X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), diffuse reflectance spectroscopy (DRS), transmission electron microscopy (TEM), Raman spectroscopic analysis, and atomic force microscopy were used to analyze the composites as prepared. The unique TiO2-FG/Sr-hexaferrite-based composite catalyst reveals superior photocatalytic properties for the disintegration of organic dyes methylene blue (MB) and rhodamine B (Rh. B) under visible-light irradiation. The result showed that the functionalized graphene with ternary structure improved the catalytic behavior of the composite due to the synergistic effect of the TiO2-FG boosted by the graphene surface to provide a fast conducting path to the photogenerated charge carrier. The markedly high photocatalytic behavior has been ascribed to the formation of the ternary structure between TiO2, FG, and Sr-hexaferrites through interface interaction. The prepared photocatalyst composite exhibited better recyclability, which further confirms its future uses as a photocatalyst in industrial waste products.Novel visible-light photocatalyst  TiO Heterostructure catalyst materials based on two semiconductors supported by a moderator have been widely used in the photocatalysis treatment process with many successful results . The isoO3, etc. ,3,4,5. SO3, etc. ,7. TransO3, etc. . There aO3, etc. . The funO3, etc.  and to hO3, etc. ,12.2 complexes have drawn a lot of attention because of the large surface area as well as better charge conductivity of graphene and the nontoxic, affordable, and ecologically friendly behavior of TiO2 [2. It is due to the π-π interaction, which greatly enhances the affinity with organic pollutants to increase the efficiency of the photocatalyst. To meet the environmental requirements, special care must be taken when creating new photocatalysts, i.e., the easy and fast synthesis route and nontoxic behavior. Because the system of heterogeneous photocatalysts works together synergistically, they can create greater catalytic activity [2-FG/Sr-hexaferrite nanocomposites supported by hexaferrite nanoparticles. For the first step, graphene oxide was taken and functionalized using chemical vapor deposition techniques. The functionalized graphene (FG) was then used as an electron mediator in a ternary system to boost the catalytic behavior of the nanocomposites [Integrated graphene/TiO of TiO2 ,14. It hactivity . Quantumactivity ,16. Despactivity . In thismposites .16H36O4Ti) used as a titanium precursor was purchased from Samchun Pure Chemical Co., Ltd., Pyeongtaek-si, Korea. Strontium nitrate tetrahydrate H8N2O10Sr and ferric hydroxide Fe(OH)2 were purchased from Daejung Chemicals Co., Ltd., Siheung-si, Korea. The supplier of ethylene glycol was Dae-Jung Chemical and Metals Co., Ltd., Siheung-si, Korea. Dae-Jung Chemical and Metals Co., Ltd., Korea provided benzene, which was utilized as a precursor to carbon. Argon gas was purchased from Samchun Pure Chemical Co., Ltd. All chemicals were used directly without further purification, and distilled water was also used in every experiment. Graphene oxide was produced using the Hummers–Offeman method [oC, was used to functionalize graphene through benzene vapors. Benzene vapors were produced prior to heating at below its boiling temperature. The control valve was used for the proper amount of benzene inlet and argon in the furnace, as reported in our previous work [oC. The benzene flow was maintained for only 10 min, and the sample was then lowered to room temperature. The functionalized graphene was then used to prepare heterostructure composite materials. Titanium (IV) n-butoxide 2 were dissolved in EG under magnetic stirring for 2 h at 60 oC to obtain the homogeneous solution. The obtained solution was then added to a Teflon-type autoclave and heated at 250 oC for 12 h. The pH was adjusted to 7 by using a NaOH drop in the solution. The obtained powder was then calcined at 850 oC. The ultrasonication techniques were used to synthesize TiOX-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM) were used to analyze the produced sample. Photocatalytic performance of all samples was evaluated for the methylene blue (MB) and rhodamine B (Rh. B) dyes under visible light irradiation. Raman investigation was carried out to look at the traces of graphene. A “labRam Aramis” Horiba Jobin Yvon spectrometer was used for the measurement. An argon-ion laser at 514 nm was utilized for the measurement. In order to conduct the morphological analysis, a field emission scanning electron microscope (FESEM (JSM)-5200 JOEL, Japan) with enhanced imaging capabilities which could be customized to performance needs was employed. High-resolution transmission electron microscopy  was used to determine the state and the sheet morphology of the prepared graphene. The number of graphene layers and their stacking condition on the individual samples were assessed using a transmission electron microscope (TEM) at an acceleration voltage of 200 kV. The photocatalytic reactor was used, and a visible light bulb (150 W) with a filter (420 nm) was used as the light source. To determine the equilibrium between absorption and desorption, the sample was held in the dark. As a result, the relative efficiencies of each substance were assessed. A UV-visible spectrophotometer was used to measure the dye concentration .2 and strontium hexaferrites, as prepared, have a diffraction pattern that resembles the M-type hexagonal structure and anatase crystal phases, as shown in 2 referred to (JCPDS PDF#: 00-021-1272) [2-FG/Sr-hexaferrite nanocomposites as compared to TiO2 and bare Sr-hexaferrites. On the surface of the FG sheet, we suppose that the suppression of peaks causes the crystalline phase of semiconductor materials to deform. The phonon confinement effect is produced by the interaction of nanoparticles with a graphene surface by reducing the formation of spherical-shape nanoparticles [The results of the analysis using XRD techniques to identify the crystal phases are shown in articles . Therefoarticles .FSEM was used to study the morphological structure of our nanocomposites, as seen in 2-FG nanocomposites show the graphene’s plate-like formation loaded with TiO2. The overall images in 2-FG/Sr-hexaferrites at various magnifications in order to find the morphological information of the composite. After the functionalization of the graphene surface, retaining the sheet’s original morphology, the FG surfaces are covered with TiO2 and Sr-hexaferrite nanoparticles [2 nanoparticles reported elsewhere. We assumed that the graphene surface that is host to the Sr-hexaferrite nanoparticles also includes TiO2. The SEM images of the sample after degradation are shown in 2 and Sr-hexaferrite nanoparticles is supported by TEM images.The use of FESEM to examine the surface topography of our nanostructured materials is shown in articles . Functio2-FG nanocomposites. The images shows that TiO2 nanoparticles are well distributed on the graphene sheet. Small-magnification TEM pictures were collected to reveal the extensive surface structure, distribution of nanoparticles, and graphene sheet composition. 2-FG/Sr-hexaferrites, as shown in 2 nanoparticles were found supporting Sr-hexaferrites on the graphene sheet. Thus, partial agglomeration of these images may be due to the ferrite’s attachment on FG sheets [G sheets .2 nanocomposite, which exhibit a distinct G band at 1580 cm−1 and D band at around 1350 cm−1. The G and D bands in 2-FG and TiO2-FG/Sr-hexaferrites, which further supports the functional density on the FG surface. There is a small 2D band observed in functionalized graphene which further confirms the layer-type morphology of FG sheets. The literature makes it abundantly evident that the D band results from the flaw caused by the functional groups on the carbon’s basal planes [2 vibrational modes [l planes . There aal modes . The othal modes . 2-GO and TiO2-FG is given in 2 gives lower absorbance than our nanocomposites. The TiO2-GO sample was only included to check and compare the absorbance level of the two composites. This improved absorbance may be credited to the optimum loading and consistent distribution of nanoparticles on the graphene sheet. The sheet type morphology of graphene is better for providing a large number of reactions sites. 2-FG in our graphene-based nanomaterials, its sharp edge in the visible range has been improved. The unpaired π electron in FG may interact with metal or semiconductor nanoparticles, shifting the band edge and causing more light to be absorbed in the visible spectrum [The DRS spectra of the composites are shown in spectrum . The shispectrum .g are the known constant, i.e., Planck constant, the frequency of vibration, the absorption coefficient, and the band gap, respectively, and A is a proportional constant. The quantity F(R∞) is related to the absorption coefficient and dedicated to the vertical axis of the spectrum. The α in the Tauc plot is replaced with the function F(R∞). The actual relational expression becomesWe transformed the reflectance plot using the following equation: 2 was plotted against the hν. The specific band gap of our nanocomposite is defined by the curve with (hν F(R∞))2 and hν on the vertical axis [2-FG/Sr-hexaferrites, TiO2-FG, and TiO2-GO, respectively. The obtained plots are given in Using the KM function, the (hν F(R∞))cal axis . The pre2-FG/Sr-hexaferrite composites was measured using AFM topography. 2-FG/Sr-hexaferrites. The sample provides the sheet-type graphene surface with some dot regions that are thought to be valleys between the graphene domains. The image illustrates some tube-type sharp edge on the graphene surface; this may be Sr nanorods, very useful for creating links with nanoparticles and their attachment. The TEM image has some minor evidence of variable shape in the Sr-hexaferrite composite. The surface architecture of the TiO2-FG/Sr-hexaferrites exhibited by AFM lead to the creation of nano heights, a distinctive and possibly complex phenomenon. The temperature inside the CVD, the reducing environment, the decomposition of oxides during the process, and benzene disintegration are just a few of the contending elements that could lead to the creation of such a structure [The height and thickness of the TiOtructure .Two organic dyes, rhodamine B (Rh. B) and methylene blue (MB), were catalytically decomposed by the nanocomposite as prepared under the effect of visible light in order to ascertain the material’s catalytic performance. Three crucial processes are involved in the degradation and photocatalysis mechanisms; adsorption of pollutants onto catalyst materials, quick transfer of charges to produce radical species, and absorption by the photocatalyst are all necessary for the oxidation of organic pollutants. Due to its two-dimensionality and conjugated structure, FG serves as an adsorption and fast-transfer material in our nanocomposites ,38. The 2 and H2O as a result of the free radicals’ reaction with it. 2-FG/Sr-hexaferrite composite. The degradation can be seen as a gradual decline in peak intensity of the Rh. B dye at 554 nm and the MB dye at 595 nm [2 nanoparticles (by offering a high number of reaction sites) and the synergistic interaction between FG and Sr-hexaferrites as cocatalysts are responsible for the drop in concentration and better catalytic effect [To achieve adsorption and desorption equilibrium, the catalytic chamber was left in the dark for two hours. The chamber was then exposed to vis-light for 30 min after adsorption–desorption equilibrium was reached. The sample was collected for analysis, and the solids were collected using a centrifuge. A UV-vis spectrometer was used to analyze the sample activity. The photogenerated electrons reacted with the adsorbed oxygen to produce free radicals ,41,42,43t 595 nm ,45. The c effect ,47,48. 2-FG/Sr-hexaferrite composite. 2-FG/Sr-hexaferrite photocatalyst has remarkable stability and was not photocorrodable throughout the catalytic oxidation of the Rh. B and MB molecules. Thus, it is possible that the composite photocatalyst composed of TiO2-FG and Sr-hexaferrites will be used in actual environmental purification procedures [2-FG/Sr-hexaferrites can be utilized as an effective catalytic material [Cyclic tests were carried out to highlight the stability of the TiOocedures ,50. By ematerial .2-FG/Sr-hexaferrites were synthesized using a simple and easy hydrothermal method. Successful CVD modifications were made to functionalized GO. The spherical TiO2 and Sr-hexaferrite dispersed well on the FG surface, as supported by TEM images, and were found beneficial for the degradation of organic dyes. The presence of minor 2D and some metallic peaks in the Raman spectra supports the existence of ternary TiO2-FG/Sr-hexaferrite nanocomposites. The functionalized graphene in the system serves as a stable heterogeneous photocatalyst material and electron mediator/absorber to retain the ternary system. The optical properties show red shift towards the visible region of the electromagnetic spectrum by their respective band gaps from DRS spectra. The strength and effectiveness of our recently created, ternary, graphene-based nanocomposites are demonstrated by the outstanding photocatalytic effect employed to degrade the organic dyes Rh. B and MB. The present work provides a new route for functionalizing graphene as a support material for the attachment and distribution of nanoparticles on the surface of two-dimensional sheets. In summary, in order to observe enhanced adsorption capacity of the nanocomposites, the FG-based composite may be the ideal material for future environmental remediation.In conclusion, ternary TiO


