Saccharomyces cerevisiae, using a library of 5154 gene knock-out strains expressing Aβ1–42. We identified 81 mammalian orthologue genes that enhance Aβ1–42 toxicity, while 157 were protective. Next, we performed interactome and text-mining studies to increase the number of genes and to identify the main cellular functions affected by Aβ oligomers (oAβ). We found that the most affected cellular functions were calcium regulation, protein translation and mitochondrial activity. We focused on SURF4, a protein that regulates the store-operated calcium channel (SOCE). An in vitro analysis using human neuroblastoma cells showed that SURF4 silencing induced higher intracellular calcium levels, while its overexpression decreased calcium entry. Furthermore, SURF4 silencing produced a significant reduction in cell death when cells were challenged with oAβ1–42, whereas SURF4 overexpression induced Aβ1–42 cytotoxicity. In summary, we identified new enhancer and protective activities for Aβ toxicity and showed that SURF4 contributes to oAβ1–42 neurotoxicity by decreasing SOCE activity.Alzheimer’s disease (AD) is known to be caused by amyloid β-peptide (Aβ) misfolded into β-sheets, but this knowledge has not yet led to treatments to prevent AD. To identify novel molecular players in Aβ toxicity, we carried out a genome-wide screen in  The aggregation of amyloid β-peptide (Aβ) into oligomers and fibrils is one of the main hallmarks of Alzheimer’s disease (AD) ,2. Aβ is2O2 and OH·, that generate oxidative stress, which oxidizes proteins, lipids and nucleic acids [Much is known about the toxic effects of Aβ oligomers and fibrils: (i) they produce reactive oxygen species (ROS), such as Hic acids  and affeic acids ; (ii) thic acids ,17;  ic acids ,22. In aSaccharomyces cerevisiae to perform a genome-wide screen to identify regulations of amyloid toxicity. The choice of this model system was based on its reduced complexity and on the similarity of most of its molecular signaling pathways to mammals, including those regulating autophagy, apoptosis, mitochondrial function, cellular trafficking and protein homeostasis [In the present work, we used eostasis .1–42 is toxic for S. cerevisiae [We previously demonstrated that Aβrevisiae . Other srevisiae ,26. Thesrevisiae .S. cerevisiae, we overexpressed wild-type (WT) human Aβ1–42 in those cells, which is the most aggregative and pathogenic isoform of Aβ. Cytosolic expression of Aβ fused to GFP (Aβ-GFP) is known not to induce toxicity [S. cerevisiae MFα. In addition, the Aβ construct contains a GFP tag at the C-terminus of the Aβ sequence connected by a linker nucleotide sequence that enables detection of the GFP signal [GAL1 promoter, so that Aβ could be induced by growing the cells in galactose medium (1–42 mutants termed Dutch (E22Q) and Arctic (E22G) were also cloned and used in this report. Dutch and Arctic Aβ are human genetic mutations very prone to aggregate, and thus induce an early onset of AD [1–42 constructs, and GFP expression was assessed by Western blot in cells cultured in inducing medium  for 6 h at 30 °C (1–42 fused to GFP results in a band of ~41 kDa when the MFα is not yet processed (MFα-Aβ1–42-GFP), and a band of ~31 kDa when the protein is mature and MFα has been processed (Aβ1–42-GFP). In addition, a ~45–50 kDa band is observed, which may represent a glycosylation modification of MFα, according to a previous report [1–42 cellular localization by confocal microscopy images of cells grown in inducing medium for 6 h  in inducing medium. In addition, we observed an increased cytotoxicity induced by both Dutch and Arctic Aβ1–42 compared to WT Aβ1–42, suggesting that Aβ1–42 toxicity depends, at least partially, on the aggregative proneness of the peptide, in agreement with previous findings [S. cerevisiae containing the described Aβ1–42 construct is an appropriate model to evaluate Aβ toxicity.Since the main objective of this study is to investigate the effect of Aβ peptide D. Quanti peptide E. We foufindings . These d1–42, from now on referred to simply as Aβ. Modifiers of Aβ toxicity were identified by screening 5154 mutants from a S. cerevisiae genome-wide deletion library, which were mated with an Aβ-containing strain, and strains containing the mutation and the inducible Aβ construct were selected using an automated system [The peptide used in the present study to screen for Aβ toxicity modulators is the WT Aβd system  followinAQP9 [HSPA1B [HSPA1L [OSBPL10 [AP3M2 [GAS7 [KMO [RAB5A [UBE4A [VSP28 [The pathophysiological relevance of these findings is supported by the fact that among the regulatory genes for amyloid toxicity identified, there are 10 genes that have been proposed in GWAS as playing an important role in AD: AQP9 ,35, HSPA [HSPA1B ,36, HSPA [HSPA1L ,37, OSBP[OSBPL10 ,38, AP3M0 [AP3M2 ,39, GAS7M2 [GAS7 ,40, KMO AS7 [KMO ,41, RAB5O [RAB5A ,42, UBE4A [UBE4A ,43 and VA [VSP28 .We next performed an interactome analysis to identify the intracellular pathways involved in toxicity and protection. Two main approaches were used for this analysis. One of them was to perform pathway enrichment for the mammalian genes, and the second one was to analyze the network components in the protein–protein interaction network constructed by the mammalian genes.p-value  and p-value .Homo sapiens. Out of the 238 mammalian orthologues identified, 81 of these were enhancer genes of Aβ toxicity  and 157 Furthermore, we identified densely interconnected nodes (modules) in the network. We performed pathway enrichment of the genes highlighted in yellow and we observed that mitochondrial translation-related pathways have an FDR less than 0.05. Specifically, mitochondrial translation elongation, mitochondrial translation initiation, mitochondrial translation termination, mitochondrial translation and translation are enriched, respectively, according to their FDR values .SURF4, STIM1, ORAI1 and CRACR2. The search resulted in several genes associated with them, including pathways of ubiquitination, KIF5A, SARAF and SGK1. Then, we used GUILDify v2.0 and the human interactome to extend and analyse the subnetwork linking these genes .Next, we carried out a text-mining analysis of the reported genes , findingse genes . The anaSURF4 , an important mechanism that controls cellular calcium homeostasis . To elucSURF4 A or a plSURF4 D, which SURF4 A or incrSURF4 D, respecF4 siRNA B,C and aF4 siRNA E,F.SURF4 is silenced ” play a protective role. Modules in biological networks are subnetworks within the whole network that are highly interconnected . The detReactome pathway enrichment of all the 238 mammalian orthologues and the module demonstrated that mitochondrial translation is the major significant pathway related to these genes. The mitochondrial translation is a term that encompasses initiation, elongation and termination . All theOn the other hand, there are multiple proteins for which their function in the cell is poorly understood. This is the case of SURF4, a housekeeping protein of 37 kDa present in the endoplasmic reticulum and proposed previously to regulate SOCE . CalciumCalcium is known to be relevant for cellular function, especially in neurons due to its role in neurotransmission. In the present paper, we highlight the importance of SURF4 as a regulator of intracellular calcium by identifying it as a novel player in Aβ-mediated cell death. A higher expression of SURF4 contributes to higher Aβ neurotoxicity related with a decrease in calcium entrance by SOCE. Therefore, we propose that SOCE contributes to neuronal protection against Aβ, as demonstrated when we silenced SURF4.In summary, we found a set of neuroprotective proteins against Aβ toxicity, and a complementary set of proteins that contribute to Aβ toxicity, such as SURF4. The maintenance of SOCE integrity seems to be crucial to protect neurons against Aβ toxicity. Further work is needed to elucidate the role of the other proteins found in this study regarding their capability to modulate the toxic effect of Aβ in neurons.w/v dextrose, 2% w/v peptone, 1% yeast extract), or synthetic minimal medium . Solid medium contains also 20 mg/L agar. When indicated, SD was supplemented with 0.7 g dropout medium without uracil (SD-URA). The inducing medium is SD-URA that contains 2% w/v galactose instead of glucose as carbon source. Since ammonium sulfate inhibits the function of the antibiotic geneticin (G418), synthetic medium G418 was made with monosodium glutamate (MSG) as a nitrogen source as follows: 20 mg/L agar, 1.7 g/L yeast nitrogen base w/o ammonium sulphate and amino acids, 1 g/L monosodium glutamic acid, 2 g/L amino acid dropout and 2% glucose. Medium preparation and yeast culturing was carried out according to standard techniques.Yeast strains were grown in YPD medium (2% 1–42 (Aβ42) was overexpressed in yeast as follows. S. cerevisiae strain BY5563α was transformed with a multicopy yeast-expression plasmid (pRS426) with the URA3 