The serum- and glucocorticoid-induced kinase 1 (SGK1) promotes cell survival under stress conditions and facilitates the emergence of drug resistance in cancer. The underlying mechanisms of these observations are not fully understood. In this study, we found that SGK1 activity is suppressed by the action of the S/T phosphatases PP5 and PP2A, which constantly dephosphorylate SGK1. Using newly developed anti-phospho SGK1 antibodies and inhibitors of phosphatases, we determined that the high degree of dephosphorylation is caused by two factors: the tendency of SGK1 to unfold, which makes it dependent on Hsp90 chaperone complexes composed of four proteins, Hsp90/CDC37/PP5/SGK1, and where the phosphatase PP5 persistently dephosphorylates SGK1 within the complex. SGK1 binding to PP2A regulatory subunits B55γ and B55δ brings PP2A catalytic subunit close to exposed SGK1 phosphoresidues. A further association of phosphorylated pS37-FAM122A—an endogenous inhibitor of PP2A—to the holoenzyme diminishes dephosphorylation of SGK1 mediated by PP2A. Our study also reveals that genotoxic stress can reverse the dominant impact of phosphatases over kinases by activating the DNA-dependent protein kinase, which enhances mTORC2 activity directed to SGK1. Thus, our results provide insight into a molecular pathway that enables SGK1 to gain phosphorylation and catalytic activity and promote cell survival, potentially diminishing the efficacy of cancer treatments. As the DNA damage response operates in many cancer cells and is further induced by chemotherapies, the findings of this study could have significant implications for the development of novel cancer therapies targeting SGK1. SGK1 is a protein kinase that belongs to the serine/threonine kinase family , 3, 4. BDespite their similarities, deleting or overexpressing AKT or SGK1 in mice leads to distinct phenotypes, highlighting fundamental differences between the two kinases. Abrogation of AKT1 primarily induces metabolic, growth, and proliferation effects , 10, whiHigh levels of SGK1 expression have been observed in many cancers, which are attributed to various factors, such as promoter methylation and aberrant splicing of mRNA , 18. ThiSGK1 exhibits catalytic activity upon undergoing two sequential phosphorylation events. The first event involves the phosphorylation of the carboxyterminal HM at residue S422 by mTORC2, creating a docking site for PDK1 . The secA and A commercial human SGK1 anti-pS422 antibody was employed to detect HM phosphorylation of SGK1 in cell lysate of 293T and the indicated human cancer cell lines. However, the phosphorylation of SGK1’s HM was found to be very low or undetectable even after treatment with insulin and insulin-growth factor (IGF1) A and S1.B). These results suggest that the observed phosphosignal aligns with the established upstream activation pathway of SGK1 . Calyculin-A also elevated the phosphorylation of endogenous SGK1 in HeLa cells and all three SGK1 isoforms: canonical, Δ60SGK1, and SGK1.1 (D and E). The isoforms differ in their amino termini but possess identical catalytic domains and HMs .It has been suggested that mTORC2 plays a role in phosphorylating the turn motif (TM) of AKT at T450 during protein translation . This phFig. 2A). To investigate this, alanine or aspartic acid substitutions were made to prevent or mimic phosphorylation, respectively. The alanine substitutions decreased the pS422 signal, while the S401D mutant significantly increased it (A and B). Additionally, phosphorylation of the TM and HM increased SGK1’s activity, as evidenced by increased phosphorylation of two known substrates, NDRG1 (C). However, phosphorylation of the TM only partially protected SGK1 from phosphatases, as demonstrated by the persistent and significant effect of Calyculin A on the mutants .It remains unclear whether the turn motif and hydrophobic pocket of SGK1 function similarly to those of AKT1. SGK1 has two potential phosphorylation sites in its carboxy terminus TM, namely S397 and S401 A. To inveased it , A and Bs, NDRG1  and NEDDs, NDRG1  Fig. S3A. To invA). Surprisingly, the substitution of Q130 with lysine in SGK1, as well as the replacement of the turn motif with that of AKT1 (TMAKT1), did not prevent dephosphorylation of S422; in fact, it was even more severe in these mutants . Furthermore, a chimera consisting of the AKT1 catalytic domain and the SGK1 carboxy terminus also showed a low level of pS422 (C and D). These results suggest that the phosphorylation of TM and its putative binding site in the catalytic domain do not impede phosphatase access to SGK1’s critical phosphoresidues.In SGK1, residue Q130 replaces a conserved lysine found in the phosphorylated TM-binding pocket of other similar kinases, potentially reducing its affinity to phosphorylated TM  Fig. 2AAFig. 2A.C and D), it was hypothesized that PP5 might dephosphorylate SGK1 while associated with the Hsp90/CDC37/PP5 complex. The N-terminal domain of PP5 contains three consecutive tetratricopeptide repeats that bind to the Hsp90 C-terminus tetratricopeptide repeat–binding site (EEVD) and activate PP5 (A), and knockout of PP5 in 293T increased pS422 level above that of WT cells, consistent with SGK1 being a substrate of PP5 (B). To investigate the compartment where PP5 dephosphorylates SGK1, 293T cells transfected with SGK1-HA and FL-PP5 or SGK1-HA and Strep-PP5 (active or inactive) were used for pulldowns. All four proteins of the complex  were detected regardless of whether the pulldown was conducted with SGK1 or PP5 (C and D), and SGK1 was found to be dephosphorylated when PP5 was active within the chaperone complex but not with the inactive PP5 mutant (F). These findings indicate that PP5 dephosphorylates SGK1 in addition to CDC37, a known PP5 substrate . This observation suggests that the dephosphorylation of CDC37 by PP5 facilitates its dissociation from the chaperone complex and promotes the disassembly of other components. However, when both WT PP5 and PP5-IA were present in the complexes, this effect was less pronounced (C). Importantly, related kinases such as AKT1 and p70S6K were not detected in the pulldowns (E), likely due to their higher structural stability than SGK1, which reduces their reliance on chaperones. Taken together, these findings indicate that the high propensity of SGK1 to unfold makes it dependent on the Hsp90/CDC37 complex, where PP5, one of its components, dephosphorylates SGK1 to maintain a low phosphorylated state.Complexes exclusively containing inactive PP5 displayed enhanced associations with all proteins within the complex, particularly CDC37 D. This oonounced C. Importulldowns E, likelyA and S4A); whereas among the PP2A inhibitors , only FAM122A .The impact of PP1 and PP2A on the state of SGK1 phosphorylation was examined by expressing the corresponding endogenous inhibitors of these phosphatases. Among the PP1 inhibitors tested  , 37, onlFigs. 4 FAM122A , 40, 41 A). Owing to the low expression of B55α, we could not confirm interaction of SGK1 with this subunit. We also examined whether FAM122A binds to B subunits. Co-IP assays detected robust signals of FAM122A with B55β, B55γ, and B55δ; low level of B55α expression again prevented a definitive conclusion with this subunit. None of the B56 subunits associated with FAM122A . On the other hand, direct association of SGK1 and FAM122A was not found . These results support a model wherein PP2A holoenzymes containing B55γ/δ subunits associate to and dephosphorylate SGK1, and binding of FAM122A to these complexes inhibits PP2A activity thereby increasing SGK1 phosphorylation.PP2A is made of three subunits: catalytic C, scaffolding A, and various regulatory B subunits that confer substrate specificity to the complex . We firsFig. S5C and D). We also found that a different isoform, FAM122B , also increased SGK1 phosphorylation, and the corresponding mutation S25 abolished the effect (E).It has been recently reported that the checkpoint kinase 1 (Chk1) phosphorylates FAM122A at residue S37 creating a 14-3-3 binding site that leads to its translocation from nucleus to cytosol . We askee effect E.A and B), even though endogenous Chk1 was active as indicated by its phosphorylation at residue pS345 (C).Given the functional importance of pS37, we raised and affinity purified a FAM122A anti-pS37 rabbit serum to examine the phosphorylation state of FAM122A. 293T or FAM122A KO cells were treated with bleomycin (BLM) to induce DNA breaks and activation of Chk1 or with an inhibitor of Chk1 (prexasertib). Unexpectedly, none of these treatments changed the state of p37-FAM122A phosphorylation , A and Bue pS345  Fig. 5CCA and B,D shows that FAM122A is phosphorylated in multiple sites, mainly S37 and S76, and BLM does not alter the pattern of S37 phosphorylation. Substitution of each of the phosphorylated sites by alanine did not change pS422-SGK1 or pS37-FAM122A levels, only pS37 was sufficient and necessary to induce these effects (E), indicating it is the only phosphorylated residue with functional impact on SGK1, and pS37 is independent of Chk1 activity. Furthermore, an in vitro assay that incorporates radioactive phosphate into the FAM122A peptide containing S37 did not detect phosphorylation by 245 S/T protein kinases that included Chk1/2 (KinaseFinder by Reaction Biology)  . OverallA) and simultaneously increasing abundance, consistent with phosphorylation stabilizing the SGK1 protein (C).While exploring the effects of Chk1 on FAM122A, we noticed that several drugs that induce DNA breaks in various ways such as BLM and etoposide also increased SGK1 phosphorylation in a time- and concentration-dependent manner A and sim protein C.Figure A, increased phosphorylation of the histone H2AX at pS139 (γH2AX) . As shown in Figure 6 (γH2AX)  and Chk1B). This result was confirmed in 293T cells expressing reduced levels of DNA-PK (∼20% compared to WT cells) where BLM-induced SGK1 phosphorylation was suppressed , meaning that SGK1 is downstream DNA-PK signaling.The first response induced by DNA breaks is the activation of one or more of any of three related kinases: ATM (Ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-dependent protein kinase (DNA-PK), each senses a different type of DNA lesions and responds activating distinct pathways to repair DNA or halt cell division . Their lFig. 6D). To distinguish these possibilities, mTORC1/2 were inhibited with Torkinib (E). To consolidate this result, we used genetic suppression of Rictor that is an essential component present in mTORC2 complex but not in mTORC1. In resting and BLM-treated RICTOR KO cells expressing ∼0.3 level of Rictor compared to WT cells, phosphorylation of pS422 in SGK1 and pS473 in AKT1 were undetectable (F), supporting DNA-PK working upstream mTORC2 as illustrated by the indirect pathway-1 of Figure 6D.As DNA-PK and mTORC2 are phosphatidylinositol-3-phosphate kinase related kinases, we considered whether SGK1 is a direct substrate of DNA-PK or alternatively, it could operate indirectly through activation of mTORC2 D. To disTorkinib , which dTorkinib E. To contectable F, supporG), whereas suppression of endogenous SGK1 in HeLa by siRNA produced the opposite effect, that is, it reduced survival of BLM-treated cells by ∼5% (H). To contextualize this finding, Figure 6H also shows that inhibiting AKT1 with AZD5363 leads to a decrease in viability in the presence of BLM by approximately 4%, which is comparable to that of suppressing SGK1 using siRNA. The small changes in survival imparted by SGK1 could be relevant in tumors dependent on high PI3P levels that are also treated with AKT1 inhibitors, a scenario where SGK1 would confer a competitive survival advantage and increase chances to develop resistance to chemotherapeutics.Finally, we examined the contribution of SGK1 to cell survival. 