The atypical chemokine receptor 1 (ACKR1) gene encodes the Duffy blood group antigens in two allelic forms: FY*A (FY*01) and FY*B (FY*02), which define the Fy(a+b-), Fy(a-b+), and Fy(a+b+) phenotypes. FY*BES (FY*02N.01) is a single T to C substitution at nucleotide -67 that prevents the FY*B from being expressed in red blood cells (RBCs). a and Fyb antigens and genotyped for FY*A, FY*B, FY*BSE , and FY*Bweak alleles. We evaluated 250 residents from a Brazilian malarial endemic region (RsMR). All individuals were phenotyped for FyFY*A/FY*B was found in 130/250 (52%) and the FY*A/FY*A in 45/250 (18%). The c.1-67>TC was present, in homozygosity, in 11/250 (4.4%). Among 34 individuals with the Fy(a+b-) and FYA*/FYB* mutations, 4/34 (11.8%) had homozygosity for the c.1-67T>C. One individual presented the Fy(a+b-), FY*A/FY*B, and c.1-67T>C in homozygosis, whereas the other presented the Fy(a+b-), FY*A/FY*A, and c.1-67T>C in heterozygosis. Among the 250 individuals, 209 (83.6%) reported previous malaria infection, and 41 (16.4%) did not. The Fy(a+b+) phenotype was present in 97/250 (38.8%), while the Fy(a-b-) was present in 7/250 (2.8%). The Plasmodium vivax (67.5%). We observed that 102/141 (72.3%) individuals expressing the Fyb antigen had a P. vivax infection, indicating the importance of the Fyb antigen, silenced by a c.1-67T>C mutation in homozygosis, in preventing the P. vivax infection. We showed that the c.1-67T>C mutation in the FY*A did not silence the FY*A expression on RBCs.  We reported a low prevalence of the Fy(a-b-) in persons who had previously been infected with  Malaria is a serious public health issue in Brazil, with roughly 145,000 cases recorded in 2014. The majority of occurrences occur in the Brazilian Amazonia (Amazon biome), a malaria-endemic regionHowever, recent migrations from the Amazonian region or other nations to non-Amazonian regions have resulted in secondary imported epidemics Plasmodium vivax, Plasmodium falciparum, and Plasmodium malaria are the three principal Plasmodium species linked to native human malaria cases in BrazilP. vivaxa and Fyb are antithetical antigens encoded by the co-dominant FY*A (FY*01) and FY*B (FY*02) genes. The minimal nucleotide polymorphism c.125G>A (rs12075) distinguishes these variantsThe atypical chemokine receptor 1 (ACKR1), formerly known as Duffy antigen receptor for chemokines (DARC), is a minor blood group antigen that functions as both a chemokine receptor and a receptor for the malaria parasite bwk, also known as the Fyx antigen, functions as a low-expression Fyb antigen, and there is no known anti-Fybwkbwk phenotype is caused by a single missense mutation in the FY*B gene's coding area, c.265C>T (rs34599082), which results in the amino acid change p.Arg89Cys in gp-Fya antigen has already been found when the FY*A allele expresses the two mutations 265T and 298A.FY*X is a recessive allele of the FY*B (FY*BWK) gene located at the Duffy locus. The gene does not encode the synthesis of a Duffy system-specific antigen. The Fya and anti-Fyb antibodies among Caucasians define the Fy(a+b-), Fy(a+b+), and Fy(a-b+) phenotypes that most of the time represent the genotypes FY*A/FY*A, FY*A/FY*B, and FY*B/FY*B, respectivelyThe anti-Fy,The null phenotype Fy(a-b-), also referred to as "erythrocyte silent" , has been described more commonly in Afro-Americans and Occidental Africans but is uncommon in CaucasiansP. vivax merozoites and functions as bait and scavengers for chemostatic agonistsP. vivax or P. falciparumP. vivax malaria can be discovered in Duffy-positive and-negative or those whose DARC status is unknown-The ACKR1 protein is required for the attachment of P. vivaxThese data support the notion that this parasite is quickly developing, capable of invading erythrocytes via receptors different from those found in Duffy, which might significantly influence the existing distribution of a and Fyb antigens and genotyped for the FY*A, FY*B, FY*BSE, and FY*Bweak alleles, and then the promoter and codifying regions of the FY gene were sequenced. Additionally, we associated the phenotypes and genotypes of the RsMR with prior P. vivax infection.We investigated blood samples from the risk-standardized mortality rate (RsMR) in Brazil in this study. All participants were phenotyped for the Fya and Fyb antigens, genotyped for the FY*A, FY*B, FY*ES, and FY*Bweak alleles, and then sequenced for the FY* promoter and coding areas. The study was authorized by the ethical committee of the Universidade Federal de São Paulo (UNIFESP).The study population consisted of 250 RsMR individuals in the city Presidente Figueiredo, located in the Amazon State, Brazil. This Amazonian city has an annual parasite index (API) of 301.65 malaria cases per 1,000 people, making it a malaria-risk regionFY* alleles using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniqueFY* were sequenced using the ABI Prism®Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer). All blood samples were phenotyped using the gel agglutination technique  and genotyped for the 2). Statistical significance was defined as a P-value < 0.05.The statistical analysis was performed using Pearson’s chi-square test  [97/250 (38%)], The Fy(a-b+) phenotype was found in 7/250 (2.8%) RsER  and 8%) RsER .P. vivax were analyzed in 209 RsER. The frequency of the previous P. vivax infection was significantly different among the various phenotype and genotype combinations (P < 0.001) (FY*A/FY*B and Fy(a-b+)/FY*B/FY*B combinations were found more frequently among individuals who had had previous P. vivax infection compared with those participants who had not been infected . The Fy(a+b-)/FY*A/FY*A and Fy(a-b-)/FY*A/FY*B combinations were more frequent in individuals with no previous history of P. vivax infection . We did not observe participants with the Fy(a-b-)/FY*B/FY*B combination who had had previous P. vivax infection (The comparison of phenotype and genotype combinations and their relation to previous infection by < 0.001) . The Fy(nfection . FY* gene was found in 11/250 (4.4%). Overall, we found a frequency of the c.1-67T>C mutation among the 84/250 (33.6%) RsER individuals. On the other hand, the majority of the RsER [166/250 (66.4%)] did not present the c.1-67T>C mutation ] and genotype (FY*A/FY*B). We found that 30/34 (88.2%) individuals with the Fy(a+b-) phenotype and FY*A/FY*B genotype presented the c.1-67T>C mutation in heterozygosis (W/M) and 4/34 (11.8%) in homozygosis (M/M) , but none of them carried such mutations. Four of 34 (11.8%) individuals presenting the Fy(a+b-) phenotype, the presence of the c.1-67T>C mutation, polymorphism of the FY*A and FY*B alleles, and c.265C>T and c.298G>A mutations. The FY*A/FY*B genotype and c.1-67T>C mutation in homozygosis were analyzed by nucleotide sequencing of the promoter and codifying regions of the FY* gene. In all four participants, we confirmed by DNA sequencing the results previously found using the PCR-RLFP method, i.e., the presence of the c.1-67T>C mutation in homozygosis and not the absence of the c.265C>T and c.298G>A mutations. These individuals had the Fyb antigen, but not the Fya antigen expression, silenced on their RBCs (We investigated the frequency of the c.1-67T>C mutation that causes the silencing of the Fyis (M/M) . All 34 eir RBCs . P. vivax infection and the presence of the c.1-67T>C mutation was investigated among the RsER, showing a discrepancy between the Fy(a+b-) phenotype and FY*A/FY*B genotype. The c.1-67T>C mutation not found (W/W) in 103/141 (73.0%) of previously P. vivax-infected RsER, compared with 38/68 (55.9%) of non-infected RsER. The presence of the c.1-67T>C mutation in heterozygosis (W/M) was detected in 14/141 (9.9%) and 10/68 (14.7%) of the previously infected and non-infected RsER, respectively. All RsER (n = 10) carrying the mutation in homozygosis (M/M) with no history of previous P. vivax infection (P < 0.001) (The correlation of the < 0.001) . P. vivax malaria infection, 13/68 (19.1%) showed the Fy(a+b-) phenotype and the FY*A/FY*B genotype silencing the Fyb expression on their RBCs. In this group of individuals, 10/13 (76.9%) presented the c.1-67T>C mutation in heterozygosis W/M), and 3/13 (23.1%) presented the c.1-67T>C mutation in homozygosis (M/M) (We observed that among 68 RsER with no history of previous is (M/M) . The othP. vivax, 14/141 (9.9%) showed the Fy(a+b-) phenotype and FY*A/FY*B genotype combination, together with the c.1-67T>C mutation in heterozygosis (W/M), while 127/141 (90.1%) did not present the Fy(a+b-) phenotype and FY*A/FY*B genotype combination  with previous P. vivax infection, but 10/20 (50%) had not been infected, suggesting that the c.1-67T>C mutation in heterozygosis did not provide full protection against the P. vivax infection  had not been infected.Among the 141/209 (67.5%) RsER with previous malaria infection by bination . We obsenfection . As descFY*A/FY*B genotype, and c.1-67T>C mutation in homozygosis (M/M) but also one additional participant presenting the Fy(a+b-) phenotype, FY*A/FY*A genotype, and c.1-67T>C mutation in heterozygosis (W/M). However, we could not confirm the silencing of the Fya antigen because the individual had the FY*A allele in homozygosis, and the phenotyping showed the presence of the Fya antigen. This could be due to one FY*A allele not carrying the c.1-67T>C mutation, while the other FY*A allele presented the c.1-67T>C mutation; therefore, the silencing had not occurred. On the other hand, the c.1-67T>C presence in the FY*A did not silence the Fya antigen on RBCs.The molecular studies analysis on this group of RsER allowed us to identify not only individuals presenting the Fy(a+b-) phenotype, P. vivax malarial infection and as receptors for chemokines,,P. vivax infection has