Background: Inflammation and steatosis are the main pathological features of alcoholic liver disease (ALD), in which, inflammation is one of the critical drivers for the initiation and development of alcoholic steatosis. NIK, an inflammatory pathway component activated by inflammatory cytokines, was suspected to link inflammation to hepatic steatosis during ALD. However, the underlying pathogenesis is not well-elucidated.Methods: Alcoholic steatosis was induced in mice by chronic-plus-binge ethanol feeding. Both the loss- and gain-of-function experiments by the hepatocyte-specific deletion, pharmacological inhibition and adenoviral transfection of NIK were utilized to elucidate the role of NIK in alcoholic steatosis. Rate of fatty acid oxidation was assessed in vivo and in vitro. PPARα agonists or antagonists of MEK1/2 and ERK1/2 were used to identify the NIK-induced regulation of PPARα, MEK1/2, and ERK1/2. The potential interactions between NIK, MEK1/2, ERK1/2 and PPARα and the phosphorylation of PPARα were clarified by immunoprecipitation, immunoblotting and far-western blotting analysis.Results: Hepatocyte-specific deletion of NIK protected mice from alcoholic steatosis by sustaining hepatic fatty acid oxidation. Moreover, overexpression of NIK contributed to hepatic lipid accumulation with disrupted fatty acid oxidation. The pathological effect of NIK in ALD may be attributed to the suppression of PPARα, the main controller of fatty acid oxidation in the liver, because PPARα agonists reversed NIK-mediated hepatic steatosis and malfunction of fatty acid oxidation. Mechanistically, NIK recruited MEK1/2 and ERK1/2 to form a complex that catalyzed the inhibitory phosphorylation of PPARα. Importantly, pharmacological intervention against NIK significantly attenuated alcoholic steatosis in ethanol-fed mice.Conclusions: NIK targeting PPARα via MEK1/2 and ERK1/2 disrupts hepatic fatty acid oxidation and exhibits high value in ALD therapy. Ethanol intake is harmful at any dose and its risks rise with the increasing levels of consumption Considerable evidence indicates that alcohol exposure weakens the intestinal barrier and facilitates the influx of lipopolysaccharide (LPS) Map3k14-encoded serine/threonine kinase, is aberrantly activated in the livers of mice and patients with ALD NF-κB-inducing kinase (NIK), a The present study revealed that NIK pushed aberrant fat accumulation in the liver by disrupting fatty acid oxidation during ALD, because NIK recruited and activated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) to suppress the fatty acid oxidation controller, PPARα. Therefore, NIK could be a therapeutic target to stop inflammation from promoting alcoholic steatosis.f/fNIK) in C57BL/6 background, was a present from Professor Liangyou Rui . The mouse was generated by inserting two loxp sites into intron 1 and intron 2 that flank exon 2-6 of Map3k14. To generate hepatocyte-specific NIK-deficient mice (ΔhepNIK), we utilized albumin-cre transgenic mice  to cross withf/f NIK mice. Wild-type (WT) mice in C57BL/6 background were purchased from the Animal Core Facility of Nanjing Medical University, Nanjing, China. In a pathogen-free barrier facility with controlled temperature and illumination, mice had ad libitum access to sterile water and standard food. Following a previous study, male mice aged 10 weeks received chronic-plus-binge ethanol feeding All mouse experiments were conducted on the basis of relevant institutional and national guidelines. The experimental protocol (Protocol Number: 1704009-3) was approved by the Animal Care and Use Committee of Nanjing Medical University. NIK flox/flox mouse .AML12 cells (SCSP-550) and HepG2 (SCSP-510) were purchased from the Cell Bank of the Chinese Academy of Sciences  and cultured according to the previous studies Primary hepatocytes were isolated by collagenase digestion from adult mice (8-10 weeks) according to a previously published protocol We purchased p3XFlag-CMV7.1 from Sigma-Aldrich and pcDNA3.1(+) from Invitrogen . Dr. Dongping Wei  provided pcDNA-HA3, pcDNA-HA3-CPT1α  and pcDNA-HA3-RXRα (retinoid-X receptor α). Dr. Liangyou Rui  provided pRK5-NIK, pRK5-NIK(KA), and β-gal expression vectors. We subcloned pRK5-NIK and pRK5-NIK(KA) into pcDNA-HA3, pcDNA3.1(+), or pAdeno-TBG-MCS-3FLAG . Bruce Spiegelman provided pSG5 PPARα (Addgene plasmid #22751), which was subcloned into p3XFLAG-CMV7.1 (Sigma-Aldrich) and pcDNA-HA3. Vectors expressing PPARα mutants, including PPARα , PPARα  and PPARα , were generated using p3XFLAG-CMV7.1-PPARα by Shanghai Generay Biotech Co., Ltd., Shanghai, China. A series of vectors expressing truncated PPARα  were prealbumin promoter in the pAdeno-TBG-MCS-3FLAG vector. The empty pAdeno-TBG-MCS-3FLAG vector and that containing the target genes were sent to OBiO Technology Corp., Ltd. for adenovirus packaging and purification.Fragments encoding NIK or NIK(KA) were synthesized via PCR and inserted downstream of the 10 viral particles [vp] for each mouse) through tail vein. At day 5 after infection, mice were decapitated after 18 h of fasting. Adenoviruses, with concentrations of 4 × 108 vp/well in 12-well plates or 8 × 108 vp/well in 6-well plates, infected primary hepatocytes and HepG2 cells. Cells were further cultured for 24 h before harvest.Male mice aged 10 weeks were injected with adenoviruses (2 × 10We followed our previous protocol According to our previous protocol AML12 cells were transfected with PPRE X3-TK-Luc (200 ng/well), β-gal expression vector (200ng/ well), and pSG5 PPARα (200 ng/well) plus other expression vectors (200 ng/well) as indicated using polyethylenimine (Sigma-Aldrich). Then, after growth for 24 h, cells received treatment of WY14643 , trametinib , SCH772984 , or IKK16  for another 24 h. Luciferase activity was determined with luciferase reporter assay system . β-gal activity, as the control for transfection efficiency, was measured by a kit . Results were averaged over three biological replicates.Staining by Hematoxylin and eosin (H&E) and Oil Red O (Sigma-Aldrich) were performed referring to the work of Sarmistha et. al. 3H] oleic acid  at 37 ℃ for 1 h. Then the culture media were transferred, mixed with perchloric acid (1.3 mol/L), and subjected to high-speed centrifugation. Thereafter, the supernatants were collected, neutralized by potassium hydroxide (2 mol/L) plus 3-(N-Morpholino) propanesulfonic acid (0.6 mol/L), and poured into an anion-exchange column  to get rid of ketone bodies. The radioactivity of 3H (tritium oxide) in the effluent normalized to protein amounts in the cells was determined to calculate β-oxidation rates.The β-oxidation rate was determined according to our previous study with minor modifications Far-western blotting was performed following a previously published protocol t-test. More than two groups of data were compared by one-way analysis of variance. p <0.05 indicates statistical significance.Results are demonstrated as the means ± SEM. Two groups of data were compared by two-tailed Student's ΔhepNIK), ethanol-induced TAG accumulation . Overexpression of NIK significantly upregulated liver TAG and p52 levels − as indicators for hepatic fatty acid oxidation capacity. Ethanol consumption reduced the serum β-hydroxybutyrate level Figure A and dows Figure C as wells Figure D than NIs Figure E and CPTs Figure F. CPT1α,s Figure E. Hence,A luciferase system was utilized to evaluate the role of NIK in the regulation of the transcriptional activity of PPARα, the primary controller of fatty acid oxidation in the liver To explore the regulatory mode of NIK on PPARα, we focused primarily on the phosphorylation of PPARα, as NIK is a kinase and it is reported that phosphorylation of the PPARα serine residues disrupts its transcriptional activity To identify the mediators implicated in PPARα phosphorylation by NIK, a series of currently known kinases that catalyze inhibitory phosphorylation of PPARα were screened Besides the interaction with NIK, MEK1/2 and ERK1/2 also bound PPARα . To confPPARα is consist of four functional domains, including A/B, C, D, and E/F To determine whether NIK regulates PPARα in an analogous manner in human and mouse hepatocytes, we utilized a hepatocyte cell line from human, HepG2. Similarly in mouse primary hepatocytes and liver, NIK overexpression reduced the rate of fatty acid oxidation and the mRNA levels of related genes, and enhanced the phosphorylation levels of MEK1/2, ERK1/2 and PPARα . SlightlTo further evaluate the role of NIK in PPARα phosphorylation, the kinase deficient mutant of NIK, NIK(KA), was compared with the WT one. Interestingly, NIK(KA), under the similar expression level with NIK, did not lose but retain the ability to induce the phosphorylation of MEK1/2, ERK1/2, and PPARα at a reduced level Figure A-B. TherTo assess the therapeutic value of NIK for ALD treatment, we used B022, a chemical NIK inhibitor, to treat mice fed with chronic-plus-binge ethanol diet. B022 attenuated ethanol-induced hepatic steatosis Figure A, elevatIn the present study, we have presented evidence that NIK is responsible for liver steatosis induced by chronic-plus-binge ethanol feeding in mice. Deletion of NIK in hepatocytes attenuated liver steatosis after ethanol consumption by protecting the hepatic capacity of fatty acid oxidation. PPARα, the primary transcriptional controller of fatty acid oxidation, is a regulatory node of NIK, as PPARα agonists reverse NIK-mediated hepatic steatosis and malfunction of fatty acid oxidation. NIK recruits MEK1/2 and ERK1/2 to form a complex that induces the inhibitory phosphorylation of PPARα Figure F. PharmaThe influx of gut-derived LPS induced by ethanol consumption initiates inflammation during ALD. LPS stimulates Kupffer cells to secrete proinflammatory cytokines and chemokines, such as TNFα and IL1β PPARα, the main controller of fatty acid oxidation in the liver, is regulated by NIK during ALD, considering that PPARα agonists reversed NIK-mediated suppression of PPARα activity, reduction in fatty acid oxidation, and hepatic steatosis. After screening several potential regulatory modes, we determine that serine phosphorylation contributes to the suppression of PPARα by NIK, as PPARα with triple mutations at S6, S12 and S21 is resistant to NIK-mediated suppression. However, there are conflicted reports for the activity regulation of PPARα by the phosphorylation of those serine residues. Juge-Aubry, C. E., et al. believed that the phosphorylation at S12 and S21 enhanced PPARα activity NIK does not directly phosphorylate PPARα. It integrates MEK1/2, ERK1/2, and PPARα into a complex, in which, NIK directly binds MEK1/2 and ERK1/2 but not PPARα, therefore, the phosphorylation of PPARα by NIK should be conducted through MEK1/2 and ERK1/2. ERK1/2 is the unique kinase phosphorylated and activated by NIK among those reported to catalyze inhibitory phosphorylation of PPARα Of note, the phosphorylation of MEK1/2 and ERK1/2 is not entirely driven by NIK kinase activity, as this phosphorylation catalytic capacity of NIK is partially retained by its kinase deficient mutant, NIK(KA). We speculate that the integrating function of NIK may be also contributable, because NIK and NIK(KA) have a similar ability to enhance the interaction between MEK1 and ERK2, which may promote spatial proximity of MEK1 to ERK2 and thus facilitate the phosphorylation of ERK2 by MEK1. In fact, MEK1/2 is also the substrate of ERK1/2, however, ERK1/2 catalyzes an inhibitory phosphorylation of MEK1/2 on threonine residues at positions 292 or 386 NIK is constitutively subjected to degradation mediated by a complex that includes cellular inhibitor of apoptosis 1/2 and tumor necrosis factor receptor-associated factors 2/3 in a ubiquitination/ proteasome-dependent manner In conclusion, the aberrant activation of NIK may represent the underlying pathogenesis of alcoholic steatosis in mice. NIK plays a causal role in impeding fatty acid oxidation by restraining PPARα activity in hepatocytes. We identified the NIK-MEK1/2-ERK1/2 pathway as the main signaling route through which NIK regulates PPARα. Our study suggests that disruption of NIK in hepatocytes could offer new therapeutics for the treatment of ALD.Supplementary figures and tables.Click here for additional data file.


Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development of novel analgesic drugs and is particularly important for understanding the mechanisms responsible for opioid-induced tolerance and addiction. The family of G protein receptor kinases (GRKs) play a pivotal role in such processes, mediating phosphorylation of residues at the C-tail of opioid receptors. Numerous strategies, such as phosphosite specific antibodies and mass spectrometry have allowed the detection of phosphorylated residues and the use of mutant knock-in mice have shed light on the role of GRK regulation in opioid receptor physiology. Here we review our current understanding on the role of GRKs in the actions of opioid receptors, with a particular focus on the MOR, the target of most commonly used opioid analgesics such as morphine or fentanyl. Our understanding of the function and regulation of opioid receptors (ORs) has improved dramatically since their identification in the seventies and subsequent cloning in the early 90 s. The first OR to be cloned was the δ opioid receptor (DOR) in 1992 ,4,5. A fAlthough opium has been used for millennia, morphine, the primary alkaloid of opium, was only isolated two centuries ago, and opioids such as morphine, oxycodone and fentanyl still remain the mainstay analgesics for the treatment of acute and severe pain. However, due to the expression of ORs in different regions of the nervous system, these opioids also induce several direct and acute side effects including respiratory depression, miosis (or pinpoint pupils), euphoria, drowsiness and constipation. Long-term side effects include tolerance, dependence and addiction. Altogether, these side effects severely limit the use of opioids in the clinic and have contributed, at least in part, to the current opioid epidemic . The phyTo date, the high-resolution structures of the four ORs have been described ,22,23,24Opioid receptors are expressed in multiple regions of the brain that contribute to pain perception and are involved in the ascending and the descending nerve pathways that deliver nociceptive information . In the i/o G protein family. The Gαi/o subunit released by activation of ORs causes the inhibition of cyclic adenosine monophosphate (cAMP) production by inhibiting the adenylyl cyclase (AC). Therefore, cAMP can no longer activate the cAMP-dependent protein kinase (PKA) responsible for phosphorylating downstream proteins and channels. Presynaptically, the released Gβγ subunits bind to and inhibit voltage-gated calcium channels (VGCC), preventing the release of neurotransmitters. Postsynaptically, Gβγ subunits activate G protein-coupled inward-rectifying potassium (GIRK) channels, releasing K+ from the cell and preventing depolarisation [At a cellular level, upon binding of opioid agonists, ORs act via activation of the Grisation . Other irisation .As with most GPCRs, acute MOR signaling is controlled by the phosphorylation of intracellular serine and threonine residues by GRKs and other second messenger kinases . ReceptoAfter prolonged activation by an agonist, most GPCRs present at the plasma membrane undergo phosphorylation by GRKs at specific serine and threonine residues located on intracellular loops or the C-terminus of the receptor. Those specific sites have been identified for many GPCRs and represent a distinct barcode recognized and phosphorylated by specific kinases ,50. Phos354TSST357 and 370TREHPSTANT379 [363, Thr383 and Thr394 are in between these motifs or at the distal region of the C-tail [There are 11 potential phosphorylation sites at the C-tail of MOR . AlthougSTANT379 , while Se C-tail . These s375 is a key residue that plays a crucial role in ligand-dependent phosphorylation and internalization of MOR [375 phosphorylation, the kinase mediating this modification, the ensuing phosphorylation events and subsequent β-arrestin recruitment and receptor endocytosis, are highly dependent on the intrinsic efficacy of the agonist itself [375, while adjacent residues are weakly  or not phosphorylated (354TSST357) [375 is the first essential step and immediately followed by phosphorylation of the flanking residues Thr370 and Thr379 of the 370TREHPSTANT379 motif, while phosphorylation of Thr376 (immediately adjacent to Ser375) and the 354TSST357 motif occur at a slower rate. Mutation of Ser375 to Ala strongly reduces phosphorylation of all the other residues, demonstrating its crucial role in the generation of the phosphorylation barcode [363 as well as for Thr370 and Ser375 in the 370TREHPSTANT379 motif. While the physiological relevance of such basal phosphorylation is still unclear, the kinases responsible for this have been proposed to be GRK2 for Ser375, Protein kinase C (PKC) for Ser363 and CaMKII for Thr370 [383 and Thr394, has been predicted, it has not been directly observed [Early in vitro studies suggested that similar to MOR internalization, MOR phosphorylation is highly dependent on the opioid agonist bound to the receptor. Zhang et al., (1998) showed that while morphine was unable to induce MOR phosphorylation at endogenous GRK2 levels in HEK293 cells, upon overexpression of GRK2, morphine gained the capacity to induce MOR phosphorylation and internalization . Subsequn of MOR ,54. Impot itself . MorphinTSST357) ,42,51,55r Thr370 . Finallyobserved . Underst375 upon morphine activation is mostly mediated by GRK5 [375 was not sufficient to drive multi-site phosphorylation and receptor internalization.It is now clear that the distinct phosphorylation barcode triggered by opioid agonists is a consequence of the action of different kinases. In an elegant study using siRNA to inhibit the expression of GRK2, 3, 5 and 6, Doll et al., demonstrated that while the phosphorylation induced by DAMGO in HEK293 cells is GRK2- and GRK3-dependent, phosphorylation of the Ser by GRK5 . The actMultiple studies using pharmacological and genetic inhibition, have confirmed the key role of GRK2 in MOR-induced phosphorylation by high efficacy agonists such as Met-enk, DAMGO or fentanyl ,55. Howe375 is essential but not sufficient to elicit receptor internalization. In fact, higher-order phosphorylation of the 370TREPHSTANT379 region is required for the recruitment of β-arrestin2 to drive clathrin-coated pit formation and receptor endocytosis [354TSST357 motif does not seem to be required for receptor endocytosis. Rather, it seems to be related to the dynamics of ligand and effector binding. Birdsong and colleagues showed that phosphorylation of the 354TSST357 motif facilitates the generation of the high-affinity state of the receptor upon high efficacy agonist stimulation, suggesting that phosphorylation of such motif can have an allosteric effect on ligand binding. Such phosphorylation was proposed to be a mechanism for “imprinting” the receptor with a memory of prior activation [The impact of the different phosphorylation profiles on MOR function and regulation at a cellular level is now well established. As mentioned above, phosphorylation of Serocytosis ,42,51. Itivation ,61. Moretivation .363 and Thr370 have been identified as residues that undergo both morphine and phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation by PKC [Other intracellular kinases have been described to phosphorylate MORs . Althougn by PKC ,63. Howen by PKC ,64,65, in by PKC ,42.363 and Thr358, with Ser363 acting as a primary, essential site. Thr361 has been shown to be important for kinase recognition and needed for appropriate Thr358 and Ser363 phosphorylation [344 by PKC has also been suggested [356, Thr357, Thr363, Ser369 at the C-tail of KOR, which are essential for internalization. In particular, residues Thr363 and Ser369 act as primary phosphorylation sites, and phosphorylation of either Thr363 or Ser369 is critical for phosphorylation of Ser356/Thr357 [346, Ser351, Thr362 and Ser363. This multisite phosphorylation is also hierarchical with Ser346 as primary phosphorylation site and is mediated mainly by GRK2 and GRK3 [Phosphorylation barcodes have also been described for the other members of the OR family . Of the rylation ,67,68. Puggested  althoughuggested . Site-di6/Thr357 . GRK2, G6/Thr357 , while Pand GRK3 .GPCR phosphorylation is now an established mechanism underlying acute agonist-dependent desensitization, namely the rapid decline in signaling in the continued presence of agonist. In the case of the MOR, acute desensitization has been suggested to play a role in the development of antinociceptive tolerance. In this context, electrophysiological approaches performed on different cell lines and in rodent brain slices have been extensively used . The mosMoreover, upon the identification of the phosphorylation barcode of the MOR, in 2015 two parallel studies used mutant receptors  to assess the impact of phosphorylation on MOR desensitization. To investigate the role of the STANT and TSST motifs on acute desensitization in live neurons, Birdsong et al.  injectedThe study of desensitization using neuronal recordings in brain slices has taken advantage of the ability to virally express phosphorylation-deficient mutant MOR receptors in knockout animals as well as the development of knockin animals that express mutant MOR in which C- terminus phosphorylation sites are replaced with alanine. Met-enk induced desensitization was largely blocked upon viral expression of a phospho-deficient MOR mutant in LC neurons of MOR knock-out rats . SimilarIt is important to note that, while GRKs are key components for MOR desensitization induced by most opioid agonists, it is also well acknowledged that other kinases contribute to MOR desensitization. As mentioned above, this is particularly the case for morphine and PKC. Indeed, there is a large body of literature using mostly electrophysiological approaches that supports the role of different PKC isoforms in both, MOR acute desensitization ,76,77,78375 or all Ser/Thr residues in the C-tail of MOR, results in a change of these signaling profiles [Receptor phosphorylation and the subsequent binding of arrestins has been shown to induce a second wave of signaling for some GPCRs ,84. In tprofiles . Moreoveprofiles ,86,87.Biased agonism is a pharmacological paradigm that describes the ability of different ligands, binding to the same receptor to stabilize distinct conformations of that receptor that differentially engage with a distinct signaling pathways to the relative exclusion of others. At the MOR, efforts to identify biased ligands were fueled by observations that in mice lacking β-arrestin2, morphine analgesia was enhanced and prolonged while tolerance, respiratory depression and constipation were diminished ,89 suggeThe use of genetically modified mice has provided key information on the mechanisms that mediate the physiological effects of opioids, including analgesia, tolerance, reward, dependence, withdrawal, respiratory depression and constipation. Insights into the physiological roles of MOR phosphorylation have been provided by assessing the actions of opioids in knock-in mice expressing phosphorylation-impaired MORs or knock-out mice lacking the expression of a particular GRK or β-arrestin isoform.Key early studies in β-arrestin2 knock out mice have impacted the field of opioid research for decades. These studies showed how in mice lacking β-arrestin2 (but not β-arrestin1), morphine analgesia was enhanced and prolonged, antinociceptive tolerance was diminished, reward was enhanced and dependence was unchanged ,95,96. L375 phosphorylation being involved in acute MOR desensitization. Interestingly, these mice had reduced antinociceptive tolerance to high efficacy agonists such as DAMGO or etonitazene, but not to morphine, again supporting the hypothesis that the development of antinociceptive tolerance occurs via different mechanisms depending on opioid agonist efficacy. More recently, knock-in mice expressing a phospho-deficient MOR (11ST/A) has been generated [In knock-in mice expressing the phosphorylation-deficient S375A mutant of the MOR, morphine and fentanyl exhibited greater dose-dependent antinociceptive responses than in the corresponding wild-type mice . This isenerated . As mentThe effects of opioids in mice lacking specific GRK isoforms have also been studied, however these are mostly limited to morphine’s actions.GRK2. Despite GRK2 being a key kinase for the regulation of MOR, studying the role of this kinase in vivo is still a challenge. Mice with a homozygous deletion of GRK2 are not viable, and heterozygous GRK2+/- mice show similar antinociceptive responses to morphine than the corresponding WTs. Use of conditional or inducible GRK2 knock-out animals or in vivo administration pharmacological inhibitors represent future strategies to address this.GRK3. Studies with mice lacking GRK3 suggest that this kinase plays a significant role in the development of tolerance to fentanyl both in vitro in mammalian brain slices and in the in vivo analgesic response [370, Ser375 and Thr379 by etonitazene and fentanyl was predominantly mediated by GRK3 [response . Indeed, by GRK3 . In contGRK5. In vivo, morphine has been shown to promote selective phosphorylation of Ser375 which is predominantly mediated by GRK5 [ by GRK5 . GRK5 knGRK6. Upon ablation of the GRK6 gene, morphine has been shown to induce greater locomotor activity and less constipation while not affecting morphine-induced thermal antinociception, analgesic tolerance and physical dependence [pendence . These rThere is strong ex vivo electrophysiological evidence of the importance of PKC for morphine-induced desensitization . MoreoveGRKs are major regulators of opioid receptor function. The distinct phosphorylation profiles elicited by different opioid agonists are highly dependent on their intrinsic efficacy, tightly regulating plasma membrane reorganization of receptors, desensitization and internalization. In vivo, the importance of MOR phosphorylation for the chronic effects of opioids is undisputed. However, the cell-type dependent mechanisms that are controlled by this phosphorylation are just starting to be unraveled. Future directions in this area will rely on genetic approaches that allow the selective knock-down of specific kinases in particular cells or tissues. Such understanding will help the development of opioid ligands with the optimal pharmacological properties that allow for the provision of pain-relief with minimal (or absent) acute and chronic side-effects.


