Immunoglobulin E (IgE) is a well-known key factor in allergic airway disease; however, its central role in non-allergic airway inflammation is often underestimated. In some airway diseases, IgE is produced as a result of allergic sensitization. However, in others, IgE production occurs despite the lack of a specific allergen. Although multiple pathways contribute to the production of IgE in airway disease, it is its activity in mediating the inflammatory response that is associated with disease. Therefore, an understanding of IgE as the unifying component of upper and lower airway diseases has important implications for both diagnosis and treatment. Understanding the role of IgE in each upper and lower airway disease highlights its potential utility as a diagnostic marker and therapeutic target. Further classification of these diseases by whether they are IgE mediated or non–IgE mediated, rather than by the existence of an underlying allergic component, accounts for both systemic and localized IgE activity. Improvements in diagnostic methodologies and standardization of clinical practices with this classification in mind can help identify patients with IgE-mediated diseases. In doing so, this group of patients can receive optimal care through targeted anti-IgE therapeutics, which have already demonstrated efficacy across numerous IgE-mediated upper and lower airway diseases. Immunoglobulin E (IgE) is widely accepted as an integral component of the pathogenesis of many allergic respiratory diseases, with increasing recognition of involvement in the non-allergic forms of disease. This is evident in both upper airway diseases, including allergic rhinitis (AR), non-allergic rhinitis (NAR), and chronic rhinosinusitis with nasal polyps (CRSwNP), and lower airway diseases, including allergic asthma and non-allergic asthma phenotypes  .Staphylococcus aureus–derived enterotoxins have been proposed to act as superantigens in patients with NP or asthma to produce a local polyclonal IgE response [The shared aspects of otherwise discrete airway diseases have led to a continuous or “unified airway” concept for which pathological processes of the upper airway are thought to mirror lower airway events , 3. The response , 13.S. aureus enterotoxins [Historically, the term “allergy” was used to describe the organ-specific or systemic antigen-specific IgE-mediated immune response of the skin and mucosa in response to extrinsic allergen exposures . Howeverrotoxins . The undrotoxins . In thesrotoxins . Howeverrotoxins .Therefore, applying the learnings surrounding the role of IgE in upper and lower airway diseases in the context of both allergic and non-allergic phenotypes has important implications for treatment.Many diseases of the upper and lower respiratory systems share a common underlying mechanism in the type 2 inflammatory pathway. This pathway is mediated by several key cell types, including eosinophils, mast cells, basophils, Th2 cells, type 2 innate lymphoid cells (ILC2), and IgE-producing B cells. Elevated IgE is a hallmark of type 2 inflammation, where it plays a key effector role in the propagation of the immune response . For simEvolutionarily, IgE contributes to the body’s defense against parasites such as helminths , 19. HowIn a localized allergic response, after an initial allergen exposure, antigen-presenting dendritic cells sensitize naïve T cells, promoting their development into Th2 cells. These cells produce the inflammatory cytokines interleukin (IL)-4 and IL-13, leading to B cell activation and production of specific IgE  can also occur in the local tissue , 25, 27.In healthy individuals, the respiratory epithelium serves as a physical barrier to protect the upper and lower airways from environmental exposures, including viruses, bacteria, fungi, allergens, and contaminants . EpithelAdditionally, disruption of the epithelial barrier can allow the entry of bacterial proteins, which can act as superantigens, into the bronchial tissue, leading to a shift toward Th2-mediated inflammation, with increases in eosinophilia, IL-5, and IgE , 30. SupRegardless of whether IgE is produced through allergic sensitization, activation of the innate immune system, or by exposure to superantigens, these pathways are known to converge in the IgE-mediated inflammatory signaling cascade.IgE is fundamental in the type 2 inflammatory response through its interaction with its two receptors: the high-affinity IgE receptor (FcεRI) and the low-affinity IgE receptor (FcεRII), also referred to as CD23 . IgE hasSecreted specific IgE binds to FcεRI on mast cells and basophils, sensitizing them to specific allergens ahead of future exposures . SubsequFollowing these early events, the later release of cytokines and chemokines  from mast cells and basophils recruits inflammatory cells, including eosinophils, to the site of inflammation  implicated in upper and lower airway diseases, and several opportunities for positive feedback to enhance the inflammatory response. At the heart of this inflammatory process lies the key mediator: IgE. Understanding the role of IgE in the pathobiology of each disease unifies the upper and lower airways.As described, there are several causative factors of the type 2 inflammatory pathway -exacerbated respiratory disease (N-ERD), is a form of asthma defined by three key factors: severe asthma, recurrent and severe NP, and acute respiratory response to NSAIDs . HallmarAlthough many patients with AERD have comorbid allergy, AERD also often occurs in non-allergic hosts with elevated eosinophilic inflammation and is sometimes associated with elevated total serum IgE, indicating that a pathway distinct from the antigen-specific type 2 allergic response is likely involved . In one S. aureus superantigens is observed in the local nasal tissue of patients with AERD [S. aureus IgE to AERD, emphasizing the role of local IgE in disease [Similarly to other IgE-mediated airway diseases, the presence of specific IgE to ith AERD . It is sith AERD . This is disease .S. aureus and superantigen exposure, leading to increased Th2 cytokine production and inflammation and worsening disease severity [AR is defined as rhinitis or rhinoconjunctivitis where symptoms, including nasal congestion, nasal itch, rhinorrhea, and sneezing, present following exposure to an aeroallergen, most commonly dander, mold, pollen, or residues from cockroaches and dust mites, with evidence of specific IgE to that allergen detected by SPT or serum-specific IgE testing . In patiseverity .A subset of patients with rhinitis are classified as having NAR, accounting for roughly 25% of all patients with rhinitis . In thesA further classification of rhinitis, local allergic rhinitis (LAR), describes the onset of disease symptoms with a localized allergic response in the nasal mucosa similar to that of AR; however, diagnostic tests, including SPT or serum IgE, are negative . DespiteAsthma comprises a heterogeneous spectrum of lower airway diseases, rather than a single disease. Most commonly, asthma is defined by allergy status based on whether the clinical symptoms were precipitated by exposure to a common aeroallergen, as confirmed by a positive SPT or serum-specific IgE .It is widely accepted that in response to an allergen exposure, susceptible patients experience elevated local specific clonal IgE, which is believed to be the cause of allergic airway inflammation in asthma through activation of the type 2 inflammatory cascade leading to eosinophilia , 66. In There are a considerable number of classifications of non-allergic asthma, making it more difficult to diagnose and manage , 68. TheS. aureus enterotoxins [Despite this variability in non-allergic asthma subtypes, many of the underlying characteristics are similar to those in allergic asthma, with the same treatment approaches suggested for both . These srotoxins .Aspergillus, and mucus plugging. Inhalation of Aspergillus spores has been shown to cause ABPA in allergic individuals [Aspergillus-specific IgE and IgG [Additional diseases of the lower airway have been linked to IgE, namely allergic bronchopulmonary aspergillosis (ABPA) . ABPA isividuals . Repeateividuals . Exposur and IgG .The central effector role of IgE in the type 2 inflammatory pathway across various upper and lower airway diseases supports the utility of IgE as a diagnostic marker. Proper testing is required to determine whether a symptomatic individual’s serum versus local IgE levels are elevated in order to provide an accurate diagnosis, because these details may directly impact the categorization of disease .The current diagnostic paradigm for upper and lower airway disease focuses on first differentiating between allergic and non-allergic disease. Symptomatic patients are initially assessed for patient history, with follow-up diagnostic testing geared toward identifying the presence of allergy , 73 throAn SPT represents a fairly common, relatively inexpensive test that provides immediate results and often serves as the first choice in confirmatory allergic testing , 74. DesSerum testing identifies the level of specific IgE in the blood of patients to specific selected allergens, and can be seen as complementary to SPT , 78. OveFor patients in whom diagnostic uncertainty remains after routine methodologies are exhausted, more expansive testing, such as an immuno-solid-phase allergen chip (ISAC), may be performed. ISAC tests for specific IgE against multiple allergen components using a multiplex assay . As ISACTaken together, routine IgE testing methodologies come with their own advantages, yet they are also impacted by numerous practical limitations –79, 81. As described, the current diagnostic paradigm in patients presenting symptoms of upper or lower airway disease relies on the use of routine allergy tests, which examine circulating specific or total IgE to identify the presence of an allergic component of the disease. In many upper and lower airway diseases, however, it is local IgE activity that drives symptomology through the type 2 inflammatory pathway , 72, 73.After initial suspicion of asthma from patient history, routine allergic testing methodologies are often the first employed . HoweverTraditionally, the distinction between AR and NAR is based on an SPT and serum IgE analysis . HoweverS. aureus–specific IgE in the local NP tissue [In patients with CRSwNP, testing for specific and total IgE in the serum may predict asthma comorbidity and severity . HoweverP tissue , suggestP tissue .Together, these observations suggest that the role of IgE in some upper and lower airway diseases may be underestimated by initial testing of systemic total or specific IgE, further highlighting the need for diagnosis by a localized allergic response. In support of this, it has been suggested that in patients with a negative SPT or no evidence of elevated serum-specific IgE, additional testing should be utilized to identify patients with localized allergic responses , 87.The nasal allergen challenge has been used in combination with routine allergen tests to differentiate between patients with AR (positive SPT or serum-specific IgE), NAR , and LAR  . DespiteS. aureus–specific IgE, colonization with S. aureus, and tissue eosinophilia within the NP tissue of patients with CRSwNP [The most direct method for investigating the pathology of disease tissue, including accurate measurements of local IgE levels, involves the collection of tissue via biopsy . Indeed,h CRSwNP . Howeverh CRSwNP . TherefoOne technique used to sample local airway IgE is through the collection of nasal secretions by nasal lavage and absorption techniques, although sample collection from the lower airway via bronchosorption is more challenging . StudiesAnother study pioneered the use of an allergen microarray chip in assessing levels of local specific IgE to house dust mites (HDM) in the nasal secretions of patients with AR. Here, total and specific IgE levels to HDM were measured in the serum and nasal secretions of patients with confirmed allergic sensitization to HDM using ImmunoCAP fluorescent