virtually gone in those regionsP. vivax area, the prevalence of the Fy(a-b-) phenotype is uncommonThe Duffy antigens are important in the P. vivax infection among populations living inside or outside the malarial endemic regions. As a result, we examined the Duffy phenotype and genotype distribution in one group of RsER in the current investigation . Additionally, we analyzed the association between Duffy phenotypes and genotypes with the P. vivax infection frequency in the group of RsER. Brazil has a large malarial endemic area represented mainly by the Amazon regionP. vivax malaria infection among RsERs suggests that, to some extent, a genetic selection mechanism against the parasite infection did not occur in the Amazon region, as compared with what happened in Western AfricaWe found a high frequency of the Fy(a+b+) phenotype among RsERs similar to that already reported in Caucasians but different from that detected in Asian and African populationsCavasini et al. found that the FYA/FYB genotype was the most prevalent among patients with malaria vivax in four parts of the Brazilian Amazon region, supporting the concept that persons with this genotype had a higher vulnerability to malariaP. vivax infection among RsER was not different for the FY*A/FY*A, FY*A/FY*B, and FY*B/FY*B genotypes distribution. Nevertheless, we observed that 102/141 (72.3%) of the individuals expressing the Fyb antigen had a previously acquired P. vivax infection, indicating the biological importance of the Fyb antigen, silenced by the c.1-67T>C mutation in homozygosis in preventing the P. vivax infection. In our study, the frequency rate of previous The lower prevalence of Fy(a-b-) phenotype in the RsMR than that described among the African population probably reflects Brazilian miscegenation with people of African descentFY*A/FY*A and FY*A/FY*B genotypes but not the FY*BSE/FY*BSE genotype. The investigators also described the presence of the Fyaweak antigen among individuals from Southeast Asia as an antigen weakly reacting with the anti-Fya sera; however, in the present study we did not find an analogous immunohematological variation. The Fy(a-b-) phenotype is almost undetectable in a malaria-endemic region of Southeast Asia, and the genetic foundation for this phenotype is distinct from that described in African adults with FY*BSE allele homozygosisFY*A/FY*B genotype, resulting in the suppression of Fyb antigen expression on RBCs. These blood samples were investigated for the presence of the c.1-67T>C mutation in the promoter region of the FY* gene that characterizes the FY*BSE allele and the c.265C>T and c.298G>A mutations in the coding region of the FY* gene that characterize the FY*BWEAK allele. The Fy(a+b-) phenotype was present among 88.2% of the RsMR, showing the FY*A/FY*B genotype and the c.1-67T>C mutation in heterozygosis that silenced the expression of the Fyb antigen on their RBCs. No individuals presented the c.265C>T or c.298G>A mutations. However, 12% of the RsER showed the FY*BSE allele in heterozygosis that corresponds to the most frequent allele found among Africans and Afro-AmericansWe found discrepancies between phenotypes and genotypes in 13.6% of the RsMR, including individuals showing the Fy(a+b-) phenotype but not the FY*B allele (W/M) presented with the dose-effect regarding the expression of the Duffy antigen on RBCs; therefore, expressing only 50% of their antigensP. vivax infectionIndividuals showing the c.1-67T>C mutation in heterozygosis in the P. vivax infection and Duffy phenotype and genotype discrepancy, we observed that among 27 individuals presenting the Fy(a+b-) phenotype and the FY*A/FY*B genotype, 13 (48.2%) did not have an infection, while 14 (51.8%) had had an infection. Therefore, the infection rate was similar between the two groups, suggesting a tendency for a phenotype/genotype protection against the P. vivax infection, even though the numbers of individuals tested with such phenotype/genotype discrepancy were relatively small. However, when we included the analysis of the c.1-67T>C mutation status, characterizing the FY*A/FY*BSE genotype, the tendency for protection against the P. vivax infection was not confirmed because only 3 of the 13 individuals showing the Fy(a+b-) phenotype, the FY*A/FY*B genotype and no P. vivax infection presented the c.1-67T>C mutation in homozygosis.When we analyzed this group of RsER, according to the FY*BSE /FY*BSE had no previous malaria infection, Carvalho et al. evaluated the resistance profile for P. vivax infection in Anajas, State of Pará, Brazil, among Duffy-negative and-positive individuals, found no significant difference between the two groupsDifferently from our data, in which 100% (7/7) of the individuals presenting the FY*A allele that silenced the Fya antigen expression on RBCs,a antigen production on their RBCs. We also found one individual who presented the Fy(a+b-) phenotype, FY*A/FY*A genotype, and c.1-67T>C mutation in heterozygosis. Based on the phenotype analysis, we could not confirm whether the c.1-67T>C mutation in this individual silenced the Fya expression or not because we were not able to demonstrate if the Fya presence was in a duplicated form, corresponding to two alleles (FY*A/FY*A), or if there was one antigen silencing corresponding to the allele carrying the c.1-67T>C mutation. Zimmerman in New Guinea and Kempinska-Podhorodecka in Sudan described individuals showing the c.1-67T>C mutation in heterozygosis for the P. falciparum infection and one did not disclose the malaria epidemiology status. Individuals with the Fy(a+b-) phenotype, FY*A/FY*A genotype, and c.1-67T>C mutation in heterozygosis were infected with malaria by P. vivax and P. falciparum. Despite the small number of cases in the current data, the presence of the c.1-67T>C mutation in FY*A and FY*B alleles may confer some protection against P. vivax infection, given the low expression of the Duffy antigens on RBCs in individuals carrying the c.1-67T>C mutation, a situation that may be exacerbated by the presence of a double mutation. Additional research with people carrying the c.1-67T>C FY*A allele are required to substantiate our results that the Fya antigen is not silenced in their RBCs.Two of the four patients presented no prior history of malaria infection; one had


Further electrochemical results revealed that our FeSA-NC/CNTs catalyst exhibited excellent ORR activity , superior to that of commercial Pt/C catalyst . It also has a great stability, which makes it possible to be a valuable non-noble metal electrode material that may replace the latest commercial Pt/C catalyst in the future.Single-atom non-precious metal oxygen reduction reaction (ORR) catalysts have attracted much attention due to their low cost, high selectivity, and high activity. Herein, we successfully prepared iron single atoms anchored on nitrogen-doped carbon matrix/nanotube hybrid supports (FeSA-NC/CNTs) by the pyrolysis of Fe-doped zeolitic imidazolate frameworks. The nitrogen-doped carbon matrix/carbon nanotube hybrid supports exhibit a specific surface area of 1626.814 m A. A3-0.1@pyrrolic 0.9 eV, g=O bonds .2O3 samples were also measured as reference samples. As illustrated in the Fe K-edge XANES spectra of FeSA-NC/CNTs  and extended X-ray absorption fine structure (EXAFS) were carried out at the Fe K-edge. Fe foil, FePc, and Fe-NC/CNTs a, the ad-NC/CNTs b showed -NC/CNTs , which c-NC/CNTs . Further-NC/CNTs d–f resul2-saturated 0.1 M KOH solution based on a three-electrode system under rotating disk electrode (RDE). A commercial 20 wt% Pt/C catalyst  was used as the reference for the performance comparison. All given potentials refer to the reversible hydrogen electrode (RHE). This ORR performance was compared in this work to other similar published research (onset) with a value of about 0.93 V for FeSA-NC/CNTs was slightly inferior to that of Pt/C, but much better than that of FeNP-NC/CNTs and FeSA-NC. It demonstrates that the high-performance Fe-SAC catalyst can be prepared by tuning the content of Fe(acac)3 in ZIF-8 and the pyrolytic conditions. According to 1/2 = 0.86 V) than that of Pt/C (0.846 V). The kinetic current density (Jk) of FeSA-NC/CNTs (39.3 mA cm−2) at 0.8 V was also much higher than that of Pt/C (14.4 mA cm−2), indicating its superior kinetics. To further unveil the electron transfer mechanism of FeSA-NC/CNTs, the LSV curves at different rotating rates are depicted in 2 concentration and the potential-independent electron transfer rate (2O2 and electron transfer number were confirmed by the rotating ring-disk electrode (RRDE), suggesting a superior selectivity of oxygen reduction for H2O with an electron transfer number greater than 3.9 and a H2O2 yield below 5% over the potential range of 0.2 to 0.9 V , demonstrating the FeSA-NC/CNTs electrocatalyst possessed accelerated ORR kinetics (1/2 (ca. 1 mV) after 5000 consecutive potential cycles  in the Nyquist plot compared with FeNP-NC/CNTs and FeSA-NC, indicating the lowest charge-transfer resistance at the catalyst/electrolyte interface and superior charge transport kinetics [The electrochemical impedance spectroscopy (EIS) was also measured . The Nyqkinetics . The Bodkinetics . Therefo3 in the precursors. Based on the results of HRTEM, EDS, and HAADF-STEM, it is clear that the catalyst has a large number of Fe single atoms distributed on the NC/CNT hybrid supports as a mesoporous structure with a specific surface area of 1626.814 m2 g−1. Benefiting from the high density and superb accessibility of Fe–N4 active sites, the catalyst exhibited excellent ORR performance with a half-wave potential (E1/2) of 0.86 V, which exceeded that of commercial Pt/C. It had a high kinetic current density (Jk) of 39.3 mV cm−2 at 0.8 V. The yield of H2O2 was below 5% and the electron transfer number was close to 4, indicating that FeSA-NC/CNTs had a high electrocatalytic efficiency. Besides, it showed excellent stability with little change in the ORR polarization curve after 5000 CV cycles. In short, the outstanding catalytic activity of FeSA-NC/CNTs can be attributed to the synergistic roles of abundant Fe single atoms as well as the porous NC/CNT hybrid architectures, which ensures the full utilization of Fe–N4 active sites with improved electron transfer and oxygen mass transport.In summary, we synthesized a single-atom FeSA-NC/CNTs catalyst through the cage-encapsulated-precursor pyrolysis strategy by skillfully tuning the pyrolyzed temperature and the content of Fe(acac)