293T treated with BLM and with an inhibitor of AKT1 (AZD5363) to mimic cancer treatments that suppress AKT1 activity. SGK1 conferred a small (∼12%) though significant increase in 293T survival G, whereas by ∼5% H. To conA central and unanticipated result of this study is the very low phosphorylation level of SGK1 found in steady-state and growth factor–stimulated cells, implying that SGK1 is kept mostly in an inactive state. The low degree of SGK1 phosphorylation is not due to weak activity of the two upstream kinases mTORC2 and PDK1 because AKT1 exhibited robust phosphorylation in the same cells and conditions; rather, we show that it stems from relentless dephosphorylation by phosphatases, primarily PP2A and PP5. These findings implicate cellular phosphatases and their regulation as major determinants of SGK1 activity under various cellular contexts and environmental conditions. The propensity of SGK1 to undergo dephosphorylation is due to failure of the kinase domain to adopt a compact structure wherein the two crucial phosphorylated residues S422 in the HM and T256 in the activation loop are protected from access to phosphatases. Owing to structural instability, SGK1 heavily relies on protein chaperones not only for initial folding but throughout its lifetime . We demo2+ with ATP or thapsigargin, PKA activation with forskolin, activation of AMPK with AICAR, or inhibition of cyclin dependent kinases with flavopiridol (data not shown). The molecular mechanism by which pS37-FAM122A inhibits PP2A also remains to be elucidated, though there is precedent from ENSA/Endosulfine—another endogenous inhibitor of PP2A—that turns into an inhibitor when the Greatwall kinase phosphorylates residue S67 at mitotic entry. pENSA binds much more avidly and is dephosphorylated more slowly than other substrates consequently; all other substrates retain phosphorylation for longer time and the average phosphorylation level increases , AKT (#4691), Phospho-AKT-Ser473 (#9271), Phospho-AKT-Thr308 (#13038), Chk1(#2360), Phospho-Chk1-Ser345 (#2348), Phospho-Histone H2A.X-Ser139 (#2577), NDRG1(#9395), Phospho-NDRG1-Thr346 (#3217), Phospho-NEDD4L-Ser448 (#8063), Hsp90 (#4877), CDC37(#4793), HA Tag (#3724), and FLAG Tag (#14793) were from Cell Signaling Technology. Phospho-SGK1-S422 (#44-1264G), FAM122A (#MA5-24510), V5-HRP (#R961-25) were from Invitrogen; NEDD4-2 (#ab131167) from Abcam; PP5 antibody (#sc-271816) from Santa Cruz Biotechnology; FAM122A (#NBP2-31646) from Novus Biologicals; GAPDH-HRP (#BE0034-100) from Easybio; actin-HRP (#AM8502b) from ABGENT; and secondary antibodies, anti-rabbit IgG HRP (#A16110), and anti-mouse IgG HRP (#A16072) from Invitrogen.Constructs were generated by PCR of the corresponding coding DNA (cDNA) with tags -HA, FLAG, V5, or Strep and cloned into pCDNA3.1 vector using Q5 high-fidelity DNA polymerases (#M0491). Site-directed mutations were introduced with Agilent PfuTurbo DNA Polymerase (#600257).All procedures related to animals were approved by Tsinghua Institutional Animal Care and Use Committee . SGK1 pS422 and pT256 antibodies were made to synthetic phosphopeptides corresponding to mouse SGK1 amino acid residues 416-429 (EAFLGFpSYAPPVDSC) and residues 249-262 (EHNGTTSpTFCGTPEC) with an additional cysteine in the N terminus. These peptides were conjugated to keyhole limpet hemocyanin (mcKLH)  and used as antigens for immunization of New Zealand White rabbits. Antibodies recognizing nonphospho-SGK1 were subtracted from immunized sera by passing through corresponding unphosphorylated peptide (same sequence as above without phosphorylation) chromatographic columns. Phospho-antibodies were further affinity purified from the subtracted fraction on phosphopeptide chromatographic columns . ELISA and western blots were performed to assess the antibody specificity. Lamda PP  was used to dephosphorylate SGK1 proteins according to manufacturer’s instructions. FAM122A phospho-S37 antibody was generated by immunizing rabbits with synthetic phosphopeptides corresponding to human FAM122A amino acid residues 34-40 (RSNpSAPL) (Dia-An Biotechnology).2 incubator in high glucose Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (BI), 1% penicillin, and 1% streptomycin (Hyclone).HEK293T (CL-0005) from Procell, HeLa (CCL-2), A549 (CCL-185), MCF7 (HTB-22) from ATCC, MDA-MB-231 , KPC2  were cultured at 37 °C in a humidified 5% COFAM122A, PP5, or DNA-PK KO 293T cells were generated by CRISPR/Cas9 system. sgRNA targeting FAM122A sequence: 5′-GCAGATAAGCCACTCCTGGG-3′ as previously described , sgRNA tCells were transfected with plasmid DNAs or siRNAs using Lipofectamine 2000 according to manufacturer’s directions. SiRNAs targeting SGK1 were designed as previously described .Serum starvation was carried out in Dulbecco’s modified Eagle’s medium for 20 h. Drugs used were as follows unless otherwise indicated: insulin , insulin like growth factor 1 , torin 1 , torkinib , pictilisib , rapamycin , Calyculin A , okadaic acid , tautomycetin , BLM , etoposide , prexasertib , AZD7648 , AZD5363 .g for 5 min at 4 °C to remove debris. Protein concentrations were determined with the BCA Protein Assay kit . Equal amounts of total protein from lysates were immunoprecipitated with FLAG magnetic beads , HA magnetic beads , or rabbit polyclonal antibody with Protein G agarose  and then eluted with SDS-PAGE loading buffer. Co-immunoprecipitations of PP5 and SGK1 were carried out with Strep-Tactin XT Superflow  and eluted with buffer BXT , pH 8.0) or with HA magnetic beads.Cells were washed twice with cold DPBS  and then lysed with TENT 1%  on ice in the presence of protease inhibitors  and phosphatase inhibitors cocktail . Cell lysates were centrifuged at 13, 500Equal amounts of whole cell lysate proteins or immunoprecipitants were loaded to 12% SDS-PAGE  and transferred to polyvinylidene difluoride membranes . Membranes were blocked with 5% skim milk in TBS-T  and then probed with indicated primary and secondary antibodies. Chemiluminescence was detected with Millipore Immobilon Western HRP Substrate (#WBKLS0500) in Bio-Rad ChemiDocTM XRS+.versus total peptides corresponding to that site.293T cells transfected with FAM122A-HA were treated with 30 μg/ml BLM or dimethylsulfoxide for 6 h and then lysed with TENT 1% supplemented with protease and phosphatase inhibitors. FAM122A was immunoprecipitated with HA magnetic beads and resolved on 12% SDS⁄PAGE. Bands containing FAM122A were visualized with ImperialTM Protein Stain  and excised for trypsin digestion, followed by LC-MS/MS analysis (Thermo-Dionex Ultimate 3000 HPLC system interfaced to a Thermo Scientific Q Exactive mass spectrometer). The Q Exactive mass spectrometer was operated using Xcalibur 2.2 software , and the MS/MS spectra were searched against human FAM122A from UniProt using Proteome Discoverer . Site phosphorylation ratio of FAM122A was calculated by the peak area of phosphorylated peptides Total RNA was extracted from cells using TRIzol Reagent , and single strand cDNA was prepared by reverse transcription of total RNA using ProtoScript II First Strand cDNA Synthesis Kit . Quantitative RT-PCR was carried out using RealStar Green Fast Mixture with ROX  in StepOnePlus Real Time PCR System (Applied Biosystems). Primer sequences were as follows: SGK1, F5′-GCAGAAGAAGTGTTCTATGCAGT-3′ and R5′-CCGCTCCGACATAATATGCTT-3′; GAPDH, F5′- ACAACTTTGGTATCGTGGAAGG-3′ and R5′- GCCATCACGCCACAGTTTC-3′.293T cells were infected with lentivirus packaged in 293T cells. After at least 10 days of selection with 1 μg/ml puromycin, cells were seeded in 96-well plates at a concentration of 1000 cells per well. Twenty four hours later, cells were treated with 1 μM AZD5363 and 30 μg/ml BLM for 2 days. HeLa cells transfected with scramble or SGK1 targeting siRNA were seeded at a concentration of 2000 cells per well and treated with 30 μg/ml BLM for 5 days 24 h post transfection. Cell viability was assessed using Cell-Titer Glo  according to manufacturer’s instructions.https://imagej.nih.gov/ij/). Statistical analyses and visualizations were performed by GraphPad Prism 9 (Graphpad.com). All data are represented as mean ± SD, and significance was tested using unpaired Student’s t test or one-way ANOVA as indicated.Western blot signals were quantified using Image J software (All data supporting the findings of this study are in the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Glacier and ice sheet surfaces host diverse communities of microorganisms whose activity (or inactivity) influences biogeochemical cycles and ice melting. Supraglacial microbes endure various environmental extremes including resource scarcity, frequent temperature fluctuations above and below the freezing point of water, and high UV irradiance during summer followed by months of total darkness during winter. One strategy that enables microbial life to persist through environmental extremes is dormancy, which despite being prevalent among microbial communities in natural settings, has not been directly measured and quantified in glacier surface ecosystems. Here, we use a combination of metabarcoding and metatranscriptomic analyses, as well as cell‐specific activity (BONCAT) incubations to assess the diversity and activity of microbial communities from glacial surfaces in Iceland and Greenland. We also present a new ecological model for glacier microorganisms and simulate physiological state‐changes in the glacial microbial community under idealized (i) freezing, (ii) thawing, and (iii) freeze–thaw conditions. We show that a high proportion (>50%) of bacterial cells are translationally active in‐situ on snow and ice surfaces, with Actinomycetota, Pseudomonadota, and Planctomycetota dominating the total and active community compositions, and that glacier microorganisms, even when frozen, could resume translational activity within 24 h after thawing. Our data suggest that glacial microorganisms respond rapidly to dynamic and changing conditions typical of their natural environment. We deduce that the biology and biogeochemistry of glacier surfaces are shaped by processes occurring over short  timescales, and thus are susceptible to change following the expected alterations to the melt‐regime of glaciers driven by climate change. A better understanding of the activity of microorganisms on glacier surfaces is critical in addressing the growing concern of climate change in Polar regions, as well as for their use as analogues to life in potentially habitable icy worlds. Mittiva2 into a pile, which was mixed to homogenize the sample. Separate aliquots were collected into sterile WhirlPak® bags and sterile 50 ml Falcon® tubes for genomic analyses, BONCAT, pH, conductivity, and analyses of dissolved inorganic compounds. For dissolved organic carbon (DOC) analysis, separate samples were collected with sterile metal trowels into acid‐washed  and ashed  glass jars.Snow samples were collected from the top 1 cm of the snow surface. Ice samples were collected from surface scrapings (top 2–5 cm of the ice surface) using ethanol‐sterilized ice axes and spatulas. We scraped an ice surface area of ~1 mSamples for molecular analyses, pH, conductivity, DOC, and dissolved inorganic compounds were thawed at room temperature on return to the field laboratory, and immediately processed once fully melted. For DNA and RNA analysis, 500 ml (IS19‐13), 250 ml (IS19‐14), and 500 ml (MIT5) of melted ice or snow were filtered through single use, sterile, 0.2 μm cellulose nitrate filters . Filters were removed from the units, folded with sterile forceps, and transferred into sterile 5 ml cryotubes that were immediately frozen and returned to the home laboratory in a liquid nitrogen cryo‐shipper and stored at −80°C until DNA/RNA extraction  in Citifluor AF‐1 antifading solution (Electron Microscopy Sciences). Filters were then covered with a coverslip and slides were kept in the dark until microscopy analyses. We also performed in‐situ killed control BONCAT experiments by fixing thawed samples with glutaraldehyde  before the in‐situ incubation with HPG. Following the incubation, all other steps were performed as described above. This control served to detect and measure the occurrence of non‐specific labelling .We used BONCAT to detect translationally active bacteria in‐situ on Langjökull, targeting both snow and ice supraglacial habitats. BONCAT is a non‐destructive click‐chemistry‐based method to visualize protein synthesis in uncultured environmental microorganisms in‐situ  and IS19‐14 (ice) from Langjökull, and samples of ice from Mittivakkat glacier (MIT5), were also transported frozen to the home laboratory. Samples were stored frozen (−20°C) for approximately 6 months. Then samples were thawed and incubated in singlicate at 2°C under an 18 W fluorescent light for an initial incubation period ranging in duration from 1 hour to 38 days (hereafter referred to as the “pre‐incubation” period). Following this, samples were subject to a second incubation period at 2°C in the light, lasting 24 h following the addition of HPG  (hereafter referred to as the “HPG‐incubation” period), and quenched with glutaraldehyde  as a fixing agent as described above. Here, we present total incubation time as the sum of the durations of the pre‐incubation period (prior to the addition of HPG) and the 24‐h HGP‐incubation period. Following the addition of glutaraldehyde, samples were filtered, and HPG‐containing proteins were fluorescently labelled as described above.2.4Slides were analyzed using an epifluorescence microscope (Leica DM 2000) and images were acquired with a DFC 420C camera and a FI/RH (Leica) filter system following Winkel et al., . Each fi2.5The bacterial and eukaryotic diversity of Langjökull and Mittivakkat supraglacial habitats were characterized by 16S and 18S rRNA gene sequencing, respectively. DNA was extracted in a sterile laminar flow hood from all samples using the DNeasy PowerSoil DNA Isolation kits (QIAGEN GmbH). 