enzyme immunoassay and customized microarray assays to determine whether these measurements correlated. Overall findings in this study suggested good sensitivity using the microarray approach, superior to that of ImmunoCAP fluorescent enzyme immunoassay, in the detection of local specific IgE to HDM in nasal secretions of patients with AR. This suggests the utility of microarray technology as a non-invasive alternative to measuring IgE levels in the nasal secretions of patients with AR . FinallyThe basophil activation test serves as a useful ex vivo test of allergic response, in which basophils from whole blood or isolated peripheral blood mononuclear cells are exposed to allergens of interest, and the subsequent expression of basophil activation markers in response to exposure is measured by flow cytometry , 94. AltAlthough there are many available diagnostic tools to determine the role of IgE in disease, there is still room for improvement. Negative results on the most routinely used tests  do not conclusively indicate the lack of an allergic response, and neither do they identify local innate immunity. However, more localized tests can be invasive, not broadly applicable, and are limited by the lack of routine use in practice. As technology and standardization of practice continue to improve, so too will the diagnosis of IgE-mediated disease. In the meantime, the use of a treatment that addresses the common, underlying factor in these diseases can serve to improve patient outcomes.As previously described, the common underlying pathophysiology of many upper and lower airway diseases is suggestive of a continuous or “unified” airway , 3. An IIn upper airway disease, anti-IgE therapy for NP has shown significant improvements in both clinical and patient-reported outcomes . SimilarIn patients with lower airway disease, similar efficacy has been demonstrated in response to anti-IgE therapy. In patients with moderate-to-severe allergic asthma inadequately controlled by medium- to high-dose inhaled corticosteroids, anti-IgE therapy demonstrated a significant reduction in asthma exacerbations . InteresTaken together, the clinical efficacy of anti-IgE therapy across numerous airway diseases challenges the traditional allergen-centric definition of upper and lower airway disease. Rather than distinguishing between allergic/atopic versus non-allergic/non-atopic disease, it may be more appropriate to think of disease as responses driven by the type  and location  of IgE-mediated versus non–IgE-mediated disease Table .Table 1In defining disease by the role of IgE , physicians can diagnose based on local or systemic IgE involvement as the underlying cause of disease and effectively treat at the source. Prompt reduction in IgE levels can disrupt the cascade of inflammation, including eosinophilic involvement, by using a targeted therapeutic, regardless of allergic status. Because IgE mediates the inflammatory response underlying many upper and lower airway diseases, halting its activity quickly and completely may lead to efficient symptom reduction in patients . TargetiFor omalizumab, additional studies have investigated the complexities of treating patients with anti-IgE. Although serum total IgE levels are useful, they are not predictive of clinical response to omalizumab , 125–127There is considerable evidence supporting the role of IgE in many upper and lower airway diseases as the underlying cause of damaging inflammation independent of allergy , 33. FurThis diagnostic paradigm poses a particular problem in the treatment of diseases lacking an allergic component, because evidence has accumulated in support of the efficacy of anti-IgE therapeutics in treating both upper and lower airway disease, regardless of allergy status or total IgE levels. The routine testing for the presence of specific IgE to diagnose allergy status rather than assessing local IgE levels prevents physicians from identifying the disease for which local IgE causes symptomology and which will respond to anti-IgE therapy. Similarly, when utilizing anti-IgE therapy, evidence suggests clinical efficacy regardless of total IgE levels.Rather than distinguishing between allergic/atopic versus non-allergic/non-atopic disease, it may be more appropriate to think of disease as responses driven by the type  and location  of IgE-mediated versus non–IgE-mediated disease. When interpreting test results, it is important to consider the involvement and diagnosis of local as well as systemic IgE. Doing so would allow for the effective, targeted treatment of IgE-mediated upper and lower airway disease by addressing the root cause of the disease, regardless of allergic status.


In the past decade (2010–2019), the annual maximum typhoon storm surge (AMTSS) accounted for 46.6% of the total direct economic loss caused by marine disasters in Chinese mainland, but its prediction in advance is challenging. By analyzing records of 23 tide-gauge stations, we found that the AMTSSs in Shanghai, Zhejiang and Fujian show significant positive correlations with the El Niño-Southern Oscillation (ENSO). For the 1987–2016 period, the maximum correlation is achieved at Pingtan station, where correlation coefficient between the AMTSS and Niño-3.4 is 0.55. The AMTSS occurring in El Niño years are stronger than those in non-El Niño years by 9–35 cm in these areas. Further analysis suggests that a developing El Niño can greatly modulate the behaviors of Northwest Pacific typhoons. Strong typhoons tend to make landfall in southeast China with stronger intensities and northward shifted landfall positions. This study indicates that the modulation effect by ENSO may provide potential predictability for the AMTSS, which is useful for the early alert and reduction of storm surge damages. Passages of typhoons over coasts give rise to storm surges which constitute a major type of marine disasters in China . In 2019, for example, various types of marine disasters in China led to a total direct economic loss of 11.703 billion CNY, of which 11.638 billion CNY was by typhoon storm surges. Particularly, the storm surge caused by typhoon Lekima, the annual maximum typhoon storm surge (AMTSS) event of 2019, contributed by as much as 10.288 billion CNY, accounting for the majority of the total economic loss by all marine disasters. The total direct economic loss brought by marine disasters over the past decade (2010–2019) was 100.122 billion CNY, 46.6% of which was caused by the AMTSS events. Therefore, the local AMTSS largely represents the trial of marine disasters for coastal facilities and alert/prevention strategies. To reduce its damage, prediction with a long leading time is of paramount need to support the early warning and mitigation activities. However, the prediction of storm surge is a challenging task, given the complexities in typhoon behaviors and dynamical responses of coastal ocean to typhoons4. With these regards, the deterministic portion of the AMTSS variability associated with low-frequency climate modes, such as El Niño-Southern Oscillation (ENSO), represents potential predictability for the AMTSS and is worthy of in-depth investigation.The Northwest Pacific is the home to about one third of the tropical cyclones5. In El Niño years, the genesis area of typhoons migrates southeastward, rendering typhoons with a lengthened northwestward propagation pathway and more growth time prior to the landfall. Therefore, the overall typhoon landfall intensity tends to be stronger in El Niño years7 and exerts stronger influences in East Asian countries8. Wang and Liu9 suggested that the relationship between ENSO and typhoon activity is tight in the warm phase of the Pacific Decadal Oscillation (PDO) and degraded in the cold phase. Zhao and Wang2 showed that the influence of ENSO on typhoon frequency has become significantly stronger since 1998 than before, owing to increasing La Niña and central Pacific-type El Niño events.ENSO is the most influential interannual climate variability mode over the tropical Pacific and has significant impacts on typhoon activities in the Northwest Pacific, modulating frequency, intensity, and trajectory of typhoons in a dramatic manner11. The first step to test this hypothesis is to examine the statistical relationship between ENSO and storm surge using reliable observational records. For example, Colle et al.12 analyzed the storm surge events of New York City from 1959 to 2007 and detected dramatically more minor-surge events (0.6–1.0 m) under El Niño condition than La Niña condition. Based on model simulation results, Oey and Chou13 failed to identify an evident relationship between ENSO and the AMTSS along the coasts of Northwest Pacific. Feng et al.14 showed that the duration time of strong typhoon storm surge events (> 1 m) in some areas of China is closely associated with ENSO. In addition, there has been a huge body of literature reporting the strong forcing effects of ENSO on sea-level variabilities of the western tropical Pacific20, the South China Sea23, and the East China Sea24.Given the notable modulation effect on the Northwest Pacific typhoons by ENSO, we pose a hypothesis that the AMTSS in southeast China may exhibit interannual variability associated with ENSO, although influences of topography and coastline geometry have also been suggested to be essential25. This is particularly true for the AMTSS that can directly cause disastrous damages to people residing in islands and coastal regions.In spite of existing studies reviewed above, the relationship between the storm surge of southeast China and ENSO has not been clarified. A particular investigation is required to achieve reliable statistical results and gain insights into the underlying physical processes. This study utilizes the typhoon storm surge records from 23 tide-gauge stations located in the typhoon-prone areas of the southeast Chinese mainland to examine the relationship between the AMTSS with ENSO, and the underlying causes for the correlations are also explored. Studies on the impacts of ENSO on coastal sea levels are useful for strategy-making to mitigate the sea-level change consequencesThe hourly water-level data from 23 tide-gauge stations along the southeast Chinese mainland coast Fig. a are use26. This effect can be seen in Fig. We further compute the maximum AMTSS for El Niño years at each station Fig. d. All thBesides the AMTSS, we also analyzed the distributions of surges at each station based on all the typhoons, and the results are shown in Fig. r = 0.55 during 1987–2016 , Niño-3 and EMI (El Niño Modoki Index) indexes are computed . While for the EMI index, significant correlations with AMTSS were only found at 3 stations, i.e., Shipu, Kanmen and Dongshan. This may be due to the fact that all the EP-El Niño events  were strong and exert robust impacts on AMTSSs. By contrast, CP-El Niño events during 1987–2016 were weaker and their impacts on AAMTSs are limited, leading to degraded correlations between EMI and AMTSS. This result may be affected by data length which does not cover sufficient number of El Niño and surge events  based on all the typhoons during El Niño and CP-El Niño years, and the results are shown in Fig. Figure 7 which caused most of large storm surges in Chinese mainland at landfall (Bulletin of China Marine Disaster). The observed annual-mean landfall intensity of typhoons shows a correlation of r = 0.40 with the Niño-3.4 index  tend to be normal or below normal during El Niño years. Therefore, there are more strong typhoons making landfalls in Guangdong, Guangxi, and Hainan in non-El Niño years, which can enhance local AMTSSs therein. This may partly explain the prevailing negative correlations seen in these areas  in El Niño years tend to originate from the eastern portion of the Philippine Sea and therefore have a lengthened path over the warm ocean and more time to growth. As such, their landing positions are shifted northward (24.23°N on average), and their landing intensities tend to be enhanced, as compared with typhoons in non-El Niño years (22.68°N on average in landing