IGHV1-69 and IGKV3-20) and form a bNAb epitope class. Furthermore, convergent evolution within these two lineages towards a 111.2GW111.3 motif in the CDRH3 is known to enhance neutralization potency. We have previously isolated an MPER neutralizing antibody, CAP206-CH12, that uses these same germline heavy and light chain genes but lacks breadth . Longitudinal sequencing of the CAP206-CH12 lineage over three years revealed similar convergent evolution towards 111.2GW111.3 among some lineage members. Mutagenesis of CAP206-CH12 from 111.2GL111.3 to 111.2GW111.3 and the introduction of the double GWGW motif into CAP206-CH12 modestly improved neutralization potency (2.5–3-fold) but did not reach the levels of potency of VRC42.01, 4E10 or PGZL1. To explore the lack of potency/breadth, viral mutagenesis was performed to map the CAP206-CH12 epitope. This indicated that CAP206-CH12 is dependent on D674, a highly variable residue at the solvent-exposed elbow of MPER. In contrast, VRC42.01, PGZL1 and 4E10 were dependent on highly conserved residues  facing the hydrophobic patch of the MPER. Therefore, while CAP206-CH12, VRC42.01, PGZL1 and 4E10 share germline genes and show some evidence of convergent evolution, their dependence on different amino acids, which impacts orientation of binding to the MPER, result in differences in breadth and potency. These data have implications for the design of HIV vaccines directed at the MPER epitope.Broadly neutralizing antibodies (bNAbs) that target the membrane-proximal external region (MPER) of HIV gp41 envelope, such as 4E10, VRC42.01 and PGZL1, can neutralize >80% of viruses. These three MPER-directed monoclonal antibodies share germline antibody genes  classes, which use the same germline antibody genes to target the same viral epitopes. Here, we compare four HIV Envelope MPER-directed antibodies  that despite having shared antibody genes, show distinct neutralization profiles. We show that CAP206-CH12 is dependent on a highly variable residue in the MPER, which results in low neutralization breadth. In contrast, the 4E10, PGZL1 and VRC42.01 mAbs are dependent on highly conserved residues in the MPER, resulting in exceptional neutralization breadth. Our data suggest that while shared germline genes within bNAb epitope classes are required, in some cases these are not sufficient to produce neutralization breadth, and MPER immunogens will need to trigger responses to conserved sites. The pursuit of an effective vaccine against HIV is an ongoing priority. It is generally accepted that an effective vaccine will require the elicitation of broadly neutralizing antibodies (bNAbs), capable of neutralizing multiple subtypes of HIV . Recent IGHV1-69 and IGKV3-20. While 4E10 and VRC42.01 have modest SHM , PGZL1 has high levels of SHM in both the heavy (20.9%) and light (12.6%) respectively. In addition to shared germline gene usage, 4E10 and VRC42.01 show convergent evolution within the CDRH1 involving the 25SGGSFS30 motif that is crucial for binding [IGHV1-69 alleles, with only the S28 being mutated within 4E10 and VRC42.01, since all IGHV1-69 germline alleles contain a T28 (www.imgt.org). Within the CDRH3, all three bNAbs, PGZL1, VRC42.01 and 4E10 contain a 111.2GW111.3 motif (IMGT numbering), with 4E10 having a double 111GWGW111.3 motif, which is crucial for its neutralization [The 4E10 bNAb class, including 4E10, PGZL1 and VRC42.01, are amongst the broadest antibodies described to date  ,20. Thes binding ,22. Thislization ,21.IGHV1-69 and IGKV3-20 germline genes and targets the MPER [We have previously reported the isolation of mAbs from donor CAP206 who developed broadly neutralizing plasma responses to the MPER . CAP206-the MPER . Howeverthe MPER . Here, wIGHV1-69 and IGKV3-20) from seven-time points  during infection  with an HIV-1 MPER region. We showed that plasma neutralization against the MPER was detected from 25 wpi and seen throughout infection  Fig 1A)Fig 1A). Clonally related sequences were detected at all time-points sequenced for both heavy (n = 75) and light chains (n = 53). Most of the clonally related reads were obtained at 10 wpi for both heavy and light chains, with fewer sequences detected at later time-points . Heavy aThough the CAP206-CH12 lineage light chain showed limited evolution, we identified a number of light chain sequences that were more mutated than CAP206-CH12. Two of these were selected for downstream analysis, K1 (observed at 120wpi) and K2 (observed at 198wpi) . To testIn an attempt to improve potency of CAP206-CH12 we paired K1  and K2  with the CAP206-CH12 heavy chain. Neutralization of eight heterologous subtype B and C viruses  was compared to the CAP206-CH12 wild-type . For the38 residue, the light chain does not play a dominant role in neutralization by CAP206-CH12.We next swapped out the CAP206-CH12 CDRL1 with the 4E10 CDRL1, previously identified as having key contact residues with the MPER ,27. This111.2GW111.3 motif, which is crucial for neutralization [111.2GL111.3 (orange) or 111.2GW111.3 (red)  . Notablyoded T28 .111.2GW111.3 motif within the CAP206-CH12 lineage, we selected six intermediate heavy chain sequences (H1—H6), including those with either 111.2GL111.3 . Since CAP206-CH12 has a 111.2GL111.3 motif we also mutated the L111.3 to a W111.3 (referred to as CAP206-CH12-GW) and introduced a double 111GWGW111.3 in CAP206-CH12 (CAP206-CH12-GWGW). We tested the ability of these antibodies including the UCA, CAP206-CH12.2, and wild-type CAP206-CH12 to neutralize a panel of 23 viruses that we have previously shown to be sensitive to CAP206 plasma [111.2GL111.3 motif), with CAP206-CH12-GW having the best breadth and potency of the three antibodies (111.2GW111.3 motif (H2 –H4) showed greater breadth and potency than CAP206-CH12. Similarly, H6, the most mutated mAb (15.8% SHM) but containing the 111.2GL111.3 motif, showed weaker neutralization breadth and potency compared to a less mutated mAb  present at the same time-point but with the 111.2GW111.3 motif. This data suggests that the 111.2GW111.3 motif improves neutralization potency in the CAP206-CH12 lineage, as with VRC42.01 and 4E10 [To test the influence of the  orange,  or 111.26 plasma , and com6 plasma –19. The tibodies . Furtherand 4E10 .Despite this, none of the lineage members showed the same degree of neutralization breadth or potency as 4E10, VRC42.01 or PGZL1 . To assess the role of mutations in the CDRHs, CDRH swaps were made between 4E10 and CAP206-CH12. However, these resulted in reduced or no neutralization of the chimeric antibodies compared to the wild-type 4E10 or CAP206-CH12 , which m111.2GW111.3 motif at 33 wpi (detected in H1—H4) enabled the lineage to neutralize additional heterologous viruses, with all the lineage-sensitive viruses neutralized by 80 wpi. Interestingly, with the exception of ZM109, neutralization of all lineage-resistant viruses (black) developed after 80 wpi, suggesting another lineage or specificity emerged at that time-point.We performed a longitudinal assessment of CAP206 plasma neutralization of all 23 heterologous viruses neutralized by this donor and partitioned the viruses into those sensitive (green) and those resistant (black) to the CAP206-CH12 lineage . NeutralMagnetic beads coated with the MPR.03 peptide were used to adsorb MPER neutralizing antibodies from CAP206 plasma at 159 wpi (~ 3 years post-infection). We tested the ability of the adsorbed and unadsorbed plasma to neutralize both lineage-sensitive and lineage-resistant viruses . Within Within the CAP206-CH12 lineage-resistant viruses, five  showed no reduction in neutralization following MPER-depletion, but four  did show reduced neutralization titres  and lineage-resistant (bottom) viruses highlights a single amino acid at position 674 (highlighted in grey) that distinguishes these two groups . Where lconfirms  that theIn order to compare epitopes, we introduced mutations at position 674 and other sites within the MPER known to affect sensitivity to CAP206-CH12, PGZL1, VRC42.01, and 4E10 ,27,28 in674, S674, or N674 resulting in complete knock-out of neutralization. N674 had no effect on neutralization for VRC42.01, PGZL1 and 4E10. However, the S674 or A674 mutations resulted in reduced neutralization, with VRC42.01 and 4E10 most sensitive to S674 , and PGZL1 highly sensitive to A674 (54-fold). Furthermore, VRC42.01, PGZL1 and 4E10 showed a more than 50-fold reduction or complete loss of neutralization when W672, F673, T676, or W680 were mutated, whereas these mutations resulted in modest effects on CAP206-CH12 and CAP206-CH12-GW.Strikingly, CAP206-CH12 had distinct epitope dependencies (highlighted in green) compared to VRC42.01, PGZL1 and 4E10 (highlighted in black) . Both CAwww.hiv.lanl.gov), we noted that residues 672, 673, and 680, upon which VRC42.01, PGZL1 and 4E10 are dependent are highly conserved. In contrast, amino acid 674 upon which CAP206-CH12 and CAP206-CH12-GW show dependence is highly variable, with only 53% of global viruses containing an aspartic acid  [111.2GW111.3 motif within the CDRH3 region of the heavy chain. As with VRC42.01 and 4E10 [111.2GW111.3 motif improves neutralization potency within the CAP206-CH12 lineage, but not to the same levels as seen in VRC42.01, PGZL1 and 4E10. Within VRC42.01 and 4E10 the GW and GWGW motifs are not part of the antibody paratope but have been shown to enable conformational changes within the antibodies. This may contribute to overall neutralization breadth and potency, and these residues may perform a similar role in CAP206-CH12 [38 alone significantly improving CAP206-CH12 neutralization potency. However, CDRH1 and CDRH3 swaps, known to be important in lipid viral membrane interactions in 4E10 [Antibodies CAP206-CH12, VRC42.01, PGZL1 and 4E10 all target the C-terminal helix of MPER of gp41 and use the same variable germline genes in both the heavy and light chains (GKV3-20) ,20,24,28GKV3-20) . Here weand 4E10 , this 11206-CH12 . We expl in 4E10 , between111.2GW111.3 motif which is encoded by IGHJ6. In contrast VRC42.01 and 4E10 use IGHJ4 which is much shorter and doesn’t contain the string of hydrophobic tyrosine residues. The hydrophobic residues in the CAP206-CH12 lineage may cause a steric clash between the antibody heavy chain and the hydrophobic core of the MPER, perhaps driving this lineage to favor the external solvent exposed region. Thus, the pairing of the IGHV1-69 with IGHJ6 instead of IGHJ4 may have resulted in the CAP206-CH12 lineage initially engaging the MPER in a different orientation than that of VRC42.01 and 4E10 and thereafter maturation dependence on D674, leading to narrower neutralization.The CAP206-CH12 lineage contains a string of hydrophobic tyrosine residues next to the We show that donor CAP206 contained multiple bNAb specificities. In addition to the CAP206-CH12 lineage, we have provided evidence for an additional MPER lineage as well as another bNAb specificity to an undefined epitope. We and others have previously described the development of multiple bNAb specificities within a single donor, which contribute towards the overall breadth of these donors ,39,31–38Neutralizing antibody responses to the MPER tend to be rarer than those targeting other epitopes on the HIV envelope . It is tOverall, this data