16S rRNA genes were amplified using the bacterial primers 341F (5′‐CCTACGGGNGGCWGCAG) and 785R (5′‐GACTACHVGGGTATCTAATCC) spanning the V3‐V4 hypervariable regions. 18S rRNA genes were amplified using the eukaryotic primers 528F (5′‐GCGGTAATTCCAGCTCCAA) and 706R  according to the manufacturer's protocol. TotalRNA libraries were prepared using the NEBNext Ultra II Direction RNA Library Prep kit (New England Biolabs GmbH) for Illumina. Extracted RNA concentrations were below the detection limit for all samples and therefore RNA was directly used in library preparation after extraction rather than being normalized to a standard concentration. The manufacturer's protocol was followed exactly using 12 μl of RNA directly (no dilution). The RNA was fragmented for 8 min, and 16 cycles were used for the library amplification. Libraries were indexed using the NEBNext Multiplex Oligos for Illumina. The resulting library concentrations and size distributions for IS19‐13 and IS19‐14 totalRNA were measured by qPCR and an Agilent 2100 Bioanalyzer, respectively. Equimolar pools were prepared for totalRNA, and quality checks showed the absence of primer dimers. The totalRNA pools were loaded on a V2‐flow cell for a 2×250 paired‐end sequencing run on an Illumina MiSeq according to the manufacturer's instructions. The library for sample MIT5 was measured and visualized on an Agilent 4150 Tapestation where concentration (measured on Qubit 4) and insert size were used to calculate the final molarity for sequencing. MIT5 was run as part of a larger pool on an Illumina NextSeq using the Mid Output v2.5 (300 cycles) kit.Metatranscriptomic sequences were quality checked using FastQC v.0.11.8, and subsequently, quality filtered using the “illumina‐utils” package of tools developed by Eren et al., . All Fas2.72 after UV photo‐oxidation (185 nm) in a Gräntzel thin‐film reactor following Regenspurg et al.  following methods described in McCutcheon et al. . The preg et al. .2.8B, into two pools which are distinguished by their state of activity or dormancy: B1 represents active microorganisms and B2 represents dormant microorganisms. A system of coupled ordinary differential equations  are viable but must undergo activation before they are capable of growth. Dormancy requires that an organism (i) is not growing or dividing  and (ii) has a lower metabolic demand than when it is active  can die, utilize DOC to meet maintenance power requirements, and transition between active and dormant states.Our simplified model only considers the heterotrophic bacteria on the glacier surface. Therefore, we assume that the catabolic energy of microorganisms represented by B1) is given by:t is time (hours), VB1 represents the rate of new biomass growth, DB1 denotes the death rate of B1, ξB1 corresponds to the deactivation of B1 into B2, and ϵBn is the activation of B2 to B1.The rate of change of active biomass (B1 is the concentration of active biomass (μg C l−1), vmax is the maximum growth rate of active biomass (hour−1), DOC is the concentration of dissolved organic carbon (μg C l−1), and Kv is the half‐saturation constant for microbial growth according to standard Michaelis–Menten kinetics  is given as:αBn is the mortality rate constant of biomass Bn (h−1).Microbial death of biomass pool B2) is given by:DB2 represents the death rate of B2, ξB1 corresponds to the deactivation of B1 to B2, and ϵBn is the activation of B2 to B1.The rate of change of dormant biomass (2.8.2B1), to dormant (B2). We prescribe that the deactivation and reactivation of biomass  depends on temperature. In natural environments, the conditions that determine state‐changes depend on a multitude of environmental factors, including the potential supply of catabolic energy to the cells . HoweveB1 to B2 is given by:RS,D is the specific rate of deactivation.The deactivation (ξ) of biomass ϵ) of biomass B2 to B1 is given by:RS,A is the specific rate of activation.Similarly, the activation  under unfavourable conditions when the temperature is below KS and thus θS < 0.5. Similarly, there is a net activation of biomass  when the temperature rises above KS and thus θS > 0.5.y et al. , is adap2.8.3MBn, is described as:mBn is the specific maintenance power requirement of Bn. Maintenance power requirements (mBn) are described as a proportional carbon cost per unit of biomass per hour ).Both active and dormant cells carry out maintenance from exogenous  catabolism. The rate of exogenous catabolism, 2.8.4DOC is given as:Y represents the growth yield, ∑MBn represents the sum of DOC consumption for maintenance by B1 and B2, and ∑DBn represents the sum of the dead cells that contribute DOC from B1 and B2.The rate of change of 2.8.5−1, which is representative of the average measured DOC concentration at MIT5. We used an initial bacterial biomass carbon concentration of 0.55 μg C L−1, which is based on 5 × 104 bacterial cells ml−1 and represents the measured concentration of bacteria occurring on glacier surface ice with a medium coverage of glacier algae, from the western margin of the Greenland Ice Sheet .The mathematical expressions described above and in Table 33.1We found that HPG was actively incorporated by bacteria incubated in‐situ on the glacier surface and ex‐situ in the lab. After HPG incubations, some cells acquired a distinct green fluorescence signal corresponding to the azide dye addition to the BONCAT label Figure .Image analyses from the incubated samples revealed blue DAPI‐stained cells, and green BONCAT+ cells Figure . An overp = .493, Table p = .001), and there was greater variability between replicates for the ice habitat .In all in‐situ incubations from Langjökull, approximately half of the bacterial cells were found to be translationally active  Figure . The actp = .573 and p = .259, respectively), whilst the active fraction of cells in Langjökull glacier ice was found to increase from an average of 13% at 1 day post‐thaw to 35% at 3 days post‐thaw (35%) , all samples from all three habitats (Mittivakkat ice and Langjökull snow and ice), including those incubated with HPG immediately (1 hour) post‐thaw, contained active cells Figure . On averp < .001) and ice (p < .001). The mean active fraction of cells in ex‐situ incubations of Mittivakkat ice samples was 45–70% (SD = 14%), in Langjökull snow it was 26–70% (SD = 14%), and in Langjökull ice it was 13–57% (SD = 22%). Across all ex‐situ incubations for samples from Langjökull, the active fraction of cells in the snow (IS19‐13) was found to be higher than in ice (IS19‐14) (p < .001).Longer‐term ex‐situ incubations were conducted for up to 39 days. The activity was overall higher among cells from Mittivakkat glacier ice samples than in Langjökull snow (p < .001). However, five days after thaw (ex‐situ), the fraction of active cells in Langjökull snow samples  appeared to reach in‐situ activity levels  (p = .069). Nevertheless, variation in the active‐fraction of cells across singlicate incubations and incubation times of 1–39 days was high and recurrently deviated above and below in‐situ activity levels. For samples of ice from Langjökull, the active fraction of bacteria reached in‐situ levels or higher two days after thawing.For Langjökull, we compared the active fraction of cells in‐situ with ex‐situ incubations following thaw Figure . For one3.2Leptolyngbyaceae (6%) . Alphaproteobacteria were mostly represented by the family Acetobacteraceae . Within the Bacteroidota, the genera Hymenobacter  and Ferruginibacter  were dominant. Actinomycetota were mostly represented by the families Microbacteriaceae , Nocardiaceae , and Sporichthyacea . The most abundant bacterial phyla in ice from Mittivakkat (MIT5) were also Bacteroidota (29%), Actinomycetota (20%), Pseudomonadota (19%), and in contrast to Langjökull, we also found WPS‐2 (17%). Bacteroidota were mostly represented by the genera Hymenobacter (16%) and Solitalea (9%). Actinomycetota mostly comprised the family Microbacteriaceae (14%). Within the Pseudomonadota, Alphaproteobacteria (12%) were most abundant with Rhodanobacter (2%) being the dominant genus, followed by Gammaproteobacteria (7%) with Acidiphilium (4%) as the most abundant genus.For bacteria, there was a high degree of similarity on all phylogenetic levels between the snow (IS19‐13) and ice (IS19‐14) samples from Langjökull Figure . The mos%) Table . Within Hydrurus  and Chlorophyceae . The two most abundant ASVs corresponding to Chlorophycaea shared the highest similarity with several Chloromonas species and were thus assigned to Chloromonas spp. . ASVs matching Chlainomonas sp.  and Chlamydomonas nivalis  were also present in lower abundances.The eukaryotic community composition varied fairly evenly on the phylum level across the Langjökull snow and ice samples Figure . The algus Table . ChloropAncylonema nordenskiöldii was only present at MIT5 (38%), whereas the second ASV  could also be found in low abundances in the other samples . Ancylonema alaskanum was present in both ice samples  albeit in low abundance.Phragmoplastophyta (Charophyta) were highly abundant in the ice sample from Mittivakkat glacier (56%), in contrast to the samples from Langjökull . The Phragmoplastophyta were entirely comprised of Zygnematophyceae. One ASV matching The fungal community composition comprised mostly of Basidiomycota, Chytridiomycota, and Ascomycota. Basidiomycota were most abundant in IS19‐13 (17%), followed by MIT5 (10%) and IS19‐14 (8%). Chytridiomycota showed the highest relative abundance in IS19‐14 (18%) followed by MIT5 (13%) and IS19‐13 (10%). Ascomycota were most abundant in MIT5 (5%) and below 2% in the other two samples. In addition, Ciliophora  and Cercozoa  showed a higher relative abundance in all three samples.3.3Chlorophyceae were particularly strongly represented in Langjökull snow and ice , as well as C. nivalis , and Prasiolales in Langjökull snow and ice . A. nordenskiöldii, which were found to be dominant among the Charophyta in Mittivakkat glacier ice, were strongly represented in 18S rRNA genes extracted from total RNA from Mittivakkat.Relative abundance bar plots of the recovered 16S rRNA genes from total RNA reveal diverse communities of potentially active bacteria. We found that the taxonomic classifications for both DNA and RNA were broadly similar for bacteria: the organisms that were generally abundant in DNA were also detected in the RNA reads. Specifically, there was a high representation of sequences belonging to Actinomycetota, Pseudomonadota, and Plancomycota among total RNA sequences in Langjökull snow and ice, and a high representation of cyanobacteria in RNA reads from Langjökull and Mittivakkat ice. Notably, Bacteroidota were not strongly represented in the RNA reads, despite their high relative abundance in the DNA sequences across all samples. For eukaryotes, the taxonomic classifications for DNA and RNA were broadly similar, with a high representation of DNA sequences belonging to Chlorophyta in all samples. In particular among the Chlorophyta, 3.42+ and Ca2+ ions , as well as Mn2+, whilst glacier ice, especially from Mittivakkat, had higher concentrations of DOC, Fe2+, K+, and Zn2+. NO3− and SO42− were below detection limits (50 ppb) in all samples.We found that snow and ice from Langjökull and ice from Mittivakkat had largely similar pH, conductivity, and concentrations of dissolved Mgns Table . Snow fr3.5−1  freezing, (ii) thawing, and (iii) freeze–thaw cycles. We account only for heterotrophic bacteria – which comprise most bacteria found to inhabit glacier surfaces  triggered a rapid transition of the active microbial community to a dormant state  triggered a rapid transition of initially dormant microorganisms to an active state  to 0.83 μg C L−1 (day 10), with microbial growth sustained throughout the simulation, and some consumption of DOC.We found that thawing  examined the response of an initially dormant community to a thaw period (3 days), followed by a freeze (3 days), thaw (3 days), and final freeze (1 day)  to mid/late (Iceland) summer. This is, in both cases, at the height