B-M-E, both solubility and yield of recombinant productionwere high. In this design, B is a silica-bindingpeptide, M is a highly thermostable,de novo-designed trimerization domain, and E is a hydrophilic elastin-like polypeptide. We show that the B-M-E triblock polypeptide rapidly assembles into highlystable polypeptide brushes on silica surfaces, with excellent antifoulingproperties against high concentrations of serum albumin. Given thatSBPs attaching to a wide range of materials have been identified,the B-M-E triblock design provides a templatefor the development of polypeptides for coating many other materialssuch as metals or plastics.We propose to exploit multivalent binding of solid-bindingpeptides(SBPs) for the physical attachment of antifouling polypeptide brusheson solid surfaces. Using a silica-binding peptide as a model SBP,we find that both tandem-repeated SBPs and SBPs repeated in branchedarchitectures implemented via a multimerization domain work very wellto improve the binding strength of polypeptide brushes, as comparedto earlier designs with a single SBP. At the same time, for many ofthe designed sequences, either the solubility or the yield of recombinantproduction is low. For a single design, with the domain structure  In fact, the binding ofSBPs to solids is not permanent since their dissociation constantsare typically larger than 100 nM, depending on the solution conditions.An obvious strategy to engineer stronger binding using SBPs isto introduce multivalent binding, displaying multiple SBPs on a scaffoldstructure 1a,b.18 reported the developmentof a strongly silica-binding peptide (highSP) containing quadruplerepeats of the minimal binding motif of a weaker precursor. Cho etal.19 showed that triplicate tandem repeatsof a ZnO-binding peptide led to stronger and more stable peptide coatingson ZnO particles than the single SBP. On the other hand, Seker etal.20 showed that the use of multiple SBPsdoes not necessarily lead to stronger binding. Recent findings byBansal et al.21 suggest that in the lattercase, lack of improved binding was due to the fact that the tandem-repeatedSBP could not simultaneously adopt the optimal spatial conformationfor binding for all tandem repeats.A first approach is to use tandem-repeated SBPs,connected directlyor via spacers 1a. Hasse22 star-like architectures have been especiallywell explored for SBPs , solutionswith higher ionic strengths (>0.5 M NaCl) were still able to displacethe adsorbed polymers.Here, we focuson the physical attachment of antifouling polypeptidebrushes to solid surfaces using SBPs as surface stickers, with thepotential to design self-assembling brushes suitable for a wide rangeof solid substrates. Previously, we have reported the design of aprotein-based diblock polymer domain E 1c, left.Bn-E with tandem-repeated SBPs 4 tag, and the linker polypeptide E20S = (VPGSG)20. For n = 2, the block encodes a poly-histidinetag for downstream purification, two BRT = (RTHRK)4 tags interspaced by a linker E3S = (VPGSG)3, and the linker polypeptide E20S = (VPGSG)20. For n = 3, the blockencodes a poly-histidine tag for downstream purification, three BRT = (RTHRK)4 tags interspaced by a linker E3S = (VPGSG)3, and the linker polypeptide E20S = (VPGSG)20. The synthetic fragments were designedto contain the features needed for PRe-RDL cloning, as described byMcDaniel.29 The fragment was digested with BamHI/AcuI and ligatedinto a BamHI/AcuI-digested pET-24a(+) vector containing the E20S =(VPGSG)20 sequence.For the construction of B-M-E triblocks, a synthetic gene encoding for the BRT-M block was synthesized by IntegratedDNA Technologies . The block encodes a poly-histidinetag for downstream purification, the BRT = (RTHRK)4 tag, a linker polypeptide E5S = (VPGSG)5,and the multimerization domains M. Thesynthetic fragment was designed to contain the features needed forPRe-RDL cloning, as described by McDaniel.29 The fragment was digested with BamHI/AcuI and ligated into a BamHI/AcuI-digested pET-24a(+) vector containingthe E40S = (VPGSG)40 sequence.For the construction of Escherichiacoli DH5α via heat shock. Coloniescontaining the correct DNA insert were selected and confirmed by DNAsequencing. Then, the plasmid was transformed intoE.coli T7-Express. The full DNA and amino acid sequencesof the polypeptides are reported in Tables S1 and S2.Forall constructs, plasmid DNA was transformed intoE. coli T7-Express containing the expression plasmids for the polypeptideswas cultured at 37 °C/215 rpm for 16 h in 25 mL terrific broth(TB) medium containing 50 μg/mL kanamycin. The starter culturewas inoculated in 1 L of lysogeny broth (LB) medium containing 50μg/mL kanamycin and incubated at 37 °C/215 rpm until OD600 > 0.6. When necessary, 0.5% d-glucose was added.Then, isopropylthio-β-galactoside (IPTG) was added to a finalconcentration of 1 mM and cells were incubated overnight at 18 °C/215rpm before harvesting.g at 4 °C to pellet the insolublefraction. Next, the polypeptide was purified using immobilized metalion affinity chromatography (IMAC). The sample was injected in anIMAC column and washed with extraction buffer containing 2 M NaCl to remove DNAcontamination. The polypeptide was eluted with a linear gradient fromextraction buffer to elution buffer . The purity of the polypeptides was assessed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Bacterial cells were centrifugedat 6000 rpm for 30 min at 4 °C and resuspended in 30 mL of coldextraction buffer .Phenylmethylsulfonyl fluoride (PMSF) was added to the cell suspensionto a final concentration of 1 mM. Cells were lysed via sonication. The cell lysate was centrifuged for 30min at 30,000To confirm the size of the polypeptide, its molecularweight was determined using matrix-assisted laser desorption/ionization(MALDI) mass spectrometry. The spectrum was obtained using a BrukerUltraFlextreme machine. The sample was prepared following the instructionsprovided by the manufacturer.For circular dichroism measurements,a Jasco Spectropolarimeter J-715 was used. Data was collected andanalyzed with Jasco software. For the sample preparation, the proteinwas dissolved at a concentration of 0.1 mg/mL in phosphate-bufferedsaline (PBS) pH. 7.4. The solution was sonicated for 10 min to reducethe presence of aggregates before the measurement. A quartz cuvettewith a 1 mm path was used. For the spectrum measurements, the instrumentwas set to continuous scanning mode, with a data pitch of 0.1 nm,a scanning speed of 50 nm/min, and a band width of 2 nm. Each spectrumwas accumulated 20 times. For the temperature ramp, the ellipticitywas measured at 222 nm while increasing the temperature of the samplefrom 20 to 95 °C, at 1 °C/min.T = 20 °C. The reported hydrodynamic sizes were obtainedusing the Zetasizer software version 7.13 . Nonfunctionalizedsilica microspheres with a diameter of 163 nm were purchased fromBangs Laboratories. For dynamic light scattering (DLS) on B-M-E solutions, the protein was dissolved in the appropriatebuffer (phosphate buffer with increasing concentrations of NaCl).The solutions were filtered using a 0.22 μm filter and sonicatedfor 10 min to reduce the presence of aggregates before the measurement.For each sample, continuous measurements were performed, with theduration of each measurement defined by the instrument. Each reportedhydrodynamic size is the average of 10 measurements. For measurementson silica nanoparticles coated with Bn-E or B-M-E, the polypeptide solution and the silica nanoparticles(0.01% v/v) were sonicated for 10 min. The polypeptide solution wasfiltered using a 0.22 μm filter. Increasing concentrations ofprotein were incubated with the nanoparticle solution for 10 min.For each sample, continuous measurements were done, each reportedhydrodynamic size is the average of 10 measurements, with the durationof the measurement defined by the instrument. For the ζ potentialmeasurements, the samples were prepared following the same procedure.A dip cell  was used. Each reported ζ potentialvalue is the average of 15 measurements.For dynamic light scatteringmeasurements, a ZS-Nano  instrument was used. Lightscattering was measured at a scattering angle of 173°, at 2 were obtained from Biolin Scientific and cleaned accordingto the provided instructions. For the salt dependency measurements,each sensor was equilibrated with the appropriate buffer  at a flow rate of 50μL/min until a stable baseline was reached. Bn-E or B-M-E were dissolved to a final concentrationof 10 μM and dialyzed in the previously mentioned buffers. Theprotein solutions were sonicated for 10 min prior to the measurement.Analysis of QCM-D data was performed using QSense software . For the antifouling test, each sensor was equilibratedwith PBS at a flow rate of 50 μL/min until a stable baselinewas reached. The SiO2 sensors were coated with PLL-PEG(1 mg/mL), the diblock polypeptide B-E (5 μM), and B-M-E (5 μM) until a stable value of frequency shift (Δf ) was reached. Coated and uncoated sensors were then flushedwith a solution of bovine