The capital element in the field of osteopathy and several other manual therapy methods, is the somatic dysfunction (SD), a functional imbalance that can involve different tissues and mechanisms in its genesis and maintenance. The main challenges found in the clinical scope are to understand the interaction, hierarchy, and relevance of the SD. Several manual tests are available to functionally evaluate the SD, each one with its applicability to analyze the different parameters of the SD. The so-called inhibitory tests are a category of functional manual tests that can be added to the diagnostic context of the SD. It is a particular type of test in which the evaluator applies manual mechanical stimuli to dysfunctional tissues and assesses the biological responses that occur simultaneously with the application of the stimulus. Its use can consider biomechanical and neurological principles in such a way that different conditions can be analyzed. The objective of this article is to review well-established knowledge and recent scientific discoveries about the SD and its local and global repercussions, in an attempt to offer ideas that can be applied to better understand the mechanisms that imply the use of inhibitory tests as complementary clinical diagnostic tools. It will be discussed some of the possible mechanisms involved in the physiology of the inhibitory tests, their practical applications in some distinct conditions, as well as new proposals of utilization based on the sensitization of metameric related structures under a dysfunctional state. The somatic dysfunction (SD) plays a central role in osteopathy and in several other methods in manual therapies. It is a functional disorder considered an obstacle to the inherent self-regulatory capabilities of the human body, characterized by clinical parameters that involve increased tissue “tension/density”, positional “asymmetries”, “restricted” mobility, and “tenderness” (TART) . SeveralClinical evaluation can reveal several SDs in the same patient, and some of the main challenges are to understand the relevance and hierarchy of these disorders in the individual homeostasis so that the therapeutic approach can be as accurate as possible. Several manual tests that analyze the SD parameters are described in the literature, each presenting its benefits and limitations . They muSomitogenesis: embryology of the segmentation processDue to the rapid development of the neural tube, a transverse vascular network appears through the dorsal aorta to nourish the high metabolism of ectodermal neural cells. These events are preponderant in the segmentation process . The firThe spinal cord segment and the sensitization mechanismsA spinal cord segment monitors and integrates different body tissues, promoting an intercommunication network between metameric related structures, such as organs, vessels, nerves, bones, skin, etc . When stPeripheral tissue receptors remain sensitized  due to the dorsal root reflex, lowering their activation thresholds. The consequences of this process are allodynia when a non-nociceptive stimulus causes the perception of pain; and also hyperalgesia, in which a nociceptive stimulus causes an excessive sensation of pain. Hyperalgesia is considered primary when related to tissues that initially cause afferent bombardment and the process of neurogenic inflammation, and secondary when it affects other tissues that converge to the same spinal cord segment (WDR) but are not the primary source of stress . The exaSome experimental studies have already shown that central sensitization can be primarily promoted by somatic or visceral tissue receptors and that this process can have a direct impact on other tissues that converge inputs to the same affected spinal segment through WDRs that are hyperexcited due to the influence of pain mediating substances such as glutamate and substance P -20. A frDue to these mechanisms, knowledge about the possible metameric relations between different tissues is of great importance, so that the process of searching for the interactions and degrees of relevance of the SDs can be as accurate as possible. From the potential relationships of a tissue stress/injury and its repercussions in other neurosegmentally related structures described above, it is important to consider that a visceral noxious input can affect the somatic tissues afferences of the same segments, producing symptoms in the locomotor system (viscerosomatic reflex) or in another viscera that converge their neural information  . An extrThe somatic dysfunction - its models and clinical featuresThe SD is defined as "... the impaired or altered function of related components of the somatic (body framework) system: skeletal, arthrodial and myofascial structures, and their related vascular, lymphatic, and neural elements" . DiffereDiagnostic tools for the SDs and the inhibitory testsThe process of clinical diagnosis of the SDs is performed through manual tests that initially are carried out globally in order to "scan" the body so that areas that need more detailed analysis, from global to local, are found. The segmental tissue evaluation is the final step within the analysis process. Several manual tests that analyze the SD parameters are described to assess each body structure . Among tThe inhibitory reflex evoked by the touchManual contact is often used during the SD evaluation procedures and its perception is a process that occurs through different mechanisms. In addition to the evaluator's haptic perception, touch can also be applied with the intention of temporarily modifying tissue biological activities, so that some repercussions on body systems can be analyzed . The traPossibly several mechanisms are involved in inhibitory reflexes provided by the manual soft contact on dysfunctional zones, whether they are triggered at the local tissue level, or even through the activation of different areas of the CNS. Three distinct mechanisms that interact with each other, will be discussed below, and hypothetically are triggered during the application of this category of functional test: 1. the proprioceptive touch; 2. the interoceptive touch; and 3. local and global changes in the fascial tissue.1. The proprioceptive touch: the mechanical stimulus provided by touch can activate tactile and proprioceptive mechanoreceptors that have low activation thresholds. These receptors inform the CNS by thick-type myelinated fibers of type I and II, or A𝜶 and Aβ, with high conduction speeds (30 to 75 m/s). The role of the activation of these fibers in the process of modulating nociceptive information provided by type C fibers has long been known . The lar2.The interoceptive touch: in addition to the pathways involved by tactile and proprioceptive stimuli, it is plausible to consider the role of pathways that involve the activation of mechanoreceptors innervated by type C unmyelinated fibers, the free nerve endings (FNE) that are spread across the entire body, making up about 80% of the sensory receptors of the fascial tissue . These m3. Local and global changes in fascial tissue: fascia forms a network that surrounds and invests various tissues at different levels, providing form and becoming an element of structural interconnection, nourishing, innervating and allowing the transmission of forces/movement through all body systems, from solid to liquid structures, from macro to micro -42. ThisPractical applications of inhibitory tests as diagnostic tools for the somatic dysfunctionsThe inhibitory tests are a category of manual tests that can be applied in the diagnostic scope of the SDs so that therapeutic decisions are made accurately while respecting individual needs. It aims to analyze the relevance of the found dysfunctions, searching for their hierarchy and interactions. Lunghi emphasized that inhibitory tests may also be intended to define the type of approach that will be applied . He highIn one of these proposals, Lunghi and colleagues propose the application of inhibitory stimuli in dysfunctional tissues so that their relationship with postural balance tests or also with semiological tests  can be analyzed . The autIt is also possible to consider the applicability of the test under conditions in which the evaluated individual presents the sensation of pain awakened by any body movement that can be reproduced in the clinical setting. In this case, the inhibitory test would seek to analyze the relationship of SDs previously and properly diagnosed, with the pain condition presented by the individual. To perform this “challenge”, it should identify the body movement that triggers the pain and/or decreased range of motion. Then, as shown in Figure Chauffour and Fusco proposed the use of inhibitory tests in order to establish the dominance and hierarchy of dysfunctional patterns , 49. Its2) on a region distant from the patient's main symptom, and ask the patient to quantify the pain on a scale (0 - 10). Then, the evaluator must contact and inhibit one of the previously diagnosed SDs and again apply the pressure in the selected zone previously chosen. If the inhibition of the SD causes a significant reduction or absence of the pain sensation, the test can be interpreted in such a way as to show the relationship of this SD as the general state of central sensitization  and/or skin of the sensitized spinal segment, and ask the individual being evaluated to quantify the perception of pain  at the moment. Then, the evaluator should apply an inhibitory stimulus in any dysfunctional structure metameric related to the sensitized spinal segment, and that supposedly can be responsible for a primary aberrant afferent activity in the genesis of the disorder presented. The inhibited structure can be the projection of a synovial joint, a visceral fascia, a muscle/tendon, a nerve, a cranial suture, the skin (e.g. scars), a bone, etc. It is recommended that this manual contact applied during the inhibition stimulus does not trigger the perception of pain to the evaluated individual, but only a tactile sensation, so that the CNS might not be bombarded with nociceptive information. Simultaneously with the inhibitory stimulus, the evaluator must again generate the same manual pressure at the vertebral segment initially tested. If the inhibitory stimulus is capable of promoting the disappearance or significant reduction in the perception of pain in the vertebral segment initially tested, it is proposed the interpretation hypothesis that the mechanical stimulus in the tissue primarily responsible for central sensitization could "silence" the activity of the sensitized WDR neurons, including those neurons that conduct afferences from receptors located on the skin and periosteum of the vertebral segment involved in the process. So this test could reveal a possible relevant imbalance in the genesis and maintenance of an alert state presented in the patient's CNS, as it is shown in Figure Using the principles of this inhibitory test, the clinical practitioner can also keep in mind the possibility of analyzing whether any SD, found during the evaluation process, has a relevant impact on the patient's CNS. Then, after diagnosing an SD in any structure , the evaluator should search for a sensitized spinal segment metameric related. If this interaction is present, the evaluator should seek to quantify the perception of pain in the sensitized spinal segment, then inhibit the peripheral structure in dysfunction and finally analyze the responses of the inhibitory stimulus on the sensitized spinal segment. If the test is positive (significant reduction or absence of pain), it reveals the relevant imbalance condition of the DS on the homeostasis of the assessed individual.Based on the characteristics described above, of the somatic dysfunctions and its interactions, its local and global repercussions, and some mechanisms possibly related to the inhibitory reflexes generated by the mechanical stimuli provided by the touch, it is plausible to consider the use of the inhibitory tests within the clinical diagnostic context of somatic dysfunctions as an auxiliary tool. Its distinct ways of application can help the clinician to understand issues related to the relevance, and also the hierarchy of the somatic dysfunctions found so that its approach can be specific and assertive. However, we suggest that scientific studies should be carried out to specifically evaluate the mechanisms evoked by inhibitory tests, its clinical applicability, as well as the advantages and limitations of this manual functional test.