of the melt season, when liquid water (at ~0.1°C) is widespread on the glacier surface, coinciding with high bacterial and algal activity  and Ochrophyta (Hydrurus) among the total and active eukaryotes in our samples – which is typical of red snow and yellow snow algal blooms, respectively  reaction rates, such as the incorporation of αBn; maintenance constant, mBn; half‐saturation for growth, Kv; and steepness of state‐change dependency, stS) have never been experimentally or theoretically determined for glacier surfaces. Thus, we associate a high degree of uncertainty to both parameter values and the model output – including the rates of growth and death, the production and consumption of DOC, and the rapidity of state‐transitions. Our modelling framework is designed to be flexible to allow for additional complexity to be introduced, such as (i) resolving different groups of microorganisms with varying rates of transitioning between active and dormant states, (ii) resolving additional biomass groups such as eukaryotic photosynthetic algae, and (iii) integration with albedo and melt models  following freezing (∼6 months) is illustrative of the capacity for microbial communities to respond rapidly to periodic thawing – even if this thaw is of short duration . Winter warming and rain events are likely to become more frequent occurrences as the Arctic responds to anthropogenic climate change  freeze–thaw conditions, as well as distinct seasonal cycles during which microorganisms must contend with prolonged periods of cold and darkness lasting several months or longer. Moreover, microorganisms in glacial environments must be capable of reactivation during periods of favourable environmental conditions . Here, we have taken the first steps to quantify the prevalence of active and dormant microorganisms in Arctic supraglacial habitats. We found that microbial communities in our glacial snow and ice samples were comprised of active cells (on average 45%–62%), but also contained inactive or dead individual cells (on average 38%–55%). We provide evidence that the diverse assemblages of microorganisms on glacier snow and ice surfaces responded to environmental conditions by switching between active and inactive metabolic states, which may be critical to enabling their persistence through adverse conditions. There is a need to further investigate the links between microbial state‐switching and resource availability and nutrient limitation on glacier surfaces. Crucially, our results suggest that glacial microorganisms are able to respond to short melt‐events occurring on the timescale of hours to days – which is sufficiently short that periodic melting on glacier surfaces potentially impacts the functioning of glacial ecosystems and biogeochemical cycles. Enhanced winter warming is predicted to become more prevalent as a result of future climate change and could therefore bring about ecological changes to glaciers. Progress in measuring, mechanistically capturing, and quantifying state‐switching and the associated biogeochemical consequences will be fundamental in understanding the ecological role of dormancy in glacial ecosystems, and the potential impacts of climate change. Studying the dynamic nature of microbial habitats on glaciers and ice sheets is critical to understanding them as refugia for microbial life, analogue environments of potentially habitable icy worlds, and regulators of regional and global biogeochemical cycles.The authors declare no competing interests.Appendix S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Bacillus subtilis chromosome. We followed the motion of both ICEBs1 and the chromosomal integration site in real time within individual cells. ICEBs1 exhibited a wide spectrum of dynamical behaviors, ranging from rapid sub-diffusive displacements crisscrossing the cell, to kinetically trapped states. The chromosomal integration site moved sub-diffusively and exhibited pronounced dynamical asymmetry between longitudinal and transversal motions, highlighting the role of chromosomal structure and the heterogeneity of the bacterial interior in the search. The successful search for and subsequent recombination into the integration site is a key step in the acquisition of integrating mobile genetic elements. Our findings provide new insights into intracellular transport processes involving large DNA molecules.Integrative and conjugative elements (ICEs) are mobile genetic elements that can transfer by conjugation to recipient cells. Some ICEs integrate into a unique site in the genome of their hosts. We studied quantitatively the process by which an ICE searches for its unique integration site in the  Conjugation is a horizontal gene transfer process that contributes significantly to the generation of genetic diversity in bacteria . During Although stably maintained integrated in the host chromosome, ICEs can excise from the chromosome to form a circular DNA that can then transfer from a donor cell to a recipient where it is first extrachromosomal but then integrates into the chromosome of the new host to form a stable transconjugant. Many ICEs integrate into a specific, often unique site in the bacterial chromosome. Locating and integrating into the proper site is an essential step in the lifecycle of these widespread elements, yet nothing is known about how an ICE locates the proper integration site.Bs1 is a relatively small ICE (∼20 kb) found in the chromosome of Bacillus subtilis . In the absence of its normal attachment (integration) site, ICEBs1 can integrate into secondary sites with reduced frequency, and integration into these secondary sites is detrimental to both the element and its bacterial host  or LacI-GFP (lacO sites inserted at the ICEBs1), as represented in Escherichia coli revealed that the motion of chromosomal loci is subdiffusive and consistent with fractional Brownian motion  is found and integration takes place. Our results underscore the importance of chromosomal structure in the search, the efficiency of target location within a crowded environment, and heterogeneity as an essential principle in bacterial subcellular behavior.Here, we focus on the quantitative characterization of the n motion ,17. More supplemented with l-arabinose (1%), phenylalanine (40 μg/ml), tryptophan (40 μg/ml), threonine (200 μg/ml) and methionine (40 μg/ml), as needed. Xylose (1%) was added to induce expression from Pxyl-rapI .600 of ∼0.8 to 1. Cells were then diluted into defined minimal medium with arabinose as the carbon source to an OD600 of ∼0.02. After at least three to four generations (OD600 of ∼0.2), expression of rapI from Pxyl-rapI was induced by addition of xylose to the donors. Cells were grown for another hour, to allow for ICEBs1 gene expression and excision, and then washed three times. Donors and recipients were then mixed at a ratio of ∼1 donor per three recipients at a concentration of ∼108 cells/ml. Five microliters of this mating mix were placed on an agarose pad . The pad was circular with a volume of ∼100 μl and a height of ∼2 mm. Then the pad was laid on top of a MatTek device that has coverslip glass (#1.5) built into a 35 mm dish. The effect of different treatments was studied by addition of 5 μl of 100× stock solutions on top of the agar pad, at 37°C and then tracking cells using fluorescence microscopy. The 100× concentrations of compounds and total times of cell tracking were: Chloramphenicol ; rifampicin  and 2,4-dinitrophenol (DNP) . Time-lapse images were recorded typically between 1–3 h following mating.Donors and recipients were colony purified from frozen (−80°C) stocks on LB agar plates with the appropriate antibiotics overnight at 42°C. Cells from a single colony were inoculated into liquid LB medium and grown to an optical density ODB. subtilis strains used are listed in Table Bs1 null JH642 derived strain (JMA222) (tetR-mApple was constructed by replacing the yfp coding sequence in pPSL38 (mApple (cgeD. Diagnostic PCR was used to verify integration by double-crossover. ΔyddS::tetO240 is linked to attB and was constructed by inserting the tetO array from pLAU44 (yddS and confirmed by diagnostic PCR.The train JMA2 (22). An pPSL38  with mAp  with a motorized x–y-stage (Prior). Stage, sample holder and objective  are inside a temperature box (Okolab) at 37°C. Phase contrast and fluorescence images were acquired with a sensitive camera (Andor DU-987), using the NIS-Element Advanced Research software . An experiment consists of a sequence of images taken from a sample. An image consist of 512 × 512 pixels (px) with a resolution of 109.76 nm/pixel. Images were typically acquired every about 4 s or 30 s over a total time of around 6 min or 45 min. These parameters allowed us to capture essential features of the dynamics while minimizing photo-bleaching. Cells were illuminated with light of the appropriate wavelength, produced by a light emitting diode set (CoolLED pE-4000), using appropriate filter sets for each fluorescent reporter: GFP ) and RFP .attB loci and one ICEBs1 were analysed. In the strain in which ICEBs1 was fully integrated (SAM837), two attB foci and two ICEBs1 foci were analysed in each cell. The presence of two foci indicates that that region of the chromosome has already been replicated. All image processing and data analysis were carried out using a MATLAB (MathWorks) algorithm developed in our laboratory. The code aims at finding the trajectories of ICEBs1 and chromosomal attachment sites labeled with fluorophores and characterize their dynamics. NIS-element images were converted to TIFF files and then individual cell segmentation was carried out in phase contrast images to determine the cell boundaries. The algorithm then calculates the center of mass of each cell and its centerline.Only transconjugants of strain SAM318 that displayed two Bs1 and attB positions were then determined with sub-pixel resolution by fitting a Gaussian plus a constant to pixel clusters above an appropriate threshold within the cell boundaries. Their positions were then determined with respect to the cells’ center of mass, with ICEBs1 and attB loci were followed ∼2–4 generations after mixing donors and recipients. The percentage of transconjugants was ∼5%, a mating efficiency that is comparable to previous observations . Two-dimensional trajectories of attB and ICEBs1 in representative cells are shown  found typically at ¼ and ¾ along the longitudinal axis . Plots of the two-dimensional Euclidean distance between ICEBs1 and attB trajectories with which they overlap are shown in Figure attB and ICEBs1 loci in a strain (SAM837) bearing integrated ICEBs1 that was unable to excise  of the total number of transconjugants, displayed mobile behavior without being integrated. These included 14 ± 2%  that displayed fast motion of ICEBs1 that often spanned a large portion of the cell volume. This fast motion was punctuated by episodes of more compact exploration, until eventual integration at attB , we calculated the spatial distribution of ICEBs1 along the main cell axis. We found that the distribution of freely moving ICEBs1 peaks at the cell center, and overlaps moderately with the longitudinal distribution of attB loci, allowing ICEBs1 to locate attB  axis and divides by binary fission. This structure has important functional implications for the organization of its chromosome and the segregation of copies into daughter cells -uracil (HPUra), a specific inhibitor of DNA polymerase III ; obstructed diffusion (OD); and fractional Brownian motion (FLM) . In cont} Figure . For an ocument} . Both caocument} ,44. Nextdocument},42,45:attB motion. In fact, fractional Brownian motion describes quantitatively the behavior of the velocity autocorrelation function. Plotting the attB locus is self-similar  of cells, ICEBs1 displayed highly mobile behavior spanning most of the cell (n = 868). Similarly to the untreated case, this motion is visibly episodic, with local explorations interrupted by rapid longitudinal motion . As discussed below, we believe that the structure of the bacterial chromosome  direction, in a bottle-brush configuration , as measured by the apparent diffusion coefficient. These features are consistent with ICEBs1 motion within an open, porous chromosomal mesh. The mesh size E. coli , since chloramphenicol results in stabilization of mRNAs (Bs1s mobility. In addition, a compacted chromosome may also severely reduce accessibility and search for attB due to a decrease in mesh size.Whereas the compaction of the chromosome by inhibition of translation, and the consequent availability of a larger