helps to explain why the CAP206-CH12 lineage, despite convergent evolution of variable heavy and light chain genes and similar epitope targeting, fails to develop the same level of breadth and potency as PGZL1, VRC42.01 and 4E10. Thus, while shared germline genes and convergent evolution within bNAb classes are crucial aspects of germline-targeting, they may not be sufficient to drive neutralization breadth.CAP206 is a participant enrolled in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 Acute Infection study, established in 2004 in Kwa-Zulu Natal, South Africa. The CAPRISA 002 Acute Infection study was reviewed and approved by the research ethics committees of the University of KwaZulu-Natal (E013/ 04), the University of Cape Town (025/2004) and the University of the Witwatersrand (MM040202). CAP206, an adult, provided written informed consent.CAP206, a CAPRISA Acute Infection cohort participant, infected with an HIV subtype C virus, developed plasma neutralization breadth . Stored IGHV1-69 and IGKV3-20) were designed such that forward primers bound in the leader sequence (IGHV1-69: 5’-CAGGTSCAGCTGGTGCARTCTGGG-3’ and IGKV3-20: 5’-GAAATTGTGTTGACRCAGTCTCCA-3’). Reverse primers bound to CH1 of the constant region . All primers included Illumina MiSeq barcodes to allow sequencing on the MiSeq. Samples from each time point were amplified seven times for both the heavy and light chains to ensure adequate coverage and minimize PCR bias. PCR conditions were as previously described [Total RNA was extracted from cryopreserved PBMCs at 7 different time-points  using the AllPrep DNA/RNA mini kit (Qiagen) as per the manufacturer’s specifications. Reverse transcription was carried out using Random Hexamers (Integrated DNA Technologies) and Superscript III RT enzyme (Invitrogen). Primers specific to CAP206-CH12 heavy and light chains  and sub-cloned in the CMV/R expression vector ,52. All 38) as well as CAP206-CH12/4E10 chimeras were introduced using either the QuikChange Lightning Site-Directed Mutagenesis Kit  or through Gibson Assembly  using primers that were synthesized by Inqaba Biotech. Mutations were confirmed by sequence analysis.Specific amino acid changes on envelope clones, CAP206-CH12 heavy chain CDRH3 (GW and GWGW), CAP206-CH12 light chain CDRL1 (K50) or reciprocal plasma/serum dilution (ID50) causing 50% reduction of relative luminescence units (RLU) with respect to the virus control wells (untreated virus). Starting concentrations of mAbs were at 200 μg/mL in order to determine a precise titre.Neutralization was measured as a reduction in luciferase reporter gene expression after a single round of infection of TZM-bl cells with Env-pseudotyped viruses as previously described . BrieflyStreptavidin-coated magnetic beads  were incubated with the biotinylated peptide MPR.03 (KKKNEQELLELDKWASLWNWFDITNW LWYIRKKK-biotin-NH2)  at a ratio of 1 mg of beads per 20 μg peptide at room temperature for 30 min. Sera were diluted 1:20 in Dulbecco’s modified Eagle’s medium (DMEM)-10% fetal bovine serum and incubated with the coated beads for one hour at a ratio of 2.5 mg of coated beads per mL of diluted plasma. This was followed by a second adsorption at a ratio of 1.25 mg of coated beads per mL of diluted sample. After each adsorption, the beads were removed with a magnet, followed by centrifugation, and were stored at 4°C. The separated immunoglobulin G (IgG) was removed and placed into a separate tube, where the pH was adjusted to between 7.0 and 7.4 with 1 M Tris (pH 9.0) buffer. The same beads were acid eluted twice more. The pooled eluates were then diluted in DMEM, washed over a 10-kDa Centricon plus filter, and resuspended in DMEM. The adsorbed sera were then used in neutralization assays. The reduction (>32-fold) in neutralization by the MPER-adsorbed plasma (green, top panel


The tissue cohort consisted of 68 formalin‐fixed paraffin embedded samples from 21 surgical cases (some patients having more than one case over their lifetimes), from 8 organ systems, which we examined for evidence of phosphorylated TDP‐43 (pTDP‐43) pathology. We identified pTDP‐43 aggregates in multiple cell types of the GI tract, including macrophages and dendritic cells within the lamina propria; as well as ganglion/neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non‐CNS pTDP‐43 pathology, aggregates were present prior to ALS diagnosis and in some instances preceded neurological symptom onset by more than 10 years. These data imply that patients with microscopically unexplained non‐CNS symptoms could have occult protein aggregation that could be detected many years prior to neurological involvement.Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non‐central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non‐CNS tissues, taken as part of routine clinical practice during life from people with ALS. We examined tissue from 13 people who went on to develop ALS; including sporadic ALS ( Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) are traditionally classified and managed as neurological disorders. There is, however, marked clinical heterogeneity within these disorders, which may in part contribute to the large number of failed clinical trials, and clinical presentation is not restricted to neurological dysfunction . Indeed,Systemic non‐CNS manifestations of PD and other neurodegenerative diseases are now well established , 3. FurtThe unifying proteinopathy seen in all cases of sporadic ALS (sALS) and most genetic ALS cases is the cytoplasmic accumulation of phosphorylated TDP‐43 (pTDP‐43) aggregates in the CNS . SeveralC9orf72 hexanucleotide repeat expansion, and three age and sex matched non‐ALS colon biopsies. The tissues were assessed for sufficiency by two independent histopathologists (SBP and JMG) using H&E stained sections. All clinical data, in addition to data provided by the Lothian NRS BioResource, were non‐identifiable and collected as part of Scottish Motor Neuron Disease Register and Care Audit Research and Evaluation for Motor Neuron Disease platform , and all patients consented to the use of their data during life. All anonymised demographic and clinical data are collated in Table All tissue was requested from the Lothian NRS BioResource RTB  and the Grampian Biorepository , covering use of residual tissue surplus to diagnostic requirements taken as standard of care with approval number SR1684 and TR000301. All archived formalin‐fixed, paraffin‐embedded tissue material with an NHS Lothian/NHS Grampian diagnostic code  was requested for 48 patients with known diagnosis of sALS or ALS with a Sections were dewaxed using successive xylene washes followed by alcohol hydration and treatment with picric acid for removal of formalin pigment. For pTDP‐43 and CD68 staining, antigen retrieval was carried out in citric acid buffer (pH 6) in a pressure cooker for 30 min, after which immunostaining was performed using the Novolink Polymer detection system  with phospho(409–410)‐TDP43 antibody , or anti‐CD68 . Phospho‐Tau  monoclonal antibody  was used without antigen retrieval. Slides were counterstained with haematoxylin and mounted using DPX mountant. For immunofluorescence, mouse anti‐TDP‐43 C‐terminal  was used in addition to the pTDP‐43 antibody. Secondary antibodies were anti‐mouse, Alexa 488 , and anti‐rabbit Cy5 . Sections were imaged using an Olympus BX51 with a Nikon Digital‐Sight camera and immunofluorescent images were taken using an Invitrogen EVOS colour fluorescent microscope. Bleach treatment was performed (where warranted) by incubating slides immediately post antigen retrieval in 10% hydrogen peroxide for 12 h overnight. This step was followed by the normal protocol in full, including the additional 3% hydrogen peroxide step used to block endogenous peroxidase activity, following a previously optimised protocol [C9orf72 hexanucleotide repeat expansion). All 48 patients had pathological pTDP‐43 inclusions in their CNS tissue and a diagnosis of ALS, and their CNS post‐mortem tissue has been evaluated previously in our prior studies [C9orf72 hexanucleotide repeat expansion. The final cohort consisted of tissue from 22 surgical cases (some patients having more than one sample over their lifetime), representing eight organ systems: GI tissue (n = 2), soft tissue , female gynaecological tissue (n = 5), pleura (n = 1), skin (n = 8), thyroid (n = 1), lymph nodes (n = 2), and urethra (n = 1)  compared to controls (n = 15) [per se, increased levels of highly aggregation‐prone proteins like TDP‐43 may be indicative of pathological TDP‐43 turnover that could predispose these individuals to TDP‐43 aggregation. Indeed, these data from other cohorts perhaps indicate that more than just the gut‐brain‐axis is involved by pTDP‐43 aggregation, supporting the hypothesis of meta‐synchronous aggregation, rather than physical spreading. The mechanistic consequences of the involvement of other tissues and their contribution to ALS pathogenesis clearly require further investigation.Whilst the presence of pTDP‐43 aggregates within gut tissues in our cohort raises the possibility of a similar mechanism of pathological spread to that seen in other neurodegenerative diseases , we also see pTDP‐43 in other tissues including skin, peripheral nerves, and blood vessels as well as lymph nodes that would not typically drain lymph fluid from the GI tract. Other groups have identified pTDP‐43 aggregates within peripheral nerves, with an identical staining pattern to that seen in our cohort . Several(n = 15) . AlthougTDP‐43 pathology has been noted in post‐mortem cerebral blood vessels previously , 21. HowOur data imply that peripheral non‐CNS tissues could hold promise as early indicators of ALS, prior to neurological involvement, and that tissue biopsies could also help us to understand how fluid biomarkers, such as blood and stool samples could be utilised through their association with the tissues that they derive from. Early diagnosis in this way would not just improve the chances of successful therapeutic intervention, it would also inevitably lead to a rethinking of neurodegenerative conditions from being end‐stage neurological disorders to systemic disorders with potential for population screening and early intervention long before neurological manifestations occur. Future studies assessing peripheral tissue cohorts such as these using western blotting might uncover the presence of C‐terminal fragmentation that is also a distinguishing, albeit heterogenous, feature of pathological aggregates in ALS . FurtherOur study was based on a cohort of 48 patients, only 13 of which had surgical specimens that were sufficient for histological assessment, 12 of which were sALS cases. Therefore, the conclusions of this study are subject to the caveats of small sample size and are biased towards sALS. For example, with tissues that we have seen no evidence of aggregation in our cohort, we cannot rule out that they are not involved by pTDP‐43 aggregation in a larger cohort, we can only state that they are not involved in our cohort. Furthermore, tissue cohorts such as these are necessarily subject to variations in fixation and ischaemic times during excision and post‐excision processing, all of which may affect our ability to detect proteins, particularly phosphorylated proteins, resulting in the possibility of false negatives. It is therefore important to include all relevant information and detailed methodological reporting to facilitate future meta‐analysis where more cases and cohorts can be evaluated in pooled datasets, as has been done for alpha‐synuclein . For thiSBP, MJA, MH, FMW and JMG contributed to conceptualisation, project administration, resources, supervision, validation, and visualisation. All authors contributed equally to data curation and formal analysis. JMG contributed to funding acquisition. SBP is a data pathologist and contributed to study design, cohort identification, and data blinding necessary for the use of NHS BioRepository tissues. SBP, JO'S, MJA, MH, FMW and JMG contributed to investigation and methodology. SBP, FMW and JMG wrote the first draft of the manuscript prior to other author input. JO'S, JP, OK, OR, KN and NH were involved with tissue/data handling and/or performed all tissue staining, imaging, and whole slide scanning. All authors contributed to manuscript reviewing and editing.Figure S1. pTDP‐43 aggregates are not identified in colon biopsies from non‐ALS individualsClick here for additional data file.