selectable marker and CYC1-GAL1 promoter controlling the expression of MFα-Aβ42-GFP , which was completely sequenced  instead of non-inducing medium (containing 2% w/v glucose) for at least 5 h at 30 °C.Human Aβequenced . The cone method . Transfohttp://www.ensembl.org (accessed on 7 October 2021)) and from Drosophila RNAi Screening DRSC Integrative Ortholog Prediction Tool (http://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl (accessed on 7 October 2021)). Further analyses were performed in the Saccharomyces Genome Database (http://www.yeastgenome.org/ (accessed on 7 October 2021)).The library used for the screen is constituted by 5154 homozygous knock-outs . These knocked out cells were produced in the BY4741 strain background (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and present geneticin (G418) resistance. Solutions of canavanine  and G418 were previously dissolved in water at 100 mg/L, filtered sterilized and stored in aliquots at 4 °C. The screen was performed with an automated system using the ROTOR HDA Singer Instruments  following the Synthetic Genetic Array (SGA) protocol with some modifications ,58. BrieYeast cells were harvested and lysed with TCA 85% for 10 min at room temperature (RT). Supernatants were resolved in SDS-PAGE. Gels were transferred in polyvinylidene fluoride membranes , which were blocked for 1 h in tween-tris buffer saline (TTBS) plus 5% milk or 3% bovine serum albumin (BSA). Membranes were incubated o.n. at 4 °C with the following primary antibodies (Abs): 1:500 anti-GFP  and 1:500 anti-Aβ 6E10 . Membranes were washed thrice with TTBS and incubated for 1 h with 1:2000 anti-mouse secondary Abs . Three washes with TTBS were performed and membranes were developed with Super signal West Pico and Femto Chemiluminiscent substrate . Blotting quantification was performed with Quantity One software software (v.4.6.8).Yeast strains were cultured in inducing medium 6 or 15 h at 30 °C. Then, they were incubated 30 min in poly-lysinated coverslips and digital images were taken with a Leica TCS SP5 II CW-STED confocal microscope, deconvolved with Huygens (SVI) and analyzed with Image J software (v.1.53c).Strains were grown o.n. at 30 °C in SD-URA containing glucose. Cell concentrations (OD660) were adjusted at OD660 0.3 and after 5 h growing in SD-URA containing raffinose, three dilutions 1:10 were spotted in plates with SD-URA containing glucose (uninduced) or galactose (induced). Plates were incubated at 30 °C for 3 days before analysis.The selected knock-out strains were grown in YPD G418 solid medium at 30 °C for 24–48 h plates. One colony of each strain was grown in YPD liquid medium at 30 °C o.n. Cell concentrations were adjusted at OD660 0.2 and after 4 h of growing in YPD liquid medium at 30 °C, cells were transformed with the Aβ construct or an empty vector pRS426 as previously described. Transformants were selected in SD-URA solid medium for 4 days at 30 °C. Then, two colonies of each knock-out strain were selected and used for spotting assay using the ROTOR HDA Singer Instruments as previously described. Briefly, knock-out strains transformed with Aβ construct or empty vector were grown in SD-URA liquid medium o.n. at 30 °C. Cell concentrations were adjusted at OD660 0.2 and after 4 h of growing in SD-URA, cells were pinned into either glucose (uninduced) or galactose (induced) medium and incubated at 30 °C for 2 to 4 days before scoring.Knock-out strains transformed with Aβ construct or empty vector were grown in SD-URA solid medium o.n. at 30 °C. Cells were pinned into SD-URA liquid medium containing either glucose or galactose and incubated at 30 °C for 48 h in a fluorimeter while OD660 values were being registered. OD660 results were used to obtain the growth curves of each mutant.Homo sapiens. While creating the network, “experiments”, “databases” and “gene fusion” were used as sources. The confidence was chosen to be medium (0.4), and no additional interactors were chosen.A network of the amyloid toxicity protective or activator genes was created by using StringDB . The 238Cytoscape’s  network Text search of the yeast genes was performed in Uniprot and DidGeNet to obtain related proteins published in the Net (Uniprot), thus increasing the number of relevant proteins/genes. We generated a list of AD genes reported in humans in the Net and we performed a text-mining search of this list using GUILDify v2.0 Web Server with DisGeNet data (DisGenet_Guild). Finally, we studied the overlap of the protective and activators genes with the list DisGenet_Guild using the GUILDify v2.0 Web Server.2.Human neuroblastoma cells (SH-SY5Y cells) were grown with Ham’s F12 GlutaMax  supplemented with 15% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37 °C in a humidified atmosphere containing 5% COSURF4 siRNA from Ambion or a negative siRNA control from Qiagen  with a pGFP reporter. RNAiMax transfection kit  was used to transfect cells in Opti-MEM. A total of 180 pM siRNA were added to cells plus 0.6 μg of pGFP reporter plasmid for cotransfection in 6-well plate and 72 pM siRNA plus 0.24 μg of pGFP in 24-well plate. Cells were transfected and incubated with growth media for 48–72 h.SH-SY5Y cells at ~80% of confluence were cotransfected with pCDNA3.1 YFP tagged plasmid using a sequence with EcoR1 and Xho1 as restriction sites (pCMV-SPORT6-SURF4 (SURF4) plasmid was obtained from BioCat . SURF4 gene was recombined into a on sites . Then, 81–42 wild-type  was solubilized in 250 µL of MilliQ water. The pH was adjusted to ≥10.5 with 1 M NaOH to avoid the isoelectric point of Aβ. A total of 250 µL of 20 mM phosphate buffer (pH 7.4) was added to neutralize pH and samples were sonicated for 1 min in a bath-type sonicator . Aliquots were prepared and dissolved to 0.4 mg/mL (88.6 µM) in serum-free Ham’s F12 GlutaMax  to treat cells. Aβ1–42 was incubated for 24 h at 4 °C to allow its oligomerization [1–40 were dissolved to 0.4 mg/mL (88.6 µM) in serum-free F12 medium to treat cells.A total of 1 mg lyophilized Aβrization . Aliquot4 cells/well). After 12 h, the growth medium was removed. Cells were treated with 5 or 10 µM oAβ1–42 in F12 medium without FBS for 24 h at 37 °C. In one set of experiments, we pretreated for 1 h with 500 nM RO2959 hydrochloride (Ro) to inhibit STIM/ORAI interactions. Cell survival was assayed by 3--2,5-diphenyltetrazolium bromide (MTT) reduction method. We added 10% MTT stock solution (5 mg/mL) for 2 h. Then, medium was discarded and 100 μL of DMSO was placed per well. Absorbances were measured in a plate reader  at 540 nm and 650 nm (as reference). Control cells were assumed as 100%.SH-SY5Y cells were seeded in 24-well plates (7.5 × 104 cells/well) to growth up to ~80% confluence. Then, cells were transfected to overexpress or to silence SURF4 as indicated above. Finally, the mRNA of SH-SY5Y cells was extracted by using the Total RNA Isolation kit  and quantified with NanoDrop ND-1000 (Thermo Fisher Scientific). Total cDNA was obtained from 500 μg of mRNA by retrotranscription using SuperScript III Reverse Transcriptase kit . Quantitative PCR was performed for SURF4, GAPDH and HPRT by using the fluorophore Sybr Green (Thermo Fisher Scientific) along with 10 μM cDNAs specific primers. The experiment was performed with QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). ΔΔCT was measured using GAPDH or HPRT as housekeeping genes.SH-SY5Y cells were seeded in 6-well plates (30 × 104 cells/well in 24-well plates) to growth up to ~80% confluence. Then, cells were transfected to overexpress or to silence SURF4 as indicated above. Cells were treated with 5 μM Fura 2AM (Invitrogen) supplemented with 0.02% pluronic acid for 40 min in an isotonic solution . Experiments were performed at RT using a custom-made chamber. Fluorescence images were obtained using a Nikon inverted microscope with a xenon lamp. GFP positive transfected cells were selected using 450 nm excitation and a camera managed by AquaCosmos software . Cytosolic calcium levels were obtained using 340/380 ratio. Measurement of basal calcium was obtained along 4 min recording each 5 s.SH-SY5Y cells were seeded in coverslips . Endoplasmic reticulum (ER) calcium release was measured using Fura 2AM. Positive controls with 1 μM thapsigargin, an ER calcium depletion drug, was carried out in free calcium solution until peak recovery.After basal calcium measurement, cells were bathed in a free calcium solution  was measured after ER calcium release by changing the extracellular free calcium solution with a solution containing 1.2 mM CaData are expressed as mean ± SEM of n experiments as indicated in the corresponding figures. Statistical analyses for qPCR were performed by two-way Student t-test or two-way ANOVA plus Bonferroni as post hoc test. The software used was GraphPad software. For calcium, a Student’s t-test was performed comparing means for basal calcium, and areas under the curve for reticular calcium and SOCE.


