Ascaris lumbricoides), the most common soil-transmitted helminthiasis, can induce allergic responses, and also immunosuppression. During ascariasis, antibodies for many A. lumbricoides antigens are produced; however, there is no clear information about the concurrent IgE, IgG4 and IgG production as well as their influences on the actual allergic reactions. In this study, we evaluated antibody responses to two A. lumbricoides molecules, rAsc l 5 and rAl-CPI (an anti-inflammatory product), in an A. lumbricoides endemic population and explored their relationship with the infection and asthma. Our results show that both molecules induce specific antibodies, but, in contrast to rAl-CPI, rAsc l 5 activates cells associated with allergic reactions in some individuals. All together, these data suggest that these molecules have differences in the elicited immune response.Helminth infections may have different effects on human health, including risk or protection from other diseases. Ascariasis  to two A. lumbricoides molecules, Asc l 5 and Al-CPI (an anti-inflammatory Cysteine Protease Inhibitor), in an endemic population, exploring their relationships with the infection and asthma. The two molecules were produced as recombinant proteins in E. coli expression systems. Specific antibodies were detected by ELISA. Lower human IgE, but higher IgG4 and IgG antibody levels were observed for Al-CPI than for rAsc l 5. The IgE/IgG4 isotype ratio was significantly higher for Asc l 5 than for Al-CPI. In humans Al-CPI did not induce basophil activation as has been previously described for Asc l 5. In mice, Al-CPI induced fewer IgE responses, but more IgG2a antibody titers than rAsc l 5. Our results suggest that these molecules elicit different patterns of immune response to A. lumbricoides.Immunity to  Ascaris lumbricoides is a soil transmitted parasite that causes ascariasis, one of the most prevalent helminthiases in humans [A. lumbricoides CPI (Al-CPI) ameliorates symptoms of inflammation in mice models of both dextran sodium sulphate-induced colitis and allergic airway inflammation [A. lumbricoides molecules proposed as therapeutic tools mandatory. Asc l 5 is the fourth allergen identified from A. lumbricoides [A. lumbricoides molecules [n humans . Its sucn humans , mainly n humans ,4, whichn humans  and the n humans , among tn humans ,8,9,10 tn humans ,12,13,14ammation ,8. Thus,ammation . Howeverammation ,17, somericoides . It is aricoides . The IgEolecules ,21. As solecules . In addiE. coli Origami (DE3). Al-CPI (GenBank Accession Number HQ404231.1) was cloned into a pQE30 vector and expressed as an 6xHis-tagged protein in E. coli SG13009. Bacterial cultures were grown in Luria–Bertani medium under conditions described previously [Asc l 5  was cloned into a pET-45b+  vector and expressed as 6xHis-tagged protein in A. lumbricoides extract (1000 µg/mL) was digested using the ProteoExtract All-in-One Trypsin Digestion Kit  and desalted using C18ZipTips . Resulting peptides were separated by reverse-phase nano-high performance liquid chromatography . The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 µL/min at 55 °C. The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide assignment, a top 12 MS/MS method was used with the normalized fragmentation energy set to 27%. The protein was identified with PEAKS Studio 8  using the UniProt (SwissProt/TrEMBL) sequence database. Only peptides with high confidence scores  < 0.5%) were considered in the database searches [searches . CD spectra were recorded using a JASCO J-815 spectropolarimeter fitted with a PTC-423S Peltier type single position cell holder , in 10 mM K2HPO4/KH2PO4. Samples were measured from 190 to 260 nm (CD far–UV spectrum) at resolution of 1 nm, with 1 nm bandwidth and a scanning speed of 1 nm/s. Five spectra were averaged and the background of the buffer solution was subtracted. Data were presented as mean residue molar ellipticity. Recombinants (concentration of 100 µg/mL) were evaluated at 20 and 95 °C, respectively. For calculation of denaturation temperature (Tm), the protein samples were subjected to gradual increases in the temperature from 20 to 95 °C. CD thermal denaturation curves were established. The thermal denaturation of the proteins was evaluated from 20 to 95 °C at 222 nm wavelength and a temperature ramp rate of 1 °C/min . ExcitatA. lumbricoides antigens was estimated in a rural population from the northern coast of Colombia  [N = 298, aged 1 to 88 years) had available serum samples for ELISA and data for parasitological variables (number of available samples from the participants are indicated in the results as needed). A physician interviewed participants for asthma diagnosis [A. lumbricoides infection was diagnosed by parasitological analysis by counting eggs per gram of feces (epg) [Seropositivity to Bolívar) ,22 with es (epg) . 2 was used. The reaction was stopped with 100 µL of 3N NaOH, and absorbance was measured at 405 nm in a spectrophotometer . Positive and negative control sera were used in each experiment and PBS was a control for non-specific binding of the anti-human IgE, IgG4 or IgG–alkaline phosphatase conjugates. Cut-off values to define positive or negative antibody responses to Asc l 5 or Al-CPI were calculated as the mean optical density (OD) of 9 negative controls + 3 SD for IgE or IgG4 and as the mean optical density (OD) of 7 negative controls + 3 SD for IgG. Differences in the number of controls used for calculating the cut-offs depended on the availability of negative serum samples. Seropositivity was defined as a positive specific IgE, IgG4 or IgG result detected by ELISA to any recombinant antigen. The cut-off values were: For IgE to rAsc l 5: 0.145; rAl-CPI: 0.141. For IgG4 to rAsc l 5: 0.170; rAl-CPI: 0.170. For IgG to rAsc l 5: 0.330; rAl-CPI: 0.316. Seropositivity was defined as a positive specific IgE, IgG4 or IgG result detected by ELISA to any recombinant antigen. IgE/IgG and IgE/IgG4 ratios were calculated from the end-point OD values for the three isotypes using IgE as the numerator. IgE was determined as described previously . For theHuman IgE detection by ELISA was performed before and after IgG depletion to evaluate the blocking activity of this last isotype on IgE binding. For IgG depletion, sera were incubated with protein G Sepharose  for 4 h at room temperature. After centrifugation, protein G Sepharose was added to the supernatant and again incubated. This procedure was repeated four times .A. lumbricoides or purified antigens, and then cells were stimulated with Ascaris extract (10 µg/mL), rAl-CPI or rAsc l 5 at different concentrations . Mean fluorescence intensity (MFI) results for CD203c in gated basophils were analyzed as stimulation indexes (SI), considering a value ≥2.0 as a positive result [A. lumbricoides extract and also to rAl-CPI. Histamine release was induced by incubating 200 µL of fresh blood with 200 µL of serial concentrations  of rAl-CPI or A. lumbricoides extract, allergen dilution buffer and anti-human IgE . Histamine concentrations were quantified by ELISA  following manufacturer instructions. Total histamine release was determined in cells treated with hypotonic solution and regarded as 100% release.The CD203c-based basophil activation assay was used for ex vivo evaluation of the allergenic activity of recombinant antigens as described elsewhere . Brieflye result . For basAntisera against recombinant rAsc l 5 and rAl-CPI antigens were raised in 6- to 8-week-old female BALB/c mice . Mice received intraperitoneal injections of the recombinant proteins (20 µg) suspended in 2 mg of Alum  three times in a seven-day interval, and blood was sampled 7 days after the last injection. Mice were kept in a specific pathogen-free environment  and fed with a standard pellet diet and drinking water ad libitum [For specific immunoglobulin determinations, MaxisorpTM microtiter plates were coated with 0.5 µg/well of rAsc l 5, rAl-CPI or OVA, respectively; followed by overnight incubation at 4 °C and washed 4 times with Tween 20 0.1% PBS. Wells were then blocked with PBS 1% BSA 0.05% Tween 20 for 3 h at room temperature. Plates were washed (5X) and incubated overnight with diluted mouse plasma samples at 4 °C (see Mouse IgG from rAl-CPI sensitized mice and PBS-injected mice (mock) were diluted 1:1000 in blocking buffer and incubated for one hour at RT with A. lumbricoides extract or rAl-CPI, and transferred to PVDF membranes after SDS-PAGE separation. Strips were rinsed twice and washed three times with 0.1% Tween 20 PBS. Horseradish peroxidase conjugated anti mouse-IgG  was used as a secondary antibody and incubated for 1 h at RT. The membrane was rinsed twice and washed three times with 0.1% Tween 20 PBS. Chemiluminiscence reaction was developed by the addition of a 1:1 mixture of the SuperSignal West Femto Maximum Sensitivity Substrate™ and incubation for 5 min in the dark. Images were obtained at different times in a specialized chemilumiscence image detector . A passive cutaneous anaphylaxis (PCA) model was performed as described previously for rAsc l 5 . Dye extp values < 0.05 (two tailed) were considered significant. In the analysis of mouse antibodies, for the comparison of the means of the two groups, the student’s t-test was used; for more than two groups, one-way ANOVA and Dunnett’s multiple comparison test were used to compare the means. In these cases, mean ± standard error of the mean (SEM) was used for each group. Correlograms were obtained in R, using the corrplot package. A Spearman correlation test was used due to the non-normal distribution of most variables.Analyses were conducted using SPSS version 20.0  and GRAPHPAD PRISM version 5.01 for Windows . IgE, IgG4 and IgG values of individual participants were not normally distributed; therefore, they were reported as a geometric mean and 95% confidence interval (CI). The Mann–Whitney U-test or Kruskal–Wallis test was used for comparison of continuous variables as needed. Differences between proportions of subjects with positive antibody responses (seropositive) were analyzed by a Pearson chi-square test (Taking into account the cut-off determined by ELISA). For all analyses, The secondary structure determination of rAl-CPI was consistent with the expected theoretical results for its primary sequence and solved three-dimensional structure . CD specWe also assessed, using Western Blot, if anti-Al-CPI antibodies raised in immunized mice were able to recognize the natural antigen in the A. lumbricoides extract, finding two bands in the 10–13 kDa range, which is in accordance with the expected size for Al-CPI . Al-CPI p < 0.0001), while the frequency of the IgG4+ cases to Asc l 5 was similar to the IgE response . As shown in 2: 4.9, p = 0.02) but not for Asc l 5 . Sex was not associated with IgG and IgG4 responses.There were also differences in the antibody response in regard to sex. Females showed significantly greater median