Tenofovir disoproxil fumarate (TDF) and islatravir  are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively. Therefore, these two nucleoside analogs have opposing resistance profiles and could present a high genetic barrier to resistance. To explore resistance to TDF and ISL in combination, we performed passaging experiments with HIV-1 WT, K65R, or M184V in the presence of ISL and TDF. We identified K65R, M184V, and S68G/N mutations. The mutant most resistant to ISL was S68N/M184V, yet it remained susceptible to TDF. To further confirm our cellular findings, we implemented an endogenous reverse transcriptase assay to verify in vitro potency. To better understand the impact of these resistance mutations in the context of global infection, we determined potency of ISL and TDF against HIV subtypes A, B, C, D, and circulating recombinant forms (CRF) 01_AE and 02_AG with and without resistance mutations. In all isolates studied, we found K65R imparted hypersensitivity to ISL whereas M184V conferred resistance. We demonstrated that the S68G polymorphism can enhance fitness of drug-resistant mutants in some genetic backgrounds. Collectively, the data suggest that the opposing resistance profiles of ISL and TDF suggest that a combination of the two drugs could be a promising drug regimen for the treatment of patients infected with any HIV-1 subtype, including those who have failed 3TC/FTC-based therapies. HIV is a global public health issue that continues to be a threat to public health. In 2020 alone, 680,000 people died from HIV-related causes and 1.7 million were newly infected. HIV-1 can be divided into phylogenetic groups  resulting from discrete zoonotic transfers ,4,5. CRFHIV-related deaths have decreased over the last 20 years due to highly active retroviral therapy (HAART). HAART typically consists of two nucleoside (or nucleotide) reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor or an integrase inhibitor. The current FDA-approved NRTIs are abacavir, emtricitabine (FTC), lamivudine (3TC), tenofovir (TFV), and zidovudine. The most commonly used NRTIs in the United States are FTC and tenofovir . While cThe major clinical resistance mutations to current frontline NRTIs are K65R (TFV resistance mutation) and M184V (FTC and lamivudine (3TC) resistance mutation). In addition, there are several reported subtype-specific differences in treatment response among HIV-infected individuals and HAART-treated patients. For example, subtype-C patients are more likely to fail on current standard-of-care tenofovir-based regimens, due to higher prevalence in this subtype of the K65R tenofovir resistance mutation in RT .Preclinical development of ISL has demonstrated high potency and favorable toxicity profiles in cell culture, mice, and rhesus macaques ,12,13,14ISL has three structural features that contribute to its efficacy and long-acting potential. Unlike currently approved HIV NRTIs, ISL retains a 3′-OH, which enhances efficient activation by the cellular deoxycytidine kinase to create ISL-monophosphate (ISL-MP), the first and rate-limiting activation step of almost all NRTIs . The 2-fHere, we explore how pre-existing resistance mutations affect the emergence of resistance to tenofovir and ISL combination regimens. We began passaging experiments with WT, K65R (resistance mutation in RT to tenofovir), and M184V (resistance mutation to 3TC/FTC and ISL). To confirm the resistance findings, both cell-based and endogenous reverse transcriptase assays were used to determine sensitivity to ISL and TFV. We also explored clinical resistance of K65R, M184V, and K65R/M184V in the context of diverse primary isolates: B, C, CRF_AE, and CRF_AG. In all isolates studied, we found K65R imparted hypersensitivity to ISL. M184V confers resistance to ISL that ranges from 3- to 10-fold relative to wildtype. The K65R/M184V double mutant typically maintained or decreased resistance to ISL compared to the M184V mutant alone. We further found that S68G, when combined with K65R/M184V, in some genetic contexts, conferred greater resistance than M184V alone. S68G can emerge during TFV-treatment . Collect2O. MT-2 cells [Stock solutions of ISL  and TDF (provided by AIDS Reagent Program) were prepared in distilled, deionized H-2 cells ,23 were -2 cells  and TZM-K65R and M184V mutations in RT were generated by site-directed mutagenesis on an HXB2 or xxLAI HIV-1 backbone using the QuikChange XL site-directed mutagenesis kit  according to the manufacturer’s protocols. 293-T cells were transfected with 10 μg of plasmid DNA using the PrimeFectimine mammalian transfection reagent . After 72 h incubation, media were harvested, filtered, and used to infect MT-2 cells and infectious virus harvested at ≥50% syncytium formation, determined by microscopic observation as previously described ,26. HIV-2), inspected daily for syncytia (as described above), and the media refreshed every three days. When no additional syncytia formation was noted for two days, the assay was terminated, and the Reed–Muench method was used to calculate the virus TCID50 [Fifty thousand MT-2 cells per well, in 96-well flat-bottomed plates, were infected with four-fold serial dilutions of virus stock, three replicates per dilution. Plates were incubated at 37 °C , the concentration determined using Spectronic BioMate 3 UV spectrophotometer , and 500 ng RNA was used as the template for cDNA synthesis. First-strand PCR was performed using random hexamer primers and the SuperScript III First-Strand Synthesis System for RT-PCR . The resulting cDNA was PCR amplified using HIV-1 LAI-specific primers ABR-RT-OF (1763 5′-GGAGCCGATAGACAAGGAACTG-3′) and ABR-RT-OR2 (3863 5′-GGCTACTATTTCTTTTGCTACTACAGG-3′). These primers are located in the 3′ end of gag and the 5′ end of integrase, respectively, and generate a 2127 bp product spanning the full length of the reverse transcriptase gene. PCR was performed using the Expand High Fidelity PCR System dNTPack .Approximately 100 ng of full-length PCR product was ligated into the pGEM-T Vector System , at a 3:1 molar ratio of insert: vector, and incubated overnight at 4 °C. Ligations were transformed into MAX Efficiency DH5α competent cells . Blue-white screening was used to select clones with successful ligations, and plasmids containing the full-length reverse transcriptase gene were isolated with the QIAprep Spin Miniprep kit . Primers ABR-RT-OF and ABR-RT-OR2 were used to sequence the 5′ and 3′ ends of reverse transcriptase, while an internal portion of the gene was sequenced with primer ABR-RT-IF (2211 5′-CAGAGATGGAAAAGGAAGGG-3′). A minimum of 20 clones from each sample were sequenced; the sequences were aligned with the appropriate P0 stock virus consensus in Clustal X-2, and the proportion of sequences with the novel substitutions was determined.The ELISA was an in-house assay using Costar 3922 high binding plates coated with anti-p24 antibody (abcam 9071) for 1 h at RT. Plates were blocked with 3% BSA in 1× PBS for 2 h at RT. Samples were diluted in 0.5% BSA 0.1% Triton X-100. Then, plates were washed with 1× PBS-T and lysed samples were added to the plate for 1 h. ELISA plates were then washed and primary antibody HIV-Ig . Plates were washed again with 1× PBS-T, then anti-human HRP  was added. Plates were washed again with 1× PBS-T and chemiluminescent substrate  was added. Fluorescence intensity was determined. The amount of p24 was calculated from a standard curve.50 values determined using Prism 9 (GraphPad) software.Each virus was generated as described above and quantified by p24 ELISA to standardize inputs. TZM-GFP cells were plated at 10,000 cells/well in a 96-well plate and with serially diluted ISL or TDF at 0.000381 nM to 100 nM (ISL) or 0.038 nM to 10 μM (TDF). The cells, media, and drug were incubated for 24 h and infected with virus and a 1 μg/mL final concentration of DEAE-dextran, followed by incubation for 48 h. The green fluorescent protein (GFP)-positive cells were then counted using Cytation 5 with Gen5 v3.03 software . Dose-response curves were plotted and ECTZM-GFP cells were plated at 10,000 cells/well in a 96-well plate and incubated for 24 h. Then, cells were infected with the serially diluted virus and a 1 μg/mL final concentration of DEAE-dextran, followed by incubation for 48 h. The GFP-positive cells were then counted as described above. The p24 content of each virus stock was also determined using an enzyme-linked immunosorbent assay (ELISA). Specific infectivity was calculated as infected cells divided by the amount of p24 content.2, 2.5 μg/mL melittin, 80 µM IP6, 0.004 mM dCTP, dTTP, and dGTP, 0.02 μM dATP, 100 ng capsid, and RT inhibitors, including ISL-TP and Tenofovir-DP. After 30 min incubation at 37 °C, 0.036 mM of dCTP, dGTP, dTTP, and 0.0398 mM dATP were added to the reaction and incubated at 37 °C for a further 16 h. The products were