Graphene-based sensors are of great interest in research due to their high specific surface area and high electron mobility that make them suitable for numerous advanced applications. In this paper, selective molecular detection using an antigen–antibody reaction on suspended graphene with a cavity-sealing structure was demonstrated. The suspended graphene sealed nanocavities in a pre-patterned Si substrate, which increased robustness and allowed the use of wet chemical processes for surface functionalization of the suspended graphene to achieve selective molecular binding. The selectivity was evaluated by nanomechanical deflection induced by molecular adsorption on the suspended graphene, resulting in spectral shifts in the optical interference between the suspended graphene and Si substrate. The chemically functionalized suspended graphene enables the analysis of intermolecular interactions and molecular kinetics by colorimetry using optical interference. Chemically functionalized suspended graphene with a cavity-sealing structure selectively detects biomolecules using nanomechanical deflection associated with molecular adsorption. In addition, when suspended graphene is used as a resonant membrane, a mass sensor capable of detecting single molecules can be fabricated. In surface stress sensors, adsorbed molecules induce surface stress, resulting in a deflection of the suspended membrane. In particular, surface stress sensors can analyze the kinetics of molecules by transducing the interactions of adsorbed molecules into nanomechanical deflections.15,16 We previously developed a surface stress sensor based on optical interferometry to increase stress sensitivity using an optomechanical transducing technique.17–19Various transducers, including those based on charge detection,20,21 A resistance change of a suspended graphene channel caused by adsorption of a single molecule on the bilayer graphene bridge was demonstrated. However, this sensor was based on the physical adsorption of carbon dioxide, and selective detection in an environment with contaminants is impossible. Molecules cannot selectively bind to pristine graphene as it has no dangling bonds on its surface. As molecular selectivity is necessary for the application of suspended graphene as a sensor, the graphene surface needs to be functionalized. It has been reported that a specific protein was detected using an ion-sensitive field-effect transistor (ISFET)-based biosensor modified with an antibody fragment on a graphene channel fixed to a substrate.22,23 This chemical modification was carried out using a wet treatment, where graphene was immersed in a solution with dissolved receptor molecules. Note, however, that the detection area of the ISFET-based biosensor is restricted to 2–3 nm from the device surface due to the Debye length under physiological conditions, and therefore it is difficult to detect charged macromolecules.24 On the other hand, surface stress-based biosensors are unconstrained by the molecular size because they is based on the principle of detecting repulsive forces due to intermolecular interactions of adsorbed molecules as nanomechanical deflection,25,26 resulting in the detection of biomacromolecules such as proteins. However, chemical functionalization of the graphene bridge for selective molecular detection is difficult because the liquid trapped in the gap of the structure can easily fracture the suspended graphene by surface tension forces. To solve this problem, drum-type suspended graphene can prevent solution from entering the cavity, allowing wet treatments.For chemical and biomedical sensing, the interface structure of the sensor surface is important. It has been reported that a single molecule of carbon dioxide was detected on suspended graphene.27 chemical vapor deposition (CVD) on a metal catalyst, such as copper28 or nickel,29 and epitaxial growth on silicon carbide.30 For large-area device fabrication, transfer techniques have been used, where CVD graphene was deposited on a Cu catalyst by thermal CVD, followed by removal of the Cu catalyst, and transferred to an arbitrary substrate.31,32 By transferring CVD graphene onto a Si substrate with pre-patterned cavities, the suspended graphene sealed the cavities and formed a drum-like structure.33 To obtain cavity sealing graphene with a drum structure, we proposed a low-pressure dry transfer technique.34 CVD graphene was transferred under vacuum to increase the real contact area between the substrate and transferred graphene in order to provide strong adhesion between the transferred CVD graphene and the substrate. The suspended graphene seals the cavities and provides sufficient robustness to allow wet chemical processing in order to perform molecular modification of the suspended graphene for selective molecular binding.Deposition techniques for producing graphene include mechanical exfoliation,In this paper, we demonstrate selective molecular detection using an antigen–antibody reaction with such graphene drum sensors fabricated using our low-pressure dry transfer technique. Selective molecular detection was evaluated by the nanomechanical deflection induced by molecular adsorption on the suspended graphene, which resulted in a spectral shift in the optical interference between the suspended graphene and the Si substrate. The proposed technique can analyze intermolecular interactions by colorimetry using optical interference.2 surfaces in contact under vacuum, the adhesion area increases due to deaeration of the gap between the two surfaces. By heating at a temperature higher than the glass transition temperature of PMMA, the fluidity of PMMA increases, resulting in a further increase in the real contact area. Finally, the PMMA layer is removed to leave a freestanding drum-like suspended graphene layer over the nano/microcavities, as shown in As for our previously reported low-pressure dry transfer technique, CVD graphene held by a temporary polymethyl methacrylate (PMMA) supporting sheet was transferred onto a Si substrate with pre-patterned nanocavities under vacuum . 25,26 With this sensing method, we obtained real-time responses of molecular binding in a liquid environment. Unlike a FET-type biosensor whose measurement distance from the sensor surface is restricted by the Debye length, the surface stress sensor can detect the response from biomacromolecules adsorbed far from the sensor surface, which allows the use of macromolecules, such as antibodies, as receptors. The sensitivity of the surface stress sensor is inversely proportional to Young's modulus and the square of the film thickness.36 Therefore, the use of 2D materials with an atomic-scale thickness can increase the static deflection and hence, the sensitivity of measuring the surface stress. By using a suspended graphene membrane, the sensitivity is expected to increase compared to that of conventional surface stress sensors, which have a typical thickness of 100–1000 nm, even though the Young's modulus of graphene is up to 1 TPa.37 In addition, the suspended graphene is a highly sensitive chemical and a biosensor with a small footprint, while conventional MEMS-based surface stress sensors need to have a large diameter  to improve sensitivity.38Surface stress measurements were used to evaluate selective molecular detection on the suspended graphene. The surface stress sensor measures a static deflection of a suspended membrane caused by the electrostatic repulsive force from adsorbed biomolecules.Optical interference generated in the air gap between the suspended graphene and the substrate can be used to detect nanomechanical deflection associated with molecular adsorption. Adsorbed molecules with equal electric charge produce repulsive coulombic forces that apply a compressive surface stress to the suspended graphene, resulting in upward deflection of the suspended graphene. Meanwhile, the expansion of the nanocavities causes a change in the reflected peak wavelength. Therefore, the presence of adsorbed molecules can be identified by wavelength peak shifts in the optical interferometry signal. In particular, this optical measurement method can evaluate the kinetics of adsorbed biomolecules in solution.39,40 however, graphene is a transparent material with a transmittance of 97.7%.41 Even in single-layer graphene, the optical interference can be obtained from the slight reflection from the suspended graphene, which corresponds to its nanomechanical deflection. The interference peak position is determined by the air gap d under the suspended graphene. The reflection spectrum was obtained by numerical calculations using:42R is the reflectance, λ is the wavelength, t is the transmittance, and r is the reflectance, and the suspended graphene and silicon are denoted by the subscripts g and s, respectively. Therefore, d can be evaluated by comparing the experimental and calculated peaks of the reflection spectra.A visible light filter using optical interference between a suspended double-layer graphene and a Si substrate was reported;The reflection spectrum from the graphene-based interferometer was measured using microspectroscopy  with CHF22,23 The pyrenyl group of PBSE binds to the graphene surface via pi-stacking, while the succinimidyl parts combine with the antibody. Chemical functionalization of PBSE was performed by soaking the graphene chip for 1 h in a solution of PBSE dissolved in deionized water at a concentration of 1 mg mL−1. When a chip in a dry state was brought into contact with the PBSE solution, a transient response was observed, where the suspended graphene deformed upward due to the surface tension force of the solution. To avoid this physical influence during chip insertion, the surface tension force of the solution was reduced by adding 1% Tween-20 to the PBSE solution. After functionalization, the graphene chip was rinsed in phosphate buffered saline (PBS) for 5 min. Subsequently, the graphene chip functionalized with PBSE was soaked in anti-bovine serum albumin antibody (anti-BSA) solution at a concentration of 100 μg mL−1 for 1 h, followed by immersion in PBS for 5 min. Then, we evaluated the molecular selectivity by immersing graphene chips in BSA antigen solution or human serum albumin (HSA) solution. Note that the surface stress response due to the molecular adsorption depends on the electric charge of the adsorbed molecules.25,26 For fair evaluation, it is necessary to select a molecule that has the same molecular weight and isoelectric point as BSA. Although HSA is the same albumin as the BSA antigen, HSA has no cross-activity with anti-BSA antibody  as a cross-linker to immobilize the antibodies.d between the suspended graphene and Si substrate was 380 nm, which should give a blue color by optical interference at the initial state. The interference color changed to green associated with immobilization of PBSE and anti-BSA on the suspended graphene. It is considered that the suspended graphene was deformed due to immobilization of the molecules, namely the cavity gap increased due to the compressive surface stress, resulting in a red-shift in the interference peak. Furthermore, when the antibody-modified suspended graphene was reacted in 100 ng mL−1 BSA antigen solution, the interference color changed to yellow, indicating a further increase in the cavity gap under the suspended graphene. These results clearly prove that the surface stress on the suspended graphene further increased with BSA antigen binding.