The 2015–2016 El Niño had large impacts globally. The effects were not as great as anticipated in Kenya, however, leading some commentators to call it a ‘non-event’. Our study uses a novel combination of participatory Climate Vulnerability and Capacity Analysis tools, and new and existing social and biophysical data, to analyse vulnerability to, and the multidimensional impacts of, the 2015–2016 El Niño episode in southern coastal Kenya. Using a social-ecological systems lens and a unique dataset, our study reveals impacts overlooked by conventional analysis. We show how El Niño stressors interact with and amplify existing vulnerabilities to differentially impact local ecosystems and people. The policy significance of this finding is that the development of specific national capacities to deal with El Niño events is insufficient; it will be necessary to also address local vulnerabilities to everyday and recurrent stressors and shocks to build resilience to the effects of El Niño and other extremes in climate and weather.The online version of this article (10.1007/s13280-020-01321-z) contains supplementary material, which is available to authorized users. The El Niño Southern Oscillation (ENSO) is the most significant driver of global inter-annual climate variability. The strongest El Niño events have far-reaching consequences for worldwide temperature and rainfall patterns, causing extreme droughts and flooding, mass coral reef bleaching, and major impacts on economies, health and food security Glantz . In 2015Past research on El Niño impacts has studied its influence on regional climates  systems to capture the complex processes and interlinkages that determine vulnerability  and during two research projects, SPACESWe collected data at national and county levels to capture cross-scale drivers, but the primary focus was at the local level to understand vulnerability of a social-ecological system that could be practically studied. Our case study encompasses three villages—Jasini, Jimbo, Vichigini—and Vanga town, located in Vanga sub-location, a rural administrative unit in Kwale County on the south coast of Kenya. We selected this study site for two reasons: (i) it was known to have experienced a severe flood during the 2015–2016 El Niño in April 2016, enabling us to explore a shock event; and (ii) the research team has been studying the site and surrounding area for several years, providing existing datasets to draw upon for contextual information and longitudinal social and ecological data.Vanga is located at the mouth of the river Umba. The river drains its catchment in northeast Tanzania Fig.  from itsS1). There are also many smallholder farmers, who commonly grow rice and cassava on the fertile riverbanks of the Umba and/or engage in livestock husbandry. Overall, Kwale County and Vanga perform poorly on available wellbeing indicators compared to national averages  tools  with the GPCC rainfall data for this period at 4.75° S, 38.75° E, giving confidence in utilising the product for localised rainfall estimates beyond 2002. The impacts on ecosystems were assessed using longitudinal data collected on mangroves, sea grasses and coral reefs. The 1998 El Niño event caused large-scale die-back in some mangrove areas in Kenya due to the substantial influx of sediments, carried by heavy rainfall on eroding coastal catchments  in the Umba drainage region. Strong El Niño and La Niña events were classified, using an Oceanic Niño index in the 3.4 region,S1). For example, after creating a seasonal calendar of a typical year, participants discussed how the seasons differed during the El Niño episode. A major flood in Vanga in April 2016 was experienced during the El Niño period. A census household survey  was undertaken in the three study villages to understand impacts on wellbeing and responses to the flood event. To capture impacts on a marginalized group, an additional survey  of female-headed households living in the flood-prone villages of Jimbo and Jasini, and Vanga town was conducted to understand how the multiple effects of the flood manifested and interacted to affect household wellbeing of a vulnerable social group. Local school attendance and hospital admission records was digitised and analysed to quantify health and education impacts from the flood. A further survey (HHS3) of 15 households (previously surveyed by the SPACES project) investigated household impacts, preparations and perceptions of El Niño beyond the flood event.Direct and indirect social impacts and responses to the 2015–2016 El Niño event were recorded during the forementioned CVCA focus group, three household surveys  and key informant interviews (KIIs) in November to December 2016. Thus, immediate impacts were recorded based on the recollections of research participants, about 6 months after the El Niño episode ended. At the workshop, participants discussed the El Niño event in relation to each workshop activity , focus groups (FG1 and 2) and interviews found that the floods impacted several aspects of household wellbeing Table . None ofThe census survey (HHS1) found that over 80% of households in the three surveyed villages were affected by the floods and 47% of households experienced an economic security impact Table . Out of “[The] 2016 flood effects were very severe, therefore, most people could not access the [health centre] facility due to broken bridges and submerged roads. In 2015, however, the turn up [at the health centre] was high since the floods were not severe and roads and bridges were all accessible during and even after floods. […In 2015], there was [also] an outbreak of diarrhoea in Tanzania [… that] spread to Vanga through immigrants who settled in Jasini Key informants identified those living in mud houses as especially affected by the April 2016 floods, and our household survey (1) recorded 68% of the houses damaged or destroyed by the floods as constructed of mud and thatch. These weakly constructed houses are most commonly inhabited by the very poor and female-headed households; this might explain why only 15% of the houses of male headed households were destroyed while 47% of all female-headed households lost their house during the floods. Indeed, fewer houses of married couples were destroyed compared to those of other martial statuses  purchase fish at Vanga’s auction to sell in nearby markets, local large-scale fish dealers (tajiri) export fish by road to sell to fish shops as far as Mombasa, and large-scale octopus dealers collect octopus from Vanga to be processed and sold in Mombasa  identified perceptions of how the impacts of the flood event interacted. Decreased agricultural production and disruption to fisheries trade reduced the food and income of farmers and fishers, and regional declines in agricultural production and the disruption of the road network reduced food imports to the sub-location, which, together, raised the price of food. Households whose crops failed were, therefore, forced to purchase this relatively expensive food. Furthermore, if a member of a household contracted a waterborne disease, working days were lost to sickness and income was spent on medication. These interactive impacts manifested in hunger for 5.9% of households (HHS1), and increased expenditures, thus making it difficult for some families to pay school fees . While school attendance is normally lower at the beginning of term because parents need to clear school debts, there was a more pronounced drop in attendance during the 2016 floods, especially in children from Jasini, because the collapsed bridge made travel across the river dangerous  was hosted in Vanga by the local chief, the National Drought Management Authority, and the Kenyan Red Cross (KRC) to inform residents of the predicted El Niño event. Out of the 15 households surveyed specifically about the El Niño event (HHS3), seven did nothing in response to warnings, with the rest buying mosquito nets (3) staying away from risky areas such as riverbanks (3) or strengthening (2), or digging ditches (2) around, their house. It is not clear whether these reported responses may be part of annual preparations for the wet season regardless of El Niño warnings. Similarly, while half (51) of HHS1 respondents said they had received a forecast of heavy rains in April, 80% did nothing to prepare for floods (HHS1). The data did not explain why most did not respond.Table During the floods, community-based actions were limited to assisting KRC with humanitarian needs assessments. For female-headed households (HHS2), some evacuated their destroyed home to live with a neighbour, which affected their social relations, some diverted cash normally used to restock their business to buy food and pay school fees, and others sold household possessions to buy food: “I used what I had from the business, what I would have used to buy clothes in Mombasa, to buy food and pay for school fees”. County government support only reached 7% of surveyed households (HHS1), while KRC played a critical role in the distribution of relief items, such as food, shelter and medical assistance, according to CVCA participants.“Every year when it floods, we know who will need assistance. For instance, we know that an old man living in an inundated house will need to be convinced to leave his home”Vanga has no community organisations responsible for disaster risk reduction or response, and few external interventions have sought to build resilience to the floods (FG1). This may explain the limited community level preparations and responses to the El Niño event and the April 2016 flood event, and the dependence of households on their own resources and humanitarian assistance. Eight months after the flood only just over half (56%) of households had repaired their houses, and the homes of four female-headed households remained uninhabitable. This weak capacity was attributed to a lack of financial and physical resources to reconstruct again (HHS1). Moreover, Jasini residents, a year after the flood, remain dependent on a fee-charging, privately owned temporary bridge (HHS1).The April 2016 flood demonstrated the general lack of capacity to respond to disasters and address flood risk in Vanga. During the CVCA, participants reported a few adaptations made to reduce flood risk. A seawall was constructed in 1970, and then strengthened and extended in 2005 by the World Bank, and an irrigation scheme was established to control floodwaters and supply water to rice fields. The community also said that they had sought to protect the mangroves by controlling cutting in recognition of the mangroves’ flood and storm regulation services. These adaptations have done little, however, to mitigate flood risk, with the KRC assisting flood victims every year:Kusi season) often makes the sea treacherous for small fishing vessels. The El Niño flooding, therefore, exacerbated a food insecure period of the year.The April 2016 floods coincided with one of Vanga’s most difficult periods of the year identified on the seasonal calendar at the CVCA focus group. Participants explained that the long rains is the key planting season; food availability is often low because stores from previous harvests are running out and they must wait for the new crop to mature. It is also the low season for small-scale fishers since strong southerly winds  (CVCA). While inland areas are more prone to drought than Vanga (key informant), every year, farmers are faced with the dilemma of whether to plant early in the long rainy season and risk their crop being destroyed by heavy rains and floods or wait until later in the rainy season but risk their crop perishing from drought: ‘For the farmers, you only plant once a year, if you miss the season, it is difficult for the whole year. You have to wait until the next year.’ (FG1). Farmers’ planting strategies are further complicated by inter-annual climate variability caused by El Niño, La Niña and the Indian Ocean Dipole. Uhe et al  showed tLong-term stressors may also influence the vulnerability of households to El Niño events. Overexploitation of the fisheries and degradation of coastal marine ecosystems through destructive fishing and mangrove cutting has resulted in a sustained decline in fisheries production Fig. 7. A key iThis article presents a social-ecological systems analysis of the complex human–environment interactions and social processes that determine vulnerability to, and the impacts of, El Niño events in place. The 2015–2016 El Niño event interacted with several features of the social-ecological system in Vanga.First, direct social and ecological impacts were amplified or intensified through system feedbacks and social and biophysical responses Below is the link to the electronic supplementary material.