serum albumin (2 mg/mL). Analysis of QCM-Ddata was performed using QSense software.A QSense E4 QCM-D instrument  was usedto quantify polypeptide binding to silica. QCM sensors coated withSiOAtomicallyflat silica surfaces were obtained using silicon wafers (SiltronicAG) with a 2–3 nm oxide layer due to natural oxidation withoxygen in air. Silica surfaces were cleaned with MilliQ water andethanol and plasma cleaned for 5 min. For AFM imaging, 30 μLof the protein solution was deposited on a cleaned silica surfaceand incubated for 1 min. After that, samples were gently rinsed withMilliQ water and carefully dried with nitrogen. Samples were imagedwith a Multimode Bruker AFM  using the automaticScanAsyst imaging mode. ScanAsyst air tips were used with a nominalradius < 10 nm.B-E diblocks with silica-binding blocks BRT = (RTHRK)418 and elastin-likehydrophilic blocks ES40 = (VPGSG)40 satisfythe prerequisites of simple production and purification as well asgood solubility and silica binding.28 Therefore,our current designs still use BRT as binding blocks for silicasurfaces and ES40 as hydrophilic brush-forming blocks. Wedesigned both Bn-E diblocks with tandem-repeatedbinding blocks BRT (up to three repeats) and B-M-E triblocks,with oligomerizing midblocks M of differentvalences m. As oligomerizing blocks M, we selected four naturally occurring33 and three highly stable de novo computationally designed oligomerizingdomains.34 In some cases, the N-terminalof the oligomerization blocks did not allow for suitable display ofthe binding block. For these cases, we used M-B-E triblock designs instead. Finally, we used short ESn sequences (n = 3–5) as linkers forconnecting oligomerization and binding blocks and for connecting bindingblocks to form tandem repeats. The designs are listed in E. coli, and purified usingIMAC affinity chromatography. For full DNA and protein sequences,see Tables S1 and S2.In our previous work, we foundthat Bn-E (n = 1–3)is shown in Figure S1. We found that theexpression of the tandem-repeat designs decreases rapidly with increasing n. Some improvement in expression for n = 2 and 3 was found using LB medium containing 0.5% d-glucose, suggesting thatthe expression at higher n is limited by the toxicityof the polypeptide.35 QCM-D analysis ofbrush formation by Bn-E diblocks with n = 1, 2 on silica-coated quartz sensors showed that the tandem-repeatstrategy is effective in improving the brush stability against displacementby high ionic strength solutions . Nonetheless, the tandem-repeat designs will not be further exploredbecause of the problems in the production of these polymers. Instead,we will focus on employing oligomerizing midblocks M with star-like display of the SBPs , the expressionof the constructs with de novo-designed oligomerization domains wasfound to be higher . However,preliminary investigations showed that the proteins with the de novo-designedoligomerization domains 1na0C3_3 and ank1C4_2 had lower solubilitythan the construct with the HR00C3_2 oligomerization domain. Therefore,we focus our attention on the design BRT-ES3-MHR00C3_2-ES40, containing the trimerizationdomain HR00C3_2 (PDB 5K7V) designed by Fallas et al.34 We will subsequently refer to this construct as B-M-E, with the implicit understanding that the bindingblock is B = BRT-ES3, the multimerizationblock is M = MHR0032_2, and the hydrophilic random coilblock is E = ES40.While all designs containingnatural oligomerization domains couldbe expressed . We found that this polypeptide quickly aggregated in solutionafter purification. Since the M block, B block, and B-M-E triblocksare soluble in PBS pH 7.4, we conclude that the B and M blocks coprecipitatein our B-M diblock design. We hypothesize that since the binding block B is highly cationic (+12) and the trimerizationdomain M has a high net negative charge(−24), the observed coprecipitation is very likely caused bystrong electrostatic interactions between B and M blocks .Possibly, trimerizationof the B and M blocks for the self-assembly in solution of the B-M-E triblocks. On the one hand, we anticipate thatthe long hydrophilic random coil blocks E solubilize the B-M precipitates, in line with previous observations regardingthe use of hydrophilic ELPs for the stabilization and solubilizationof macromolecules.37 On the other hand,the E blocks may not be able to completelyprevent association of B-M-E trimers in solution.Next, we explored the consequencesof electrostatic interactionsbetween B-M-E,we used dynamic light scattering (DLS). Since we hypothesize that B-M interactionsare electrostatically driven, we varied the salt concentration toestablish whether they can be screened by the addition of salt.To establish solution sizes for B-M-E solutions is highly salt-dependent, showing a sharp decreasebeyond a salt concentration [NaCl] ≈ 400 mM. As shown in DH ≈ 250 nm. Above [NaCl] ≈ 400 mM, we observeda slight decrease of the size of the large assemblies and additionallyobserved assemblies with a much smaller hydrodynamic diameter DH ≈ 22 nm (at [NaCl] = 400 mM). For [NaCl]> 400 mM, the scattering intensities corresponding to the largeandsmall assemblies are roughly equal. Given the strong dependence ofscattering intensity on particle size, this implies that at leastat [NaCl] > 400 mM, the majority of the B-M-E proteinsis part of the small assemblies.As shown in M block has a heightof 5 nm and a diameter of 7 nm. The hydrodynamic diameter of a single E block28 is 9 nm.Hence, the observed assemblies with a hydrodynamic diameter DH ≈ 22 nm could very well correspondto the B-M-E trimers, but this cannot be establishedwith certainty from the DLS measurements alone. We have attemptedto separate the putative trimers from the larger assemblies usingSEC at a salt concentration [NaCl] = 500 mM to establish their precisesolution stoichiometry but found that the proteins eluted as a singlerather broad peak covering a range of hydrodynamic sizes . At the same time, SDS-PAGE indicatedthat peak fractions only consisted of the B-M-E protein . This most likely indicates the trimers are in fast equilibriumwith the larger assemblies. Indeed, all SEC fractions showed the presenceof both the larger and the smaller assemblies in DLS.A trimer of the B-M-E triblocks withsilica surfaces was investigated using monodisperse nonporous silicananoparticles with a hydrodynamic diameter DH =163 nm. As we have shown for B-E diblocks, this method allows fora straightforward determination of both the concentration of proteinrequired for a stable adsorbed polypeptide brush and the brush heightvia dynamic light scattering (DLS).28 Theresults are shown in DH = 220 nm, leading to an estimated brush height h ≈ 28 nm.The interactionof the B-M-E triblocks. The results are shown in CB-M-E of a few μM is required to giverise to a significant change of the ζ-potential, due to theself-assembly of a polypeptide brush on the surface of the silicaparticles. The coating makes the ζ-potential less negative,increasing from around −40 to around −20 mV.Electrophoretic light scattering was used to determinethe changein ζ-potential of the silica particles coated with the B-E diblocks,28 wefound that binding with a single solid-binding peptide is not strongenough to resist displacement at higher salt concentrations, and thiswas part of the motivation to investigate multivalent SBP binding.Weused a quartz crystal microbalance to investigate whether the B-M-E triblocks can still form stable brushes on silicasurfaces at high concentrations of NaCl. The triblock polypeptidewas dissolved at a concentration of 10 μM in phosphate buffer(PB), phosphate-buffered saline (PBS), PBS with 0.5 M NaCl, and PBSwith 1 M NaCl. The kinetics of adsorption of the B-M-E triblocks at these different salt concentrations on silica was followedusing the quartz crystal microbalance. Polypeptide solutions wereflushed over the silica sensors and after a stable layer had formed,sensors were rinsed with various buffers used for the layer formation.The results are shown in Inour previous work on B-M-E trimersform stable layers on silica surfaces, with good resistance againstrinsing with both low and high salt buffers. This observation, however,does not necessarily imply that the proteins adsorb as expected, withtheir binding domains B facing the silicaand the elastin-like blocks E facingthe solution. One possibility is that the larger assemblies observedin DLS for bulk solutions adsorb on the silica and form rather inhomogeneouslayers. To exclude this possibility, we performed atomic force microscopy(AFM) of B-M-E layers adsorbed on silica. Imagingwas done in air; the results are shown in B-M-E layers are very thin (<10 nm) and very homogeneous overlarge areas of 2 μm × 2 μm. The surface morphologyof the B-M-E layers as observed in AFM is thatof densely packed dots, which we tentatively identify with the trimers.Hence, AFM rules out the possibility that the larger dynamic assembliesobserved using DLS in solution lead to inhomogeneous coatings.According to the QCM measurements, the g-PEGpolymers as a positive control for self-assembled antifouling brushes,and we also tested our previously designed B-E diblock polypeptides. QCM was usedto follow the kinetics of brush assembly on silica-coated quartz sensors.After brush formation, a high concentration of bovine serum albumin was flushed on the sensor, followed by rinsing withPBS. As a negative control, we used a bare silica sensor. QCM resultsare shown in Next to brush stability, the prevention of biologicalfouling isa key property for the successful application of adsorbed brushes.As a first step, we tested the antifouling ability against high concentrationsof serum albumin in PBS pH 7.4. We used PLL-g-PEG-coated silica sensors are as