Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago. Organisms occupying niches that experience sub-zero temperatures often produce antifreeze proteins (AFPs), which function by adhering to, and preventing the growth of, ice crystals2. Once bound, ice growth is limited to areas around the AFP, causing micro-curvatures to form on the ice surface4. This makes it energetically unfavourable for water to join the ice lattice resulting in a depression of the freezing temperature below the melting temperature, which is termed thermal hysteresis (TH) and is used to quantify an AFP’s potency.Organisms living in sub-zero environments must adapt to avoid cellular injury due to freezing. Formation of ice crystals within living tissues poses the risks of cell dehydration and the rupture of cellular membranes, leading to death5. Since then, AFPs have been observed in other fishes6, insects8, and microorganisms11. In fish, AFPs are thought to have arisen during the Cenozoic era beginning 20–40 million years ago when sea ice at the poles was present for the first time in ~ 200 million years13. In seawater, the high concentration of NaCl (~ 0.45 M) depresses the freezing temperature of seawater to ~ − 1.9 °C. As fish blood has a lower solute concentration and freezes at ~ − 0.8 °C, any contact with ice crystals could nucleate freezing and kill the fish5. Consequently, fish AFPs must be produced in adequate quantities and with sufficient activity to decrease the body freezing temperature by at least 1.1 °C. This provides a selective advantage to these fish, as they can safely hunt for food in ice-laden seas where fish lacking AFPs are at risk of freezing. There have been four types of AFPs found in fish: (1) alanine-rich type I AFP, (2) lectin-like type II AFP, (3) type III AFP derived from sialic acid synthase, and (4) antifreeze glycoproteins (AFGPs). With different folds that all perform the same function, it raises the question of how these AFPs first arose14.The first AFP characterized was from a teleost fish15. The simple repetitive AFGPs have arisen on two independent occasions including once from a trypsinogen gene by duplication and divergence16. Type II AFPs have evolved from a C-type lectin progenitor and have been spread to at least two distant taxonomic branches of fish by lateral gene transfer18. Duplication and divergence of a sialic acid synthase gene has given rise to the type III AFP gene family, which has only been found in one branch of fishes19. It is thought that these fish AFP folds have arisen within the last few tens of millions of years in response to polar glaciation20. In other branches of organisms like insects22 and microorganisms24 there are also examples where different AFP folds have independently arisen to perform the same task.Alanine-rich alpha-helical type I AFPs have independently evolved on at least four occasions25. These small organisms (most are commonly only a few millimeters long) are typically soil dwelling, but some species live in trees, on ponds or wet surfaces surrounding stones, or on glaciers. Collembola got their name from an abdominal organ, the collophore, which is involved in osmo- and ion regulation26. A second defining abdominal anatomical feature is a spring-loaded organ known as a furca, which allows them to escape predators by “jumping”. This has given them their nickname “springtails”. These primitive organisms arose around 450 million years ago and there are close to 10,000 species classified to date27. Those that live in sub-zero environments have cold tolerance mechanisms to survive in these harsh conditions, one of which is the production of AFPs. Previously a few species of Collembola have been found to have TH activity31, and cold hardiness and supercooling abilities32 but no systematic study of the AFP types present and their distribution in Collembola has been made to date.Collembola are the most abundant terrestrial arthropods and are found on all continentsHypogastrura harveyi (HhAFP)29. HhAFP was predicted to have a glycine-rich polyproline type II (PPII) helical bundle fold33 a structure that was later confirmed by X-ray crystallography35. This fold, which appears unique to Collembola, consists of two layers of antiparallel PPII helices connected by loop regions. Each rotation around the helix is exactly three residues in length with tripeptide repeats of G-X1-X2, where X1 is often glycine. The core of the protein contains the inward facing glycine residues, allowing for tight packing due to the absence of side chains33. Compact packing of the helices allows for a hydrogen bonding network to develop between the helix backbones that increases the stability of the fold36. The two layers both have an outward pointed face. One surface is flat, contains small, hydrophobic residues, and is thought to function as the ice-binding surface (IBS)37. This surface is alanine-rich, but also contains serine, threonine, and valine residues. The opposing surface is uneven and contains larger residues, some of which are polar or charged. H. harveyi also produces a larger 15.6-kDa AFP isoform that is proposed to have 13 polyproline helices38.The first collembolan AFP characterized was the small 6.5-kDa isoform from HhAFP was recently characterized from Megaphorura arctica (MaAFP) collected in Iceland31. Although the 6.5-kDa MaAFP shares high similarities in coding sequence to HhAFP, their untranslated regions (UTRs) are divergent to the extent that common ancestry could not be definitively established. Additionally, 9.6-kDa AFP isoforms from Granisotoma rainieri (GrAFP) were studied39. The structure of GrAFP was modelled to be a polyproline type II bundle with nine helices, which was confirmed using X-ray crystallography. When the UTRs of the five GrAFP cDNAs were compared, the most dissimilar shared only 69% sequence identity. Therefore, a lack of similarity between the UTRs of different species does not disprove homology.A putative homolog of Here we have characterized the AFPs from 18 species of Collembola from numerous families within two of the four extant orders of Collembola, collected from four different continents. The extent of the analysis was determined by the amount of biomass available. Small quantities of springtails (< 100 mg of freeze-dried tissue) were used to determine TH activity and ice-shaping. AFPs were purified from larger samples (> 100 mg of freeze-dried tissue) by ice affinity purification (IAP) and characterized by MALDI-MS, amino acid composition, and/or tandem mass spectrometry. Transcriptomes were generated from some species to deduce AFP sequences at the nucleic acid level, and in some cases to recombinantly express the encoded proteins. AFPs present in the different Collembola tested here all inhibited ice growth on the basal plane, suggesting that they could be hyperactive. Where more detailed analysis was possible, all the AFPs examined had the same glycine-rich tripeptide repeating pattern indicative of the PPII helical bundle. To date, this fold has only been found in Collembola and its presence across distant species suggests that the PPII helical bundle fold originated in a basal collembolan species, shortly after the group arose.M. arctica and Entomobrya nivalis, that were field collected and cold acclimated in the laboratory  to buffer (50 mM Tris–HCl (pH 7.8), 150 mM NaCl, 1 mM phenylthiocarbamide and 1 × EDTA-free Roche protease inhibitor cocktail) and homogenized by hand using a disposable plastic pestle in a 1.5-mL microcentrifuge tube. All manipulations and temporary storage of samples were done on ice or at 4 °C to prevent thermal denaturation of the AFP. The homogenates were centrifuged at 16,300×g for 30 min and the supernatant was filtered through glass wool to remove lipid. AFPs in the filtered supernatant were recovered using four rounds of ice-shell purification as previously described40. The final ice fraction for each preparation was concentrated to < 500 µL using an AmiconUltracel 3 K filter  spun in a Sorvall ST16R centrifuge at 3000×g.AFP extractions for protein characterization were performed using > 100 mg of freeze-dried animals. Tissue samples were homogenized in buffer (50 mM Tris–HCl (pH 7.8), 150 mM NaCl, 1 mM phenylthiocarbamide and 1 × EDTA-free Roche protease inhibitor cocktail) using an IKA ULTRA-TURRAX disperser . The homogenate was centrifuged at 22,000×40 and were also used to calculate protein concentration.Amino acid compositions were determined at the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre  by acid hydrolysis as previously describedMALDI-TOF MS was performed at the Protein Function Discovery Facility  using alpha-cyano-4-hydroxycinnamic acid matrix on dried droplets with a SCIEX Voyager DE Pro in Linear mode. Protein sequencing by tandem mass spectrometry was done at the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre .39. The transcriptome of C. antarcticus was assembled at the Sequencing & Genotyping Center , and those of G. rainieri and E. nivalis were assembled at the Institute for Genome Sciences .RNA samples were extracted from 30 to 50 mg of tissue stored in RNAlater . Extraction was performed as previously describedThe AFP-containing samples were injected into a grid filled with immersion oil on a Peltier unit. The temperature was controlled using a nanoliter osmometer  and a model 3040 temperature controller . The samples were flash-frozen and melted slowly until a single ice crystal remained. The temperature was held just below the melting point and then decreased at a rate of 0.075 °C/min until ice growth began. Videos of the ice crystals during TH measurements were recorded either using Panasonic WV-BL200 CCTV camera or a DMK 33UX249 USB 3.0 monochrome industrial camera .G. rainieri (QQY00623.1) and Folsomia candida (OXA44825.1) AFPs were ordered from GeneArt . The DNA encoding the signal peptide was removed and an N-terminal methionine residue was encoded to help introduce an NdeI cut site. To the C terminus, leucine and glutamate codons were introduced to add a XhoI cut site. The genes were subcloned into pET-24a vectors39. The resulting plasmids were transformed into TOP10 competent cells  for isolation using a GeneJET Plasmid Miniprep kit . DNAs were sequence checked before the plasmids were retransformed into BL21 (DE3) expression cells . Cell cultures were grown in lysogeny broth medium with 100 µg/mL kanamycin at 37 °C. Upon reaching an OD600 of 0.6–0.8 the cell cultures were cooled to 20 °C, and 1 mM isopropyl β-d-1-thiogalactopyranoside was added to induce the cell culture overnight. Cells were centrifuged at 4500×g for 30 min and resuspended in 50 mL of lysis buffer (20 mM Tris/HCl (pH 7.8), 500 mM NaCl, 5 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride, and one dissolved tablet of cOmplete™ ultra protease inhibitor cocktail). Cells were sonicated 16 times at 10 s per round and cooled to 4 °C between cycles to prevent protein denaturation.Codon-optimized, synthetic genes for 39.His-tagged recombinant AFPs were separated from the lysate supernatant using Ni-affinity chromatography. Fractions containing AFP were pooled, loaded into a 250-mL round-bottom flask seeded with an ice shell, and ice-affinity purifiedhttps://phylot.biobyte.de/).The taxonomy IDs for each species were extracted from the NCBI taxonomy database. The phylogenetic tree was assembled using phyloT . In contrast, ice crystals in the buffer control were disk-shaped and kept growing with the same shape, while the burst with type I AFP occurred along the c-axis  of two of the homogenates suggest that, as in other arthropods such as Tenebrio molitor41, most Collembola produce hyperactive AFPs. Additionally, the consistent differences in ice shaping and the burst between collembolan AFPs, and type I AFP from winter flounder  have been previously reported39. Here, AFP extracts from three additional species  were also subjected to amino acid analysis 29. In contrast, F. candida has one main peak consisting of four sub-peaks that differ by ~ 16 Da , and two with incomplete coding sequences (GR870234.1 and FF278983.1). The full-length transcripts encoded a 108-amino-acid protein (8.5 kDa) after the removal of the 19-amino-acid signal peptide  with a 19-amino-acid signal peptide and a 187-amino-acid mature protein  and CaAFPb-6 (15.5 kDa) closely match peaks 15,158 and 15,463 Da seen by MALDI , herein called FcAFP, resembling other PPII helical AFPs. FcAFP shares 57% sequence identity with GrAFP-4  was 86% identical to the 11.2-kDa isoform (EnAFP-3) with a 29-amino-acid deletion that likely removes two helices. The other sequences had between 64 and 81% identity.Ceratophysella communis (VNWX01004235.1) The gene was predicted to contain a single intron51, and the resulting 732-bp open reading frame encoded an AFP predicted to have a 23-amino-acid signal peptide52. The mature protein was 220 amino acids in length and can be modelled to have 12 PPII helices 54. Yet, when using 16S, 28S, and cox1 sequences the positions of Neelipleona and Symphypleona were reversed and Symphypleona was basal55. Regardless of the exact phylogenetic relationship, mitochondrial dating suggests that the four orders diverged between 437 and 421 million years ago56. This period coincides with the Andean-Saharan glaciation, an ice age that lasted from 460 to 420 million years ago57. Additionally, the lineages corresponding to extant families diverged between 414 and 184 million years ago56, in which the Karoo glaciation occurred, between 360 and 255 million years ago58. Diversification and the need for freeze resistance in the same timeframe would allow for radiation of the species expressing PPII helical AFP.The glycine-rich PPII helical bundle was predicted to be the AFP fold of eight species of Collembola, spanning five families and two orders. The presence of PPII helical AFPs in both Entomobryomorpha and Poduromorpha suggests that this protein family originated prior to their divergence Fig. . The exaSinella curviseta, Heteromurus nitidus, Lepidocyrtus violaceus and three species from the genus Orchesella did not display antifreeze activity AFPs, while E. nivalis did  during an interglacial period, only to evolve a replacement to cope with a new ice age. For this reason, it is worth noting the different amino acid composition of an AFP previously identified in the Antarctic collembolan species, Gomphiocephalus hodgsoni30. This AFP contained high percentages of histidine and cystine that set it apart from the PPII-type AFPs (Supplementary Table H. harveyi and C. communis) produce glycine-rich PPII helical AFPs.Unlike teleost fish, where AFPs did not originate until the Cenozoic era (~ 30 million years ago) following the Paleocene–Eocene thermal maximum15. Fortunately, the origin of the flounder type I AFPs was traced via their UTRs20. It is possible that convergence also played a role in the evolution of collembolan AFPs. However, this seems unlikely, as all but one species examined to date produce glycine-rich AFPs and the known sequences form39, or can be modelled , introns, and repetitive sequences replete with potential glycine codons, make identification of AFP genes difficult. Therefore, identification of these AFPs is heavily reliant on tissue extraction. Unfortunately, to the best of our knowledge, laboratory culturing of Symphypleona and Neelipleona is difficult, complicating detailed studies on AFPs in these two orders.Supplementary Information.


The HFr increased thiobarbituric acid reactive substance (TBARS) levels in plasma and urine, and Ex decreased the HFr-increased TBARS levels in plasma. The HFr increased the neuronal and endothelial NO synthase (nNOS and eNOS) expressions, and Ex enhanced the HFr-increased eNOS expression. The HFr inhibited the eNOS phosphorylation at serine 1177, and Ex restored the HFr-inhibited eNOS phosphorylation. The HFr increased xanthine oxidase and NADPH oxidase activities, and Ex restored the HFr-increased xanthine oxidase activity but enhanced the HFr-increased NADPH oxidase activity. The HFr increased the nitrotyrosine levels, and Ex attenuated the HFr-increased levels. These results indicate that although Ex enhances the HFr-increased eNOS expression and NADPH oxidase activity, an HFr inhibits renal eNOS phosphorylation and NO bioavailability, whereas Ex ameliorates them.Chronic exercise (Ex) exerts antihypertensive and renoprotective effects in rats fed a high fructose diet (HFr). To elucidate the mechanisms, the impacts of an HFr and Ex on the nitric oxide (NO) system and oxidative stress in the kidney were examined. Rats were fed a control diet or an HFr, and a part of the HFr-fed rats underwent treadmill running for 12 weeks. The HFr did not affect nitrate/nitrite (NO Fructose is a monosaccharide sugar that is presented in fruits and vegetables. It has been widely used in manufactured food products in Western countries since the middle of the 20th century. Consuming sugar is important for the epidemics of lipid disorders, abdominal obesity, and type 2 diabetes ,2,3. Numx) excretion [x excretion and decreases blood pressure in HFr-fed rats [The renal impacts of fructose have been reported. HFr upregulates sodium and chloride transporters, resulting in salt absorption, and induces salt-sensitive hypertension . HFrs alxcretion ,14. AddiFructose has also been reported to induce oxidative stress in the kidney. An HFr increases nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and thiobarbituric acid reactive substance (TBARS) levels, which is an index of oxidative stress-induced lipid peroxidation in rat kidney . Howeverx excretion in spontaneously hypertensive rats (SHRs), Wistar Kyoto rats (WKYs), and Zucker diabetic rats [Chronic exercise (Ex) has been shown to exert antihypertensive and renoprotective effects in animals and humans with hypertension and CKD . Ex withtic rats ,24. Ex atic rats , the XO tic rats , and bottic rats . HoweverMale Sprague Dawley rats  at 5 wk of age were used. The rats were housed in an animal care facility with a controlled temperature of 24 °C under a 12:12 h light-dark cycle with food and water ad libitum. The protocol was carried out after a one-week acclimation. All involved rats were reviewed and received prior approval by the Animal Welfare Committee of Tohoku University.n = 8 in each group). The sample size calculations are provided by G Power 3.1. The projected total sample size needed with power = 0.80 is approximately n = 21. Thus, our proposed total sample size of N = 24 will be adequate for the objective of this study. The standard lab chow pellets were replaced by a control diet  or an HFr , which induced metabolic syndromes as previously reported [After the acclimation, the rats were divided into 3 groups  was measured every 2 weeks from the start of Ex using a tail-cuff monitor  described previously . The ratThe urine samples of rats kept in metabolic cages were collected on ice for 24 h and stored at −80 °C for further analysis. The rats were returned to their cages after the 12th week exercise and kept still for 48 h prior to the sacrifice. After being anesthetized with pentobarbital , the plasma samples were collected via decapitation. Urine and plasma samples were centrifuged at 2000 g for 15 min; the supernatant was collected for further analysis. To evaluate renal function, urinary albumin and plasma creatinine were measured . The kidneys were quickly removed, and the cortex was homogenized in a buffer described previously . The prox and TBARS levels in plasma and urine were measured by commercial kits  described previously [x and TBARS levels and nmol/day for urinary NOx and TBARS excretion.The NOeviously . Data arS1177, 612392, RRID: AB_399750; p-eNOST495, 612706, RRID: AB_399946; BD Biosciences, USA); Akt and p-Akt at serine 473; AMP-kinase (AMPK) and p-AMPK at threonine 172 ; and XO and nitrotyrosine . The intensities of the bands  were quantified using Image J software  and normalized to those of the reference protein β-actin at 40 kDa, and the mean intensity of bands for the CON-Sed group was assigned a value of 1. The phosphorylated proteins were represented as the ratio of phosphorylated proteins to their total protein expressions.Western blot analyses were performed to elucidate the protein expression and phosphorylation as described previously . Renal cThe activity of NADPH oxidase and XO were measured by lucigenin-enhanced chemiluminescence and pterin-based fluorescence assay, as described previously ,28. For p < 0.05 was considered to indicate statistical significance. Data were analyzed by GraphPad Prism .Data are presented as means ± SE. The data were analyzed using two-way repeated-measures ANOVA or one-way ANOVA, followed by a Tukey test for multiple comparisons among the groups. The HFr significantly increased systolic blood pressure, and Ex significantly inhibited the HFr-induced elevation A. The HFx levels in plasma or urine, and Ex significantly increased the NOx levels in plasma and urine  by 40% [x excretion in rats [Previous studies have reported the impact of an HFr on renal NOS expression in HFr-fed rats ,13,15. Nxcretion . Gordish) by 40% . Hasegaw in rats . Ex enha in rats ,24.2+ and the interaction with substrate and co-factors, as well as adaptor and regulatory proteins and and protein phosphorylation. The eNOS phosphorylation of serine 1177 enhances activity, whereas the phosphorylation at threonine 495 inhibits activity [x excretion in spite of the increase in the renal eNOS and nNOS expressions, suggesting an inhibition of eNOS activity by the HFr. Therefore, this study also examined the renal eNOS phosphorylation and shows that the HFr inhibited the eNOS phosphorylation at serine 1177 and that Ex restored the HFr-inhibited eNOS phosphorylation. In contrast to the present results, Galleano et al. reported that an HFr (10% fructose in drinking water) for 8 weeks stimulated the NOS activity and the eNOS phosphorylation at serine 1177 in the kidney and aorta [NO production is substantially adjusted by NOS activity, which is regulated by fluid shear stress and numerous agonists via cellular events, including increases in intracellular Caactivity . The HFrnd aorta ,32. Medend aorta . Stanisind aorta .2+/calmodulin-dependent protein kinase II [The phosphorylation of eNOS at serine 1177 is mediated by Akt, AMPK, protein kinase A, and Cainase II . This stinase II , and Gu inase II . Fructosinase II . Wahba einase II .This study shows that the HFr increased the renal NADPH oxidase activity and that Ex enhanced the HFr-increased NADPH oxidase activity. In agreement with the present results, an HFr (10% fructose in drinking water) for 8 weeks increased the renal NADPH oxidase activity , and fruIn a previous study, Price et al. also examined the impacts of an HFr on antioxidant enzymes, including SOD, glutathione, and catalase, in the kidney . HFr inc2+/calmodulin-dependent protein kinase II. Third, we did not fully clarify the interaction between the renal NO system and oxidative stress in the renal eNOS and nNOS expression. Fructose induces the eNOS uncoupling via oxidative stress and inhibits eNOS activity [This study had several limitations. First, we examined impacts of an HFr and Ex on only the renal NADPH oxidase and XO as the source of superoxide anions. Exercise has been reported to increase the production of reactive oxygen species and result in oxidative stress in numerous tissues ,40. We aactivity . In endoactivity ,42. We pactivity  and enhaactivity . A furthIn conclusion, an HFr inhibits the renal eNOS phosphorylation at Ser 1177 in spite of the increases in eNOS and nNOS expressions, and Ex enhances HFr-increased eNOS expression and restores HFr-inhibited eNOS phosphorylation, suggesting that Ex increases renal NO production. Ex enhances the HFr-increased NADPH oxidase activity, whereas Ex restores HFr-increased XO activity and attenuates renal nitrotyrosine levels. Ex exerts antihypertensive and renoprotective effects probably by increasing renal NO production and NO bioavailability in HFr-fed rats.