Rotavirus; in developing countries, it is a primary cause of deaths in children less than five years of age. This study was planned to find out the etiologic agents of acute watery diarrhea. In this study, 1465 stool samples were analyzed with the symptoms of acute diarrhea. Demographic data analysis showed no. of episodes of diarrhea, vomiting, and fever. All samples were checked by ELISA technique for the presence of Rotavirus circulating strains. More than 6% patients were found to be positive with Rotavirus. Common Rotavirus genotypes, including G2P4, G2P6, G3P4, G8P4, G8P6, G9P4, and G10P4, were detected in patients through RT-PCR. This study concluded that detection of rotavirus strain diversity and management of diarrheal patients may identify assortment of emerging strains and reduce emergence of antimicrobial resistance and repeated episodes of diarrhea, which may also help to avoid and manage the essential nutrients lost leading to malnutrition and stunted growth, as well as to reduce high mortality rate in young children less than five years.One of the common viral pathogens in infectious diarrhea is  Rotavirus is considered the major cause of severe gastroenteritis ranges from asymptomatic self-limiting watery diarrhea to life threatening infection in children worldwide. Malnutrition and stunted growth can occur if mismanaged in symptomatic cases . Am). AmRotac cases . At 50°C for 60 min, the process for reverse transcription was performed in a heating chamber. The termination of reaction was at 85°C for 5 min. Cooling of mix, 2 U of RNase H in 1 μl followed by incubation 20 min at 37°C. 50-μl processed for PCR containing 2.0 μl of the RT-PCR products. Amplification buffer 5 μl, 1.5 μl of forward and reverse primer each adding, 2.5 U DNA polymerase. Denaturation of Template , 30 cycles of PCR were set. PCR cycle set for denaturation step (94°C for 30 s), annealing (55°C for 30 s), and extension (68°C for 3 min for gene 4 and for 2 min for gene 6). Additional incubation  followed by PCR. PCR purification kit (Qiagen) to collect purified DNA. By using RT-PCR as templates, VP4 and VP6 gene sequences were determined.Reverse transcriptase PCR was performed by taking genomic RNA 1 Rotavirus are circulating in the community of Karachi as population belongs to nearby rural areas, where human interaction with animals makes it possible for the assortment for human and animal strains and emergence of new strains.The current study found that many diversified strains of human Rotavirus infection prevalence rate in Eastern countries reported 40.5%-60.5% [Rotavirus in stool samples of children under five years of age—i.e., G9P8, G1P8, and G1P4, were of prime importance in addition to G2P4 (6%), G4P6, G9P6, and G9P4, whereas 2.4% mixed infection caused by more than one strain was also identified [In the present study, a prevalence of 6%  RVA in d5%-60.5% . Similar5%-60.5% . In Karaentified , 15.In another study from Pakistan, G1P8, G2P4, G1P6, G9P8, G9P6, G12P6, G6P1, and mix infection with other pathogens were reported as causative etiological agents. These findings are indicative that RV genotype prevalence can be in various combinations and seasonal variation does not affect the prevalence of infection . RotavirRotavirus infection due to assortment of genes, several G types  and circulating P-types are a cause of mix infection in a single patient in multiple cases [Rotavirus subtypes G2P4, G10P4, G9P4, and G3P4 (Figures In le cases . It is ale cases . Similarle cases . By usin Figures .Pathogenesis might be the result of several assortments of human and animal strain resulting in origin of G11; however, from Pakistan, most of the focus was on VP7 and VP4 genotyping studies from different cities , 20. It Rotavirus was detected in our population beside common circulating strains around the Globe. Rotavirus genotypes which were detected in our patients were G10P4, G2P4, G2P6, G3P4, G8P4, G8P6, and G9P4.In this study, it is noted that the presence of new mix strains of Spectrum of etiological agents is involved in diarrhea which is a huge health burden worldwide as well in Pakistan. The data of the current study indicate that by conducting prevalence studies and identifying risk factors disease can be timely prevented or managed. Thus, morbidity and high mortality in infants can be reduced. This issue warrants broad-spectrum investigation.


Acinetobacter baumannii is a major public health threat, causing difficult-to-treat nosocomial infections. We identified two virulence pili independently critical to A. baumannii catheter-associated urinary tract infections, the chaperone-usher pathway pili Abp1 and Abp2. Mechanistically, these pili contribute to A. baumannii biofilm formation. Further, their respective tip-adhesive proteins, the adhesins Abp1D and Abp2D, make use of large, distally oriented binding pockets to adhere to glycoproteins like fibrinogen, a wound-healing protein important in CAUTI pathogenesis.Multidrug-resistant  Acinetobacter baumannii infections are an urgent clinical problem and can cause difficult-to-treat nosocomial infections. During such infections, like catheter-associated urinary tract infections (CAUTI), A. baumannii rely on adhesive, extracellular fibers, called chaperone-usher pathway (CUP) pili for critical binding interactions. The A. baumannii uropathogenic strain, UPAB1, and the pan-European subclone II isolate, ACICU, use the CUP pili Abp1 and Abp2  in tandem to establish CAUTIs, specifically to facilitate bacterial adherence and biofilm formation on the implanted catheter. Abp1 and Abp2 pili are tipped with two domain tip adhesins, Abp1D and Abp2D, respectively. We discovered that both adhesins bind fibrinogen, a critical host wound response protein that is released into the bladder upon catheterization and is subsequently deposited on the catheter. The crystal structures of the Abp1D and Abp2D receptor-binding domains were determined and revealed that they both contain a large, distally oriented pocket, which mediates binding to fibrinogen and other glycoproteins. Genetic, biochemical, and biophysical studies revealed that interactions with host proteins are governed by several critical residues in and along the edge of the binding pocket, one of which regulates the structural stability of an anterior loop motif. K34, located outside of the pocket but interacting with the anterior loop, also regulates the binding affinity of the protein. This study illuminates the mechanistic basis of the critical fibrinogen-coated catheter colonization step in A. baumannii CAUTI pathogenesis.Multidrug-resistant  Acinetobacter baumannii is a gram-negative, opportunistic pathogen able to colonize a variety of human host tissues . Catheterization is a common medical procedure in a hospital setting, wherein tubing is inserted into the bladder lumen to allow for the voiding of the bladder , is attenuated in CAUTI (prpA-D) and CUP2 (cupA-D), respectively. These pili belong to the γ4 clade of the CUP pilus family, of which over 100 members have been identified in Gram-negative bacteria  possessing abp1D and 51%  possessing abp2D. 31%  of the A. baumannii genomes possessed both abp1D and abp2D. Only 37%  of 5,201unique genomes contain neither abp1D nor abp2D. Thus, these genes are prevalent and co-occur frequently. Among Multidrug-resistant (MDR) isolates containing both pili are the uropathogen UPAB1  strain is attenuated in a mouse CAUTI model (abp1 and UPAB1Δabp2) compared to WT (abp1 and ACICUΔabp2) being attenuated in colonizing the catheter and bladder in the CAUTI model (abp1Δabp2) with either abp1ACICU or abp2ACICU shows that each individual pilus is capable of fully restoring both bladder and catheter colonization . Further, both ACICUΔabp1 and ACICUΔabp2 strains are deficient in binding to fibrinogen compared to WT . UPAB1Δabp2 and UPAB1Δabp1Δabp2 each exhibited a defect in their ability to form biofilms in vitro with and without fibrinogen at 37 °C compared to WT  and Abp2D (referred to as “Abp2DRBD”) from ACICU were cloned, expressed, purified, and crystallized. The primary amino acid sequences of the Abp1D and Abp2D RBD are >50% identical to one another  (RBD (2.1 Å) (8DF0)  and Abp2DRBD (from ACICU) (1.3 Å) (8DEZ)  reveal canonical β (beta)-strand jelly-roll structures. Circular dichroism (CD) reflects the secondary structure observed in the crystallographic models for both Abp1DRBD and Abp2DRBD with a spectral peak consistent with β-sheets elements  located at the distal end of the RBD relative to the linker to the pilin domain  . This anterior loop motif is conserved between Abp1DRBD and Abp2DRBD. Interestingly, other lectin domains in the γ4 clade, including MrkD  and GafD , contain an analogous loop. In these structures, this anterior loop contributes to the binding pocket architecture. Below, we show that the conserved loop motif also contributes to the binding of Abp1D and Abp2D, and thus, we refer to this region bound by this loop as the binding pocket.The RBD of Abp1D  . The strelements B, as expelements A. In AbpRBD crystal structure, a citrate molecule from the crystallization buffer is located within the putative binding pocket and is, in part, bound by the anterior loop (described above). The carboxylic acid residues of the citrate are coordinated by the side chains of the amino acids R86, Y100, R102, and Y141  using biolayer interferometry (BLI). The R102 and Y141A mutations reduced binding relative to Abp2DRBD WT by approximately ~70% and ~90%, respectively. Surprisingly, R86A Abp2DRBD maintained adherence to BSM. We next inverted the side chain charge at position 86, creating R86E, to attempt to actively repel adherence. This