance increases sharply.The physiologic saline electric impedance was also measured during heating (melting) from −48 to 0 °C. 3O+ and OH−, and the Bjerrum or valence defects.The motion or the kinetic energy of molecules and ions directly depends on temperature. Different composition, therefore, explains that temperature effects on electrical properties can differ among materials. With rising temperature, inter-ionic attraction decreases resulting in reduced viscosity and increased mobility . In addition to this, with temperature increment the degree of dissociation of electrolyte increases, hence the number of ions also increases for conduction . At low If the ice contains any impurities, then the electrical properties largely depend on those impurities . Thus, tIn the last 50 years, the suggestion of electrical point defects in ice, i.e. Bjerrum L (bond with no hydrogens on it), D (bond with two hydrogens) defects, and ionic defects, shaped our understanding of the dielectric relaxation and electrical conductivity of ice. Bjerrum defect states (DL) have mobilities in the same order of magnitude as that generally occurring for ionic transport. This suggests that thermal activation over a potential threshold facilitates the transfer of protons through the jumping of Bjerrum defect states to a neighbouring bond. Therefore, in the ice phase, the impedance of the results can be characterized by the single dispersion Debye relaxation phenomenon.A is a coefficient, E is the activation energy barrier of the dielectric relaxation (eV), k is the Boltzmann constant (8.618 · 10−5 eV/K), and T is the absolute temperature (K).A method to characterize the Debye relaxation is using the Debye relaxation time constant , which iThe frequency and temperature dependences of pure ice (ice of de-ionized water) that we observed here were very similar to the results obtained by other authors. In the impedance, for de-ionized water, a dispersion is only found for the high frequency range but, in the low frequency range, such a dispersion is not observed. However, both low frequency and high frequency dispersions were observed in the impedance of the ice of the de-ionized water. In the liquid physiological saline solution impedance, no dispersion was observed. Nevertheless, the dispersion started to appear in the ice state at lower temperature. 2O) and a ‘conduction’ band (excess protons fluctuating in H bonds), which exhibit increasing impedance with decreasing temperature [Both the frozen solid salt water and de-ionized water behave like a semiconductor where electrons enter conduction bands as temperature increases, the charge carriers (protons) between a ‘valence’ band  formed from platelets of pure ice separated by ordered rows of brine-filled pockets or cells. Brine is also located between the grains. This brine remains unfrozen until the temperature reaches below −20 °C and then start the crystallization. This implies that the presence of this brine plays an important role in shaping the behaviour of the substance. In fact, according to Assur , the temIn accordance with interpretation presented in the literature , during It is apparent from our limited observations, that the electrical properties of physiological saline ice and deionized ice are appreciably different from those of meat ice (frozen meat). The greatest difference appears in the impedance change rate (increasing and decreasing rate as a function of temperature). In all the three materials, impedance increases sharply during initial ice crystallization. Temperature and ice crystal structure appear to play a more important role in controlling the electrical parameters. In addition, at higher frequencies the effect of temperature is considerably less. We have also shown that pure ice has two dispersion spectrum related to temperature. Saline ice has two different temperature points where crystallisation occurs.