Reflecting climatological conditions, the cave ice undergoes repeated freeze-thaw cycles and regular yearly deposition of fresh ice. Three distinct ice block samples, collected from the frozen lake in May 2016, were analysed to obtain data on ice physicochemical properties and the composition of associated microbiota. Isotopic composition of the ice samples  and a local meteoric water line (LMWL) constructed for monthly precipitation at Postojna were used to estimate the isotopic composition of the water that formed the ice, which had high values of deuterium excess and low concentrations of chloride, sulphate and nitrate. The values of total organic carbon (1.93–3.95 mg/l) within the ice blocks fall within the range of those measured in karst streams. Total cell count in the ice was high and the proportion of cell viability increased along the depth gradient and ranged from 4.67 × 104 to 1.52 × 105 cells/ml and from 51.0 to 85.4%, respectively. Proteobacteria represented the core of the cave-ice microbiome (55.9–79.1%), and probably play an essential role in this ecosystem. Actinobacteria was the second most abundant phylum (12.0–31.4%), followed in abundance by Bacteroidetes (2.8–4.3%). Ice phylotypes recorded amounted to 442 genera, but only 43 genera had abundances greater than 0.5%. Most abundant were Pseudomonas, a well-known ice dweller, and Lysobacter, which previously was not reported in this context. Finally, two xanthophytes, Chloridella glacialis and Ellipsoidion perminimum, known from polar environments, were cultured from the ice. This indicates that the abundance and ecological role of phototrophs in such environments might be greater than previously deduced.Paradana is one of the biggest ice caves in Slovenia, with an estimated ice volume of 8,000 m In recent years, cold habitats such as glaciers and ice sheets2, sea ice3, lake ice4 and permafrost5 have received most attention as regards microbiological studies, mainly because of their sensitivity to climate change. Also, there is a growing research interest related to frozen ecosystems as analogues of extraterrestrial habitats6. Even if the study of these habitats is supported strongly by the rapid development of new technologies  more fundamental ecological research is needed urgently. In this respect there is a lack of information on the dynamics and functions of microbial communities, their management and predictive models, interactions between micro- and macroorganisms in cold habitats, linkage of biodiversity, climate, and function and finally an integrated view encompassing micro- and macroorganisms and the physical environment1.Cold ecosystems include deep oceans, polar regions, high mountains and subterranean caves with a large variety of aquatic and terrestrial ecosystems10. Besides, ice strata of different ages  from Scărişoara Ice Cave in Romania revealed microbiota as an integral part of the ice in terms of diversity, quantity, and activity14. Low temperatures in this cryosphere slow down or stop some metabolic and environmental processes and preserve the habitats as “time capsules”, sources of nutrients and assemblages of organisms. Besides microbiota, ice deposits in caves represent an invaluable archive of paleo-environmental data15. Data from Scărişoara Ice Cave served to construct a model of Holocene winter climate variability in Central and Eastern Europe16. Reconstruction of past climates is not the only application of ice-cave data. Tritium and 14C data from ice caves allow discrimination of the period of ice formation (before or after the 1960s) and age of ice19. Stable isotopes of oxygen and hydrogen and chemical composition provide information on the origin of the ice, e.g. local precipitation, karst and/or surface water20.Ice deposits in caves have been poorly studied as microbial habitats, despite providing important ecological information on microbiota living close to what are normally considered boundary living conditions for some species. The few available studies on the cave-ice microbiome all indicate its high diversity21, investigation of ice caves is now crucial as a step towards collecting and preserving essential climatic and geological data, as well as information about its associated life and related genetic pool. Particularly threatened with decay and disappearance is cave ice, which represents only a fraction of the terrestrial cryosphere. Its impending disappearance is especially evident in karst caves at lower altitudes in temperate zones.Because of the accelerated trend of ice-melt worldwideThe study presents details of the physicochemical characteristics of cave ice, and focuses on the structure of the associated microbiome in ice from the largest ice cave in Slovenia, as a potential basis for future research and comparison with other ice caves. Here, the detail of cave-ice deposition is largely impacted by snow levels, the presence of external organic debris, the influence of cave percolation water, and the effects of strong air circulation. Such extreme environmental conditions could direct the compartmentalization of microbial habitats.22, and above the minimum values reported for surface streams, i.e. 0.1–36.6 mg/l23, indicating a significant input of organic matter for the underground ecosystem. TOC indicates an available in situ source of carbon for the ice microbiome. Nitrogen expressed as nitrate did not exhibit high values in ice samples , hardness and alkalinity, less nitrate, and more sulphate. This could indicate that this ice stratum includes a higher proportion of percolation water, which contains more ions than rain and snow as shown by the differences between the percolation water from the cave Planinska jama (that was used for preparing growth media) and the ice, as shown in Table 25. In the current samples, the pH effect on the microbial community structure is less evident because all the values are rather similar  with the local meteoric-water line (LMWL) constructed for the precipitation station at Postojna , provided the δ18O value − 6.3‰ for the original water before freezing. It represents relatively enriched water, but such a value is not uncommon in daily precipitation in Slovenia27. The ice lake in Paradana is presumably formed by the refreezing of water from melting snow accumulated during the winter months20, with some contribution of water dripping from the cave ceiling. November and December 2015 had only a few days with precipitation in Postojna . However, January and February 2016 had 12 and 20 days with precipitation and monthly totals were high, 152 mm and 312 mm, respectively. The air temperature data adjusted for the elevation difference between Postojna and the Trnovski gozd karst plateau (about 600 m) indicate that about one third of the precipitation in January and one half in February probably fell as snow. The rest was probably a mixture of solid and liquid precipitation, but heavy rains could have occurred as well . Isotopic composition of precipitation varied significantly between and also during individual events. It is known that snow cover can preserve the isotopic composition of the original snowfalls for long periods28. However, individual snowfalls can mix at the entrance of the cave and the isotopic composition of snow accumulated in the cave can also be influenced by thaws caused by temporary increases of air temperature or rainfall. The isotopic composition of snowmelt water that eventually refreezes in the cave is therefore the result of many processes. Further research with better temporal and spatial resolution of samples and sampling of snowmelt water would be needed to improve knowledge on the dynamics and sources of ice formation. LMWLs known from the literature for other precipitation stations in Slovenia, i.e. Kozina, Portorož and Ljubljana that are given in Supplementary Fig. 18O values for the original water, which we consider too high . Postojna is the closest precipitation station to the Paradana and the data on isotopic composition of precipitation cover the period of ice sampling . Although such isotopically light precipitation occurred in just two of the 27 months of the observation period, the two values changed the LMWL intercept significantly. However, because they did occur, they cannot be disregarded in the LMWL construction. Daily precipitation data indicate that in both cases monthly values were influenced dominantly by precipitation that fell during just one day . The LMWL intercept at Postojna without those two months would be 8.3, i.e. closely similar to values in Ljubljana and Kozina. Long-term data from Ljubljana show that the δ18O value of monthly precipitation was lower than − 16.0‰  in only 5 months in the years 1981–2010. Thus, precipitation with notable isotopically light values, as observed in Postojna between 21 and 23 November 2015 (92% of the precipitation fell on 22 November) appears to be rare in the study area. Nevertheless, it was observed, and it influenced the intercept of LMWL significantly.When analysed in more detail, results obtained using the approach described above  also revealed the sensitivity of the constructed LMWL, the length of data series and extreme values. This is illustrated by records of isotopically very light precipitation in November and December 2015  of water from the autumn/winter months, with precipitation from the Eastern Mediterranean air masses having particularly high d-excess (up to 22‰). The Western Mediterranean air masses have d-excess of about 14‰, whereas air masses from the Atlantic have values of only about 10‰29. Late