quantified by qPCR with SYBR green detection using the following primers: 5′-GAGCTCTCTGGCTAACTAG-3′ and 5′-TGACTAAAAGGGTCTGAGGGATCT-3′ adapted from [RT protocols were adapted from ,29; reacThe statistical significance was determined using Prism 9. A one-way analysis of variance (ANOVA) was performed, as well as a Dunnett’s or Tukey’s multiple-comparison test (see figure legends).50 values), the WT virus required 97 days to escape two passages and could only survive the initial combination of 360 nM TDF and 3.6 nM ISL. The virus isolated had no mutations except M184I at 9.5% , escaping from final concentrations of 14.4 nM TDF and 14.4 nM ISL, over the course of 34 days. M184I was detected in 71.4% of the passage 6 (P6) sequences and M184V in 28.6%. When the TDF:ISL ratio was increased to 10:1, the WT virus escaped from only five passages over 65 days, with final drug concentrations of 144 nM TDF and 14.4 nM ISL; M184V was present in 100% of the final passage . At 100: at 9.5% .The M184V strain progressed through five passages in 1:1 TDF:ISL, spanning 87 days and reaching final concentrations of 130 nM TDF and 130 nM ISL, where K65R/M184V was found at 9.5%, M184I was 4.8%, and a reversion to M184 was also found in 4.8%. M184V was unable to escape from even a single passage in 10:1 or 100:1 TDF:ISL.The K65R virus survived seven passages in 1:1 TDF:ISL over 32 days, escaping from a final combination of 7.2 nM TDF and 7.2 nM ISL. K65R/M184V took over the final population of 95.7% at passage 7. K65R/S68G/M184V was found at 4.3% prevalence at passage 7. In 10:1 TDF:ISL, the K65R strain progressed through seven passages over 36 days and escaped a final combination of 72 nM TDF and 7.2 nM ISL. K65R/M184V appeared in 100% of the population . The K65To evaluate the contributions of the individual mutations and combinations of mutations—identified during viral passaging experiments, we introduced mutations into the HIV-1 NL4-3 lab strain and determined sensitivity to ISL and TDF A. M184V To determine whether the mutations identified had effects on viral fitness, we used a single-round replication assay to determine the specific infectivity of viral stocks. Specific infectivity was determined by amount of infection per amount of p24 or capsid. The only significant changes in specific infectivity compared to WT were S68N/M184V and K65R/S68N, which had a minor increase and decrease, respectively B.To further verify the effects of ISL and TDF resistant mutations, removed from the context of infection in the context of endogenous RT, we implemented an endogenous RT assay. We used isolated virions supplemented with dNTPs, IP6, and phosphorylated ISL  and tenofovir , and RT products using qPCR. The resistance phenotypes broadly recapitulated those observed in cell-based assays ; K65R an50 of ISL was 0.80 nM. TDF was also effective at inhibiting subtypes with a mean EC50 of TDF of 87 nM; however, subtypes A, B, C, and D had 1.3-, 2.2-, 2.2-, 1.6-fold change from NL4-3, respectively  to ISL than M184V (3-fold). This was unexpected as K65R/M184V double mutants displayed an intermediate phenotype in all other isolates examined, presumably as a consequence of the enhanced resistance conferred by M184V and the enhanced sensitivity of K65R C. B, C, Inspection of the CRF_AE sequence revealed Gly68, rather than the Ser68 present in the other isolates and the majority of isolates circulating globally. A S68G/N mutation also emerged during passaging experiments with ISL and TDF combinations . To testTo evaluate if Gly68 affects fitness of the CRF_AE isolate, we performed specific infectivity assays. Gly68 reduced specific infectivity in the context of WT CRF_AE, but when combined with other mutations , it played no role in specific infectivity B. Some pNRTIs have been fundamental in HAART , but dueM184V/I confers very high resistance to 3TC/FTC (>100-fold resistance) ,55,56,57Previous studies demonstrated that ISL resistance mediated by M184V/I is conferred by steric hinderance of the β-branched amino acids , which may perturb the hydrophobic pocket into which the 4′E of ISL binds . In addiK65R/M184V (9-fold resistance to ISL) compared to CRF_AEM184V (3.5-fold resistance to ISL). Upon examination of the RT genome of the isolates studied, G68 was present rather than the more common S68; this mutation (S68G) was also selected during our passaging experiments. In the context of CRF_AEK65R/M184V, G68 appeared to confer greater resistance to ISL, relative to CRF_AEK65R/M184V carrying S68. Nevertheless, the resistance observed to ISL by CRF_AES68G/K65R/M184V remained under 10-fold relative to the WT. It is also important to note that we did not see the same resistance pattern in NL4-3, suggesting that other residues  play a role in the resistance observed. We also see specific infectivity defect of G68 present in CRF_AE WT as compared to S68; however, in the context of K65R, M184V, and K65R/M184V, we saw no significant differences between Ser68 and Gly68. A previous study also found S68G in a CRF_AE isolate, where it was identified as a potential compensatory mutation for K65R [The hypersensitivity to ISL of RT_K65R across all subtypes examined suggests that the previously identified mechanism for hypersensitivity, a decrease in the excision efficiency of ISL-MP from the 3′ terminated primer, likely holds true in all isolates . With M1for K65R . Similarfor K65R . Our datOur results highlight the high genetic barrier to resistance of TDF/ISL combinations, suggesting high efficacy for this combination if used therapeutically on a global scale. We demonstrate that TDF/ISL have opposing resistance profiles and that mutations that have been shown to confer resistance to these antivirals individually remain susceptible to ISL and TFV in combination. Taken together, these two drugs hold promise for a global combination regimen due to their high barrier to resistance, opposing resistance profiles, and efficacy against a broad range of subtypes.


B-M-E triblock protein designed to self-assemble into antifoulingcoatings.Previously, we have shown that the design performs well on silicasurfaces when B is taken to be a silica-bindingpeptide, M is a thermostable trimerdomain, and E is the uncharged elastin-likepolypeptide (ELP), E = (GSGVP)40. Here, we demonstrate that we can modulate the nature of the substrateon which the coatings form by choosing different solid-binding peptidesas binding domain B and that we canmodulate antifouling properties by choosing a different hydrophilicblock E. Specifically, to arrive atantifouling coatings for gold surfaces, as binding block B we use the gold-binding peptide GBP1 (with thesequence MHGKTQATSGTIQS), while we replace the antifouling blocks E by zwitterionic ELPs of different lengths, ZEn = (GDGVP-GKGVP)n/2,with n = 20, 40, or 80. We find that even the B-M-E proteins with the shortest E blocks make coatings on gold surfaces with excellentantifouling against 1% human serum (HS) and reasonable antifoulingagainst 10% HS. This suggests that the B-M-E triblockprotein can be easily adapted to form antifouling coatings on anysubstrate for which solid-binding peptide sequences are available.We analyze modularity for a  A key approach for controlling the interactions of surfaceswith biomolecules is the application of a surface coating that canmodulate the interactions with the biomolecules and in this way remedythe shortcomings of using the bare surface, such as undesired adsorptionfrom biomacromolecules or microorganisms.Interfacing synthetic materials with livingorganisms, cells, orbiological fluids is required in many technologies, from in vitrobiosensing to in vivo biomaterial implants, and often requires combiningdifferent disciplines such as materials science and bioengineering.12 Both chemical and physical attachments of the coatings have beenexplored, with chemical attachment—either grafted from brushesor grafted to brushes13—generallyleading to more stable coatings of higher functionality,14 while physical attachment generally allows forsimpler and more easily scalable coating processes. Particularly,successful antifouling coatings consist of brushes of uncharged hydrophilicor zwitterionic flexible polymers, and detailed systematic studiesthereof exist.15 For example, well-knownuncharged antifouling polymers are polyethylene glycol (PEG)19 and poly (N-(2-hydroxypropyl) methacrylamide).20 Brushes of the PEG polymer can be chemicallygrafted onto surfaces with high density and in this way promote interfacehydration which prevents biomolecular adsorption.22 Also, zwitterionic polymers, such as polycarboxybetaine methacrylate,have been shown to lead to excellent antifouling when grafted as abrush.23In many cases, interfacingsynthetic materials with living organismsboils down to first preventing any unwanted interactions of the syntheticsurface with the adjacent