nucleoid-free volume might have led to greater mobility of ICEof mRNAs , and in of mRNAs ,76. The Bs1 to changes in the compaction state of the bacterial chromosome induced by antibiotics, we anticipate that the dynamics of the search process might also be affected by the size of the ICE itself. ICEBs1 is ∼21 kb, but ICEs range from ∼12 kb to ∼500 kb (Based on the observed response of ICE ∼500 kb ,78. ICEs ∼500 kb . UnderstBs1 differs substantially from that used by lambda phage to find its attachment site during lysogenization (Bs1s, which can span all a recipient's interior until the integration site attB is found, phage DNA remains confined near its point of entry primarily near the poles in an infected cell. There, the integration site approaches it, driven by the replication and segregation of the host genome (Bs1 motion may be required to enable encounter with its integration site, as ICEBs1 can likely enter a recipient cell at any point along the cell's contour (The search mechanism used by ICEnization . In shart genome . Thus, bt genome , in orde contour , in cont contour , and yetBs1 differs considerably from that of lambda phage, our experiments following the search at the single-cell level reveal that the efficiency of target location by ICEBs1 for an integration site is very high. Indeed, the fractal dimension associated with the trajectory of an ICEBs1 prior to integration is Bs1 is space-filling, a crucial feature to ensure a thorough search for the attB target. In the unlikely case in which an integration site is not found, the rapid cell to cell spreading of ICEBs1 along chains, common in microbial communities on surfaces, increases further the efficiency of conjugation, amplifying the number of cells that acquire conjugative mobile genetic elements (While the variegated dynamical behavior of ICEelements ,83,84.The task faced by an ICE as it searches for a unique site of integration along a host chromosome is highly challenging. Bacterial chromosomes are typically millions of base pairs long, and the number of possible addresses is thus very large. Furthermore, the search is compounded by the complex nature of the crowded, viscoelastic bacterial interior and its non-equilibrium, active nature. Compared to transcription factors searching for cognate binding sites, the small number of ICEs in a recipient is small and their size is about an order of magnitude larger. Yet, the search is efficient and target sites are located within physiologically relevant timescales. How is this task accomplished?attBs motion, to active systems in which the anisotropy might prove crucial for uncovering details that are lost in the steady-state (Our results provide key insights about the strategy used by integrating conjugating DNA elements in their search. First, far from being a diffusive process, the search is episodic, allowing for compact local explorations interspersed by periods in which ICE motion is fast, crisscrossing the cell within seconds-long intervals. This highly heterogeneous dynamic behavior and the attendant search strategy, is consistent with bacterial chromosome structure, and underscores the fact that heterogeneity is essential in bacterial subcellular organization, despite the fact that a bacterium consists of only one compartment. Second, the motion of both the integration site and ICE is subdiffusive and remarkably, highly anisotropic. Our observations indicate that the anisotropy reflects bacterial chromosome structure and not simply the stadium-like, elongated shape of a cell and its boundaries. From a theoretical perspective, our work stimulates the quest for an anomalous diffusion model that can characterize the anisotropy in the diffusion exponents. The interest extends beyond the specific context of ICEs and dy-state ,86.Bs1, or other long DNA molecules imported into a cell during horizontal gene transfer processes been tuned by evolutionary pressures? Answering this question is critical to optimize the use of ICEs for the genetic modification of undomesticated bacteria (Bs1 and its access to the integration locus. To what extent is anisotropic dynamical behavior advantageous to search process? Future studies could address these questions and the extent to which other aspects of the search were honed by evolution.Our results raise many fundamental evolutionary questions. For example, has the size of ICEbacteria . Has thehttps://github.com/ferinat/Bacillus-Analysis-Public.The software for segmentation is available at gkad068_Supplemental_FilesClick here for additional data file.


Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disease with multiple genetic and a variety of environmental risk factors. Although current drugs significantly aid in controlling the disease, many people have led to the application of complementary therapies due to the common belief that they are natural and safe, as well as due to the consideration of the side effect of current drugs. Curcumin, cannabinoids, wheatgrass, Boswellia, wormwood and Aloe vera are among the most commonly used complementary medicines in UC. However, these treatments may have adverse and toxic effects due to unintended interactions with drugs or drug-metabolizing enzymes such as cytochrome P450s; thus, being ignorant of these interactions might cause deleterious effects with severe consequences. In addition, the lack of complete and controlled long-term studies with the use of these complementary medicines regarding drug metabolism pose additional risk and unsafety. Thus, this review aims to give an overview of the potential interactions of drug-metabolizing enzymes with the complementary botanical medicines used in UC, drawing attention to possible adverse effects. Inflammatory bowel diseases (IBDs) are idiopathic inflammatory disorders that have a chronic course characterized by exacerbation and remission throughout an individual’s lifetime. It is thought that a genetic predisposition as well as environmental factors play a role in the manifestation of these diseases. IBDs are classified into two types: ulcerative colitis (UC) and Crohn’s disease (CD) , 2. UC aAlthough the burden of the disease is increasing, therapy for UC is challenging due to the absence of clearly defined medical treatments to manage periods of recurrence and remission with long-term consequences. However, drugs such as 5-aminosalicylate, azathioprine, 6-mercaptopurine, cyclosporine, and antitumour necrosis factor monoclonal antibodies have been utilized to treat UC . The maiThe microsomal cytochrome P450 system is a product of the CYP superfamily of genes, and it is a key electron transport chain in the membrane of the endoplasmic reticulum of the cell. These enzymes are critical in the oxidative activation, inactivation, and excretion of the majority of xenobiotics and drugs, as well as in controlling the degree of their toxicity, among other functions –24. CytoHumans express a total of 57 CYP proteins; of these, seven are found in mitochondria, where they catalyse particular oxidation events associated with steroid metabolism. Only six genes —CYP1A2, CYP2C9, CYP2B6, CYP2C19, CYP2D6, and CYP3A4— make substantial contributions to drug clearance out of the remaining 50 genes studied. It has been estimated that approximately 60 % of all drug-related material provided to patients is cleared by these six CYPs. The CYP3A subfamily is responsible for metabolizing approximately half of the medicines that have been cleared by CYPs –30. The A computerized search of PubMed, Web of Science, and Scopus for randomized controlled trials involving ulcerative colitis was conducted to identify the natural plants used by patients to treat ulcerative colitis. The unfavourable effects of these natural products on cytochrome P450-mediated drug metabolism were reviewed in this review without addressing the therapeutic effectiveness of these natural compounds. A promising choice for curing ulcerative colitis are natural plants and products; however, they are not as safe as the public believes and may lead to serious consequences in both short-term and long-term usage. The individual plants are discussed in detail in alphabetical order below.Aloe vera is a plant that belongs to the Liliaceae family and is extensively used in traditional medicine worldwide. Aloe vera is considered the most widely studied and commercialized plant species. It has been used as a folk remedy for the treatment of a wide range of diseases, including skin problems and gastrointestinal disorders ,34. The It has been observed that aloe vera contains 94.8 mg per kilogram of dry matter of quercetin . QuercetFurthermore, anthraquinones are the most important type of components in aloe species, including aloeemodin, rhein, emodin, chrysophanol, physcion and their glucosides ,53. StudRhein is another anthraquinone metabolite that is abundantly present in aloe vera and other plants . Rhein iIn addition, aloe-emodin inhibited the levels of N-acetylation of sulfamethoxazole and p-aminosalicylate, as well as the expression of NAT1 mRNA and protein in A375 cells. S2 cells . Inhibit[34,In conclusion, aloe and its phytochemical ingredients have been shown to inhibit all six main CYP families, which are responsible for drug metabolism. Additionally, they seem to block NAT enzymes, which are involved in the metabolism of mesalamine, which is one of the most widely prescribed medications for ulcerative colitis patients. These findings highlight the need for more research on aloe vera, such as hazardous chemical discovery, mechanism-based suppression of CYP activities, and toxicokinetic interactions in both metabolically competent cell and animal models. As a result, aloe is a natural herbal substance that is far from being safe for human use, as suggested in the literature 34,,46. WhenAnanas comosus) is a tropical plant whose edible fruit is one of the most important tropical fruits in the international trade market [Pineapple , which were activated by human xenobiotic-metabolizing enzymes produced in immortal mammalian cells, and the results are very promising. They employed genetically modified metabolically active lung fibroblasts of Chinese hamster V79 cells that included human CYP1A2, NAT2*4 and SULT1A1*1, which were obtained from hamster V79 ,78. DespThus, the inhibitory impact on CYP2C9 is thought to be the major mechanism underlying pineapple juice interactions, while minor interplay with CYP1A2 and NAT should also be considered. It is reasonable to conclude that pineapple is the safest herbal product to consume among all others; nonetheless, even slight fluctuations in plasma concentrations owing to pineapple interactions may be harmful to individuals taking drugs that are metabolized by the enzyme CYP2C9.Andrographis paniculata, a member of the plant family Acanthaceae, is one of the most prominent medicinal herbs in the world and has been used for centuries in the treatment of a wide range of diseases throughout Asia, America, and Africa. In addition, it contains a number of photochemical compounds with distinct and fascinating biological features. Randomized, double-blinded and multicentre studies investigating the A. paniculata extract have suggested similar efficacy to mesalamine for patients with mild to moderate ulcerative colitis [ colitis ,79–82.Andrographis paniculata extract and the major component andrographolide greatly alter both the expression and activity of important P450 isoforms, such as CYP1A1, CYP1A2, CYP2A4, CYP2B9, CYP2B10, CYP2C9, CYP2E1 and CYP3A4, in human hepatocytes [Andrographis paniculata extract (APE) inhibited the activities of CYP1A2 and CYP2C in rat and human liver microsomes, but this inhibition was not confirmed in vivo [Studies have reported that atocytes –90. Ther in vivo –94. Furt in vivo .Muthiah and colleagues  investigAndrographis paniculata at a concentration of 100 g/mL significantly inhibited CYP3A4 (>85%). This result might be attributed to andrographolide, which had good fitting scores in the docking experiment, as well as the presence of various phenolic compounds in the studied extracts [Andrographis paniculata and its phytochemicals, such as andrographolide, bacoside A, and asiaticoside, were investigated for their ability to inhibit pooled microsomal CYP450s as well as human recombinant CYP3A4, CYP2D6, CYP2C9, and CYP1A2 enzymes, followed by the determination of the inhibitory concentration (IC50). The most potent inhibitory action was shown by A. paniculata against CYP3A4 and CYP2D6 [Aqueous extracts of extracts . In addid CYP2D6 ,94,98. Fd CYP2D6 .As a result, several different types of CYP isoforms