R86E mutation abolished Abp2D binding to BSM (RBD similarly abolished binding to BSM as measured by enzyme-linked immunosorbent assay (ELISA) . Appropriate folding of the R86E variants was demonstrated by both size exclusion chromatography (SEC) for Abp2DRBD and CD for both Abp1DRBD and Abp2DRBD . This is also relevant to other γ4 clade adhesins. R105 in MrkD is placed in an analogous location as R86 in Abp1D and Abp2D, and R105E also eliminates MrkDRBD binding  unit. By comparing these two molecules, we identified dynamic regions. The structures of the two molecules of Abp1DRBD had a backbone rmsd value of 0.176 Å  simulations to perform the goal-oriented sampling scheme, FAST  to the top (residues 104 to 110) of the binding pocket (loop opening distance) to define “open” vs. “closed” and provide a FAST objective. A more “open” pocket has a larger loop opening distance and a “closed” pocket has a smaller loop opening distance. Subsequent analysis showed that equilibrium populations of Abp2DRBD skewed toward a larger pocket opening distance, indicating that the anterior binding pocket loop in Abp2DRBD is flexible but predominantly favors a more open conformation . The electron density of the anterior loop is sparse, starting at position 86, even though the rest of the protein is well-modeled. This indicates that the loop is flexible and oriented in different conformations between different molecules throughout the crystal. However, we were able to model the R86E anterior loop, finding that it is displaced dramatically up and into the binding pocket by 11.6 Å relative to the structure solved for Abp2DRBD WT   to form a salt bridge in both Abp1DRBD molecule A or Abp1DRBD molecule B and the loops are more closed than in the Abp2DRBD structure . Therefore, we hypothesized that the salt bridge in Abp2DRBD is restricting the movement of the anterior loop in our simulations. By comparing simulations of an K34A variant of Abp2DRBD to simulations of WT, we found that the K34A mutation releases the anterior binding pocket loop. The K34A FAST structural ensemble favors a smaller loop opening distance and a more closed conformation compared to WT . R86E mutations in both Abp1DRBD and Abp2DRBD uniformly decrease the binding of the protein construct to each of the tested glycoproteins. Thus, our mutagenesis work demonstrates that Abp1D and Abp2DACICU share a common mechanism of adhesion to multiple ligands via analogous binding pockets, which share conserved residues critical for binding in Abp2D (R102 and Y141A). Though Abp1D and Abp2DACICU share residues critical for adherence , Abp2DRBD has a higher affinity for fibrinogen than Abp1DRBD  separately contributes to uropathogenesis by mediating adherence to the host factor fibrinogen. We found that Abp1 and Abp2 are present in a high percentage of A. baumannii strains, and here, we have unveiled their role in device-associated infections. Whether they are important in other types of infections outside of the urinary tract remains to be studied. We hypothesize that this may be the case, as both Abp1DRBD and Abp2DRBD recognized receptors containing the NA2 glycan, which is a common N-linked glycan present on various human glycoproteins, including fibrinogen  and closing (high binding) of the binding pocket in part via a salt bridge interaction. Interestingly, this loop motif is found in several other γ4 clade RBD, and R86, at the base of Abp1D- and Abp2D-binding pockets, is located in an analogous region to the pockets of other γ4 clade receptor binding domains. We discovered that an R86E mutation attenuates fibrinogen binding. The crystal structure of R86E Abp2DRBD and FAST simulations of R86E and K34A revealed that such mutations destabilize the anterior loop in Abp2DRBD, allowing it greater flexibility. For R86E, the electrostatic consequences of the side chain charge inversion, necessary to eliminate binding at this position, could be contributing to the flexibility of the anterior loop motif. K34 forms a salt bridge with E87, which controls the opening (low binding) and closing (high binding) of the pocket by regulating the movement of this anterior loop. Eliminating the electrostatic interaction of K34-E87 via K34A dysregulates the anterior loop by allowing it to flip up. This would form a more closed binding pocket for a ligand and, functionally, results in a higher affinity protein compared to WT.The crystal structures of Abp1D and Abp2DE. coli adhesin, FimH, which is located at the tip of the type 1 pilus. FimH is a two-domain CUP adhesin composed of a receptor-binding domain and a pilin domain that connects it to the pilus (similar to Abp1 and Abp2). FimH exists in an equilibrium between relaxed and tense conformations  or decrease (T52I) the binding of the pilus depending on the substituted residue  and sucrose counterselection as previously described . Assembl8 CFUs bacteria in 50 μL via transurethral inoculation. At 24 h postinfection (hpi), mice were sacrificed, and kidneys, bladders, and implants were aseptically removed. The bacterial load present in each tissue was determined by homogenizing each organ in PBS and plating serial dilutions on LB agar supplemented with antibiotics when appropriate. All CAUTI studies were performed in accordance with the guidelines of the Committee for Animal Studies at Washington University School of Medicine, and we have complied with all relevant ethical regulations. Mice were housed with a cycle consisting of 12 h of light and dark with access to standard food and water ad libitum.Six- to eight-wk-old female C57BL/6 mice were obtained from Charles River Laboratories. Mice were transurethrally implanted with a small piece of silicone tubing (catheter implant) and inoculated as previously described . Briefly600 of 0.05 in human urine (IRB: 201901098) or human urine with human fibrinogen (150 µg/ml) (Sigma) and inoculated into sterile, round-bottomed, polystyrene 96-well plates (Corning Inc.) with 200 µL final volume per well. Plates were incubated for 24 h under static conditions at 37 °C with humidity to prevent evaporation. After incubation, planktonic cells in the culture were removed and transferred to another 96-well plate and OD600 readings were taken using a BioTek microplate reader. Bacteria bound to the wells of the overnight cultures were washed 5 times with water and stained with 300 µL of 0.1% crystal violet solution dissolved in water for 15 min. After staining, wells were washed 5 times with water and left to completely dry overnight. Crystal violet staining was dissolved in 200 µL of 30% acetic acid, and absorbance was measured at 550 nm.Overnight static cultures were normalized to an OD600 of 0.5, and then washed and resuspended in PBS. A total of 100 µL of bacteria was added to the coated wells and incubated for 2 h at 37 °C, followed by PBS washes to remove the unbound bacteria. Next, bacterial cells were fixed with formalin for 20 min at room temperature, followed by three washes with PBS containing 0.05% Tween 20 (PBS-T). Then, the plates were blocked overnight at 4 °C with 1.5% BSA-PBS, followed by three washes with PBS-T. After the washes, the plates were incubated for 2 h at room temperature with rabbit anti–Acinetobacter antisera . Plates were washed with PBS-T, incubated with the Odyssey secondary antibody  for 45 min at room temperature, and washed with PBS-T (three times). The plates were scanned for infrared signals with the Odyssey Imaging System (LI-COR Biosciences).Flat-bottom microplates (Thermo Fisher) were coated overnight at 4 °C with BSM (10 µg/ml) (Sigma) and human fibrinogen from plasma (150 µg/ml) (Sigma). The plates were blocked for an hour with 1.5% BSA in PBS, followed by washing with PBS (three times for 5 min each). Bacterial strains were grown overnight in LB broth, normalized to an ODk-mer database of 661 K curated genomes was utilized using the COBS search index   , with a 7c030bb) .https://www.ebi.ac.uk/Tools/msa/clustalo/) on default settings (http://sts.bioe.uic.edu/castp/index.html?2was)  . Volume ml?2was) . SecondaMD simulations were run with Gromacs 2020.1 at 310 K using the AMBER99SB-ILDN force field with explicit TIP3P solvent –44. WildThe FAST algorithm  was usedα, Cβ, N, and O) and clustered with a k-centers algorithm based on rmsd to generate 3,000 cluster centers. Following clustering, an MSM was built for each variant by row-normalizing the observed transition counts, at a lag-time of 2 ns, with a small pseudocount as a prior (Markov State models (MSMs) were built from the FAST simulation data using enspara . An MSM  a prior .N-linked oligosaccharides from fibrinogen were enzymatically removed with PNGase F (New England BioLabs) following the manufacturer’s protocol. Briefly, 100 µL of glycoprotein denaturing buffer was added to 1 mL of 1.5 mg/ml fibrinogen in PBS. The mix was incubated for 10 min at 100 C. After 100 µL glycobuffer 2, 100 µL of 10% NP-40, and 3 µL PNGase F were added to the mix and incubated at 37 °C for 2 h. The glycosylation status was checked by gel electrophoresis and Coomassie blue staining of the samples. For protein-binding studies, fibrinogen was adhered to 96-well ELISA plates and then treated or mock-treated with PNGase F generally in line with its described protocol.A. baumannii gDNA preparations, using a 5′ primer containing an EcoRI recognition site and homologous to sequences upstream of the signal sequence and a 3′ primer containing a BamHI recognition site and a 6× His tag. The PCR product was digested with EcoRI and BamHI and ligated into a similarly cut pTRC99a vector and transformed into competent cells. Expression plasmid constructs were confirmed by sequencing. Amino acid