Dear Editor,1 Despite extensive biochemical and cellular studies on ATM phosphorylation, these phosphorylation sites are rarely mutated in cancers, challenging the pathophysiological relevance of ATM-mediated phosphorylation in the disease setting.Accumulating evidence supports that, as a critical genome guardian mechanism, the DNA damage response (DDR) is a barrier to early tumorigenesis. As a crucial DDR pathway, Ataxia Telangiectasia Mutated (ATM)-mediated phosphorylation of downstream targets is essential for activation of cell cycle checkpoints, DNA repair, and programed cell death in the presence of DNA damage.2 Recent studies have shown that SPOP plays a critical role in the DDR, and clinical data support that SPOP mutations are associated with genomic instability.3 Among SPOP mutation sites, we found that the Serine 119 mutation to asparagine (S119N), one of the clinically relevant mutations, resulted in a radiosensitive phenotype. S119N mutated prostate cancer cells showed hypersensitivity to IR , an E3 ubiquitin ligase adaptor, is among the most frequently mutated in prostate cancer. IR Fig. –c and a  IR Fig. . We also IR Fig. . We also IR Fig. . In nonm IR Fig. , inducti4 This endogenous interaction was confirmed in PC-3 cells  caused a dramatic reduction in phosphorylation signals Fig. . To asseTo further investigate SPOP’s function in the DDR, we aimed to identify proteins that SPOP interacted in response to IR. We performed the Stable Isotype Labeling by Amino acids in Cell culture (SILAC) assay, and found that proteins with increased SPOP interactions in response to IR are involved in cell division , a component of the MCM helicase complex for validation. However, despite the dissociation from the SPOP complex after IR, the MCM5 protein level did not change Fig. . Howeverp = 0.0001)  Fig. . These rIn conclusion, we demonstrate that a prostate cancer-relevant mutation of SPOP on Serine 119 causes prolonged DNA repair and hypersensitivity to ionizing radiation. We prove that Serine 119 is required for the SPOP interaction with ATM, and demonstrate that ATM phosphorylates SPOP on Serine 119. Further, we identify the MCM5-MCM3 complex is among SPOP interacting proteins in response to DNA damage. We demonstrate that Serine 119 phosphorylation is required for the SPOP-MCM3 dissociation and it inhibits MCM3 ubiquitination and degradation Fig. . Taken tSupplemental information


In 2014, a telemedicine system was established in 24-h routine use in the emergency medical service (EMS) of the city of Aachen. This study tested whether the diagnostic concordance of the tele-EMS physician reaches the same diagnostic concordance as the on-site-EMS physician. The initial prehospital diagnoses were compared to the final hospital diagnoses. Data were recorded retrospectively from the physicians’ protocols as well as from the hospital administration system and compared. Also, all diagnostic misconcordance were analysed and reviewed in terms of logical content by two experts. There were no significant differences between the groups in terms of demographic data, such as age and gender, as well as regarding the hospital length of stay and mortality. There was no significant difference between the diagnostic concordance of the systems, except the diagnosis “epileptic seizure”. Instead, in these cases, “stroke” was the most frequently chosen diagnosis. The diagnostic misconcordance “stroke” is not associated with any risks to patients’ safety. Reasons for diagnostic misconcordance could be the short contact time to the patient during the teleconsultation, the lack of personal examination of the patient by the tele-EMS physician, and reversible symptoms that can mask the correct diagnosis. At the same time, there is a steadily increasing shortage of on-site-EMS physicians, especially in rural areas of Germany5. In order to counter this problem, the EMS needs to be optimised. Examples of optimisation are the introduction of a structured emergency call query by the dispatch center6, a new law for training of paramedics or the implementation of telemedicine in the EMS. Telemedicine in the EMS is currently being introduced in some regions in Germany8, the first holistic system was implemented in the city of Aachen, Germany in 20149. The telemedicine system allows an immediate consultation with an EMS-physician at any time and, consequently, enables a higher quality of emergency medical care.Due to the demographic change, prehospital emergencies increase by 3.7% annuallyDuring the years after implementation, the number of telemedicine consultations for prehospital emergency patients increased. The tele-EMS physician can offer support for paramedics especially regarding diagnosis.10.The diagnosis in the prehospital phase often determines the prehospital therapy and influences the choice of the hospital, if hospitalisation is needed. Therefore, it is important to know whether a distant tele-EMS physician can determine the correct diagnosis from the given information by the paramedics. As there are no predefined diagnoses for both, on-site-EMS and tele-EMS-physician, the tele-EMS physician needs to determine the correct diagnosis based on the information provided by the paramedics using clinical findings. The tele-EMS physician primarily supports the paramedics, but can also speak to the patient himself by phone if necessary. As the correct diagnosis lays the foundation for further treatment at hospital, it could be hypothesised that these diagnoses influence the prognosis and outcome of patients by preventing unnecessary delays to therapy initiation. Nevertheless, there are hardly any evaluations of the diagnostic concordance of on-site-EMS physicians and none of tele-EMS physicians12.Therefore, this study aims to analyse whether the diagnoses of a tele-EMS physician is as precise as those of an on-site-EMS physicianIn this retrospective cohort study. Data were recorded from emergency protocols as well as from hospital information systems in order to compare prehospital diagnoses with the final diagnoses determined by the hospital.Patients who were transported to the University Hospital RWTH Aachen were included in the study. Besides patients’ diagnoses, demographic data, such as age, gender, date of discharge, or death, were extracted from the prehospital protocols and the hospital administration system.The local ethics committee granted an analysis of the data for quality assurance purposes and waived the requirement of informed consent . All data were recorded and saved anonymously.13. This system was implemented as an additional structure into the regular 24/7 routine EMS of Aachen in April 201416. In the city of Aachen, there are twelve ambulances, two EMS-physicians, three primary care hospitals und an university hospital. Before the system was implemented, an EMS physician was involved in approximately 30% of all missions , after the implementation the on-site EMS physician was involved in approximately 20% and the tele-EMS-physician in approximately 10% of all missions.The Department of Anaesthesiology of the University Hospital RWTH Aachen developed from 2007 to 2013 a holistic telemedicine system as an addition to the conventional prehospital emergency systemThe two main components of the telemedicine system are the teleconsultation centre and the telemedically-equipped ambulances. A mobile communication unit is attached to the monitor-defibrillator-unit  and allows data transmission from the following components: audio communication between paramedics and the tele-EMS physician; real-time vital data transmission; 12-lead ECG transmission; picture transmission from a connected smartphone; real-time video streaming from a 360° camera in the ambulance; printing of a teleconsultation report inside the ambulance; and position of the ambulance using GPS technology.The teleconsultation centre consists of a 180° computer workstation with four standard monitors, on which the transmitted data are displayed using a specifically developed software named TM-Doku .The tele-EMS physician is, in comparison to the on-site-EMS physician, available immediately. The average consultation time at the beginning of the implementation, i.e. at the time of the presented study, was 11 min, currently it is 9 min. The average duration of missions supervised by on-site-EMS physicians is 43 min.The professional qualification of all tele-EMS physicians is based on former experience as an on-site-EMS physician from at least 500 on-site emergency missions combined with further training in the telemedicine system, as well as in communication with the EMS teams. All tele-EMS physicians fulfil the standard of at least being in their 5th year of residency in anaesthesiology.17. Nevertheless the tele-EMS physician decides whether transportation to a hospital is necessary or not, since the physician is the responsible leader of the scene itself. The telemedicine system allows an immediate consultation with an EMS-physician at any time, consequently the teleconsultation eliminates the physician-free period and enables an immediate start of medical treatment.The tele-EMS physician can offer support for paramedics and can delegate the administration of drugs to achieve a better quality of care for patients, as the paramedics must not administer any medication by law. Furthermore, he can bridge the time of diagnosis and treatment for life-threatening diseases and injuries until the on-site-EMS physician arrives on the scene. The paramedics decide in accordance with the SOP (standard operating procedure) when to consult the tele-EMS physician and the paramedics can only take live-saving actions such as defibrillation without the delegation of a physician if there is acute risk to life. In future, paramedics will have more skills due to the Emergency Paramedic Act passed in 2014 and the associated expansion of training from 2 to 3 yearsAs teleconsultation allows the tele-EMS physician to attend multiple emergency cases at the same time, the finite resourced of an on-site-EMS physician can be used wisely and called out only in life-threatening emergencies. Therefore, this function is more often available for patients who really need an on-site-EMS physician for invasive advanced life support measures.Tele-EMS physicians' data were evaluated from January to September 2015. To enable an evaluation of the same diagnostic collective, a control group of on-site-EMS physicians’ data was chosen from January to March 2014, a period before the introduction of the telemedicine system. For analysing a comparable number of tele-EMS and on-site EMS missions, 3 months were included for the on-site EMS-cases and 9 months for the telemedicine cases.To analyse comparable collectives, the following exclusion criteria were applied: patients under the age of 18 years, suicidality, resuscitation, polytrauma, psychosis, forced admission, rampage threat, premature birth, transportation to a palliative ward and dislocation of a tracheostomy tube. These exclusion criteria were based on diagnoses which are not found to be suitable for teleconsultation, but suitable for an on-site-EMS physician. In case of CPR for example, the missions are not supervised by a tele-EMS physician as this time critical treatment leads no room for a tele-consultation. Consequently, it is necessary that an on-site-EMS physician is on scene.The physician’s diagnoses were analysed based on the standardised tele-EMS and on-site-EMS physician protocols. Up to three initial diagnoses per case were recorded from the tele-EMS and on-site-EMS physician protocols. These were extracted from the designated fields for diagnoses as well as from the free text entries and, afterward, manually digitised by the study group. There is only one diagnosis written in the designated field, however, a maximum of two further diagnoses were extracted from the free