Coronaviridae family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis.Belonging to the In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/HRM, we did the sequencing of the partial S1 gene.It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain.Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool. Avian infectious bronchitis (IB) serves as a major threat to the worldwide poultry industry, and it is a disease endemic to Jordan, causing respiratory, renal, and reproductive problems in chickens ,2. IB viThe IBV genome has an approximate size of 27,500 nucleotides and is organized in the following order: UTR5’-POl-S-3a-3b-E-M-4b-4c-5a-5b-N-UTR3’ ,9. In paIBV strains can be classified into six genotypes that together comprise 32 distinct viral lineages, the majority of which belong to genotype 1 . In the High-resolution melting (HRM) curve analysis is a newly established PCR-based technique that has been used to differentiate related strains of the same animal or avian virus -19. ThisApproval from the Jordan University of science and technology animal care and use committee (JUST-ACUC) is not applicable as these samples were collected from recently dead chickens in private farms, and then samples were submitted to our laboratory.IBV vaccines were purchased from local Jordanian markets. The most commonly used IBV vaccine strains in Jordan are Mass-type strains  as well as the 4/91 (793/B) vaccine strain . These vg for 5 min. After that, 150 µl of the supernatant was mixed with 250 µl of Trizol. A SuperScript® IV First-Strand Synthesis kit  was used according to the manufacturer’s instructions to make cDNA for both the vaccine and field samples.The IBV vaccine vials were rehydrated with 1 ml of 1× PBS, then mixed by vortexing to dissolve the vaccines. A 150 µl of the rehydrated vaccine was added to 250 µl of TRI Reagent  and suspended for 5 min in a 50°C dry bath. Then, 400 µl of absolute ethanol was added, and RNA extraction was performed using the Direct-zol RNA MiniPrep kit . IBV field samples, mostly consisting of tracheal and kidney tissues that were submitted to our laboratory for IBV diagnosis, were homogenized and centrifuged at 3000× 2, 0.1 µl Taq DNA polymerase, 1 µl cDNA, and 18.15 µl H2O. Thirty-five cycles of amplification were carried out in a thermocycler, with each cycle consisting of denaturation for 45 s at 94°C, annealing for 1 min at 50°C (for both rounds), and extension for 2 min at 72°C, and a final extension for 7 min at 72°C. Electrophoresis of amplified products was carried out using 1.5% agarose gel, which was visualized and photographed under UV light with a 100 bp ladder. Nested PCR primer sequences for the S1 gene were as previously reported [Nested RT-PCR amplification was performed using the synthesized cDNA to amplify S1 gene fragments of the IBV genome. The first and second rounds nested PCR was performed in a reaction volume of 25 µl that contained 2.5 µl of 10× PCR buffer without magnesium, 1 µl dNTPs, 1 µl of each reverse and forward primers, 0.75 µl MgClreported .For this assay, the nested RT-PCR for the amplification of S1 fragment was repeated. However, the S1 RT-PCR products from the first round of nested RT-PCR were used as a template in the HRM protocol. A Type-it HRM PCR Kit  was employed according to the manufacturer’s instructions on the Qiagen Rotor-Gene Q 5plex HRM Platform . The qRT-PCR 25 µl reaction volume contained 12.5 µl of 2× HRM PCR master mix, 1.75 µl of each 10 µM S1 gene internal primers for the second round, and 2 µl of S1 RT-PCR product from the first round of nested PCR. The optimized cycling protocol for HRM analysis on the Qiagen Rotor-Gene Q 5plex HRM consisted of the following conditions: An initial PCR activation step for 5 min at 95°C followed by 40 cycles involving denaturation for 10 s at 95°C, annealing for 30 s at 55°C, and extension for 10 s at 72°C. HRM is for 2 s from 65 to 95°C with 0.1, 0.2, and 0.3°C increments.Two softwares were used for the analysis of the HRM results. The operating HRM software installed on the Rotor-Gene Q 5plex machine and the Rotor-Gene ScreenClust HRM software  was used to analyze the results of HRM and to assign the different IBV strains into clusters according to their HRM patterns. The HRM analysis using the operating software was done by setting a reference IBV genotype either as 4/91, Mass, or variant II genotypes, after normalizations, the HRM genotype confidence percentages (GCPs) were obtained. For the ScreenClust software, the unsupervised model was used and then data were normalized and