expected: the self-assembled PLL-g-PEG brushes effectively prevent the adsorption of BSAon the silica surface (For the negative control on bare silica, a strongQCM signal isobserved upon injecting BSA. Only a small part of the adsorbed BSAis removed in the PBS rinsing step that follows, demonstrating theBSA has adsorbed irreversibly. The results for the PLL- surface 9a. DurinB-E diblock show that brushesare gradually rinsed off by PBS (B-E brushes are considerably less stablethan the PLL-g-PEG brushes with respect to rinsingwith both PBS and BSA in PBS.The results for silica sensors coatedwith the f by PBS 9b. Upon B-M-E triblock are shown in Finally, the results for sensorscoated with the B-M-E coatings,all binding blocks B are properly orientedtoward the silica surface, as designed. In fact, for the more weaklybound B-E diblocks, we observed strong displacement caused by BSA, indicatingthat the negatively charged BSA molecules compete effectively withthe negatively charged silica. This also indicates a strong interactionbetween the B blocks and BSA. In the B-M-E coatings, any exposed binding blocks B would have also led to strong interactions withBSA, which we did not observe.Since we do not see changesin the signal upon flushing with concentratedBSA, we can also conclude that, in the BRT-ES3-MHR00C3_2-ES40 has very promising properties,forming highly stable and antifouling polypeptide brushes on silicasurfaces.Our work shows that it is possibleto design, produce, and purifyself-assembling polypeptide brushes that harness multivalent bindingof SBPs for attachment to solid surfaces. The number of successfuldesigns is strongly limited by practical constraints such as polypeptidetoxicity, low expression levels, and low solubility. However, we alsoshowed that the design with sequence B-M-E designas an antifoulant against concentrated solutions of BSA in PBS, thefollowing step would be to evaluate complex biological fluids suchas diluted serum. In fact, the antifouling behavior against a singleprominent serum protein such as BSA does not guarantee similar resultswith a complex mixture of biomolecules.39 Hence, furtherchanges to our original design may still be necessary. These couldinclude changing the length or nature of the E block.Given the initial success of our g-PEG brushes arealready adequate formany applications, we believe that recombinant antifouling polypeptidesmay offer advantages for specific cases. In particular, the B-M-E triblock sequence can be directly used as a combinedadhesion and antifouling tag for displaying proteins on surfaces.Also, given the wealth of data on SBPs suitable for other surfaces,we expect it will be relatively straightforward to develop B-M-E triblocks that strongly adhere to, for example,metals and plastics.While PLL-


N-retinylidene-N-retinylethanolamine (A2E) accumulates in retinal pigment epithelium (RPE) cells and generates oxidative stress, which further induces cell death. Polyphenols are well known for their antioxidant and beneficial effects on vision. Chrysanthemum boreale Makino (CB) flowers, which contain flavonoids, have antioxidant activity. We hypothesized that polyphenols in ethanolic extracts of CB (CBE) and its fractions suppressed A2E-mediated ARPE-19 cell damage, a human RPE cell line. CBE is rich in polyphenols, shows antioxidant activity, and suppresses intracellular accumulation of A2E and cell death induced by A2E. Among the five fractions, the polyphenol content and antioxidant effect were in the order of the ethyl acetate fraction (EtOAc) > butanol fraction (BuOH) > hexane fraction (Hex) > dichloromethane fraction (CH2Cl2) > water fraction (H2O). In contrast, the inhibitory ability of A2E accumulation and A2E-induced cell death was highest in H2O, followed by BuOH. In the correlation analysis, polyphenols in the H2O and BuOH fractions had a significant positive correlation with antioxidant effects, but no significant correlation with cell damage caused by A2E. Our findings suggest that substances other than polyphenols present in CBE can suppress the effects of A2E, and further research is needed.In age-related macular degeneration,  Lipofuscin is a yellow-brown pigmented auto-fluorescent granule composed of lipids, oxidized proteins, and metals as a heterogeneous by-product of intracellular lysosomal degradation ,2. OxidaN-retinylidene-N-retinylethanolamine (A2E), a pyridinium bis-retinoid produced due to an abnormal vitamin A visual cycle, is a major component of RPE lipofuscin -β-d-glucopyranoside, chlorogenic acid, 3,5-di-O-caffeoylquinic acid, and apigenin, were identified in CBE [2O fraction of CBE. Quercetin is detected in the H2O fraction from methanolic extract of Syzygium cumini (L.) seed [2O fraction, there is a possibility that non-polyphenols also have a protective effect against A2E. Although follow-up studies are needed to identify polyphenol and non-polyphenol components that have cytoprotective effects against A2E in H2O fraction of CBE, CBE itself has been shown to inhibit A2E-induced cell damage. As A2E is a biomarker in AMD patients, CBE could be a medicinal plant with protective effects against AMD.The five fractions of CBE also contained TPC and TFC and had antioxidant activity but with different degrees of efficacy. TPC and TFC were abundant in the EtOAc > BuOH > Hex > CHifferent . The ants report . Among fd in CBE ,48. In ad in CBE . Chrysand in CBE ,51. Anthd in CBE ,53. The L.) seed . CyanidiL.) seed . However2O fraction, in which no flavonoids were detected. Finally, we propose that the non-polyphenolic components present in CBE may effectively prevent AMD.Overall, the results suggest that CB can be used as a functional plant to attenuate AMD development and progression. CBE, which contains a large amount of phenolic acid and flavonoids, has antioxidant effects and effectively inhibits A2E accumulation and A2E-induced cell death. In the CBE fraction experiment, the EtOAc fraction, containing a large amount of polyphenol, also had high antioxidant activity, but no cytoprotective effect against A2E was found. The protective effect against A2E was most potent in the H


PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs  While lng niche .+ cells in GBM and that PDPN identifies a population of aggressive-treatment-resistant GSCs that may be contributing to poor GBM patient outcome.Expression of the type-I integral membrane glycoprotein podoplanin (PDPN), also known as OTS-8, PA2.26, gp36, gp38, RANDAM-2, T1-α, and aggrus, correlates with defining glioma stem cell characteristics –11. In cin vitro and in vivo and found that its expression is correlated with CD133 expression, but not vice versa. In xenograft tumorigenesis experiments, we discovered that PDPN expression greatly influences tumor growth.In the present study, we examined PDPN expression across large glioma cohorts in addition to The Cancer Genome Atlas (TCGA) and found PDPN to indeed be an independent prognostic marker among glioma patients. We experimentally characterized the function of PDPN using patient-derived GSCs RNA-Seq by Expectation–Maximization (RSEM) normalized  TCGA RNAImmunohistochemical (IHC) analysis of the prognostic significance of PDPN in GBM was performed on a group of patients for whom sufficient formalin-fixed and paraffin-embedded (FFPE) tissue remained from specimens previously examined for expression of proteins in the RAS signaling pathway and for the secreted protein YKL-40 , 17 in aDetermination of radiation therapy (RT) response was performed by comparing the enhancing tumor size between the postoperative magnetic resonance imaging (MRI) and the first post-RT MRI in the manner previously reported . RT resp473 Akt (p-Akt) and p-Thr202/Tyr204 MAPK (p-MAPK), have been previously described (Immunohistochemistry (IHC) staining of FFPE tissue was performed as previously described . Detailsescribed , 18. StaT was calculated as the difference between the mean CT of the tumor cDNA and the mean CT of four reference genes . Primer and probe combinations consisted of the following: PDPN, forward 5’GGGTCCTGGCAGAAGGAG3’, reverse 5’CGCCTTCCAAACCTGTAGTC3’, Universal Probe #20; GAPDH, forward 5’GGAAGCTTGTCATCAATGGAA3’, reverse 5’TTGATTTTGGAGGGATCTCG3’, Universal Probe #9; TFRC, forward 5’CCAGAGCTGCTGCAGAAAA3’, reverse 5’TGTTTTCCAGTCAGAGGGACA3’, Universal Probe #12; and RPLPO, forward 5’CTGGAAAACAACCCAGCTCT3’, reverse 5’GAACCAAAGCCCACATTCC3’, Universal Probe #74. GUSB amplification was performed using an Assays-on-Demand gene expression assay according to the manufacturer’s instructions (Life Technologies). For cell lines, 20-µ reaction mixtures were performed in triplicate and included 1 µl cDNA template and 1 µl each of primer and probe mix and TaqMan Universal PCR master mix (Life Technologies). Amplification proceeded as follows: denaturation at 95°C for 15 min and 40 cycles at 94°C for 1 min and 60°C for 1 min. ΔCT was calculated as the difference between the mean CT of the tumor cDNA and the mean CT of the reference gene beta actin (ACTB). Primers and probes combinations consisted of PDPN #Hs01089983 and ACTB #Hs99999903 (Life Technologies).TaqMan qRT-PCR was performed as previously described  using TaPDPN shRNA  was cloned at Cellecta using the Cellecta pRSI-EF1a-TetRep-2A-Puro-H1Tet-(sh) vector cut at the BbsI restriction site. shRNA sequences were as follows: shLuc 5’-ACCGGCGCTGAGTACTTTGAAATGTTGTTAATATTCATAGCGACATTTCGAAGTACTCAGCGTTTT, MD5 5’-PDPN ACCGGGCTCCTCTTAAACATTTGTTGTGTTAATATTCATAGCACAGCAAATGTTTAGAGGAGCTTTT, MD7 5’-ACCGGCCAGGAGAGTAACAACTTAACGTTAATATTCATAGCGTTGAGTTGTTGCTCTCCCTGGTTTT. For miRNA experiments, miRNA was generated using the PDPN GenBank sequence NM_006474 in the Block-iT RNAi Designer (Life Technologies) to achieve the NM_006474_441_top 