biological fluid (“antifouling”)and then adding the desired specific interactions required by theapplication. Therefore, the antifouling coating preventing non-specificinteractions of the surface with biomolecules, microorganisms andcells, have been investigated in many studies.24 with peptides havingbeen isolated that bind strongly to different surfaces, such as metals,26 minerals,28 plastics,30 and even semiconductors.31Using synthetic materialsallows for tailor-made functionalization,but the synthesis of the required components typically has a largerecological footprint than required for the in vivo synthesis of biomacromoleculeswith similar functions. Biomolecules, in particular proteins, canbe precisely manipulated at the DNA level. Very large peptide andprotein libraries can be generated for screening potential interactionswith natural materials. In fact, surface-binding peptides selectedfrom various types of libraries have proven to be useful tools inmaterials science,32 we have argued that proteindesign should, in principle, allow for the creation of proteins thatself-assemble into antifouling polypeptide brushes that are not onlyeasy to apply but also highly stable and functional. This earlierwork considered silica as a model surface. A B-M-E modular design consisting of three domains was proposed, where B is a solid-binding peptide for anchoringto the surface, M is a multimerizationdomain for increasing the overall protein binding strength to thesurface through multivalency, and E isan uncharged hydrophilic elastin-like polypeptide (ELP)34 serving as an antifouling block. Compared to first-generation coatingswith the simpler B-E design,35 the multivalent B-M-E designs assembled into highly stable coatings that couldnot be displaced, neither by high salt buffers nor by serum albuminprotein solutions. Also, the coatings were highly antifouling againsthigh concentrations of serum albumin. While we hypothesized that the B-M-E protein design should be highly modular and allowfor swapping out binding domains B andantifouling domains E to lead to coatingswith different functionalities for different materials, we did notdemonstrate this in our previous study.Previously,B-M-E design by testing it with bindingblocks B for gold as opposed to silicaand for antifouling polypeptides E ofdifferent lengths and with different sequences. Gold is chosen asa model surface since it is chemically very different from silica,is an important surface material in biosensing, and a number of well-characterizedsolid-binding peptides are available for gold surfaces. To test theantifouling properties, we here more stringently challenge the coatingswith human serum (HS) of various dilutions, as opposed to only single-proteinserum albumin solutions.Therefore, in the currentwork, we explicitly explore the modularityof the 38 and which has previously beenreferred to as GBP1,26 a non-cysteine peptidewith a single-letter amino acid sequence MHGKTQATSGTIQS. Previously,32 we have used E = (GSGVP)40 for the antifouling domain. Here, we explorea series of three different lengths of the zwitterionic ELP sequence, E = EZn = (GKGVP-GDGVP)n/2 with n = 20, 40, or 80.To replace the original silica-bindingdomain, we chose a gold-bindingpeptide that has been extensively studied both by experimental workand by computer simulations,6) for later nickel affinity purification.The three B-M-E protein constructs in thisstudy are abbreviated as B-M-En, where n = 20, 40, or 80 is the number ofpentapeptide repeats of the E block.All protein constructs used in this study carry a His-tag  as the guest residue, E = ES40 = (GSGVP)40. The multimerizationdomain MHR00C_3_2 was a thermostabletrimer previously de novo designed and characterizedby Fallas et al.39 A short ES3 linker, ES3 = (GSGVP)3, was used to connect the solid-binding peptideto the trimerization domain. Plasmids used in the construction ofprevious silica proteins are used here to construct the gold-bindingversions, H6-BGBP1-ES3-MHR00C_3_2-EZ20, H6-BGBP1-ES3-MHR00C_3_2-EZ40 and H6-BGBP1-ES3-MHR00C_3_2-EZ80, where BGBP1 = GBP1 = MHGKTQATSGTIQS is the gold-bindingpeptide sequence. All the plasmids mentioned above contain necessaryfeatures for recursive directional ligation by plasmid reconstruction(PRe-RDL) cloning, as described by McDaniel et al.40Previously,41 was purchased from IntegratedDNA Technologies . Gibson assembly was usedto insert this gene fragment into the linearized plasmid for H6-BRT-ES3-MHR00C_3_2, yielding an expressionplasmid for H6-BGBP1-ES3-MHR00C_3_2. Primersused for obtaining the linearized plasmid for H6-BRT-ES3-MHR00C_3_2 from a previously designed plasmid32 are given in Table S1. The DNA and amino acid sequences for the gene fragment encoding B-M-E proteins are shown in Tables S1 and S2.A gene fragment encoding GBP1, with suitable overhangsfor Gibsonassembly,EZ40 and EZ80. A plasmid encoding EZ20 flanked by sequences necessaryfor gene oligomerization via PRe-RDL was purchased from TwistBioscience.Restriction enzymes and DNA modifying enzymes for the PRe-RDL procedurewere purchased from New England Biolabs.PRe-RDL was used to obtainoligomers of the genes for EZ20 gene to obtain the EZ40 gene using PRe-RDL,the plasmid for EZ20 gene was digested with AcuI and BglI. The sameplasmid was also digested with BseRI and BglI. The two digested fragmentswere ligated to obtain the EZ40 plasmid. The gene for EZ40 was duplicatedin the same above-mentioned way using PRe-RDL to obtain the plasmidwith the EZ80 gene.For duplication ofthe 6-BGBP1-ES3-MHR00C_3_2 was digestedusing AcuI and EcoRV, and plasmids containing genes for EZ20, EZ40, and EZ80 were digested with BseRI and EcoRV. Pairs of digestedfragments were ligated to obtain plasmids encoding for H6-BGBP1-ES3-MHR00C_3_2-EZ20, H6-BGBP1-ES3-MHR00C_3_2-EZ40, and H6-BGBP1-ES3-MHR00C_3_2-EZ80.Next, the plasmid for HEscherichia coli . Thetransformed strains were cultivated in 250 mL Erlenmeyer flasks with25 mL of terrific broth medium containing 50 μg/mL kanamycinat 37 °C/215 rpm for at least 16 h as the start culture. Thestart culture was diluted in a 2 L Erlenmeyer flask with up to 1 Lof autoclaved lysogeny broth (LB) medium . When the culture OD600 reached0.6–0.8, IPTG  was addedto LB at a final concentration of 1 mM. Next, bacteria were incubatedfor protein expression at 18 °C/215 rpm > 21 h before harvesting.After overnight protein expression, cultures were centrifuged at 4°C/6000 rpm for 30 min to pellet the cells. Bacterial pelletswere resuspended in 30 mL of cold lysis buffer . Then, 300 μL of PMSF (phenylmethylsulfonylfluoride) was added to the bacteria containing lysis buffer to a finalconcentration of 1 mM. Next, resuspended cells were sonicated usinga Q125 Sonicator (Qsonica) with a 2 s on/off duty cycle at 85% amplitude.After sonication, the bacterial lysate was centrifuged at 4 °Cand 30 000 × g for 30 min to obtain asupernatant with soluble proteins. Overexpressed proteins were isolatedfrom the supernatant using gravity-immobilized metal-ion affinitychromatography (IMAC) columns . Before elution,the column was washed with 10 column volumes of lysis buffer. Elutionwas done using 1 column volumes of elution buffer . A final polishing step was doneusing size exclusion chromatography (SEC). 1 mL of samples from IMACpurification was filtered using a 0.22 μm filter  and then injected into a Superdex 200 Increase 10/300GL column. The SEC purification process was performed with aflow rate of 0.75 mL/min in phosphate-buffered saline (PBS) bufferpH 7.4 on a 1260 Infinity II HPLC (Agilent). The purity of the proteinsthroughout the purification process was monitored using SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis). Analytical SECof purified proteins was performed on a Superose S6 10/300 gl  column with a flow rate of 0.5 mL/min.All plasmidswere sequenced before starting protein expression. Plasmids containingthe desired DNA sequences were transformed into T7-ExpressThe molecular masses of the proteins were verifiedusing matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)mass spectrometry. All samples were desalted before measurement usinga dialysis device (Thermo Scientific Slide-A-Lyzer MINI) with a cut-offof 3.5 kDa. Then, the dialyzed protein samples were concentrated to1mg/mL. Next, matrix solutions for measurement are prepared as follows:5 mg of DHB  was dissolved in 200 μLof solution .Then, 1 μL of the matrix was placed on a target plate , followed by 1 μL ofprotein solution. After that, the mixed samples were gently driedusing a hair dryer. Mass spectra were obtained using a Bruker UltraFlextreme. The data were processed using Bruker FlexAnalysis(version 3.4).A Jasco SpectropolarimeterJ-715 was used to record circular dichroism (CD) spectra. Sampleswere dialyzed to Milli-Q before measurement using a dialysis device(Thermo Scientific Slide-A-Lyzer