substitution constructs were generated via quick-change PCR, with a WT template. Protein was expressed and harvested in a large-scale fermenter format from C600 containing expression plasmids, grown to logarithmic phase, and induced with IPTG for ~1 h. The culture was subsequently harvested, and the periplasm isolated generally as described previously . Microarray assays were performed at 5 and 50 µg/mL on the v5.4 microarray.RBD from ACICU and Abp1DRBD from UPAB1, each in 20 mM MES 5.8 + 50 mM NaCl, were concentrated to 9.218 and 4.690 mg/mL, respectively. In a 1:1 drop, Abp2DRBD from ACICU was crystallized over 0.1 M LiSO4, 0.1 M Na citrate, and 15% ethanol. Crystals took approximately 1 wk to appear and formed long, narrow rods. Crystals were looped and cryoprotected in 0.1 M lithium sulfate, 0.1 M Na citrate pH 5.5, 15% ethanol, and 20% glycerol. In a 1:1 drop, Abp1DRBD crystallized over 0.8 M AmSO4 and 0.025 M citric acid pH 3.56. Crystals took approximately 1 wk to appear and formed trapezoidal-prism-like shapes. Crystals were looped and cryoprotected in 0.8 AmSO4, 0.025 M citric acid, and 20% glycerol. Abp2DRBD R86E was crystallized at 8.84 mg/mL in 20 mM MES 5.79 + 50 mM NaCl over a well solution of 0.2 M AmSO4, 20% Isopropanol, 16% PEG 4000, and 0.1 M HEPES 7.5. Crystals were looped and cryoprotected in ~0.18 M AmSO4, ~18% isopropanol, ~0.9 M HEPES 7.5, ~15% PEG 4000, and ~18% glycerol. Data were collected at ALS 4.2.2 macromolecular crystallography beamline. The dataset was solved by molecular replacement and refinement performed in Phenix, using MrkD (3U4K) for Abp2D, Abp2D (8DEZ) for R86E, and a trimmed model of Abp2D (8DEZ) for Abp1D.Abp2D2SO4. Wells were read at 450 nm.Ligand glycoprotein was added to either HBX4 (Immulon) or Microlon600 (Greiner) plates and allowed to dry overnight onto the well bottom. Commerically available human fibrinogen and human  Collagen IV were used. The wells were then blocked for ≥ 4 h to overnight at 4 °C with a blocking buffer of 1× PBS + 2% BSA + 0.05% Tween-20. Protein was added and allowed to incubate with adhered ligand overnight at 4 °C diluted in a blocking buffer. Wells were then washed 3× with wash buffer of 1× PBS + 0.05% Tween-20. Streptavidin-HRP was added at 1:1,000 dilution in a blocking buffer and incubated for ~1 h at 4 °C. Wells were washed 2× with wash buffer and then 1× with 1× PBS. Wells were developed with 1:1 tetramethylbenzidine development reagent and quenched with ~1 M HRBD protein constructs were used at 2.07 µM. The assay was conducted by the following: baseline in blocking buffer was collected for 60 s (seconds), loading of the biotinylated BSM (in 1× PBS) for 300 s, baseline in blocking buffer collected for 60 s, association with protein collected for 300 s, and dissociation in blocking buffer for 300 s. Data were collected on the Octet Data Acquisition 9 software. Baseline data were subtracted from the protein sample curves in GraphPad Prism to generate the data.BLI was conducted on an Octet Red 96-well unit. Streptavidin pins were soaked for at least 10 min in a blocking buffer of 1× PBS + 2% BSA + 0.05% Tween-20. The assay was conducted with all analytes and ligands diluted in a blocking buffer. Abp2DPurified samples (at 50 µg/mL) of the RBD of Abp2D WT, R86E, and R86A were prepared in 20 mM phosphate pH 7. Then, 150 µL of sample in a Starna Cell 16.100F-Q-10/Z15 or similar cuvette was used. Three sample curves and three buffer curves were collected for each sample. The buffer curves were subtracted from the sample data. The data were collected on a Chirascan spectrometer unit and data were analyzed using Prism GraphPad software.RBD WT, R86E, and R86A were prepared in 20 mM MES 5.79 + 50 mM NaCl. The Superdex 200 sizing column was brought to equilibrium with 20 mM MES 5.79 + 50 mM NaCl. The samples were manually injected, and the flow rate was 0.5 mL/min. All samples were conducted in sequence on the same day.Purified samples of Abp2D4 overnight at 4 °C, then spun down to pellet the precipitated protein. That protein was resuspended in ~30 mL of 1× PBS 7.4 and dialyzed against ~500 mL of 1× PBS 7.4. The product was then spun down again to remove the remaining undissolved precipitate and passed through a 0.22-µm filter to preserve the product.Urine protein was isolated as described in Conover et al. 2016  and collAppendix 01 (PDF)Click here for additional data file.


A. pseudoglehnii (APE) against influenza virus infection. Two chlorophyll derivatives, ethyl pheophorbides a and b, isolated as active components of APE, exerted virucidal effects with no evident cytotoxicity. These compounds were effective only under conditions of direct incubation with the virus, and exerted no effects on the influenza A virus (IAV) surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Interestingly, virucidal activities of ethyl pheophorbides a and b were observed against enveloped but not non-enveloped viruses, suggesting that these compounds act by affecting the integrity of the viral membrane and reducing infectivity.Screening of the antiviral and virucidal activities of ethanol extracts from plants endemic to the Republic of Korea revealed the inhibitory activity of a 70% ethanol extract of the whole plant of  Influenza viruses belong to the Orthomyxoviridae family and are divided into A, B, and C types . Among tInfluenza virus infects approximately 5 million patients, resulting in 250,000–500,000 deaths each year . Over thNumerous antiviral drugs have been developed against influenza virus that target the ion channel (M2), NA, RNA polymerase, and inosine 5′ monophosphate (IMP) dehydrogenase proteins. Among these, only antiviral agents targeting M2 and NA have been approved by the FDA ,15. AmanA. pseudoglehnii (Compositae), a perennial plant native to Ulleung Island, is an endemic species to the Republic of Korea . Co. Con-hex2 was also isolated as a green oil. Data from LC-ESI-MS revealed an [M + 1]+ peak at m/z 621.4 , 9.68 , 10.31 , and 11.17  in the low-field region , 6.38 , and 8.02 ], two ethyl groups , and three methyl signals  were observed. 1H and 13C NMR spectra of 2 were very similar to those of 1  of 0.086 ± 0.072 µg/mL and 0.094 ± 0.058 µg/mL for IAV  and the NA inhibition assay was performed A. ZanamiNext, to ascertain whether the two ethyl pheophorbide compounds inhibit HA, two assays using chicken red blood cells (cRBC) were employed. HA is a dimer composed of HA1 and HA2. HA1 contains a domain that binds sialic acid of cells and HA2 interacts with the cell membrane via structural rearrangement induced by low pH during fusion of the viral and cell membranes . First, 50 values of ethyl pheophorbide a against HCV, HCMV and JEV were 3.04 ± 1.69 µg/mL, 0.45 ± 0.26 µg/mL, and 8.66 ± 1.44 ng/mL, respectively. The IC50 values of ethyl pheophorbide b against HCV, HCMV and JEV were 2.19 ± 1.12 µg/mL, 0.46 ± 0.30 µg/mL, and 10.17 ± 1.79 ng/mL, respectively. Ethyl pheophorbides a and b clearly inhibited all enveloped viruses but had no effect against non-enveloped adenovirus , human cytomegalovirus (HCMV), Japanese encephalitis virus (JEV), and non-enveloped adenovirus were treated with the compounds. At 1 h after treatment, viral infectivity was determined using plaque assays for HCMV, JEV and adenovirus, or immunofluorescence assay for HCV. Both ethyl pheophorbide compounds significantly reduced replication of HCV, JEV and HCMV A–C. The enovirus D. Based A. pseudoglehnii is an edible plant endemic to the Republic of Korea with low reported toxicity and multiple beneficial pharmacological properties. In this study, pre-treatment of IAV with APE induced a virucidal effect and the chlorophyll derivatives ethyl pheophorbides a and b were isolated as bioactive chemical constituents.Chlorophyll is responsible for absorbing light during photosynthesis in plants. However, excessive photosynthesis could function as a photosensitizer and cause damage to cells. Therefore, synthesis and degradation of chlorophyll is an important process in preventing cytotoxicity, and various intermediates are generated during chlorophyll degradation . Ethyl pChemical compounds isolated from plant extracts with virucidal activity against enveloped viruses have been previously reported. For instance, curcumin inhibits the activity of enveloped viruses by damaging the viral membrane structure, leading to increased membrane permeability. Upon insertion of curcumin into the phospholipid bilayer of the membrane, its phenol ring interacts with the hydrogen bond site of the membrane and induces structural changes . Ethyl p50 values of ethyl pheophorbide a against IAV and IBV were 0.085 and 1.02 µg/mL, respectively, and antiviral activity against IBV was relatively lower. The reasons underlying the distinct virucidal activities of ethyl pheophorbides a and b against different enveloped viruses require further investigation. Interestingly, however, examination of the anti-influenza virus activity of commercially purchased pheophorbide a devoid of an ethyl group revealed similar IC50 values against IAV and IBV (data not shown). We could not effectively compare the virucidal activities of commercially synthesized compounds with those of isolates from plant extracts. To establish whether the ethyl group serves as a critical moiety, it is necessary to synthesize these compounds to the same levels of purity and compare their virucidal effects.In this study, IC3–104 PFU/mL with buffer  and injected into the allantoic cavity of eggs. After culturing in an incubator (48 h for IAV and 72 h for IBV), allantoic fluid was obtained and the virus titer was determined using the plaque assay. MDCK cells were inoculated with influenza virus, with