O.D. values for specific IgE determinations to Asc l 5 and Al-CPI, respectively. This difference was also observed in the number of cases with a positive IgE test for Al-CPI . In contrast, specific IgG and IgG4 responses to Al-CPI were higher than those to Asc l 5  after adjustment for sex and age. Regarding IgG to rAsc l 5, there was no association with severe infection in a similar multivariate model . We also explored the relationships between egg counts (e.p.g.), antibody-related variables and age (p = 0.02) and also with subjects’ age . Age inversely correlated with IgE/IgG ratios to rAl-CPI  and to Asc l 5 .The frequency of infection in the study sample was 67.2% . Positive responses  to Al-CPI or to Asc l 5 were not associated with an nfection . In addinfection . However and age , findingConsidering the potential confounding effect of age in the relationships between antibody responses and egg counts, we ran multivariate generalized linear models to account for the effect of this covariate . As reported in Mouse models of experimental immunization have been useful for analyzing the allergenic activity and type of immune response to putative allergen molecules. Ovalbumin (OVA) was used as a positive allergenic control. rAsc l 5 induced the highest levels of IgG1 A, and rAA. lumbricoides is a source of immunomodulatory molecules and allergens, both with IgE-binding properties [A. lumbricoides allergen that we have recently registered at the WHO/IUIS allergen nomenclature sub-committee. Knowing the antibody binding properties of parasite antigens can help us to understand the mechanisms of their influence on allergic diseases like asthma, and also to prevent unwanted allergic reactions induced by parasite-derived immunomodulatory products intended for therapeutical purposes or vaccine candidates [A. lumbricoides exposed populations. Helminth infections are well-known modifiers of allergic response , and mosoperties ,8,18,30.ndidates ,31. In andidates  and protAnisakis simplex and Fel d 3, a cystatin-like protein described in cats. The sequence similarity between Al-CPI and these allergens stands at 42% and 30%, respectively, and the likelihood of cross-reactivity is minimal. Ani s 4 has the capability to trigger basophil activation [Our results show a lower IgE response to Al-CPI and higher levels of IgG4 and IgG than Asc l 5. In addition, the frequency of IgG4 seropositivity to Al-CPI was almost double the IgE seropositivity  while thtivation , and, totivation ,14,34,35tivation  and argutivation . In conttivation . HoweverA. lumbricoides molecules, which would expand the knowledge of mechanisms underlying the antibody pattern we have observed in both humans and mice. It is a matter of increasing research into the involvement of human IgG antibodies in the control mechanisms that have been proposed to explain why only a minority of sensitized people are symptomatic . Some stAnisakis simplex, Ani s 4) [Anisakis simplex allergens. For example, the allergen Ani s 4 even resists autoclave treatment (at 121 °C) used in thermal industrial steps of fish processing, retaining its IgE-binding properties and allergic activity [A. lumbricoides extract. High coverage of the mature sequence was detected. Since other CPIs from nematodes have been described as secreted proteins, our results show that Al-CPI is present in the somatic extract of the parasite and can induce immune responses during natural infection. This cystatin was found to be an abundant protein in the analyzed extract. The influence of abundance on modulation of IgE/IgG responses is not well-defined.We found that rAl-CPI and rAsc l 5 have different thermal stabilities as detected by CD. Al-CPI is a thermostable protein as are other members of the CPI protein family of nematodes (including its homologue from Ani s 4) . In contAni s 4)  studies activity ,52. ThusA. lumbricoides induces immunological responses influenced by the load of infection. Here, we found that specific IgG antibodies to Al-CPI are correlated with egg burden and subject’s age, probably as a result of a prolonged exposure. Interestingly, IgE/IgG4 ratios to rAl-CPI were inversely associated with A. lumbricoides egg load after adjustment by age and sex. Similar results were reported by McSharry, C., et al., who found that, in endemic areas, people with significantly more IgE antibodies to ABA-1, an antigen found in the pseudocelomic fluid of Ascaris, which have a lower burden of adult parasites [A. lumbricoides and A. suum. Therefore, the potential effects of the IgE/IgG4 to Al-CPI on resistance to ascariasis deserve further investigations.In the studied population , the prevalence of ascariasis has been between 56 and 62.5% for 10 years, as reported in two separate studies with sample collections in 2004 and 2014 ,53. Thisarasites . This suarasites ,55,56, pSeveral limitations should be stated. Although their physico-chemical characterization and the evaluation of their biological activity support a good folding, since natural antigens were not isolated and compared to the recombinant counterparts, the obtained results with rAsc l 5 and rAl-CPI may not completely reflect natural exposure. Second, although IgG depletion experiments did not change IgE binding to the recombinant antigens, the effects of this isotype response on degranulation assays were not explored. Third, although rAl-CPI induced low IgE production in mice under routinary protocols for immunization, we cannot rule out the fact that it could take place after longer exposure or in the presence of other parasite components that serve as adjuvants. Our conclusions are limited to the statement that, under the same protocol of immunization, rAl-CPI induced lower IgE production than rAsc l 5  as well During ascariasis, helminth-derived components induce different types of humoral immunity. Our findings strongly suggest that the immunomodulatory protein Al-CPI has low allergenic activity, which supports its potential use as an anti-inflammatory product in humans. In contrast, Asc l 5 induces histamine release and the degranulation of cells typically activated by IgE. The clinical impact of this allergenic activity is not clear since sensitization to Asc l 5 is not associated with asthma presentation. Comparing the types of immune responses to parasite antigens is an essential step in understanding the dual effect of helminth infections on allergic diseases.


Furthermore, a comprehensive quality assessment is presented. Both wastewaters show a wide range in terms of CODt, CODs, TN and NH4-N, whereby condensates show a greater variability with no direct relation to dryer type or temperature. In the slurries, CODt shows a relative standard deviation of 106% (mean 21.1 g/L) and NH4-N of 33% (mean 2.29 g/L), while in condensates it reaches 148% for CODt (mean 2.0 g/L) and 122% for NH4-N (mean 0.7 g/L). No inhibition of ammonium-oxidizing-bacteria was detected in the slurries, while two out of five condensates showed an inhibition of >40%, one of >10% and two showed no inhibition at all. Since the inhibition could be avoided by mixing, deammonification can be recommended for condensate treatment. For slurry treatment, the importance of employing some form of solid-liquid-separation as a pretreatment was noted due to the associated COD.Deammonification is a well-established process for sludge liquor treatment and promising for wastewaters with high nitrogen loads because of its low energy demand compared to nitrification/denitrification. Two wastewaters with high NH Pig slurry can contain up to 5 g/L NH4-N . With this intermittent aeration, the development of the OUR indicating AOB activity could be registered over the course of the experiment. Each reactor received a different composition of substrate consisting of the slurry/condensate sample in different dilutions with tab water (TW) maintaining the same volume and maintaining the same NHThe experiments were run until a continuous decrease in OUR indicated substrate depletion. To be able to only compare the autotrophic respiration rates responsible for nitrification, the heterotrophic OUR registered in reactor (C) was subtracted fully and partially (50%) from the registered OUR in (A) and (B), respectively. In each reactor, the VSS concentration was determined following DIN 38409-1 (DIN) to calculate the Specific Oxygen Uptake Rate (SOUR) for each reactor [mgO2/gVSS/h]. To assess any inhibition the maximum SOUR reached in reactor (A) and (B) were compared to the maximum SOUR in the reference (D). The maximum SOUR being the mean SOUR registered for a prolonged period at the highest level. Any negative deviation of the maximum SOUR of greater than 5%, compared to the reference, was considered an inhibition.4-N concertation in C-1, it was not possible to ensure a sludge loading of 0.06 gN/gVSS for the inhibition test without diluting the sludge to an unreasonable degree. Therefore, the setting was done differently than described above. In accordance with the other experiments, reactor A received an undiluted sample of C-1 resulting in an unusually low sludge loading. Instead of supplying reactor B with a 1:2 diluted sample of C-1, reactor B received an undiluted sample of C-1 topped with NH4Cl to match the sludge loading of 0.06 gN/gVSS. The reference (D) was also set to a sludge loading of 0.06 gN/gVSS to be comparable with the other batch tests.Because of the low NHt, CODs, TN and NH4-N vary widely. TS ranges from 5.4 g/kg to 125.1 g/kg, CODt from 3.13 to 89.50 g/L, CODs from 0.79 to 19.40 g/L, TN from 0.72 to 8.84 g/L and NH4-N from 0.65 to 3.26 g/L. The CODs makes up between only 1% and 88% of the CODt, also showing a great variability. High values for TS not only suggest a high CODt but also a tendency of high TN and TP which can be observed in the considered data. LOI lies between 51.2% and 81.9% originating from different amounts of inorganic material in the slurries, underlining the influence of stabling, washing and storage on slurry composition stated by Møller, Sommer et al. [3-N was detected with maximum 35.8 mg/L, NO2-N was only detected in one slurry (0.5 mg/L). The pH is the most stable parameter with a range from 7.16 to 8.07.In the considered pig slurries TS, CODr et al.  and Choir et al. . While Nt is present in all slurries, it does not translate directly into a degradable fraction, for some of it is linked to larger particles. To estimate the biodegradability, CODs is used instead. The ratio of CODs:NH4-N ranges from 0.1 to 6.6. Using CODs as a substitute for CODb, only 1 out of 12 samples reaches the recommended ratio of 5.8 for nitrification/denitrification, encouraging the use of a less COD-dependent treatment. For the deammonification to function optimally the anammox needs a ratio of NH4-N to NO2-N of 1.32 [4-N in the slurry needs to be oxidized to NO2-N. As the nitritation process releases protons (H+) and no protons are bound in further steps as is the case