MINI) with a cut off of 3.5 kDa.All samples prior to CD measurements were diluted to 0.1 mg/mL andplaced into a sonication bath for 10 min to minimize potential aggregation.All measurements were performed in a quartz cuvette  with a 1 mm path. For spectra, the continuous scanningmode was used, with a wavelength step size of 0.1 nm, a scanning speedof 50 nm/min, and a bandwidth of 2 nm. Spectra shown are averagesof 15 acquired spectra. For temperature ramp measurements, ellipticityat 222 nm was monitored continuously from 20 to 95 °C at a rateof 1 °C/min.B-M-E protein solutions were diluted to 10 μM in PBS buffer,then filtered with a 0.22 μm pore size filter, and placed ina sonication bath for 10 min prior to use. For measurement, both proteincoating formation and antifouling test were performed at a flow rateof 50 μL/min. First, a stable quartz crystal microbalance witha dissipation monitoring (QCM-D) baseline was obtained by prolongedflushing of the QCM-D channels. This was done until frequency variationswere less than ∼2 Hz. Next, the gold-binding B-M-E protein was flushed onto the sensors for 30 min, followedby a 15 min PBS wash step. Finally, for analyzing the antifoulingbehavior of the coating, bovine serum albumin ,1% HS, and 10% HS were injected for 30 min, followed by 15 min ofPBS wash step. The QCM-D data was analyzed using the QSense Dfindversion 1.2.7. .Gold-coated quartz sensors (QS-QSX301) were purchased from BiolinScientific, Sweden. QCM data was obtained using a Q-Sense E4 QCM-Dinstrument . B-M-E20 protein in PBSwas applied to the clean gold surface and then left to incubate for3 s and 5 min, respectively. After incubation, the samples were againrinsed with MQ water and dried using nitrogen gas. Next, the goldsensors were imaged using both a Multimode AFM with the ScanAsyst imaging mode in air and X-ray photoelectron spectroscopy(XPS). Scanasyst Air cantilevers (Bruker) were used with the followingspecifications: thickness 650 nm, length 115 μm, width 25 μm,resonance frequency 70 kHz, and spring constant 0.4 N/m. Data wasanalyzed by NanoScope Analysis version 1.5 (Bruker). XPS measurementswere performed using a JPS-9200 photoelectron spectrometer  with a focused monochromated Al Kα X-ray source (spotsize of 300 μm) radiation at 12 kV and 20 mA, with 10 eV asthe analyzer pass energy.Commercial gold sensor chips (dimensions: 12 × 7 mm) wereused as an atomic force microscopy (AFM) substrate. Gold sensor chipsare traditional glass slides with a 45–50 nm gold thin filmdeposited onto an adhesion layer .Gold surfaces were first plasma-cleaned followed by extensive rinsingusing Milli-Q water and drying using nitrogen gas. To prepare samplesfor AFM imaging, 50 μL of the 0.5 μM and 5 μM 9 GNPs/mL. Protein samples in PBS buffer at 0.1 μM were filteredusing a 0.22 μm pore size filter. The protein samples were placedinto a sonication bath for 10 min prior to use. The protein samplesand GNPs were mixed in a series of protein volumes: GNPs with a volumeratio of 1:49, 5:45, 15:35, 25:25, or 35:15, each time reaching afinal volume of 50 μL. Protein:GNP mixtures were incubated for5 min before starting the measurements. All measurements were performedin a quartz cuvette  with a lightpath of 3 mm at 20 °C. Each reported particle size is an averagevalue of 15 independent measurements. The reported hydrodynamic sizeswere obtained using the Zetasizer software version 7.13 . The reported values were obtained from a distribution fit performedby the Zetasizer analysis software. In all cases, a single peak dominatedthe scattering intensity, and values for this peak are reported.A ZS-Nano  instrumentwith a scattering angle of 173° was used to measure the hydrodynamicsize and zeta potential of gold nanoparticles (GNPs) before and afterthe protein coating. GNPs were purchased from Sigma-Aldrich . According to the productinformation provided by the manufacturer ,the concentration of gold nanoparticles is 1.9 × 1032 we designed a B-M-E triblock protein that spontaneouslyassembled on silica surfaces into coatings with good antifouling properties.Here, we redesigned the silica coating protein to instead coat goldsurfaces. A precise domain organization of the sequences of the originalsilica-coating protein and of the sequences for the redesigned goldcoating proteins are shown in B-M-E protein shown in Inour previous study,B-M-E20, B-M-E40, and B-M-E80 , where the length is expressed in terms of the number ofelastin-like pentapeptide repeats to arrive at the new designs. Wereplaced the silica-binding sequence BRT in the original design by the gold-bindingsequence BGBP1 and replaced the original elastin-like antifouling block withserine (S) as a host residue in the elastin-like pentapeptide motif, ES40, with a zwitterionic elastin-like block EZn with alternating aspartic acid (D) and lysine (K) as guestresidues and the number of pentapeptide repeats n = 20, 40, or 80.We designed a series of gold-coating proteins withincreasing lengthsof the antifouling block that we here refer to as BGBP1 is a 14-amino-acidgold-bindingpeptide referred to as GBP1.26 Many gold-bindingpeptides have been reported in the literature,43 but GBP1 is particularly well characterizedboth experimentally and with computer simulations.47 Also, GBP1 could bind to goldsurfaces even at high salt concentrations.48 Moreover, GBP1 is a peptide without cysteine (C) residues. In previousstudies,51 peptides or polymers containing a sulfhydryl group(-SH) were used to form a thiol bond with gold atoms to anchor peptidesor proteins on gold surfaces. Here, we chose a cysteine-free peptideas a surface anchor to show the potential of the B-M-E construct as a non-covalent coating. Unless specified, the B mentioned in below text is BGBP1.In our new designs, the surface anchor 32 the multimerizationdomain M is a well-characterized thermostabletrimer previously computationally designed by Fallas et al.39 and referred to as HR00C_3_2 (PBD ID: 5K7V).As in ourprevious design,55 Zwitterionic peptides and polypeptides have also been demonstratedto have good antifouling properties. For example, repeats(single-letteramino acid) of EK, DK, ER, and DR have been used in antifouling peptidesfor gold surfaces.50 Also, zwitterionicELPs with repeated sequences of VPKEG have been shown to have verylow interactions with blood proteins.56 For the antifouling domain, an improvement was sought by using azwitterionic ELP sequence with guest residues of the elastin-likepentapeptide being alternately D and K, rather than an uncharged hydrophilicsequence with the guest residue S. Here, we explore three zwitterionicpolypeptide sequences of type EZn = (GDGVP-GKGVP)n/2, for n = 20, 40, or 80.Recent studies have linked the strong interactionbetween zwitterionicpolymers and water molecules with their good antifouling properties.B-M-E20, B-M-E40, and B-M-E80 proteins were synthesized and cloned into avector with a T7 promoter system. Proteins were expressed in E. coli and purified using IMAC, followed by SEC. B-M-E20, B-M-E40, and B-M-E80. Additional data is shown in Figure S1–S3. SDS-PAGE analysis of the final purifiedproteins shows a single band (B-M-E withestimated molar masses of 129 kDa (B-M-E20), 155 kDa (B-M-E40), and 207 kDa (B-M-E80), respectively. To more precisely establish the mass of the purifiedpolypeptides, we used MALDI-TOF mass spectrometry. Results for B-M-E20 are shown in B-M-E40 and B-M-E80 are shown in Figures S2b and S3b). We find thatthe mass experimentally determined for B-M-E20 (43 088.1 Da) matches the theoretically expectedvalue (43 064.3 Da) within the error of the measurement, andthe same holds for B-M-E40 and B-M-E80 as shown in Synthetic genes encoding M domain is exclusivelyα-helical,39 and if the experimentalCD spectra are consistent with that of mainly a α-helical protein,we can have good confidence that the trimerization domain M is correctly folded within the full-length B-M-E20. Results for the CD spectrum of B-M-E20 are shown in M domain is correctly folded within the full lengthof B-M-E20. The M domain has previously been reported to be highlythermostable.39 We have also found thisto be the case for the M domain in thecontext of our previously designed silica-coating proteins.32M domain in the gold-coating proteins: the spectrado not change when heating from 20 to 95 °C and cooling backto 20 °C (20 °C rev). Indeed, Next, we investigated the protein secondary structureusing CDspectroscopy. The B-M-E proteins on goldsurfaces. In our earlier work on silica-coating proteins,35 we found that B-E designs with only one silica-binding domain could still berinsed off with high salt buffers or displaced by serum proteins.However, this was no longer the case for the B-M-E designs with multivalent surface anchorage. Here, we use QCM-D toinvestigate whether the new series of B-M-E triblocks,redesigned to bind to gold