position). These typhoon events cause stronger AMTSSs along Shanghai, Zhejiang, and Fujian coasts in El Niño years. By contrast, the majority of strong typhoons that made landfall in Guangdong, Guangxi, and Hainan, were generated in the South China Sea or near the Philippine coasts, thus they receive limited modulation effect by ENSO along their shortened propagation pathway toward China. As such, the correlations between the AMTSS and ENSO in these provinces are largely insignificant. In addition, Fig. These relationships are largely explained by the impact of ENSO on the intensities and landing positions of the Northwest Pacific typhoons that make landfalls in southeast China. Strong typhoons  and major-surge (> 1 m) events. The correlation coefficients between Niño-3.4 index and the annual numbers of the two types of surge events were provided in Fig. Our results show that there are generally more northward landfalls in El Niño years for all the WNP typhoons, but the difference is insignificant in statistical sense. Yet, the difference for strong typhoons is significant. To gain further insights, following Colle et al.This study postulates ENSO as a remote forcing regime for the AMTSS in the typhoon-prone coastal areas of Chinese mainland. Since useful prediction for ENSO can be achieved by several months ahead by the present generation of forecast systems, this can be rather useful for enhancing the probabilistic prediction of storm surges and provide a precious time window for warning, prevention, and mitigation steps. In other words, precursors of El Niño over the tropical Pacific may be used as predictors for strong AMTSSs in Shanghai, Zhejiang, and Fujian, and proper preparation can be carried out to reduce the losses caused by storm surges. We are also aware that ENSO is only one of the many processes modulating typhoons and storm surges. Extreme typhoons and storm surges can also take place in non-El Niño years see Fig. .26. So, in future research, carefully designed numerical simulation studies are required to clarify the key mechanisms of storm surge response to ENSO-modulated typhoon events. In addition, in this study, different impacts of EP and CP type El Niño on the storm surge were explored, and the conclusions are in accordance with the previous studies that both EP and CP-El Niño events can modulate the typhoon intensities and tracks39. The possible impacts of ENSO diversity on the northwest Pacific typhoons2 and China storm surges are also worth further in-depth investigation to improve our understanding for the linkage between coastal marine disasters and climate change/variability.This study describes the relationship between the AMTSS in southeast China and ENSO index and explores the underlying modulation processes. Yet both typhoon storm surge and ENSO are complex in dynamics, and more in-depth investigation is required to understand the impact of ENSO on China’s typhoon storm surges and incorporate into existing prediction systems. The AMTSS at Lvsi, Jiangsu province was not significantly affected by ENSO, although it locates near the high-correlation region Fig. a. This mThe hourly water-level data from 23 tide-gauge stations located at the typhoon-prone coastal areas of Chinese mainland was used to calculate the storm surge during typhoon processes. The spatial distribution of the tide-gauge stations and data duration are shown in Fig. 36. So, to investigate such impacts on the storm surge, we adopted the Niño 3 and EMI (El Niño Modoki Index) indexes to represent the EP and CP type El Niño events respectively. Niño 3 index is calculated by the average SST anomaly over the region . The SST data used for calculating Niño-3.4, ONI and Niño 3 was the Extended Reconstructed Sea Surface Temperature, Version 537. The EMI index is calculated by the method of Ashok et al.38 and can be obtained at Japan Agency for Marine-Earth Science and Technology.The typhoon data used in this study was obtained from the China Meteorological Administration-Shanghai Typhoon Institute (CMA_STI), which provides typhoon locations and intensities at 6-h intervals for the period of 1987–2016. The Niño-3.4 and ONI (Oceanic Niño Index) indexes were adopted to represent the overall ENSO variability and can be obtained from the NOAA  Physical Sciences Laboratory. Niño-3.4 index (ONI index) is calculated by the average (3 month running mean) sea surface temperature (SST) anomaly over the NINO34 region . As previous studies have shown that eastern Pacific (EP) and central Pacific (CP) type El Niño have different impacts on WNP typhoon activities such as the genesis locations and tracks2. Both student’s t-test and Wilcoxon–Mann–Whitney test30 were used to test the statistical significance of the results. The identification of the EP-El Niño and CP-El Niño years for the period of 1987–2016 adopts the results of Zhao and Wang (2019). 1987, 1997 and 2015 are identified as EP-El Niño years, 1991, 1994, 2002, 2003, 2004, 2006 and 2009 are identified as CP-El Niño years.Correlation coefficients of the AMTSS and ENSO index were calculated at the 23 tide-gauge stations to quantify the covariance between the typhoon storm surge and ENSO. The average ENSO indexes for the extended typhoon season of May–November is used to represent the ENSO condition of a year and compute the correlations with typhoon intensity and AMTSS


Mycobacterium tuberculosis complex (MTBC) from Sikkim, India. A total of 399 Acid Fast Bacilli sputum positive samples were cultured on Lőwenstein–Jensen media and genetic characterisation was done by spoligotyping and 24-loci MIRU-VNTR typing. Spoligotyping revealed the occurrence of 58 different spoligotypes. Beijing spoligotype was the most dominant type constituting 62.41% of the total isolates and was associated with Multiple Drug Resistance. Minimum Spanning tree analysis of 249 Beijing strains based on 24-loci MIRU-VNTR analysis identified 12 clonal complexes (Single Locus Variants). The principal component analysis was used to visualise possible grouping of MTBC isolates from Sikkim belonging to major spoligotypes using 24-MIRU VNTR profiles. Artificial intelligence-based machine learning (ML) methods such as Random Forests (RF), Support Vector Machines (SVM) and Artificial Neural Networks (ANN) were used to predict dominant spoligotypes of MTBC using MIRU-VNTR data. K-fold cross-validation and validation using unseen testing data set revealed high accuracy of ANN, RF, and SVM for predicting Beijing, CAS1_Delhi, and T1 Spoligotypes (93–99%). However, prediction using the external new validation data set revealed that the RF model was more accurate than SVM and ANN.In India, tuberculosis is an enormous public health problem. This study provides the first description of molecular diversity of the  Advances in molecular technology have helped us understand the genetic structure of Mycobacterium tuberculosis complex (MTBC) providing insights regarding the population dynamics and spread of MTBC locally and globally. The information obtained by molecular typing of MTBC isolates is essential for understanding TB epidemics and preventing TB4. Current studies also indicate that the outcome of TB infection may be related to strain diversity of MTBC6. For example, "Beijing strain" of MTBC has been reported to be more virulent in animal models and is often reported to be responsible for causing outbreaks8. Moreover, knowledge of the genetic diversity of MTBC is very useful for assessing the impact of the TB control program9. Although numerous studies on the genetic diversity of MTBC have been conducted in India41 yet no such types of studies are available from the hill state of Sikkim where the prevalence of MDR strains of MTBC is high42.In India, the burden of tuberculosis (TB) is enormous. According to the latest estimate of the World Health Organisation (WHO), the largest number of incident cases in 2018 were from India  accounting for 27% of global cases43. To better understand the genetic diversity of multidrug-resistant (MDR) and Non-MDR MTBC circulating in Sikkim, we characterized 399 MTBC isolates from Sikkim using spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem Repeats (MIRU-VNTR) typing. Spoligotyping is a PCR-based reverse-hybridization blotting technique based on polymorphisms in the presence or absence of "spacers" in the Direct Repeat (DR) locus of MTBC44. Typing of MTBC using spoligotyping led to the creation of the database "SpolDB4" in 2006 which gave the first overview into the global diversity and phylogeography of MTBC spoligotypes44. Subsequently, SITVIT web & SITVIT2 databases were created.Sikkim is a small hilly state in the North East region of India, adjacent to three neighbouring countries like China, Nepal, and Bhutan. India TB report showed a high incidence of TB cases48. Our study aimed to understand the genetic diversity of clinical isolates of MTBC from pulmonary tuberculosis cases from Sikkim a remote state in North-eastern India where the burden of tuberculosis is an emerging public health concern.A more robust genotyping method, namely 24-loci MIRU-VNTR typing is being used for the genotypic characterization of MTBC isolates. Numerous studies have shown that 24-loci MIRU-VNTR genetic markers have high discriminatory power, provide deep insight into MTBC Spoligotypes and sub-Spoligotypes and thus can be used as a very good alternative method for IS6110 restriction fragment length polymorphism (RFLP) which has numerous limitations49. However, due to technical difficulties and possibility of carryover contamination of the hybridization membrane, numerous researchers have developed alternative methods like mass spectrometry50, Luminex MagPlex magnetic microspheres51, multicolour melting curve analysis52 for carrying out spoligotyping. In this study we tried to predict dominant spoligotypes prevalent in Sikkim using 24-loci MIRU-VNTR profile using Artificial Intelligence.Spoligotyping of MTBC strains is widely used in the epidemiological studies on tuberculosisThe 399 MTBC isolates from Sikkim were found to be representing 58 different spoligotypes shown in Fig. In the present study, 362  isolates occurred in clusters. The Hunter Gaston Discriminatory Index of spoligotyping was low (HGDI = 0.5977) in MTBC of Sikkim  showed lower discriminatory power in Beijing MTBC isolates from Sikkim as compared to Non-Beijing isolates from Sikkim.Table The 399 MTBC isolates from Sikkim were found to represent 394 24-loci MIRU-VNTR profiles out of which 389 profiles were unique, i.e. each type is represented by only one MTBC isolate and 5 MIRU-VNTR types formed clusters and the clustering rate was 2.51%  i.e. MTBC isolates having similar MIRU-VNTR profiles but differ only at a single locus. Neighbor-Joining phylogenetic tree of 249 Beijing and 150 Non-Beijing MTBC isolates from Sikkim based on spoligotyping data and 24-MIRU-VNTR profile is also given  isolates were distributed in 12 Clonal Complexes (CCs), 10 isolates formed 5 identical clusters that are having identical MIRU-VNTR profiles, and the remaining 205 (82.3%) isolates were unique. The largest clonal complexes (CC1) and (CC2) include 9 and 3 isolates, respectively  were Multiple Drug Resistant (MDR) in contrast to 7 (4.7%) out of 150 Non-Beijing isolates. Beijing isolates had more than 8.6 times higher risk of being MDR  than Non-Beijing strains and this difference was statistically significant , SVM type was C-classification, Kernel type was Radical Basis Function. Hyperparameter optimisation revealed that best parameters for Epsilon was = 0 and Cost = 4.The results of SVM analysis are given as a confusion matrix showing predicted and observed Spoligotypes of Beijing, CAS1-Delhi & T1 Spoligotypes for training data set and testing data set  algorithm was first trained on a training data set (70% randomly selected data) to learn predictive patterns and subsequently applied to testing data set (30% data which was kept aside and not used for model training) for evaluation of classification accuracy, sensitivity and specificity. We used tenfold cross-validation