5’TGCTGACTTATAGCGGTCTTCGCTGGGTTTTGGCCACTGACTGACCCAGCGAACCGCTATAAGT3’ and NM_006474_441_bottom 5’CCTGACTTATAGCGGTTCGCTGGGTCAGTCAGTGGCCAAAACCCAGCGAAGACCGCTATAAGTC3’ miRNA oligo sequences, which were annealed and ligated into the Life Technologies Block-iT Pol II miR RNAi EmGFP expression vector per kit protocol (Life Technologies).2.The GSCs examined in this study have been published previously , 24. GSC5/well in a coated six-well plate on day 1. The cells were infected with lentivirus containing the shRNAs (MD1-MD12 and Luc) at multiplicity of infection (MOI)=5 in the presence of 0.4 µg/ml polybrene the following day. On day 3, the medium containing the lentivirus and polybrene was replaced with fresh regular medium to let the cells recover from infection. Finally, infected cells were cultured and expanded to T-25 and T-75 flasks. PDPN shRNAs identified as MD5 and MD7 was used in PDPN knockdown experiments. For experiments with adherent cell lines, U87 lines were grown to 80% confluence in 10-cm plates and transfected with 24 μg of appropriate vector containing either PDPN miRNA or corresponding empty vector (control), using Lipofectamine 2000 in Opti-MEM (Life Technologies). U87 transfectants were selected and maintained using blasticidin.For PDPN shRNA transduction experiments, GSCs were prepared as single-cell suspensions and seeded at 5×10FACSAria (BD Biosciences) was used to analyze and sort GSCs based on PDPN and CD133 expression using a PE-conjugated mAb to PDPN  alone or in combination with an APC-conjugated mAb to CD133 . Staining was performed per recommended manufacturer protocols.4 GSCs (sorted sub-populations) or 5×105 GSCs (PDPN knockdown experiments) were directly injected into brains of athymic nude (nu/nu) mice of both sexes at age of 6–8 weeks using a stereotactic apparatus under anesthesia and with analgesics. Animals that showed signs of distress or were moribund were euthanized and autopsied. Doxycycline (Sigma) was administered via drinking water (2 mg/ml) containing 5% sucrose. Water was changed every 3 days. For in vivo bio-luminescent imaging, GSCs were engineered to express luciferase. On the day of imaging, animals were treated with luciferin . Tumor growth was monitored using IVIS 200 system bio-luminescent imaging, and tumor volume was measured using Living Image 4.7.3 software. All mice were cared for according to the guidelines and under the supervision of the Institutional Animal Care and Use Committee.For survival experiments, 2.5×10in vitro limiting dilution clonogenic survival assay  values for treated cells were normalized to that of the control (non-irradiated) plates. The surviving fraction was determined by dividing the PE of treated cells by the PE of controls.GSC neurosphere formation and radiation response was determined using the al assay . Prior t3, 1 mM dithiothreitol (DTT), and 1 µM phenylmethylsulfonyl fluoride (PMSF) (Roche Applied Science). Thirty micrograms of protein was loaded for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using the standard protocols. Upon electrophoresis, protein was transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore) and incubated in 10% nonfat dry milk blocking solution for 30 min. Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN  and vinculin  at 4°C. Membranes were subsequently washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rat and anti-mouse secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h and prepared for chemiluminescent detection using the ECL Plus Western Blotting Detection System kit  according to the manufacturer’s protocol.Whole cell lysates were prepared from cultures using a standard NP-40 lysis buffer (Life Technologies) with 1× protease inhibitor tablet, 0.1 mM NaVOTotal RNA was isolated from FACs-sorted PDPN+ and PDPN− subpopulations using the MasterPure Complete DNA/RNA Purification kit (Epicentre). The RNA samples were processed on Affymetrix U133A 2.0 microarray chips (Affymetrix). Raw microarray data were processed by affy  and limmPDPN expression grouped by IDH status or grade showed normally distributed strata, and p-values were subsequently obtained using two-sample Student’s t-test. Normalized read counts were log2 transformed and showed a bimodal distribution for PDPN expression. Clinical data consisted of tumor grade, histology, vital status, and follow-up time. Patients living at the time of this study had OS censored at the time of last follow-up. Nested models were compared using the likelihood ratio test (LRT). Analysis was conducted in R (v 3.1.2) using the survival package  and PDPN-low (n=18) groups were 37.0 and 240.7 weeks, respectively  of patients underwent gross total resection (GTR) and 98 (48%) of patients underwent sub-total resection (STR) or biopsy only Table 1.PROM1 gene and is present on leukemic and solid tumor cells  days, while mice that received PDPN-CD133+ cells did not succumb to tumor formation (n=3)  revealed sharply contrasting gene expression signatures in PDPN+ and PDPN− GSCs , and VerTo understand if PDPN may be playing a role in tumor aggressiveness and radioresistance in additional cell lines and contexts, we performed a series of experiments using adherent cell lines, which are grown in serum . We first tested for PDPN expression in multiple adherent human glioma cell lines, using normal human astrocytes (NHA) as a negative control and found that PDPN was highly expressed in U87 and LN319 cells . Single-colony selection or sorting of PDPN− cells from knockout lines were not performed, as GSCs are very heterogeneous and have highly variable intra-cellular growth rates and characteristics . The prognostic relevance of PDPN was independent of both extent of surgical resection and of age, which is one of the strongest predictors of outcome for GBM patients. Furthermore, PDPN expression increased from 70% in low-intermediate-grade astrocytomas to 87% in high-grade astrocytomas (GBMs). Mishima et al. found PDPN expression to be absent in WHO grade II tumors while present in 36% of WHO grade III and 53% of WHO grade IV tumors , and the role of PDPN and other cellular markers within those niches remains to be defined –58. MorePDPN silencing has been shown to reduce the invasive capacity of GBM cells  can be found in the article/The animal study was reviewed and approved by NYU School of Medicine Animal Ethics Committee. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.EE, QW, ES, RV, RE, JG, LG, TL, KB, LL, ZY, and YD conducted the experiments. AM, EE, QW, ES, RV, FL, and KA conceptualized the experimental design and methodology. AM, EE, QW, ES, RE, and RV performed the analyses. AM, EE, QW, ES, and RV performed the writing, reviewing, and editing. FL and KA provided resources. All authors contributed to the article and approved the submitted version.This work was supported by the NIH/NCI under award numbers R01CA190121 , P50CA127001 , P01CA085878 (RV), and P30CA016672 Cancer Center Support Grant ; by the American Brain Tumor Association (ES); by the National Brain Tumor Society Defeat GBM Research Collaborative ; by the Cancer Prevention and Research Institute of Texas under award number RP120256  and RP140606 (RV); and by the Broach Foundation (FL)The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Identifying T cell epitopes on pancreatic ductal adenocarcinoma (PDAC) associated antigens or neoantigens has been a challenge. In this study, we attempted to identify PDAC T cell epitopes by mass spectrometry (MS).We isolated HLA class I (HLA-I) and HLA class II (HLA-II)-restricted peptides, respectively, from tissues of human PDAC by using the pan-HLA-I or pan-HLA-II affinity purification column and identified T cell epitopes by peptidome analysis with MS.Through peptidome analysis, we identified T cell epitopes shared by multiple patients with different HLA types and those containing sequences of both anti-HLA-I and HLA-II antibodies-affinity purified peptides. The identified epitopes bound non-matched HLA molecules and induced T cell response in peripheral T cells from both HLA-type matched and non-matched patients. Peptides containing both HLA class I and class II epitopes were able to induce polyfunctional cytokine responses in peripheral T cells.T cell epitopes in PDAC can be discovered by the MS approach and can be designed into vaccine and TCR-T cell therapies for both HLA-type matched and non-matched patients.The online version contains supplementary material available at 10.1186/s13045-022-01373-6. To the editorIt was a challenge to identify T cell epitopes in pancreatic ductal adenocarcinoma (PDAC) largely due to lack of knowledge on immunodominant antigens  and effeγ or granzyme B or both was significantly stimulated by these peptides not only in the PBMC from at least one of two patients whose tumors were used for identifying these peptides, but also in those from HLA-type unmatched patients (Additional file p < 0.01, Fig. p < 0.05, FSurprisingly, T cell response as demonstrated by the expression of either IFN-γ, TNF-α, and IL-2 expression from T cells in HLA-type unmatched PBMCs (Fig. After we used the pan-HLA class I affinity purification column to bind the HLA-I peptides in the lysate of PDAC tissues, we subjected the flow-through for HLA-II peptide isolation with the pan-HLA-II affinity purification column. The numbers of HLA-II peptides with different lengths peaked at 14–16 amino acids Fig. B. Next, Our study is the first one to examine HLA class I and class II-restricted peptidomes in human PDAC. Previously, similar studies in few other malignant diseases were successfully conducted and reported , 10. ThiAdditional file 1: Methods, Supplemental Figures S1–12, and Supplemental Tables S1, S3–5.Additional file 2: Supplemental Table S2, PDAC peptidome.