clusters were generated to contain all IBV isolates. The typicality and probability of each sample was recorded.The S1 RT-PCR products of the nested RT-PCR amplification were treated with ExoSAP-IT PCR Product Cleanup Reagent  according to the manufacturer’s instructions. Briefly, 2 µl of ExoSAP enzyme were added to 5 µl of the product then incubated at 37°C for 15 min to degrade remaining primers and nucleotides followed by another incubation at 80°C for 15 min to inactivate the ExoSAP-IT reagent. The cleaned RT-PCR products were sent to a sequencing facility  to be sequenced by chain termination technology. S1 sequences were viewed on the BioEdit software and edited by the Edit Sequence interface of the Lasergene package. Sequence alignment, calculation of the sequence nucleotide similarities, and construction of the phylogenetic tree were done by the MegAlign interface of the Lasergene package. The partial sequences of S1 gene of all IBV isolates included in this study were deposited in GenBank under the accession numbers; MK680008-MK6800034.To detect the field and vaccine IBV strains, we used a nested RT-PCR assay to detect the S1 gene. Twenty-two out of the 23 samples were positive for the S1 gene after nested RT-PCR. Those 22 samples, along with the five IBV vaccine reference strains, were subjected to qRT-PCR targeting the S1 gene followed with HRM curve analysis. The first cluster consisted of the IBV 4/91 and IBV 4/91-like strains. Three vaccines and three field isolates were located in this cluster. The IBV strain 4/91 can be found both as a vaccine and as a field strain and many IBV infections in different countries have been linked to 4/91 field strains. The IBV field samples  belonged to cluster 1, representing the 4/91 and IBV 4/91-like strains, respectively. Based on HRM analysis, the melting temperature of the 101216D sample (78.30°C) was close to that of the 4V (1/96) strain (78.09°C), while the melting temperatures of the 7P6 sample (77.49°C) and the 3V (CR88 variant) strain (77.09°C) were similarly close. Despite the variation in melting temperatures between the vaccine and field strains in cluster 1, HRM and ScreenClust analysis were still able to identify the similarities between them by classifying them into the same cluster.The second cluster contained the IBV Mass and Mass-like strains. Two IBV vaccine strains were located in this cluster along with 10 field IBV isolates, three of which were found to be of a variant II-like (1S/1494/06-like) strains on sequencing . While cluster 2, which includes IBV mass and mass like strains, has a closer range between the melting temperatures (78.24-78.51) with less variation up to 0.27. This indicates that the viruses of the field samples share close sequences to the Mass vaccine strain.The third cluster contained the IBV variant II-like (1S/1494/06-like) strains, and only field isolates were found in this cluster. This cluster has the highest difference in the melting temperature that reaches 0.75. Eight samples were of variant II-like (1S/1494/06 like) IBV strain representing 34% of the total IBV samples.The fourth and final cluster included one IBV field strain (072017), the latter of which was found to be Q1-like strain on sequencing.HRM analysis has been used to detect and genotype fowl adenoviruses, avian influenza virus, and avian nephritis virus, among others ,21,22. TRegarding cluster 3, the variant II-like (1S/1494/06-like) stain is an endemic IBV strain in Jordan that is involved in the respiratory, kidney, and reproductive tract problems ,24. ReceConcerning cluster 4, sample 072017 was found to belong to the unique Q1 strain, which was first reported in China, in 1996, and then in Italy during a 2011 outbreak in broilers ,27. EvenOur qRT-PCR with HRM/spike gene assay was able to genotype all vaccine IBV strains and cluster them into separate clusters according to their distinct genotypes . Most of the IBV field samples belonged to the variant II-like (1S/1494/06-like) strain. Those variant II-like (1S/1494/06-like) strains were all classified into one cluster expect for three samples that were clustered within the Mass and Mass-like IBV strains. The current qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains which are confirmed by S1 gene nucleotide sequencing, making it a rapid and cost-effective tool.MA and OA designed the experiment. OA performed the technical assays. MA and MBA supervised the technical assays, MA and OA analyzed the results. MA and OA prepared the manuscript. All authors read and approved the final manuscript.