autumn to early winter precipitation in Slovenia (October to December) regularly exhibits high d-excess27. Unfortunately, the available data are insufficient to support analysis of the reason for high deuterium excess of the ice in detail. Study samples also display far lower concentrations of chloride, sulphate and nitrate than samples collected by Carey et al.20.It is worth noting that the δ4–15.15 × 104) to that recorded in the Pivka River (SW Slovenia) at the ponor connecting to the karst underground, i.e. 4.29 × 104–12.38 × 1030. A large proportion (51.0–85.4%) of entrapped microbes in the ice were viable, showing that they were able to survive ice formation and melting, or even several freezing–melting cycles. A relatively high cell viability can be linked to the availability of compatible solutes, indicated by correspondingly high TOC  or depth (0–25 m) and the total number of cells or viability14.The upper ice stratum represented by Ice-1 and Ice-2 had comparable microbial load expressed in total ATP concentration and total cell counts, whereas the Ice-3 block exhibited significantly higher values  and cultivated at 10 °C produced the highest colony counts . Another 35 OTUs had relative abundance above 0.5% and the remaining 76 OTUs had relative abundance below 0.5%. The unique OTUs probably contribute to the variability due to internal variations within the ice block caused by incoming snow or the freezing of percolation water. For example, samples Ice-2 and Ice-3 were cut from the same ice block in a vertical ice profile, but differed in their content of dark, particulate, organic inclusions.A Venn diagram of the distribution of 441 distinct OTUs found in the three studied samples is presented in Fig. Members of 29 bacterial phyla were detected in the cave ice microbiome Fig. . All sam43. Indeed, Proteobacteria are commonly associated with water influx into the karst underground46. The prevalence of Proteobacteria in the ice samples coincided with the decreasing trend of TOC concentration in samples Ice-2, Ice-3 and Ice-1  over Proteobacteria (33.5%), but Proteobacteria were assigned as the largest group of metabolically active microbes14.The second most abundant phylum in the studied samples was Actinobacteria, a group associated with cave sediment communitiesC. glacialis and E. perminimum, were obtained via cultivation.In Paradana the relative abundance of Bacterioidetes phylotypes was comparably lower than that of the other two and was 4.3% in Ice-1, 3.2% in Ice-2, and 2.8% in Ice-3. Three other bacterial phyla represented > 1% of phylotypes in at least one sample and corresponded to Firmicutes, Cyanobacteria and Gemmatimonadetes. Phototrophic bacterial phylotypes belonging to Cyanobacteria were recovered from all three samples. They represented 1.3% of phylotypes in sample Ice-1, but only 0.6% and 0.3% in samples Ice-2 and Ice-3 respectively, from where algae, Phyla whose relative abundance was less than 1% were grouped together and classified as “Rare phyla”. These phyla comprised 2.2%, 1.5% and 1.2% of Ice-1, Ice-2, and Ice-3, respectively. Their relative abundance is presented in Supplementary Fig. 14. Actinobacteria represented the second most abundant group of phylotypes, with its relative abundances declining from 30.8% in Ice-1 to 26.2% in Ice-3 and 11.7% in Ice-2. Other notably abundant classes were Alpha- and Betaproteobacteria, whose abundances ranged from 9.6 to 26.3% and from 6.9 to 12.3%, respectively.Among the 31 classes detected in this study, members of Gammaproteobacteria were most abundant and represented 20.1% (Ice-1), 45.3% (Ice-2) and 42.5% (Ice-3) of total detected phylotypes  and Bacilli (0.6–2.1%), while the abundance of Gemmatimonadetes (0.3–1.8%), the only class of the phylum, was similar to that described in soil, around 2.0%14. The most abundant genera in the Paradana samples were Pseudomonas and Lysobacter (Gammaproteobacteria); however, their abundance varied within the samples.At the genus level, ice cave sequences from three samples corresponded to 442 genera, but only 43 genera had abundances greater than 0.5% in all samples , yet was scarce in Ice-1 (0.5%) and Ice-2 (0.2%). In a contrast, Lysobacter phylotypes are usually found in waters and soils56 and were dominant in Ice-1 (23.6%) and Ice-2 (56.6%), but represented only a minor portion of phylotypes in Ice-3 (0.7%). Lysobacter members are pigment producers; for example, L. oligotrophus synthesises a water-soluble melanin pigment57, which could be partly responsible for dark coloration of the ice.Sphingomonas spp. represented the third most abundant phylotypes. Members of the family Sphingomonadaceae were commonly found in cave drips58. In Scărişoara Cave, members of Sphingomonas were particularly common in recently formed ice strata 14. In Paradana the abundance of Sphingomonas phylotypes followed that of Lysobacter spp. and was 19.2% and 14.5% in Ice-1 and Ice-2, but only 1.1% in Ice-3.Lysobacter is commonly know for its antagonism towards other bacteria under nutrient-poor conditions, but in a direct assay of Lysobacter predation, the population of Pseudomonas fluorescens was not affected59. Furthermore, plant-colonizing Sphingomonas protect plants against pathogenic P. syringae61 but there are reports that Sphingomonas can be outcompeted by fast growing Pseudomonas62.It is possible that the above mentioned dominant bacteria interact with each other, but the nature of any interactions is not yet elucidated. For example, Polaromonas phylotypes are particularly abundant in polar and high-elevation environments owing to the dispersal of dormant cells by air currents63. In cave ice, Polaromonas phylotypes represented a minor proportion of the diversity. They were most abundant in Ice-3 (6.9%), and scarce in Ice-1 (1.1%) and Ice-2 (0.7%). Other notable genera whose abundance exceeded 0.5% were Flavobacterium (abundance range 2.7–3.6%), Nocardioides (abundance range 0.8–3.7%), Pseudonocardia (0.6–3.3%) and Pedobacter (0.5–2.9%), which are common in a variety of natural environments. In particular Flavobacterium is common in a variety of cryospheric habitats64.It is important to mention specific genera that represented a minor portion of diversity. Pseudomonas and Lysobacter were the most abundant genera identified in Paradana. The roles and abundance of phototrophs might also be significant, particularly in caves or parts of caves with available sunlight. It remains unconfirmed, however, whether specific microorganisms or concentrations of microorganisms can provide reliable boundary markers for regular episodes of ice formation in caves.Study of recently formed ice in Paradana revealed no evidence of either uniform depositional conditions or uniform microbial representation, even in the same ice stratum, indicating compartmentalization within a larger ice block. Paradana, and caves having similar geological settings and climatological conditions, are subjected to repeating cycles of freezing–melting events, and ice deposition, that influence the microbiome. In general, ice as a habitat in karst caves at lower altitudes in temperate zones is currently prone to wasting. Soluble organic compounds transported by percolation water and melted snow mitigate osmotic stress, reduce the environmental extremity and support the dynamic nature of the cave-ice microbiome. Proteobacteria, particularly from the class Gammaproteobacteria, represent the core of the cave-ice microbiome in this recently formed ice ecosystem. The studied ice cave Velika ledena jama v Paradani  is situated on the Trnovski gozd karst plateau (1130 m a.s.l.) at the foot of the Golaki ridge . Annual precipitation on the plateau averages 1700–3200 mm, with its maximum in October and November, and with snow-cover present from November until the end of April. The mean annual temperature on the plateau at 1200 m a.s.l. is around 4.5 °C.65. The cave is more than 7,000 m long and 850 m deep. Because of the distinctive funnel-shaped morphology of the cave entrance, snow, soil, leaves, and tree branches slide into the cave. Permanent ice appears at a depth of about 35 m inside the cave. Most of the cave-ice forms in late winter from water that percolates through the ceiling and meltwater from the snow that accumulates in the entrance area. Water flows for some distance along the chilled passage floor before it freezes. Snowmelt produces additional water that partly inundates the passages and creates a frozen lake. This lake was used as a source of ice for cooling and food preservation until the beginning of the twentieth century. The commercial exploitation of ice was documented as early as 1867. Based on available documentation, the ice volume around 1917 was comparatively greater than currently. In the period from the 1950s to 1977 the cave entrance was completely ice-filled, and in 1978 the ice melted and further exploration of the cave became possible. During the following decade, the ice body changed its volume notably. Since 2000, the ice block has predominantly been shrinking in volume and surface area66. Relative mild winters in recent years accelerated the ice melt trend markedly.The entrance to Paradana is at the bottom of a large, 50 m-deep doline, where snow accumulates during the winter and persists until summer due to cold-air circulation from the cave. This creates favourable conditions for temperature and vegetation-zone inversion3. The cave’s continuation beyond Velika ledena dvorana leads to smaller passages, followed by vertical pits and ramps. In this part of the cave, ice is