in denitrification, sufficient total alkalinity (TA) is crucial to a stable deammonification. For each milligram of NH4-N converted, 0.14 mmol of TA are used up [4-N, a minimum TA:NH4-N ratio of 0.0784 mmol/mg in the wastewater is needed. In the considered slurries, TA:NH4-N ranges from 0.07 mmol/mg to 0.12 mmol/mg allowing for a theoretical oxidation of at least 56% of the NH4-N present in 11 out of 12 samples. Even though a high COD of 1.32 . This me used up . So, to t, CODs, TN, NH4-N, NO2-N, NO3-N, TP, PO4-P and TA can be found in t, CODs and NH4-N with the literature and slurry monitoring is presented under All data on the slurries including TS, LOI, pH, CODt follows the pattern described by the TS, CODs varies independently. On average, the CODs made up 43.6% of the CODt, with the LOI averaging at 50.5%. Except for NO2-N and NO3-N, all measurements fit well with the results of the slurry characterization. The maximum for NO2-N lies at 2.79 mg/L and for NO3-N at 67.00 mg/L. The CODs:NH4-N ratio ranges from 1.3 to 4.1 with an average of 2.4, not reaching the recommended 5.8 for nitrification/denitrification. TA:NH4-N ratio varies between 0.06 to 0.18 mmol/mg with an average of 0.13 mmol/mg allowing for a theoretical oxidation of over 56% of the present NH4-N in 28 of 30 samples. Over the course of the monitoring period, EC and TA seem to be following the pattern described by NH4-N. Even though a broad variation in almost all parameters is present, no clear seasonal pattern as described by Choi, Kim et al. [t, CODs, NH4-N, TA, pH and EC over the 9-month monitoring period. All measured values can be found in the The pig slurry shows a rather broad variation in almost every parameter over the course of the 9-month monitoring period. The pH is an expectation, with a relative standard deviation of only 1.3%. m et al.  could be4-N and TA is most important. For TA, only 5 values could be obtained in the literature and linked to NH4-N [4-N ratio of 0.0784 mmol/mg [t values in this study range from 3.13 g/L (S-5) up to 89.50 g/L (S-11), both being one-time samples. The monitoring data show that even a continuous piggery operation produces a wide range in CODt from 6.775 to 29.750 mg/L with an average of 13.144 g/L. With this, the measured CODt fits the literature well. t in this study set in context with the literature values. In the literature, CODt ranges between 1.453 g/L [t. The results from the slurry characterization and monitoring obtained in this study show a broad range in different parameters. Assessing the deammonification potential COD, NHto NH4-N ,37,38, w mmol/mg . CODt va.453 g/L . Still, .453 g/L ,40,41,42.453 g/L ), seem tt is often achievable [s making up as little as 13.7% (S-1) or 15.9% [t. An extreme finding is sample S-9, with only 1.0% of the CODt being CODs. Considering CODs, values up to 59.7 g/L and 62.98 g/L are reported in literature [s. s values in comparison to the values found in the literature.Using solid–liquid separation, a good reduction in CODhievable . This poor 15.9%  of CODt.terature ,45. The terature ,40,42,464-N does not vary as vastly as CODt and CODs but still shows a broad variation. The examined samples range from 0.65 g/L (S-6) to 3.26 g/L (S-8), while the range found in the literature goes from 0.802 g/L [4-N content of the examined slurries to the values found in the literature.NH.802 g/L  up to 5..802 g/L . Of the .802 g/L ,37,38,41.802 g/L ,47,48,49.802 g/L ,47 with t, CODs or NH4-N in pig slurry can be derived.Often the animal type, feeding and stabling conditions are not directly or consistently mentioned in literature. Due to insufficient data, no quantitative connection between the influencing factors and CODEven though a small negative deviation in activity (<5%) is present in most tests with slurry samples, which is a normal phenomenon in microbiological experiments, no inhibition of the nitritation process could be observed. Testing slurry S-1, the maximum SOUR of the reference reaches 14.4 mgO2/gVSS/h. Reactor A (100% sample) reaches a maximum SOUR of 14.4 mgO2/gVSS/h while reactor B (diluted sample 1:2) reaches 18.4 mgO2/gVSS/h. This translates to a deviation to the reference of 2.3% for reactor A. Reactor B even shows a 28.0% higher activity than the reference. In the batch test with slurry S-2, reactor A (100% sample) with a maximum SOUR of 63.4 mgO2/gVSS/h lies 4.2% below the maximum SOUR registered in the reference (66.2 mgO2/gVSS/h)—not yet considered an inhibition. Reactor B (diluted sample 1:2), registering at 66.5 mgO2/gVSS/h, lies on the same level as the reference with a SOUR elevated by 0.4%. Surprisingly, the activity in the reactors supplied with slurry S-4 exceeds the reference in both cases (undiluted and diluted 1:2). Here, the baseline of the reference for maximum SOUR lies at 49.0 mgO2/gVSS/h. Reactor A shows a maximum SOUR of 55.3 mgO2/gVSS/h, which is 12.7% higher than the reference. As in S-1, the diluted sample scores even higher with 66.0 mgO2/gVSS/h, 34.5% higher than the reference. t can be observed in samples with an elevated TSS, with CODt ranging from 70 mg/L up to 8950 mg/L. CODs makes up between 42.7% and 98.3% of CODt. With NH4-N making up between 68.5% and 94.9% of TN, the range varies vastly from 59 to 2300 mg/L. The CODs:NH4-N ratio varies between 0.2 and 1.8, not reaching 5.8 in any sample underlining the need for a carbon-independent treatment. TA:NH4-N ratio ranges from 0.04 to 0.19 mmol/mg, allowing for a theoretical oxidation of over 56% of the containing NH4-N in 4 out of 6 samples. With a pH of up to 10.18, some samples need to be especially considered for FA concentrations if selected for treatment [The considered samples show a great range in every parameter. Suspended solids vary widely between the samples, being as low as 0.5 mg/L and as high as 700 mg/L, with shares of organics represented by a LOI of 33.3–86.0%. The samples higher in TSS are the samples with a higher degree in drying, supporting the claim that a high degree of drying results in dustier vapors . The tenThe data supplied spans over the years 2018, 2021 and 2022. As in slurry monitoring, the composition varies with almost every parameter expect the pH which is also fairly stable in the condensate. 5) and total Kjeldahl nitrogen (TKN) do not differ drastically in terms of their variation and annual mean when comparing the three years considered. While mean TSS is similar in all years, the maximum measured in 2018 lies far below the maxima of 2021 and 2022. Moreover, a rise in CODt from 2018 to 2021 is apparent, as well as a small drop in mean NH4-N. Unfortunately, no information on any operational changes in this time period was supplied. Noticeably, there is also a drop in CODt between 2021 and 2022 of 740 mg/L in the annul means, a 36.7% decrease. t in 2021 and 2022, as well as the annual means represented by a dotted line. The data shows two fairly stable levels with a period of greater variability around the turn of the year. In November 2021, the degree of drying was reduced from 42% to 39% and in the beginning of 2022 the polymer, used to facilitate sludge dewatering, was changed. Moreover, the use of glycerol as a co-substrate was drastically reduced in the beginning of 2022. The decrease in degree of drying is not pronounced enough for a reduction in CODt, as described by Brautlecht [t:NH4-N ratio in the condensates ranged from 0.4 to 1.7 in 2018, from 0.5 to 1.7 in 2021 and from 0.2 to 1.9 in 2022, with an annual mean of 0.8, 1.0 and 0.6, respectively. As in the samples of the characterization, this favors a carbon-independent treatment. Additionally, the CODt seems not to be readily degradable as the mean BOD5: CODt ratios of 0.21 (2018), 0.15 (2021) and 0.23 (2022) suggest.The pH, EC, LOI, the Biological Oxygen Demand after 5 days  and a maximum range of 5.375 g/L [t reported from lab experiments by Deng, Yan et al. [t [t in condensates derived from thin-film dryers score lowest in the comparison with a maximum of 0.628 g/L [When comparing the CODss broad ,51,52,53ss broad  as a genWilderer  of a con.107 g/L  as its l.375 g/L . Regardin et al.  and Yan,n et al.  is dwarft al. [t . Noticea.628 g/L . On the .628 g/L  and a mat is still achievable with de-dusting or filtration. While most of the condensates where data on CODt and CODs are available have a portion of up 80 to 98% CODs in CODt, there are condensates with as little as 21% CODs [s in the condensates range less widely than the CODt. The lowest value measured in this study is 0.053 g/L (C-3), while the lowest reported in the literature is 0.109 g/L [s in the literature, mostly being reported as one-time samples, it makes for a difficult placement of the measured values in this study. s measured in this study and CODs presented in the literature, differentiating by dryer type. The CODs in condensates derived from disc dryers lie between 1.0 and 2.5 g/L with six one-time samples  and .621 g/L . Except .621 g/L  with a C4-N values in the literature spread over a wide range from 0.039 g/L to 3.11 g/L [4-N in condensates of 0.3 to 1.5 g/L, stated by DWA [4-N values considered lie below 2.5 g/L. This also includes the values reported in lab experiments [4-N minima and maxima in the recorded data span over a range of 1.31 g/L (C-8 ‘18) and 1.30 g/L  and lie in between the ranges of 0.674 g/L [t and CODs, the range in large-scale operations seem to dwarf the effect of drying temperature on the NH4-N content of condensates as reported by Deviatkin, Havukainen [t and CODs, thin-film dryers score noticeably low.As the other parameters do, NHd by DWA , these vd by DWA ,29,59 ord by DWA ,52,59 reeriments ,60,61,62.674 g/L  up to a vukainen . Out of In contrast to inhibition testing with pig slurries, three condensates posed an inhibition to the nitritation process by different degrees. In all inhibitory samples, the effect of inhibition decreases by 54% to 59%, when diluted 1:2. 