text in order to be able to depict the diagnosis more holistically. This was only the case when clear differential diagnoses were defined by the on-site- and tele-EMS physicians.18. Subsequently, the precise ICD-code of the University Hospital RWTH Aachen was compared to the ICD-codes of the diagnoses of the tele-EMS and on-site-EMS physicians.The suspected diagnoses of the tele-EMS and on-site-EMS physicians were compared with the diagnoses, which were extracted from the administrative data of the University Hospital RWTH Aachen at the end of the stay in the hospital. Therefore, in a first step, in order to guarantee a blinded process, the tele-EMS physician and on-site-EMS physician diagnoses were manually coded according to the ICD-groups , whereas the diagnoses of the university hospital RWTH Aachen were found as a defined ICD-10-code and used as the reference diagnosis in this studyAfter data collection and processing, the diagnoses made by the tele-EMS and on-site-EMS-physicians were analysed and compared. Additionally, tracer diagnoses, leaning on the consensus document on emergency care, "acute coronary syndrome" (ACS), "stroke", “cardiac diseases”, "hypertension", "epileptic seizure", and “chronic obstructive pulmonary disease” (COPD) were identified and analysed individually in relation to diagnostic miscordances. In addition, all diagnostic miscordances were analysed and reviewed in terms of logical content by two experts.19.The use of the camera system, which is implemented in every ambulance, was also analysedStatistical significance was tested using the Fisher exact test and the unpaired t-test using SPSS Statistics, Version 25.0 . The probability of a type 1 error below 1% was considered as significant.All authors confirm that all methods were carried out in accordance with relevant guidelines and regulations of the Declaration of Helsinki.There were no significant differences between included patients treated by tele-EMS physicians and on-site-EMS physicians in terms of demographic data, such as age and gender, as well as regarding the hospital length of stay, as shown in Table The prehospital diagnoses of 584 patients treated by the tele-EMS physician and 634 patients treated by the on-site-EMS physician were analysed. Due to the applied exclusion criteria, the cohorts and diagnoses are comparable in content and structure Fig. .Figure 1After comparing the diagnoses using the ICD-10-system and after the double expert review, which compared the diagnoses content-wise and logically, there was no significant difference between the accuracy of the correct diagnoses in general (p = 0.0193). Consequently, the correct diagnosis was chosen in 82.0% (n = 520) and 76.5% (n = 447) of the patients treated by the on-site-EMS physician and tele-EMS physician, respectively.As shown in Fig. Six patients receiving an initially incorrect diagnosis by the tele-EMS physician died during further treatment in the hospital. Looking closer at diagnoses and cause of death, the following was found: two patients were diagnosed with an ACS, of which one died of complications following a diaphragmatic hernia, and the other of pneumonia. The third patient was diagnosed with pneumonia and died due to heart failure. With these three patients, no correlation could be drawn in terms of the diagnoses and the cause of death. Diagnoses did not lead to a lack of therapy or prehospital intervention. The fourth patient, diagnosed with pneumonia, died of sepsis as a consequence of pneumonia. The fifth patient, who died of a urinary tract infection, was initially diagnosed with sepsis. The sixth patient was diagnosed with pulmonary edema and died because of an ACS. In those three cases, the causes of death showed a causal connection to the tele-EMS-diagnoses, even though they were not matching exactly in terms of wording.Five patients who were wrongly diagnosed by the on-site-EMS physician also died during hospitalisation. Two of those died due to heart failure and were diagnosed by the on-site-EMS physician with pulmonary edema and tachycardia. The third patient was diagnosed with pneumonia and died of sepsis. The fourth patient died because of an open wound of the pelvis and was diagnosed with a femoral neck fracture. These four diagnoses can obviously be seen as directly related to the cause of death. The only diagnostic misconcordance with no correlation to the cause of death was a patient who died because of an acute renal failure and was diagnosed with COPD.There was no significant difference between tele-EMS and on-site-EMS physicians in terms of duration between the time of admission and the time of death . None of the deceased tele-EMS-patients and only two of the patients treated by the on-site-EMS physician died after less than 24 h of hospitalisation (p = 0.490). No significant differences could be observed in these patients in terms of gender  and age .In 54.6% (n = 319) of all teleconsultations (n = 584), information about the use of the video camera was found. Video streaming was used in 43.9% (n = 140) of those cases; in 56.1% (n = 179) video streaming was not used. Among the 36 cases where the tele-EMS physician misdiagnosed the patients with seizures, the camera was used in 25.0% (n = 9). In the cases where the tele-EMS physician correctly diagnosed those patients with epileptic seizures (n = 17), the camera was used in 35.3% (n = 6) of the cases. Altogether, the camera was not used significantly more often (p = 0.5196) on correctly diagnosed patients with seizures than on misdiagnosed patients.This study aimed to investigate whether there is a difference in diagnosis quality between a tele-EMS physician and an on-site-EMS physician. Statistically higher accuracy of the one-site EMS-physicians could not be shown.When comparing both systems, it was shown that the diagnostic concordance regarding tracer diagnoses does not differ significantly between tele-EMS physician and on-site-EMS physician, with the exception of the diagnosis "epileptic seizure". The mortality within 24 h after admission in the hospital does not differ significantly as shown in Table 20. The rate of diagnostic misconcordance concerning “cardiac diseases” is high in both groups (54.5% vs. 52.9%) because of the multiple other diagnoses with similar symptoms and frequent combination of ACS, heart failure, lung edema, and tachycardia. In terms of COPD it is also high, as the diagnostic possibilities are very limited in the EMS. In the case of the epileptic seizure-patients, tele-EMS and on-site-EMS physicians both have high diagnostic misconcordance rates. In 88.9% (tele-EMS physicians) or 60.0% (on-site-EMS physicians) of those cases, the patients were diagnosed with "stroke", an important differential diagnosis of seizure. Due to the short time of preclinical treatment and the short time of patient contact, it is possible that reversible symptom complexes  were present during the prehospital phase but not in the hospital setting, thereby masking the correct diagnosis. Also, it can be assumed that the prioritised diagnosis of stroke  is preferably selected by tele-EMS or on-site-EMS physicians. A comparable study by Arntz et al. describes the underestimation of the severity of neurological disorders and discusses the use of support, such as, for example, video-assisted care for complex neurological diseases21. However, our data show that complex neurological conditions can be better understood by the on-site-EMS physician, who can observe and examine the patient personally. Therefore, we analysed the present data according to the use of video cameras, which was used in only about 25.0% of all teleconsultations. Most tele-EMS physicians think that the phone transfer of information via headset without the use of the video camera is sufficient. We detected no significant difference of misdiagnosed or correctly diagnosed patients with epileptic seizures. It should be mentioned that the video stream is only possible in the ambulance itself, whereas most of the treatment starts outside the ambulance. Moreover, as a consequence of this study in the standardised questionnaire of the tele-EMS physician, questions regarding epileptic seizures were implemented to be asked by the tele-EMS physician, which should lead to a higher correct diagnosis rate.This confirms the efficiency of telemedical therapy, which was already shown in 2016 by Brokmann et al. for ACSThe correct prehospital diagnosis is particularly important when a special department is required for the treatment; for example catheterisation laboratory for ACS-patients or endovascular intervention for stroke-patients, which is not available in every basic and standard care provider. A diagnostic misconcordance with the selection of the wrong hospital can consequently lead to a possibly life-threatening therapy delay.22.Prehospital diagnostic options are limited  due to restricted apparatus and laboratory diagnostic features. Furthermore, working under time pressure, on-scene might also limit the prehospital diagnosis. Thus, the tele-EMS physician, who is, in comparison to the on-site-EMS physician, available immediately, has an advantage of being shielded from the emergency scenery and, therefore, can apply maximum concentration. He can also easily use the provided SOP and review the latest recommendations of various medical societies and, therefore, work based on up-to-date algorithms. In order to come to the right diagnosis, the tele-EMS physician is highly dependent on accurate information from the paramedics’ team on-site, whereas the on-site-EMS physician has the possibility to examine and consult the patient himself. Patients with respiratory problems for example are very hard to examine as the tele-EMS physician needs to trust the paramedic on information about breath sound, speed and depth. Yet, it has to be said, that diagnostic mistakes occur in prehospital as well as in intrahospital settings24. The establishment of clinical prediction rules helps the paramedics classify patients with strokes in order to receive endovascular intervention as fast as possible. These studies rather investigate the time-opimised patient care in isolation than considering differential diagnoses25.Studies on stroke-patients show the reliability and accuracy of telemedical support. It provides a faster access to specialised treatment and accelerates the door-to-needle-time28.According to the emergency paramedic law, a training curriculum could be helpful. A standardised and structured procedure could lead to an improvement of taking the patients’ medical history and consequently improve the transfer of information to the tele-EMS physician. Structured basic education, training, and further education of tele-EMS and on-site-EMS physicians are critical to achieving a high diagnostic concordance30. Such a system could inform the tele-EMS physicians and on-site-EMS physicians, as well as the EMS paramedics, about the final diagnoses and thus help to verify the initial diagnoses, thus providing an opportunity for subjective evaluation of possible diagnostic errors21. This could, consequently, serve as a regular, personal re-evaluation. Likewise, a correct diagnosis can be confirmed and also be regarded as a personal learning process that could be transferred to future cases by the attending physician.Since the prehospital care of the patients usually ends with admission to the appropriate hospital, a feedback system would be required, similar to the response number of the EMS in the province of HessenIn conclusion, it could be shown that the ICD-codes are not an optimal classification for comparing diagnoses; especially not for preclinical diagnoses that are not already available as an ICD-code. Because there are several ICD-codes for the same diagnosis, this procedure can easily lead to errors. The results are still very meaningful and significant despite