present only to a depth of about − 200 m.The floor of the Velika ledena dvorana cave chamber contains a huge ice body lying over collapsed boulders Fig. . Here, t67. The temperature of the Trnovski gozd karst massif and of the air inside its caves is relatively stable of about 4–5 °C. In winter, when the external temperature falls below 4 °C, an airflow is created leading to transfer of cold air from the surface into the cave. This air re-emerges from the karst massif at higher altitude (about 1400 m a.s.l.) where numerous patches of melting snow-have been observed. Initially the snowmelt is due to the effect of displaced air. The cold air flows through a system of interconnected cavities within the Golaki ridge. The height difference of about 270 m is great enough to create a “chimney effect”68. In the summer, the air temperature at the surface rises above the temperature within the cave system and the airflow reverses its direction. It moves from the lower parts of Paradana, passes the ice body, is cooled to about 1 °C, and exits the cave. Both winter and summer temperature regimes and flow directions are stable. During the summer months, cold airflow creates and maintains a large, stable, volume of cold air in the doline that holds the cave entrance.Both the formation and the preservation of ice have been attributed to the effects of air circulation2 s, < 0.1 W/m2, LI-1500, LI-COR). Ice-2 was cut aseptically from the ice block Ice-2–Ice-3 to obtain the same ice stratum (approximately 15 cm thick) as in Ice-1. Compared to Ice-1 and Ice-2, the Ice-3 block was less transparent, with more dark inclusions of particulate organic material, and it was formed before the Ice1–Ice2 stratum.The exact micro-locations for ice sampling were selected specifically, away from the margins of the large ice body, to ensure comparable ice strata in the samples and to avoid material such as branches, stones and other detritus that are scattered in the ice block. Two ice blocks, Ice-1 and Ice-2–Ice-3, approximately 1.5 m apart, were cut with a chain saw from the frozen lake in Velika ledena dvorana on 09 May 2016 Fig. . At the 69 were also examined.Monthly composite precipitation samples were collected in Postojna between August 2015 and October 2017  and analysed for stable isotopes in order to construct the local meteoric water line (LMWL). Additionally, mean daily air temperature and daily precipitation were measured at Postojna. LMWLs calculated for other precipitation stations in SloveniaContact surfaces of the ice blocks were sterilized using a gas burner before melting in the laboratory. Complete ice melt occurred after 48 h of incubation at room temperature, with the blocks yielding 3.5–5.0 litres of water. All ice samples included layers containing dark inclusions of organic matter, which affected the transparency of both the ice and the water after melting. The water was sub-divided aseptically to provide aliquots for subsequent analyses: 500 ml was used for chemical analyses, 5 ml for cultivation of microbes, total ATP concentration and flow cytometry, 5 ml for isotopic analyses, and 1.5 litre of sample water was used to extract each total community DNA. DNA was isolated from filters after membrane filtration of water through 0.22 µm pore-size filters.−), nitrate (SM 4500-NO3−), ortho-phosphate (SM 4500-P), sulphate (SM 4500-SO42−), and total organic carbon (SM 5310 TOC) were determined following standard methods70. Electrical conductivity (EC) and pH were measured using a portable WTW MultiLine P4 meter.Alkalinity (SM 2320), hardness (SM 2340), and concentrations of chloride  with respect to Vienna Standard Mean Ocean Water. The analytical accuracy was better than 0.2‰ for δ18O and better than 1.0‰, for δ2H. The modified intersection method was used to obtain the isotopic composition of the precipitation that formed the cave ice73. Correlation of δ2H and deuterium excess d (d = δ2H–8δ18O) in water from ice was first checked to exclude kinetic fractionation during water freezing. Then, intersection of the freezing line determined by stable isotopes in samples Ice-1 to Ice-3 and the LMWL constructed for precipitation at Postojna provided the probable isotopic composition of the water that formed the sampled cave ice.Stable isotopes of oxygen and hydrogen were measured by Picarro 2120i and 2130i water isotope analysers using previously described protocols74.Microbial load in the samples was measured as total adenosine triphosphate (ATP) , and expressed in Relative Light Units (RLU). Total cell counts and cell viability were determined by flow cytometry. The Cell Viability Kit with Liquid Counting Beads (BD Biosciences) was used according to the manufacturer's instructions on a FACS Canto Flow Cytometer (Becton Dickinson) equipped with three lasers. At least 30,000 events were acquired. Data analysis was carried out using BD FACSDiva 6.1.2 software (Becton Dickinson)3 mg/l; and alkalinity 256 CaCO3 mg/l. All plates were incubated under aerobic conditions. In addition, a subset of RIDACOUNT plates were incubated anaerobically using GENbag anaerobic airtight bags (Biomérieux). Plates were cultivated at 37 °C for 2 days, 20 °C for up to 14 days, 10 °C for 28 days, and 5 °C for 28 days.Colony counts in water samples were expressed as colony-forming units (CFUs) per ml using the following media: RIDACOUNT Total Aerobic Count (R-Biopharm); RIDACOUNT Yeast&Mold Rapid (R-Biopharm); nutrient agar ; R2A agar (Merck); malt extract agar ; tap water agar (TWA) and percolation water agar (PWA). The PWA contained cave percolation water from Planina Cave  with the following physicochemical characteristics: pH 8.27; EC 525 μS/cm; chloride 1.3 mg/l; nitrate 15.7 mg/l; sulphate 4.3 mg/l; ortho-phosphate 0.0 mg/l; hardness 261 CaCO75.To estimate bacterial r/K growth strategy, water samples were inoculated onto R2A agar plates and incubated at 20 °C. Microbes that grew after 3 days were classified as r-strategists (fast growing opportunistic species at low population densities), and microbes that grew within an additional 4–7 days as K-strategists34. For cell-size estimation, 30 randomly selected cells were measured, and the average size and standard deviation were calculated  and incubated in a laboratory with moderate natural illumination and at room temperature. After 33 weeks, algae were identified by examination under a Nikon Eclipse TE 300 phase-contrast microscope equipped with a digital camera and Nikon software NIS Elements. Cells were identified according to the specialized key of Kol and Flint260/A280), were used for subsequent sequencing.Water (1.5 l) from melted ice blocks was filtered through two 0.22 μm pore-size filters . Filters were used to isolate total community DNA using a MoBio PowerWater DNA Isolation kit. DNA purity and concentration were determined spectrophotometrically using standard absorbance at 260 nm and 280 nm on a Perkin Elmer Lambda 25 UV–VIS Spectrometer equipped with UV CellTray . The 260/280 absorbance ratios ranged from 1.63 to 1.74. The best DNA results from the duplicates, in terms of quantity and purity . Each sample was sequenced once. Ion 16S Metagenomics Kit  was used to amplify V2-4–8 and V3-6, 7–9 regions of 16S rRNA in two separate PCR reactions consisting of initial denaturation at 95 °C for 10 min, 25 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 20 s, and polymerisation at 72 °C for 20 s, followed by a final extension step at 72 °C for 7 min.Amplified DNA was purified using Agencourt AMPure XP . Purified PCR products were quantified on LabChip GX Touch using HT DNA High Sensitivity Reagent Kit . Fifty nanograms of amplicons were processed to make the DNA library using Ion Plus Fragment Library Kit and barcode adapters Ion Xpress Barcode Adapters Kit . Size distribution and number of processed libraries were evaluated with the LabChip GX instrument . Each sample was adjusted to 20 picomolar concentration. Equal volumes of all samples were combined and processed in emulsion PCR and enrichment steps with One-Touch 2 and One-Touch ES systems using the Ion PGM Hi-QT View OT2 Kit . Finally, the libraries were sequenced in a 316 v2 chip on the Ion Personal Genome Machine (PGM) using the Ion PGM Hi-QT View Sequencing Kit following the manufacturer’s protocol .76. Primer sequences were removed from the reads, and reads shorter than 150 bp were removed from the dataset. The sequences were aligned against the Curated Greengenes 16S reference library v13.5. Minimum alignment coverage was set to 90% and the read abundance filter set to 10 reads. Genus-level OTUs were determined at 97% sequence identity and species-level OTUs at 99% sequence identity. Rarefaction curves were determined based on Chao1 metrics. Vegan Community Ecology77 and Bioconductor78 packages were used within an R free-software environment79.Raw reads were analysed using cloud service Ion Reporter Software v.5.10, which implements QIIME's open-source bioinformatics pipelineto calculate the alpha-diversity measures Chao1, ACE and Shannon index, to produce heatmaps, and to generate a Venn diagram.raw sequencing reads were deposited in the NCBI Sequence Read Archive under the BioProject accession number PRJNA635015 corresponding to BioSample accession numbers of SAMN15015198 (sample Ice-1), SAMN15015199 (sample Ice-2) and SAMN15015200 (sample Ice-3).Supplementary Information