surfaces, form protein brushes on goldsurfaces in PBS buffer. The QCM-D results for coating formation by B-M-E20, B-M-E40, and B-M-E80 on gold surfaces areshown in B-M-E protein solutions, the quartzcrystal oscillation frequencies decrease, indicating protein layerformation, with the magnitude of the frequency drop being proportionalto the length of the E block. Accordingto the Sauerbrey equation,57 the frequencydrop is only linearly related to adsorbed mass in QCM-D for adsorbedlayers with low dissipation. Previously, we have found that the silica-binding B-M-E proteins form highly hydrated polypeptide brusheswith strong dissipation.32 Rather thantrying to fit our data with complicated models fully accounting forthe dissipation, we here simply use the frequency drop as a qualitativemeasure for adsorbed mass.Next, we tested the formation ofprotein coating by the B-M-E proteins, the frequency shift signal saturates,indicating that no additional protein is bound. Next, QCM-D sensorswere flushed with PBS. This did not lead to any observable changein the QCM signal, indicating that no washing off of any potentiallyweakly bound B-M-E proteins took place, nordid the PBS buffer displace bound B-M-E proteins.We therefore conclude that the trivalent binding of the GBP1 peptideis sufficiently strong.Before injecting proteins, a flat baseline wasobtained by flushingthe gold sensor with PBS for 20 min. Then, the frequency signal decreasedwhen proteins were injected into the QCM channels, which means proteinswere binding to surfaces. Around 10 min after starting to inject the B-M-E proteins adsorbed on gold surfaces.To verify that the proteins coat thegold surface homogeneously,we performed AFM imaging of dried layers of the B-M-E trimers adsorbas independent units and do not adsorb in clustersor as aggregates. This is best studied at conditions leading to lowercoverage than the conditions used in the QCM-D coating formation experiments.Dried samples were imaged in air. Representative AFM images for B-M-E20 are shown in B-M-E20 for 3 s or 5 μM B-M-E20 for 5 minare shown in More specifically, wewant to verify that the Figure S3 and Table S4. These showed that onlyfor the samples incubated with protein, and not for the bare surfaces,there are clear carbon and nitrogen signals, next to the Au signalthat is present for all samples.To confirm that the additional feature visiblein the AFM imagesare indeed due to the adsorbed protein, we performed XPS measurementson samples identical to those used in AFM. XPS results are shown in B-M-E polypeptide brushes self-assembledonto gold surfaces, we used dynamiclight scattering (DLS). As shown earlier for silica nanoparticlesand the silica-coating B-M-E polypeptides,32 bridging interactions may lead to particle clusteringat very low concentrations, but at higher concentrations, a fullysaturated layer develops, leading to a small but measurable increasein the particle hydrodynamic diameter, from which we can estimatethe layer thickness.To determine the layer thicknessof the d =83 nm. Dilute suspensions of the GNPs were incubated for 5 min atdifferent concentrations of the gold-binding B-M-E proteins. Results for DLS size measurements and zeta potential measurementsfor the case of B-M-E20 are shown in We used GNPs with a hydrodynamic diameter B-M-E20 concentrations,DLS suggests significant bridging-induced GNP clustering similar towhat was observed earlier for B-M-E proteinsand silica nanoparticles. Bridging could be caused by interactionsof the GNPs with any of the three domains of the B-M-E protein, as indicated in the inset of As shown in D stabilize at a value somewhatlarger than the diameter d of the uncoated GNP. Thediameter of B-M-E20-coated goldnanoparticles is D = 107nm, suggesting a layer thickness h for the B-M-E20 coating of h = (D – d)/2 = 12 nm. Results for B-M-E40 and B-M-E80 were similar, although, as expected, the layer thicknessincreases with the length of the E block.These DLS data are shown in Figures S5 and S6.At high concentrations, the measuredhydrodynamic diameters B-M-E20 using smaller GNPs with a hydrodynamic diameter d = 42 nm . We finda very similar layer thickness of h = 13 nm. Dueto the lower scattering of the smaller GNP, at high protein concentrations,one can now also observe a peak in the DLS signal due to the dissolvedproteins. This peak corresponds to a hydrodynamic diameter d = 14 nm for the B-M-E trimer,which is of the same order of magnitude as the layer thicknesses wefind.To probe for any dependence of layer formation on GNP size,wealso performed the DLS measurement for B-M-E20 proteins are shownin B-M-E20, the zeta potentialbecomes somewhat less negative, changing from −10 mV for uncoatedGNP to −6 mV for coated GNP.Results for the zeta potential of GNPs and GNPs coatedwith 58 Therefore, we next tested the extent to which the B-M-E coatings can prevent non-specific protein adsorption.In our previous study,32 we found thatthe silica-binding B-M-E protein performed well against1 mg/mL BSA. Here, we investigate biofouling for B-M-E layers assembled on gold surfaces against diluted HS .Our aim is to render the goldsurfaces antifouling and, at a later stage, to make the coating layersfunctional, for example, for use in biosensor surfaces.B-M-E layer was formed and extensively rinsed with PBS. Next, theprotein foulant was injected to the QCM-D channels and finally rinsedagain with PBS. Results for 1% HS and 10% HS are shown in 59Representative QCM-D results of antifoulingtests with diluted HS are shown in B-M-E proteins are trimers of ratherhigh molar mass and they adsorb not as rigid flat layers but as thick,hydrated polymer brushes, as we discuss in more detail in furtherparagraphs.The signal for serum proteins adsorbing to thebare gold sensorsis smaller than that for the coating proteins adsorbing to the goldsensors since the B-M-E proteins,hardly any fouling can be detected for the case of 1% HS , EZ used here is differentfrom the serine-containing ELP block ES used for the silica-bindingversions of the B-M-E protein studied before.32 To check whether the change to zwitterionicELP E blocks indeed improves the antifoulingperformance, we also constructed the gold-binding B-M-E20 protein with E = ES as the antifouling block. The antifouling performance of B-M-ES20 and B-M-EZ20 layersadsorbed on gold against 10% HS is compared, and the results are shownin Figure S8. We find that the zwitterionicELPs indeed offer a better antifouling performance, as hypothesized.The sequence of the zwitterionicELP block 61). Also, for the incubationtimes with the coating proteins used in B-M-E40, we tested longer incubation times with coating proteins,as well as longer incubation times with 10% HS . Results suggest that longerincubation times with the coating proteins, as expected, lead to aQCM signal that eventually becomes stable and remains stable afterflushing with PBS . After longerincubation with 10% HS, we observe for the uncoated gold surface  but not for the coated gold surface that during the final PBS rinse,there is somewhat more displacement than observed for shorter incubationtimes with 10% HS.It is well known that protein adsorption from HS onto solid surfaceshas complex kinetics with faster adsorbing components with low affinitygradually being replaced by more slowly adsorbing species with highaffinity  layers on gold surfaces.50 Cysteinescould potentially be engineered into the binding blocks B, but we have shown that even without cysteines,the trivalent gold-binding peptides offer sufficiently strong binding.Moreover, an advantage of using gold-binding peptides may be thatbinding is not influenced by the presence of reducing agents.In this work, we have relied on a non-thiol-containinggold-bindingpeptide sequence for surface anchoring to gold. A complementary andfrequently used approach is to use thiol groups, for example, in cysteineresidues, to strongly anchor molecules to gold surfaces.B-M-E triblocks offer a multi-material solution that can be usedto modify multiple materials in an identical manner. Indeed, a widerange of sequences for solid-binding peptides are available. For example,sequences for metal-binding peptides,63 mineral-bindingpeptides,28 plastic-binding peptides,30 and even for semiconductor-binding peptides.31 The potential for analogous B-M-E design-basedprotein coatings is therefore large.The main advantage, however, is that the modular 64 Somewhat to our surprise, we found no difference in thedegree of antifouling for the B-M-E proteinswith different lengths of the E block.In particular, we found that B-M-E20, with the shortest antifouling block, works just as well as theones with longer E blocks, B-M-E40 and B-M-E80.In the literature on antifoulingpolymer brushes, it is generallyreported that the degree of antifouling, at a given grafting density,increases with the length of the