the different presentation of the diagnoses (free text vs. ICD-Code), as this leads rather to a shift of the results at the expense of diagnostic misconcordance. However, another type of comparison should be considered in future research. Any other method would be expected to give better results in terms of diagnostic concordance.There are a few limitations.Only patients admitted to the university hospital RWTH Aachen were included in the study because analysing the data of the three other hospitals in Aachen was not possible due to privacy policies, but more than 50% of all patients are transported to the university hospital RWTH Aachen, which is the only hospital in Aachen with a department for neurology and a cardiac catheterisation laboratory.Secondary transports were not investigated; these will be evaluated in further studies. This may reveal patients who were initially transported to the wrong hospital. Missions where the tele-EMS physician was bridging the time until an on-site-EMS physician was on scene were also not investigated. An on-site-EMS physician from neighbouring areas, when no other on-site-EMS is available in the city of Aachen, is required about 30 times a month. They take about 10 min longer to be on scene. In these cases the TNA can bridge the gap. Those missions will also be investigated in further studies.The telemedical and emergency medical diagnoses were coded by diagnostic keywords in ICD-groups. By contrast, the diagnoses documented in the hospital administration system were available as a precisely defined ICD-code. It was shown that a tele-EMS physician’s or on-site-EMS physician’s diagnosis was considered wrong, but instead was identical to the diagnosis documented in the hospital administration system, as the ICD-groups are broader and several diseases of one symptom complex are summarised, as well as the fact that there exist several ICD-codes for one and the same diagnosis.Due to the handwritten recording of patient data, transmission errors when filling out the application protocols and when manually entering the protocol data into a digital table cannot be ruled out and might be the cause of missing or incomplete patient data, which then led to the exclusion of those patients.In addition, the missions without a physician involved were not evaluated because an EMS physician, on-site-EMS or tele-EMS physician, must always be involved in every potentially life-threatening emergency. This is laid down by law, since paramedics in Germany are not allowed to administer drugs. Accordingly, a physician is always involved when medical therapy is required.The rare use of the video camera in those cases with patients with seizures must also be mentioned.It has to mentioned, that in spite of the many obvious advantages of telemedicine in general, the implementation of a telemedical system in all areas of medical care is hardly realisable due to costs, technical implementation, technical know-how and data protection regulations as privacy policies. Thus its generalisation can be limited.No significant differences in patient outcomes in terms of length of stay, morbidity and mortality could be found in this study. It was shown that the diagnostic concordance  does not differ significantly between tele-EMS and on-site-EMS physicians, with the exception of the diagnosis "epileptic seizure". Regarding the diagnosis of "epileptic seizure", both the tele-EMS physician and on-site-EMS physician often made the mistaken diagnosis of "stroke", which does not contribute any added risk to patient.To avoid diagnostic misconcordance in cases of neurological diseases, it might be helpful to make more frequent use video-assisted aids to help the tele-EMS physician to examine the patient even more systematically and holistically. As a consequence of these findings, the questionnaire in case of assumes strokes, posed by the tele-EMS physicians to the ambulance staff, has been extended by the question, whereas the anamnesis shows any signs for epileptic seizures or anticonvulsant medication. As a consequence of the presented results, however, the procedural instructions were changed, so that the use of video is mandatory for all patients with neurological conditions, as far as they agree.13.Future investigations will be needed to show whether the video consultation and the extension of the questionnaire lead to a further improvement of the diagnostic concordanceIn summary, this study shows that telemedicine is definitely relevant to the future prehospital medical care and that a patient-oriented and highly qualified EMS can no longer be imagined without telemedical support.


Despite the unique physiology and metabolic pathways of microbiomes from cold environments providing key evolutionary insights and promising leads for discovering new bioactive compounds, cultivable bacteria entrapped in perennial ice from caves remained a largely unexplored life system. In this context, we obtained and characterized bacterial strains from 13,000-years old ice core of Scarisoara Ice Cave, providing first isolates from perennial ice accumulated in caves since Late Glacial, and first culture-based evidences of bacterial resistome and antimicrobial compounds production. The 68 bacterial isolates belonged to 4 phyla, 34 genera and 56 species, with 17 strains representing putative new taxa. The Gram-negative cave bacteria (Proteobacteria and Bacteroidetes) were more resistant to the great majority of antibiotic classes than the Gram-positive ones . More than 50% of the strains exhibited high resistance to 17 classes of antibiotics. Some of the isolates inhibited the growth of clinically important Gram-positive and Gram-negative resistant strains and revealed metabolic features with applicative potential. The current report on bacterial strains from millennia-old cave ice revealed promising candidates for studying the evolution of environmental resistome and for obtaining new active biomolecules for fighting the antibiotics crisis, and valuable cold-active biocatalysts. Thus, cold-adapted microbiomes are very important for the global ecology, while their biological activities maintain the nutrient flux in the environment and contribute to the global biogeochemical cycles, being also sensitive indicators of climate changes3. Moreover, their unique physiology and metabolic pathways provide essential knowledge for understanding the adaptation mechanisms to low temperatures and a valuable source of biomolecules for biotechnological applications. To overcome the challenges of life in harsh conditions 4, cold-adapted microorganisms have developed a wide range of adaptations, from cellular envelope  and metabolic adaptation , to cryoprotectants  and increased variety and number of chaperons production, and novel metabolic capabilities , that can be exploited to develop novel biotechnological perspectives5. Despite the constantly increasing data on the microbial composition and diversity in Polar ice sheets and glaciers9, Polar soil and permafrost11, and aquatic environments13, microbiomes entrapped in perennial ice from caves still remain a largely undiscovered life system14.Earth is a primarily cold planet, with more than 85% of the biosphere being exposed to permanent temperatures below 5 °C and with frozen habitats covering up 20% of our planet surface3 perennial ice block dated about 13,000-years before present (BP) being one of the oldest and largest in the world18.Among the explored ice caves, Scarisoara Ice Cave (Romania) constitutes a unique cold environment, its 100,000 m22 and fungi24 in cave ice deposits. The microbial diversity and geochemical dependence of uncultured microbiome from underground frozen habitats highlighted complex bacterial communities in sediments from Antarctic volcanic ice caves from Mount Erebus25, and both sediments and ice from Hawaiian lava tubes26. A rich uncultured prokaryotic diversity was also found in Scarisoara cave ice strata of different ages28, in addition to a complex fungal community29. The distribution of the total and putatively active bacterial and fungal communities appeared to be modeled by the climatic characteristics during ice deposition and the organic carbon content of the ice substrate29.Recent investigations revealed the presence of cultivable bacteriawww.who.int)30. AR is an old natural phenomenon, the antibiotic biosynthetic pathways and the resistance genes evolving over millions of years35. More evidence indicated environmental resistome as the reservoir for resistance genes found in clinical pathogens37. Due to their multiple physiological roles in natural environments, antibiotics acted as selecting and accelerating factors of AR evolution39. Bacterial strains highly resistant to known antimicrobial compounds usually have the potential for antimicrobial activity against other bacteria9.Antimicrobial resistance (AR) is nowadays in top ten of global threats, concerning the animal and human health, food security and affecting the entire societal development , in addition to Micrococcaceae (6 strains), Nocardioidaceae (3 strains) and Dietziaceae (1 strain) (Supplementary Table Microbacterium (13), Bacillus (5), Arthrobacter (4), Brevundimonas (4), Chryseobacterium (4), Cryobacterium (3), and 2 isolates of Aeromicrobium, Paralcaligenes, Pedobacter, Phyllobacterium, Pseudarthrobacter, Pseudomonas and Sporosarcina genera . In addition, two different Bacillus safensis homologs were retrieved from old strata , and different B. thuringiensis strains from 400-years BP ice. The isolates from the oldest cave ice layers also comprised Aeromicrobium panaciterrae, Paenibacillus amylolyticus, Pseudomonas brenneri, and P. grimontii homologs among the Late Glacial cave ice bacterial strains  and Proteobacteria (19), with a lower representation of Firmicutes (7) and Bacteroidetes (6) , and 4–20 °C range (Flavobacterium glaciei SC1A.2 and Arthrobacter sp. SC41AB.3). The remaining 64 strains were psychrotrophs, with minimum growth temperatures of 4 °C and 10 °C. Among these, a variable number of Actinobacteria (10), Proteobacteria (7), Firmicutes (2) and Bacteroidetes (1) strains were cultivated between 4 and 25 °C, Phenylobacterium sp. SC71.1B and Eoetvoesia caeni SC41AB.2 could grow between 10 and 25 °C, and Pedobacter bambusae SC1A.4 within the 4–28 °C temperature interval  intervals , Cryobacterium (3 strains), Leifsonia (1 strain), Glaciihabitans (1 strain), Salinibacterium (1 strain), Psychrobacter (1 strain), Paenisporosarcina (1 strain) and Flavobacterium (1 strain). Among these, F. glaciei SC1A.2, S. xinjiangense SC80A.3, and C. levicorallinum SC21E.1 were psychrophiles. The remaining 8 isolates  were classified as psychrotrophs based on their growth temperature range  (25), extended-drug (XDR) (8), and pan-drug (PDR) (2) resistance phenotypes, according to the definitions proposed by Magiorakos et al.Microbacterium pygmaeum SC83A.2, Pseudarthrobacter oxydans SC70B.1 and Dietzia sp. SC61A.5B displayed a XDR phenotype, while G. tibetensis SC21E.3, Mycetocola manganoxydans SC51A.2 and A. psychrolactophilus SC7AB.1 were susceptible to most of the tested antibiotics  and Bacillus sp. SC8A.7 (19 antibiotics)  Fig. , while SE. caeni SC41AB.2, Caulobacter sp. SC61A.5 and P. glaciei SC80A.2 were only resistant to 14, 18 and 16 antibiotics, respectively  and P. bambusae SC1A.4 the most limited AR (16 antibiotics).More than 50% of the Gram-negative cave ice bacteria were resistant to all tested antibiotic classes, excepting rifampin, tetracyclines and carbapenems in the case of Proteobacteria Table . The majThe highest resistance rate to broad-spectrum aminoglycosides was displayed by Bacteroidetes 100%), Proteobacteria (96.5%) and Actinobacteria (88.5%) strains, while only 62.9% of Firmicutes were resistant to this class  while only 11.1% to nitrofurantoin, and completely susceptible to carbapenems and tetracyclines , five Gram-negative Proteobacteria , and the Firmicutes B. toyonensis SC86E.3 strain  was recorded for Delftia sp. SC71.5.Screening of enzymatic activities using API ZYM test systemCaulobacter sp. SC61A.5, M. ginsengiterrae SC1A.5 and P. brenneri SC97A.1. Only four isolates were able to metabolize urea. Gelatin hydrolysis was limited to B. toyonensis SC86E.3 and M. ginsengiterrae SC1A.5. A. psychrolactophilus SC7AB.1, M. ginsengiterrae SC1A.5, M. pygmaeum SC65A.2 and Pseudarthrobacter sp. SC86E. Four cave bacteria tested positive for β-galactosidase, and cytochrome oxidase activity was measured for five strains . Meanwhile, a reduced assimilation capacity was observed for Delftia sp. SC71.5 and B. toyonensis SC86E.3 testing positive for 4 and 5 substrates, respectively  showed activity against 9 pathogens. No antimicrobial effect was recorded against one Enterobacter, two methicillin resistant S. aureus, one Klebsiella, and all Acinetobacter sp. strains  focused on their growth temperature, antibiotic resistance, enzymatic and antimicrobial activity profiles.22 as compared to glacier ice53, the cave isolates exhibited a large diversity, covering the main phyla commonly found in frozen habitats, i.e. Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes43. Among these, an important fraction (17 strains) showed a low 16S rRNA gene identity with previously reported bacteria  appeared unrelated to other representatives of these genera. Interestingly, all of these LGP-originating bacteria were psychrotrophs able to grow above 30 °C.Despite the lower microbial density found in Scarisoara ice strata55, the Cryobacterium spp. Scarisoara isolates are, to our knowledge, the first retrieved from ice accumulated in caves60 and Salinibacterium xinjiangens63 was cultivated for the first time from ice deposits. Amongst Proteobacteria, Pseudomonas  and Psychrobacter glaciei (8000 years old ice) strains were isolated for the first time from the ice core of this cave, adding this habitat to other frozen environments colonized by these bacteria67. Interestingly, the Phyllobacterium cave isolates from 700 and 7000-years old ice homologous to plants-associated species69 could originate from the abundant vegetation above the cave70. Firmicutes strains belonging to Bacillus and Sporosarcina genera were retrieved from different Scarisoara ice strata, confirming the resilience of these genera in recent and old icy habitats75. The presence of Flavobacterium glaciei in 100-years old Scarisoara ice, previously reported in China No.1 glacier76, highlighted a wide geographical distribution of this psychrophilic species. In addition, the recovery from 100-years old ice of Pedobacter strains closely related to oil and aquatic strains78 confirmed their adaptation to frozen habitats79. Meanwhile, the scarce recovery of Bacteroidetes strains from Scarisoara ice suggested the high sensitivity of this phylum67 to environmental variations.From Actinobacteria, a prevalent microbial taxa in cold environmentsPseudomonas species originating from 13,000-years old ice, similar to Arctic9 and Antarctic80 homologs.Scarisoara ice cave isolates showed high resistance to different antibiotic classes including the ones drastically affected by the emergence of resistant strains in the clinical sector. Most Scarisoara isolates exhibiting MDR and XDR phenotypes thrived in ice strata formed at least 1000-years ago. Bacterial strains with the broadest (PDR) resistance belonged to Arthrobacter psychrolactophilus SC7AB.1 isolated from 400-years old ice was resistant only to nalidixic acid and cefixime,  and Bacteroidetes (1), while Dietzia sp. SC61A.5B (XDR) as compared to their homologs D. maris83 and D. papillomatosis84 susceptible to most antibiotics. The two XDR Paralcaligenes cave strains SC18B.3 (900-years BP) and SC71.3 (7000-years BP) appeared to have an opposite phenotype as their homologs isolated from soil resistant to only lincomycin, oleandomycin, rifampicin, and vancomycin85. Other four XDR cave isolates belonging to Flavobacterium, Paralcaligenes, Phenylobacterium and Microbacterium genera isolated from up to 9000-years old ice  and XDR P. loti SC71.2 (7000-years BP) susceptible to different antibiotics . Interestingly, Delftia sp. SC71.5 from 7000-years BP cave ice shared a similar resistance profile with clinical homologs89, although the low 16S rRNA gene identity (95%) could indicate a more distal cave strain ancestor.Similar susceptibility patterns were observed for 81, suggesting a correlation between AR and cold-adaptation mechanisms via horizontal gene transfer (HGT)90. In our study, the high incidence of XDR and PDR phenotypes recovered from layers of old ice sealed between 1000–13,000 years could be associated with the high relative abundance of phyla Actinobacteria and Proteobateria18. Actinobacteria are known for their ability to produce bioactive compounds and for their multiple resistance to antibiotics91, and Proteobacteria constitute a recognized group susceptible to acquire AR genes by HGT93. In some cases, MDR could be conferred by cross-resistance as observed for cave isolates resistant to tetracycline, various β-lactams, all aminoglycosides and almost all macrolides. A similar pattern of cross-resistance was observed in methicillin-resistant S. aureus strains94.The prevalence of MDR found in the 68 isolated strains revealed the existence of highly diverse resistome in old samples  from permafrost, Arctic soil, and a deeper cave site disconnected from the surface for over 4 Myr28 and fungal29 communities entrapped in perennial ice accumulated in Scarisoara ice cave, in particular during the cold and dry Little Ice Age , and the warm and wet intervals Medieval Warm Period (MWP) (AD 800–AD 1250) and Mid-Holocene Warm Period 95. In this respect, uncultured prokaryotic communities from LIA and MWP formed ice strata of this cave appeared to be dominated by Firmicutes or Actinobacteria28, while Proteobacteria dominated both the total and potentially active bacterial communities contained in MHWP ice18 accumulated during to Sahara Desert formation period96.Climate variation during ice deposition appeared to model the composition of bacterialThe current data Fig. A revealeC. bauzanensis SC8A.3, R. argentea SC7AB.4 and M. hydrocarbonoxydans SC8A.4, Bacillus spp. SC8A.7, SC8A.1 and L. antarctica SC7AB.2, while A. psychrolactophilus SC7AB.1 (Actinobacteria) was highly sensitive to most of the antibiotics , and Firmicutes (Sporosarcina sp. SC21C.3) species  showed a high antimicrobial activity against 7 and 14 pathogens, respectively, the later one with extensive preference for Gram-negative clinical isolates. Considering their PDR phenotype, these strains could constitute important candidates for studying the resistome of millennia-old bacteria and AR evolution. Moreover, their positive testing for acid phosphatase and leucine arylamidase activities with applications in food industry101 and alkaline phosphatase, commonly used for clinical diagnostics and dairy industry102 constitute promising leads for new biocatalysts.Among cold habitats bacteria, Dietzia sp. SC61A.5B showing a XDR phenotype also inhibited the growth of Gram-negative pathogens. Similar to Pseudomonas cave isolates, this Scarisoara strain showed high leucine/valine arylamidase and acid phosphatase activities, important catalysts for food processing101. Moreover, M. pygmaeum SC83A.2 isolated from 9000-years BP cave ice displayed a high antimicrobial activity mainly against Gram negative pathogens, associated with XDR phenotype. The broader AR of this strain as compared to Artic isolates9 uncovered a valuable bacterial candidate for exploring new antimicrobial mechanisms.The 5000-years old Caulobacter sp. SC61A.5 cave isolate (5000-years old ice) was also one of the strains with broader antimicrobial activity against Gram-negative pathogens and broad AR phenotype, unlike other representatives of this genus41. This psychrotrophic ice bacterium also presented α- and β-glucosidase activities with putative extensive applications103. Similar to Pseudomonas and Dietzia cave isolates, a high Leu/Val arylamidase and acid phosphatase activity was measured for this strain.Arthrobacter psychrolactophilus SC7AB.1 showed a particular AR to fluoroquinolone and cephalosporins, and antimicrobial activity against Gram-negative and Gram-positive pathogens, in addition to a variety of enzymatic activities as putative source of cold-active catalysts. The XDR phenotype of Delftia sp. SC71.5 isolated from 7000-old cave ice, unlike the homologous soil strain susceptible to broad spectrum antibiotics105, and the broad antimicrobial activity against Gram-negative pathogens recommend theses strains as novel candidates for studying the environmental resistome and for isolation of putative active biomolecules.101. Cold-active lipase activity for a wide range of applications in biofuels and pharmaceutical industry106, detergents107, and food processing108 was also identified in most of the cave ice isolates.Most of the cave isolates tested positive for acid phosphatase activity used in food industry, constituting valuable catalyst candidates for low temperature food processing22, the current study adds to the knowledge on functional characteristics of isolated bacteria from this underground perennial ice core. The isolated bacterial strains belonging to the four major phyla ubiquitous for frozen environments confirmed their viability along the 13,000 cal BP cave ice, and revealed their extended antibiotic resistance profiles, as a pioneering survey for this type of habitat for understanding the environmental resistome evolution.In addition to our previous reports on cultivable bacteria from ice layers formed during the last 900-years in Scarisoara ice caveAlthough with a lower microbial density as compared to glacier ice, our cave isolates belonging to Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes exhibited a large diversity encompassing putative new representatives for this habitat or worldwide. Many of these strains preserved in millennia-old ice from a secluded type of icy habitat exhibited a large spectrum of antimicrobial resistance, highlighting their important contribution to the environmental resistome, and the potential risk of releasing antibiotic resistant bacteria in the water and soil due to temperature increase and ice melting. However, further studies are needed to identify the associated AR genes, their localization and transferability potential. In addition, the psychrotrophic isolates highlighted significant antimicrobial and catalytic activities, being thus valuable candidates for biomedical and biotechnological applications. Overall, due to their extended AR profile as compared to that of the reported homologs and the antimicrobial activity of all isolates, in particular against the Gram-negative pathogens raising global treatment limitations, these cave bacterial strains retrieved from up to 13,000-years old ice could provide important clues for understanding the evolution of natural and clinical resistance and for developing novel antimicrobial strategies.18. All ice samples were collected under aseptic conditions by flaming the drilling equipment and using sterile 1-L plastic bags, transported to the laboratory under permanent frozen conditions where they were stored at − 20 °C until processed18.Ice sampling was performed by vertical drilling to a depth of 25.33 m into the perennial ice block of Scarisoara Ice Cave resulting in 97 ice core fragments covering a chronology of up to 13,000 calibrated years before present  ice, as previously described18 were thawed at 4 °C in the absence of light, and inoculated in 10 mL R2B liquid media . The enriched samples were incubated at 4 °C and 15 °C in shaker incubators (160 rpm) for up to 120 days. The enriched cultures (100 µL) were used to inoculate R2A Agar  plates