Lichen sclerosus (LS) is a chronic inflammatory skin condition characterized by hypopigmented atrophic macules, papules, patches, plaques, and erosions. About 85% of LS cases involve the anogenital region, while the remaining case are extragenital.A 29-year-old man with no history of skin or autoimmune disease presented with a 6-months history of an intermittently itchy rash consisting of light colored macules that began on his wrists and then spread up his arms. On initial presentation, he was treating the rash with triamcinolone 0.05% cream twice daily from his primary care physician with no improvement after weeks of use. He was not taking any medications or supplements. Physical examination revealed hypopigmented atrophic coalescing macules with keratotic plugs reminiscent of open comedones on the bilateral volar forearms  extendinA 4-mm punch biopsy was performed on the left volar forearm. The specimen showed epidermal atrophy with mild vacuolar changes of the basal layer of the epidermis and follicular plugging. In the superficial dermis, there was sclerosis with homogenization of collagen and melanophages. Perivascular lymphohistiocytic inflammation was present in the superficial to mid dermis . These hTreatment options including phototherapy and topical corticosteroids were discussed with the patient, and he was prescribed topical betamethasone dipropionate 0.05% ointment twice daily and recommended to start phototherapy. The patient was lost to follow up for 7 months, at which point he contacted the clinic with worsening lesions, not having started the topical corticosteroids or phototherapy. He was then started on narrow band ultraviolet B phototherapy 2-3 times weekly using the clinic’s standard psoriasis protocol for skin type IV. He was recommended to add alternating triamcinolone acetonide 0.1% ointment twice daily and tacrolimus 0.1% ointment topical twice daily to his treatment regimen along with the phototherapy. After 31 phototherapy treatments, he had marked improvement, and plans to continue treatment until the LS was cleared.The prevalence of LS varies from 0.1% to 1.67%, with the extragenital form comprising only 15% to 20% of cases.Extragenital LS most commonly involves the trunk, proximal extremities, breasts, neck, shoulders, and buttocks.Dermoscopy can be a useful tool in the diagnosis of LS. Hypopigmented plaques with comedo-like openings in the center have been previously described as the dermoscopic pattern of extragenital LS.None disclosed.


Climate-related phenomena in Peru have been slowly but continuously changing in recent years beyond historical variability. These include sea surface temperature increases, irregular precipitation patterns and reduction of glacier-covered areas. In addition, climate scenarios show amplification in rainfall variability related to the warmer conditions associated with El Niño events. Extreme weather can affect human health, increase shocks and stresses to the health systems, and cause large economic losses. In this article, we study the characteristics of El Niño events in Peru, its health and economic impacts and we discuss government preparedness for this kind of event, identify gaps in response, and provide evidence to inform adequate planning for future events and mitigating impacts on highly vulnerable regions and populations. This is the first case study to review the impact of a Coastal El Niño event on Peru’s economy, public health, and governance. The 2017 event was the third strongest El Niño event according to literature, in terms of precipitation and river flooding and caused important economic losses and health impacts. At a national level, these findings expose a need for careful consideration of the potential limitations of policies linked to disaster prevention and preparedness when dealing with El Niño events. El Niño-related policies should be based on local-level risk analysis and efficient preparedness measures in the face of emergencies. El Nino-Southern Oscillation (ENSO) is a global-scale climate variability phenomenon. It is caused by atmosphere-ocean interactions in which the tropical atmosphere responds to fluctuating ocean temperatures in the equatorial Pacific Ocean. ENSO events occur on a timescale of 3 to 7 years , which aIn 1983 and 1998, ocean warming from the coast of South America to the central equatorial Pacific developed into extreme El Niño events and developed according to well-established ENSO mechanisms, albeit with enhanced ocean-atmosphere feedbacks associated with westerly winds along the equator , 5. ThesIn general, ENSO events have wide-ranging impacts on global climate conditions and the frequency of hydro-meteorological anomalies, such as floods and droughts . ConsequIn recent years, the scientific community has started focusing on the diversity among ENSO events, particularly concerning the spatial distribution and strength of the warming or cooling patterns in the equatorial Pacific , as thesVery intense and recurrent rainfall on Peru’s northern and central coast was observed in 2017. Above-average precipitation led to increases in seasonal river flows, with up to a 50-fold increase compared to historical averages in the Rimac basin located in Lima, and with seasonal river flows above 250% of the average in several other basins and resulted in landslides along the whole length of the Peruvian coast. The event was categorised as a “Coastal” El Niño due to the anomalies in the SST in Zones 1+2 (quadrant that covers the northern part of the Peruvian sea) having its effects on the Pacific coast of South America .The 2017 measured precipitation and subsequent increase in river flows had a significant impact on the health and socioeconomic wellbeing of an estimated 1.9 million Peruvians (approx. 6% of the population) –25. It cWe describe relevant climatic conditions during the Coastal El Niño 2017, differentiating the development and impacts of that event from those of the well-known extreme global-scale El Niño events of 1982–1983 and 1997–1998, which featured the strongest warming in the far eastern Pacific since 1900, far exceeding other events in terms of the warming strength in this region . On the Using publicly available climate, health, and economic data, as well as historical literature on global ENSO events, we also assessed the national and sub-national economic impacts of Peru’s 2017 Coastal El Niño, described, and compared the epidemiological data and recorded rates of mandatory-reporting infectious diseases, and analysed the associated government preparedness and response during the event. Lessons learned from the 2017 Coastal El Niño can provide a good basis for better preparedness and responses for future events.The analysis and findings of this study are relevant for broader disaster and risk management in Peru. Considering that beyond El Niño events, scenarios of heavy rains and floods linked to a changing climate are also expected, this case study aims to highlight the urgency of adopting preparedness and response measures to prevent future impacts on Peru’s public health and the economy as those experienced during and after the 2017 Coastal event.The 2017 Coastal El Niño strongly affected the coastal regions of Peru, from Tumbes to Ica. . These rWe assessed the impact of the 2017 Coastal El Niño on four components: climate, economy, health, and policy. For the climatic characterisation, we searched historical climate, atmospheric, and oceanographical records in Peru and literature reviewing the distinctions between ENSO phases and circumstances to define the Coastal El Niño event. For the economic analysis, we used a vector autoregressive (VAR) methodology to determine the economic impact of the 2017 Coastal El Niño event and presented the economic valuation of direct losses by the economic sector measured by the Government.For the health component of this study, we compiled publicly available national data on cases of mandatory-reporting infectious diseases previously associated with severe weather events. We compared reported infection rates during the 2017 Coastal El Niño with those observed during equivalent periods in the previous five years. For the policy dimension of this case study, to identify potential bottlenecks, limitations and to understand and assess the response of the Peruvian government to the emergency, we conducted in-depth interviews with individuals who led response efforts to the event and reviewed national legislation and policies for disaster emergency response and preparedness at the national, sub-national (regions) and local geopolitical levels (provinces).We used the following observational data: (i) monthly Sea Surface Temperature (SST) data for Puerto Chicama, located in La Libertad department on the northern coast of Peru  for the 1925–2017 period, from the University of Washington’s Joint Institute for the Study of the Atmosphere and Ocean (JISAO) and the Peruvian Ocean Institute ; and (ii) the monthly Piura river discharge measured at Sanchez Cerro bridge in the city of Piura 1925–2020 from the National Meteorological and Hydrological Service (SENAMHI), JISAO, the National Water Authority , the Chira-Piura Special Project  and the study of Takahashi and Martínez  and GPCPTo determine the economic impact of the 2017 Coastal El Niño event and the economic valuation of direct losses by the economic sector, we used economic data from three national agencies: (i) regional Gross Domestic Product (GDP) data for 1998 from the Institute of National Statistics (INEI), (ii) macroeconomic data related to aggregated GPD , terms of trade, 1982–1983 total GDP and agriculture and livestock production GDP from the Peruvian Central Bank (BCR) and (iii) ENSO 2017 economic valuation of direct losses of public infrastructure by sector in US$ million from the Special Authority for Reconstruction (RCC) –40.To describe the epidemiological context before, during and after the El Niño 2017, we used surveillance data on weekly cases of mandatory-reporting infectious diseases  from 2012 to 2017 from the Peruvian Ministry of Health’s Center for Epidemiology, Disease Control and Prevention (CDC-MINSA).To collect the data, we conducted in-depth semi-structured interviews with key stakeholders who were involved in the 2017 response. The interviewees were related to the Ministry of Health (MINSA), the Presidency of the Council of Ministers (PCM) and the private industry and provided context and further understanding of the institutional response to the event. These in-depth interviews were tailored to each stakeholder focused on the issues within their jurisdiction. The questions were framed under the main measures applied in the emergency. All interviews were taped-recorded with prior participant consent and took detailed notes. Interviews were transcribed verbatim in Spanish and compared with the notes supporting the transcription process. We completed the assessment by searching and reviewing technical legislation and policy documents regarding disaster response and preparedness from 2010 to 2017.Details and sources used for this manuscript can be seen in The climatological mean (1981–2010) of the monthly time series of different climate variables was calculated separately. A monthly series descriptive analysis of the SST of Puerto Chicama, the El Niño 1+2 index, and the Piura River discharge for the El Niño years 1925, 1983, 1998, and 2017 were described with their respective climatological values. We also contrast the El Niño 1+2 index with the reference threshold of 25ºC as an indicator of the onset of heavy rainfall and strong river discharge on the northern coast of Peru. Finally, we compared the spatial patterns of SST and precipitation anomalies in the Tropical Pacific for two El Niño events, 1998 and 2017.The economic impact of the 2017 Coastal El Niño event was estimated using a Vector Autoregression (VAR) methodology , there was a country-wide increase in key weather-sensitive infectious diseases cases under mandatory reporting surveillance , compared to the average of previous, recent and most comparable years (2012–2016). Then, we evaluated whether observed cases of dengue and diarrheal disease, respiratory illness, and pneumonia among children <5 years old increased during and after the onset of Coastal El Niño, both for all of Peru and each of the four northern coastal regions most affected . We compared weekly 2017 cases grouped in five-week periods versus the mean of the equivalent weeks in 2015–2016 and used analysis of variance to determine whether cases increased above expected levels of previous years and, if so, when upon the El Niño onset. Comparisons may slightly underestimate the impact of the 2017 Coastal El Niño, as there was a mild El Niño event between 2015 and 2016, potentially increasing incidence rates in that period.We followed a semi-structured guide for the interviews to assess the appropriateness of the 2017 Coastal El Niño response at different governmental levels and the effect of the governance structure on this response. By using the international guidance documents on risk management, “Disaster risk management systems analysis: A guide book” from the Food and Agriculture Organization, and the “Information management and communication in emergencies and disasters: Manual for disaster response teams” from the Pan American Health Organization, we assessed and adapted three key capabilities: (i) information management, (ii) rapid adaptation in response to changing conditions, and (iii) coordination between governmental agencies , 42. In The ethical review board of Universidad Peruana Cayetano Heredia reviewed and approved the study . All participants who agreed to participate in the study provided verbal consent before being interviewed.SST data from Puerto Chicama  showed tIt is important to note that, rather than SST anomalies, it is the absolute SST that matters most for precipitation. The reference threshold of around 25°C can be taken roughly as the minimum SST required to trigger precipitation in northern Peru , althougIn contrast, the onset of the 1925 and 2017 Coastal El Niño was abrupt and of shorter duration according to the Puerto Chicama time series SST , with thDespite similarities in the impacts on the northern coast of Peru in terms of high SST and heavy rainfall , the 199El Niño events of high intensity (such as those registered in 1982–1983 and 1997–1998) are among the climate phenomena events that have recently caused the greatest negative impact on economic activity and living conditions in Peru . In both"huaicos", “huaycos”) that were observed in Tumbes, Piura, Lambayeque La Libertad, and Lima, which affected the economic activity [In 1983, Peru’s GDP fell by 10%, and estimates suggest that nearly half of the contraction in economic activity was explained by weather conditions related to the 1982–1983 El Niño . The Impactivity .The northern coast registered a 3% contraction in its GDP level. One of the most affected economic sectors, fishing, recorded a GDP fall of -35.6% in 1998 compared to 1997’s output . AccordiDespite the severity of infrastructure damage, the estimated impact of the 2017 Coastal El Niño on national economic activity–measured in 2017 GDP percentage points lost–was lower than the recorded effect of previous severe El Niño events. This could be explained by the significant diversification of the Peruvian economy in the past 20 years and important innovations in sectors such as agriculture, driven by the increase in international trade and investment . The natFurthermore, the 2017 Coastal El Niño was not an event of a national-level impact but mainly a regional event with an intense, focal impact on the northern coast of Peru. This could also explain why its effects were lower than expected. This region only accounts for 20% of Peru’s population, 16% of the GDP, and 18% of household spending. Regional total GDP fell in the main regions of the northern coast during 1Q2017: Piura (-3.6%), Ancash (-2.6%), La Libertad (-1.1%) and Lambayeque (-0.2%). Particularly, the agriculture and livestock GDP component remained affected in some of these regions throughout 2017: Piura fell by -20.2% and Ancash by -0.2%, while La Libertad and Lambayeque presented small growth figures 0,6% and 0.7%, respectively.Our econometric vector autoregression (VAR) analysis of macroeconomic and climate variables isolated the effects of weather anomalies and used data before the Coastal El Niño (fourth quarter of 2016) to produce a referential 2017 economic activity forecast (baseline). The differences between the baseline forecast and the actual observed data can be decomposed by individual shocks related to the outcome of each variable after the prediction. For example, a private investment shock shows how much of the aggregate forecast error can be explained by the performance of this variable, which in our results is negative, probably due to a loss of business confidence in this period. Similarly, a negative consumption shock can be understood as a loss in consumer confidence, while a positive term of trade shock can be related to higher-than-expected main export prices. Finally, the sea temperature anomalies and the Piura River water flow negative shocks represent how much of the prediction error is explained by the unexpected climatic conditions of the Coastal El Niño.Our estimates showed that the Coastal El Niño had a negative effect of -4.5 and -1.1 percentage points on private investment growth and private consumption growth, respectively (obtained by addition of rows (a) and (b)). Considering that in 2016 these economic aggregates represented 18% and 64% of GDP, respectively, we can give an approximate aggregate GDP impact by multiplying each negative effect by the individual weight of these indicators on GDP. These results translate into an estimated aggregated negative impact of -1.5 percentage points on 2017 GDP growth. So, GDP growth in 2017 would have reached 4.0% if the Coastal El Niño had not occurred, instead of the officially recorded 2.5% growth.Despite the well-known impacts of El Niño-related floods on public infrastructure such as roads, bridges, schools, and healthcare facilities, investments to prevent damage in the most vulnerable areas have remained insufficient. It was no surprise that the intense rainfall and mudslides of the 2017 Coastal El Niño brought about such expected damage to main roads and agricultural areas and floods in urban and rural areas of the northern coast. In total, 1,159 health facilities were affected, of which 32 collapsed, and 39 were declared inhabitable in a context where 1,644,879 people were affected and 505 injured .In addition, 3,703 schools were damaged, 141 collapsed, and 315 were categorised as inhabitable . TranspoAlmost 49,000 housing units were affected or damaged, and streets and sidewalks were severely affected. Vulnerable areas, due to their proximity to riverbanks, were worse off. Repairing all damaged public infrastructure in urban and rural areas and installing prevention infrastructure would cost approximately US$ 7.7 billion, approximately 3,5% of Peru’s GDP and 11% of the total GDP of the five most affected regions .Required investments to complete such massive reconstruction could take many years, considering Peru’s track record of deficient public investment. Between September 2017 and April 2019, only 27% of the total approved budget related to the reconstruction works was used . Since tIn 2017, the CDC-Peru documented over 68,000 dengue cases, representing Peru’s largest dengue outbreak in history and a two-fold increase from the maximum annual cases observed between 2012 and 2016 . The fouth (week 12). While the timing of the peak in cases followed dengue’s typical seasonal patterns, cases in 2017 exceeded by 4.9 standard deviations the 2012–2016 annual mean . Weekly cases increased substantially both countrywide and in Piura following the flooding in the city of Piura on March 25ual mean , a highlReported countrywide leptospirosis cases in 2017 were also 4.9 standard deviations above the 2012–2016 annual mean (probability < 0.00005%), mainly due to a sizable increase between January and June 2017 in Piura and Tumbes . In contMean weekly cases of diarrheal disease, acute respiratory infections and pneumonia increased significantly after the El Niño onset in Piura , in addition to other specific increases in specific regions  conformed in the Law 29664  is respoCivil society´s engagement and adequate information management are crucial steps in disaster risk reduction. Language barriers and inadequate or untimely information communication may lead to disinformation and confusion during an event. According to all the interviewees, situations where the citizens did not understand the forecast and early warning systems due to language limitations and inappropriate dissemination strategies hindered the appropriate response."Despite the warnings from the early warning system, many districts failed to carry out preparedness works and communicate effectively to their residents."(PCM representative)Moreover, local governments were not prepared for the event despite available annual preparedness funds. Subnational governments (region and province-level) do not have the capabilities to create an integrated disaster risk plan. Furthermore, the interviewee pointed out many authorities lack the political will to apply for and manage funds, mainly due to the rigidity in spending and a lack of human capacity. Usually, this context leads local governments to prioritise initiatives with short-term immediate results and higher visibility and political impact rather than integrated approaches with more considerable, albeit long-term, public gains.“Very few mayors and regional presidents used the budget allocated in the previous year to prepare for the phenomenon of El Niño”(PCM representative)In addition, by restricting the funding schemes to one year, the central government has substantially limited the possibility of developing integrated approaches needed for this kind of climate event. A PCM representative stated that despite knowing the possibility of an El Niño event, the magnitude of the event surpassed the prevention efforts put in place by the local governments. Once the event was on course, the central government declared a state of emergency to access the emergency funds. Thirteen of the 24 departments of Peru , corresponding to 109 provinces and 879 districts, were declared to be in a state of emergency.“States of emergency allow local governments to make use of different budget items and overcome control barriers established by the comptroller to speed up the spending required by the emergency.”(PCM representative)A PCM representative mentioned that changes in the government structure in December 2016 led to slower bilateral coordination and created bottlenecks in the initial emergency response. This led to a centralised response, where the central government coordinated directly with the National Emergency Operation Centre (COEN), while the ministers of the different portfolios were sent to the regions to coordinate directly with the Regional Emergency Operation Centres (COER). Efficient and effective coordination and communication among all sectors were slow, but sending national representatives allowed faster decision-making and coordination between the municipalities, armed forces, police, firefighters, regional health, agriculture, transportation and communications directorates and private companies.“There were cases of critical patients, which require immediate transfer, who were mobilised thanks to coordination with the armed forces”(MINSA representative)The disaster risk response drove local stakeholders  to try and bridge some of the governmental gaps. They donated “goods” during the emergency and were involved in the early reconstruction effort through the mechanism of “works for taxes”, led by the “Pro Inversion” programme of the central government .