brush polymers.B, which favor a brush density that is as high as possible.If the surface is not saturated with binding blocks, and the lateralbrush pressure of antifouling blocks E favors a density as low as possible. Possibly, in the present case,this competition leads to significantly higher brush densities forthe B-M-E20 polymer, such thatantifouling properties are maintained, even though the length of theantifouling block is short.Quite likely, the brush density of the self-assembled brushes arisesfrom a competition between the binding blocks B) and antifouling domains (E) of our original silica-coating triblockprotein,32 we have demonstrated here thatthe B-M-E design that allows the formationof highly stable proteincoatings is highly modular. For the current gold-binding peptidesas the binding domain, excellent antifouling could be obtained usinga zwitterionic E domain, E = EZ.By changing the sequences of the bindingdomain (It would be interesting to test whethereven shorter antifoulingblocks still lead to acceptable results. This would be highly relevantfor the particular case of gold surfaces since especially for sensingschemes relying on local surface plasmon resonance, any coating needsto be as thin as possible.B-M-E designs provideexcellent antifouling against 1% HS on gold surfaces. In at leastsome sensing applications, a 100-fold dilution with buffer may beacceptable or desired, but a much broader range of applications wouldbe possible with an increased antifouling against, for example, 10%HS. This very likely requires coatings with a further increased surfacedensity of more hydrophilic E blocks.This could possibly be achieved through a further optimization ofboth M and E blocks, and work toward this is ongoing.Our current E blocks, e.g., with capture molecules forbiosensing applications. Especially, if the functional groups to beadded are proteins or peptides, the protein nature of B-M-E is a great advantage, allowing for either directinclusion of the functional domains in fusion designs or indirectattachment via one of the many available immobilization tags.Finally, many applicationswill involve functionalization of the


E-C was found to be very effective for the treatment of rural domestic sewage. The removal rate of TP in rural domestic sewage was 91–95.9%. After adsorption treatment, the discharge of TP in rural sewage met the second-grade (TP < 3 mg/L) and even first-grade (TP < 2 mg/L). This study provides an experimental basis for efficient P removal by E-C adsorbent materials and suggests possible applications in rural domestic sewage.In order to promote the improvement of the rural living environment, the treatment of rural domestic sewage has attracted much attention in China. Meanwhile, the rural regions’ sewage discharge standards are becoming increasingly stringent. However, the standard compliance rate of total phosphorus (TP) is very low, and TP has become the main limiting pollutant for the water pollutants discharge standards of rural domestic sewage treatment facilities. In this study, waste eggshell (E) was employed as a calcium source, and waste peanut shell (C) was employed as a carbon source to prepare calcium-modified biochar adsorbent materials (E-C). The resulting E-C adsorbent materials demonstrated efficient phosphate (P) adsorption from aqueous solutions over the initial pH range of 6–9 and had adsorption selectivity. At an eggshell and peanut shell mass ratio of 1:1 and a pyrolysis temperature of 800 °C, the experimental maximum adsorption capacity was 191.1 mg/g. The pseudo second-order model and Langmuir model were best at describing the adsorption process. The dominant sorption mechanism for P is that Ca(OH) China is a big agricultural country with nearly 500 million rural people. Due to population growth and the improvement in social and economic living standards, the increase in rural domestic sewage is becoming one of the main environmental problems in rural areas . In rece2O) is one of the most typical schemes in China. However, some reports have observed that the efficient removal of COD, NH3-N, and total phosphorus (TP) is hard to realize [The most conventional treatment methods for domestic sewage in rural regions consist of an operational sequence of aerobic/anoxic/aerobic processes. Anaerobic–anoxic–oxic  present in wastewater ,6. AmongThe removal of P in wastewater from pig farm, cattle farm, pond and human urine by Ca-modified biochar has been reported ,10. So f2PO4, HCl, NaOH, KCl, KNO3, K2SO4 and KHCO3) were of analytical grade and purchased from Aladdin Industrial Corporation .The peanut shells were obtained locally in Jiaxing (China). Before usage, they were air-dried and then ground to pass through 40 mesh sieves (0.425 mm). Waste eggshells were obtained from a mess hall in Jiaxing. They were dried at 60 °C in an oven after being cleaned three times. Additionally, there were then ground to pass through 40 mesh sieves (0.425 mm) before use. All chemical reagents  in a shaker bath  at pH 7 and 25 °C. The E-C adsorbent dose was 250 mg/L. Then, the suspension was filtered through a 0.45 μm PES syringe filter, and the filtered solution was used to detect the phosphorus concentration. A total of 0.1 mol/L NaOH or HCl was used to adjust the initial pH values of the solutions. KH0 rpm in To evaluate the equilibrium adsorption parameters, adsorption isotherm experiments were conducted as follows: E-C dose, 250 mg/L; initial P concentrations of 5, 10, 25, 50, 100, 150 and 200 mg/L; initial pH, 7.0; and adsorption time, 24 h.Adsorption kinetic tests were conducted as follows: E-C dose, 250 mg/L; initial P concentration, 100 mg/L; initial pH, 7.0; and adsorption time, 1–1440 min.−, NO3−, SO42− and HCO3−) on P removal by E-C were evaluated as follows: E-C dose, 250 mg/L; initial phosphate concentration, 100 mg/L; Cl−, NO3−, SO42− and HCO3− concentrations, 100 and 1000 mg/L; and adsorption time, 24 h.The effect tests of common competing substances  process (named as F1), and the design capacity was 50 t/d. The second centralized rural sewage treatment facility was an A2O process (named as F2), and the design capacity was 20 t/d. Every three days, a sample of rural home sewage was taken from the secondary sedimentation tank. A total of 40 mL of rural home sewage was collected in 100 mL glass vials with stoppers containing 0.01 g of E-C adsorbent materials, and the vials were oscillated (at 180 rpm) in a shaker bath for 4 h at 25 °C.Rural residential sewage from two centralized rural sewage treatment facilities in Jiaxing (Zhejiang province) were utilized to assess the E-C adsorbent’s suitability for actual phosphorus sewage. The first centralized rural sewage treatment facility was the anaerobic–anoxic–oxic  and adsorption capacity at different times t  was calculated as follows:0 (mg/L) represents the initial P concentration, Ce (mg/L) represents the P concentration at equilibrium, and Ct (mg/L) represents the P concentration at time t; V (L) is the volume of the solution; and m (g) is the adsorbent mass.All experiments were repeated three times. The equilibrium adsorption capacity  according to the standard method . The sur2/g, average pore size of 2.95 nm, and pore volume of 0.083 cm3/g; the corresponding values for E-C were 64.956 m2/g, 6.46 nm and 0.105 cm3/g, respectively.By weighing the samples before and after pyrolysis at 800 °C, the resulting mass loss (49.3%) during the preparation of the E-C adsorbent material was achieved. The basic physicochemical properties and major elements of the E-C and BC adsorbent materials are shown in 2 and CaO during high-temperature pyrolysis [2 broadens the material’s pore size as an activation substance, and it can therefore be used to produce activated carbon [The eggshell decomposed into COyrolysis . The ford carbon . In addid carbon .3 in eggshells was successfully introduced into the E-C adsorbent materials.The pore volume and surface area of the E-C were higher than those of the BC. Biochar’s adsorption performance largely depends on its porosity and specific surface area. The larger the adsorbent’s specific surface area and higher the porosity, the more surface adsorption sites there will be and the higher the adsorption performance ,22,23. At is the P adsorption amounts at time t, and qe is the P adsorption amounts at equilibrium time; k1 is the first-order kinetics’ adsorption rate constant, and k2 is the second-order kinetics’ adsorption rate constant. The results showed that the coefficient of determination simulated with the pseudo-second-order model (R2 = 0.99) is higher than the pseudo-first-order model (R2 = 0.83)  , which se (mg/g) is the equilibrium adsorption amount, KL (L/mg) is the Langmuir adsorption equilibrium constant, qmax (mg/g) represents the maximum adsorption capacity, Ce (mg/L) represents the P equilibrium concentration, KF (mg(1−1/n)L1/n/g) represents the Freundlich adsorption constant representing the adsorption capacity of the adsorbent materials, and n represents an indication of linearity.In this study, the isothermal experiments were carried out with different initial P concentrations . Langmuir and Freundlich adsorption isotherm models were used in the study of E-C adsorbent’s adsorption isotherms for P in aqueous solutions:max = 195.8 mg/g, KL = 0.39 L/mg, and R2 = 0.95. The corresponding fitting parameters for the Freundlich model are as follows: KF = 47.3 mg(1−1/n)L1/n/g, 1/n = 0.32 L/mg, and R2 = 0.91. Furthermore, the regression coefficient 2R of the Langmuir model is higher than that of the Freundlich model. it is further confirmed that the Langmuir isotherm model adequately describes the experimental data of P adsorption by E-C, implying that the effective adsorption surface of E-C is homogeneous and a monolayer during the adsorption of P. this is consistent with the above adsorption kinetic results described by the pseudo-second-order model. Similar P adsorption behavior has been reported in other research of Ca-based adsorbents [The fitted adsorption isotherms of P onto the E-C adsorbent materials are shown in sorbents ,26,27,28max of the E-C, through calculating the Langmuir isotherm, was 195.8 mg/g, which was consistent with the experimental maximum adsorption capacity of 191.1 mg/g, indicating that the E-C adsorbent materials in aqueous solution had excellent adsorption properties for removing P. The E-C adsorbent material’s adsorption capacity for P was higher than that of the Ca-biochar adsorbent prepared from eggshell and peanut shells through ball milling and chemical impregnation methods (the maximum P adsorption capacity was 130.57 mg/g) reported by Liu et al. [The qu et al. .3, KNO3 and K2SO4) were added into the adsorption system. These common anions have different levels of effect on the P adsorption capacity of the E-C adsorbent materials, following the order HCO3− > SO42− > NO3− > Cl−  on the adsorption of P onto the E-C was examined. The P adsorption amount onto the E-C increased from 182.6 mg/g at pH = 6 to 191.1 mg/g at pH = 7 . AdditioIn this study, calcium-active components contributed to the adsorption of P onto the E-C adsorbent material by strong chemical interaction mechanisms, resulting in the high adsorption capacity of the E-C for P over the pH range (6–9).−1 (C-O) and 1420 (C=C) were observed for the adsorbent before and after adsorption, suggesting that the C-O and C=C groups did not contribute to the adsorption of P. Before adsorption, the peak at 3641 cm−1 (O-H) was attributed to Ca(OH)2 for the E-C [−1 vanished, showing that the -OH took part in the chemical adsorption process of P. Furthermore, the peaks at 563, 601 and 1027 cm−1 (P-O) were significantly enhanced and attributed to phosphates, showing that P was adsorbed onto the E-C adsorbent materials. Similar results were reported by other researchers [The adsorbent materials before and after P adsorption were analyzed by XRD, SEM and FTIR, and the adsorption mechanisms of P onto the E-C adsorption materials were discussed. Before and after P adsorption, the characteristic peaks of the BC had no significant change in the FTIR spectra analysis A. This w the E-C . After aearchers ,20.3 characteristic peaks  and Ca(OH)2 characteristic peaks . Ca(OH)2 groups are the main active sites in the E-C adsorbent materials for P adsorption. CaCO3 groups in eggshells were not fully decomposed at 800 °C. In Lee’s study, at a calcination temperature of 800 ℃, the mineral composition of eggshell still contained 35.7% CaCO3 [The diffraction patterns of the E-C before and after P adsorption showed a significant change in the XRD spectra analysis . Before 2 diffraction peaks vanished, and the E-C had Ca5(PO4)3OH characteristic peaks . The Ca5(PO4)3OH, generated from the reaction of phosphate with Ca(OH)2, was responsible for the removal of P from the water, consistent with the FTIR and kinetic findings. In addition, the CaCO3 (PDF #05-0586) characteristic peaks still existed. It was pointed out that CaCO3 did not participate in the chemical reaction with P. CaCO3 might adsorb a small amount of P by physical adsorption.After adsorption, Ca(OH)3 and Ca(OH)2. In addition, the E-C adsorbent materials had a porous structure before adsorption. After adsorption of P, a lot of flocculent precipitates appeared on the materials’ surfaces, and the pores of the E-C were blocked. The above results show that during the adsorption of P onto the E-C adsorbent materials, a good deal of flocculent precipitates were generated on the surface of the E-C adsorbent materials and in the pores. Therefore, the porosity and effective active sites of the E-C were reduced, and finally the adsorption saturation of the E-C adsorbent materials was induced. From 5(PO4)3OH.Before and after P adsorption, the morphologies of the BC had no significant change though SEM images A,B. Befo2 reacts with phosphate to form Ca5(PO4)3OH precipitates, and this result is similar to other studies [According to the above results, the mechanisms of the E-C to adsorb P include physical adsorption, surface precipitation, and electrostatic attraction. The dominating sorption mechanism of the E-C for P is that Ca(OH) studies ,25,31.3-N, COD and pH in rural sewage of F1 was 12.1–22.8 mg/L, 42–85 mg/L and 6.8–7.2, respectively. The discharge of NH3-N, COD and pH in rural sewage of F2 was 6.8–19.1 mg/L, 35–76 mg/L and 6.7–7.2, respectively. The discharge of NH3-N, COD and pH in rural sewage of two centralized rural sewage treatment facilities met the second-grade Discharge Standard (DB33/973-2021)  [It was observed from  pH 6~9) . PhosphoThe TP content in the rural domestic sewage of F2 was 3.15–10.6 mg/L B, the TPMany complex components may be present in actual wastewater which can interfere with P removal by the adsorbent. In this study, the E-C adsorbent materials also exhibited a high P adsorption capacity from actual rural sewage. Therefore, the E-C adsorption materials have great potential in the treatment of large-scale rural domestic sewage containing P.5(PO4)3(OH) is produced on the E-C adsorbent materials; furthermore,, it also has the physical properties of biochar. Therefore, the P-adsorbed E-C adsorbent materials may be further used as soil regulators and fertilizer. This method can realize the reuse of E-C and realizes the virtuous cycle of P resources in the ecosystem [The recovery of adsorbents is an important economic parameter. However, the operation procedure of recycling P adsorbents is very complicated . The maicosystem ,20,30,332 on the E-C adsorption materials reacting with phosphate to generate Ca5(PO4)3OH precipitate. E-C effectively removed P of rural domestic sewage. The removal rate of TP in rural domestic sewage was 91–95.9%. After adsorption treatment, the discharge of TP in rural sewage met second-grade Discharge Standard (DB33/973-2021) (TP < 3 mg/L), and even met the first-grade Discharge Standard (DB33/973-2021) (TP < 2 mg/L). This study provides the experimental basis for the efficient removal of phosphorus by E-C adsorbent materials, and it provides the possibility for its application in rural domestic sewage.In this study, Ca-modified biochar (E-C) adsorbent materials were prepared utilizing eggshell and peanut shell waste as raw materials. The E-C adsorbent materials were used to remove P from aqueous solutions, and the results show that the prepared E-C adsorbent materials exhibited an excellent adsorption capacity over the initial pH range of 6–9 and adsorption selectivity. An experimental maximum adsorption capacity of 191.1 mg/g was obtained by the E-C sample that was prepared with a mass ratio of 1:1 and a pyrolysis temperature of 800 °C. The dominant sorption mechanism for phosphate was due to Ca(OH)