All anthocyanin-3-glucosides showed a mixed inhibition with a strong competitive proportion, Kic < 134 µM and Kiu < 270 µM; however, the impact of the B-ring substitution was not statistically significant. UV/Vis detection failed to examine the inhibitory effect of acylated cyanidins isolated from black carrot (Daucus carota ssp. Sativus var. Autrorubens Alef.). However, ITC measurements reveal a much stronger inhibitory effect compared to the cyanidin-3-glucoside. Our results support the hypothesis that anthocyanins are efficient α-amylase inhibitors and an additional acylation with a cinnamic acid boosts the observed effect. Therefore, an increased consumption of vegetables containing acylated anthocyanin derivatives might help to prevent hyperglycemia.An inhibitory effect on α-amylase and α-glucosidase is postulated for polyphenols. Thus, ingestion of those secondary plant metabolites might reduce postprandial blood glucose level (hyperglycemia), which is a major risk factor for diabetes mellitus type II. In addition to a previous study investigating structure−effect relationships of different phenolic structures, the effect of anthocyanins is studied in detail here, by applying an α-amylase activity assay, on the basis of the conversion of 2-chloro-4-nitrophenyl-4-O-ß-galactopyranosyl maltoside (GalG Anthocyanins are water-soluble natural red and blue pigments found in flowers, fruits, and vegetables. They are polyphenols and belong to the subgroup of flavonoids , consistAnthocyanins are often found in foods or coloring extracts added to foods for coloring purposes . ParticuDaucus carota ssp. Sativus var. Autrorubens Alef.) is a popular source for the preparation of coloring extracts. The five major anthocyanins of black carrot are cyanidin-based structures , followed by UV/Vis spectroscopic detection of the released CNP. As anthocyanins and, in particular, acylated anthocyanins exhibit a strong self-absorption at the corresponding detection wavelength (405 nm), isothermal titration calorimetry (ITC) was used additionally for higher anthocyanin concentrations and the predominantly acylated structures obtained from black carrot. In contrast to the frequently used multiple injection experiment, which provides binding constants between the polyphenols and proteins  = 0−2000 µM) in MES+ buffer, inhibitor [I] and 50 µL enzyme solution  were mixed in a microtiter plate . For blanks, the enzyme solution was substituted with MES+ buffer. The final volume per well was 250 µL. To quantify the reaction product, a linear calibration with eight different CNP concentrations in the range of 0−35 µM was performed for each plate. Dilutions of the anthocyanin stock solutions (0.1% HCl) were equilibrated at pH 6.1 for one hour. Final inhibitor concentrations were limited due to its strong absorption at 405 nm to 12.5, 25, and 50 µM. For each anthocyanin structure, the assay was conducted on two to three different days with freshly prepared stocks of substrate, enzyme, and inhibitor.The enzyme activity assay, based on the hydrolytic cleavage of GalG-workers  and alre-workers . To enabm) and the maximal conversion rate (vmax) values  were calculated by fitting the data by two approaches: (i) According to the mixed inhibition equation (Equation (4)) or competitive inhibition equation (Equation (5)), applying an optimal global fit containing data from all inhibitor concentrations including the control ([I] = 0 µM); and (ii) by an independent fit according to the Michaelis Menten equation (Equation (6)), providing the apparent values Kmapp and Vmaxapp (In addition to the Michaelis Menten constant (K) values , inhibit Vmaxapp  for each50 values of the different anthocyanins were derived from the inhibition constants for different substrate concentrations by the Cheng−Prusoff equation (Equation (9)) [The ICion (9)) .(9)IC50M: Michaelis Menten constant (µM)Kmax: Maximal conversion rate of the uninhibited reaction (µM/min)v0: Observed conversion rate at given substrate and inhibitor concentration (µM/min)v[S]: Substrate concentration[I]: Inhibitor concentrationic: Inhibitor constant, reflecting the competitive inhibition in the equation (µM)Kiu: Inhibitor constant, reflecting the uncompetitive inhibition in the equation (µM)Kmaxapp: Calculated maximal conversion rate, when the Michaelis Menten equation is used on an inhibited curve (µM/min)vmapp: Calculated Michaelis Menten constant, when the Michaelis Menten equation is used on an inhibited curve (µM)K50: Half maximal inhibitory concentration  (µM)ICp < 0.05. Data were processed using Microsoft Excel® (2010).For data analyses and calculations, Origin 2018b  was used. The Levenberg−Marquardt algorithm procedure was chosen for iteration. All assays were performed in two independent replicates at different dates. Values are expressed in arithmetic means ± average deviation (AD). The statistical significance and differences between groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s test. Differences were considered significant when 2CNP conversion to CNP was monitored by the released heat. According to the previous study, ITC measurements were carried out in a single injection experiment set-up on a MICROCALTM PEAQ-ITC calorimeter  ) plotted versus the inhibitor concentration shows a linear trend, indicating a complete inhibition. Linearized plots such as Lineweaver−Burk and Hanes  and (8), ic, Kiu, and their ratio do not differ significantly among the anthocyanidin-3-glucosides (2 (ic ˂ Kiu) with a high competitive portion (Kic/Kiu 0.31−0.45 (i); 0.26−0.58 (ii)). This implies an interaction of the anthocyanin-3-glucosides with both the enzyme and the enzyme-substrate complex. Nevertheless, substrate binding to the enzyme hinders the binding of the inhibitor, and the competitive part of the inhibition dominates. Based on the IC50 values calculated from the inhibition constants according to the Cheng−Prussoff equation (Equation (9)), minor differences in the inhibitory strength are obvious ) and competitive inhibition equation (Equation (5)) with Origin enables the determination of the inhibition constants K and Kiu . Both vaucosides . The lowsides  in the presence of the complex anthocyanin mixture obtained by SPE ), containing five different anthocyanins (BC 1−5)  , exceede control A. A v0 sive HPLC  at 12.5 ive HPLC B,C. As tive HPLC A, contaimax) in experiments with BC 4 is always lower than the control. Therefore, despite improper determination of the conversion rate, analyses of the inhibitor type by linearization plots according to Lineweaver−Burk and Hanes  and a Kiu value markedly exceeding Kic also support a predominantly competitive inhibition. The Kic value of 20 µM should only be a rough estimation due to the general error  in the UV/Vis detection. However, it indicates an improved inhibition compared to the non-acylated anthocyanin-3-glucosides  and (5), respectively . The higucosides . ic, Kiu), the ITC data demonstrate that the inhibition strength of acylated anthocyanins isolated from black carrot overtakes non-acylated compounds. Furthermore, the impact of a mono- or disaccharide seems to be negligible.Due to the simulated increase in enzyme activity by addition of black carrot anthocyanins determined by UV/Vis spectroscopy, the inhibitory effect was additionally investigated by ITC to prove the assumed stronger inhibition of α-amylase by acylated structures than by non-acylated anthocyanins. The thermogram, as well as the extracted Michaelis Menten plot, shows a significantly stronger inhibition of BC 4 compared to the non-acylated BC 2 and Cyd-3-glc . For thein vitro and in silico for flavonols and (iso)flavones by Piparo et al. [50 180 ± 20 µM, Mlv-3-glc IC50 675 ± 73 µM [50 24 ± 3 µM, Peo-3-glc IC50 75 ± 7 µM [Our data reveal that the substitution pattern on the anthocyanin B-ring seems to have little impact on α-amylase inhibition. Previous STD-NMR investigations of our group on the interaction epitope of various polyphenol structures point toward a major impact of the conjugated system . Thus, io et al. . For the ± 73 µM ; Cyd-3-g5 ± 7 µM ). Howeve5 ± 7 µM .50 180 ± 20 µM; Cyd-3-rut IC50 200 ± 24 µM) [50 24 ± 3 µM; Cyd-3-rut IC50 31 ± 7 µM) [The effect of the glycosidic side chain is discussed controversially in the literature. Our data based on ITC experiments suggest no significant difference  between ± 24 µM)  and Sui  ± 7 µM) .H value); nevertheless, the pKH values of the non-acylated anthocyanin-3-glycosides in aqueous solution are comparatively similar around 3 . Thus, ycosides .H above 4.3), and, thus, decay is reduced. The improved inhibitory strength of the acylated structure might be explained by the additional aromatic system of the acid and, therefore, stronger interaction with the enzyme . Furtheriu values (ic ˂ Kiu).A competitive inhibition type has been characterized for Cyd-3-glc and Peo-3-glc by Sui et al.  and for u values  and pooru values  support As anthocyanins are also promising glucosidase inhibitors , an incrOur data show a marked inhibition of porcine intestinal α-amylase by anthocyanin-3-glucosides, independent of the B-ring substitution, and the presence of mono- or diglycosides at position C-3. The UV/Vis-based enzyme activity assay was unfeasible for acylated anthocyanins isolated from black carrot. However, reaction monitoring by ITC indicates a markedly stronger inhibition of the α-amylase by the acylated structures compared to anthocyanin-3-glycosides. To the best of our knowledge, the effect of acylated anthocyanin structures on α-amylase activity has been studied for the first time, indicating that a diet with an increased proportion of vegetables containing acylated anthocyanins might be a successful approach to prevent hyperglycemia and diabetes mellitus type II.