Bacillus velezensis strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners  = 0.011, 0.005, 0.056, and 0.005 mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the B. velezensis lipopeptides would have great potential to act as lipase inhibitors.Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a  Obesity is a complex, multifactorial disease that arises from the interaction of excessive caloric intake, sedentary lifestyle, metabolic disorder, and genetic predisposition,,Dietary and lifestyle modifications such as calorie restriction and physical exercises are the common strategies adopted to control body weight; further, these methods have limited anti-obesity effectsBacillus is described as an efficient source of lipopeptide biosurfactants. The Bacillus lipopeptides are divided into three different families, including iturins, surfactins, and fengycins, consisting of a cyclic lipoheptapeptide or decapeptide with a long hydrophobic fatty acid moietyBacillus lipopeptides possess high biodegradability, biocompatibility, and high stability towards extreme environments. These remarkable properties make lipopeptides potent candidate drugs for therapeutic medical applicationsInterestingly, surfactants were found to produce inhibitory effects on the lipolytic efficiency of lipase by generating inactive aqueous enzyme-surfactant complexes or by blocking the congregation of enzymes at the lipid/water interfaceBacillus subtilis SPB1 could significantly reduce the body weight of obese rats and relieve hyperlipidaemia without apparent side effects,,B. subtilis SPB1 lipopeptides consist of iturins, surfactins, fengycins, and other lipopeptide isoforms. Moreover, the surfactins were speculated to be the major contributor to the anti-obesity effects of B. subtilis SPB1 lipopeptide. However, it is still unknown whether all the types of lipopeptides display the inhibition effect on the lipase. Considering the structural differences between different lipopeptide families, the comparative studies of lipase inhibition activities of surfactin, iturin, and fengycin would be important for the application of lipopeptide as lipase inhibitor. The aim of this article is to report a new lipopeptide-produced Bacillus velezensis strain FJAT-52631 that could coproduce iturin, fengycin, and surfactin and to evaluate the inhibition activity of each type of lipopeptide. Furthermore, the action modes of lipopeptide on lipase catalysis was carried out.It had been reported that lipopeptides of 2.2.1.Mucor miehei lipase (EC3.1.1.3), 4-Nitrophenyl palmitate (4-NPP), iturin, and surfactin were purchased from Sigma-Aldrich . Acetonitrile, hydrochloric acid (HCl) and Tris were purchased from Sinopharm .Lyophilised powder of The strain FJAT-52631 (CCTCC No. M 2019760) was isolated from a soil sample from Wuyi Mountain, Fujian Province, China and it was identified through whole genome sequence analyses.2.2.A single clone of the strain FJAT-52631 was inoculated in a 25-ml sterile tube with 5 ml liquid culture media  and incubated for 25 h at 30 °C and 170 rpm. The pre-culture was inoculated (1%) into 250-mL flasks with a 50 mL potato dextrose broth culture medium and then cultivated for 48 h in a rotary shaker at 30 °C, 170 rpm. After fermentation, the cells were removed by centrifugation (6000 g for 5 min) and the lipopeptide in the culture supernatant was precipitated by adding 3 N HCl to achieve a final pH of 2. The precipitates were dissolved in a phosphate buffer and then lyophilised for anti-lipase activity tests and liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) analyses.2.3.18 solid phase extraction method with methanol/water (v/v) as an elution solvent. Each elution fraction was evaporated at a reduced pressure , dissolved in water and then lyophilised.The qualitative and quantitative analyses of lipopeptides produced from the FJAT-52631 were carried out using the LC-QTOF-MS/MS method described in our previous studies2.4.The lipase inhibition was determined according to the method described by Liu et al.m versus the inhibitor concentration.The inhibition mechanisms were studied by fixing the concentration of substrate and changing the lipopeptides and enzymes to monitor enzymatic reaction. The inhibition types were determined based on the Lineweaver-Burk plot2.5.Ultraviolet (UV) wavelength scanning spectra of the 4-NPP hydrolysis product was measured in the absence and in the presence of lipopeptide using a model 1510 spectrophotometer . The 1 mL reaction mixture contained 0.75 mmol/L 4-NPP and 0.015 mg/mL of lipopeptide in Tris-HCl buffer (pH 7.8). The final concentration of the lipase was 0.4 µg/mL.sv) was calculated according to F and F0 are the fluorescence intensities with and without lipopeptide, respectively; further, [I] is the concentration of the lipopeptide. The binding constant (KA) and the binding affinity (n) were calculated according to the Fluorescence quenching spectra of the lipase and the lipopeptide were carried out according to the method described by Liu et al.2.6.The interactions between lipopeptide and lipase were modelled using molecular operation environment software (MOE). The energy between the lipase and the ligand was minimised before docking. The docking parameters were set according to a previous