addition of infection medium  after incubation at 37 °C for 1 h. Baby hamster kidney cells (BHK21) were purchased from the Korean Cell Line Bank  and Japanese encephalitis virus  obtained from the National Culture Collection for Pathogens . BHK21 and 293A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) supplemented with 10% FBS and 1X P/S. BHK21 cells were inoculated with JEV at 37 °C for 1 h, followed by the addition of DMEM containing 2% FBS and 1X P/S. After 48 h, JEV was obtained from the supernatant. Recombinant adenovirus expressing GFP was generated by inserting the GFP gene into an adenoviral vector. The GFP gene was amplified using pEF-GFP vector (Addgene #11154) as a template. The primer sequences used were 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTATGGTGAGCAAGGGCGAGGAGCTGT-3′ (forward) and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTACTTGTACAGCTCGTCCATGCCGA-3′ (reverse), and PCR was performed using 5X HOT FIREPol® Blend Master Mix . The amplified GFP gene was cloned using the Gateway system ), followed by transfection into 293A cells, as described previously +, 1H and 13C NMR data: Green oil; LC-ESI-MS (positive mode) Viruses and cells were subjected to three different treatments. In the first method, IAV was treated with the extract for 1 h at room temperature and Madin-Darby canine kidney (MDCK) cells were infected with the mixture at a multiplicity of infection (MOI) of 0.01. At 1 h after infection, the mixture was removed (pre-treatment). In the second method, IAV was treated with the extract at room temperature and MDCK cells were immediately infected with the mixture at a MOI of 0.01. At 1 h after infection, the mixture was removed (adsorption). In the third group, MDCK cells were infected with IAV at a MOI of 0.01, which was removed after 1 h. Cells were subsequently treated with the extract (post-infection). At 24 h after infection, cells were harvested to extract total RNA, and viral RNA transcript levels were evaluated via qRT-PCR.50) values were calculated using GraphPad Prism 7 .IAV and IBV were treated with different concentrations of ethyl pheophorbide a or b and incubated at room temperature for 1 h. MDCK cells were infected with the mixtures and the supernatant was obtained at 48 h after infection. MDCK cells were infected with serially diluted supernatant, and after 1 h, incubated with a 1:1 mixture of 2X DMEM  and 2% agarose containing 1 µg/mL TPCK-trypsin. Adenovirus, HCMV and JEV were treated with different concentrations of ethyl pheophorbide a or b (0–10 µg/mL) and the mixtures were incubated at room temperature for 1 h. BHK21 cells were infected with serially diluted JEV for 1 h, followed by incubation in medium  containing 0.5% agarose. At 48 h after incubation, cells were fixed with 10% formaldehyde at room temperature and stained with 0.3% crystal violet. 293A cells were infected with serially diluted adenovirs for 6 h, followed by incubation in medium  containing 0.4% agarose. At 48 h after infection, medium containing 0.4% agarose was added. At 4 to 6 days after infection, Thiazolyl Blue Tetrazolium Bromide  solution was added to the medium at a concentration of 0.5 mg/mL and plaques were observed 1 h after incubation at 37 °C. The plaque assay for HCMV has been described in a previous report . The numCells were fixed with a 1:1 mixture of methanol and acetone for 10 min at −20 °C, washed with 1X PBS, and blocked with 1% BSA at room temperature for 1 h. Subsequently, cells were incubated with primary antibody overnight at 4 °C, followed by secondary antibody at room temperature for 1 h. After staining of cells with 4,6-diamidino-2-phenylindole (DAPI) , fluorescence was examined under a Nikon TS100-F fluorescence microscope  equipped with a digital camera. Fluorescence images were analyzed using Nikon NIS-Elements microscope imaging software. An antibody against HCV core protein was purchased from Anogen . Alexa 488-labeled secondary antibody was purchased from Thermo Fisher Scientific .MDCK cells were treated with different concentrations of ethyl pheophorbide a or b (0–10 µg/mL) for 48 h and subsequently incubated with 0.5 mg/mL MTT solution at 37 °C. After a 4 h period, tetrazolium crystals were dissolved with DMSO and absorbance was measured at 560 nm.Cells were fractionated and transferred to nitrocellulose membrane as described previously . Antibod® EvaGreen qPCR mix Plus (Solis BioDyne) and specific primers. The primer sequences used for amplification were as follows: IAV M1, 5′-GACCAATCCTGTCACCTCTGAC-3′ (forward) and 5′-AGGGCATTTTGGACAAACCGTCTA-3′ (reverse); Canis lupus familiaris glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CCAGGGCTGCTTTTAACTCTGG-3′ (forward) and 5′-ACTGTGCCGTGGAATTTGCCG-3′ (reverse).To determine viral transcripts levels, total RNA was extracted using a HiGene™ Total RNA Prep Kit . RNA was reverse-transcribed using a TOPscript™ cDNA synthesis kit  to synthesize complementary DNA (cDNA), which was amplified using the StepOnePlus Real-Time PCR system  with HOT FIREPol5 PFU using 1X Assay buffer (33.3 mM 2-(N-morpholino) ethanesulfonic acid (MES), 4 mM CaCl2, pH 6.5) containing 0.1% NP-40 and treated with ethyl pheophorbide a (1 µg/mL), ethyl pheophorbide b (1 µg/mL) or zanamivir (1 µM) at room temperature for 45 min. The mixture was subsequently treated with 0.3 mM 2′-(4-Methylumbelliferyl)-α-d-N-acetylneuraminic acid , a substrate of NA, and incubated at 37 °C for 1 h. After the reaction was terminated with stop solution , fluorescence was measured at excitation and emission wavelengths of 355 nm and 460 nm, respectively, using an EnSpire fluorescence analyzer .IAV was diluted to 8 × 10IAV was serially diluted using 1X PBS. After incubation with chicken red blood cells  for 1 h at 4 °C, aggregation was confirmed. The lowest virus titer causing aggregation was 1 HA unit (HAU). For the experiment, 4 HAU was used. IAV was treated with DMSO, ethyl pheophorbide a (1 µg/mL), or ethyl pheophorbide b (1 µg/mL) with shaking at room temperature for 45 min. Following the addition of cRBCs (0.5%), aggregation was confirmed after 1 h at 4 °C.8 PFU/mL) was treated with DMSO, ethyl pheophorbide a (1 µg/mL) or ethyl pheophorbide b (1 µg/mL) at room temperature for 1 h. Mixtures were incubated with cRBCs (2%) at 37 °C for 30 min, followed by sodium acetate–acetic acid  at 37 °C for 30 min. After centrifugation at 800× g for 5 min, absorbance of the supernatant was measured at 560 nm.IAV (10


Then, the samples were investigated as OER and HER bifunctional electrocatalysts in alkaline media. Compared with other catalysts, LaCo0.94Pt0.06Oδ3− had good stability and presented more activity at a lower overpotential of 454 mV (at 10 mA cm−2), a lower Tafel slope value of 86 mV dec−1 and a higher mass activity of 44.4 A g−1 for OER; it displayed a lower overpotential of 294 mV (at −10 mA cm−2), a lower Tafel slope value of 148 mV dec−1 and a higher mass activity of −34.5 A g−1 for HER. The improved performance might depend on a larger ECSA, faster charge transfer rate and higher ratio of the highly oxidative oxygen species (O22−/O−). Furthermore, the eg orbital filling of Co approaching 1.2 in the B site might play a leading role.In this study, a series of perovskite oxides LaCo x1−PtxOδ3− catalysts, LaCo0.94Pt0.06Oδ3− proved best for catalyzing OER/HER, with η = 454/294 mV, which might be attributed to LCP6 having the eg orbital filling of Co closest to 1.2.Among the perovskite LaCo Among them, non-noble metal perovskite oxides, with plentiful reserves, low cost, excellent redox performance and stable structure, have gradually become the topic of interest for researchers all over the world.12With the rapid depletion of fossil fuels and the resulting environmental problems, researchers are working hard to search for sustainable alternative energy sources and energy storage and changeover methods.3, where the larger cation A is commonly a lanthanide, rare-earth metal or alkaline earth metal which has 12-fold coordination with O2− anions and the smaller cation B is a transition metal in corner-sharing octahedra with O2− anions. The composition and structure lead to easily doping elements or making deficiencies in A or B sites, which greatly affect the valence and structure of the transition metals, further improving the OER and HER performances of the materials.13–17 Shao and Haile confirmed that the doped perovskite Ba0.5Sr0.5Co0.8Fe0.2Oδ3− (BSCF) showed excellent OER activity in alkaline conditions, even exceeding that of IrO2.13 Afterward, BSCF and Pr0.5BSCF were also proved to be good electrocatalysts for HER.14 In addition, oxygen vacancies caused by the changing valence of the transition metals in perovskite catalysts such as BaTiOδ3−,18 SrCoOδ3−,19 and Lax1−SrxCoOδ3−  perovskite  were prepared via the citric acid–ethylenediaminetetraacetic acid (CA–EDTA) complexing sol–gel method. Among these doped perovskite oxides, LaCo0.94Pt0.06Oδ3− (LCP6) shows the highest OER and HER activities, demonstrating that LaCo0.94Pt0.06Oδ3− (LCP6) could be a promising candidate as an OER and HER bifunctional electrocatalyst in an alkaline condition.In the present paper, we report our findings on enhancing OER and HER activities by doping minor precious metal Pt into perovskite oxide LaCoO2.2.1.3)3·6H2O , Co(NO3)2·6H2O , KOH , citric acid , ethylenediaminetetraacetic acid , and ammonia hydroxide  were purchased from Kermel. Pt(NO3)2  was bought from Aladdin. Nafion solution  was bought from Sigma-Aldrich. Carbon black  was purchased from Cabot, USA. All chemicals were used directly without further purification.La3·6H2O, Co(NO3)2·6H2O and Pt(NO3)2 were dissolved in deionized water to form a clarified purple solution, with CA and EDTA added to the above solution in the molar ratio of total metal ions : CA : EDTA = 1 : 2 : 1. Then 25% ammonia hydroxide was dropped into the stirring solution until pH = 6.0–7.0. The mixture was magnetically stirred in water at 80 °C to form a transparent purple wet gel, followed by drying at 120 °C for 24 h. The obtained xerogel was calcined at 400 °C for 3 h to remove organic impurities and then at 850 °C for 5 h to form the final perovskite structure.A series of perovskite oxides LaCo2.3.