Allergic responses are mainly caused by IgE, which is often located on the cell surface. The current diagnostic method detects both allergen-specific IgE and total IgE levels, but a number of allergic patients have a normal serum IgE level, which is a poor clinical correlate for allergy. Here, we developed a simple method to detect the level of cell-bound IgE by dissociating it from blood cells with lactic acid. Dissociated cell-bound IgE and plasma IgE levels were detected using the same ELISA kit at the same time. We established two clinical cohorts: an allergic patient group and a healthy participant group. In general, cell-bound IgE correlated well with plasma IgE; however, some patients exhibited high cell-bound IgE levels but low plasma IgE levels. We recommended 350 ng/mL peripheral blood total IgE (cell-bound IgE + plasma IgE) as the cut-off value for allergy diagnosis. Using this indicator, 90.32% of our allergic patients were correctly diagnosed. The peripheral blood total IgE level is a promising clinical diagnostic indicator in allergic patients and will provide more guidance for allergy diagnosis and therapeutic evaluation. The half-life of free IgE in the blood is only 2–3 days, while IgE bound to the high-affinity receptor FcεRI on mast cells or basophils is stable for several weeks5. Most IgE is bound to cells through its receptors, leaving only a small proportion free in the plasma6. Serum IgE levels are very important for the diagnosis of allergies and generally correlate with disease severity7. However, the clinical detection of IgE is limited to free serum/plasma IgE, which ignores the large contribution of cell-bound IgE8. A number of allergic patients have normal serum IgE levels, which is why the World Allergy Association does not recommend the use of total IgE as a diagnostic guideline for allergy9. The level of free IgE in the blood is usually measured by ImmunoCAP10.The concentration of IgE in the circulation is very low ; it is the least prevalent antibody type, with a level much lower than the normal level of IgG (5–10 mg/ml)11. Not all allergens that are in extracts have been defined at the molecular level yet. Other allergens have been well characterized but have not been produced at the quality level required for component-resolved diagnostic tests. The skin prick test is the gold standard for diagnostic allergy tests and is used to confirm allergic sensitization to suspected allergens and provide guidance for the treatment of patients. While this test can be uncomfortable for patients, it also has an occasional risk of infection, though it is relatively safe.Allergen-specific IgE is the causative agent of allergic disease. Several studies have reported that specific IgE levels correlate well with the severity of allergy; however, a relatively high number of molecules must be defined and produced at a sufficient quality to cover all clinically important allergen specificities7. FcεRII, also known as CD23, is a low-affinity receptor. Studies have shown that CD23 is expressed on B cells, monocytes, T cells, dendritic cells, platelets and neutrophils8. Eleonora Dehlink et al. investigated the relationships among the levels of serum IgE, cell-bound IgE, and IgE receptors in a pediatric population. The authors analyzed FcεRI, CD23 and cell-bound IgE on peripheral blood cells by flow cytometry, measured total serum IgE concentrations by ELISA and found that cell-bound IgE on FcεRI+ cells correlated well with serum IgE generally; however, some patients exhibited high amounts of cell-bound IgE but low total serum IgE levels12.There are two IgE receptors: FcεRI and FcεRII. FcεRI is a high-affinity receptor that is ubiquitous on mast cells and basophils. Other types of cells, such as dendritic cells, Langerhans cells, monocytes/macrophages, eosinophils, and platelets, have lower FcεRI expression14. The density of CD23 molecules on B cells has been shown to correlate with total IgE levels8. In addition to its transmembrane forms, CD23 has been found as a soluble protein in human serum, and several reports have shown that the severity of allergy correlates directly with serum levels of soluble CD23 (sCD23)16. However, CD23 is approved as a prognostic parameter only for B-cell chronic lymphocytic leukemia17.Soluble IgE receptors may mediate IgE-mediated immune responses and are important in the understanding of allergic responses. Soluble FcεRI (sFcεRI) is found as a soluble free IgE receptor and in complex with IgE. An association between sFcεRI and allergy severity has been reported, but some individuals with normal IgE levels have high levels of sFcεRIHere, we developed a new and simple method for determining cell-bound IgE levels. We collected samples from 93 allergic patients and 102 healthy people and analyzed the relationship between serum IgE levels and cell-bound IgE levels to provide more guidance for allergy diagnosis and therapeutic evaluation.Two clinical cohorts were established: an allergic patient group (n = 93) and a healthy participant group (n = 102). Patient personal information, such as age, sex and clinical diagnosis, was obtained. We obtained peripheral blood samples for flow cytometry analysis and IgE level measurements by ELISA at the time of enrollment.The inclusion criteria for the allergic patients depended on the doctors’ clinical diagnosis and confirmation of the allergy information and allergic symptoms in clinical diagnostic reports at the Shenzhen Second People’s Hospital. The patients selected as allergic individuals had a positive CAP result in a test with 21 different allergen extracts.We obtained information on the allergic symptoms of the patients from the clinical diagnostic reports at the hospital. Personal information, such as age and sex, was collected through a questionnaire, and written informed consent was provided by the patients.The project was approved by the ethical committee of the Shenzhen Second People’s Hospital (No. 046), which voted unanimously for its completion, and conformed to the basic principles of medical ethics. All the methods were performed in accordance with the relevant guidelines and regulations.Fresh whole blood from allergic patients and healthy people was collected with EDTA as the anticoagulant. One milliliter of whole blood from each sample was used after centrifugation at 500 g for 15 min , and the upper plasma layer was collected. Then, 1 mL of ACK lysis buffer  was added to the cell precipitate on ice for 10 min to lyse the red blood cells. After centrifugation, the cell precipitate, which contained all white blood cells (WBCs), was collected, and 0.5 mL of lysis buffer  was added. The samples were then vortexed for 5 seconds, followed by an incubation on ice for 5 min to dissociate IgE from the cell membrane. The samples were then centrifuged, and the supernatant was removed for renaturation with 0.5 mL of 5 mM NaOH, resulting in a total volume of 1 mL. The collected plasma and cell-bound IgE were used for IgE quantitative determination using the Elecsys IgE II Immunoassay . The IgE concentrations in the healthy, nonatopic test subjects were greatly dependent on age, and the recommended threshold value for adults is 240 ng/mL.in vitro assay for the detection of human IgE antibodies to airborne and food allergens in the serum or plasma. The test kit contains test strips coated with parallel lines of 21 different allergen extracts. The test strips were first moistened and incubated with patient serum in the first reaction step. Positive samples showed specific IgE antibodies binding to the allergens. The readout for the EUROLINE kit is shown in Supplementary Table The EUROLINE test kit  was used as a semiquantitative The antibodies used for flow cytometry were mouse anti-human IgE , mouse anti-human CD23 , and mouse anti-human FcεRI . Flow cytometric analysis was performed on a FACSCalibur . Forward scatter (FSC) and side scatter (SSC) signals were recorded in the linear mode, and fluorescence signals were recorded in the logarithmic mode. Data were analyzed using CellQuest software .Data were analyzed with SPSS for Windows .A paired t-test was used to determine statistical significance. A p-value less than 0.05 was considered significant.Two clinical cohorts were established as shown in Table To learn more about the allergic patient group, we used the EUROLINE test kit to detect human IgE antibodies specific for 21 different airborne and food allergens in allergic patient serum. The readout for the EUROLINE kit is shown in Supplementary Table We developed a new and simple method for detecting cell-bound IgE: whole blood was first centrifuged to separate peripheral blood cells from the plasma, red blood cells were lysed with ACK buffer to obtain WBCs, and cell-bound IgE was dissociated from its membrane receptors. The collected plasma and cell-bound IgE were used for IgE quantitative determination using an ELISA kit. To confirm that all cell-bound IgE was dissociated from the cell membrane, we used anti-IgE PE-conjugated antibody staining and flow cytometry to analyze WBCs. As shown in Fig. We compared the cell-bound IgE level with the plasma IgE level in each group and observed that the cell-bound IgE level was significantly higher than the plasma IgE level in the allergic patient group (p < 0.01). Consistent results were also found in the healthy participant group Fig. . The datTo illustrate the relationship between the cell-bound IgE and plasma IgE levels, we created an X-Y graph in which each point represents a sample with the X axis representing the plasma IgE level and the Y axis representing the cell-bound IgE level Fig. . Per theFurthermore, in the population of allergic patients with a plasma IgE level >240 ng/ml (Q2 + Q3), 65.95% (Q3/(Q2 + Q3)) had a cell-bound IgE level >240 ng/ml. Among the healthy participants, 2.94% (Q2 + Q3) had a plasma IgE level >240 ng/ml, and only 0.98% (Q3 + Q4) had a cell-bound IgE level >240 ng/ml. Therefore, the cell-bound IgE level is a better clinical diagnostic indicator of allergy than the plasma IgE level.At present, the cut-off value for plasma/serum IgE levels as a clinical diagnostic indicator of allergy is 240 ng/ml; however, as low as 33.57% of the allergic patients were diagnosed with allergy using this indicator. However, using 240 ng/ml cell-bound IgE as a cut-off value, 60.00% of the allergic patients were diagnosed with allergy Fig. . We analin vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses.We measured the concentrations of the soluble forms of the two IgE receptors in the samples by ELISA and found that the soluble FcɛRI (sFcɛRI) values in the allergic patients were significantly higher than those in the healthy people Table . The sCD17, and the serum IgE concentration does not accurately reflect the levels of systemic IgE19. At present, the current diagnostic method detects both allergen-specific IgE and total IgE levels, which is poorly correlated with allergy determination20. A new method is needed to investigate the IgE pool in the peripheral blood. There have been some reports on measuring human cell-bound IgE levels using flow cytometry12. Cell-bound IgE levels are calculated using the percentages of FcεRI+IgE+ and FcεRII+IgE+ cells among all whole-blood leukocytes, with the percentage of IgE+ cells in the receptor-bearing cell population multiplied by the mean fluorescence intensity (MFI) of IgE to generate a unit of IgE/cell. This reported method is simple and convenient, and the percentage of IgE+ cells reflects the cell-bound IgE level. However, this method does not quantitatively analyze the distribution of the whole IgE pool in the blood. In this method, serum IgE is detected by ELISA, while cell-bound IgE is detected by flow cytometry. Other defects in this method are that the MFI of IgE cannot be converted into the actual content unit (ng/mL) and that quantitative standards are lacking.IgE plays a critical role in immediate-type allergic reactions. Most somatic IgE is bound by its receptors13. We measured the concentrations of the soluble forms of the two IgE receptors in samples by ELISA and found that the sFcɛRI values in allergic patients were significantly higher than those in healthy people  also show elevated total IgE values. These patients would be diagnosed as allergic by using our method. In this particular case, only the detection of helminth- specific or allergen-specific IgE together with an analysis of patient history would be useful to give an adequate diagnosis.In our study, all the cell-bound IgE was dissociated from blood cells, and the dissociated cell-bound IgE and plasma IgE levels were detected using the same ELISA kit at the same time; therefore, the data are consistent, and the units for both measurements are ng/mL. Our new recommended method is not only simple but also convenient to carry out because it uses a kit already used in the clinic. Any IgE detection kits used in hospitals can be applied to detect cell-bound IgE levels with the addition of several pretreatment steps. This method is simple to perform and can be used in the analysis of large numbers of clinical samples.Supplementary Information.