study3.3.1.Bacillus velezensis CBMB205T was 99.95%. This value was above the threshold ANI value of 95–96% used for delineating prokaryotic species, suggesting that strain FJAT-52631 is a strain of the species B. velezensisA lipopeptide-produced strain FJAT-52631 was isolated from the soil sample, which was further accurately identified through the whole genome sequence analyses. The genome sequence of strain FJAT-52631 contains 3929791 bp (GenBank accession number: CP045186). The G + C content of the chromosomal DNA for strains FJAT-52631 was 46.5 mol %. The genome-based similarity calculated based on the OrthoANIu between strain FJAT-52631 and the type strain 3.2.2 spectral data, and identification results were summarised, as shown in 14–C16 iturin A; the surfactins consisted of C12–C16 surfactin A, and C16 surfactin A derivative; further, the fengycins consisted of C16/C18 fengycin A, C16 fengycin A2/B2, C16–C17 fengycin B, and C15 fengycin A/B derivatives. The iturin, fengycin, and surfactin content in the supernatant were calculated as 2.66 ± 1.50, 86.95 ± 4.08, and 35.93 ± 2.28 mg/L, respectively. The results demonstrated that fengycin was the most abundant lipopeptide family that was produced by the strain FJAT-52631.The lipopeptide production from Bacillus spp. firstly depends on itself. For example, the B. subtilis SPB1 strain have the ability to coproduce iturins, fengycins, and surfactins and highest content of surfactin in lipopeptide mixture was observedThe lipopeptides produced from the strain FJAT-52631 were identified using the LC-QTOF-MS/MS method. Results revealed that three sets of homologue molecules with retention times in the range of 12.2–21.5, 27.0–38.7, and 45.2–54.0 min could be categorised as iturins, fengycins, and surfactins, respectively. Peptide sequences of each lipopeptide group were determined based on previous literature reportsThe crude lipopeptide from strain FJAT-52631 was further purified by solid-phase extraction method, and characterised by LC-TOF-MS analysis. Results show that the purified fengycin in the crude lipopeptide was obtained by elution with 80% methanol , while t3.3.Bacillus group have been proved to be excellent surfactants and could reduce lipase activity in plasma from alloxan-induced diabetic ratsM. miehei and human pancreatic lipase are serine proteases, which possess the same active site and catalytic modeM. miehei exhibits much higher enzyme activity, better purity, and more mature processes than the human pancreatic lipaseM. miehei lipase was selected for use in the present study.As reported in literatures, surfactants could inhibit lipase activity at certain concentrations due to their special property50) of crude lipopeptide, purified fengycin, iturin, and surfactin standards were 0.011, 0.005, 0.056, and 0.005 mg/mL, respectively. We have previously reported that furoic acid and oxalic acid inhibited lipase with IC50 of 0.242 and 1.425 mg/mL, respectively50 = 0.004 mg/mL)Results showed that lipase inhibition was dose dependent. After adding these four inhibitors, the relative enzyme activity decreased significantly with increasing concentration of effectors. The half maximal inhibitory concentration ; this indicated that the fluorescence quenching mechanism of lipase is static when the lipopeptide is presentF0−F)/F] = 1.429lg[I] − 0.108 (R2=0.9255) was obtained from A and binding site n from the intercept of the above equation and the slope were calculated as 1.429 L/g and 0.78, respectively. This result indicates that the interaction between lipase and lipopeptides is quite intensive and there is only one binding site between themThe fluorescence emission spectra of crude lipopeptides on lipase are shown in obtained . The val3.6.M. miehei lipase have three active site residues Ser, His, and Asp that forming an oxygen negative ion hole, which are covered by an “alpha-screw lid”,The M. miehei lipase activity by direct interact with the active site, which are closely related to the amphiphilic molecular structures of lipopeptides.Studies showed that the small molecules are easily to enter the gap in the oxygen negative ion to interact with the catalytic site of the enzyme. For example, the furoic acid and oxalic acid can directly act on active amino acid of Ser, while the big molecular orlistat does not contact with the active amino acids of, Ser, His or Asp4.B. velezensis against lipase in vitro. The lipopeptides produced from B. velezensis FJAT-52631 were categorised as iturin, fengycin, and surfactin. The crude lipopeptides, iturin, fengycin, and surfactin exhibited strong inhibition activities against lipase with dose-dependence. The lipopeptide inhibition catalytic reaction was reversible and the inhibition type was competitive. The fengycins and surfactins directly interact with the residues of the enzyme’s active centre, while iturins do not. Fengycins were the most abundant compounds among the crude lipopeptides, which were considered to contribute significantly to the inhibited activities against lipase. Our results suggest that these lipopeptides could be used directly as lipase inhibitors and the in vivo anti-obesity effects of lipopeptides will be explored in further studies.We report here on the strong inhibitory activity and action modes of lipopeptide from