4-N concentration, reactor A (500 mL of pure sample) only reaches an initial NH4-N concentration of 14.5 mg/L, translating to a sludge loading of 0.01 gN/gVSS. The maximum SOUR in reactor A registers at 10.1 mgO2/gVSS/h, 51.2% lower than the reference with 20.7 mgO2/gVSS/h. Reactor B, where the undiluted sample of C-1 was topped with NH4Cl the maximum SOUR at 23.0 mgO2/gVSS/h), lies 11.0% higher than the reference. The lack of activity in reactor A seems to be due to a deficient supply of ammonium and not the contents of the condensate itself. Testing condensate C-1 with a low inherent NHIn the batch test with condensate C-2, reactor A (100% sample) shows a 60.5% lower maximum SOUR than the reference with 15.6 mgO2/gVSS/h compared to 39.4 mgO2/gVSS/h. In reactor B with the diluted sample (1:2), the maximum SOUR lies at 29.7 mgO2/gVSS/h, 24.5% lower than the reference. This shows a clear inhibition of the nitritation process by C-2. The dilution of C-2 (1:2), however, decreases the inhibitory effect by 59%. In testing condensate C-3, only a slight inhibition is present in reactor A (100% sample) with its maximum SOUR being 5.8% lower than the reference, with 36.5 mgO2/gVSS/h compared to 38.7 mgO2/gVSS/h. Diluting C-3 at a 1:2 ratio results in a decrease of its inhibitory effect as shown by the maximum SOUR in reactor B (38.2 mgO2/gVSS/h) which lies 1.3% under the reference, and is hence not considered to be inhibitory. Condensate C-6 results in an inhibition of the nitritation process by 40.3% with a maximum SOUR of 30.8 mgO2/gVSS/h in reactor A (100% sample), compared to 51.7 mgO2/gVSS/h in the reference. Like in C-2 and C-3, diluting the sample at a 1:2 ratio shows a positive effect on the attainable maximum SOUR, which lies at 42.0 mgO2/gVSS/h in reactor B, 18.7% lower than the reference. This translates to a reduction of the inhibition by 54%. The discussion of the obtained results follows the three main objectives of this study: substrate quality, AOB inhibition and deammonification potential.t, CODs and NH4-N reported. Only the CODs:NH4-N ratio found in this study, ranging from 1.3 to 4.1, lies clearly lower than the ratios that could be obtained from the literature. Of eight values, only one one-time sample reported by Boursier, Béline et al. [s:NH4-N ratio favorable for nitrification/denitrification is not to be taken for granted, even if the current literature suggests otherwise. One influencing factor for the variability in pig slurry quality that is mentioned in the literature is the pigs’ diet. A diet drastically reduced in N can reduce the TN of the pig slurry by up to 0.97 g/L and also reduces NH3 emissions [4-N of 1.046 g/L observed in the slurry monitoring (1.87–2.92 g/L) of a continuous piggery operation suggests that peripheral influence factors, such as stabling, storage and use of cleaning water, may have a higher influence in comparing the quality of slurries from different piggery operations [t, TP and TN, with the latter potentially being hydrolyzed to NH4-N during slurry storage. As the TS, CODt and CODs measurements of pig slurries in this study suggest, a first reduction in CODt can be easily reached via solid–liquid separation. These influencing factors have been investigated for specific piggery operations but not for a categorization of pig slurry qualities, which is helpful in planning possible treatments. From this study, the key figures for pig slurry quality can be derived, though it is not possible to categorize the characteristics in regard to the animal type  as the peripheral conditions have a higher influence on the quality. The results of this study on the characteristics of pig slurries fit well with the literature and underline the high variability in CODe et al.  showed ae et al. ,40,45. Tmissions ,64. Stilerations ,32. In pt, CODs and NH4-N is apparent. While Friedrich and Heindl [4-N concentration of 2 g/L as an extreme example, this study shows that this value is exceeded regularly. In total, 12 of the 52 values (including lab tests) exceed this concentration and additionally 10 values are exceeding 1.5 g/L. The variability of the parameters seems to be greater than often assumed in literature. This variation is not only true when comparing condensates from different drying facilities but also for the long-term operation of one particular dryer. For CODt, Brautlecht [t which is supported by the reported influence of drying temperature on CODs by Deng, Yan et al. [t of up to 2.418 g/L, collected from a medium temperature drier, compared to the condensates from a high temperature drier with a CODt of only up to 0.373 g/L. This supports the claim that other influence factors mask the influence on drying temperature on CODt when comparing different drying operations. For NH4-N, the degree of anaerobic stabilization of the sewage sludge has a major influence on NH4-N concentration in the condensates, while the temperature does not seem to have any effect [t, CODs and NH4-N. Nevertheless, there is not enough evidence to conclude that the use of thin-film dryers always results in lowly loaded condensates, for no information about the condensers used could be obtained. These numerous influence factors make it virtually impossible to predict condensates quality only based on dryer type, temperature and degree of drying; only tendencies may be concluded. For a better categorization of condensate qualities—needed for better treatment planning—systematic research is direly needed.Considering condensates from sewage sludge drying, an even greater variability in parameters as CODd Heindl  marked aautlecht  and Deviautlecht  reportedn et al.  and Yan,n et al. . Regardin et al. , this inn et al.  also repy effect ,65. Thisy effect . While tThis study does not show any inhibitions of AOB activity by pig slurry. A direct comparison with the literature is not possible, for no studies systematically examining the influence of raw pig slurry on AOB activity could be found. Even though no inhibition of AOB is to be expected in the treatment of raw pig slurry, the inhibition of AOB reported by Zhang, Lin et al.  as a resThe inhibitions of AOB recorded for condensates in this study fit well with the findings of Beier, Mahnig et al.  where, os:NH4-N ratios of up to 12.4 for pig slurries in the literature suggest a good suitability for a treatment via nitrification/denitrification, in this study extremely low values of down to 0.1 were measured, suitable for deammonification. By using CODs as a substitute for CODb in this study, the biodegradability might even be overestimated. The TA:NH4-N ratios measured in this study and derived from the literature suggest that an oxidation of approx. 56% an NH4-N in the slurries—essential for al substantial nitrogen elimination via anammox—is not of great concern. However, process control measures need to be implemented where an excessive oxidation of ammonium might shift the NH4-N:NO2-N ratio to be unfavorable for anammox. This could be a halting of the oxidation or, if a two-stage process is used, the supply of ammonium to the anammox via a bypass. From a nutrient perspective pig slurry seems to be well suited for a treatment with deammonification. But in certain cases, the high amount of organics does pose an obstacle for anammox efficiency [t elimination via nitritation and anammox can also hinder certain treatment goals. Therefore, a coupling of the deammonificiation process with some form of COD elimination seems reasonable [. A first step should be proper solid–liquid separation. Also, currently, the combination of nitritation/anammox with a denitrification is being investigated for anaerobically treated pig slurry/manure with process set-ups and control strategies [4-N) in combination with denitritation might also be of use.While CODficiency ,21. In arategies ,26. Due 4-N and low CODs content in condensates, they are well suited for deammonification, even though for a full treatment some form of COD removal might be recommended in certain cases [4-N described in this study implies that for any treatment with a net H+ production some form of acid capacity might need to be supplied. Another problem for a biological treatment of condensates poses the inhibition of AOB by certain condensates that could be observed in this study and was also reported by Beier, Mahnig et al. [4-N of condensates might be used for NOB suppression in co-treatment. Nevertheless, the production of lowly loaded condensates that do not pose any inhibitions on AOB activity is possible as C-1 shows.Due to the potentially high NHin cases . Becauseg et al. . No cleag et al. . Apart fg et al. . A combig et al. . For slug et al. . The post and NH4-N, whereas the quality of pig slurries seems more predictable with its connection to stabling and collection and is set in a narrower range. The COD:NH4-N ratio in pig slurry is often unsatisfactory for treatment via nitrification/denitrification. It could be demonstrated that the deammonification process can be beneficially applied for the reduction in nitrogen, especially when COD is removed in a pre-treatment via solid–liquid separation and used to enhance biogas production. A competition between denitrifying bacteria and anammox bacteria for NO2-N can be avoided by using a two-stage process where partial nitritation and anammox are separated, especially as no inhibition of AOB was detected with raw pig slurry. In fact, the TA in pig slurries generally supports the deammonification process. Due to the high amount of TA in certain pig slurries, the partial nitritation may exceed the oxidation of 56% of ammonium needed for anammox, shifting the desired NH4-N:NO2-N ratio of 1:1.32 towards NO2-N. The ratio can easily be adjusted by bypassing the nitritation stage with fresh wastewater.As shown in this study, pig slurry and vapor condensates show great variability in COD4-N ratio found in condensates favors the treatment via deammonification, with no need for an additional COD removal and strong potential for an energy-efficient nitrogen removal process. A problem in condensate treatment is posed by the determined inhibition of up to 60.5% on AOB activity. The method used in this study for inhibition testing gives a quick and reliable result for the necessary classification of the condensate as the inhibitory effects are not easily predictable. In the experiments, with a dilution of 1:2, inhibitory effects could be reduced by more than half; therefore, a combined treatment of condensates and sludge liquor is favorable. The variability in quality of the condensates exceeds even the variability shown in pig slurries with no direct relation to dryer type or temperature. Concerning the nitrogen removal efficiency, the COD:NHTo summarize, general key figures for pig slurry and condensate quality can be drawn from this study as given above, but for a specific implementation of a deammonifcation process  for pig slurry or condensate treatment it is necessary nevertheless to examine the particular wastewaters. This is especially true for possible inhibitory effects in condensates. In pig slurries and condensates, the up- and downstream processes play a major role in composition and quality, holding potential for quality optimization and assessment. Both effluents are well suited for treatment by deammonification. However, for pig slurry treatment, the process technology needs to be directed towards a solid–liquid separation and/or an integration of COD removal  in the deammonification process, possible in a two-stage system (as is being studied in the BMBF-funded research project “KompaGG-N” (grant number: 02WQ1516C)). The ammonium concentration of condensates is much lower than in pig slurries but there is a much higher chance of AOB inhibition. Therefore, it is recommended to treat the condensates together with another lower-loaded part stream, which can also compensate the low total alkalinity of condensates and thus strengthen the nitrification.