have been observed to interact with AP when tested in vitro. It was discovered that this herb inhibited CYP1A2, CYP2C9, and CYP3A4 more than CYP2D6, CYP2E1, and CYP2B isoforms, indicating that this plant had a distinct inhibitory pattern.Artemisia absinthium L., is an important perennial shrubby medicinal plant native to Asia, the Middle East, Europe, and North Africa [Artemisia absinthium for six weeks [Wormwood, or h Africa . A. absih Africa –104. Desh Africa . In an oix weeks . After 6ix weeks ,107.Wormwood contains a variety of phytochemicals, including kaempferol, quercetin, and coumarin, which have been demonstrated to inhibit either cytochrome P450 isoform 3A4 or 2B6, which are responsible for the metabolism of its main ingredient artemisinin in humans ,108,109.Eupatilin and jaceosidin are pharmacologically active flavones discovered in Artemisia species. Although they are not reported in A. absinthium, similar methoxylated flavones have been reported ,115. EupA. annua, A. afra, A. abrotanum, A. absintium, A. apiacea, A. herba alba, and A. pontica) tested. The amount of CYP3A4 inhibition observed with all Artemisia samples was unexpectedly large, reaching up to 6 times the levels observed in ketoconazol or diluted grapefruit juice. Because grapefruit juice has a reputation for being the most powerful CYP3A4 inhibitor derived from a plant, this is shocking to learn that all Artemisia samples exhibit this high level of CYP3A4 inhibition.The modulation of cytochrome P450 activities of Artemisia plant extracts (water or ethanolic) on human cells, as well as the antiinflammatory activity, have been researched by pioneering researchers ,118. TheIn conclusion, the DDI prediction indicated that Artemisia might result in both induction and inhibition. Additionally, combination medicines (including artemisinin) cleared by CYP2B6 and CYP3A4 and, to a lesser extent, CYP1A2 and CYP2C9 may need a dosage adjustment to sustain treatment or prevent adverse effects. In addition, considering its well-known potential toxic effects  highlighBoswellia serrata tree is a moderately large deciduous tree that is native to India, the Middle East, and Northern Africa. When the paper-thin bark of the Boswellia tree is peeled back, sticky oleoresin is revealed. Extracts of this oleoresin have been utilized in Ayurvedic medicine to treat inflammatory diseases and gastrointestinal issues for its antiinflammatory, antiseptic, astringent, and stimulating action [The g action –122. Stug action ,124. Clig action ,125,126.Withania somnifera, Boswellia serrata, Zingiber officinale, and Curcuma longa, was investigated for its toxic effects and cytochrome P450 enzyme inhibition activity [Boswellia serrata extract play an important role [A type of Ayurvedic medicine, BV-9238, a proprietary formulation of ant role .Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata) was determined using LC/LC/ESI-MS. The boswellic acids 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid showed the highest inhibitory activities for the CYP enzymes 2C8/2C9 and 3A4, with IC50 values in the range of 5 to 10 μM. Since the gum resin of B. frereana also showed a comparable strong inhibition of the applied CYP enzymes, the boswellic acids are obviously not primarily responsible for the potent inhibitory activity of the frankincense extracts. Therefore, phytoconstituents other than boswellic acid and its metabolites exert P450 inhibitory actions. Since herbal extracts of B. serrata are widely used for the treatment of inflammatory diseases, further in vitro and in vivo studies are needed to clarify the clinical relevance of these findings [The inhibition of the applied CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4 by the frankincense extracts  inhibitors based on the modulation of Pgp-mediated efflux. Thus, it also affects drug transport in the gastrointestinal tract as well as drug metabolism [The authors provided case reports of suppression of metabolic pathways  . Incidenefficacy ,130,131.tabolism .It became apparent that Boswellia extracts and frankincense contain several phytochemicals that modulate CYP2C19, CYP3A4 and CYP2C9 as well as Pgp, suggesting unavoidable adverse interactions that may pose serious health consequences. It is obvious that more research is needed to obtain definitive conclusions about Boswellia toxicities in the future.Cannabis sativa, usually known as Indian hemp, is an herbaceous annual plant that has been grown mostly in Central Asia (India and China) since antiquity [ntiquity . C. satintiquity . It inclntiquity ,136. Δ9-ntiquity ,137–143.Numerous in vitro and in vivo investigations suggest that cannabis may impact P450 isoenzymes to alter drug metabolism. The comprehensive analysis implicated the role of P450 in the metabolism of various exogenous cannabinoids, including tetrahydrocannabinol , cannabidiol , and cannabinol  –148. CanCannabis, particularly CBD, has a strong inhibitory effect on CYP1A2 activity. Competitive inhibition of 7-ethoxyresorufin O-deethylase activity catalysed by recombinant hCYP1A2 was observed in the presence of these cannabinoids. CBD was shown to be the most effective inhibitor of CYP1A activity. CBN was also efficient at decreasing the activity of CYP1A2. However, THC was a less powerful inhibitor of CYP1 activity and less selective against CYP1 inhibition than CBD and CBN . In contTHC, CBN, and CBD were found to be highly effective inhibitors of the CYP2A6 and CYP2C9 enzymes but a weaker inhibitor of CYP2B6. THC and CBN both displayed mechanism-based inhibition of the enzyme CYP2A6. The cannabinoids THC, CBD, and CBN suppressed the coumarin 7-hydroxylase activity of recombinant CYP2A6 in a noncompetitive manner, with apparent Ki at approximately 30–40 μM. THC, CBD, and CBN inhibited recombinant CYP2B6 7-dependent benzoxyresorufin O-debenzylase activity in a mixed fashion in a dose-dependent manner, with very small Ki values at approximately 2 μM, suggesting stronger inhibitory action on CYP2B6 than CYP2A6 . SimilarA competitive inhibition study of CYP3A revealed that THC, CBD, and CBN were all effective inhibitors, with CBD being the most potent at a concentration comparable to that found in typical cannabis inhalation. CBD was shown to be the most powerful inhibitor of CYP3A4 and CYP3A5 among the three main cannabinoids tested, and it was found to be competitive. The IC50 values for THC or CBN for CYP3A4 or CYP3A5 were quite high. In addition, CYP3A7 activity was suppressed to a comparable level by these cannabinoids but in a mixed manner ,157. MorCYP2C9 was found to be inhibited by major cannabinoids, with the inhibitory effect of CBD being dependent on the substrates used in the experiments. Most of the cannabinoids reduced the CYP2C9-mediated metabolism of tolbutamide by more than 50%, and as a result, they were further investigated. The inhibitory concentrations (IC50) of CBD, CBDV, cannabigerolic acid and THCA varied between 2.5 and 6.4 μM . CannabiIn summary, the effects of cannabis and its phytoconstituents on CYPs are undeniable. There is enough evidence to draw firm judgement on the safety of cannabis or cannabidiol in people with UC. To obtain clearer findings on the safety of cannabis usage, studies with greater methodological quality and a larger number of participants with long-term follow-up are necessary. Patients should be told that the use of cannabis and cannabinoids may result in DDIs that have an impact on the effectiveness, tolerability, and safety of their medication.Curcuma longa L.) is a perennial herb that is a member of the Zingiberaceae family of plants. It is one of the most important Indian spices and natural colouring agents due to its rhizomes. From ancient times to the present, this plant has been widely used for a variety of purposes, including for therapeutic purposes in foods [Turmeric . Likewise, other curcuminoids were found to inhibit CYP1A2, which accounts for approximately 13% of human liver cytochromes, and biotransform arylamine drugs such as theophylline and lidocaine [Curcumin is a competitive inhibitor of CYP1A2 with an IC50 ranging from 40–100 μM according to in vitro testing involving human CYP enzymes produced in the idocaine ,181–186.idocaine .Curcumin at concentrations ranging from 0.9 to 100 μM was shown to be a competitive inhibitor of the CYP2B6 enzyme, a member of the CYP2 family that plays an important role in chemotherapeutic drug metabolism. For example, cyclophosphamide ,188–191 In human epithelial colorectal adenocarcinoma cells, turmeric extracts were found to inhibit the activity of the intestinal CYP3A4 isoform by approximately 50% . Pure cuThe effect of curcumonoids on CYP enzymes  involved in the steroidogenic pathway was also investigated in another research study. Both the 17-hydroxylase and 17,20-lyase activities of CYP17A1 and the aromatase activity of CYP19A1 were considerably inhibited ,208. AnaAs a result, curcuma and its main constituent curcumin interact with the drug-metabolizing enzymes CYP1A2, 2B6, 2C9 and 3A4, NAT, CYP17A1 and 19A1. It affects the pharmacokinetics of medications taken concurrently in conjunction with it, despite its general public safety perception.Germinated barley foodstuff (GBF), which is composed of dietary fibre and glutamine-rich protein that serves as a probiotic, has been shown to lessen the recurrence rate and clinical reduction in disease activity in patients –214. On There have been no reported interactions between Hordeum vulgare and Triticum aestivum with any of the P450 isoforms that are involved in the metabolism of drugs. As a result, they are considered to be the safest alternatives to conventional drugs for the treatment of UC.Indigo naturalis (IN) is an herbal medication made from the leaves and stems of plants such as Indigofera tinctoria, Strobilanthes cusia, and Polygonum tinctorium. It is used to treat a variety of diseases, including UC. IN is an herbal medicine that is quality controlled in China if it contains more than 2% indigo and 0.13% indirubin, which is the legal limit. IN that originates in Fujian is often considered to be of the best quality. In randomized, placebo-controlled clinical trials, IN was shown to be useful in the treatment of ulcerative colitis [ colitis –224. Unf colitis .There are numerous unknowns about both the efficacy of IN for UC and the interaction of IN with drug metabolizing enzymes. The data that are currently available, even if limited, indicate that IN may offer a considerable threat when used in conjunction with currently utilized pharmaceuticals ,226. CYPThus, all these limited findings of IN suggested that drugs administered in conjunction with IN may need special consideration in terms of drug-drug interactions, and further detailed studies are required.Bletilla striata, Capparis ovata, Commiphora wightii, Cynara scolymus, Fumaria officinalis, Glycyrrhiza glabra, Glycyrrhiza uralensis, Hypericum perforatum, Mentha piperita, Momordica charantia, Oenothera biennis, Plantago ovata, Plantago psyllium, Potentilla erecta, Sanguisorba officinalis, Sophorae flavescentis, Sorbus domestica, Strobilanthes cusia, and Withania somnifera [A number of other herbal products exhibiting antiinflammatory properties are reportedly used in the treatment of UC, and various preclinical and clinical studies are currently available. Among the most notable plants that have been utilized are omnifera ,234–251.The most notable characteristics of traditional herbal medicines are undoubtedly their multicomponent and multieffect natures. Overall, the clinical relevance of the CYP system is well known in drug-drug interactions as well as herbal medicine-drug interactions because various xenobiotics, including those found in traditional herbal medicines, can influence their activity and thus have an impact on systemic drug exposure, i.e. efficacy and toxicity. As shown by the studies examined in this review, herbal medicines used to treat ulcerative colitis are not exempt from this phenomenon .In conclusion, despite their possible antiinflammatory properties, herbal products should not be prescribed due to their interactions with drug-metabolizing enzymes and adverse events, according to the canonical primum nonnocere  medical concern. Moreover, considering the polymorphisms in human CYP genes, which largely alter the function of the enzymes, the prediction of adverse effects is almost impossible. Thus, physicians should encourage their patients to declare their use of complementary medicines to avoid significant consequences that may result from concurrent use of conventional pharmaceuticals.