−1 HSA solution, as shown in via an antigen–antibody reaction, leading to a deflection double that of suspended graphene with the HSA physically adsorbed. Hence, we successfully demonstrated selective molecular detection by chemical functionalization on cavity-sealed suspended graphene using optical interferometry.To demonstrate the selective detection of target molecules on the suspended graphene, reflection spectra were measured by immersing the anti-BSA-conjugated suspended graphene chip in 10 μg mL36 as a function of the drum diameter. The measured values were consistent with a quadratic curve, indicating that the nanomechanical deflection at saturation depended on the square of the diameter. It is suggested that the nanomechanical deflection of the suspended graphene depends on the surface stress associated with molecular adsorption.To evaluate the diameter dependence of the membrane response, we performed the same measurements with diameters of 6–10 μm. From the reflection spectra, we determined deflections for diameters of 6, 8, and 10 μm during the antigen–antibody reaction of 39, 70, and 110 nm, respectively, as shown in −1 (ESI Fig. S4−1, no spectral shift was observed before and after the dropping of the BSA antigen solution, indicating that the lower limit of detection was determined to be 10 ng mL−1.For the evaluation of concentration dependence, the deflection of a 6 μm-diameter drum to the antigen–antibody reaction was measured with BSA antigen concentrations of 1–1000 ng mL18 By applying suspended graphene to the optical interferometric sensor, a single sensor pixel including a buffer amplifier can be configured with 10 × 10 μm2 or less while the conventional surface stress sensor with the design rule of several hundred microns has no size compatibility with the CMOS image sensor. Therefore, a highly sensitive graphene-based surface stress sensor allows miniaturization and a high-density sensor array for multi-biomarker detection, resulting in an increase to 106 pixels per cm2.In summary, we demonstrated selective molecular detection using a graphene drum, which had sufficient strength to allow wet chemical functionalization. Real-time molecular adsorption in solution was performed by colorimetry using optical interference associated with nanomechanical deflection of the suspended graphene. In addition, the kinetics of the molecular interaction could be evaluated using the suspended graphene based on the detection of surface stresses. Our proposed technique for selective molecular detection achieves highly sensitive chemical sensing and biosensing and offers charge detection, resonant mass detection, and surface stress detection methods. We anticipate that the suspended graphene-based sensor could detect single molecules without labelling. Moreover, the optical interferometric surface stress sensor employs a CMOS image sensor technology for a light receiver and signal processor.There are no conflicts to declare.NA-002-C9NA00788A-s001


Background: Nurses and nursing students experienced an emotional burden while working during the COVID-19 outbreak. During the COVID-19 outbreak three questions for nurses working under these extreme circumstances were formulated: 1. What today’s events do you remember? 2. How do you feel ? 3. Do you have enough support? The purpose of this study was to obtain insight into whether nurses and nursing students perceive that the use of the three-questions-method contributes to effective coping with the emotional burden during the COVID-19 outbreak. Methods: Focus group interviews were held with hospital nurses (n = 11) and nursing students with internships in mental health care (n = 2), hospital (n = 9), and homecare/nursing home care (n = 3) in September 2020 followed by twenty semi-structured interviews one year later. Results: Almost all nurses and nursing students named factors that contributed to the emotional burden: fear, powerlessness, frustration, lack of knowledge about COVID-19, and pressure to pass the internship. Participants indicated that using the three-questions-method can help to effectively cope with the emotional burden during and after the COVID-19 outbreak. Conclusions: Using the three-questions-method offers added value in coping with emotional burden and can be used in education as well as in practice. The coronavirus crisis in the Netherlands was part of the global coronavirus crisis, which was caused by the outbreak of the infectious disease COVID-19 in 2019–2020. At the beginning of 2020, SARS-CoV-2—the coronavirus that causes COVID-19—was also detected in the Netherlands. Although the first infection was officially detected on 27 February 2020, a number of Dutch people had already been infected.The outbreak led to measures that were unprecedented in the Netherlands, whereby social traffic was largely halted. Schools, universities, libraries, cafes, hairdressers, museums, cinemas, and restaurants had to close their doors by government directive. High school exams, sports matches, and many other events were cancelled. Dutch people were advised to keep a distance of 1.5 m from each other and to refrain from traveling abroad. In addition, people were asked to stay indoors as much as possible and to work from home. Foreigners were instructed to only travel to the Netherlands if strictly necessary The feeling of frustration was caused by the attitude of the outside world and media reports, according to the participants. As a result, they stopped following news reports and current affairs programs to protect themselves. In addition, the lack of knowledge and experience about the new syndrome led to frustration in practicing the profession.It also made me angry. But also the aggression, they write about a lot in the newspaper now. Yes, daily you get a big mouth and threats to your head from visitors because you address them about the COVID policy.[#F2]Nursing students mentioned that they felt pressure from complying with the assignments from the university. In addition to COVID-19 patient care, they were responsible for their learning and development, such as completing the internship and conducting hands-on nursing research.I still have an internship, I still have to do certain things, I feel the pressure from university.[#F4]Factors related to the physical burden were also mentioned. Almost all participants reported being overtired. In addition, they explained that they had no time to drink or eat during the work shift. Moreover, they indicated that, due to the long working days, they were physically exhausted after a shift.I’d come home at, I don’t know, six o’clock at night or something after a shift, and I’d lie down in my bed and I’d fall asleep until the next morning. And then I hadn’t even eaten, because I was exhausted and I just couldn’t take it anymore.[#F2]Peer support or support from colleagues was mentioned as a way to reduce the emotional burden, but also support from family and friends was seen as helpful. However, participants also stated that family members often did not know what was going on in the hospital.Well, we often talked about it with colleagues. How it was for us as nurses. And we still have quite a few conversations about it.[#F3]Although nursing students felt supported by teachers and fellow students, they explained that online education limited the interaction with lecturers and students.That you actually see each other and it’s different anyway, because online you’re going to take into account that someone else might want to say something. You’re going to hold back because you’re afraid you’re going to talk through someone and then you end up saying nothing.[#F4]Nurses but also other healthcare professionals reacted quickly to the pandemic and readied themselves to collaborate in patient care. Nurses’ adaptability and sense of belonging during work were perceived as positive.We all stood for the same thing and everyone for the same thing. The cleaners or the people from the catering: everyone for the same goal. And there was a great togetherness. I liked that.[#F3]Phase 1: Potential Benefits of Using Three-Questions-Method.During the focus group interviews in phase 1, participants indicated that the three-questions-method could be used proactively and informally within education, for example during intervision meetings, work supervision, or student career counseling. They also suggested that—in nursing practice—the three-questions-method could be used during the coffee or lunch break, handover moments, or evaluation sessions. According to the focus group participants, both supervisors and colleagues could ask the questions back and forth.“I think that if you ask people those three questions that you do open up a conversation more quickly. First of all, that people start to express themselves more easily than with the question “how are you?”[#F2]In phase 2, 15 Dutch nurses and 5 nursing students participated in the interview sessions, in which we investigated the extent to which the three-questions-method was used and the perception of the effectiveness of using the method. A year after the introduction, 6 of the 20 participants used the three-questions-method.Phase 2: Perceived Effectiveness of Using Three-Questions-Method.All respondents (n = 20) indicated that the COVID-19 period had impacted them, both physically and emotionally. In particular, the emotional burden took a substantial toll on the respondents. They explained that there was not enough time or space to express their emotions sufficiently. A large proportion of the respondents indicated the need for stability and a safe working environment in which they can talk to trustworthy colleagues.Six participants mentioned having a positive experience with the use of the three-questions-method. According to them, the method ensures that experiences are made discussable, as team members have the opportunity to express their feelings. According to some respondents, feelings of stress and insecurity also diminish by using the three-questions-method. Three users were very positive about the three-questions-method because of its easy use. According to them, the three-questions-method is simple and straightforward to use, which gives everyone the opportunity for a short evaluation.Three respondents mentioned gaining more insight into their colleagues’ experiences, which could provide a handle on how to deal with problems in the team. In addition, they mentioned that the issues raised while using the method could create a sense of trust and security in a team in the long run.It’s actually a bit of insight into each other’s heads. That’s how I actually see it. And where you can help each other and where you can support each other as a team.[#I19]The respondents pointed out that both facilitating and hindering factors influence the appropriate use of the three-questions-method.Working with familiar colleagues was perceived to be a facilitating factor when using the three-questions-methods. Respondents explained that sharing emotions with familiar colleagues was easier in comparison with new colleagues. Using the three-questions-method in smaller groups has a stimulating effect, because a small group encourages sharing experiences, according to three respondents. However, some respondents indicated to prefer one-on-one conversations with colleagues, due to feelings of unsafety within a group.Well, if you are not much of a talker, it is difficult.[#I9]Trusting colleagues and feeling safe within the group facilitate the use of the three-questions-method, according to six respondents. In addition, they explained that the method can be used in a low-key way, which was considered to be pleasant....that relationship of trust is essential and if the safety in the group is not there, you are not going to share in that.[#I11]Phase 2: Use of Three-Questions-Method in Healthcare Organisations.Two respondents indicated that they used the three-questions-method in multidisciplinary groups with—for example—a social worker and a pastoral caregiver.Most respondents indicated that they use the three-questions-method at three fixed moments during the shift: at the start, halfway, and at the end of the shift. One respondent mentioned that the three-questions-method was not used structurally, but approximately once a month. However, the respondent suggested that this was not enough to be effective. In addition, one respondent indicated that the method was used less in a team with many new colleagues, because the feeling of trust was expected to be lower in such a team.Three respondents indicated that the three-questions-method was mainly used in groups, but that it was also possible to use it with one or two others. Two respondents indicated that the effect of the three-questions-method depends on the context. They indicated that it was important to use the three-questions-method more often during difficult periods, such as the COVID-19 pandemic. Particularly due its simplicity, the method offers an easy-to-use tool to cope with the emotional burden, according to six respondents.The interviews revealed that several respondents would also use the method after the COVID-19 outbreak and in multidisciplinary settings and meetings.And so I think it’s not just for nurses but actually for many more health care professionals.