Electrophysiological analysis confirmed the existence of synaptic inputs into the pACC by pIC GABAergic cells. Optogenetic stimulation of these pathways did not change mechanical nociception, but induced conditioned place preference behavior responses. Our results suggest the presence of inhibitory projections between the pIC and the pACC which are able to selectively modulate affective aspects of neuropathic pain.The insula and anterior cingulate cortex (ACC) are brain regions that undergo structural and functional reorganization in neuropathic pain states. Here, we aimed to study inhibitory parvalbumin positive (PV+) posterior insula (pIC) to posterior ACC (pACC) projections, and to evaluate the effects of direct optogenetic manipulation of such projections on mechanical nociception and spontaneous ongoing pain in mice with Spared Nerve Injury (SNI). CTBThe online version contains supplementary material available at 10.1186/s13041-023-01052-8. The pain modulatory system comprises anatomical pathways that include inhibitory circuits originating from cortical areas such as the insula and anterior cingulate cortex (ACC) . The pos488 was injected into the pACC region of PV-tdTomato mice, and overlap between CTB488 and tdTomato label in slices from the insular cortex visualized and quantified. The green retrotracer-labeled GABAergic cells in the pIC are shown in Fig. 488 injection was verified by examination of pACC slices . We also examined the degree of c-Fos activation in GABAergic cells projecting to the pACC in the presence and absence of SNI . Hence, SNI does not appear to induce increased activity in GABAergic pIC to pACC projections.We first examined to what extent the insular cortex and pACC are linked by inhibitory connections. CTBces Fig. b. Quantices Fig. c revealsTo complement our retro-tracing experiments, we used electrophysiology on pACC slice preps with opto-stimulation of synaptic inputs from the pIC. AAV-EF1a-DIO-ChR2-H134R was injected in the pIC of PVCre transgenic mice to express ChR2 in PV+ neurons . PVCre SHAM and SNI mice were subjected to optostimulation with a blue laser for selective ChR2 activation in PV+ projections from the pIC to the pACC. There was no effect of optostimulation on mechanical withdrawal thresholds in SHAM mice  (Fig. Several interconnected brain regions have been implicated in neuropathic pain, including the prefrontal cortex , 11, amyOnly a small portion of pIC to pACC projections were activated under basal conditions, and the proportion of activated cells appeared even lower after SNI surgery. c-Fos labeling has limitations, including a lack of quantitative output of neuronal activity, and subtle activity alterations within a specific subset of already active neurons due to SNI may not have been resolved. Our sample size was, however, limited, and thus putative activity differences between SHAM and SNI surgery may not have been fully resolved.In summary, our results support the idea that inhibitory pathways between the pIC and pACC can modulate affective-motivational aspects of pain, highlighting these areas as potential targets for the management of neuropathic pain.Additional file 1. Materials and Methods


Inhibition is a pillar of cognitive control, i.e., the ability to select, regulate, and coordinate mental representations of internal states and sensory evidence to choose the most appropriate behavioral strategy, overcoming inappropriate habitual actions. About ten years ago, Braver  postulatThe relationship between inhibitory control and clinical disorders characterized by poor urge control has been the object of several studies. Impulsivity has been frequently ascribed to deficient inhibitory control, and not seldom the two terms have been used interchangeably. However, evidence from behavioral studies is exceptionally mixed . As niceThis point brings us to another open challenge. While there are validated measures of reactive inhibition, e.g., the stop signal reaction time (SSRT), which is computed when using the stop signal task (SST, , howeverThe first two methods assessing proactive inhibition described above provide results that have been replicated several times; thus, there is no doubt about their validity. However, they are not very sensitive because they show little or no modulation, e.g., , thus prMost of the studies investigate movements performed with a single effector; however, in real life, we often use multiple effectors simultaneously. Thus, while the computational and neural mechanisms mediating movements initiation and inhibition made with single effectors have been widely studied, much less is known when more than one effector has to be employed to respond. The review by Jana et al.  nicely aThe other papers deal with the neural underpinnings of reactive and proactive inhibition. It is known that the network subserving this core cognitive function is extended, including both cortical and subcortical brain regions of both hemispheres . The papOn the one hand, experimental studies of this rich collection of articles provide lab-based evidence supporting the existence of proactive and reactive inhibition as two separate domains of inhibitory control. On the other hand, theoretical papers underline the importance of such parcellation for understanding the performance of healthy people in different contexts and for characterizing the phenotypes of diseases with pure urge control. Having said this, it is very likely that in real life, core cognitive functions operate in concert. Future studies aimed at understanding the interplay of the core cognitive functions are warranted.


Ice streams flowing into Ross Ice Shelf are presently responsible for around 10% of the mass flux from West Antarctica, with the noteworthy exception of Kamb Ice Stream, which stagnated in the late 1800s. The subsequent reduction in ice supply led to grounding-line retreat at the coastal margin where Kamb transitions into the floating Ross Ice Shelf. Grounding-line migration is linked to broader changes in ice-sheet mass balance and sea level, but our understanding of related ice, ocean and seafloor interactions is limited by the difficulty in accessing these remote regions. Here we report in situ observations from an underwater vehicle deployed at Kamb that show how fine-scale variability in ice and ocean structure combine to influence a diversity of ice–ocean interactions. We found a stratified water column within a tenth of a degree of freezing at the ice base and mapped basal crevasses with supercooled water and active marine ice formation. At the seafloor, we interpret parallel ridges as crevasse impressions left as the ice lifted off during grounding-line retreat. These observations from a recently ungrounded sub-shelf environment illuminate both the geomorphological signatures of past grounding-line retreat and the fine-scale sensitivity of ongoing ice–ocean interactions to ice topography. Observations from a remotely operated underwater vehicle reveal crevasse refreezing and the fine-scale variability in ice and ocean structure at the Kamb Ice Stream grounding line in West Antarctica. GLs are dynamic transition zones where ice sheets flow into ice shelves, and GL positions reflect a balance of glacial, marine and geologic factors. Changes in GL position are linked to broader changes in ice-sheet mass balance5; for example, ice-shelf thinning due to reduced ice flux can cause GL retreat7, or alternatively, increased ocean melting can cause ice-shelf thinning, GL retreat and accelerated ice flow that compound ice-sheet mass loss10.Along Antarctica’s coast, floating ice shelves buttress the grounding-line (GL) positions of their tributary ice streams and glaciers3. The West Antarctica ice streams that feed RIS flow at speeds of hundreds of metres per year but are known to cyclically slow, stagnate and reactivate on century timescales12. Of these, Kamb Ice Stream (KIS) has been stagnant since ~186515. The shift from flow speeds of 350 m yr−1 in the main KIS trunk16 to now near-zero surface velocities at the KIS GL17 is thought to be due to a change in the subglacial water system18. Stagnation initiated an ongoing accumulation of ice upstream19 that presently offsets 25% of the mass loss from West Antarctica20. Reactivation22 could increase Antarctica’s current contribution to sea-level rise23 by 12% (ref. 24). Along the KIS coast, reduced ice flux post-stagnation also led to thinning and GL retreat until at least the 1980s15, but a lack of in situ observations from remote GL environments limits our understanding of ice, ocean and seafloor interactions as GLs migrate during cycles of ice-stream activity.Earth’s largest ice shelf by area is the Ross Ice Shelf (RIS), which represents a third of all floating glacial ice26 , in conjunction with Aotearoa/New Zealand’s Ross Ice Shelf Programme and Antarctic Science Platform. We completed three missions  Icefin27 that evolved post-flotation. Upstream (eastern) crevasse walls were more steeply sloped than downstream walls, and asymmetric water circulation was measured within a crevasse cavity , ranged from +0.02 to +0.08 °C in the upper layer and from +0.25 to +0.34 °C in the lower layer  and fresh glacial meltwater (GMW). Dense HSSW is generated in the Ross Sea polynyas as brine is rejected during sea-ice formation, and a portion subsequently advects below the RIS, where it drives melting at the GL31. Resultant production of GMW at the colder, pressure-depressed freezing point (which decreases ~0.1 °C per 130 dbar) freshens and cools the HSSW. The stratification between the two layers reflects different GMW concentrations, which are quantified by their position along the Gade mixing line29 49. The 30 m water column was separated into two layers, with a colder upper layer that limited heat flux from the warmer lower layer to the ice base throughout this survey, within ~3.30 km of the contemporary GL. We mapped multiple crevasses in the ice-shelf base and observed supercooled water and marine ice refreezing within a crevasse. Crevasse distribution and refreezing processes are linked to overall ice-shelf mechanical strength39 but are difficult to observe with remote-sensing techniques. These in situ observations demonstrate the sensitivity of ice–ocean interactions to decametre-scale topography and temperature variability of thousandths of a degree.We used an ROV to map ice, ocean and seafloor interactions in the recently formed ocean cavity seaward of the KIS GL46 and establish a relationship that may inform interpretation of the geomorphological record in other deglaciating regions42. Previous work at KIS measured GL retreat at 28 ± 5 m yr−1 in the decade following 197449, and the modern satellite record shows that the GL has been static at the contemporary location ~4.2 km east of the KIS1 site since then50. Combined with the relatively lower ice-shelf melt rates of 0.26 m yr−1  estimated here, this evidence reinforces the role of grounded ice-stream dynamics in driving the variability of cold-cavity3 ice-shelf GLs7.At the seafloor, impressions left by basal crevasses as the ice lifted off support post-stagnation GL retreatThe survey reported here contributes a unified perspective on extant and past GL processes and demonstrates sub-kilometre-scale environmental variability below ice. This motivates continued efforts to pair the spatial context provided by ROV systems with traditional profiling instruments and long-duration mooring installations, which together can provide a complementary spatial and