for SVM and ANN models. The testing data sets are non-overlapping. Finally, k-ML models are generated. et Table . The senA multilayer perceptron network was used for ANN. K-fold cross-validation was also performed for ANN analysis. The input layer consisted of 24 factors (24-MIRU-VNTR types). The number of units includes 24 (excluding the bias unit). The number of hidden layers was 1 and the number of units in the hidden layer were7 (excluding the bias unit). The activation function used was Hyperbolic Tangent. The output layers included one dependent variable for Spoligotypes (Beijing or CASI Delhi or TI). The activation function used was SoftMax and the error function was cross-entropy.https://www.ibm.com/in-en/analytics/spss-statistics-software). The normalized importance values are computed by dividing importance values by the largest importance value and expressed as percentage.The result of tenfold cross-validation for predicting Beijing or CAS1-Delhi or T1 Spoligotype using Artificial Neural Network analysis for testing, training datasets are given in Table The optimal number of decision trees was found to be 3000 and optimal number of variables used at each split was found to be four. The out of bag (OOB) estimate of error of final tuned model based on training data set was 3.78%. The confusion matrix for training and testing data set is given in tables 7and 8, respectively.We used MTBC dataset from different region (Assam) for validating of ML models. All ML models used in the present study viz., RF, SVM and ANN models were trained to predict Beijing or CAS1-Delhi or T1 Spoligotypes using data from MTBC isolates obtained from Sikkim. However, to validate the performance of RF, SVM & ANN models new data set used was based on MTBC data generated from Assam. The accuracy, sensitivity and specificity of RF, SVM and ANN models against external new data set are given in Table 53.India has still the highest burden of TB despite intense national efforts to control and eliminate it . TB is difficult to control and eliminate in India probably due to its vast geographical and socio-economic diversity. Recent global studies have shown that high genotypic diversity of MTBC strains is an important factor in the pathogenesis of TB by affecting virulence, transmissibility, host response and the emergence of drug resistance31. Till date, only limited studies have been conducted on 24-loci MIRU-VNTR and spoligotyping based method to characterize MTBC strains in India31. Our study based on 24-loci MIRU-VNTR and spoligotyping of 399 MTBC isolates provides the first insight into the population structure of MTBC isolates from the hill state of Sikkim. According to this study the Beijing spoligotype was found to be the most dominant Spoligotype responsible for tuberculosis transmission in Sikkim, followed by CAS1_Delhi. The Delhi/CAS Spoligotype is effectively confined to India, Western Asia and Eastern Africa14. The Beijing strains, first described by Van Soolingen et al., in the Beijing area in 199554. The Beijing Spoligotype of MTBC is dominant in countries from Eastern Asia, Central Asia, Northern Asia and South-Eastern Asia although Beijing strains have also been reported from Austral Africa, Austral Asia, Southern Asia, Western Asia, North America, Central America, Northern Europe and Southern Europe. In India except NE region Beijing/Beijing-like strains of MTBC are less prevalent and their frequency ranges from 3 to 7%35. The Beijing strain of MTBC is more dominant in Sikkim, about 62.41% (present study) and 35.45% in Assam19. The dominance of Beijing genotype in Sikkim is a matter of great concern as this genotype has been associated with the high frequency of the drug resistance55; and treatment failure57. Moreover, the Beijing genotype is known to cause epidemic outbreaks in several countries because of their high adaptability and also this strain is considered to be less sensitive to BCG vaccination59. Our present study has revealed that Multiple Drug Resistant tuberculosis (MDR-TB) is more prevalent in Beijing strains 29.7% (n = 249), whereas in Non-Beijing strains of MTBC prevalence of MDR was 4.7% (n = 150) only. Multiple Drug Resistance thus appears to be associated with the Beijing strains in North Eastern region of India as it has been previously observed in other Southeast Asian countries like Vietnam, Thailand and also in South Africa64.Recent advances in MIRU-VNTR profiling and spoligotyping methods have provided powerful tools to determine various MTBC strains circulating in TB patients and to understand transmission dynamics of tuberculosis in a regionBeijing isolates in Sikkim indicates that more attention is needed to be given to the TB control program in this region to prevent the spreading of this dominating genotype in the community. Recent studies have shown that the modern Beijing strains of MTBC are spreading throughout the world because of their high degree of transmission potential65 and BCG vaccination has been found to favour the positive selection of Beijing strains66.The predominance of Beijing family strains, we also identified strains belonging to other families such as CAS1_Delhi (15.79%), T1 (5.76%), Orphan (4.26%), CAS (1.75%), T4 (1.50%), CAS2, H3, LAM6 (1.00%), H1, MANU2, UNKNOWN (0.75%), EAI7-BGD2, EAI5 T3, T5 (0.50%), The Less frequent strains belonged to EAI3-IND, LAM9, URAL-2, X1 and X2 (0.25%). MIRU-VNTR profiling (24-loci) was more discriminatory (HGDI = 0.9999) of genotyping method as compared to spoligotyping method (HGDI = 0.59).In addition to Beijing genotype. However, more elaborate longitudinal studies are needed to be undertaken in this region to understand the transmission dynamics of MTBC, and also to get an insight into the efficiency of the TB control program in Sikkim.In this study we tried to predict the main spoligotypes of MTBC in Sikkim, India using 24-loci MIRU-VNTR profiles. Two-dimensional scatterplot of MTBC isolates indicates that 24-loci MIRU-VNTR data can group MTBC isolates according to their spoligotype  and molecular characterization of MTBC isolates. Biosafety level 3 was used for culture and DST, and BSL level 2 facility was used for molecular experiments. Modified Petroff's method was used to decontaminate sputum samples, and all the samples were subjected to culture on solid LJ media at 37 °C for 6–8 weeks. The 67for all first-line anti-TB drugs like rifampicin (RIF), isoniazid (INH), streptomycin (STR), ethambutol (EMB) and pyrazinamide (PZA).DST was done using the proportion method68.DNA was extracted from fresh cultures by the cetyl-trimethyl ammonium bromide (CTAB) method69. using a commercially available kit . Briefly, the direct repeat (DR) region was amplified with primer pair Dra, 5′-GGTTTTGGGTCTGACGAC-3′ (biotinylated 5′ end) and DRb, 5′-CCGAGAGGGGACGGAAAC-3′. The DNA amplification was carried out in GENEAMPPCR system 9700 of Applied Biosystems. The amplified PCR products were hybridized with nitrocellulose membrane having covalently linked 43 spacer oligonucleotides following the standard procedure69. The hybridized fragments were detected using an enhanced chemiluminescence system  and subsequent exposure in X-ray film in darkroom70. The spoligotypes were initially reported as 43 digits binary representation of 43 spacers; one was scored for positive hybridization and zero for no hybridization.For the detection of presence or absence of 43 spacers was done on all isolates as described by Kamerbeek, et al46. The PCR products' fragment sizes were determined in the LAB CHIP  or agarose gel electrophoresis. The copy number of the tandem repeats was calculated as a function size of the PCR product and interpretation based on the reference table46. In doubtful cases, the experiment was repeated for confirmation. For quality control, Mycobacterium tuberculosis H37Rv and one Beijing strain were used in every batch of the experiment.MIRU-VNTR typing was performed by amplifying 24 hypervariable MIRU loci of all 399 isolates of MTBC from Sikkim. These 24 MIRU loci used for typing in this study are MIRU02, Mtub04, ETRC, MIRU04, MIRU40, MIRU10, MIRU16, Mtub21, MIRU20, QUB11b, ETRA, Mtub29, Mtub30, ETRB, MIRU23, MIRU24, MIRU26, MIRU27, MTUB34, MIRU31, Mtub39, QUb26, QUB4156 and MIRU39. The details of primer pairs and PCR reaction conditions are given by Supply et al., 2006plus (https://www.miru-vntrplus.org/) and SITVIT2 (http://www.pasteur-guadeloupe.fr:8081/SITVIT2/) were used for assignment of MTBC species, Spoligotypes, and genotypes by comparing with international reference database strains71. Spoligotypes were identified by a similarity search in MIRU-VNTRplus and SITVIT2. As on 3rd April 2020, the SITVIT2 database contains 1,11,635 entries from 177 countries. In this database, the spoligotypes are designated as Spoligotype International Type (SIT) if isolates share them from two or more patients, and if a spoligotype is from a single patient, it is designated as orphan70.Web tools MIRU-VNTRBeijing strains (n = 249) and Non-Beijing strains (n = 150) to determine their Clonal Complexes (CC). We allowed single-locus variants (SLVs) to be included in clonal complexes and identical patterns of MIRU-VNTR. Clonal Complexes identified genetically closely similar strains sharing common transmission link72. Unknown spoligotypes/orphan strains were subjected to phylogenetic tree analysis using the Neighbour Joining (NJ) tree and categorical coefficient to predict these isolates' Spoligotype. The discriminatory power of spoligotyping and MIRU-VNTR typing system was calculated using the Hunter Gaston Discriminatory Index (HGDI)73.Phylogenetic genetic analysis of 399 isolates and the international reference strains was done using Neighbour joining (NJ) tree method based on combined analysis of spoligotypes & 24-MIRU-VNTRs implemented by MIRU-VNTRplus web tool. A Minimum Spanning Tree (MST) using 24-loci MIRU-VNTR dataset was also constructed for N = the total number of strains in the sample population, S is the total number of types described, and the NJ tree is the number of strains belonging to the jth type.Where RStudio version 1.3.1093 and R version 4.0.3 was used for Principal components analysis (PCA). Built in R function ‘prcom’ and libraries ‘devtools’, ‘ggplot2′,‘plyr’, ‘scales’ and ‘grid’ were also used. First two PCs were used for plotting MTBC isolates (n = 335) in 2-D scatter-plot to get an idea if these MTBC strains tended to group according to their spoligotype . The MTBC isolates were colour coded by Spoligotype type to visualize possible clustering in three groups.Target input variable was categorical representing three classes of MTBC Spoligotypes viz., Beijing, CAS1_Delhi and T1 and was encoded as 1, 2 or 3 representing three classes. Independent input variables were 24 in number, and all were numeric. Numeric variables were normalized to have values ranging between 0 and 1. No data was missing.79 were used to predict dominant spoligotypes of MTBC using MIRU-VNTR data. Random forest is a robust supervised classification/regression machine learning technique. It is an ensemble classifier based on constructing 100 s of decision trees at training time. The bootstrap samples are used to grow numerous decision trees, and a random sample of independent predictors are used at each node. We used two parameters in RF for tuning viz., ‘ntree’ number of trees to grow and ‘mtry” number of variables to select at a node split. RF model is robust and does not overfit the training data. RF analysis was conducted using R package (V 4.0.3). Libraries ‘caret’, ‘lattice’ & ‘ggplot2′ were used for RF analysis.In the present study, artificial intelligence-based machine learning methods such as random forests, support vector machines and artificial neural networksk-fold cross-validation (10-fold for Beijing or CAS1-Delhi and T1 MTBC Spoligotype) to test the performance of the model. The single dependent variable was categorical (‘1’ for Beijing ‘2’ for CAS1/Delhi and ‘3’ for T1) and 24 independent variables (24-loci MIRU-VNTR profile) were numeric. We used ‘caret’ and ‘e1071’ libraries