[#I11]This study demonstrated that nurses experience an emotional burden due to their work during the COVID-19 outbreak. This manifests itself in powerlessness and frustration, partly caused by the lack of knowledge and experience about the new disease and its clinical manifestation. The pressure of successfully completing the internship increases the emotional burden for nursing students substantially. Fatigue and physical complaints also contribute to the physical burden. These findings are in line with nurses’ experiences during previous epidemics of SARS, MERS-CoV, and Ebola, during which nurses developed physical and mental disorders, such as loneliness, anxiety, fear, fatigue, and sleep disorders ,17,18.Peer support is an effective intervention in reducing work stress during intense situations ,20,21,22Nursing students collaborated with nurses to deliver patient care during the COVID-19 outbreak. As a result of university closure and the switch to online education, they experienced a pause in their education. Both nurses and nursing students had to cope with mental and emotional issues, including stress, anxiety, and fear ,24. In oThis research presents several strengths. The researchers conducted the interviews via online video calls, as they could not visit the healthcare organizations and universities, due to the implemented safety measures during the COVID-19 outbreak. This method allowed for both verbal and nonverbal data to be acquired. The interviewers were trained in interviewing skills. Due to the heterogeneity of the sample , the results demonstrated a broad spectrum of experiences with the three-questions-method.The study also had some limitations. Precise and generalizable results could not be obtained, because of the qualitative design and the restricted sample size. In addition, the study was conducted in a short period of time. Longitudinal research would offer a valuable avenue for future exploration.The results show that both nurses and nursing students experienced an emotional burden from their work during the COVID-19 outbreak. This was caused by a combination of factors, including fear, helplessness, and frustration. For nursing students, the perceived pressure of obtaining their internship also played a major role. Fatigue, physical complaints, and poor self-care also contributed to the emotional burden.It is important that healthcare and educational organizations pay attention to the emotional and physical health of nurses and nursing students while working in crisis situations, such as the COVID-19 outbreak. The three-questions-method provides nurses and nursing students an easy-to-use and effective tool to cope with the emotional burden during work. If we want to retain current and future nurses, it is essential that we support them in staying emotionally and physically healthy, now and in the future.


Long-time environmental protection of metallic materials is still required in the manufacturing and engineering applications. Nickel-graphene nanocomposite coatings have been prepared on carbon steel using sodium dodecyl sulfate (SDS) as a dispersant in the electrolyte by an electrochemical co-deposition technique. In this study, the effects of surfactants on graphene dispersion, carbon content in the coatings, surface morphology, microstructures, microhardness and corrosion resistance properties of the nanocomposite coatings are explored. The results indicate that the reasonably good graphene dispersion, coarser surface morphology and reduction in grain sizes are achieved upon increasing the surfactant concentration in the electrolyte. The surfactant also influences the preferred orientation of grains during electrodeposition; the (200) plane is the preferred orientation for the nanocomposite produced with SDS in the bath electrolyte. The microhardness, adhesive strength and corrosion performance of the nickel-graphene nanocomposite coatings are found to increase with the increasing concentration of sodium dodecyl sulfate in the deposition bath. Moreover, the influencing mechanism of surfactant concentration on the properties of nanocomposite coatings has been discussed. Long-time environmental protection of metallic materials is still required in the manufacturing and engineering applications. A variety of hard particles ,6 such as SiC,7 WC,8 diamond,9 TiO2,10 Al2O3,11 SiO2,12 CNTs13 and graphene nanosheets,14–17 owing to their enormous mechanical and excellent electrochemical properties18 can be the desired reinforcement particles to be incorporated into a metal matrix. As electrochemical co-deposition is a process largely used for the reinforcement of submicron and nanosized metallic particles, polymer additives or nonmetallic compounds in the metal matrix, it is advantageous over other techniques, for example, this process involves a simple and controllable setup, it can be performed at about room temperature, it is capable and effective to coat large, complex and irregular-shaped components and it is a cost-effective process.19–22 Recently, electrochemically deposited composite coatings have attracted significant attention due to their extensive properties.23 In fact, the morphology, structure and properties of composite coatings fabricated by the electrochemical co-deposition process are greatly affected by different deposition parameters including electrolyte composition, deposition current density, pH, additives, bath temperature and characteristics of reinforcements.24–29 Moreover, much effort has been devoted to study the effects of the operating parameters on the co-deposition mechanism and properties of the deposit.30–32Electrodeposition of nickel and its composites is a widely applied technique in industries to tailor the surface properties of substrate materials for the engineering applications. For several decades, various attempts have been made to achieve superior properties and better performance of coatings 33 Moreover, poor mechanical properties of coatings are obtained due to the agglomeration of nanoparticles. Therefore, several attempts have been made to achieve a high co-deposition content and its uniform distribution. Hence, many chemical and physical methods have been performed by different researchers;34,35 among them, the addition of organic surfactants is an effective method to overcome the agglomeration problem in coatings.36As it is well-known, the agglomeration of submicron and nanosized particles existing in the electrolyte during the deposition process is due to high ionic strength of the electrolyte and the surface free energy of particles.et al.37 have reported the effect of the surfactant cetyltrimethylammonium bromide (CTAB) on alumina dispersion, weight fractions and properties of composite coatings. They obtained better alumina dispersion in the electrolyte and improved corrosion resistance properties when the concentration of CTAB was increased. Lin and Duh38 have investigated the effect of sodium dodecyl sulfate (SDS) on the surface roughness and wettability of Ni–P coatings. Chao and others studied the effect of cationic (CTAB) and anionic (SDS) surfactants on the properties of nickel carbon nanotube composite coatings and found that both cationic and anionic surfactants greatly influenced the mechanical and corrosion resistance properties of the prepared deposit.39 M. Rezaei-Sameti with group40 conducted a study on the effect of sodium dodecyl sulfate (SDS) and sodium saccharin on the surface morphology, microhardness and wear resistance of Cr-WC nanocomposite coatings. Their results indicated that as the content of WC was increased in the coatings, higher hardness and better wear resistance of the coatings were obtained with the increasing concentration of SDS in the electrolyte. Kartal et al.22 reported the effect of the surfactant SDS on the properties of Ni-WC composite coatings and found that the homogeneity of the WC dispersion increased and the mechanical and tribological properties of the composite coatings were improved significantly with the increasing concentration of SDS in the deposition process. A. Zarebidaki and S. R. Allahkaram41 investigated the influence of the SDS surfactant on the deposition behavior of Ni–P-CNT composite coatings. They demonstrated that the good dispersion and uniform distribution of CNTs in the coatings were achieved when the SDS concentration was increased in the electrolyte, and SDS was responsible for higher microhardness and better corrosion resistance of the coatings. Kyle Jiang and others15 observed that the addition of the anionic surfactant sodium dodecyl sulfate (SDS) increased the dispersion of graphene platelets in the deposition solution. An anionic surfactant (SDS) is mainly considered because it resists pin pores and assists the formation of metallic ion complexes.42Liu The use of an appropriate concentration ratio of surfactant and graphene in the electrolyte is highly significant in the preparation of nickel-graphene nanocomposite coatings. In this study, the effects of SDS at different concentrations in the deposition bath on the dispersion of graphene, surface morphology of the coatings, and weight percentage of the deposit are explored. Furthermore, the microstructure, mechanical properties and corrosion resistance behavior of the nanocomposite coatings affected by different surfactant concentrations in the electrolyte have been investigated.2.Natural graphite of 325 mesh was purchased from Qingdao Ruisheng Graphite Company Ltd. All other chemicals were of analytical grade and purchased from Beijing Chemical Works. The substrate material was Q235 carbon steel, and its chemical composition is provided in Table S1.†2.14 was added gradually to a mixture of 180 mL H2SO4 and 20 mL H3PO4, and a slight exothermic reaction occurred 40 °C; then, the reaction mixture was heated to 55 °C and stirred for 12 h. After this, ice water (400 mL) along with 30% H2O2 (3 mL) was added to this mixture. The obtained mixture was kept at room temperature for 24 h for the purpose of sieving. The suspension was centrifuged and washed several times to decant the supernatants, and it was then dried at 60 °C in a vacuum desiccator for 24 h. Finally, the solid product GO was obtained.43 Graphene oxide was reduced into graphene by a chemical reduction method using the reducing agent hydrazine hydrate.44 The synthesized graphene was used in required concentrations in the electrodeposition process.Graphene oxide (GO) was synthesized by an improved Hummers method. Chemical exfoliation of graphite powder is a general approach to produce graphene oxide. In brief, 1.5 g graphite powder with 9 g KMnO2.22SO4 solution to remove surface impurities, oil and oxide layers from the substrate. The basic bath composition and electrodeposition parameters are shown in −1, 0.2 g L−1 and 0.4 g L−1 in the electrolyte are characterized.The electrodeposition process was carried out to fabricate Ni/graphene nanocomposite coatings. The samples of carbon steel grade Q235 with a size of 20 × 10 × 2 mm were used as substrates (cathode) for deposition. Moreover, two plates of nickel (anode) with size 70 mm × 40 mm × 1 mm placed at both the ends of the bath solution served as the anode, and carbon steel serving as the cathode was adjusted between the two anode plates. Prior to electrodeposition, the samples were prepared by grinding different grades of abrasive emery papers  to obtain smooth and uniform surfaces. The substrate was first dipped in 10% HCL and then in a 5% H2.3λ = 1.5406 Å). Bruker Multimode atomic force microscopy (AFM) was