Aspergillus niger was studied. Among them, copper nanorods had the most obvious inhibitory effect on the growth of Aspergillus niger. The phase of copper nanorods was modified by chitosan, and its inhibitory effect on the expansion of Aspergillus niger was measured. 1. Preparation of copper nanorods and chitosan@copper nanorods: Copper nanorods with a diameter of about 300–350 nm and a length of about 100–800 nm were prepared by the liquid-phase reduction method. The chitosan solution was prepared by using the characteristics of chitosan dissolved in dilute acid to prepare chitosan@copper nanorods and modify the phase of copper nanorods. 2. Determination of the inhibitory effect of various copper nanomaterials on the growth of Aspergillus niger, including Cuprous Oxide nanoparticles, copper nanorods, nano copper oxide, and copper hydroxide, which have certain inhibitory effects on the growth of Aspergillus niger. Among them, copper nanorods have a better effect. On this basis, chitosan@copper nanorods are obtained by modifying the phase of copper nanorods with chitosan. The measured antibacterial effect is that the EC50 value is 344 mg/L.In this paper, the inhibitory effect of various nanomaterials on the growth of  Aspergillus niger is a common species of Aspergillus fungi in the subgenus Trichosporae, order Trichosporae, and family Trichosporae. It is widely distributed in China and the world and is most commonly found in food, crop products, and soil. Aspergillus niger (Aspergillus niger) mainly causes post-harvest disease (post-harvested disease). High temperatures and humid environments are prone to cause it, especially in the sugar content of fruit and grain crops, which causes more serious damage. In grapes, for example, mould contamination can cause losses of up to 30–40% of the total yield of grapes and other bulk fruit and vegetables [Aspergillus niger is a common pathogen on grapes, the fruit and tissues are slightly brownish after damage, gradually becoming soft and rotten, and the fruit stalks and fruit surface often grow a thick layer of mould, the conidia and conidia of the pathogen. The mould layer is white and thin when it starts to appear and becomes greenish-black and thicker when it forms in large numbers. In severe cases, most of the grape grains on the whole bunch are partially discoloured, or even yellowish white, and eventually the affected fruits lose water and crumple. Aspergillus niger is one of the dominant moulds in stored rice, and its strong mould-causing ability and fungal toxin production potential pose a serious threat to the safety of rice. In recent years, innovative use of tea tree essential oil [Aspergillus niger has been reported [Aspergillus niger on this basis in order to explore other materials with good inhibitory effects on the growth of Aspergillus niger. The experiments were carried out to explore other materials with good inhibitory effects on the growth of Aspergillus niger.getables . Where Atial oil , antagonreported , but silAspergillus niger in this paper. Since the discovery of carbon nanotubes by Lijima et al. in 1991 [Nanomaterials are materials with at least one-dimensional size between 1 and 100 nm, with tiny size and new structure between crystalline and non-crystalline , under t4-5H2O and NaOH as the main materials and a hexa-aliphatic alcohol as the reducing agent and adjusting n(OH−):n(Cu2+) = 4 at 180 °C for 20 h under high temperature and high pressure conditions. In this experiment, the temperature, the amount of alcohol, and n(OH−):n(Cu2+) all had an important influence on the results, and the high temperature and the polyol were favourable to the production of copper nanorods, which gave us important ideas for our experiments. Due to the hydrophobic nature of copper nanorods, their poor dispersion in water and their tendency to agglomerate and settle limit their application to a certain extent. Therefore, the surface modification of copper nanorods by chitosan.There are many methods for the preparation of copper nanorods, including vapour phase deposition and hydrothermal synthesis. Among them, hydrothermal synthesis is an excellent experimental method that is simple and easy to operate and requires low experimental conditions. Large-scale production of copper nanorods offers the possibility of preparing copper nanorods, usually under high temperature and pressure conditions, in a liquid-phase system in a closed vessel. Qingchun Chen prepared copper nanorods  with a wThe chemical name of chitosan is polyglucosamine (1-4)-2-amino-b-d glucose . It is gAspergillus niger was measured, from which nanomaterials with a better inhibition effect on the growth of Aspergillus niger were selected, and the reasons for the difference in inhibition were briefly analysed to provide some reference for the research and application of metal nanomaterials in agricultural fungicides.In this experiment, copper nanorods were formulated by liquid-phase synthesis. Based on the characteristics of hydrophobicity and poor water dispersion of copper nanorods, chitosan was used to modify their surface, i.e., chitosan@copper nanorods, which greatly enhanced their water dispersion performance and improved their bacterial inhibition ability to a certain extent. In addition, the inhibition effect of various metal nanomaterials on the growth of Ethylenediamine, sodium hydroxide (flake), copper nitrate trihydrate, chitosan , ice acetic acid, and anhydrous ethanol were acquired from Chengdu Kolon Chemicals Co.  Magnesium hydroxide nanoparticles, copper oxide nanoparticles, copper nanoparticles, and copper nanorods were all prepared in our laboratory.4-3H2O were added to the conical flask, placed on a magnetic stirrer, and stirred well to obtain a clear blue solution. Next, 300 µL of ethylenediamine and 22 µL of hydrazine hydrate were added in rapid succession while stirring and stirring stopped when the solution changed colour from blue to dark blue and gradually to blue-violet. The conical flask was transferred to an oven at 85 °C, and the reaction was carried out for 1 h. The solution was observed to be red in colour, with a fluffy, pore-like red-brown substance emerging from the surface and a little crumbly substance at the bottom. After cooling to room temperature, the solution was transferred to a centrifuge tube, centrifuged at 10,000 rpm for 5 min, washed three times with anhydrous ethanol, freeze-dried, and stored at 25 °C at room temperature.Copper nanorods were prepared under alkaline conditions using copper sulphate trihydrate as the copper source, hydrazine hydrate as the reducing agent, and ethylenediamine as the complexing agent, following the method of Liu et al.  with sliA 1% acetic acid solution was prepared by mass by taking 100 mL of acetic acid solution in a beaker and adding 1 g of chitosan (CS). The mouth of the cup was covered with sealing film, placed on a magnetic stirrer, and stirred vigorously until it dissolved completely. A transparent yellow solution (chitosan solution) was be obtained and set aside.Twenty mg of copper nanorods were weighed and placed into a 100 mL beaker with a magnetic stirrer added in advance. Then, 20 mL of anhydrous ethanol solution was added, sealed with sealing film, put into a sonicator, sonicated evenly, and placed on a magnetic stirrer after the sealing film was removed. Then, 20 mL of the configured chitosan solution were added dropwise while stirring. Stirring continued for half an hour after the addition to make it wrap evenly. After half an hour, transfer to a centrifuge tube, centrifuge at 10,000 rpm for 5 min, wash once with pure water, freeze-dry, and store at 25 °C at room temperature.Aspergillus niger: Aspergillus niger was inoculated in the centre of a potato glucose agar medium (PDA plate) incubated at 28 °C for 7 d with light. An appropriate amount of mycelium was picked from the plate, placed in a seed-keeping tube with 15% glycerol, and placed in a refrigerator at 4 °C for cold storage. For long-term storage, it is placed in a freezer at −80 °C for freezing.Preservation of Aspergillus niger: When using, an appropriate amount of Aspergillus niger mycelium is picked in the centre of the PDA plate, placed upside down in a constant temperature incubator at 28 °C, and incubated until the colony is just as long as the edge of the Petri dish before use.Culture of 2 nanoparticles, CuO nanoparticles, and Cu nanorods were diluted 10 times with sterile water . 9 mL of PDA medium was added to a 50 mL conical flask, sterilised, and cooled to 70 °C. 1 mL of the drug solution was added to it and mixed well to make the PDA medium with the drug. Three plates were set up for each nanomaterial and different drug concentrations, with an additional set of blank controls (1 mL of sterile water in PDA medium), and each concentration treatment group was repeated three times.The toxicity of magnesium hydroxide nanoparticles, copper oxide nanoparticles, copper nanoparticles, and copper nanorods was determined in vitro using the plate with drug method . MagnesiAspergillus niger by the single-spore isolation method (refer to Buy New Red’s method with slight modifications) [Aspergillus niger strain was placed in the light at 28 °C for about 7 d, with colonies just to the edge of the Petri dish, and colonies covered with black spores were dipped in the mould layer and scribed on the plate to separate the conidia with sufficient spacing. The plates were incubated at 28 °C for 7 d. When inoculated with single spores, the plates were removed and set aside. The surface of the scalpel was disinfected by dipping it in 75% alcohol and burning it on the outer flame of an alcohol lamp, which was repeated 3 times, and then cooled to room temperature. Under a 40–45X dissecting microscope, the tip of the scalpel was used to pick the germinated single conidia from the scribing plate and transfer them to the medicated plate. Equation (1) is used to calculate the growth inhibition rate of black moulds.Inoculation of cations) . Ten mL/In the above equation,X—growth inhibition rate;d1—control colony diameter;d2—colony diameter of the treatment group.Aspergillus niger by the above four nanomaterials were measured at a concentration of 1000 mg/L as follows.The results of the inhibition of the growth of (data results processed using SPSS 26).The prepared copper nanomaterials were characterised using X-ray diffraction (XRD) and infrared spectroscopy (FT-IR) at zeta potential, and the morphological characteristics of the copper nanomaterials were analysed using transmission electron microscopy (TEM). The above process was commissioned by an external institutional academic research council, and the results obtained are fed back as follows.Homogeneous copper nanorods with diameters of 100–350 nm and lengths of 100–800 nm were prepared by liquid phase reduction and modified with chitosan. The modified copper nanorods, i.e., chitosan@copper nanorods, had diameters of approximately 200–400 nm and lengths of 400–2000 nm. The prepared copper nanomaterials were characterised by X-ray diffraction (XRD) and infrared spectroscopy (FT-IR) at zeta potential, and the morphological characteristics of the copper nanomaterials were analysed by transmission electron microscopy (TEM), both of which demonstrate that chitosan was successfully encapsulated on the surface of the copper nanorods.Aspergillus niger was significantly different, among which the inhibition effect of copper nanorods on the growth of Aspergillus niger mycelium was more