temporal understanding of the environments and processes below ice shelves.a, turbidity, dissolved organics and current speeds. For optical imaging, Icefin had two standard-definition forward cameras, one standard-definition aft camera and a 1,080p (progressive-scan) downward-looking camera with 5 cm scaling lasers. For acoustic imaging and sounding, Icefin had a forward imaging multibeam, upward-looking single-beam altimeter and a downward-looking Doppler velocity logger (DVL) for navigation with acoustic Doppler current profiling and altimetry. Real-time telemetry and data were relayed topside during missions through a Kevlar-reinforced 4.3 mm neutrally buoyant fibre-optic tether (Linden Photonics SPE7094) and recorded using Greensea Systems Balefire software with Lightweight Communications and Marshalling for data handling51. Uncompressed video was captured at 30 frames per second.The ROV Icefin -derived latitude and longitude52 at the surface to generate a mission track. A navigational challenge at high latitudes with near-vertical magnetic-field inclinations and high declinations is accurately determining the initial vehicle heading53. For this work, we employed a method by which heading was found on the surface as the bearing between two GPS-derived points aligned to the vehicle, using a long baseline (≥50 m) to reduce error to ~1°.Vehicle navigation and data georeferencing are accomplished by dead reckoning with the DVL, which determines 55.Navigational error accumulates in the position solution over the duration of a mission, and maximum error can be estimated as the distance between the borehole location in software and the actual borehole location the vehicle returns to. This distance was typically <50 m for missions up to 1 km in range  was established in November 2019. The Antarctica New Zealand team began drilling at HWD-1 on 11 December and completed a 22-cm-diameter pilot hole on 14 December. Ice-shelf thickness was 583.4 m, with a density-corrected surface elevation (correction described in Reference datums) of 76.9 m. Later reaming widened the borehole to at least 35 cm to enable ROV Icefin deployment. The hole was maintained by regular reaming until 23 December. As one component of the broader science programme, Icefin completed three missions under ice between 17 and 21 December . This permits Icefin to operate in supercooled waters and to be stored ‘on ice’ outdoors between missions, which greatly eased operational complexity and impact to the larger scientific programme.58 Toolbox z_from_p function considering sample latitude. Correcting measured freeboard (65 m) in the freshened borehole water column to mean seawater density at that depth  casts completed in McMurdo Sound) yields a freeboard, or surface elevation, of 77 m. The mean World Geodetic System 1984 ellipsoid surface elevation at camp was 27.5 m, so we note an ellipsoid–geoid offset of approximately −49.5 m at the KIS1 site. While ROV navigation underwater is referenced to sea pressure , this offset is noted for comparison with data referenced to ellipsoid elevation. Horizontal distances are indexed to GL position by compressing the 1.5 km composite section .Elevations (m) are reported with respect to local sea level, where the sea surface is 0 dbar (sea pressure), and are converted using the Gibbs Seawater (GSW)–1. For these missions, the applied offsets were +0.0175 °C and +0.249 mS cm–1.The NBOSI cabled conductivity and temperature (CT) sensor does not include a pressure sensor, so temperature and conductivity data were processed using the pressure calculated after correcting for the mounting offset to the primary vehicle pressure sensor and vehicle pitch. To obtain sea pressure, atmospheric pressure recorded just above the sea surface before each mission is subtracted. Despite routine calibration, previous fieldwork in McMurdo Sound established a linear temperature and conductivity offsets (biases) between the NBOSI cabled CT sensor when compared with the adjacent dissolved oxygen optode thermistor and an RBR Concerto3, where the latter two sensors consistently agreed. We treat this operationally by conducting an independent CTD cast immediately before or after each Icefin mission and then applying a constant offset based on observed water-mass properties by each sensor. Temperature offsets generally on order of 10–30 mK have persisted through pre- and post-season calibration efforts. Offsets in conductivity space are more difficult to constrain as the NBOSI CT is the only onboard conductivity sensor, but through co-calibration at KIS1 with two RBR Concerto3 profiling CTDs , we observe it to range between +0.01 and 0.25 mS cm57 using the Python3 implementation of the GSW Toolbox58 to derive SA, Θ, the potential density anomaly (σ0) and finally thermal driving. Contour plots and vertical profiles are generated by down sampling and averaging observations to 1 Hz, followed by applying a 4 s rolling mean and linear interpolation across a regularly spaced grid. Oceanographic visualizations follow best practices described in ref. 59.Conductivity values are further quality controlled for low readings resulting from low vehicle velocities (causing poor CT flushing) or the passage of ice or sediment particulate, which reduces the bulk conductivity of the sensing volume. Corrected and filtered data were then processed according to the Thermodynamic Equation of Seawater 2010SA and Θ endmember properties, following Wåhlin et al.60, after Gade28 (equation (29).Tocean and Socean are HSSW temperature (−1.903 °C) and salinity (34.925 g kg−1), respectively, LF is the latent heat of fusion for ice and CP is the specific heat capacity of water at HSSW temperature, salinity and pressure. These properties are taken from a range of HSSW observations made at similar depths during past Icefin missions in McMurdo Sound (near the RIS terminus and polynya). Fresh GMW concentrations of the KIS water-column layers are calculated from the slope of the Gade line.Mixing in the KIS water column falls along a Gade line calculated using HSSW equation ; see alsX, Y and Z velocities of the vehicle (Icefin) by pinging a static surface, where X, Y and Z are the respective major, minor and vertical axes of the vehicle. As the DVL calculates the vehicle’s velocities, it also retrieves water-column velocities in 2 m bins at a variable start distance from the vehicle. The minimum nominal altitude from an interface for current profiling to occur is 10 m. Gradients in vehicle pitch, roll, heading and speed dictate the distance of the first bin from the vehicle and the sampling frequency, which can reach a maximum of 5 Hz although we subsample velocities to a fixed rate of 1 Hz. The manufacturer-stated accuracies of ADCP current velocities are 1% of the vehicle’s velocity in that direction. As Icefin travels at speeds of ≤50 cm s−1, typically in the x direction, the uncertainty in vehicle X velocity recorded by the ADCP is ≤5 mm s−1. Water-column X velocities are differenced from the vehicle velocity, resulting in an uncertainty of ≤1 cm s−1. Vehicle velocities in the Y and Z directions are typically much lower than in the X direction, so associated water-column uncertainties are likewise lower.Ocean current speeds come from the LinkQuest NavQuest 600 Micro DVL on board Icefin, which doubles as an acoustic Doppler current profiler (ADCP). The DVL function calculates the X and Y velocities are analysed in this work. The following post-processing steps were independently applied to each velocity component:Remove measurements if bin depth is below seafloor depth.−1.Remove raw measurements of NaN or exactly 0 m sRemove measurements if absolute value of vehicle pitch or roll is greater than 30°.Convert measurements from vehicle reference frame to geographic reference frame.Apply 30 s mean filter.Filter measurements for gradients greater than 1 standard deviation from the mean in vehicle speed, pitch, roll and individual bin velocity.Collate bins by each up and down vehicle swoop into 1 m vertical bins, then remove measurements if they exceed 1 standard deviation of the mean for that 1 m depth range.Only X and Y velocity components were combined into current speeds.After completing these post-processing steps, the Seafloor elevations are derived from the average range of the four ADCP beams and corrected for tidal variation as recorded by a surface Global Navigation Satellite System station. Overhead ice-ceiling elevations were measured with a single-beam upward-looking altimeter and re-projected to correct for vehicle roll, pitch and yaw with a rotation quaternion.To enable accurate determination of the orientation and scale of ice and seafloor features, ambient ocean properties were input to determine the local speed of sound. Mosaics Fig.  are gene61 and recommendations in ref. 62, with calibration coefficients determined in ref. 63. Seawater oxygen saturation values to derive percentage of saturation for each observation are calculated using the GSW O2sol function 58. During the third mission, an offset of 8.01 μmol kg−1 was applied to an anomalous section of data from 21 December 2019 11:20:46 to 21 December 2019 15:10:30 utc on the basis of values for the same water mass observed during previous missions. The anomalously offset readings may have been due to transient ice formation or interference with the sensing foil.Dissolved oxygen data are corrected to compensate for pressure effects and salinity following manufacturer’s procedures a and fluorescent dissolved organic material data are output in counts, which are corrected per the manufacturer’s calibration by the scale value after subtraction of dark (zero) counts. Chlorophyll a and dissolved organic material had no systematic variation in the KIS1 water column. The pH and redox signals are output in analogue voltages and calibrated per manufacturer’s procedure. A pH signal, while on the order of sensor resolution, was detectable, and relatively higher, in the upper water layer. Redox voltages had no systematic variation in the KIS1 water column.Raw turbidity, chlorophyll Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41561-023-01129-y.Supplementary InformationSupplementary Sections 1 and 2 and references.Supplementary Video 1−1, and ‘range’ is the straight-line distance between the vehicle and the borehole. ‘Z_ice’, ‘Z_veh’ and ‘Z_bed’ indicate the elevation of the ice base overhead, vehicle and seafloor, respectively. ‘CT’, ‘SA’, ‘thermal driving’ and ‘DO’ indicate Conservative Temperature , Absolute Salinity (g kg−1), thermal driving (Θ - Θfp) and pressure- and salinity-corrected dissolved oxygen concentrations (µmol l−1), respectively. Laser scale is 5 cm when present.This video is a clip from a synchronized composite of Mission 2 showing seafloor Imprint A. Forward-looking multibeam sonar, forward-looking upper and lower analogue cameras and high-definition downward-looking camera streams are shown. Overlays  indicate the vehicle attitude in degrees of roll, pitch and yaw. ‘SOG’ indicates speed over ground in m sSupplementary Video 2This video clip depicts basal scours in the seafloor imaged during Mission 2. Layout and overlays are as defined for Supplementary Video 1.Supplementary Video 3This video is a clip from Mission 2 showing sediment-laden basal ice at the ice-shelf base. Layout and overlays are as defined for Supplementary Video 1.Supplementary Video 4This video is a clip from the crevasse survey at the end of Mission 3. Layout and overlays are as defined for Supplementary Video 1.