λ = 1.5406 Å) in the range of 20–80° with a scan rate of 6° min−1. Scanning electron microscopy (SEM) was carried out on a JEOL JSM-7800F thermal field emission scanning electron microscope with an accelerating voltage of 15.0 kV. Transmission electron microscopy (TEM) was performed on a JEOL JEM-2100F field emission transmission electron microscope with a 200.0 kV accelerating voltage. X-ray photoelectron spectra (XPS) were examined by a Kratos-Axis Ultra DLD photoelectron spectrometer equipped with an Al Kα (hν = 1486.6 eV) X-ray source.Powder X-ray diffraction (XRD) was recorded on a D8 Advance diffractometer employing Cu Kα radiation  pre-treated in nitric acid to clean the surface. The active carbon fiber paper coated with ink was air dried in the surrounding environment for 2 h. The catalyst mass loading on the carbon fiber paper was nearly 0.3 mg cm2.5.2 electrode in saturated KCl solution as the reference electrode and a Pt wire for OER and a graphite rod for HER as the counter electrodes. The supporting electrolyte was 0.1 M aqueous KOH solution. The polarization curves were recorded using linear sweep voltammetry (LSV) under potential windows from 0 to 1.0 V and −1.8 to −0.9 V (vs. Hg/HgCl2) for OER and HER, respectively, with a scan rate of 5 mV s−1. The overall water splitting was tested by a two-electrode electrochemical cell in 0.1 M aqueous KOH solution at a scan rate of 5 mV cm−2 with the mass loading of catalyst at 0.6 mg cm−2. The double layer capacitance (Cdl) was acquired via the cyclic voltammetry (CV) method with different scan rates of 5, 20, 40, 60 and 80 mV s−1 and a potential window from 0.3 to 0.4 V (vs. Hg/HgCl2), in which no faradaic processes happened. Slopes of the curves between half of the anodic and cathodic current difference centered at 0.35 V (vs. Hg/HgCl2) and scan rates represented the double layer capacitance.5 Electrochemical impedance spectroscopy (EIS) was carried out at 0.7 V and −1.5 V (vs. Hg/HgCl2) for OER and HER, respectively, within a frequency range of 0.1 to 105 Hz. The overall water splitting test was carried out in a two-electrode system.Electrochemical measurements were performed in a conventional three-electrode electrochemical cell using a Hg/HgCl3.3.1.d = 0.271 nm and 0.270 nm for LCP6 and LC, respectively, corresponding to the (1̄10) crystal plane of the perovskite structure (JCPDS no. 84-0848). Furthermore, no lattice fringes of platinum or platinum oxides were found, which corresponded to the XRD patterns. The above results indicated the successful synthesis of doped perovskite.The particulate morphologies of the samples were investigated by SEM . All of 2−), ∼529.6 eV (highly oxidative oxygen species for O22−/O−), ∼531.3 eV (hydroxyl groups or surface adsorbed oxygen for –OH or O2) and 532.9 eV (adsorbed molecular water for H2O).5,14 The O 1s XPS peak deconvolution results of all prepared catalysts are shown in 3/2 and Co 2p1/2 at ∼780.1 and ∼795.2 eV belong to Co3+, whereas the peaks of Co 2p3/2 and Co 2p1/2 at ∼782.0 and ∼797.3 eV belong to Co2+.33 The ratios of Co3+ to Co2+ based on peak area intensity are listed in 7/2 and 4f5/2 are shown in Fig. S3c in the ESI.7/2 and 4f5/2 could be deconvoluted to two peaks assigned to Pt4+ and Pt2+.34,35The species on sample surfaces were examined through high-resolution X-ray photoelectron spectroscopy (XPS). As shown in 3.2.−1 in 0.1 M KOH aqueous solutions at room temperature. Compared with other samples, the OER polarization curve of LCP6 exhibited the lowest onset potential of ≈1.59 V vs. RHE, implying a better start of LCP6 for catalyzing OER. Because of the connection to solar-to-fuels conversion, the overpotential (η) at the current density of 10 mA cm−2 is an important parameter for solar cells.36 Obviously, the LCP6 showed the lowest overpotential of 454 mV at the current density of 10 mA cm−2, followed by LCP4 (474 mV), LCP2 (488 mV), LCP8 (507 mV) and LC (541 mV), suggesting the best OER activity of LCP6. In addition, the Tafel slope (−1) was the smallest among all the samples, presenting the enhanced OER activity of LCP6 over the others. In addition to activity, stability is equally important for electrocatalysts. LCP6, which has the lowest overpotential, was tested by cyclic voltammetry for 500 cycles at a scan rate of 50 mV s−1 and by chronopotentiometry at the current density of 10 mV cm−2 continuously for 20 h to evaluate stability. After cycling, the linear sweep volt–ampere curve only showed a minor voltage increase of about 20 mV at the current density of 10 mA cm−2 (2 (346 mV in 2. vs. RHE). LCP6 had the highest value of 44.4 A g−1, 4-times higher than that of LC (9.3 A g−1), demonstrating that the doped perovskite oxide LCP6 has promising OER activity, while the mass activities of LCP2, LCP4 and LCP8 were 23.6 A g−1, 31.0 A g−1, and 17.3 A g−1, respectively. Next, the turnover frequencies (TOFmass) of the catalysts were calculated to understand the improved activity. Based on common sense, only the B-site metals in the octahedral sites of perovskite material were used as the active sites.37 Formula (2) in the ESImass. As can be seen in mass value of 2.92 × 10−2 s−1, 4-times higher than that of LC (0.59 × 10−2 s−1), confirming the excellent OER activity of LCP6 as an electrocatalyst. The TOFmass values of LCP2, LCP4 and LCP8 were 1.52 × 10−2 s−1, 2.02 × 10−2 s−1, and 1.15 × 10−2 s−1 respectively.As shown in el slope  for LCP6 mA cm−2 . The chr mA cm−2  showed tvs. RHE, has the best performance to catalyze HER, followed in sequence by LCP4, LCP8, LCP2 and LC. At the current density of −10 mA cm−2, the required overpotential for LCP6 was only 294 mV .5,16 The overall response begins with the adsorption of OH− and finishes with the desorption of the OH−. In contrast, HER proceeds through two steps.31,40 The first step is a Volmer reaction (H2O + e− → Hads + OH−), which is the reduction of water to H atoms and OH− species, then the intermediates are adsorbed onto the surface of the catalysts. The second step is either a Heyrovsky reaction (H2O + Hads + e− → H2 + OH−) or Tafel reaction (Hads + Hads → H2), where the former is an electrochemical desorption step and the latter is a chemical desorption one. The Tafel slopes are 120, 40 and 30 mV dec−1 when the HER is determined by the Volmer, Heyrovsky and Tafel processes, respectively.31 As shown in −1, indicating that the Volmer reaction might be the determining step of HER catalysis.From the results presented above, LCP6 has proven to be a promising OER and HER bifunctional electrocatalyst. As is well-known, the widely accepted OER mechanism on perovskite oxides in alkaline media is the proposed adsorbate evolution mechanism (AEM).33 However, in this work, the crystalline phase may be a secondary factor, because LCP6 showed the best electrocatalytic performance.The XRD patterns showed that the prepared perovskite particles exhibit a crystalline phase transition from tripartite phase to cubic phase with the increase of platinum doping amount. A previous paper proposed that perovskite oxides with a cubic phase may present enhanced electrocatalytic performance.via cyclic voltammograms  was much larger than those of LCP4 (0.93 mF cm−2), LCP2 (0.74 mF cm−2), LCP8 (0.68 mF cm−2) and LC (0.65 mF cm−2). Additionally, double layer capacitance is frequently used to reflect the electrochemical active surface area (ECSA) of the catalysts. Thus, the above result suggests that LCP6 has a larger ECSA, indicating a relatively high amount of active sites involved in OER and HER.41 The charge transfer resistance during OER and HER was evaluated by the electrochemical impedance spectra (EIS). The Nyquist plots  of all the catalysts st plots  implied 22−/O−) compared with the remaining samples. Previous studies have shown that highly oxidative oxygen species (O22−/O−) are beneficial to both OER and HER.21,25,30,42 From 3+ to Co2+ of each sample. Generally, the spin state of Co3+ is intermediate-spin  and Co2+ is high-spin .24 Thus, the eg orbital filling of Co was calculated to be 1.29 in LCP6.22 Compared with the other samples, the electronic number 1.29 of LCP6 is closest to 1.2. According to the eg orbital filling descriptor theory,21,25 LCP6 should have the best electrocatalytic performance for OER and HER. As shown in Fig. S3c,5/2 peak and 4f7/2 peak of LCP6 have shifted to higher binding energy than others, implying the presence of a lower oxidation state of Pt in LCP6. After analysis, the ratio of Pt4+/Pt2+ and the average valence of Pt in samples are shown in − is an important step.30,33 Also, the higher oxidation state in the B site is beneficial to the adsorption of OH−, which could hasten the initial response of OER and HER. Therefore, LCP6 possessed an enhanced activity.To further understand the differences in electrochemical performance, the results of XPS were analysed in depth. From 4.x1−PtxOδ3−  were prepared by the citric acid–ethylenediaminetetraacetic acid (CA–EDTA) complexing sol–gel method and investigated as OER and HER bifunctional electrocatalysts in alkaline media. The LaCo0.94Pt0.06Oδ3− catalyst exhibited the best activity and stability in catalyzing the oxygen and hydrogen evolution reactions with overpotentials of 454 mV and 294 mV (10 mA cm−2), respectively, compared with other catalysts. The enhanced performance might be attributed to a larger ECSA, faster charge transfer rate, higher ratio of the highly oxidative oxygen species (O22−/O−) and the eg orbital filling of Co approaching 1.2 in the B site. In summary, the LaCo0.94Pt0.06Oδ3− perovskite is an efficient OER and HER bifunctional electrocatalyst.In summary, a series of perovskite oxides LaCoThere are no conflicts of interest to declare.RA-009-C9RA05491J-s001