obvious. Therefore, a smaller concentration gradient was set on this basis, and chitosan was used to modify the copper nanorods to make them more stable in order to further investigate the inhibition effect of copper nanorods on the growth of mycelium.From the above results, it can be seen that when the concentration of the agents was 1000 ppm, the inhibition effect of the four agents on Chitosan@Cu nanorods were subjected to an in vitro indoor virulence assay using the drug-carrying plate culture method. Chitosan@Cu nanorods were dissolved in sterile water, treated with a cell crusher to homogenise them, and configured into a 10,000 mg/L master mix for reserve (ready-to-use). The master mix was diluted 10 times the mass concentration gradient in As can be seen from Aspergillus niger by the single spore isolation method was carried out as described in The inoculation of Aspergillus niger were obtained according to From Aspergillus niger. The inhibition rate of the four materials was measured by the strip plate method with all four solutions at 1000 mg/L, in which the inhibition effect of copper nanorods was significantly better than the other materials. Based on this, CS@CuNRs were obtained by surface modification of copper nanorods through chitosan to enhance their bacterial inhibition effect and stability in water. The regression equation of the virulence of CS@CuNRs on the growth of Aspergillus niger was further measured by using the liquid culture method with drugs. The virulence regression equation for CuNRs against Aspergillus niger was 362 mg/L for EC50 and 862 mg/L for EC90. The virulence regression equation for chitosan@CuNRs was y = 0.0014 + 0.0186. The virulence regression equation for chitosan@copper nanorods was 344 mg/L for EC50 and 630 mg/L for EC90. It is evident that the inhibition effect of copper nanorods was enhanced by the surface modification of chitosan. However, this test did not involve the in vivo inhibition test, and the inhibition mechanism of copper nanorods is not known, which needs to be further investigated.In this part, we firstly discussed the inhibitory effect of several nanomaterials, including copper nanorods, copper nanoparticles, copper oxide nanoparticles, and magnesium hydroxide nanoparticles, on the growth of Aspergillus niger by various nanomaterials, including copper-based nanomaterials, copper nanorods, copper oxide nanoparticles, copper nanoparticles, and magnesium-based nanomaterials, magnesium hydroxide nanoparticles, was tested using the plate banding and single spore inoculation methods. The results show that copper nanorods significantly inhibit the growth of Aspergillus niger mycelium. The surface-modified chitosan copper nanorods were more effective in inhibiting the growth of Aspergillus niger than the surface-unmodified copper nanorods, but this paper only compares the size of the inhibition concentration of the two; the mechanism of the difference in their inhibition effect is not yet clear, and further research is needed. Secondly, only the inhibition effect of copper nanorods on the growth of isolated Aspergillus niger before and after surface modification was measured; on the one hand, no in vivo inhibition test was conducted, and on the other hand, the safety of the two materials was not assessed. A difficult problem is that due to the certain bactericidal properties of CuNO3, if not washed thoroughly, the residual Cu+ will cause certain errors in the experimental results. Although we have undergone repeated washing, we may still be unable to avoid trace amounts of Cu+ residue. How to minimise the residual amount of Cu+ is also a problem that needs to be optimised in the future. Finally, field use is the ultimate goal of pesticide screening, as mould rot caused by Aspergillus niger is the dominant post-harvest disease, so the application scenario is mostly for post-harvest crop protection, but copper nanorods are dark reddish-brown in colour and direct application will affect the fruit appearance to a certain extent. Overall, although agents with good inhibitory effects on the growth of Aspergillus niger have now been screened, further research is needed in many aspects, from the inhibition mechanism to the in vivo inhibition effect to the final use, but I believe that the future is promising.In this paper, a copper nanorod with a diameter of approximately 300–350 nm and a length of 100–800 nm was prepared using a liquid-phase reduction method in a strongly alkaline solution system with copper nitrate trihydrate as the copper source, ethylenediamine as the complexing agent, and hydrazine hydrate as the reducing agent in a hydrothermal system at 80 °C. The surface modification of the copper nanorod using chitosan can reduce the surface oxidation rate of the copper nanorod, increase their water solubility, and improve their antibacterial effect. The inhibition of the growth of isolated


N-arylalkyl derivatives altered at C-5 is reported. Their inhibitory activity and selectivity toward four GH38 α-mannosidases  were investigated. 6-Deoxy-DIM was found to be the most potent inhibitor of AMAN-2 (Ki = 0.19 μM), whose amino acid sequence and 3D structure of the active site are almost identical to the human α-mannosidase II (GMII). Although 6-deoxy-DIM was 3.5 times more potent toward AMAN-2 than DIM, their selectivity profiles were almost the same. N-Arylalkylation of 6-deoxy-DIM resulted only in a partial improvement as the selectivity was enhanced at the expense of potency. Structural and physicochemical properties of the corresponding inhibitor:enzyme complexes were analyzed by molecular modeling.A synthesis of 1,4-imino-ᴅ-lyxitols and their  Iminosugars are analogs of monosaccharides in which the endocyclic oxygen atom is replaced with a nitrogen atom –5. TheseNatural iminosugars can be monocyclic or bicyclic compounds since the presence of the nitrogen atom allows for a formation of an additional cycle. In synthetic iminosugar analogs, various structural modifications are possible and many of these compounds inhibit glycoprocessing enzymes –14.Canavalia enciformis  which operates in the pH range 4–5. This class II mannosidase is inhibited by swainsonine very effectively (IC50 = 1–5 × 10−7 M)  and interacts with the hydrophobic pocket created by Tyr727, Phe206 and Trp415. Also, the binding position of this side chain is in a good overlap with the hydroxyethylene part of the piperidine ring of swainsonine. This indicates that the (R)-1-hydroxyethyl group of 29 could mimic the interactions of the hydroxypiperidinyl moiety of swainsonine in the active site of dGMII.In order to better understand the results of our inhibition study, the inhibitor:enzyme interactions were analyzed by molecular modeling. Structures of the inhibitors were docked into an X-ray structure of dGMII and geometries of the resulting inhibitor:dGMII complexes  ,39 -1-hydroxyethyl substituent of 29 and other derivatives synthesized in this work, pair interaction energies between the bound inhibitors  and amino acid residues of dGMII were calculated at the quantum mechanics level (FMO-PIEDA-MP2/6-31G*) in an active-site model of the inhibitor:enzyme complexes optimized at the hybrid QM/MM level (BP86/LACVP*:OPLS2005). The FMO-PIEDA results are compiled in 29:dGMII were analyzed: the overall interaction energy (ΔEI-E) between 29 and the enzyme is −563.6 kcal mol−1, towards which the interaction energy between the (R)-1-hydroxyethyl group of 29 and the enzyme  contributes only 9%. As can be seen in Elinker-E contributors are Tyr727  and Asp472 . Both of these amino acid residues use their side chains to interact with the hydroxy group of the (R)-1-hydroxyethyl moiety of 29. The interaction with Trp95 is also significant but to a lesser extent , and the overall interaction of 29 with the Phe206-Trp415 hydrophobic pocket is insignificant . Therefore, the main contributors remain Tyr727 and Asp472 (two hydrogen bonds). A similar conclusion can also be made for the other calculated inhibitors with one  or two (DIM) hydroxy groups at C-5. This explains why the inhibitory activity of 29 [Ki(AMAN-2) = 0.19 µM] is only slightly better than the previously synthesized derivative 30 (with a hydroxymethyl moiety at C-5) [Ki(AMAN-2) = 2.3 µM] [To evaluate the inhibitory effect of the . The inhibitory activities of these derivatives increased in the following order: 10 [IC50(AMAN-2) = 415 µM] < 20 [IC50(AMAN-2) = 48 µM] < 28 [IC50(AMAN-2) = 22 µM] < 30 [IC50(AMAN-2) = 2.3 µM] < DIM [IC50(AMAN-2) = 0.81 µM] < 29 [IC50(AMAN-2) = 0.24 µM]. Interestingly, almost the same trend was found for the overall FMO-PIEDA interaction energies (ΔEI-E) and the energies (ΔElinker-E) between the side chain at C-5 and the active site  < 20  < 28  < 29  < 30  < DIM ; and 10  < 20  < 29  < 30  < 28  < DIM , respectively. The calculations predict that the functional group attached to C-5 of the inhibitor must bear one or two hydroxy groups . The methyl group itself (in 10) contributes repulsively and decreases the overall interaction energy. Thus, only hydroxymethyl, (R)-1-hydroxyethyl and (R)-1,2-dihydroxyethyl are suitable substituents at the C-5 position of 1,4-imino-ᴅ-lyxitols. The calculations further predict that alkylation of the nitrogen atom may further increase the interactions of the (R)-1-hydroxyethyl group at C-5 with the enzyme (N-2-naphthylmethyl in 28). However, this alkylation also decreases the interactions of the lyxitol core with the enzyme  and the overall ΔEI-E for 28 became lower than for 29, which is in agreement with the measured inhibitory activities of these compounds. This is a surprising result because N-alkylation of 30 (structure 31 in R)-1-hydroxyethyl group at C-5 of 29 compared to the hydroxymethyl at C-5 of 30 allows for a different conformation of the N-2-naphthylmethyl group in the active site of α-mannosidases , which is the major component of the overall interaction energy between the inhibitor and the enzyme. This assumption was further supported by additional FMO-PIEDA calculations for 31. In both 31 and 28, the 2-naphthylmethyl group is attached to the ring nitrogen of the inhibitor. The interaction energy between the N-2-naphthylmethyl moiety of the inhibitors and the enzyme was almost the same -1-hydroxyethyl group at C-5).To evaluate the inhibitory effect of the structural moiety at C-5 of the 1,4-imino-ᴅ-lyxitols, FMO-PIEDA interaction energies were calculated for another five derivatives  provided the least effective inhibitors of the target Golgi enzymes. The comparison between iminosugars 17–20 and their C-5 epimers 26–29 showed that the hydroxy group must adopt an R-configuration in order to exhibit a strong inhibition profile. Also, unsubstituted 6-deoxy-DIM 29 turned out to be the best inhibitor out of all the analogues synthesized in this study as its N-arylalkylation significantly reduced potency and barely improved selectivity. This is in sharp contrast to the previously synthesized N-2-naphthylmethyl 1,4-imino-ᴅ-lyxitol which showed a high selectivity toward Golgi-type α-mannosidases [N-2-naphthylmethyl is too bulky for the 6-deoxy-DIM core and causes weaker binding of the inhibitor ring to the enzyme. Therefore, the next efforts should be focused on the identification of a novel N-substitution pattern of the DIM skeleton that would have more beneficial effects on the inhibition profile.1,4-Imino-ᴅ-lyxitols and their C-5 altered osidases . The FMOFile 1Experimental .File 2Copies of NMR spectra.File 3Optimized QM/MM complexes (inhibitor:enzyme).