in R for SVM analysis. We tested ‘radial’, ‘linear’, polynomial’ and ‘sigmoid’ kernel functions to determine best function suitable for classifying three dominant MTBC Spoligotypes. The optimal value for ‘Epsilon’ and ‘Cost’ were determined using ‘tune’ library. Artificial Neural Networks (ANN) are currently popular and powerful machine learning tools that are biologically inspired computational models that imitate brain neurons and solve complex problems. The ANN typically consists of the three-layered network  consisting of artificial neurons or nodes and interconnected by connections (synaptic weights). ANN require training data  for model building. The dependant and independent variables are given as input (training phase) that information will be used for the system to learn using the back-propagation learning algorithm to predict outputs. The Multilayer Perceptron (MLP) was used to build ANN. K-fold cross-validation was also used for the evaluation of ANN. We used SPSS v26 for ANN analysis. SPSS software has the provision to manually choose parameters such as number of hidden layers, number of units in hidden layers, activation functions (hyperbolic tangent or sigmoid), and output layer activation functions like identity, SoftMax, hyperbolic tangent & sigmoid. Instead, we opted for automatic architecture selection to select optimal parameters with a number of hidden layers one to fifty. The accuracy of the ANN model was best as revealed by ROC analysis.Support Vector Machines (SVM) are advanced nonparametric machine learning data mining techniques based on supervised and kernel-based methods. SVM is used for classification, prediction and regression problems. Learning in the SVMs is achieved by finding an optimal linear hyperplane using appropriate kernel functions, maximizing the margin between the classes. The classification can be binary or multiclass. Prediction of spoligotype using 24-loci MIRU-VNTR profile of MTBC is an example of the multiclass classification task. In the present study, the data set of 335 MTBC isolates from Sikkim having information on three dominant spoligotypes and 24-loci MIRU-VNTR profiles. We used Sensitivity, specificity, and accuracy were calculated to measure the performance of SVM and ANN predictions. Sensitivity was measured by the formula TP/(TP + FN), specificity was measured as TN/(FP + TN), and accuracy by (TP + TN)/(TP+TN+FP+FN) where TP, TN, FN and FP represent true positive, true negative, false negative and false positive, respectively. The Receiver Operating Characteristic Curve (ROC) analysis was also used to determine ANN classifiers' performance, where x-axis represents 1-specificity and the y-axis represents sensitivity and the value ranges between 0.0 and 1.0.For external validation, 132 isolates of MTBC collected from different geographical areas (the state of Assam) were also processed for spoligotyping and 24-loci MIRU-VNTR typing. To check for overfitting, we used blind external data (obtained from MTBC isolates from Assam) to evaluate model performance of all ML methods i.e. RF, SVM and ANN.Excel 2016 of Microsoft office was used for calculations related to performance measure of RF, SVM and ANN classifiers. This was done by generating confusion matrices generated for training, testing and external new data sets.Binary logistic regression analysis was used to find the association between multiple drug resistance (MDR) status and MTBC Spoligotype. The dependant variable used was MDR status of MTBC isolate and the independent variable used was whether the MTBC isolate belonged to Beijing or non-Beijing Spoligotype. The Wald test was used to find statistical significance of Independent variable. The strength of the Association between MDR and Spoligotype was determined using the odds ratio and 95% confidence interval of the odds ratio.This study was approved by the Ethical Committee of ICMR-Regional Medical Research Centre, North-East Region, Dibrugarh. All processes were performed in accordance with the related regulations and guidelines. Written informed consent was obtained from all the participants or their guardians in the case of minors who provided sputum samples. Patients found positive for AFB were referred to the nearest DOTS centre for treatment.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.


Tenebrio molitor mixed with a seed  and a pseudocereal (Chenopodium quinoa) to strawberry–cranberry ice cream. The objective was to increase micronutrients, macronutrients, and antioxidants, thus rendering the product a food complement. Four ice cream formulations were manufactured: the control strawberry–cranberry ice cream and three experimental mixtures, one of them with an addition of Tenebrio larvae (HT) and two others with a combination of Tenebrio larvae, chia (HTC), and quinoa (HTQ). The ice creams were submitted to proximate chemical analysis: mineral, fatty acid, vitamin, and one antioxidant (cyanidin 3 glucoside) determination. The strawberry–cranberry ice cream was used as a control formulation to evaluate if there were significant differences among nutrients, to which a Dunnett test with a critical value of α = 0.05% was applied. The three formulations that were studied showed a significant increase in the analyzed micronutrients and macronutrients compared to the control formulation. We observed increases of up to 62% in lipid content in the HTC formulation, while an increase of 41% in the protein content of the HT formulation was observed. We quantified an increase and enrichment of vitamins and minerals in the manufactured products, so that their nutritional value was significantly enhanced. In the determination of cyanidin 3 glucoside, we found that the formulation to which chia had been added showed an increase of 74% as compared to the control ice cream; this is important because anthocyanins are a group of flavonoids that stand out for their antioxidant and antimutagenic capabilities.Functional ice creams were developed by adding larvae of the insect  Nutrition relies on different factors, such as place of residence, surrounding habitat, available alimentary resources, religion, ethnic group, education, migration, colonization, trade, and the ways in which food is cooked or preserved and stored, which have their origins in ethnic footprints . BesidesIn this context, the search for functional foodstuffs appears to be urgent. Bello  defines Tenebrio molitor, the mealworm, as the protein ingredient of interest, in which the presence of good-quality nutrients has been demonstrated  and phenylalanine + tyrosine (5.21 g/100 g protein). Overall, insects contain higher amounts of lysine and threonine, but lower amounts of methionine/cysteine .T. molitor larvae, adults, exuviae, and excreta, respectively. The mealworm larvae presented an average fat content of 32.7%, a quantity that is greater than that of locusts (21.5%) and grasshoppers (3.8%) but less than that of termites (61.1%)  .In fatty acid composition, they have a high component of oleic acid (C18:1), linoleic acid (C18:2), and palmitic acid (C16) in larvae, with values of 43.17, 30.23, and 16.72%, respectively. Also, the amount of ω-6 was determined to be significant.These essential fatty acids demonstrate that insects can be used for many other purposes such as feeding of domestic animals and as food supplements for human beings. Besides, it has been reported that insects' caloric value is 50% higher than that of soybeans, corn, and beef . ConsideFurthermore, larvae have among their fats lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid .In mineral composition, as in other insects, mealworm contained poor sources of calcium which were 434.59, 484.39, 801.14, and 1,537.97 mg/kg for larvae, adult, exuviae, and excreta, respectively. Even when the calcium content is considerably low in mealworm, it contains instead high levels of P .Therefore, in terms of nutritional content, edible insects  have been stated to have more of it when compared to other conventional foods.There will be a huge economical change involved if insects become more commonly considered as an acceptable food source for both human beings and domestic livestock in industrial countries .Insects have potentially been used as a human nutrient source in traditional food among indigenous people throughout history . InsectsThe production and innovation of food based on insects have become a trend in recent years, and in many parts of the world, it is something completely normal; such is the case in Germany, Belgium, Brussels, Costa Rica, Spain, and Holland, among others. In the Netherlands and China, insects are used as food ingredients and are progressively viewed as an option for meat .Insects have an excellent nutritional value, and their flavor is also fabulous; that is to say, they are even a delicacy; mealworm is widely used in various types of foods, for example, with the larvae, ice creams were prepared at the insect experience festival in Wageningen, The Netherlands .Tenebrio, van Huis et al.   ; and min−1) .T. molitor has been used as a source of protein with different alimentary purposes throughout the world  the purchase of the ingredients for the ice cream foundation: water , pasteurized cow's milk cream, powdered whole milk , sugar , gums  , and the raw materials to provide it with flavor and functionality ; the ice cream foundation was then left to mature for 24 h at 4°C; and (b) the selection and cleansing of the insect larvae: these were obtained from the Institute of Biology, UNAM, National Autonomous University of Mexico, where they are cultured in controlled conditions at a temperature of 26–27°C and a relative humidity of 60%, being fed on wheat bran for a period of 90 days; the larvae were dipped in boiling water for 5 min—this was done in order to avoid them changing color during the procedure—and afterwards they were stored in freezers at −4°C; and (c) quinoa's preparation: seed conditioning achieved by boiling it for 10 min and drying it in a stove at 80°C for 6 h.T. molitor larvae; HTC, larvae mixed with chia; and HTQ, larvae mixed with quinoa. These were manufactured according to the specifications contained in NMX-F-714-COFOCALEC-2012  and the conventional ice cream (control ice cream) whose basic formulation was used to prepare all the ice creams, as well as to verify if there was an increase in the nutrients when these were assessed by means of diverse types of chemical analysis.Tenebrio larvae ingredients were done with respect to the control formulation to observe how the nutrients varied and to verify that the objective of the investigation was achieved.The substitution and inclusion of the seed (chia), pseudocereal (quinoa), and In this case, we considered the amount of matured ice cream base  and the adjustment of solids with respect to the NMX-F-714-COFOCALEC-2014 standard to consider it milk ice cream.T. molitor were in fact analyzed.In the third phase, we carried out the chemical analyses to elicit the chemical composition of each of the ice creams: (a) proximate chemical analyses , (b) liposoluble and hydrosoluble vitamin determination, (c) fatty acid quantification, and (d) assessment of the antioxidant known as cyanidin chloride. Strawberries, cranberries, chia, and quinoa were not analyzed; nevertheless, for chia and quinoa, we used the data from several reliable bibliographic sources published in the United States or even by the FAO . The larThe general process of the ice cream manufacture was as follows. The ingredients were weighed, mixed, and pasteurized at 68°C for 30 min. The addition of the strawberry–cranberry pulp, seeds, and larvae compromised the aging of the foundation at 4°C/24 h, which was then whipped for 10 min and frozen at −4°C.To carry out chemical determinations, official published and standardized methods were used: for humidity , we deteThe methods employed for the quantification of micronutrients in the manufactured formulations were the following. The mineral profile was quantified by the AOAC 985.3 method that assesses minerals in ready-to-feed milk-based infant