Nephthea sp. and isolated a strain that was sub-cultured in three different media . Actinomycete isolate’s phylogenetic analysis of the 16S rRNA gene revealed that it belongs to the genus Rhodococcus. In vitro screening of the antimalarial activity for three extracts against Plasmodium falciparum was carried out. Non-targeted metabolomics for the chemical characterization of the isolated actinomycete species UA111 derived extracts were employed using high-resolution liquid chromatography–mass spectrometry (LC-HR-MS) for dereplication purposes. Additionally, statistical analysis of the vast LC-MS data was performed using MetaboAnalyst 5.0. Finally, an in silico analysis was conducted to investigate the potential chemical compounds that could be the source of the antimalarial potential. The results revealed that ISP2 media extract is the most effective against Plasmodium falciparum, according to antimalarial screening (IC50 8.5 µg/mL), in contrast, OLIGO media extract was inactive. LC-HRMS-based metabolomics identified a range of metabolites, mainly alkaloids, from the genus Rhodococcus. On the other hand, multivariate analysis showed chemical diversity between the analyzed samples, with ISP2 extract being optimal. The docking analysis was able to anticipate the various patterns of interaction of the annotated compounds with three malarial protein targets . Among all of the test compounds, perlolyrine (11) and 3097-B2 (12) displayed the best docking profiles. In conclusion, this work demonstrated the value of the established method for the metabolic profiling of marine actinomycetes using the data from liquid chromatography–mass spectrometry (LC-MS), which helps to streamline the difficult isolation stages required for their chemical characterization. In addition, the antimalarial efficacy of this strain has intriguing implications for future pharmaceutical development.Malaria is a persistent illness with a great public health concern. To combat this fatal disease, developing effective antimalarial medications has become a necessity. In the present study, we described the actinomycetes associated with the Red Sea soft coral  Rhodococcus is a member of the taxonomic group actinobacteria and is a member of the Nocardiaceae family and the order actinomycetales; they are classified as aerobic, Gram-positive, non-motile, non-sporulating, mycolate-containing, and non-cardioform actinomycetes _ , in accordance with the molecular formula C17H26O4 was recognized as octahydro-7α-methyl-1-(1-methyl-2-oxo-propyl)-5-oxo-1H-indene-4-propanoic acid that was previously isolated from Rhodococcus corallines [m/z 174.1022 [M_H]_ , corresponding to the molecular formula C10H9NO2, was suggested to be indole-3-acetic acid, which was previously isolated from Rhodococcus sp. BFI 332 [m/z 155.0237 [M_H]_ , in accordance with the molecular formula C7H8O4, was recognized as 2,5-Dihydro-3-methyl-5-oxo-2-furanacetic acid (S-form), which was previously isolated from Rhodococcus rhodochrous + , was characterized as rhodinodohyde, which was previously described from Rhodococcus sp. UA13 + ; this compound was previously reported from Rhodococcus sp. UR59 [m/z 116.0497 [M_H]_  for the predicted molecular formula C8H7N was distinguished as 2-Phenylacetonitrile, which was isolated before from marine Streptomyces sp. GWS-BW-H5 [m/z 131.0707 [M_H]_ , for the suggested molecular formulas C6H12O3 was identified as 3,4-Dihydroxy-3-methylpentan-2-one that was previously obtained from Streptomyces griseorubens sp. ASMR4 [m/z 160.0395 [M_H]_ , corresponding to the proposed molecular formula C9H7NO2, was identified as Indole-3-carboxylic acid, which was earlier obtained from Streptomyces sp. TK-VL_333 [m/z 166.0503 [M_H]- , with the molecular formula C8H9NO3, was recognized as 2-Hydroxy-5-methoxybenzamide that was previously isolated from marine Streptomyces sp. [m/z 265.0973 [M_H]+ , in accordance with the molecular formula C16H12N2O2, was recognized as Perlolyrin, which was previously isolated from a terrestrial Streptomyces sp. GW11/1695 _ ; this compound was previously reported from marine Streptomyces sp. + (2.4039) was identified as p-tolyl-3-aminopropanoate (C10H13NO2) [m/z 164.07093 [M_H] , corresponding to the proposed molecular formula C9H11NO2, was identified as Anthranilic acid ethyl ester [m/z 182.08152 [M_H]+ , in accordance with the molecular formula C9H11NO3, was recognized as tyrosol carbamate [m/z 152.07104 [M_H]−  corresponding to the molecular formula C8H9NO2, was suggested to be streptokordin [m/z 211.08715 [M_H]+ , in accordance with the molecular formula C13H10N2O, was identified as penicinoline E [m/z (retention time in min) 308.13568 [M_H]+ (2.36095) and 210.0403 [M+H]− (2.304102) were putatively identified as antibiotic A 53868A (C11H22N3O5P) and antibiotic T0007 B2 (C9H9NO5) ..m/z) werRhodococcus and the production of bioactive antimalarial compounds. The observed variations emphasize how crucial it is to use a diverse range of media to improve the isolation effectiveness of metabolites from actinomycetes associated with the marine environment. Based on prior experience and published reports, the actinomycete cultivation media M1, ISP2, and OLIGO were selected [P. falciparum species, we ran molecular docking simulations within the crystal structures of various proteins found in P. falciparum species, such as the Plasmodium falciparum cell cycle regulator and non-human-derived cyclin-dependent kinase, called P. falciparum kinase . Additionally, the P. falciparum cytochrome bc1 complex , and finally, P. falciparum lysyl-tRNA synthetase . These simulations revealed a moderate to strong docking score  with all test molecules within the three active sites used in this study  revealed that both molecules 11 and 12 succeed in having binding interactions  active site mediated through H-bond donor and acceptor interactions with key amino acid residues lining the active site , as listed in Moreover, both molecules 11 and 12 showed an interesting binding mode within the P. falciparum lysyl-tRNA synthetase  showed the highest and best possibility of being a target by such compounds and could be used to illustrate their antimalarial biological activities. In brief, the docking simulations within lysyl-tRNA synthetase resulted in the best docking score and number of binding interactions within the active site of lysyl-tRNA synthetase crystal structures, especially with molecules 11 and 12, which have a high and comparable docking score within all other dereplicated molecules and to the co-crystallized ligand (9X0), as shown in Finally, the docking scores and the docking accuracy of the test molecules within the crystal structure of http://www.swissadme.ch/ accessed on 7 August 2022). Starting with the role of five (Lipinski’s RO5) [Investigating the oral bioavailability of such dereplicated molecules was achieved by measuring the physicochemical descriptors, pharmacokinetics, drug-like nature, and medicinal chemistry friendliness of six of these dereplicated molecules  using the SwissADME free web tool (i’s RO5) ,34, all Briefly, having H-bonding centers (either acceptor or donor) helps in enhancing water solubility and H-bond formation with various amino acid residues lining the target’s active sites, in the same way, having an acceptable number of rotating bonds helps in the adaptation and flexibility alignment of such molecules within the target active sites. Additionally, having a partition coefficient  within −0.5 to ≤5, and both TPSA and MR values below 140 and 130, respectively, generally indicates a good probability of penetration through cell membranes, gastrointestinal penetration, and hence bioavailability. Fortunately, all of the test molecules (except molecule 11) were found to not be a substantial substrate for permeability glycoprotein  and having an Abbott oral bioavailability score above zero, indicating their high probability of medicinal impact and biological activities in clinical trials . Finally, all the test molecules pass the medicinal chemistry filters of Lipinski. In the same way, all the test molecules also pass (except molecule 6) the Pan-assay of interference compounds (PAINS), indicating their high probability of being a drug candidate for drug discovery and development, as shown in Codonopsis pilosula. It was also reported in rye grass (Lolium perenne L.) [Codonopsis lanceolata, Panax gingseng [Streptomyces species [Streptomyces strain SA3097 and showed cytotoxic activity in vitro against the cancer cell lines, human lung carcinoma (LU99) and human breast cancer (MCF-7) [Streptomyces species isolated from the soils of Nepal using an untargeted metabolomics study [Perlolyrine (11) is a β-carboline alkaloid that was previously isolated from the roots of enne L.) , Codonopgingseng , and rec species . On the  (MCF-7) . Moreovecs study .Rhodococcus sp., an infrequently studied marine bacterium, was investigated utilizing the established process using LC-HR-ESI-MS-based analytical techniques. Herein, the OSMAC technique was used to isolate and subculture the actinomycete, Rhodococcus associated with the Red Sea soft coral Nephthea sp. in three different media . The extracts were then examined for their antimalarial activity and chemical diversity. The ISP2 extract showed substantial antimalarial efficacy. On the other hand, several metabolites of various structural kinds have been identified as a result of our study of this species, belonging to different classes, mainly alkaloids. In silico studies revealed that the metabolites perlolyrine (11) and 3097-B2 (12) have a high and comparable docking score within all other dereplicated molecules. This study emphasized the unique function of actinomycetes associated with marine organisms as an unexplored source of active metabolites for the creation of effective natural antimalarial drugs. By combining the analytical, statistical, and dereplication approaches for examining their bioactive principles, it is possible to effectively shorten or replace the time-consuming bioassay-guided isolation schemes and complex chromatographic procedures. Biochemometrics approaches have been widely used in natural product research as an initial evaluation step in the process of finding new drugs from actinomycetes. Targeting the bioactive antimalarial metabolites in complicated crude extracts can therefore be more successful with greater integration with in silico technologies such as neural network-based virtual screening. Using elicitation approaches, cryptic gene clusters in microorganisms could be a new approach to finding new antimalarial drug leads.