used to measure the surface roughness of the composite coatings.Microhardness was determined using a Fischer HM2000 micro hardness tester. A load of 200 mN and a dwell time of 12 s were applied. The average hardness was obtained by testing five different positions of uniform distribution for each sample. The spectra for graphene were obtained using a Renishaw inVia RM1000 Raman microscope at a laser excitation wavelength of 532 nm. The adhesion strength of the nanocomposite coatings was measured using a DeFelsko (S/N AT05268CE) gauge meter. A Hitachi S-4700 scanning electron microscope was used for the observation of surface morphology, and an EDAX micro analyzer attached with a Hitachi S-4700 SEM was used to measure the elemental composition of nanocomposite coatings. The X-ray diffraction (XRD) patterns were obtained using a D/MAX-2500 X-ray diffractometer, with Cu Kα radiation  was used as the reference electrode. The scanning rate was 0.5 mV.Electrochemical impedance spectroscopic (EIS) measurements were performed using a PARSTAT 2273 electrochemical workstation (Princeton). The perturbation was 10 mV, and the applied scanning frequency range was 100 kHz to 10 mHz. The working electrode with a surface area of 1 cm3.3.1d spacing of 0.8 nm as reported elsewhere.43 After reduction, (001) eventually disappeared, and a new peak (002) was observed at 2θ = 25.4° corresponding to an interlayer distance of about 0.4 nm, which was highly consistent with a previous report43 and indicated good arrangement of the interlayer distances of graphene. Furthermore, for confirmation, notable structural changes during the chemical reduction of GO are shown in −1 and G peaks appeared at ∼1590 cm−1, which ensured lattice distortions.44SEM images of graphene nanosheets are shown in 3.2−1 graphene. The comparison analysis of the composite coatings obtained from electrolytes with SDS and without SDS and the effect of anionic surfactant on the surface morphologies are also demonstrated. It can be seen that the addition of the anionic surfactant SDS has increased the roughness of the composite coatings significantly.−1 in the basic bath solution.The surface morphologies of composite coatings were observed to be much coarser when the surfactant SDS was added to the depositing electrolyte, as shown in Fig. S1,−1 in the electrolyte. It can be seen perceptibly that when the SDS concentration is high in the deposition bath, large bulge morphology45 that comprises graphene nanosheets and nickel is observed in the coatings. The expected reason for the effect of the surfactant SDS can be explained as follows: during the electrodeposition process, nickel ions move and deposit on the cathode surface, and graphene sheets also tend to incorporate into a freshly grown nickel matrix. Moreover, the agglomeration of graphene sheets is increased, and bulge formation is observed due to the high conductivity46 and small size of graphene nanosheets because current density is higher around the graphene sheets than that in the other areas. Thus, the faster deposition of nickel ions would be around graphene sheets that would lead to the formation of higher bulge morphology in the composite coatings. Furthermore, a higher concentration of SDS facilitates better dispersion of the graphene sheets in the electrolyte, which is supportive for higher co-deposition of graphene sheets because the adsorption of surfactants on the graphene sheets causes electrostatic repulsion between graphene sheets that hinders the aggregation and promote fast and uniform deposition. In addition, some negative functional groups due to the presence of SDS in the electrolyte were likely to get adsorbed on the graphene sheets and raise the nickel ion deposition around the graphene sheets.The SEM images show bulge morphologies  of nicke−1 SDS concentration in the deposition bath solution.The cross-sectional image is shown in EDS was performed to evaluate the composition of nanocomposite coatings. 3.347 revealed the cluster size and the mean cluster size calculations, and they reported that the results calculated from XRD using the Scherrer's method and also TEM observations were relatively constant. Scherrer's formula is given below to calculate the average grain size of the composite coatings:48D is the average crystalline size, K is the Scherrer constant, β is the FWHM, θ is the Bragg angle and λ is the wavelength with a value 0.15406 nm.X-ray diffraction patterns of the composite coatings obtained using different concentrations of SDS in the basic solutions are shown in 15,49 given as follows:I(hkl) is the reflection intensity of the tested samples and Io(hkl) is the refection intensity of the standard oriented sample.The preferred orientations were found by determining the texture coefficients using equationet al.50 reported that the coatings produced by face-centered cubic (fcc) metals showed a linear increase in the strain energy density of grains oriented at (hkl) with an angle between (100) and (hkl). In this observation, the analyzed results indicate a reduction in strain energy density with the increasing SDS concentration in the co-deposition process; this is mainly liable for the decrease in grain sizes and the relevant preferred orientations of the nickel-graphene nanocomposite coatings.Table S23.4−1, 0.2 g L−1 and 0.3 g L−1 graphene sheets. The hardness of the obtained coatings prepared from the deposition bath containing SDS was increased when compared with that of the coatings produced without SDS in the electrolyte. This increasing tendency of microhardness of the composite despite higher concentration of SDS can be explained by the effect of surfactants on the deposition mechanism of reinforcement particles and metallic matrix of the nanocomposite; essentially, the adhesion strength between the matrix and the reinforcement particles plays an important role in the strengthening effect of any formed composite. It is expected that the surfactant is incorporated into the deposit after electrodeposition and can be supportive for the properties of the obtained coatings.51 In the present study, it may be justified that negative groups adsorb on the surfaces of graphene sheets, which are beneficial to increase the nickel ion attraction and develop a good combination between the nickel deposition layer and graphene sheets. Additionally, a higher carbon content in the composite is achieved as the SDS concentration reaches a maximum value to 0.4 g L−1, and this is also a vivid reason for the enhanced hardness and adhesive strength. The adhesion strength shown in via a similar trend as microhardness because the increased content of graphene sheets in the deposit due to higher SDS concentration in the electrolyte is largely liable for the tremendous mechanical properties of the fabricated nanocomposite coatings.Adequate mechanical properties of nickel-graphene nanocomposites are greatly important to obtain superior corrosion resistance and other surface properties of materials. The corrosion performance of nickel-graphene nanocomposite coatings is measured using polarization curves and electrochemical impedance spectroscopy in a 3.5% NaCl solution. −1 SDS in the electrolyte has the highest impedance and possesses better corrosion resistance.Moreover, the superior corrosion resistance performance is due to the addition of graphene nanosheets that have tremendous mechanical properties and act as perpetual reinforcement in the nickel matrix. In fact, from the EIS results, we can evaluate the anti-corrosion property of coatings from the diameter of the semicircles, and the larger diameter ensures higher impedance to corrosion; we have found that the coating produced with 0.4 g L4.−1, 0.2 g L−1 and 0.4 g L−1. The results indicate that the maximum carbon content and excellent mechanical properties of composite coatings are obtained due to higher concentration of anionic surfactant SDS in the electrolyte. A coarser surface morphology is observed owing to bulge formation on the surface of Ni/graphene nanocomposite coatings, and the average grain sizes are also decreased with the addition of SDS in the deposition process. Electrochemical tests demonstrate that the composite coatings exhibit more positive corrosion potentials (−0.129 V), lower corrosion current density (1.425 × 10−6 A cm−2) and higher impedance when 0.4 g L−1 concentration of SDS is present in the deposition bath that is responsible for the significantly superior corrosion resistance properties due to uniform distribution of graphene sheets in the nickel matrix during electrochemical co-deposition.In summary, nickel-graphene nanocomposite coatings have been synthesized on carbon steel substrates from nickel sulfamate electrolyte containing graphene sheets with SDS surfactants at different concentrations of 0 g LThe authors declare that there is no competing financial interest.RA-008-C7RA13651J-s001


ATyr56, leading to the formation of an axisymmetric tunnel-shaped pocket, which is an important structural basis for improving the binding affinity of symmetric inhibitors with PD-L1. Furthermore, after inhibitor dissociation, the conformation of ATyr123 and BMet115 rearranged, which blocked the entrance of the binding pocket, while the reverse rearrangements of the same residues occurred when the PD-L1 monomer was complexed with the inhibitors, preparing PD-L1 for dimerization. Overall, this study casts a new light on the inhibitory mechanism of BMS inhibitors targeting PD-L1 dimerization and provides an idea for designing novel small molecule inhibitors for future cancer immunotherapy.Inhibitors blocking the PD-1/PD-L1 immune checkpoint demonstrate impressive anti-tumor immunity, and small molecule inhibitors disclosed by the Bristol-Myers Squibb (BMS) company have become a hot topic. In this work, by modifying the carbonyl group of BMS-202 into a hydroxyl group to achieve two enantiomers (MS and MR) with a chiral center, we found that this is an effective way to regulate its hydrophobicity and thus to reduce the negative effect of polar solvation free energy, which enhances the stability of PD-L1 dimer/inhibitor complexes. Moreover, we studied the binding modes of BMS-200 and BMS-202-related small molecule inhibitors by molecular dynamics simulation to explore their inhibitory mechanism targeting PD-L1 dimerization. The results showed that the size exclusion effect of the inhibitors triggered the rearrangement of the residue  Programmed cell death protein 1 (PD-1), an inhibitory receptor on activated T cells, can suppress the activity of activated T cells and maintain immune tolerance by binding to its ligand, namely programmed cell death 1 ligand 1 (PD-L1) ,3,4. How50) determined by homogeneous time-resolved fluorescence (HTRF) method ranged from the nM to µM level , which turned the pocket into a hydrophobic tunnel .,22.50) dµM level , which bµM level . In addic tunnel . This sic tunnel ,28,29. Bc tunnel . The resAlthough the above experimental results provided the vital structural basis for a structure-based drug design, the mechanism of PD-L1 dimerization induced by BMS small molecule inhibitors is not well understood. Molecular simulation could be a unique way to approach the dimerization mechanism at a molecular level. Ahmed et al. investigated the conformational dynamics of the existing crystal PD-L1 structures by classical and accelerated molecular dynamics simulations, and the result of principal component analysis showed that the C’’D loop contributed to the maximum structural displacements and the conformation of PD-L1 was consistent with the conformational selection mechanism during the binding process . Lim et Inspired by the above studies, in this work, we tentatively regulated the hydrophobicity of BMS-202 by modifying its carbonyl group into a hydroxyl group, which might weaken the negative effect of the polar solvation free energy, thus improving the stability of the PD-L1/inhibitor complexes . It shouAPD-L1 and BPD-L1 monomer and marked as the SA and SB system, respectively, while the other was made only by the PD-L1 dimer, named the Dimer system  simulations. In