formulations by atomic absorption spectrophotometry . For the fatty acid profile, we applied the gas chromatography method GC-FID  (AOAC 969.33).To assess the liposoluble vitamin profile, the method of Chotyakul et al.  was usedTo quantify hydrosoluble vitamins, we used the method from Gliszczynska and Rybicka  and RokaFor the mineral profile, we used AOAC 985.35. And for cyanidin quantification, we applied a modified method from Martínez-Cruz et al. ; we inteTo evaluate if there were significant statistical differences between the nutrients thus determined, we carried out a one-factor analysis of variance (ANOVA) between the four formulations, as well as a Dunnett test to identify which macronutrients or micronutrients show variations with respect to the control ice cream. The Dunnett test is used to create confidence intervals between the mean of each factor level and the mean of the control group. In such a way, this test compares each formulation finding if there are any or no differences between each of them, but also with respect to the control with a confidence level of 0.05 .We must initially point out that all the results presented in our tables are the mean obtained from three repetitions (that mean had a standard deviation and a variation coefficient or error lower than 5%) and that the values in the rows with different letters signal that between them there exists a significant difference; only the values of carbohydrates were obtained by difference, and in each table, the highest quantified values are indicated in bold.According to NMX-F-714-COFOCALEC-2012 , the proTenebrio. In the same table, reducing carbohydrates or reducing sugars are defined as sugars that can be oxidized by weak oxidizing agents; for example, all monosaccharides are reducing sugars.We observed significant differences in composition between the control ice cream and the functional formulations: these are due to the addition of chia, quinoa, and the insect larvae. In this case, the fiber content corresponds to the formulations of elaborate ice cream, that is, to the total mixture of all the ingredients. Consequently, it does not refer to The experimental samples  have significant differences in their lipid contents that enhance their nutritional value and consistency, with the HTC formulation being the one that shows the highest proportion by the addition of such ingredients. Ramos-Elorduy et al.  mentioneAs When carrying out the study, the lipids quantified by gas chromatography are classified as saturated or unsaturated, and these are simultaneously reclassified into monounsaturated and polyunsaturated; they are added, and these are the values reported in T. molitor, chia, and quinoa—being ingredients with high levels of unsaturated fatty acids—diminished the percentage of the saturated ones. The predominant fatty acids in the ice creams were palmitic acid, which varied between 29 and 32% and was the most abundant fatty acid in the HT and HTC formulations, and oleic acid, which ranged from 22 to 25% and was the only unsaturated acid present in important quantities in the experimental formulations.In The latter is due to the fact that the foundation to manufacture the ice cream is made up of dairy products in which saturated fats make up 70% of the total fat weight, with palmitic acid being the most common  in the rThe adequate proportion of these fatty acids, ω-6/ω-3, can be considered as another determining factor of the high quality and nutritional value maintained in the investigated formulations ; for exaThis is a macronutrient, and the ice cream with added chia and quinoa showed significant change with respect to HCOo. Nevertheless, the addition of larvae to the formulations had a positive impact, as it increased its content due to the fact that larvae have a high protein content (58%). When combining larvae with the seed or pseudocereal, a decrease was seen in the protein percentage, as these last two ingredients provided a higher quantity of dietary fiber, fat, and/or carbohydrates. All of the manufactured ice creams  had a greater protein content than commercial ice cream. Our manufactured ice cream provided 7% to 10% of the protein required daily, so it could be considered a good dietary complement.The addition of larvae and its combination with the seed and pseudocereal diminished the proportion of carbohydrates; HTQ ice cream was the exception. This harbored the greatest quantities of carbohydrates even in comparison to HCOo, which is due to the fact that quinoa has a proportion of starch of 53.1% .In dry basis analyses, it was observed that only the HT formulation had significant differences compared to the control, as these sugars found in the ice cream derive from strawberries, cranberries, and milk.As Bunaeopsis aurantiaca and Imbrasia oyemensis are rich in calories; B. aurantiaca, I. oyemensis, and Rhynchophorus phoenicis have similar protein amounts to beef; Cirina forda, wheat flour, and the different formulations of ice cream are high in nitrogen-free extract, and with respect to ash, the highest amount corresponds to C. forda.In R. phoenicis and wheat flour are enriched in proteins by 130%, since wheat flour only has 10.89. In addition, there are published data that point out that the nutritional value of mealworm is comparable to that of beef . HTQ ice cream was the one that had the highest Fe concentration; this is because quinoa has 1,416.7 mg Fe/100 g upon a dry basis. However, its content does not reach a whole milligram in the final product; that is, ice cream provides 25% of the RDI according to NOM-051-SCFI/SSA1-2010, 17 mg. Therefore, we recommend the consumption of iron-rich foodstuffs to comply with this requirement.In this case, it was observed that all of the formulations had an increase due to the addition of the ingredients. Nonetheless, the highest value was found in the HTC formulation, due to the fact that this vitamin is the most abundant in the seed with 44 μg/100 g . That isAccording to the FAO , quinoa In this research, the highest concentrations were found in the ice cream to which chia had been added (HTC). Nevertheless, for the ice cream added with quinoa, a high content of vitamin D was also obtained. The NOM-051-SCFI/SSA1-2010 indicates an RDI of 5.6 μg; as can be seen, all of the ice creams surpassed this figure, but the occasional consumption of these ice creams would not affect consumers' health.It was quantified in a high proportion in the formulations, as they showed a higher quantity than the control ice cream. Nonetheless, when only larvae were added, the increase of vitamin E was minimal, which is due to the fact that larvae have very low concentrations of this vitamin (1.9 mg/100 g) as compared to chia and quinoa . On the Quinoa is also an abundant source of vitamin E, whose concentration ranges from 3.7 to 6.0 mg/100 g; however, when the pseudocereal is mixed with the larvae, vitamin E concentration diminishes because—as both ingredients have low levels of tocopherols—the combination of both dilutes them. The NOM-051-SCFI/SSA1-2010 points out that according to the RDI of vitamin E, one must consume 11 mg of it. As In In All of the experimental formulations showed a significant increase. However, HTC is the one that provides the highest quantity of this vitamin, because, as Muñoz et al.  have repThe results in Niacin was the vitamin that had the highest concentrations in the ice cream, even in the control formulation, since milk contains tryptophan—which is considered niacin's precursor—in a significant quantity: 7 mg/g protein. According to In all of the experimental formulations, significant increases were obtained when compared with HCOo. The fortification of this vitamin in the ice creams can be appreciated, as when an extra ingredient or their combination is incorporated, an increase of up to 87% is observed. As −1), there was a decrease as compared to that of the HCOo; that is, the larvae favored only a dilution of this vitamin in the formulations. Unfortunately, there are no data about the quantity of this vitamin in these ingredients that would enable us to draw a quantitative comparison. Notwithstanding this situation, the quantities of B12 in the ice creams effectively cover the RDI of 2.1 μg (0.0021 mg) that the NOM-051-SCFI/SSA1-2010 recommends.In In this case, the nutritional value of the ice creams prepared with insect addition was improved in macronutrients and micronutrients, as compared to the control batch. Therefore, in this research, we focus on the potential application of ice creams with diverse functional properties.The results shown with the experimental batches of ice cream are an important contribution in the elaboration of nutritious and functional foods in the future .Clanis bilineata tsingtauica , T. molitor Linnaeus, 1758, Acheta domesticus , and Apis mellifera; they were compared with beef, pork, and chicken. Details of the methods are found in Payne et al.  have been used, as well as non-parametric tests and Bonferroni adjustments to verify nutritional differences. Of the available insects studied, the ones that are commercially important are e et al. .According to the Ofcom model, insects are not healthier than meat products, and with the results of the NVS method, crickets and yellow worms are significantly healthier than beef and chicken.In this publication, it is reported that crickets and bees are richer in iron compared to beef, pork, and chicken and higher in calcium and riboflavin than meat and meat offal, but there is no indication that the insects investigated are statistically healthier than meat. However, their results are not conclusive at this time and should therefore be treated with reserve.In any case, the nutritional quality of the insects studied is “relatively” unimportant since in the daily diet through the use of mixed diets, the phenomenon of complementation/supplementation between different proteins occurs. Food supplementation is a concept used to design diets or foods in which different sources of protein are mixed in order to improve the quality of the resulting combination.The three formulations—HT, HTC, and HTQ—showed a noteworthy augmentation in the previously examined macronutrients and micronutrients in comparison to the control formulation. After a thorough examination, it could be observed that the HTC formulation has proven to be the best one due to containing the upmost values of essential nutrients such as protein, fat, dietary fiber, ash, minerals, cyanidin, and vitamins. According to the formulations of the ice creams made, the amount of protein increased by 41%; that is, these larvae are ideal to enrich various types of conventional foods such as ice cream, and this will be a success in the future. Likewise, ice creams improved significantly in fatty acids, vitamins , and minerals ; therefore, the larvae have a high potential to supplement conventional foods.To sum up, we can conclude by saying that the importance of creating new formulations of functional food ice cream lies not only in its nutritional value but also in the functional macronutrients and micronutrients, which, all in all, effectively prevent any sort of disease.T. molitor larvae such as ice cream are a novel and emerging food industry. Therefore, it may be developed as a sustainable source of alternative resources for human consumption and widely used in many other investigations such as animal feeding and the elaboration of some functional foods.For the aforementioned reasons, the food industry is looking for alternative nutritional sources such as insects, for the elaboration of products; such is the case of the nutraceutical ice creams analyzed and the protein enrichment of various conventional foods, among which we have “jungle bars,” cookies, tortillas, cakes, chocolates, and sausages. At present, various foods made with The benefits of using insects as human food are diverse; moreover, based upon this premise, we can claim that insects have a high potential of becoming a new sector in the constantly innovative food industry of Mexico.