The microhardness of Zn coating was significantly decreased from 70.8 ± 5.6 HV0.025 to 43.9 ± 12.5 HV0.025, with the heat temperature from the ambient temperature to 350 °C, and was slightly decreased with the heat time at 250 °C. Although the tensile strength of Zn coating was slightly increased by heat treatment, with the highest value of 40.9 ± 3.9 MPa at 150 °C for 2 h, excessive heat temperature and time were detrimental to the tensile strength, with the lowest value of 6.6 ± 1.6 MPa at 350 °C for 2 h. The heat temperature and heat time presented limited effects on the corrosion current and corrosion ratio of the Zn coatings, and Zn coatings before and after heat treatment effectively hindered the simulated body fluid from penetrating into the substrate. The corrosion behavior of Zn coatings was discussed in terms of corrosion products and microstructures after immersion.The influence of post-process heat treatment on cold-sprayed Zn coatings on the Mg alloy substrate was investigated at different temperatures  and times . Phase, microstructure, microhardness, and tensile strength of Zn coatings were analyzed before and after heat treatment. Corrosion properties of Zn coatings after heat treatment were investigated in simulated body fluid by using potentiodynamic polarization and immersion testing. Results show that although the heat treatment presented little effect on phase compositions of Zn coatings, the full width at half maxima of the Zn phase decreased with the heat temperature and time. Zn coatings presented comparable microstructures before and after heat treatment in addition to the inter-diffusion layers, and the inter-diffusion layer was dependent on the heat temperature and time. Both the thickness and the microhardness of inter-diffusion layers were increased with the heat temperature and time, with the largest thickness of 704.1 ± 32.4 μm and the largest microhardness of 323.7 ± 104.1 HV Magnesium alloys present excellent biodegradability and suitable mechanical properties to natural bones, which have been considered novel potential implants . The curMg, Zn, and Fe are considered biodegradable metals, and Zn and Fe present higher corrosion resistance than Mg ,7. Zinc Cold spray is a novel and environmentally-friendly technology, which can produce metal, ceramic, cermets, and organic coatings on the surface of different materials ,10. UnliCold-sprayed metal coatings are formed by plastic deformations of metal particles due to the high-velocity impact. The inter-particle bonding interface and the coating/substrate interface are weak and mechanically interlocked, which results in overall poor ductility with brittle fractures commonly occurring ,17. To aThis work aimed to investigate the influence of heat temperature and time on microstructure, mechanical, and anti-corrosion properties of cold-sprayed Zn coatings on Mg alloy substrates. The formation of the intermetallic layer at the Zn coating/Mg alloy substrate interface and corrosion behaviors of Zn coatings after heat treatment were discussed.2O3 grit (200 μm) for 5 min and then cleaned in ethanol. The Zn coatings were deposited on AZ31B substrates by a cold-spraying system assembled by Xi’an Jiaotong University. Both the primary and carrier gases were made of nitrogen. The primary gas pressure and temperature were 2.0 ± 0.1 MPa and 260 ± 20 °C, respectively. The spray distance was 20 mm, and the transverse speed of the torch was 50 mm/s. After coating deposition, zinc coatings were put into a silica tube. Then the tube was sealed in a vacuum and heated up to 150 °C for 2 h, 250 °C for 2, 8, 16 h, and 350 °C for 2 h.Spherical zinc powder  was used as the original material of Zn coatings (as shown in reference ). SpheriKα radiation of 1.5418 Å, an operating voltage of 35 kV, an operating current of 35 mA, a 2θ range of 20–90°, and a scan rate of 0.1°/s. Cross-sectional morphologies of different Zn coatings were investigated by scanning electron microscopy . The chemical elements were examined by an energy-dispersive spectrometry (EDS) module integrated within SEM.The phase structures of different Zn coatings were identified by X-ray diffraction  at CuMicrohardness of Zn coatings before and after heating was obtained by Vickers microhardness tester  at the load of 25 gf and the dwell time of 20 s. Tensile strengths of Zn coatings before and after heating were measured using a standard tensile test method (ASTM-C633) designed for thermal sprayed coatings. The end surface of the cylindrical sample with Zn coatings was boned on grit-blasted facings of the loading fixtures, being the same size and shape as the sample, using a special adhesive glue  with a tensile strength of about 70 MPa. The assembly was held perpendicularly and placed in an oven at 100 °C for 2 h. After the bonding glue was cured and hardened, the assembly was loaded into the machine  at a head speed of 1 mm/min. The bond strength of coatings was evaluated by the average value of at least three sample test results. After tensile testing, the surface and cross-sectional microstructures of fractured Zn coatings were examined by SEM.+, 5.0 mM K+, 1.5 mM Mg2+, 2.5 mM Ca2+, 103.0 mM Cl−, 10.0 m HCO3−, 1.0 mM HPO42−, and 0.5 mM SO42−. Before testing, the Zn coatings before and after heating were immersed in SBF for 1, 7, and 14 days. Polarization curves were then carried out on an electrochemical workstation  from voltages of −2 V to 1 V (vs. SCE) at a scan rate of 1 mV/s. At least three tests were carried out for each sample. After immersion for 14 days, the surface and cross-sectional microstructures, and phase constitutions of Zn coatings were examined by SEM and XRD, respectively.The potentiodynamic polarization tests were carried out in simulated body fluid (SBF) at the temperature of 37 ± 1 °C . The SBF contained 142.0 mM Na21Zn25, Mg2Zn3, MgZn2, and Mg2Zn11. The Mg/Zn atom ratios at different regions along the inter-diffusion layers were presented in 2Zn11 phase, the middle region was the MgZn2 and Mg2Zn3 phase, and a very thin transition layer was closed to Mg substrate. According to the Mg-Zn phase diagram [There were obvious inter-diffusion layers between Zn coatings and the Mg alloy substrate. The formation of a diffusion layer is reported for a Mg alloy brazed joint using Zn-based filler metal ,31,32,33 diagram , the eut21Zn25, Mg2Zn3, MgZn2, and Mg2Zn11) exhibit significantly higher microhardness compared with the pure Zn and Mg [n and Mg . It is an and Mg . The incn and Mg . It can The fracture surfaces of the tensile samples were investigated as shown in 2 and Zn5(OH)8Cl2·H2O existed on all the surfaces of Zn coatings, as shown in 2, and Zn5(OH)8Cl2·H2O could be formed by the Zn dissolution and chemical reaction between Zn2+, Cl-, and OH- [2that existed on the Zn coating was heated at 350 °C for 2 h, which could be formed by the corrosion of the Mg alloy substrate. and OH- ,40. In a0.025, with the heat temperature from the ambient temperature to 350 °C, and was slightly decreased from 65.1 ± 12.7 to 58.6 ± 4.7 HV0.025, with the heat time from 250 °C for 2 h to 250 °C for 16 h. The inter-diffusion layer presented the lowest microhardness of 125.2 ± 24.6 HV0.025 at 150 °C for 2 h and the highest microhardness of 323.7 ± 104.1 HV0.025 at 350 °C for 2 h. The tensile strength of Zn coatings after heating was relative to the inter-diffusion layers and was decreased with the heat temperature and time. The highest tensile strength was 40.9 ± 3.9 MPa at 150 °C for 2 h, and the lowest thickness was 6.6 ± 1.6 MPa at 350 °C for 2 h. Zn coatings before and after heat treatment showed lower corrosion currents and lower corrosion rates than the Mg alloy substrate, but the effect of heat treatment was limited. The lowest corrosion current and corrosion rate were 0.62 ± 0.23 mA/cm2 and 7.3 ± 3.5 mm/a for the Zn coating heated at 150 °C for 2 h after immersion for 14 days, respectively. Except for the Zn coating heated at 350 °C for 2 h presenting cracks at the coating/substrate interface, Zn coatings before and after heat treatment effectively hindered the simulated body fluid penetrating into the substrate with porous corrosion products on the Zn coating surfaces after immersion for 14 days.The influence of post-process heat treatment on microstructure, mechanical, and anti-corrosion properties of cold-sprayed Zn coatings was systematically investigated. The Zn coatings presented similar phase compositions of Zn and ZnO before and after heat treatment, but the full width at half maxima of the Zn phase was decreased. Except for the heat condition of 350 °C for 2 h, Zn coatings presented comparable microstructures after heat treatments. Inter-diffusion layers with different thicknesses were formed at the coating/substrate interfaces. The lowest thickness was 2.2 ± 0.4 μm at 150 °C for 2 h, and the largest thickness was 704.1 ± 32.4 μm at 350 °C for 2 h. The Zn coatings, the substrate, and the inter-diffusion layers exhibited different evolutions of microhardness with the heat temperature and time. The microhardness of Zn coatings was significantly decreased from 70.8 ± 5.6 to 43.9 ± 12.5 HV