order to verify that the docking method was appropriate, the BMS-202 inhibitor was redocked to the PD-L1 dimer, and the conformation of the molecule in the docking and native crystal were almost superposed . The rooThe initial structures obtained by molecular docking were performed 200 ns MD simulations, and the RMSD of protein heavy atoms and inhibitors were used to assess whether the systems reached the equilibrium state . As showAPD-L1 and BPD-L1 for comparative analysis. As depicted in APD-L1 and BPD-L1 in all the systems basically overlapped, which suggested that the regional flexibility of PD-L1 in all systems was basically the same whether it was in the form of a dimer or monomer, with or without an inhibitor. Specifically, four dense peaks at 40–53 and 68–78 appeared in the residues of APD-L1 and BPD-L1, respectively. According to the secondary structures of PD-L1, these peaks mainly appeared in the random coil regions with high flexibility, which were far away from the binding pocket and barely affected the binding process.In order to determine whether the protein of these systems had similar regional flexibility, root mean square fluctuation (RMSF) was calculated to evaluate the fluctuations of the protein structure referring to its average structure at each frame, and the results were divided into Considering the balance between computational cost and accuracy, the binding free energies of these systems were calculated by the MM-PBSA method to evaluate the binding affinity between PD-L1 and small molecule inhibitors . With a Gnonpl,total) was favorable to the binding between PD-L1 and BMS small molecule inhibitors. Furthermore, the ΔGnonpl,total can be divided into Van der Waals energy (ΔEvdw) and non-polar solvation free energy , where Van der Waals interaction was the main driving force during the binding process. In contrast, the polar binding free energy  consisting of electrostatic energy (ΔEele) and polar solvation free energy  was overall unfavorable to the binding process, mainly due to the serious negative effects of ΔEpol,sol.Specifically, the binding free energy (ΔG) was divided into polar and non-polar parts. Due to the hydrophobic surface of PD-L1, the non-polar binding free energy , and the last 50 ns trajectories were then projected onto the two largest principal components (PC1 and PC2) to characterize the free energy landscapes of the binding pocket. The results of all systems are shown in BMet115 and ATyr123 in DIM-Conf 1 and 2 showed a downward flip, leading to the reduction of the cavity of the binding pocket. As a consequence, the entrance of the binding pocket was completely blocked. On the contrary, when a PD-L1 monomer was complexed by the inhibitors, the PD-L1 monomer would undergo reverse rearrangement and prepare for dimerization with nearby PD-L1.It was found that the side chains of the four Tyr residues were on the same side in the MS system. For the DIM-Conf 1 and 2 conformations, due to inhibitor dissociation, Tyr56 and Tyr123 have extra space to stagger up and down, which weakened the size exclusion effect and led to a new stable conformation. In addition, the side chains of residue The PD-L1/BMS-200 and PD-L1/BMS-202 crystal complexes  were downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank ,26,39. TMolecular docking is a technique of placing an inhibitor into the binding pocket to obtain a series of matching patterns of interaction. Autodock Vina was used for docking small molecule inhibitors into the PD-L1 dimer . We builMolecular dynamics simulation is a powerful tool for analyzing the conformational change of protein and the kinetic characteristics of small molecule inhibitors at an atomic level. For all the systems above, the MD simulations were carried out using the Gromacs 2016 software package . The GAFcom, Gpro, and Glig are the free energy of the PD-L1/inhibitor complex, PD-L1 dimer, and small molecule inhibitors, respectively. The energy (EMM) calculated by molecular mechanics consists of the energy of van der Waals (Evdw) and electrostatic interaction (Eele). The solvation free energy (Gsol) is comprised of the polar solvation free energy  and non-polar solvation free energy . ΔSASA denotes the solvent-accessible surface areas, and all the empirical parameters, including γ, are set to default. Considering that the entropy contribution (ΔS) is computationally expensive and tends to have a large margin of error that introduces significant uncertainty to the results, the ΔS of all systems were ignored [For the MD trajectory of biomolecular systems, the MM-PBSA method is commonly used to calculate the protein–inhibitor binding free energy (ΔG) to determine the binding affinity between protein and inhibitor ,51. In t ignored ,54. In aAPD-L1 and BPD-L1 monomer, respectively, while the other was made only by the PD-L1 dimer.In this work, to explore the effect of the hydrophobicity of the inhibitors on its binding affinity, we modified the carbonyl group of BMS-202 into a hydroxyl group, which generated a pair of enantiomers (MS and MR) and was complexed with the PD-L1 dimer. Meanwhile, we compared the binding modes of BMS-200, and these BMS-202-related small molecule inhibitors by molecular dynamics simulation to investigate their inhibitory mechanism targeting PD-L1 dimerization. Moreover, three control groups were also established for comparison. Two of them were constructed by complexing the S-enantiomer (MS) with the ATyr56, and the size exclusion effect triggered the rearrangement of the side chain of ATyr56, resulting in the formation of an axisymmetric tunnel-shaped pocket, which is an important structural basis for improving the binding affinity of symmetric inhibitors. Moreover, the downward conformational rearrangements of residues ATyr123 and BMet115 completely blocked the entrance of the binding pocket after inhibitor dissociation, such that other molecules could not access it, while the reverse rearrangement occurred when the PD-L1 monomer was complexed with the inhibitors, elucidating the key mechanism of PD-L1 dimerization.It was found that the chemical modification could effectively improve the hydrophobicity of BMS-202 and greatly reduced the negative effect of polar solvation energy, which enhanced the stability of the PD-L1 dimer/inhibitor complexes, providing an idea for the structural modification of small molecule inhibitors. Regarding the inhibitory mechanism of BMS inhibitors targeting PD-L1, it can be concluded that the staggered phenyl rings were the key fragment of the inhibitors, which occupied a large space and formed numerous non-polar interactions with surrounding residues, allowing the inhibitors to be firmly anchored in the pocket. Complementally, the linear flexible tail of the inhibitors caused hydrogen bond interactions to match the uneven surface of the binding pocket. Moreover, both a T-stacking interaction and a size exclusion effect existed between the inhibitors and Overall, this study casts a new light on the inhibitory mechanism of BMS small molecule inhibitors targeting PD-L1 dimerization and provides an idea for the design of novel small molecule inhibitors.


Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disorder of the central nervous system caused by opportunistic infection of the JC virus (JCV).A 58-year-old Japanese woman was admitted to our hospital for aphasia. She had a 5-year history of untreated sarcoidosis and was a human T cell lymphotropic virus-1 (HTLV-1) carrier. Serum angiotensin-converting enzyme, soluble interleukin-2 receptor, lysozyme, and calcium levels were elevated. JCV-DNA was not detected in cerebrospinal fluid by PCR testing. Skin biopsy revealed noncaseating granuloma formation. Bilateral multiple nodular lesions were present on chest X-ray. Brain magnetic resonance imaging showed left frontal and temporal lesions without gadolinium enhancement. As we suspected that systemic sarcoidosis had developed into neurosarcoidosis, we started steroid and infliximab administration. After treatment, the chest X-ray and serum abnormalities ameliorated, but the neurological deficits remained. At 1 month after immunotherapy, she developed right hemiparesis. Cerebrospinal fluid was positive for prototype (PML-type) JCV on repeated PCR testing. Brain biopsy revealed demyelinating lesions with macrophage infiltration, atypical astrocytes, and JCV antigen-positive cells. We diagnosed her with PML and started mefloquine, leading to partial remission.+ T cells, and may developped the patient’s PML. The comorbidity of sarcoidosis, PML, and HTLV-1 infection has not been reported, and this is the world’s first report of PML associated with HTLV-1 infection and sarcoidosis.Sarcoidosis and HTLV-1 infection both affect T cell function, especially CD4 Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by opportunistic infection with the JC virus (JCV). PML is induced by a dysfunction of cellular immunity and can be triggered by various conditions, such as a human immunodeficiency virus (HIV) infection, hematologic malignancy, and disease-modifying drugs for multiple sclerosis . Here, wA 58-year-old Japanese female with a 5-year history of untreated sarcoidosis was admitted to our hospital due to progressive aphasia lasting for 4 months. She was an HTLV-1 carrier, but was not affected by leukemia. She was a current smoker with a Brinkman index of 560. Physical examination revealed fine crackles on the lower back and a brownish skin lesion on the left knee Fig. A–H. Thes+ phagocytic macrophages , considering the possibility of PML based on the MRI findings. We administered steroid pulse therapy , followed by oral prednisolone 45 mg/day) with taper. After steroid treatment, infliximab (200 mg) was administered for the neurological symptoms. After initiating immunotherapy, her serum and chest X-ray findings were ameliorated, with the normalization of angiotensin-converting enzyme 9.8 IU/L), lysozyme 14.6 μg/mL), serum calcium (8.8 mg/dL), and soluble interleukin-2 receptor (356 U/mL) levels. However, her neurological conditions showed no improvement. She was transferred to a rehabilitation hospital with the continuation of 15 mg/day oral prednisolone. At 1 month later, right-sided total paresis appeared and she was re-admitted to our hospital. Brain MRI revealed scattered multifocal lesions in the left frontal lobe with a “milky way appearance” [5 mg/day  mg/day,  μg/mL, s IU/L, ly+ T cell deficiency, such as HIV infection or idiopathic CD4+ cell deficiency syndrome [+ T cell counts reportedly correlate with the prognosis of PML [+ T cell reactivity is associated with the prevention and resolution of PML [Here, we report an HTLV-1 carrier with untreated sarcoidosis who developed PML after treatment for sarcoidosis. PML is a fatal central nervous system disease caused by the opportunistic infection of JCV due to the suppression of cellular immunity, which leads to subacute neurological symptoms [syndrome . In addis of PML . Conversn of PML .+ T lymphocytes and reduces host immunocompetence, even in the non-leukemic state [tax gene. Previous reports showed that JCV promotes intercellular proliferation via the tax gene [+ lymphocytes. These mechanisms may increase the risk of developing PML. Previous reports have shown that there is no relationship between HTLV-1 infection and sarcoidosis [In this case, HTLV-1 infection was also thought to have contributed to the development of PML. HTLV-1 mainly infects CD4ic state . In additax gene , leadingcoidosis , 15. In Concerning the relationship between sarcoidosis and PML, sarcoidosis is reported to be a risk factor for the development of PML, whether treated or not . Clinici+ T cells, and may have been involved in the development of PML. The comorbidity of sarcoidosis, PML, and HTLV-1 infection has not been reported, and is considered a rare event.Here, we report a case of PML associated with untreated sarcoidosis and HTLV-1 infection. JCV-DNA was not detected in the initial CSF examination, but was detected after immunotherapy for sarcoidosis. Sarcoidosis and HTLV-1 infection both influence T cells, especially CD4