The results showed that MOF-1 had good acid–base stability and solvent stability. Furthermore, MOF-1 had excellent green fluorescence and with different phenomena in different solvents, which was almost completely quenched in acetone. Based on this phenomenon, an acetone sensing test was carried out, where the detection limit of acetone was calculated to be 0.00365% (volume ratio). Excitingly, the MOF-1 could also be used as a proportional fluorescent probe to specifically detect tryptophan, with a calculated detection limit of 34.84 μM. Furthermore, the mechanism was explained through energy transfer and competitive absorption (fluorescence resonance energy transfer (FRET)) and internal filtration effect (IFE). For antibacterial purposes, the minimum inhibitory concentrations of MOF-1 against Escherichia coli and Staphylococcus aureus were 19.52 µg/mL and 39.06 µg/mL, respectively, and the minimum inhibitory concentrations of MOF-2 against Escherichia coli and Staphylococcus aureus were 68.36 µg/mL and 136.72 µg/mL, respectively.Two triple interpenetrating Zn(II)-based MOFs were studied in this paper. Named [Zn Among many amino acids, tryptophan is one of the amino acids that make up human proteins, and abnormal metabolism of this amino acid can lead to anxiety, dysphagia, hallucinations, and delusions ,2,3. In 2O)2]·DMF}n with high solvent, pH, and thermal stability for the first time to detect two amino acids n (MOF-1) and [Zn31.5(DDBA)3]n (MOF-2) with three-layer interpenetration structure were designed and synthesized. MOF-1 had good stability and fluorescence characteristics, which can not only rapidly and sensitively detect acetone (LOD = 0.0036%), but also recognize tryptophan (LOD = 34.84 μM). In addition, the antimicrobial properties of MOF-1 and MOF-2 were compared. The results showed that the inhibitory zone of MOF-1 was significantly larger than that of MOF-2, and the inhibitory zone of Escherichia coli was larger than that of Staphylococcus aureus. In addition, the inhibitory zone of MOF-1 was larger than that of MOF-2 according to the minimum inhibitory concentration, which was consistent with the inhibitory zone experiment results. The antibacterial dynamic curves also reflected the broad-spectrum antibacterial properties of the two materials. In summary, this paper provides a useful reference for the detection of acetone and amino acids by fluorescence spectroscopy, and also provides considerations for the development of new antibacterial materials.In this paper, two kinds of metal–organic skeleton materials [Zn


Cow's milk allergy (CMA) is the most common food allergy in early childhood. Children with CMA require a precise and punctual diagnosis. Oral food challenge (OFC) is the gold-standard procedure for diagnosing allergies, but it is laborious and requires a particular setting. The aim of the study was to identify the cutoff value of serum allergen-specific IgE values able to predict a positive response to OFC. α-lactalbumin, β-lactoglobulin, and casein were measured.  Children with suspected CMA performed OFC with cow's milk (CM) or derivatives. Total IgE and specific IgE to raw CM, p = 0.03), α-lactalbumin (p = 0.013), β-lactoglobulin (p = 0.09), and casein (p = 0.019). The cutoff was, respectively: 5.13 kUA/L for raw CM, 1.47 for α-lactalbumin, 1.35 for β-lactoglobulin, and 4.87 for casein.  Seventy-two children performed OFC, and 30 (41.6%) had a positive response. The significant predictive factors were sensitization to raw CM extract ( This study allowed us to define a set of cutoff values for CM protein-specific IgE. However, these cutoffs should be interpreted not as a diagnostic tool for CMA but only predictive of response to OFC in a specific territory. Thus, the practical message may be that a value above the cutoff allows a good approximation to identify children to be started on OFC. Food allergy represents a relevant health concern, mainly in childhood; in particular, cow's milk allergy (CMA) is the most relevant in infancy and early childhood, affecting up to 5% of children . Howeverα-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), and casein (Bos d 8). The sensitization prevalence of these molecules is hugely variable, whereas the allergy prevalence in sensitized subjects is about 20% [From an immunological point of view, cow's milk (CM) consists of different allergenic molecules. The most relevant allergenic molecules are in Bos d , β-lactoin Bos d , and casAnother essential characteristic of CMA is the natural history: the onset usually occurs before 12 months of age, at the same time as the introduction of CM . MoreoveSymptoms usually occur immediately after CM ingestion and involve mainly the skin, gastrointestinal and respiratory tract, and, more rarely, the cardiovascular system . FurtherConsequently, CMA patients require adequate management based on thorough workup and close follow-up. Namely, an early and precise diagnosis is mandatory for prescribing a tailored diet and avoiding useless and potentially dangerous milk avoidance. The correct diagnosis of CMA requires a suggestive history, the documentation of sensitization, i.e., production of specific IgE, and consistency between history and sensitization .Sensitization may be investigated by skin prick test, with allergen extracts or fresh milk, and/or serum IgE assay. However, serum assay is more precise and reliable than skin testing . MoreoveThough, OFC should be performed only in particular settings, characterized by trained staff and equipped with adequate tools. Therefore, predictive tools that facilitate CMA suspicion can be handy in clinical practice. In this regard, several studies have attempted to define cutoffs of specific IgE levels, mainly molecular-specific, that could identify subjects with suspected CMA as a first step –28. HoweThis study retrospectively evaluated all children consecutively admitted to performing OFC from 2013 to 2022. The data were extracted from an electronic platform containing demographics, clinical history, and biological data.α-lactalbumin, β-lactoglobulin, and raw milk.Laboratory data included peripheral eosinophils, total serum IgE, and allergen-specific IgE to casein, IgE was measured using ELISA assays provided by Thermofisher . The total serum IgE normal level is <100 kU/L. Sensitization is defined when the value of allergen-specific IgE is >0.35 kUA/L.The inclusion criteria were the need to perform OFC for suspected CMA, suggested by symptom occurrence after CM ingestion, sensitization to CM extract and/or milk molecules, and pediatric age. Exclusion criteria were concomitant diseases and medications that could interfere with interpreting results.The OFC was performed using the following procedure: patients started from doses between 0.05 and 1 mL, progressively incremented. The OFC ended when a clinical manifestation occurred or a cumulative dose of 80 mL had been reached.The Ethics Committee of the IRCCS Istituto Giannina Gaslini approved the procedure . Parents signed informed consent.A univariate logistic regression model was applied to identify which variables best predict response after OFC. Subsequently, a receiver operating characteristic curve was used to assess the accuracy of different IgE classes in predicting the response. For each receiver operating characteristic curve, the area under the curve with the corresponding 95% confidence interval was calculated with the best cutoff point, which provided both the highest sensitivity and specificity.p value of 0.05 was considered significant. All statistical analyses were performed using SPSS 23.0 .A 2-tailed Seventy-two children, 52 males (72.2%) and 20 females (27.8%), mean age of 71.6 months, performed the OFC to CM, as reported in The mean total IgE level was 476.4 kU/L, the data concerning sensitization to milk and its molecules are in detail reported in p = 0.03), α-lactalbumin (p = 0.013), β-lactoglobulin (p = 0.09), and casein (p = 0.019). The cutoff was, respectively: 5.13 kUA/L for raw CM, 1.47 for α-lactalbumin, 1.35 for β-lactoglobulin, and 4.87 for casein. In addition, area under the curve (AUC), specificity, and sensitivity values are reported in detail in Thirty (41.6%) children were positive for OFC with milk . The anaCMA is the most frequent food allergy in children under the age of 3 years. Children with CMA require hardworking management by the physician and constant attention from the parents. However, it is quite common to observe children on unnecessary and often harmful exclusion diets at an age when milk is an essential nutrient for growth. Therefore, a precise and accurate diagnosis is the fundamental premise for properly managing CMA. In this regard, OFC remains the gold standard for providing a certain CMA diagnosis . HoweverThe present study derived from previous research showing that children with anaphylaxis to CM proteins had higher levels of specific IgE than children with mild CMA . In partAs we recently reported data concerning children with CMA and undergoing oral immunotherapy , the preα-lactoglobulin, 1.35 for β-lactalbumin, and 4.87 for casein. However, these cutoff values are only partially reliable considering the single AUC scores, specificity, and sensitivity outcomes. Moreover, these findings are partially consistent with the most recent literature data, as reported in α-lactalbumin , 1.85 for β-lactoglobulin , and 1.47  [α-lactalbumin, 1.59 for β-lactoglobulin, and 2.33 for casein [α-lactalbumin, 1.6 for β-lactoglobulin, and 0.95 for casein [α-lactalbumin, 7 for β-lactoglobulin, and 7.3 for casein [The results highlighted that the predictive cutoff for raw CM was 5.13 kUA/L, 1.47 for ity 98%) . A Danisr casein . A Spanir casein . Finallyr casein . In addir casein  reviewedTherefore, there is no complete agreement between the various cutoffs defined by the various studies. In addition, considering the criteria for validity , all the more reason that each study population has different outcomes. Consequently, the proposed cutoff values can have a somewhat relative reference value primarily because of the country under consideration. In this regard, the relative inconsistency among these studies could depend on different populations and the lack of sIgE/tIgE relationships for a more precise analysis.Therefore, the results should always be interpreted cautiously and related to the survey location. Furthermore, based on the validity criteria of the tests, it seems necessary to emphasize that the diagnosis of CMA mandatorily requires the performance of OFC.This study had several limitations, especially the limited number of subjects and the failure to perform a double-blind OFC with a placebo. In addition, the ratio between total IgE and allergen-specific IgE was not performed. On the other hand, this study reflects what can occur in real-life in a level 3 pediatric allergy center.In conclusion, this study allowed us to define a set of cutoff values for CM protein-specific IgE. However, these cutoffs should be interpreted not as a diagnostic tool for CMA but only predictive of response to OFC in a specific territory. Thus, the practical message may be that a value above the cutoff allows a good approximation to identify children to be started on OFC.