Reconstructed palaeoprecipitation reveals a persistent SCAND-like circulation and southerly storm track during the Younger Dryas. The Younger Dryas (YD) was a period of rapid climate cooling that occurred at the end of the last glaciation. Here, we present the first palaeoglacier-derived reconstruction of YD precipitation across Europe, determined from 122 reconstructed glaciers and proxy atmospheric temperatures. Positive precipitation anomalies (YD versus modern) are found along much of the western seaboard of Europe and across the Mediterranean. Negative precipitation anomalies occur over the Fennoscandian ice sheet, the North European Plain, and as far south as the Alps. This is consistent with a more southerly and zonal storm track, which is linked to a concomitant southern location of the Polar Frontal Jet Stream, generating cold air outbreaks and enhanced cyclogenesis, especially over the eastern Mediterranean. This atmospheric configuration resembles the modern Scandinavian (SCAND) circulation over Europe (a blocking high pressure over Scandinavia pushing storm tracks south and east), and by analogy, a seasonally varying palaeoprecipitation pattern is interpreted. The Younger Dryas (YD) is recognized as a period [12.9 to 11.7 thousand years ago (ka)] of rapid climate change , annual accumulation  equals annual ablation (snow and ice melt). The ELA is linked to atmospheric conditions via the interplay between temperature (ablation) and precipitation (accumulation) . The res10Be exposure ages and for 14C-dated organic samples . Only moraines with an age that fell within the time span of the YD were selected for further analyses. The palaeoglaciers were then reconstructed from the dated frontal moraine, following an equilibrium profile flowline approach. This was extrapolated to a three-dimensional (3D) ice surface, from which ELAs were calculated, all using bespoke, semiautomated toolboxes in ArcGIS . These data generated a regional, theoretical, ELA surface (ELAthl) . Mean summer temperature at the palaeoglacier ELAs was determined using the same free-air lapse rate and used to calculate the annual “potential palaeoprecipitation” at the ELA (PPPELA) (ELA) have been calculated, compared to the modern precipitation , to reveal the regional picture of enhanced and reduced precipitation across Europe during the YD , unlike previous attempts to use palaeoglacier ELAs to study atmospheric circulation ((ELAthl) . Cosmoge(PPPELA)  (16), asg the YD .thl surface . Assuming that the PPPELA and PPAELA patterns reflect air mass advection at synoptic to sub-synoptic scales, i.e., storm tracks, they can be used to assess the prevailing YD atmospheric circulation across Europe.The ELA palaeoprecipitation , which directed storm tracks along the western Atlantic margin  and into the Mediterranean. Following a circulation weather type approach, the YD precipitation pattern most closely resembles the modern Scandinavian (m-SCAND) circulation, although here shifted further south and formed under different boundary conditions. m-SCAND, predominantly a winter circulation  (−1 (fig. S3E) to 100 to 600 mm a−1 . A shallower precipitation gradient (400 to 600 mm a−1) with two higher-precipitation centers over Corsica–Northwest Italy and the eastern Mediterranean just west of Crete is evident in  . The mod in  for the 1785 m elevation difference across the 130 km between the sites. An estimate for the modern precipitation gradient, using data from ] in Cantabria, Spain, where YD precipitation is estimated to be 1050 mm aELA . Where these were not available, they were requested from the laboratory that undertook the analyses. This rigorous quality control allowed maximization of the number of sites that were dated either directly or by morphostratigraphical relationship to the YD. Note that the techniques used to constrain the ages of the moraines have a range of uncertainties . Although these uncertainties are quite large for cosmogenics, the target was moraines that are formed during readvances/stillstands, i.e., during climatic periods favorable for glacier existence/stability. Provided the age range for the moraines overlapped with the time window of the YD, here taken to be 12.9 to 11.7 ka before the present (B.P.), they were selected as sites for palaeoglacier reconstruction. The moraines meeting all our quality controls were accepted as YD and were used in subsequent workflows, providing a final dataset of 122 palaeoglaciers  placed the site outside of the YD time window or sites did not meet some of the other assessment criteria. For a dated moraine to be used for palaeoglacier reconstruction, it also had to either appear on a map with coordinates or be readily identifiable on available satellite/aerial imagery. On the basis of the original paper/s and an assessment of free-to-access imagery, any glacier that appeared to have had the possibility of any significant mass loss from calving was excluded. Sites were then assessed for the fidelity of the chronology, by review of the dating context, the landform and sample/s dated, and the dating technique. All relevant information was captured in a database to facilitate subsequent recalibration using IntCa13 for y is the yield stress, τd is the driving stress, ρ is the density of ice, g is gravity, H is ice thickness, and h is the ice surface elevation; b is the bed elevation; τav is the average basal shear stress; Δx is the step length; F is the shape factor; ρ, g, and H as above; and i is the iteration (step) number. The shape factor F takes account of the lateral drag imposed by the constraining topography, should it exist, i.e., it is small if the valley is narrow and deep, and high if it is wide and shallow, reaching 1 for unconstrained ice masses, e.g., plateau ice fieldsA is the glacier cross-sectional area and p the perimeter length of the cross section . F is calculated automatically or at used defined intervals along the length of the glacier flowline (F) were applied where glacier flow was topographically constrained. The centerline ice surface elevation generated from 3 (−8%) and −2.53 km2 (−9%), and for Griesgletscher, they were 0.014 km3 (10%) and −0.44 km2 (−8%).To remove any potential bias/errors in the reconstructed palaeoglaciers from the original papers, an equilibrium profile modeling approach was applied. Glacier thickness is reconstructed along a central flowline, assuming perfect plasticity τy=τd=ρgH moraine  bn is the net mass balance gradient; A is area. The ELA for each glacier is calculated in ArcGIS using a bespoke tool  2533:1975], these errors equate to a ±0.325°C and ±0.65°C temperature difference, respectively, at the ELA. The numerical approach used here for the glacier reconstructions and application of the AABR for ELA calculation, which accounts for the glacier hypsometry associated with both simple and complex glacier geometries, is believed to have reduced the overall error. Taking the two test glaciers from above and using an AABR of 1.7, the ELA for Folgefonna was determined to be +3 m from that estimated for the extant icefield surface  associated with the PFJS to be identified. The identification of the breakpoint used a piecewise regression for the south-north ELA transect , in R, following two approaches. The first implemented an iterative searching approach, within a user-defined range encompassing the breakpoint, and identified the combination that returned the lowest mean squared error. Thus, the two regressions are not forced to touch or to be continuous and returned the breakpoint at 55.02°N. The second approach used the segmented package in R, and here, the segments are continuous and touching. This approach located the breakpoint at 54.66°N. These values should not be taken as an absolute location of the PFJS during the first half of the YD. Rather, this is the location of the transition zone south of which there is a significant maritime influence and substantial total annual precipitation, north of which the environment becomes polar with less total annual precipitation.thl). They are theoretical because a true ELA will only exist where there is sufficient topography above the ELAthl, for a glacier to form  where summer equates to June, July, and August , the mean temperature of the coldest month (MTCM), or the mean temperature of the warmest month (MTWM). YD palaeotemperatures for the study area are available from multiple proxies. These may be terrestrial, providing estimates for summer temperature , MAAT (permafrost/ice wedge casts), or MTCM (ice wedge casts) . The chronologies for the different proxies are variable, in some instances limited to a single YD record, while in others, a well-constrained age-depth model is available. To maximize the number and spatial coverage of data points available across the region, as far as possible, we obtained (or generated) a single (mean) value temperature for the first 500 years of the YD, i.e., from 12,900 to 12,400 B.P. Where a single value only was available, this was used, and where none were available for the first 500 years, the nearest one, within the YD, was selected. The chronological data in the original publications were used as reported, and the calculation of a mean temperature for the 500-year time window, for the first half of the YD, limiting any potential issues with age range errors. We chose the first half of the YD because the glacier chronologies are limited by the uncertainties associated with cosmogenic exposure ages, this is generally viewed as the coldest and most stable part . We did not use the YD temperature reconstruction in . The data showed a strong north-south trend, as reported elsewhere (SLE between 31°40′N and 56°41′N (fig. S1).MAATSLE dataset was derived using chironomids, coleoptera, diatoms, foraminifera, and pollen and generated 92 data points (data file S5). First-degree detrending was applied, and it was then interpolated using kriging. The resulting dataset provided a gridded MTWMSLE between 40°49′N and 71°8′N (fig. S1).The MTWMSLE dataset only reached 56°41′N, and the dataset covered neither the Moroccan Atlas Mountains nor the easternmost part of Turkey. To accommodate for this, the following procedures were undertaken to extend the gridded temperature datasets to the outer ranges of the spatial extent of the palaeoglacier ELA reconstructions.Ideally, the two gridded temperature datasets should have the same spatial coverage as the palaeoglaciers. The MAATSLE, calculated at the locations of palaeoglacier sites in the British Isles located south of 56°41′N. This produced a very strong [R2 (coefficient of determination) = 0.997] negative northward correlation (fig. S4).1) For Scotland, a second-order polynomial was fitted through the MAATSLE from the full dataset provided in data file S4. This produced a very strong (R2 = 0.959) negative northward correlation (fig. S5).2) For the sites in Norway, a second-order polynomial was fitted through the SLE MAATSLE for the area south of 40°49′N, a second-order polynomial was fitted through the dataset (data file S5). This produced a very strong (R2 = 0.753) negative northward correlation (fig. S6), generating the southward projection of MTWMSLE for palaeoglacier sites in Spain, Morocco, Greece, and Turkey.3) To generate the MTWMSLE was determined from the MAATSLE and the MTWMSLE by fitting a sine curve through these three points (fig. S7) and taking the mean for June, July, and August temperatures. For the final part of the workflow, the MSATELA was then determined from the MSATSLE by applying the 0.0065°C m−1 lapse rate for all the palaeoglaciers.The MSATet al.  than the original paper, and the resulting equation for the potential total annual palaeoprecipitation (snow and rain) at the ELA (PPPELA) in millimeters water equivalent isT is the MSATELA in °C.A reanalysis of the dataset of Ohmura ELA data points were interpolated, as above (kriging), to generate a regional map of YD potential palaeoprecipitation, and it must be remembered that this is precipitation at the glacier ELA  have been calculated usingELA. “This downscaling method still suffers from the lack of weather station density inherited from both input datasets” (ELA calculated for the Dinaric Alps, specifically over Montenegro, which is a region of extreme precipitation gradients in the present day. The data from (ELA calculation (ELA reconstructed in ELA. For these two regions, the PPPELA provides a better characterization of the YD climate. Additional complications arise due to the difference in grid resolution between the modern precipitation data at 1 km (To better contextualize the pattern, potential palaeoprecipitation at the ELA standard anomalies (PPAculation , but thi