“An alliance was made between other companies and a conversation with the regional government was made and they presented a list of priority works, and after a brief discussion, we decided to collaborate in the reconstruction of the bridge”(Private industry)A clear example of the existing gaps could be seen in managing supplies (water and food) and shelter for the displaced people. The response to re-building the lost infrastructure was slow and inexistent in some areas. Health services could not cope with the increased flow of patients from other flooded areas. Health staff initially turned away patients because of insufficient personnel to treat them or lack of identification cards and public insurance . Many health workers lost their homes and were themselves displaced.“Many health establishments denied care to people because they did not have an ID. In the same way, there were attention problems with social security because in the absence of the identity document they could not be registered”(MINSA representative).In addition, the existing health system did not allow for access to health care treatment in camps for displaced populations. In these camps, people had to deal with additional difficulties concerning violence, mental health, reproductive health, and access to care for underlying conditions.“Many patients with cancer, tuberculosis or HIV lost their medicines and had no access to new ones.”(MINSA representative)The last topic that surfaced during the interviews was reducing risks in illegal housing, pointing toward the need for local-level risk evaluations, particularly in vulnerable populations. In recent years, urban expansion has continued to grow disproportionately and often informally, with the most disadvantaged populations occupying the land in dangerous and marginal areas of the cities that have an increased risk of landslides and flooding.“There are many people who are exposed to risks generated by the environment. On the central coast many people occupy unsuitable spaces, but with the approval of the municipality… (…) The municipalities do not always carry out prevention work in high-risk areas, many wait for an emergency to occur before acting.”(MINSA representative)The 2017 Coastal El Niño event was intense and abrupt, with a rapid onset. It did not correspond to a warm ENSO phase but coincided with neutral to cool ENSO conditions and was confined to the far-eastern Pacific. Because of this intensity and abruptness, climate models used for projections could present biases, particularly in the far-eastern Pacific, which should be considered with care. Despite the short duration of this event, restricted to February-April, a ten-fold increase in heavy rainfall and river flooding did incur important economic losses and health impacts, similar to those of the 1983 and 1998 severe warm ENSO (El Niño) events.The econometric analysis based on macroeconomic variables and sea temperature anomalies showed that the Coastal El Niño event impacted more than just infrastructure, harming private consumption and private investment, with an aggregate negative impact on GDP growth of -1.5 percentage points in 2017. Higher event frequency than the normal 3–7 year cycle would have a dire effect on Peruvian economics and the country’s ability to mitigate the potential impacts, prepare accordingly and bounce back.On the other hand, public health impacts with increases in infectious and vector-borne diseases in disaster settings are not unexpected during El Niño events. The health assessment shows that CDC-Peru recorded the largest dengue outbreak ever, with 76,093 confirmed cases, 68,630 (90% of reported cases) in the 2017 Coastal El Niño-affected areas. The analysis also displays an increase in reported leptospirosis cases in Piura, one of the regions most affected by extreme weather. Other immediate impacts on health outcomes, particularly on infectious diseases, may have been difficult to assess with surveillance data due to the interruption of healthcare services and disease reporting during the most severe weeks of the event.The 2017 Coastal El Niño event also highlighted the challenges in Peru’s disaster response system. It showcased the gaps and potential failures in the health system’s structure and function and the information management and dissemination system. Furthermore, it portrayed how the state responded under the pressure of a disaster situation to protect disenfranchised and vulnerable populations. Preparedness is a key topic, particularly for the different climate change scenarios and their effect on extreme weather events like ENSO or Coastal El Niño events.Based on the findings, we conclude that the necessary improvements to Peru’s disaster preparedness and response under all climate change scenarios should span a broad set of issues at different geopolitical scales and impacts tailored to the diversity of vulnerabilities. From territorial zoning, strategic health facility placement in the Regions, and versatile information and communication strategies to governance structures and financial mechanisms. Moreover, since the frequency of ENSO events will likely increase and intensify with unabated greenhouse gas emissions , 16, theS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Overactive Janus kinases (JAKs) are known to drive leukemia, making them well-suited targets for treatment. We sought to identify new JAK-activating mutations and instead found a JAK1-inactivating pseudokinase mutation, V666G. In contrast to other pseudokinase mutations that canonically lead to an active kinase, the JAK1 V666G mutation led to under-activation seen by reduced phosphorylation. To understand the functional role of JAK1 V666G in modifying kinase activity we investigated its influence on other JAK kinases and within the Interleukin-2 pathway. JAK1 V666G not only inhibited its own activity, but its presence could inhibit other JAK kinases. These findings provide new insights into the potential of JAK1 pseudokinase to modulate its own activity, as well as of other JAK kinases. Thus, the features of the JAK1 V666 region in modifying JAK kinases can be exploited to allosterically inhibit overactive JAKs. Janus kinases (JAKs) are at the forefront of cytokine signaling, working immediately downstream of cytokine receptors, which lack intrinsic catalytic activity. The JAK family consists of four tyrosine kinases ), and through paired combinations with seven signal transducers and activators of transcription (STATs), they orchestrate cell-specific cytokine signaling events that ultimately dictate patterns of gene expression . JAK1 anAggressive forms of acute lymphoblastic leukemia (ALL) are believed to be driven by mutated JAKs ,11 or toThe majority of pipeline JAK inhibitors target the ATP-binding site and are classified as Type I and Type II inhibitors, depending on whether the kinase is active or inactive, respectively. High conservation of the ATP-binding site among the JAK family and across human kinases allows such inhibitors to act broadly. Although these pleotropic effects by current JAK inhibitors may be beneficial in shutting down multiple signaling pathways utilized by cancer cells, this feature may also be contributing to the unwelcome adverse effects that have raised concerns for their clinical use . To overThe structure of JAKs consists of four functional domains harboring seven Janus Homology (JH) motifs, including the carboxyl-terminal JH1 Tyr kinase domain and the JH2 pseudokinase domain. The amino-terminal end houses the 4.1, Ezrin, Radixin, Moesin (FERM) domain and the Src Homology 2 (SH2) domain, jointly spanning JH3–JH7. The bulk of activating mutations in JAK family members occurs within functional “hot spots”  found acInitially, we sought to identify novel activating JAK mutations driving ALL, but instead found an inactivating JAK1 JH2 mutation and explored its potential in modifying kinase activity. Specifically, in vitro kinase assays revealed that the V666G mutation resulted in a JAK1 kinase-dead-like phenotype. A dominant negative effect of inactive JH1 and JH2 JAK1 mutants occurred on wild-type (WT) JAK family members, as observed by changes in Tyr phosphorylation. The JAK1 V666 region can modify its own intrinsic catalytic activity and that of dimerized JAK2 and JAK3 kinases as well. Perhaps most interesting, JAK1 V666G decreased Tyr phosphorylation of the ALL-associated JAK3 constitutively active mutants, M511I and A573V. These findings demonstrate that JAK1 V666G exerts a dominant negative role on JAK3. Likewise, these findings support the notion of shared trans-inhibitory interactions between the JAK1 JH2 and hyperactive JAK3 JH1, suggesting that the V666 belt of the JAK1 pseudokinase may be exploited by therapies that aim to inhibit overactive JAKs in an allosteric manner.Nine ALL patient (P1-P9) specimens, analyzed by whole exome sequencing (WES) and reported previously , containTo investigate the impact of V666G on JAK1 kinase activity, auto-activation via overexpression in the JAK1-deficient cell line, U4C, was coupled to in vitro kinase assays, and Tyr phosphorylation of JAK1 and STAT5 were measured. Auto-activation is defined as the phosphorylation that occurs between the same JAK kinases and is not facilitated by cognate receptors. Compared to JAK1 WT, auto-activation of JAK1 V666G was similar to that of the kinase-dead control, JAK1 K908A, with a significant observable decrease in Tyr phosphorylation b. Thus, To investigate whether JAK1 V666G could be activated by WT JAK family members, JAK1–MYC constructs were co-expressed with either WT JAK1–GFP, JAK2 or JAK3, and examined for cross-activation indicated by Tyr phosphorylation of both JAKs. Cross-activation here is defined as the phosphorylation between distinct JAK kinases not facilitated by cognate receptor proteins. Similar to kinase-dead JAK1 K908A, JAK1–MYC V666G was not phosphorylated by JAK1–GFP WT protein a. HoweveIt seemed plausible to expect that the dominant presence of JAK1 V666G on JAK3 WT protein observed in the kinase overexpression studies would affect the IL-2 signaling pathway. Whether JAK1 V666G could disrupt cell-signaling, phosphorylation via trans-activation of JAK3 and subsequent activation of STAT5, ERK and AKT downstream of IL-2, were assessed. Trans-activation under these conditions refers to phosphorylation occurring between distinct kinases facilitated by intermediate proteins such as cognate receptors. First, U4C cells were transfected with IL-2Rβ and γc to reconstitute the IL-2 receptor complex with JAK3 and JAK1 constructs. JAK1 V666G showed reduced Tyr phosphorylation compared to WT JAK1, even in the presence of JAK3 and other IL-2-signaling components a. In thiAs discussed previously, JAK3-activating mutations M511I and A573V have been shown to drive T-ALL ,30,34. TThe JAK1 JH2 single-point mutation, V666G, exhibited an inhibitory effect on auto-activation, cross-activation, and IL-2 induced trans-activation. To explore the structural significance of this site on intramolecular JH1–JH2 interaction, the solved full-length mouse JAK1, bound to the cytokine receptor structure, was utilized  and a hoWithin the homology model of the closed human JAK1 structure d, and poJAK1 is an essential mediator of IL-2 receptor signaling. However, it is unclear whether the absence of JAK1 or the presence of inactive JAK1, either as a classical JH1 kinase-dead mutant (JAK1 K908A) or via JH2 mutation, would affect JAK3 activation and IL-2 signaling. Deciphering these outcomes could provide support for trans-inhibition between JAK kinases. In agreement with earlier reports ,43, we dJH2-activating JAK3 mutants, including A573V and