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Glutathione-S transferases (GSTs) are part of a ubiquitous family of dimeric proteins that participate in detoxification reactions. It has been demonstrated that various GSTs induce allergic reactions in humans: those originating from house dust mites (HDM), cockroaches, and helminths being the best characterized. Evaluation of their allergenic activity suggests that they have a clinical impact. GST allergens belong to different classes: mu , sigma (Bla g 5 and Asc s 13), or delta (Per a 5). Also, IgE-binding molecules belonging to the pi-class have been discovered in helminths, but they are not officially recognized as allergens. In this review, we describe some aspects of the biology of GST, analyze their allergenic activity, and explore the structural aspects and clinical impact of their cross-reactivity. Glutathione-S transferases (GSTs) are a part of a ubiquitous family of dimeric proteins that have triggered the interest of different biology-related disciplines due to their involvement in drug resistance, carcinogenesis, and allergy. Most of these isoenzymes participate in detoxification reactions, coupling reduced glutathione (GSH) to xenobiotics and/or endogenous substances, such as bilirubin and steroids. They have allergological importance for multiple reasons: several of them are epidemiologically relevant as environmental sensitizers and due to their sequence and structural conservation even among phylogenetically distant species, they may mediate cross-reactivity among invertebrate allergen sources, such as cockroach, house dust mites (HDM), and helminths. Also, probably because of their abundance and immunogenicity, some helminth GSTs have been evaluated in pre-clinical studies and clinical trials as vaccines to prevent helminthiasis , 2. AltoAscaridia galli with homology to a GSH-dependent prostaglandin-H D-isomerase and high activity and specificity in the GSH-dependent isomerization of prostaglandin H to prostaglandin E, a lipid mediator associated with suppression of host immunity  and were annotated as mu-like GSTs. Characterization of their biochemical features by ProtParam indicated that their pIs also ranged from five to nine as the results previously observed at the proteomic level  RNA expression in bone marrow-derived dendritic cells at even higher levels than Der f 1 or Der f 2  in UniProt, and two reported IgE-binding frequencies of 10 and 80% in Singapore and Colombia, respectively , 58. Them is low . In summm is low , 60 and,m is low , 62.Tyrophagus putrescentiae by affinity chromatography. It is a 26 kDa protein sharing 83% of amino acid sequence identity with Der p 8. IgE seroprevalence against Tyr p 8 was 45.3% (48/106) among Taiwanese allergic patients who were sensitized to the mite extract. Pre-adsorption of all sera with D. pteronyssinus extract reduced the rate of IgE recognition to 18%, which suggests cross-reactivity among GSTs from these sources. There is evidence of basophil histamine release sensitized with sera from Tyr p 8-allergic patients, confirming its allergenic activity  ethane and 4-hydroxynonenal were used as substrates, but its IgE-binding frequency was lower (17.9%) compared to the sigma class (20.5%) in cockroach-sensitized patients from Seoul, Korea. There is no evidence of its allergenic activity .B. germanica allergens are found in low-income dwellings, as detected with Bla g 2 in the United States  of IgE reactivity to this isoform. Both recombinant versions induced basophil activation as measured by the CD63-based flow cytometry assay  can induce IgE antibodies in infected individuals  purified by Acevedo et al. contained six isoforms that bind IgE  conducted in a deprived community living in Cartagena, Colombia, indicated that ~20% of children were sensitized to rAsc l 13 at a young age (6 months old), increasing to 45% at 3 years of age. A positive IgE response to Ascaris GST was associated with housing features related to poor hygienic conditions and Ascaris infection . Also, aA. lumbricoides had IgE against GSTO with a similar frequency to rAsc l 13  has only 90.3% of identity to rGSTO.To characterize other allergenic isoforms of Ascaris ssp. in our laboratory, we identified the GST genes predicted by the Davis laboratory at the University of Colorado and expressed them as recombinant proteins. We have analyzed the enzymatic activity and humoral response of an omega-class isoform. The recombinant GST omega-class (rGSTO) has dehydroascorbate reductase and thiol transferase activity reported as well for other omega-class GSTs . More thAsc l 13 . DespiteA. lumbricoides using a strain from the Republic of Ecuador. Based on this work, we identified 15 GST sequences of A. lumbricoides reported in the UniProt database. The sequence and tertiary structure were verified to confirm that these proteins belonged to this protein superfamily. Most predicted products from these sequences had features of the cytosolic subfamily, as the thioredoxin-like fold domain and the GST N-terminal and C-terminal domains. There was only one sequence identified as kappa subfamily GSTs, due to the presence of the DSBA-like thioredoxin domain, and one as microsomal subfamily GST  of A. lumbricoides did not have a similar sequence in A. suum and two sequences in A. suum (F1LBW9 and F1LCY4) were not identified in A. lumbricoides. The A. suum kappa-GST (F1LDD6) had an identity of 68.3% with an A. lumbricoides DSBA domain-containing protein with a match length of 182 of 254 amino acids.In 2018, the Parasite Genomic group at the Wellcome Trust Sanger Institute made the draft genome assembly and gene predictions of mily GST . In A. s in 2018 , we iden classes . At the  classes . It is gSeveral investigations have pointed out different parasite GSTs as immunomodulatory proteins, including molecules derived from helminths and protists . The parF. hepatica have been found over mouse BMDCs, reducing their inflammatory response to LPS and inhibiting its capacity to induce Th17 polarization , which is majorly comprised of mu-class GST isoforms (96%), in a septic shock mouse model. It was observed that nFhGST significantly suppressed the LPS-induced TNFα and IL1β in vitro production by macrophages and the pro-inflammatory cytokine/chemokine storm within C57BL/6 mice elicited by lethal doses of LPS (Immunomodulatory properties of sigma-GST (fhGST-si) from rization . Stimulas of LPS .Schistosoma haematobium has shown to induce a strong mucosal immune response, associated with IL-10 and Th2 cytokine production in animal models and humans  in experimental colitis. It was also found to induce Th2 polarization, characterized by local and systemic production of IL-13 and IL-5. An eosinophilic infiltration in colonic tissue was evidenced in GST-immunized mice, and eosinophil depletion was associated with a loss of the ameliorating effects observed for Sh28GST , whose humoral and cytokine response to Sh28GST-Alum was investigated, it was observed that immunization induced IgG1, IgG2, IgG3, and IgG4 antibodies, in contrast to IgE whose titers did not significantly raise in comparison with baseline. Production of IL-5 and IL-13 was significantly induced after two administrations of rSh28GST in six out of eight vaccinated adults  in individual sera collected from cockroach-allergic patients. Modeling and sequence analysis indicated that both molecules may share a lineal epitope in their N-terminal domain ; although with a similar global structure, determined by key conserved amino acids and positions, there is low to moderate sequence conservation among them .B. germanica, observed in nature, is needed. Defining the allergenic activity will provide better information about the clinical impact of these IgE-binding molecules. In addition, extending the studies about their mechanisms of action for inducing allergic inflammation is necessary to improve our knowledge of allergy pathophysiology and to discover other options of allergy treatment. Their biological activity may be involved in Th2 polarization, which makes it interesting to explore other non-IgE mediated mechanisms that promote allergic responses (Several IgE-binding GSTs have been evaluated for their allergenic activity, being Bla g 5 the best characterized at the recombinant level, but more information on the complete repertoire of GSTs from esponses .Schistosoma-GSTs seem to induce a protective IgE response, the Ascaris-GSTs may induce allergic reactions (Ascaris. On the contrary, it is also possible that due to the nature of the infection, Schistosoma spp. has a more immunosuppressive net effect that reduces the chance of potential allergenic molecules, such as GSTs, to elicit IgE-mediated reactions leading to a topic manifestations. Thus far, most of the studies tend to support that schistosome infection reduces skin test responses and allergic responses (Helminth-GSTs can induce different immune responses in the host; while eactions . A possieactions . There ieactions , 91. In eactions , 92. In esponses , 94.The current data suggest that cross-reactivity is not frequent among GST allergens and those detected seem to be non-clinically relevant; however, more research is needed to understand how molecules with a low degree of sequence homology exhibit cross-reactivity. It is expected that GSTs from different classes are not cross-reactive, but an important exception has been described . Since lJZ and LC: conceived this manuscript. AL and JZ: performed bioinformatic analysis and developed figures. All authors contributed to manuscript writing, reviewing, and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pine Island Ice Shelf (PIIS) buttresses the Pine Island Glacier, the key contributor to sea-level rise. PIIS has thinned owing to ocean-driven melting, and its calving front has retreated, leading to buttressing loss. PIIS melting depends primarily on the thermocline variability in its front. Furthermore, local ocean circulation shifts adjust heat transport within Pine Island Bay (PIB), yet oceanic processes underlying the ice front retreat remain unclear. Here, we report a PIB double-gyre that moves with the PIIS calving front and hypothesise that it controls ocean heat input towards PIIS. Glacial melt generates cyclonic and anticyclonic gyres near and off PIIS, and meltwater outflows converge into the anticyclonic gyre with a deep-convex-downward thermocline. The double-gyre migrated eastward as the calving front retreated, placing the anticyclonic gyre over a shallow seafloor ridge, reducing the ocean heat input towards PIIS. Reconfigurations of meltwater-driven gyres associated with moving ice boundaries might be crucial in modulating ocean heat delivery to glacial ice. Glacial melt can modify heat transport, and therefore ocean processes, associated with ice front retreat, as revealed by direct observations from the Pine Island Bay region of Antarctica. Many West Antarctic glaciers have recently been losing mass due to ice shelf thinning6 and rapid grounding line retreat7, thereby seriously threatening their stability. The heat transported by the relatively warm modified Circumpolar Deep Water (mCDW)11 is the major heat source melting the West Antarctic ice shelves. Pine Island Ice Shelf (PIIS) is among the most rapidly melting ice shelves, whose feeding glacier is responsible for ~40% of the net ice mass loss from West Antarctica6.Antarctic ice shelves buttress the ice sheet and restrain the speed of the ice flow, dampening ice discharge to the ocean14. The variations in PIIS melting have been observed over weekly to decadal timescales16, under the influence of ocean heat fluxes at the continental shelf break11 and in Pine Island Bay (PIB)16. The thermocline depth in front of PIIS is considered to determine the heat transport into the ice shelf cavity (i.e. ocean below the ice shelf) by adjusting the mCDW thickness16. Although continental shelf break processes dominate the heat influx from the Southern Ocean11, local (within PIB) sea-ice production and associated salt gain and heat loss have been reported as the forcings that deepen the thermocline depth at seasonal timescales16. In addition, frictional response to local wind forcing in front of PIIS has been proposed to dominate short-term variability in ocean heat flux12.The basal melting rate of West Antarctic ice shelves varies owing to the variability in oceanic forcing at multiple timescales19. Thus, the oceanic area in front of PIIS lengthened and narrowed   Fig. . In 200920, an anticyclonic gyre with a radius of ~16 km was identified in 2020 20.To identify the influences of PIIS front retreat on ocean circulations in PIB, hydrographic profiles were obtained in PIB during January–February 2020 Fig. ; Methods020 Fig. . Its cen020 Fig. , and the 20 Fig. . The cyc21. Due to the PIIS front retreat, the double-gyre moved southeastward by more than 20 km in 2020 compared with 2009 and 2014; that is, the location of the western limb of the cyclonic gyre in 2020 matches that of the eastern limb of the cyclonic gyre in 2009  source in the vicinity of the ice shelf front, produce a gyre-train.  