using a 100–10–10 serial dilution, and incubated at 4 °C and 15 °C for 2 to 4 weeks. Morphologically different colonies were isolated and purified under the same growth conditions.A series of 28 core ice samples covering an age span of 92 ± 26 cal BP to 13,098 ± 29 cal BP at 100–300 years old interval21. After purification , the amplicons were sequenced using the amplification primers .Bacterial isolates were identified by 16S rRNA gene sequencing. Total DNA was extracted using DNeasy Blood & Tissue Kit  and the 16S rRNA gene fragments were amplified by PCR as previously describedwww.codoncode.com), and the closest phylotype was assigned by BLAST alignment109.Chimera analysis of the sequences and low-quality aligned regions were eliminated using CodonCode Aligner version 8.0.1  solid medium.110. The isolates were cultivated on R2A agar and TSA agar plates at 15 °C for 2–5 days. The antibiotic susceptibility profile was determined after incubation at 37 °C for 24 h, using Stahylococcus aureus ATCC 25923  as control strain110. Based on the number of antibiotics with inhibitory effect, the strains were assigned as MDR, defined as non-susceptible to at least one agent in three or more antimicrobial categories, XDR as non-susceptible to at least one agent in all but two or fewer antimicrobial categories, and PDR, defined as non-susceptible to all agents in all antimicrobial categories. According to these definitions, our strains exhibiting a MDR phenotype were resistant to 20–25 antibiotics, from at least three different classes, the XDR ones were resistant to 26–27 antibiotics, and those exhibiting a PDR were resistant to all tested 28 antibiotics51.The antimicrobial susceptibility analysis was carried out using the disk diffusion method111.Functional characterization of selected bacterial isolates was performed using API ZYM and API 20NE strips  according to the manufacturer protocol, with incubation at 15 °C. The enzymatic activity was evaluated based on the number of nmol of hydrolyzed substrate as (0): no activity, (1 and 2): low activity , (3): moderate activity (20 nmol), (4 and 5): high activity Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 , and 20 clinical isolates from the Research Institute of The University of Bucharest Microbial Collection (Supplementary Table 112. The cave isolates were cultivated in 20 mL Tryptic Soy Broth (TBS) medium  at 15 °C, under agitation (160 rpm), for up to 14 days, and the cells were harvested by centrifugation at 7500 rpm for 20 min. The supernatant was filtered-sterilized using 0.22 µm syringe filters , and concentrated to 1 mL with a vacuum Concentrator Plus Complete system . The pathogen (test) strains were inoculated on Mueller–Hinton agar (MHA) solid medium  and grown at 37 °C for 18 h. One colony from each test strain was resuspended in physiological saline solution to 0.5 McFarland turbidity, and further plated on MHA solid medium covering uniformly the plate surface. Immediately after pathogen plating, 5 µL samples of concentrated extracts of cave bacteria were added as single drops in separated quadrants (6 per plate). After incubation at 37 °C for 18 h, the presence/absence (±) of antimicrobial activity was estimated based on the presence of an inhibition zone inside each quadrant. The assay was performed in duplicate. A negative control comprising 20 mL of sterilized TSB medium concentrated to 1 mL was used for each pathogen test strains following the same protocol.The antimicrobial activity of the ice cave isolates was tested against two reference strains, i.e. Supplementary Infomation.


The ongoing COVID pandemic has disrupted the ability to hold scientific conferences in person. Despite this, over 200 people from across the world attended The First International Symposium on the Chaperone Code, which was held virtually on October 28 to 29, 2020. The meeting highlighted the many ways that posttranslational modifications (PTMs) on molecular chaperones regulate their function to control proteostasis in diverse organisms.in vitro folding mechanisms. With the exponential improvement in proteomic technologies over the past 10 years, a huge number of PTMs have been uncovered on chaperones including phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination. Despite the identification of these sites, the respective roles and roles of the modifications . While chaperones have been extensively studied for over 40 years, the majority of studies have focused on chaperone actions, ATPase cycle regulation, and Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding, stability, and activity of more than 300 proteins also known as clients. Many of these clients are involved in various maladies including cancer, neurodegenerative and infectious diseases , 2. TherLen Neckers  showed that phosphorylation of a highly conserved Tyr313 in the middle domain of human Hsp90α is important for initial binding of Aha1  demonstrated the use of computational design in deciphering the chaperone code. Using this approach, it is possible to identify regulatory amino acids that are subject to PTMs and consequently impact the conformational dynamics and ATPase activity of Hsp90 both in vitro and in vivo  is instrumental in metabolic regulation and cell survival. Andrea Rasola,  showed context-dependent PTMs of TRAP1. More specifically, mutation and inactivation of neurofibromin, a Ras GTPase-activating protein, induce an oncogenic metabolic switch via mitochondrial ERK-mediated phosphorylation of the molecular chaperone TRAP1  discussed how secreted kinase signaling could impact extracellular Hsp90 (eHsp90) chaperone function. She showed that the first secretory eHsp90 co-chaperone TIMP2  described a new phosphorylation site (S530) on Candida albicans Hsp90, targeted by casein kinase 2 (CK2), which results in the inhibition of Hsp90 function and blocking expression of key virulence traits. She showed that the phosphomimetic mutant S530E (but not S530A) abolished C. albicans survival at high temperatures, supported a switch to filamentous growth, a morphological change that is important for fungal virulence, and rendered C. albicans susceptible to both antifungal (fluconazole) and Hsp90 (radicicol) drugs  drugs . These sDaliaBarsyte-Lovejoy  described how characterization of a novel inhibitor of the arginine methylase PRMT7 led to the discovery that Hsp70 is methylated on R469, a highly conserved amino acid present on the client-binding domain. Intriguingly, this PRMT7-catalyzed methylation can only occur when Hsp70 is in its ATP-bound “open” conformation. R469 methylation appears to be important for a wide variety of chaperone processes that include stress granule response to proteasome inhibition and the general response to heat stress (The molecular chaperone heat shock protein 70 (Hsp70) is involved in folding, stability, and quality control of proteins , 13. Hspt stress .Magnus Jakobsson  discussed the impacts of mono, di, and trimethylation of Hsp70 K561 promoted by METTL21A. The trimethylated K561 Hsp70 may have the greatest functional relevance, as it is the major form found in both the nucleus and the cytosol  demonstrated a novel role of Hsp70 in the mitochondrial import of the superoxide dismutase-2  appropriate to the levels of ROS inside the mitochondria. This process is driven by AKT1-catalyzed phosphorylation of Hsp70 on S631, which alters Hsp70 structure and ability to bind the E3 ubiquitin ligase, CHIP. Several avenues of investigation remain to be explored, including the role of adjacent Hsp70 phosphorylation sites S633 and T636, which are also phosphorylated by AKT1 but do not impact the mitochondrial import of SOD2  described the innovative use of expanded genetic codes in bacteria to produce recombinant Hsp70 phosphorylated on S66. This phosphorylated form was crystallized and revealed that S66 plays a role in Hsp70 interdomain communication. At the cellular level, this Nek6-mediated phosphorylation of Hsp70 is required to maintain a functional mitotic spindle, although the key clients involved are yet to be determined  tested the hypothesis that functionally relevant sites of modifications on Hsp70 chaperone impact its allosteric mechanism. One important site identified was T495 on mammalian Hsp70, a site previously identified in yeast as a functional hotspot as well as being activated in mammalian cells by the legionella pneumophila kinase LegK4 as part of its program of infection .nfection . She is Seema Mattoo  described how Huntingtin yeast interacting protein  is able to AMPylate the ER-resident Hsp70 BiP at T366 and T518 impacting the BiP ATP cycle. In contrast to many of the other talks demonstrating activation of chaperones through addition of PTMs, her message was that site-specific AMPylation has differential effects on BiP’s ATPase activity and provides a way for cells to stall BiP function in preparation for future stresses  provided evidence that although AMPylation of BiP in C. elegans does not alter animal survival, it does reduce Aβ toxicity, an effect that can be mimicked by directly silencing Hsp70 chaperones Hsp1, 3, and 4 simultaneously  discussed the extensive stress-induced PTMs of HSFs, exploring their ability to both activate and repress transcription. Using Precision Run-On sequencing (PRO-seq), a technology that maps nascent transcription at a nucleotide’s resolution  family of proteins, which regulate the expression of chaperones and co-chaperones under a variety of cellular stresses . Lea Sissolution , she shosolution .O-GlcNAcylation is regulated by the concerted actions of the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) that add and remove O-GlcNAc to serine or threonine amino acids, respectively. Natasha Zachara  presented work on decoding the role of glycosylation in stress response. She showed that AMPKα and sequestosome modification by O-GlcNac and O-GlcNAcylation responds to oxidative stress. Additionally, O-GlcNac levels potentiate autophagy in an AMPKα-dependent manner. This is a perfect example of how one PTM such as O-GlcNAcylation can be used by cells to sense and respond to stress  discussed the consequences of chaperone code failure in the eye lens, loosely interpreting the “code” as the job definition of a chaperone to protect other proteins. Buchner explained that the current model for cataract formation assumes that damaged crystallin proteins assemble into light-scattering aggregates. Chaperones are thought to counteract this by sequestering misfolded crystallin proteins. He showed that in the lens, point mutations in α- (with a chaperone activity), β-, or γ-crystallin proteins are substantially reduced and that the mutant proteins do not accumulate in the water-insoluble fraction. As the mutant lenses show changes in protein composition and the spatial organization of crystallin, this suggests that the imbalance in the lenticular proteome results in changed crystallin interactions as the basis for cataract formation, rather than the aggregation of the crystallin mutants. This work ultimately helps in designing pharmacological treatments to prevent cataract or delay its onset  closed the meeting by describing the history of research uncovering the roles of PTMs in chaperone–client interactions (in vitro and in vivo) of these modifications. The second level is more complex, determining the interplay between different sites and functional hierarchy involved. In the third level of the Code, evolutionary considerations are taken into account—how do sites of PTM appear on chaperones over time and understanding why different chaperone paralogs have evolved to have different PTMs. At the final level, there was discussion of how all of this information can be integrated into the global concept of the Chaperone Code (ractions . He follone Code . Given tThe authors declare that they have no conflicts of interest with the contents of this article.