M511I, have been reported to require IL-2 receptor and JAK1 for their transforming ability . PreviouFurthermore, it is unclear whether the inactivating JAK1 V666G mutation presents a selective advantage in ALL. One possibility could be that JAK1 V666G inactivation helps subvert detection by the immune system. For example, cancers with high mutational burden have JAK1 loss-of-function mutations . The negStructural studies of JAK kinases  make it Previous models of JAK1 from partial JH2–JH1 proposed JH2 trans-inhibition suggest that dimerized JAKs interact between JH2 and JH1 domains, where the JH2 domain of one JAK inhibits the JH1 domain of the other JAK ,52. V666To date, FDA-approved JAK kinase inhibitors primarily target the ATP-binding pocket in JH1 domains , but areGenomic DNA was collected from a small cohort of seven healthy donor controls and nine patients diagnosed with acute lymphoblastic leukemia (ALL), as described and reported previously . BrieflyThe human JAK1 MYC tag , JAK1 GFP tag , JAK2 , and JAK3 (Invitrogen pcDNA3.1) plasmids were purchased. IL-2Rβ, γc, STAT5B, and mutant M511I and A573V JAK3 were generated and reported previously ,58. MutaThe JAK1-deficient human U4C cell line (ATCC) was grown in DMEM, supplemented with 10% fetal bovine serum , 2 mM L- glutamine , and 1% penicillin/streptomycin (Corning). Cells used for non-cytokine transfection experiments received complete media, while cells used for cytokine experiments were made quiescent by receiving 1% FBS media, and incubated for 24 h post-transfection. When cytokines were used, cells were stimulated with 400 IU/mL rhIL-2 for 10 min at 37 °C, prior to immunoprecipitation and Western blot analysis.2, 5 mM MnCl2) alone, or supplemented with 100 μM ATP at 32 °C for 20 min, as previously described [U4C cells were transfected according to the manufacturer’s instructions . For all auto-activation and cross-activation studies, cells were transfected with 15 μg of indicated JAK plasmid, along with 10 μg of STAT5B. For auto-activation experiments, MYC-tagged JAK1 WT or JAK1 constructs, K908A or V666G, were transfected with STAT5B and used for overexpression and in vitro kinase assays. Briefly, cells were harvested 24 h post-transfection and subsequently pelleted, lysed, and clarified, as described previously . A samplescribed ,60. All Similarly, for cross-activation experiments, cells were transfected to overexpress the JAK1 WT GFP tag, JAK2 WT, or JAK3 WT with STAT5B, either alone or with the JAK1 WT MYC tag, or JAK1 MYC tag constructs, K908A or V666G. Samples were immunoprecipitated with either Origene antibody against GFP tag (TA150041), or Santa Cruz antibodies against c-MYC (sc40), JAK1 (sc376996), JAK2 (sc390539), or a polyclonal antibody generated against the carboxyl terminus of JAK3, described previously .For IL-2 signaling experiments, U4C cells were transfected with 5 μg IL-2Rβ, and 5 μg γc to reconstitute IL-2 receptor. For Hyperactive JAK3 mutation experiments included 15 μg of JAK3 WT, JAK3 M511I, or JAK3 A573V, expressed with either JAK1 MYC WT or V666G for immunoprecipitation. Complementary to this scheme, a separate set of experiments used for total cell lysate, was examined for IL-2-induced activation of STAT5.Samples were separated by 7.5% or 10% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane  for Western Blot analysis, as previously described ,60. For The open and closed structures of wild-type human JAK1 were developed by aligning the crystallographic structure of mouse JAK1 (PDB ID: 7T6F) and human TYK2 domains (PDB ID: 4OLI) as templates, respectively. Swiss-Model was used to diminish clashes and minimize the energy for the final structures. All molecular graphics and analyses were performed with UCSF Chimera . BioRendwww.graphpad.com. Similarly, two-way ANOVA and a Tukey’s multiple comparisons were used under the guidance of the UTEP BBRC Statistics Core team for analysis.One-way analysis of variance (ANOVA), followed by a Tukey’s multiple comparison test, were performed and graphed using the GraphPad Prism version 7.00 for Mac OS X, GraphPad Software,


The shiitake mushroom is the most commonly cultivated edible mushroom in the world, and is rich in protein. This study aims to obtain the peptides with α-glucosidase inhibition activity from shiitake mushroom protein hydrolysate. The conditions of enzymatic hydrolysis of shiitake mushroom protein were optimized by response surface test. The results showed that the optimal conditions were as follows: the E/S was 3390 U/g, the solid–liquid ratio was 1:20, the hydrolysis temperature and time were 46 °C and 3.4 h, respectively, and the pH was 7. The active peptides were separated by gel filtration and identified by LC-MS/MS analysis and virtual screening. The results indicated that fourteen peptides were identified by LC-MS/MS. Among them, four new peptides  with the higher docking score were selected and chemically synthesized to verify their inhibition activity. The IC50 values of EGEPKLP, KDDLRSP, TPELKL, and LDYGKL for α-glucosidase inhibition activity ranged from 452 ± 36 μmol/L to 696 ± 39 μmol/L. The molecular docking results showed that the hydrogen bond and arene–cation bond were the two major interactions between four peptides and 2QMJ. The hydrogen bonds were crucial to the inhibition activity of α-glucosidase. The results indicate the potential of using the peptides from shiitake mushroom protein as functional food with α-glucosidase inhibition activity. Diabetes mellitus (DM) is a widespread metabolic disorder that affected 537 million adults globally in 2021 ,5,6.The most effective treatment for T2DM is to reduce hyperglycemia, especially postprandial hyperglycemia, by slowing down the carbohydrate metabolism. The membrane-bound α-glucosidase, located in the small intestine’s epithelial mucosa, is a major digestive enzyme involved in carbohydrate metabolism. It can cleave the glycoside bonds in carbohydrate to free the glucose ,8. ThereRecently, some plant or animal protein hydrolysates with α-glucosidase inhibition activity have been found, such as egg ,15, beanLentinus edodes) is the largest edible mushroom in China and is also the second most commonly cultivated edible mushroom in the world [Shiitake mushroom , pepsin , alkaline protease (200 U/mg), acid protease (50 U/mg), papain (800 U/mg), and flavor protease (20 U/mg) were purchased from Qiyi Biotechnology  Co., Ltd. α-glucosidase (10 U/mg) and 4-nitrophenyl a-D-glucopyranoside  were purchased from Sigma-Aldrich . Acetonitrile  was purchased from Fisher Chemical . Formic acid  was purchased from Thermo Scientific . Other reagents were of analytical grade and commercially available.An Thermo Easy-nanoLC system was connected to a Thermo Scientific QE mass spectrometer, USA Thermo Fisher Scientific. Other devices used were as follows: Analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd. . AMR-100 automatic enzyme label analyzer, Hangzhou Aosheng Instrument Co., Ltd. . MTYK-MI805 pH meter, Beijing Zhonghui Tiancheng Technology Co., Ltd. . HZQ-B Constant Temperature Culture Shaker, Suzhou Weier Laboratory Supplies Co., Ltd. . H-1650 high speed centrifuge, Changsha Xiangyi Centrifuge Instrument Co., Ltd. . Freeze-drying Instrument, Shanghai Haozhuang Instrument Co., Ltd. .Extraction of shiitake mushroom protein: A certain quantity of dried shiitake mushroom powders was dissolved in deionized water with the mass–volume ratio of 1:20. The solution pH was adjusted to 9.0 with 1 mol/L NaOH. The mixture was sonicated at 40 KHz and 50 °C for 60 min, then centrifugation was used to separate the supernatant for 15 min at a speed of 4000 rpm. The supernatant pH was adjusted to 3.5 by 1 mol/L HCl. Then, after centrifugation at 5000 rpm/min for 20 min, the precipitates were collected.Hydrolysate preparation: Two grams of protein powder was dissolved in deionized water according to With the E/S of 4000 U/g, the solid–liquid ratio of 20, the pH of 7.0, the hydrolysis temperature of 45 °C, and the hydrolysis time of 4 h as the basic fixed factors, one of the factors was changed and the single factor test was carried out. The gradient design of each factor was as follows: E/S , solid–liquid ratio , pH , hydrolysis time , hydrolysis temperature . The α-glucosidase inhibition activity of hydrolysate with different conditions was compared.The hydrolysis conditions of shiitake mushroom protein were improved using CCD. The inhibitory activity of α-glucosidase acted as the response value. According to the single factor experiment, E/S (A), temperature (B), and time (C) were selected to be optimized for response surface optimization. 2CO3 solution. The absorbance value was measured at 405 nm using an AMR-100 automatic enzyme label analyzer. The results were calculated using the following formula:A quantity of 100 μL α-glucosidase solution (0.2 U/mL) was premixed with 50 μL sample solution in the enzyme label plate, and incubated at 25 °C for 10 min; 50 μL of PNPG (5 mmol/L) solution was added to start reactions, and incubation was continued for another 30 min at 37 °C. The reaction was terminated with 50 μL 0.67 mol/L NaThe hydrolysate solution was separated by ultrafiltration membranes of 1 k Da, 3 k Da, and 5 k Da. The fraction was named UF-I (>5 k Da), UF-II (3–5 k Da), UF-III (1–3 k Da), and UF-IV (<1 k Da), and collected separately.The fraction F-IV obtained by ultrafiltration was further separated by gel filtration according to Chen et al.’s method . The samThe fraction with maximum activity obtained by gel filtration chromatography was analyzed by LC-MS/MS according to the method of Chen et al. .The peptides were diluted in a 20 μL solution of 0.1% formic acid and 5% acetonitrile and separates with an Acclaim PepMap RPLC C18  linked to a PepMap RSLC C18 in an LC-MS/MS system. The mobile phase was as follows: mobile phase A (0.1% formic acid) and mobile phase B (80% acetonitrile with 0.1% formic acid); the gradient elution was performed with a gradient of 3–99% B (0–34 min) and the flow rate was 500 nL/min. m/z in ESI positive mode. Resolution was 120,000, AGC target was 4 × 105, and maximum IT was 50 ms. MS/MS scanning conditions were as follows: resolution was 30,000, AGC target was 1 × 105, maximum IT was 100 ms, TopN was 20, and NCE/stepped NCE was 32.MS data were acquired over the range from 100 to 1550 Peptide sequences were identified using PEAKS Studio X software in combination with a shiitake mushroom protein database search.The semi-flexible molecular docking was performed between the identified peptides and the crystal structure of the human α-glucosidase (PDB code: 2QMJ) using MOE software according to Chen et al.’s method . The cryTo obtain the receptor molecules required for docking, the water molecules from 2QMJ were eliminated, the molecules were protonated, and energy was minimized. The receptor pocket of the 2QMJ was used as the docking target for molecular docking, and the peptides were used as the ligand. Each coupling was performed 30 times. The peptide with a better docking effect was screened from the obtained results for further analysis.2O/1,2-ethanedithiol (EDT)/triisopropylsilane (Tis) = 95/1/2/2, v/v/v/v) was added to the resin to cut the peptide. The synthetic peptides were purified by HPLC. The molecular weight of the purified peptide was confirmed by mass spectrometry. The screened potential α-glucosidase inhibitory peptides were chemically synthesized by China Peptides Co., Ltd. . Fmoc solid-phase synthesis was used to obtain the raw peptide. Briefly, chlorine resin was swelled by dichloromethane (DCM). Fmoc-AA (amino acid)-OH, O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU), and N,N-diisopropylethylamine (DIEA) were added to the drained resin for 30 min with a nitrogen bubble reaction to accomplish the condensation reaction. The resin was then washed with DMF to remove Fmoc. After being drained, cutting solution (trifluoroacetic acid (TFA)/Hp < 0.05) was analyzed by DPS. Response surface data were analyzed using Design-ExpertV8.0.6 software.All experiments had three repetitions. Data are expressed as mean ± standard deviation. Statistical significance . Therefore, 3000 U/g was selected as the appropriate enzyme concentration.The effect of enzyme concentration on the α-glucosidase inhibitory activity of shiitake mushroom protein hydrolysates was investigated. With the increase in the enzyme–substrate ratio (E/S), the α-glucosidase inhibitory rate increased first and then slightly decreased A. The α-Temperature is a key influencing factor for enzymatic reaction. Increasing temperature can speed up the reaction rate, but excessive temperature can cause protease inactivation. The effect of different temperatures on the α-glucosidase inhibition activity of enzymatic hydrolysates was investigated. With the increase in temperature, the inhibitory rate of α-glucosidase first increased and then decreased. When the temperature reached 45 °C, the maximum inhibitory rate of α-glucosidase was attained B. TherefThe substrate concentration is a key parameter of enzymatic reaction. When the solid–liquid ratio was 15, the inhibitory rate of enzymatic hydrolysates was lower. When the solid–liquid ratio was 20, the α-glucosidase inhibitory rate of enzymatic hydrolysates increased and then decreased slightly C. It mayThe inhibition activity against α-glucosidase was significantly influenced by the enzymolysis time. With the increase in enzymolysis time, the inhibitory rate of α-glucosidase first increased and reached the highest level at 3 h, and then decreased D, which With the increase in pH, α-glucosidase inhibitory rates of hydrolysates rose first and then decreased. When pH was 7, the inhibitory rate of α-glucosidase reached the highest level E. There In the actual process of enzymolysis, various factors may interact with each other. To further investigate the significance of the influence of various factors on the target value, E/S, temperature, and time were taken as independent variables, and α-glucosidase inhibitory rate as the response value, and a CCD experiment was conducted. The results are shown in p < 0.0001). The R2 was 0.9877, which indicates that the test value and the fitting value had a high correlation. The regression equation model fitted the data well because the p value of the lack of fit was not significant. Consequently, it is possible to forecast the test results using the model. It can be seen from According to the variance analysis results , the regThe optimal conditions predicted by the model were as follows: the E/S was 3390 U/g, the solid–liquid ratio was 1:20, the hydrolysis temperature and time were 46 °C and 3.4 h, respectively, and the pH was 7. Under optimal conditions, the predicted value of the α-glucosidase inhibitory rate was 65.2%. To verify the reliability of the predictive value of the model, three validation tests were carried out under optimum conditions, and the average α-glucosidase inhibitory rate of hydrolysate was 64.6 ± 1.2%. The result indicates that the optimized parameters of the model were accurate and reliable.The molecular weight of the peptide is closely related to the α-glucosidase inhibitory activity. As shown in Peptides are frequently purified by gel filtration. The UF-IV fraction was further separated by a gel filtration column (Sephadex G-10). The results are presented in The sequences of fraction F5 were determined by HPLC–MS/MS with de novo sequencing. Fourteen peptides were identified from fraction F5 (score > 80). The molecular weight of the fourteen peptides ranged from 699 Da to 829 Da .The binding energy and binding sites of peptides to receptor proteins are related to their biological activity. To further screen active peptides from these 14 peptides, the peptides docking with the crystal structure of α-glucosidase (2QMJ) were performed using MOE software. Peptides with binding energy below −14 as well as more than four binding bonds were selected, as shown in The α-glucosidase inhibition activity of these peptides was obtained by molecular binding with residues in the active center. DM is a typical metabolic disease with chronic complications ,5,6. WitRecently, many studies have confirmed that α-glucosidase inhibitory peptides prepared from food protein have excellent α-glucosidase inhibitory potential . With thUltrafiltration and gel chromatography can be used to further isolate and purify active peptides from the hydrolysates. In this study, low molecular weight peptides (MW < 1 k Da) had stronger α-glucosidase inhibition activity than other fractions , which is consistent with the results of previous reports ,37. WangThe amino acid composition, molecular weight, and chain length of the peptides influence biological activity of α-glucosidase inhibitory peptides ,40,41. TThe IC50 values of EGEPKLP, KDDLRSP, TPELKL, and LDYGKL for α-glucosidase inhibitory activity ranged from 452 ± 36 μmol/L to 696 ± 39 μmol/L. In previous studies, the IC50 value of RVPSLM with α-glucosidase inhibitory activity identified from egg white protein was 23.07 μmol/L . The fouThe peptides with more hydrophobic amino acids had better inhibitory activity of α-glucosidase ,45. In pFurther study of the interactions between the peptide and α-glucosidase showed that EGEPKLP interacted with the 2QMJ through four hydrogen bonds. KDDLRSP and LDYGKL formed five hydrogen bonds with 2QMJ. TPELKL formed five hydrogen bonds and an arene–cation interaction with 2QMJ. The α-glucosidase inhibition activity is mainly dependent on hydrogen bonding, which agrees with the results of previous research ,48.In this study, the optimal conditions for preparation of α-glucosidase inhibitory peptide from shiitake mushroom were established. Four new α-glucosidase inhibitory peptides  were identified from shiitake mushroom protein hydrolysates using LC-MS/MS and virtual screening. TPELKL exhibited a higher α-glucosidase inhibitory activity than EGEPKLP, KDDLRSP, and LDYGKL. The molecular docking results demonstrated that the hydrogen bond and arene–cation bond were the two major interactions between the four peptides and 2QMJ. The hydrogen bond was crucial to the α-glucosidase inhibition activity. The results suggest that shiitake mushroom may be a reliable source of α-glucosidase inhibitory peptide and provide a feasible strategy for further utilization of shiitake mushroom protein. To improve the possibility of using these peptides as supplements, future studies are required, including examination of the effects of the peptides on other enzymes linked to starch digestion, the antidiabetic mechanism of these peptides, and their safety and stability in vivo.


Helminth infections modify the natural history of allergic diseases, by either decreasing or increasing their symptoms. Several helminth components are involved in the increasing of the allergic response and symptoms, overcoming the concomitant immunosuppression of helminthiases. However, the role of individual IgE-binding molecules in this process remains to be defined.A. lumbricoides allergen has been discovered, with potential use in molecular diagnosis.We updated the list of helminth allergens and IgE-binding molecules, their effects on asthma presentation, and their impact on allergy diagnosis. Data from genetic and epigenetic studies of ascariasis are analyzed. A new species-specific Most helminth IgE-binding components are not officially classified as allergens in the WHO/IUIS database, although there is evidence of their influence increasing allergic manifestations. Further immunological characterization of these components is needed to better understand their mechanisms of action and evaluate the ways in which they can influence the diagnosis of allergy. Ascaris lumbricoides, Strongyloides stercoralis, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenalis), filarial nematodes , and platyhelminth flukes . During infection, helminths produce a variety of molecules for their metabolism and survival as parasites, some of them stimulate the synthesis of specific IgE, and a small number induce allergic symptoms and/or increase the frequency and severity of allergic diseases. The clinical effects of this pro-allergenic activity depend on the modulation of host immunity exerted by the parasites, which means that they must overcome the concomitant immunosuppression to be apparent.Most parasite allergens come from helminths, including those causing soil-transmitted infections  has led to an increase in allergic diseases . Therefolsewhere  and willA. lumbricoides infection (ascariasis); however, these and other clinical manifestations also occur during other helminthiases. These infection-induced symptoms are present in a minority of the population, probably because it is genetically predisposed to overreact to parasitic infection, and the degree of concomitant immunosuppression is not sufficient to reduce the manifestations of allergy, especially if the infection is acute and mild. Numerous studies have evaluated the pro-allergenic effects of helminthiases on allergic diseases, and a recent meta-analysis concluded that helminth infections may increase the risk of bronchial hyperreactivity in children and atopy in adults; most of these effects associated with A. lumbricoides infection [The first observed allergy-like manifestations induced by helminths were symptoms of respiratory distress or hives in children suffering nfection ••. HowevHelminthiases have been controlled in most developed countries but are still endemic in tropical regions; for example, ascariasis has decreased during the last 60 years in industrialized countries, and currently, the most severe infections are present in some rural tropical villages. In tropical urban areas, hygienic conditions are better and regular deworming campaigns are carried out, which diminish the frequency and intensity of infections. Therefore, the effects of helminth allergens on allergic diseases are mostly observed in urban zones of developing countries, although they could also be present in some temperate regions •. UrbaniA. lumbricoides cystatin (Al-CPI) do not have relevant allergenic activity but strong immunomodulatory effects [Helminth allergens are the result of normal type 2/IgE immune responses during infections. Animal models have shown that specific IgE antibodies, together with eosinophils, can destroy helminth larvae, and other type 2 immune mechanisms have been revealed using diverse helminth species in animal models of infection , 2, but  effects . In thisA. lumbricoides induces respiratory symptoms similar to asthma, and sometimes Löffler’s syndrome. Also, there is epidemiologic evidence that ascariasis increases the frequency and symptoms of asthma. This potentiating effect can be detected at the population level, as it has been repeatedly observed that sensitization to A. lumbricoides is associated with a higher prevalence of asthma [During larval pulmonary transit, f asthma , 11 and f asthma , 12, 13,Specific IgE to HDM is the most important risk factor for asthma in the tropics. Thus, any condition that increases the allergic response against them could also increase the symptoms and severity of this disease. Since HDM exposure is high and permanent in tropical environments and induces IgE sensitization at a very early age Fig. , the croAscaris-sensitized children had stronger IgE to B. tropicalis and those sensitized to the Ascaris allergen ABA-1 had increased IgE responses to Blo t 5 and Blo t 12, two species-specific allergens of B. tropicalis, ruling out the effect of cross-reactivity [A. lumbricoides and HDM, where subjects sensitized to ABA-1 (a marker of ascariasis) had at least twice the odds of being sensitized to HDM [Ascaris-sensitized patients had significantly higher IgE levels to the HDM-specific allergens Der p 2 and