also strengthen the cyclonic gyre.Meltwater-rich glacially modified water (GMW) that buoyantly flows out from the ice cavity is a driver for cyclonic gyre formation in PIB, as it imparts cyclonic vorticity25 according to the buoyant coastal plume theory26 applied to the Southern Hemisphere. After the separation of the GMW from the southern coast, GMW is entrained and circulates in the double-gyre. According to the 2020 observations, this can be identified by the relatively high meltwater fraction above the mCDW layer  along the ice front (>10‰) and the interface between the western rim of the cyclonic gyre and eastern rim of the anticyclonic gyre (>7‰)  Fig. . Meltwat7‰) Fig.  and 2b, 3 lighter than the pure Winter Water (WW) produced during the previous winter22 as observed in 2020 ed Figs. . The eased Figs. . Thus, t16) and the mCDW layer thickness at the entrance of PIB  in 2009  in 2020 owing to the lower OHC and reduced velocity at the mCDW layer . In 2009, convex upward isopycnals similar to those shown at L4 were observed near L3 Fig. . Due to The OHC at 400–700 m increased at L4 Fig.  with theOur observations demonstrated that the previously observed double-gyre relocated to the newly exposed region due to the recent PIIS front retreat Figs.  and 3. T9 and equivalent to that in 2012 (37 Gt/yr)9 and 2014 (40 Gt/yr)21. The 2012–2014 period was characterised by consistently lower mCDW temperatures and melting rates than those in the 2009 period. Therefore, the 2012–2014 period was considered a relatively cool period21. Such a low meltwater flux in 2020 may result from step (v) in the negative meltwater feedback. However, we only partially elucidated the underlying mechanism using limited ship-based observations.The meltwater flux from PIIS in 2020  may induce the deactivation of step (i) and (ii) in the negative meltwater feedback. In the future, the negative meltwater feedback may play a more crucial role in the basal melting of PIIS depending on PIIS front retreat distance as there are seafloor ridges (including a 400 m high seafloor ridge27) underneath PIIS, which are regarded as a modulator of ocean heat transport towards the grounding line27. Thus, the negative feedback mechanism warrants future investigations on ocean circulation in the PIB through year-round monitoring and/or numerical models that reflect geographic boundary changes and high-resolution bathymetry28.The negative meltwater feedback may control the ocean heat input and basal melting rate of PIIS on weekly or monthly scales31. However, as proposed herein, a large amount of meltwater can occasionally produce negative feedback, which may moderate the basal melt rate depending on the frontal migration changes. Thus, this study has an important implication for other Antarctic ice shelves that the local ice-ocean interactions controlling the basal melting of ice shelves could be altered by geographic conditions linked to the frontal migration. The circum-Antarctic ice shelves have been retreating over the recent decades influenced by diverse environmental factors including, but not limited to ice front migration32, yet our understanding of influences of these retreats on local ice shelf-ocean interactions remains poor. Understanding changes in the local ice shelf-ocean interaction caused by the retreat will be one of the key elements to improving capabilities for predicting future changes of ice shelves and global sea-level rise. Therefore, the Antarctic ice shelves that export a large amount of meltwater (such as the Thwaites Ice Shelf33) and are retreating rapidly32 need to be continuously monitored based on comprehensive observations that consider mCDW pathways and detailed bathymetry. In addition, to improve the prediction of future sea-level rise due to Antarctic melting, ice shelf frontal migration and related meltwater distribution should be well incorporated into the models.Enhanced meltwater input in a warming world may trigger a redirection of the coastal current leading to the frequent intrusion of warm water into the Antarctic continental shelf region, thereby accelerating ice shelf melting 34, was estimated to be ~6 km in PIB. The spacing of the 2020 observations (~7 km) is comparable to the first baroclinic Rossby radius of deformation, indicating that our sampling space is sufficiently small to capture the ocean gyre circulations at horizontal scales of a few tens of kilometres in this area. The four meridional lines nearly cover the region where the cyclonic gyre was detected in 200920 .We conducted full-depth conductivity-temperature-depth (CTD) and Lowered Acoustic Doppler Current Profiler (LADCP) casts at 29 stations along four meridional lines  sensor data were calibrated using the following equation:The CTD data from both surveys were processed using the method recommended by Sea-Bird Electronics Incorporated2 value for the correlation between the bottle samples and sensor values was 0.999.Equation  is based36, and de-tiding was not applied to the LADCP data because the observed current velocities were significantly stronger than the tidal velocities (<1 cm/s) in PIB20. The horizontal currents in PIB were also observed by ship-based ADCP (SADCP) from 29 January to 25 February 2020. The NBP had two Teledyne RDI ADCPs37, both Ocean Surveyor models (phased array) operating at 75 kHz in narrow and broadband modes and at 38 kHz in narrowband mode. The lower-frequency ADCP could reach 1000 m in good conditions. However, the range was typically less in the PIB environment. A thick ice-protection window impacted the high-frequency ADCP. Therefore, it only reached 100–150 m in broadband mode and 400–450 m in narrowband mode. The University of Hawaii Data Acquisition System (UHDAS) combined ADCP and navigational data streams and used Common Ocean Data Access System (CODAS) processing to incrementally build a dataset of averaged (15 min) edited ocean velocities for each ADCP and ping type specified. The SADCP data, which had percent-good (percentage of available pings in an ensemble) values lower than 90%, were removed from this analysis. We used the horizontal ocean currents obtained by averaging the currents observed at three frequencies. In addition, historical SADCP data observed in 200920 and 2014 were also used to identify horizontal ocean circulations in PIB  images obtained on the same dates.The PIIS front in 2009 and 2014 was manually digitised from Moderate Resolution Imaging Spectroradiometer (MODIS) Antarctic ice shelf image39. Finally, the horizontal ocean surface current vectors were calculated by dividing the travel distance by the time difference between the two images. Considering that sea ice drifted owing to both the surface ocean current and wind drag, we selected the images obtained when the wind was not strong (<5 m/s) to estimate the sea-ice motion dominantly forced by the ocean currents. Considering the 250 m pixel size of the MODIS image and the time interval of ~100 min, the resolution of the sea-ice motion estimation was <4 m/s with the sub-pixel resolution ability of the optical flow technique. The directional accuracy of the optical flow technique was <4° for a synthetic image sequence39. The estimated sea-ice motion at a 250 m resolution was re-gridded at an ~4 km resolution, as shown in Supplementary Fig. Horizontal ocean surface currents can be inferred by sea-ice motion derived from MODIS images  can be estimated using three independent tracers :40. The mCDW and WW endpoints were selected based on observational data  and the typical values of PT, S and DO for the ice water were approximately –90.75 °C, 0 and 28.46 mL/L, respectively41 , modified Circumpolar Deep Water (mCDW) and meltwater from PIIS . Layers with unreliable meltwater fractions were excluded from this calculation 42 using CTD data from the PIB. The dynamic height anomaly indicates the geostrophic stream function. The anomalies in Fig. During the geostrophic velocity calculation, dynamic height anomalies with respect to 500 m were also estimated following TEOS-103 and 27.75 kg/m3 isopycnals at the location of the cyclonic gyre were simulated based on the linear relation between the density gradient and volume transport. The volume transport of the cyclonic gyre in 2009 was 1.5 Sv20, and the slope of the concave 27.47 kg/m3 (27.75 kg/m3) isopycnal was ~7.2 m/km (5.7 m/km) . Based on these assumptions, the 27.47 kg/m3 and 27.75 kg/m3 isopycnals were simulated from the western limb of the cyclonic gyre to the PIIS front  Fig. . The avekm) Fig. ; therefo4. The OHC was estimated using the following equation:f is the in situ freezing temperature, Z1 is 400 m for 400–700 m OHC and upper boundary of the mCDW for OHC at the mCDW layer, Z2 is 700 m for 400–700 m OHC and bottom depth for OHC at the mCDW layer, ρ is the ocean density (kg/m3) and Cp is the ocean heat capacity (J/kgK). The in situ freezing point was estimated following TEOS-1042. Both ρ and Cp were calculated based on T and S. The ocean heat content estimated based on shallow stations (<550 m) cannot represent the values in the 400–700 m layer; therefore, they were excluded from the estimation of the mean value for each line.The OHC was calculated using the temperature above the in situ freezing point in the 400–700 m depth range and the mCDW layer. The 400–700 m layer indicates the depth range between the ice draft and the top of the ridge beneath PIISThe average OHC was calculated for each line using the weighted arithmetic mean. The weight parameter for each value was determined using the number of data points used for the OHC calculation for each station . The ocean is restored to the ISOMIP ‘warm’ conditions at the western wall (X = 1000 km), and the other two walls are rigid boundaries. The restoring timescale varies linearly from 1 h at the grid adjacent to the boundary to 1/2 day at the fifth grid cell inside the domain. In this simulation, the only sources of PV are the meltwater and the restoring boundary; using a long domain, with the restoring boundary located far (~800 km) from the ice shelf, we could ensure that the PV near the ice front is dominated by meltwater alone.We simulated the impact of meltwater using the Massachusetts Institute of Technology general circulation model (MITgcm)47. We used the standard drag coefficient of 2.5 × 10–3 for the ice shelf in the momentum balance adjacent to the ice shelf, while the drag coefficient that enters the three equation formulations is calibrated to 1.0 × 10–3 to ensure that the total melt flux in the simulation  closely matches the estimated observed value in 2020 . The simulation was performed for 70 model years from an initial state where the domain is entirely filled with cold (−1 °C) water, and reached a steady-state after 30 model years or so. In the Supplementary, we only showed the simulation result averaged over the final 2 model years (Supplementary Fig. In the MITgcm, melting is parameterised using the three equation formulations with velocity-dependent transfer coefficientsSupplementary InformationPeer Review File