Polaromonas was ubiquitous, but differed genetically among diverse niches. Surface glacier-ice was dominated by Cyanobacteria, while the perennially ice-capped, well-mixed water column of Thores Lake had a unique assemblage of Chloroflexi, Actinobacteriota, and Planctomycetota. Species richness increased downstream, but glacier microbes were little detected in the lake, suggesting strong taxonomic sorting. Ongoing climate change and the retreat of Thores Glacier would lead to complete drainage and loss of the lake microbial ecosystem, indicating the extreme vulnerability of diverse cryohabitats and unique microbiomes in the Last Ice coastal margin.Arctic climate change is leading to sea-ice attrition in the Last Ice Area along the northern coast of Canada and Greenland, but less attention has been given to the associated land-based ecosystems. Here we evaluated bacterial community structure in a hydrologically coupled cryo-ecosystem in the region: Thores Glacier, proglacial Thores Lake, and its outlet to the sea. Deep amplicon sequencing revealed that  The Canadian Arctic Archipelago (CAA), one of the most glaciated areas of the Arctic, has experienced rapid ice attrition over the past decades, especially at the coastal margin of the Last Ice Area (LIA). Wind and currents push the sea ice up against the coast of the far northern islands in the CAA, resulting in some of the thickest sea ice of the Arctic Ocean  and persPolaromonas are known to thrive in these cold environments ” in combination with either of the following keywords: “ice”, “glacier”, “Arctic” and “Antarctica”. This produced a set of 855 unique Polaromonas sequences. From these sequences, whole genomes as well as plasmids were removed, and we retained only sequences from the 16 S rRNA gene (376 sequences). To these we added the 16 S sequences from uncultured bacterium reported to be Polaromonas identified by Darcy et al. [Polaromonas in our pipeline. These sequences were aligned with mafft (v7.453) [Polaromonas 16 S genes or gene fragments. The phylogenetic tree was produced as described in the previous paragraph. For accession numbers and references of sequences obtained on NCBI, see Supplementary Table y et al.  (55 sequ(v7.453) , the ali(v7.453) , and non(v7.453) , produciComamonadaceae , and represented 19% of reads in the cryosphere samples and 8.2% of lake sequences.The core microbiome across the Thores glacier-lake-outflow system contained 141 amplicon sequence variants (ASVs), with around one tenth belonging to the Polaromonas ASVs were common to all compartments of the glacier-lake continuum  dominated other members of the genus, accounting for over 25% of reads per sample , closely related to P. cryoconiti, was also detected in all compartments, but at much lower relative abundances   Fig. . Two phyica Fig. . Two lowens Fig. , were unens Fig. . All phyens Fig. , and theice Fig. .Polaromonas phylotype in our analyses (pol1), i.e. the ASV present in the greatest fraction of samples and at the greatest abundance  grouped closer to the root of the tree  groupedPolaromonas dominated, they were only part of the rich assemblages identified in these cryohabitats. The Deinococcota phylum (including Deinococcus) , Bacteroidota  , Pseudanabaenales  and the Bdellovibrionata order 0319-6G20  were significantly more characteristic of the cryosphere than lake and river water  on the other hand contained a large proportion of sequences whose taxonomy at the phylum level could not be assigned  and Pseudanabaenaceae (Cyanobacteria) than ice , Anaerolineaeceae (Chloroflexi), Gemmataceae, Phycisphaeraceae and Pirellulaceae (Planctomycetota)  were also consistently present throughout the water column  Fig.  and wereinterface) was dominated by Proteobacteria , and a decreasing proportion of unassigned taxa , with cryosphere-associated samples clustering along Axis 1, except for the bottom of lake ice  . This genus is ubiquitous in the polar regions and other cold environments [Polaromonas ASVs between habitats in the Thores glacier-lake-river continuum.Cold-dwelling aw ponds  and in Law ponds , a largeronments  and has ronments , consistPolaromonas phylotypes identified in Thores were generalists, i.e. organisms that appear to survive under diverse conditions, consistent with the metabolic versatility found within this genus [Polaromonas genus in Thores, was also ubiquitous. This phylotype was closely related to P. ginsengisoli, which was initially isolated from soil, and shares 98.6% of its 16 S rRNA with P. eurypsychrophila [Polaromonas genus are generalists with broad tolerances that likely endure year-to-year, as data presented here were collected over two consecutive summers.Many of the is genus . This inhrophila , but hashrophila . The conP. eurypsychrophila, which was originally isolated in glacier ice on the Tibetan Plateau [Polaromonas have been suggested to operate on glacial surfaces, including sorting by local habitat conditions, but also community interactions with other prokaryotes and with eukaryotic microbes [Phylotypes pol3 & pol5 were consistently found in the lake, as well as in lesser abundances in other compartments downstream of the lake. These organisms were closely related to  Plateau . These pmicrobes , which mPolaromonas phylotypes specific to the glacier-lake interface implies that this is a mixing and transition zone, with insufficient time or selective forces to result in detectable genetic differentiation within this taxon. A limnological study of Thores Lake showed that this zone has many distinct properties, including in nutrients, conductivity and turbidity [Polaromonas) that were observed in the present study to be unique to this zone, the interface also contained a distinct phytoplankton community, with a higher proportion of green algae and the chlorophyte pigment chlorophyll b relative to the main body of the lake, which was characterized by chrysophytes and fucoxanthin [Leptolyngbya and Pseudanabaena that dominated the glacier samples, indicating strong selection against them, or reduced dispersal abilities of these biofilm-forming taxa. As sampling occurred over two different summers, there may be inter- or intra-year variation patterns that were not observed. However, Cyanobacteria have been found to exhibit long-term persistence in perennially cold habitats, suggesting robust assemblages on the glacier surface [The lack of urbidity . Consistoxanthin . We foun surface .Polaromonas in the mixing zone suggests that these phylotypes have sufficiently broad tolerances to persist in such conditions, and to resist microbial loss processes during their short residence time in the transition zone, while other groups like Cyanobacteria that originate from the glacier are more likely to be filtered in the interface.The presence of both lake and glacier Polaromonas in the littoral mixing zone. In the present study, we were unable to identify the source of most taxa, especially in the lake, and the percentage of taxa whose source was identified was lower than at Ward Hunt. This may be due to the long term stability of Thores Glacier [Source tracking has been used in many instances to model microbial connectivity across glaciers , 59, 60, Glacier  and its  Glacier  that allPolaromonas phylotypes in Thores Lake but also by the distinctive assemblage of other taxa. Actinobacteriota accounted for approximately 20% of reads throughout the water column; cells of this phylum are typically small in size and are often abundant in oligotrophic freshwaters [Gemmataceae, is known to include psychrotolerant taxa [Polaromonas was only detected at the bottom of Lake Hazen where glacial meltwater joins the water column [The strong in-lake effect on microbiome composition was indicated not only by the unique shwaters . Planctoshwaters , and oneant taxa . The higant taxa , Japan, ant taxa , USA. Thr column , whereasr column ; and BacCyanobium in the bottom of Thores Lake ice since this picocyanobacterial genus was abundant in the surface waters of the lake.Another cryospheric compartment of interest in the Thores Lake system is the perennial ice cover, which would protect the lake from direct airborne seeding. There are likely, however, to be complex interactions between the lake ice and the underlying lake waters. While dissolved compounds  are excluded from lake ice during its formation, particles can be retained in the new ice . This frThe results obtained here underscore the high bacterial species richness in the Thores ice-water ecosystem, and the diverse array of community assemblages that segregate among cryohabitats. The presence of ASVs does not necessarily imply that the cells are actively growing, but transcriptomic studies of glacier microbiomes elsewhere have indicated that a large proportion of the observed community has the potential for metabolic activity . HoweverPolaromonas may evolve into novel ecotypes, potentially losing cold-adaptation functions and genetic diversity.Arctic ice-dwelling microbes are at the forefront of climate change, and are threatened by habitat extinction. There is therefore an urgent need to understand this unique biodiversity before it is lost to further warming , 75. MicPolaromonas, will likely persist in this refuge into the immediate future, but will be prone to more severe effects of ongoing climate change.The Last Ice Area is projected to remain ice-covered into the middle of the century when summer ice will have largely disappeared from the rest of the Arctic Ocean . The perSupplementary Information