Blo t 5 [A. lumbricoides infection includes responses not only to helminth antigens but also to HDM allergens. However, the role of A. lumbricoides components, the host genetic background, and the epigenetic modifications that influence this boosting effect are only beginning to be unraveled.Trying to further explore this boosting effect in human ascariasis, we studied the evolution of the immune responses in children of the FRAAT birth cohort , findingactivity . This nod to HDM . Further Blo t 5 . These fCHIA) and chitinase 3 like 1 (CHI3L1) were significantly enriched in high IgE responders to the resistance marker ABA-1; in addition, SNPs in the tumor necrosis factor superfamily member 13b gene (TNFSF13B) encoding the cytokine B cell activating factor were associated with high levels of total IgE [A. lumbricoides–infected and non-infected subjects, finding significant associations between H4Ac levels in the IL4 and CHI3L1 genes and IgE levels to A. lumbricoides. In addition, the levels of specific IgE antibodies to HDM were associated with H4Ac levels in the TNFSF13B gene. These results suggest that acariasis induces histone acetylation of immune response genes, which in turn increases the IgE response to both helminth and HDM allergens [In a search for genetic variants supporting the IgE hyperresponsiveness of ascariasis, we found that SNPs of the genes coding for acidic mammalian chitinase , CD203c-based basophil activation tests, and a passive cutaneous anaphylaxis mouse model [A. lumbricoides, B. tropicalis, and D. pteronyssinus extracts, suggest that it is A. lumbricoides species-specific [Immunochemistry analyses of mponents ; howevermponents , 22. Morse model ••. Furthspecific .Ascaris-HDM cross-reactivity [Ascaris GST (Asc l 13) has not been properly defined, its recombinant form induces skin wheal and flare reaction in subjects with ascariasis. The frequency of IgE reactivity to this molecule is low (< 20%) but may be clinically relevant in cases where there is also sensitization to mite and cockroach GSTs [Ascaris and other allergenic sources is supported by the structural homology between Asc l 13 and allergenic GSTs in cockroaches (Bla g 5) and HDM (Der p 8 and Blo t 8) [Ascaris spp. was ABA-1 (Asc s 1), discovered in Ascaris suum. It is a polyprotein abundant in the pseudocelomic fluid of both A. suum and A. lumbricoides. In humans, IgE and IgG responses to ABA-1 have been associated with protection [Ascaris infection.Tropomyosin, a well-known pan-allergen, is involved in activity , 25. Speactivity , as it iactivity . Althougach GSTs . The allBlo t 8) . A comprBlo t 8) •. The fiotection  rather tStrongyloidiasis can be asymptomatic , but in S. stercoralis involves type-2 pathways, including IgE antibodies. Both somatic antigens and excretory/secretory products (ESPs) are recognized by IgE, trigger IgE-dependent histamine release from basophils, and induce positive skin test reactions [Wuchereria bancrofti–infected individuals [S. stercoralis IgE-binding recombinant protein rA133 is recognized by IgE from 100% of infected patients but not from healthy controls or individuals infected with other parasites [Immunity to eactions . Howevereactions , and shaeactions , 38. Streactions . Anti-stividuals . Finallyarasites .Schistosoma eggs to induce type-2 inflammation [Schistosomiasis has been regarded as a protective factor against atopy and asthma , 42, depammation , suggestSchistosoma components are recognized by IgE, including a 22.6-kDa molecule from S. mansoni  that is associated with human resistance to reinfection [S. japonicum and S. haematobium respectively [S. mansoni tegumental-allergen-like proteins (TALs) that are differentially expressed throughout the parasite lifecycle [Some nfection , and theectively , 49. Twoifecycle . In termifecycle .S. mansoni–infected individuals, with a higher IgE-binding frequency after anthelmintic treatment [S. mansoni IgE-binding proteins are the IL-4-inducing principle from S. mansoni eggs , a glycoprotein that induces IL-4 release from basophils [S. mansoni and 0% in uninfected controls [Trematode “venom allergen-like proteins” (VALs) are similar to the wasp venom allergen Ves v 5. SmVAL6 is recognized by IgE from reatment . Besidesreatment . Other Sasophils , 54, andcontrols .S. mansoni ATP diphosphohydrolases (SmATPDases) also have allergenic properties [S. mansoni has IgE-binding homologs to well-known allergens like tropomyosin, GSTs, profilin, lipocalin, cyclophilin, and phosphoglycerate kinase [In addition, operties , and somoperties , 58. Furoperties . It is ae kinase .Toxocara spp. are more likely to have asthma compared to non-infected children [T. canis [There is experimental and epidemiological evidence that toxocariasis increases asthma susceptibility . A meta-children . Human sT. canis ; howeverN. americanus can induce basophil activation and degranulation in the absence of detectable specific IgE, probably due to early basophil sensitization or an effect mediated by hookworm-secreted proteases [Ancylostoma caninum, can penetrate human skin and produce an intensely pruritic, erythematous, serpiginous eruption that is characterized histologically by an important eosinophilic infiltration, called larva migrans [N. americanus calreticulin, which induces basophil histamine release and has little cross-reactivity with its human counterpart [Most studies about these nematodes suggest that they are more associated with immunosuppression than with an increase in allergic symptoms , althougroteases , 67. Fur migrans . Althoug migrans , 71. Onenterpart , 73.Na-ASP-2, is abundantly secreted by N. americanus larvae upon entry into the host and is structurally similar to vespid venom allergens and SmVALs [Na-ASP-2 can elicit positive skin test results [Na-ASP-2 have been studied as anti-hookworm vaccines [Na-ASP-2 induced generalized urticaria in three of seven individuals who received a single dose of a recombinant version of this protein, all of which had pre-existing Na-ASP-2-specific IgE [A. caninum, Ac68 and Ac-ASP-1 are IgE-binding molecules from this parasite [Another hookworm IgE-binding molecule, d SmVALs , 76. Na- results , supportvaccines , 78, butific IgE . Regardiparasite , 80.W. bancrofti in the lungs can cause an allergic reaction that resembles asthma, a rare condition known as tropical pulmonary eosinophilia (TPE), which presents with cough, dyspnea, wheezing, chest pain, systemic manifestations (including fever and weight loss), peripheral blood eosinophilia > 3000/µL, and elevated total IgE > 1000 IU/mL. Patients with TPE have higher levels of filarial-specific IgE than those with other manifestations of filarial infection [Human filariasis has been associated with allergic manifestations and atopic sensitization. Microfilaria of nfection , and thinfection , 83.O. volvulus molecules Ov27, OvD5B, and OvGalBP [W. bancrofti and B. malayi components trigger histamine release from human basophils, whereas B. malayi antigens also induce positive skin test reactions [B. malayi have been identified, including transthyretin-related protein (TTR), WbSXP-1, macrophage migration inhibitory factor, and gp15/400 [B. malayi IgE-binding component with a potential role in the pathogenesis of TPE given that it is recognized by IgE present in the bronchoalveolar lavage fluid and serum of patients with this condition [B. malayi γ-GT [Filariae induce a long-lasting IgE response and contain several IgE-binding components that are poorly characterized  such as  OvGalBP , 86. W. eactions , 84, 87.gp15/400 •. Interegp15/400 •. Sensitgp15/400 •. Bm23-2ondition . Bm23-25ondition , 90. In ayi γ-GT .B. tropicalis trypsin (Blo t 3) shares amino acid sequence similarity with its W. bancrofti counterpart [D. pteronyssinus and OvTrop from O. volvulus), as well as between cockroach and filarial GSTs  [B. malayi and W. bancrofti  [Many filarial proteins have homologs in allergenic sources such as mites and cockroaches, which can lead to cross-sensitization , 93. Forncrofti) , 95. Furd WbVAH) , 97.A. lumbricoides and HDM allergens (tropomyosin and GST) may confuse the diagnosis because sensitization to both organisms is very frequent [A. lumbricoides [Since allergic responses and helminth immunity have several similarities, it is possible that some diagnostic parameters for allergy can be confounded by helminthiases. In rural areas of the tropics, severe helminthiases can diminish allergen skin testing results , 99, evefrequent . In addifrequent , 102. Fofrequent . Anotherricoides , 105.A. lumbricoides infection [Helminthiases could also limit the usefulness of type 2 asthma markers such as total IgE, fractional exhaled nitric oxide (FeNO), and blood eosinophils, which can impact decisions regarding the use of biologics for treatment. Both asthma and helminthiases run with high total IgE levels; in tropical zones, children reach high total IgE levels at the age of 3 years  nfection  A. lumbricoides parasitized children in Ecuador [Ascaris-sensitized and non-sensitized patients [n = 99) from a rural village in Colombia. Preliminary unpublished results  showed no difference in FeNO levels between infected and non-infected children. Cases with lung migratory phase parasites, such as A. lumbricoides or A. duodenalis, had FeNO levels below 20 ppb  has been demonstrated experimentally, although more research is needed to identify the molecules involved and the underlying mechanisms. Still, this information supports clinical and epidemiological evidence demonstrating the diverse pro-allergenic effects of helminthiases in humans, especially in areas where helminthiases are frequent. The modifications of the natural history of allergic diseases induced by helminth infections also impact their diagnosis and management.


Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration–response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α1B-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.The function of the α Polyethyleneimine was obtained from PolyScience . Nitrocellulose membranes were obtained from Bio-Rad , and SuperSignal West Pico Chemiluminescence kits were obtained from Thermo Fisher Scientific . Agarose-coupled protein A was obtained from Merck-Millipore, Darmstadt, Germany)). Antiphospho-ERK 1/2 (Thre202/Tyr204)  and anti-total ERK (p42/44) antibodies  were obtained from Cell Signaling Technology , and monoclonal anti-GFP was obtained from Clontech-Takara  , with polyclonal anti-GFP generated in our laboratory Pi. Labeled cells were stimulated as indicated, washed with ice-cold phosphate-buffered saline, and solubilized for 1 h in lysis buffer as described previously [Receptor phosphorylation was performed as previously described . In brieeviously . The extIntracellular calcium concentrations were assessed as previously described . Briefly1B-AR constructs were incubated for the times indicated, then fixed, and images were obtained. The plasma membrane was delineated utilizing differential interference contrast imaging. Each cell’s intracellular α1B-AR-eGFP fluorescence  was quantified as the “integrated density”, employing ImageJ software . The procedure is described in detail in “Corrected total cell fluorescence” ). Usually, 8–10 images of the indicated number of different cultures were taken for each condition. Bars in all microscopic images indicate 10 µm.Cells expressing the αnce”  for 2 h, followed by two washes with PBS (pH 7.4), and subjected to a second Western blotting using the anti-total ERK 1/2 antibody. No baseline phospho-ERK was detected; therefore, the maximal response for each αp < 0.05 was considered statistically significant.Data are presented as the mean ± standard error of the mean. Statistical analysis of comparable groups was performed using an ordinary one-way ANOVA with the Bonferroni post test. GraphPad Prism software (version 8.4.3) was employed for these analyses. A value of