Freezing resistant plant organs are capable to manage ice formation, ice propagation, and ice accommodation down to variable temperature limits without damage. Insights in ice management strategies are essential for the fundamental understanding of plant freezing and frost survival. However, knowledge about ice management is scarce. Ice crystal localisation inside plant tissues is challenging and is mainly based on optical appearance of ice in terms of colour and shape, investigated by microscopic methods. Notwithstanding, there are major uncertainties regarding the reliability and accuracy of ice identification and localisation. Surface light reflections, which can originate from water or resin, even at non-freezing temperatures, can have a similar appearance as ice. We applied the principle of birefringence, which is a property of ice but not of liquid water, in reflected-light microscopy to localise ice crystals in frozen plant tissues in an unambiguous manner.Buxus sempervirens leaves, large ice needles occupied and expanded the space between the adaxial and abaxial leaf tissues. In Galanthus nivalis leaves, air-filled cavities became filled up with ice. Buds of Picea abies managed ice in a cavity at the bud basis and between bud scales. By observing the shape and attachment point of the ice crystals, it was possible to identify tissue fractions that segregate intracellular water towards the aggregating ice crystals.In reflected-light microscopy, water was clearly visible, while ice was more difficult to identify. With the presented polarised cryo-microscopic system, water, including surface light reflections, became invisible, whereas ice crystals showed a bright and shiny appearance. Based on this, we were able to detect loci where ice crystals are accommodated in frozen and viable plant tissues. In Cryo-microscopy in reflected-polarised-light allowed a robust identification of ice crystals in frozen plant tissue. It distinguishes itself, compared with other methods, by its ease of ice identification, time and cost efficiency and the possibility for high throughput. Profound knowledge about ice management strategies, within the whole range of freezing resistance capacities in the plant kingdom, might be the link to applied science for creating arrangements to avoid future frost damage to crops. Low-temperature resistance of plants is highly diverse, ranging from chilling susceptible species in tropical biomes to extreme freezing resistant species of the boreal zones that even survive immersion in liquid nitrogen in the dormant state. Stress caused by low temperature limits global plant distribution, threatens crop survival and by this can lead to tremendous economic damage . A challFor the investigation of ice management processes in plant tissues various methods have been employed , 3: diffPrecise temperature measurements can detect ice formation, as the phase transition from liquid water to ice releases measurable heat, the so-called freezing exotherm. Based on this principle DTA compares the sample temperature with a reference temperature and allows to identify freezing, but cannot detect the initial site of ice nucleation or the direction of ice propagation . In contThe supercooling water in plant samples exposed to freezing temperatures can be quantified by NMR spectroscopy , 22, whiIce crystals can be visualised and located inside of plant tissues by cryo-SEM and/or light microscopy. Microscopic methods require sectioning at a certain point in time; this entails the potential risk of ice crystal formation during incision. Further, temporal monitoring of ice formation in a specimen is not possible . HoweverTo directly detect and observe ice in plant tissues, optical light microscopy techniques have been used since the 19th century . Continu20. The optical property “birefringence” is a feature of ice but not of liquid water. While water in the liquid state is known to be optically isotropic, ice crystals are optically anisotropic [Cornus officinalis overwintering buds [Lactuca sativa and Celastrus orbiculatus seeds [To improve visibility of ice in plants, a prerequisite is a reliable distinction between the solid and liquid aggregation state of Hsotropic . Howeversotropic . The preing buds , Lactucaus seeds .Buxus sempervirens), in leaves of an herbaceous species  and in overwintering buds of conifers (Picea abies).In this study, we tested whether ice can unambiguously be distinguished from liquid water using polarised light in reflected-light microscopy. To rule out disruptive effects during preparation of thin cross sections and the necessity of a liquid sample carrier for transmitted-light microscopy, we used reflected-light microscopy to inspect frozen plant organs immediately after cutting. Inspections were performed in structural different plant organs from three freezing resistant species: in leaves of an evergreen shrub  is demonstrated for three species in Fig. Identification of ice in plant tissues using cryo-microscopy in reflected-polarised-light  and the abaxial leaf side (spongy tissue) was found  µm)  Fig. c. When fµm) Fig. d. The icPicea abies have a complex architecture  when the filters are crossed. However, this only holds if there is isotropic material between the polariser and the analyser. If there is birefringent material in between, this will result in a wave inclined at some angle compared with the original plane wave , which wrpl, ice could be identified more clearly, whereas liquid water could be made invisible. Water surface reflections can be deceptive in reflected-light microscopy as their distinction from ice is often hardly possible. With CMrpl, water surface reflections disappeared under crossed polarisers ruling out potential uncertainties. Similar to water, resins from pines can cause deceptive surface light reflections in reflected-light microscopy. As resins did not show birefringence (unpublished Stegner and Neuner), surface light reflections from resins could also be extinguished by crossed polarisation filters. The only obstacle were birefringent tissue components. However, their potential misleading interpretation can be ruled out by investigation of unfrozen control samples.With CMrpl, it is possible to gain detailed new insights into ice accommodation and segregation strategies in plant tissues. The system allows a rapid inspection of a high number of samples. The acquisition costs are comparably low and operation expenses are negligible. Besides the determination of ice crystals in frozen plant tissues, CMrpl offers additional valuable information about ice aggregation. The resolution is high enough to identify the shape of the apoplastic ice crystals, which can be valid information to understand how they are formed. Additionally, in all three examples  > the unaided eye > DTA [rpl has major benefits in ease of ice identification, less time requirement, inspection of a high number of samples and cost efficiency.To understand how plant species with different freezing tolerance limits accommodate ice masses in their organs, without becoming damaged, will be essential for the fundamental understanding of frost survival. Unambiguous ice identification at a high resolution with CMye > DTA ). The reBuxus sempervirens L.), in leaves of an herbaceous geophyte  and in overwintering buds of a gymnosperm tree (Picea abies (L.) H. Karst.). All plants were taken during winter from the field: Leaves of B. sempervirens were sampled in December from a single individual in the Botanical Garden of the University of Innsbruck , twigs with buds of P. abies were sampled in January from seven individuals in the Alpine Garden on Mt. Patscherkofel , and leaves of G. nivalis were sampled in January from two individuals that were cultivated in pots in the Botanical Garden of the University of Innsbruck. All experiments were repeated at least 7 times for each species. For the experiments, samples were either immediately used after collection or, in case of samples from the Alpine Garden, stored for a maximum of 3 days at + 5 °C.Localisation of ice in plant tissues was studied in leaves of an evergreen shrub (Buxus (lamina with petiolus) and Galanthus (lamina with leaf basis), and about 10 cm long shoots (Picea), which were defoliated around the terminal buds, were used. For sectioning under the microscope a special sample holder was designed , leaves were cross sectioned with a razorblade guided by the sample holder surface. Buxus and Galanthus leaves or shoots of Picea were cooled down to the respective species specific sublethal target temperatures. To avoid artificial supercooling, cooling rates were limited to a maximum of 3 K/h [Pseudomonas syringae van Hall 1902) were added into the Eppendorf tubes. Upon reaching the target temperatures, leaves and buds plus shoots were sectioned in the frozen state inside the cooling compartment with a similarly cooled razorblade by use of insulated gloves that are build-in in the lid of the freezer  was used: Reflected-light illuminator U-KMAS mounted on BXFM-ILHS, single port tube with lens U-TLU and a video camera mount adapter U-CMAD3. Illumination was provided by an external cold light source . For magnification, the microscope was equipped with the following objective lenses: MPLFLN5X , MPLFLN10X , LMPLFLN20X  . Focusing was conducted by a motorised stage M-MFD-MX51 . Images were taken with a 9 megapixel digital camera (UC90), which can be fully controlled by the image analysis software cellSense Standard . For the investigation of optical properties (e.g. birefringence) a polariser (U-PO3) and a rotatable analyser (U-AN360-3)  were used.The microscopic system except for the camera unit was placed inside of a laboratory chest freezer  Fig.  which is