Reference target protein sequences with 100% identity were retried from UniProt following NCBI BLAST. The types and locations of RAASs were identified and visualized by AlphaFold and PyMol. Linux-r-base/R-studio was used for the data presentation. Multi-drug-resistant variants of NS3/4A in genotype 1 (n = 9) and genotype 5 (n = 5) along with DAA-specific NS3/4A, NS5A, and NS5B variants were identified pan-genotypically. A total of 27 variants (RAASs) of all the targets were identified. Fourteen genotype 1-specific substitutions: V1196A, V1158I, D1194A/T/G, R1181K, T1080S, Q1106R, V1062A, S1148G, A1182V, Y2065N, M2000T, and L2003V were identified. The most frequent substitutions were V1062L and L2003M, followed by Q2002H. L2003V, Q2002H, M2000T, Y2065N, and NL2003M of NS5A and L2003M of NS5B conferred resistance to daclatasvir. S2702T NS5B was the sofosbuvir-resistant variant. D1194A NS3/4A was triple DAA  resistant. The double-drug resistant variants R1181K , A1182V and Q1106K/R , T1080S , and single drug-resistant variants V1062L (telaprevir), D1194E/T (simeprevir), D1194G (asunaprevir), S1148A/G (simeprevir), and Q1106L (Boceprevir) of NS3/4A were determined. The molecular phenomenon of DAA resistance is paramount in the development of HCV drug candidates. RAASs in NS3, NS5A, and NS5B reduce the susceptibility to DAAs; therefore, continuous RAAS-dependent resistance profiling in HCV is recommended to minimize the probability of DAA therapeutic failure.Hepatitis C virus (HCV) is a hepatotropic virus that affects millions of human lives worldwide. Direct-acting antiviral (DAA) regimens are the most effective HCV treatment option. However, amino acid substitution-dependent resistance to DAAs has been a major challenge. This study aimed to determine the increasing risk of DAA resistance due to substitutions in DAA target non-structural proteins . Using a Sequence Retrieval System (SRS) at the virus pathogen resource (ViPR/BV-BRC),  HCV and hepatitis B virus (HBV) are the two major hepatotropic viruses , which aApart from the side effects of the standard therapy, another challenge that impedes the effective management of HCV infection is its chronicity and potential to progress to HCC. Moreover, attaining sustained virologic response (SVR) for at least 3 or more months is a crucial parameter for measuring the effectiveness and success of the therapy . The pooVirus Pathogen Database and Analysis Resource , USA. Multiple query datasets were generated based on the retrieved sequences to execute the risk assessment analyses. To ascertain the risk of resistance to DAAs , the protein sequences of the drug target nonstructural proteins  of hepatitis C virus were retrieved from Bacterial Viral Bioinformatics Resource Center (BV-BRC)/Nonstructural proteins  sequences were retrieved from across the HCV genotypes (from genotypes 1 to 7) by applying the in-built Sequence Retrieval System (SRS) at ViPR, now known as BV-BRC Bacterial and Viral Bioinformatics Resource Center | BV-BRC. Nonstructural protein sequences without any genotype designation (genotype unclassified/genotype UC) in the database were also retrieved, evaluated, analyzed, and included in the present study. While applying SRS, specimen attributes such as blood, plasma, serum, ascitic fluid, and cell supernatant were considered. Host attributes (across gender), virus attributes (across HCV genotypes), infection class , and time attributes (beyond the year 2011) were also kept under consideration while executing using SRS. n = 26533), d2 = genotype 2 (n = 371), d3 = genotype 3 (n = 2738), d4 = genotype 4 (n = 154), d5 = genotype 5 (n = 31), d6 = genotype 6 (n = 444), d7 = genotype 7 (n = 03), and d8 = genotype UC (n = 2489) before processing them (n = 25162), d2 = NS5B (n = 5413), and d3 = NS5A (n = 2188) before being processed  with their booster protease inhibitors (ritonavir), NS5B/RdRp inhibitors (sofosbuvir), and NS5A inhibitors . The Food and Drug Administration (FDA) approved various combinations of two or more antivirals from these three groups of DAAs at different times from 2011 onward for treating HCV infections caused by different genotypes . As the ing them b. Based rocessed c.AlphaFold Protein Structure Database (ebi.ac.uk). The locations of the RAASs (mutations) were ascertained and visualized by mapping the folded 3D structure of DAAs’ target proteins by employing PyMOL PyMOL | pymol.org. Data visualization and graphical context were generated by the R-package for the WSL2-based R-studio. The execution of the analytical methods has been demonstrated by a self-explanatory diagram (I determined the DAA resistance risk by employing an in-built sequence feature variant type (SFVT) of ViPR’s antiviral-resistance assessment tool, which is based on a computational algorithm. Each query dataset was purified, prepared, and separately analyzed via SFVT to gain insight into the resistance-associated amino acid substitutions (RAASs). The reference sequences for NS3 and NS5A/NS5B polyproteins used during the analyses were sequence IDs AAA72945.1 and NP_671491.1, respectively. The result was obtained following the sample run, which encompassed the enhanced resistance to any one or multiple DAAs with their allied RAASs in each query sequence of the datasets. Some RAASs without any phenotypic effect on resistance to the DAAs were excluded from the study. To access the locations of the RAASs on the respective DAA targets, each reference sequence was blasted using NCBI Protein Basic Local Alignment Search Tool BLAST: Basic Local Alignment Search Tool (nih.gov) to select NS3 and NS5A/NS5B sequences with 100% percent identity and query coverage. Based on the sequence IDs of selected sequences, I retrieved sequences from UniProt UniProt to achieve a 3D protein structure (UniProt ID for NS3:P27958 and for NS5A/NS5B: P26663). The retrieved sequences were folded for a 3D protein structure using AlphaFold2/MMSeqs2  diagram .n = 23) genotype 1, forty-seven (n = 47) genotype 2, one (n = 1) genotype 3, five (n = 5) genotype 4, twelve (n = 12) genotype 5, and four (n = 4) in genotype unclassified sequences (n = 4), one (n = 1), twenty-six (n = 26), one (n = 1), and one (n = 1), respectively (n = 55), twenty-six genotype 4 (n = 26), and one unclassified genotype (n = 1), which is summarized in n = 9) genotype 1 and five (n = 5) genotype 5 mutated NS3 protein sequences exhibited enhanced resistance (ER) to multiple DAAs (2-3 DAAs) while the rest of the mutated NS3, NS5A, and NS5B sequences were associated with resistance to a particular DAA  RAASs in total across all the DAA target proteins under consideration: S2702T, V1196A, I1158V, D1194E, D1194T, D1194G, R1181K, T1080S, Q1106R, D1194A, V1062A, V1062L, Q1106K, S1148G, A1182V, S1148R, L2003M, Q2002R, Q2002H, Q2002L, Q2002S, S1148A, Y2065H, Q1106L, Y2065N, M2000T, and L2003V (n = nine different positions) of unique resistance-associated substitutions in NS3 , NS5A , and NS5B  target proteins were spotted and detected using PyMol  in target proteins for DAAs have been a major concern ,41,42. IIn the current study, the resistance-associated amino acid substitutions in all the target proteins of DAAs, NS3 , NS5A , and NS5B , have been identified, which corroborates reports in other studies ,45,46,47Substitution V1062L was evaluated pan-genotypically , which is validated by the results of Keiffer et al. , and LinMoreover, Stedman et al. highlighted the inhibitory effect of sofosbuvir on NS5B polymerase across the HCV genotypes . S2702T On the contrary, Wyles et al. demonstrated that there was no influence of Q to K HCV polymorphism on the treatment outcome of simeprevir and sofosbuvir in combination . BocepreDAAs are the most effective therapeutic options for the treatment of chronic HCV infections. However, since the DAA has been approved for clinical applications by the Food and Drug Administration (FDA), resistance-associated mutations in DAA target proteins have been the focus in order to avoid the possibility of therapeutic failure. In the present study, a wide range of amino acid substitutions in NS3A, NS5A, and NS5B target proteins for DAAs, which were associated with resistance to various FDA-approved DAAs for the treatment of HCV infection, were determined across the HCV genotypes. Multidrug-resistant NS3/4A variants in genotype 1 and genotype 5 were determined. Amino acid substitution-dependent resistance to NS3/4A protease inhibitors, NS5A, and NS5B inhibitors was assessed across the HCV genotypes. V1062L and L2003M were observed to be highly frequent, followed by Q2002H. These RAASs may impact the efficacy of the DAA-based HCV treatment, leading to virologic failure even in patients treated/retreated with multiple DAAs due to the emergence of multidrug-resistant variants of HCV. HCV-infected patients in which DAA treatment fails due to these RAASs are categorized as difficult-to-treat patients. Combination therapy SOF/VEL/VOX or SOF/GLE/PIB plus RBV for 12 weeks or without RBV for 16–24 weeks are the treatment choices for such difficult-to-treat patients. Therefore, the continuous assessment of pan-genotypic RAAS profiling in HCV is recommended to ensure the success of the DAA treatment and minimize the probability of therapeutic failure during the management of chronic HCV infection. Moreover, the execution of large-scale targeted next-generation sequencing of NS3A, NS5A, and NS5B proteins of isolated HCV genotypes/subtypes, especially from difficult-to-treat patients, could be considered for future drug design along with the current strategy of identifying in vivo and in vitro effective combinations of DAA.


Scientific Data 10.1038/s41597-023-02365-y, published online 12 July 2023Correction to: In this article, the grant number NRF2022R1A5A102641311 relating to National Research Foundation of Korea was omitted. The original article has been corrected.