ABCG2, CD44, SPP1) and milk production performance on Chinese Holstein cattle. The results showed that the different genotypes of these 10 SNPs from the ABCG2, CD44, and SPP1 genes significantly affected the milk production performance of Chinese Holstein cattle in terms of milk yield, milk fat percentage, milk protein percentage, somatic cell score, and urea nitrogen content. The ABCG2, CD44, and SPP1 genes could be selected for marker-assisted selection, which is of great significance for future precise molecular breeding.Chinese Holstein cattle are the main breed of dairy cows in China. This work performed an association analysis between polymorphisms of three candidate genes  were screened in this study for SNPs association analysis with production traits in 999 Holstein cattle. In this research, flight mass spectrometry genotyping was used to detect the polymorphism of SNP seats. It was shown that four, four, and two single nucleotide polymorphisms (SNP) loci were detected for the ABCG2, CD44, and SPP1 genes, respectively, and the different genotypes of these 10 SNPs significantly affected the milk production performance of Chinese Holstein cattle in terms of milk yield, milk fat percentage, milk protein percentage, somatic cell score, and urea nitrogen content. Among them, ABCG2-G.80952G > T locus, ABCG2-G.120017G > A locus and CD44-G.2294G > C locus had significant effects on somatic cell score (p < 0.01). Cows with GG genotypes at ABCG2-G.80952G > T locus, AA and GG genotypes at ABCG2-G.120017G > A locus, and GG genotypes at CD44-G.2294G > C locus had lower somatic cell scores. The present study elucidated that ABCG2, CD44, and SPP1 could be selected for marker-assisted selection and will benefit for future precise molecular breeding.Based on our results of genome-wide association analysis, we performed gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis; three candidate genes ( The performance of dairy cows mainly refers to milk production traits. Milk-producing traits have always been the more concerned quantitative economic traits in dairy farming, which are jointly controlled by micro-efficiency polygenes and are susceptible to environmental influences . Due to SNP is the third generation of molecular markers after restriction fragment length polymorphism (RFLP) and microsatellite polymorphism (MPP) . SNP refIn our previous work, we used the recorded data of 86,281 test days of 8580 Holstein cows in Jiangsu, and we used the random regression measurement day model to estimate the genetic parameters of Holstein somatic cell scores (SCS). At the same time, the adjusted phenotype was used to perform a genome-wide association analysis (GWAS) of dairy cow SCS using the FarmCPU method to screen for significant SNPs and candidate genes ,8.ABCG2, CD44, SPP1) were selected in this study for SNPs association analysis with production traits in Holstein cattle. The present study elucidated that ABCG2, CD44, and SPP1 closely related to dairy cattle performance were selected for the SNPs screening, which contributed to the marker-assisted selection and is of great significance for future precise molecular breeding.Based on the results of GWAS, we performed gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis; three candidate genes  and the same parity (parity 2). The collected samples were sent to Neogen Biotechnology Co. Ltd , and DNA was extracted and genotyped from the hair follicles of 999 cows.https://david.ncifcrf.gov, accessed on 4 November 2022) and the bovine reference genome ARS-UCD1.2 in the UCSC database were used, and other online tools have carried out the GO  annotation and KEGG  pathway enrichment analysis of candidate gene functions. DNA purity and concentration were tested as follows: using NanoDroP1000 spectrophotometer can directly detect the ratio of DNA OD260/OD280, OD260/OD230 and the concentration of DNA, using the following formula to calculate the concentration of DNA: DNA concentration (ug/mL) = 50 × OD260, when the ratio of OD260/OD280 is between 1.7–1.9, it indicates that the concentration of DNA is relatively ideal.The database (DAVID) online software  for sequencing. The sequencing results were compared with Vector NTI Advance 11 software to find out the mutation sites and their specific positions. The amplification procedure was as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, renaturation at 68 °C for 30 s, and extension at 72 °C for 2 min 10 s, 17 cycles. Denaturation at 94 °C for 30 s, renaturation at 51 °C for 30 s, and extension at 72 °C for 2 min 10 s, 20 cycles. The final extension was at 72 °C for 10 min and stored at 4 °C.The screened mutation loci were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for typing detection. The genotyping detection system adopts the time-of-flight mass spectrometry biochip system (MassARRAY® MALDI-TOF System) developed by Sequenom, Inc. . This system has been widely promoted and used in the Human Genome Hapmap Project.2 ) analysis were performed for SNP sites after ABCG2, SPP1, and CD44 genotyping by SHEsis software  [ABCG2, SPP1, and CD44 gene SNPs loci. The reduced linear model excluded fixed effects of age and paternity. The specific models were as follows:ijklmnopY is the observed value of the trait, μ is the population mean, and iC is the fixed effect of calving season. jM is the fixed effect of the lactation stage, kP is the fixed effect of parity, lJ is the fixed effect of measured season, mN is the fixed effect of the test year, nG is the fixed effect of genotype, oF is the cattle field effect, and ijklmnope is the random error. Multiple comparisons between genotypes were obtained using Duncan’s method.The Hardy-Weinberg equilibrium test (HWE), genotype and allele frequency analysis, and linkage disequilibrium  . Multi-fThe profiles of three genes were in the biological process (BP), cell component (CC), and molecular function (MF) categories by GO analysis, respectively. The BP analysis showed that the three genes were classified into the cellular process, localization, metabolic process, response to stimulus, and single-organism process. The CC annotation revealed that the three genes were involved in the cell, cell part, and organelle. The MF classification of genes suggested that three genes were involved in binding. GO annotation analysis elucidated that the three genes participated in the regulation of production performance by multiple indispensable activities A.Furthermore, we performed KEGG pathway enrichment analysis to explore the most active pathways of these genes; the results revealed that these genes were related to signaling molecules and interactions, as well as the immune system and infectious diseases, which are partially but not completely involved B.ABCG2 and CD44: ABCG2-g.57261A > G, ABCG2-g.80952G > T, ABCG2-g.94683A > G, ABCG2-g.120017G > A, CD44-g.2263A > G, CD44-g.2294G > C, CD44-g.86895A > G, CD44-g.86978G > A. SPP1 was found two SNP loci SPP1-g.50265G > A and SPP1-g.50315C > T. ACG2, CD44, and SPP1, respectively.By comparing the sequencing results with the three gene sequences, four SNP loci were found, respectively, in ABCG2, CD44, and SPP1 genes with SHEsis software, and the results were obtained by D′ value and r2 value. The D′ > 0.7 and r2 > 1/3 might suggest a sufficiently strong LD to be available for plotting [2 values among the different SNP loci of the ABCG2, CD44, and SPP1 genes listed in ABCG2 gene ABCG2-g.57261A > G and ABCG2-g.94683A > G, ABCG2-g.120017G > A, ABCG2-g.80952G > T and ABCG2-g.120017G > A are in a highly linked state , respectively, and the linkage degree between the remaining loci is relatively low , and there is no high degree of linkage between other loci chain relationship   C.ABCG2 gene, ABCG2-g.57261A > G and ABCG2-g.80952G > T were located in intron 1, ABCG2-g.94683A > G in intron 5, and ABCG2-g.120017G > A in intron 13. ABCG2 gene has three genotypes for each SNP locus The dominant genotypes of ABCG2-g.57261A > G, ABCG2-g.80952G > T, ABCG2-g.94683A > G and ABCG2-g.120017G > A were GG, GT, AG, and AG, respectively. After χ2 test, the 4 SNPs of ABCG2 gene all reached HWE (p > 0.05). Among the four SNPs of CD44 gene, CD44-G.2263a > G and CD44-G.2294G > C were located in intron 2, and CD44-G.86895A > G and CD44-G.86978G > A were located in exon 17. CD44-g.2294G > C, CD44-g.2263A > G, CD44-g.86895A > G and CD44-g.86978G > A also had three genotypes, respectively. The dominant gene of CD44-g.2263A > G, CD44-g.86895A > G and CD44-g.86978G > A was AA, and the CD44-g.2294G > C was CG. Similarly, the two SNP loci of SPP1 also had three genotypes, respectively, and the dominant genotypes were GG and CT. After χ2 test, all SNP loci of the three genes were in HWE (p > 0.05) (As shown in  > 0.05) .ABCG2-g.57261A > G locus on milk fat rate reached a significant level (p < 0.01). The milk fat rate of GG- type cows was the highest, and the AA genotype was the lowest. The ABCG2-g.94683A > G locus had a highly significant effect (p < 0.01) on the urea nitrogen content in milk, which was significantly higher in the milk of AA-type cows than in AG and GG types. The ABCG2-g.80952G > T locus had a highly significant (p < 0.01) effect on milk yield, milk protein rate, somatic cell score, and urea nitrogen in cows, with TT-type cows having significantly lower measured daily milk yield than GG and GT types, while TT-type cows had significantly higher milk protein rate, somatic cell score, and urea nitrogen than GG and GT types. ABCG2-g.120017G > A had significant effects on milk yield, milk protein percentage, somatic cell score, and urea nitrogen (p < 0.01) and had significant effects on milk fat percentage (p < 0.05). The milk protein percentage, milk fat percentage, somatic cell score, and urea nitrogen of AG cows were significantly higher than those of AA and GG cows. A square visual diagram of ABCG2 gene difference expression is shown in The correlation analysis results of the four SNP loci of the BCG2 gene with different genotypes of the Holstein cattle somatic cell score, urea nitrogen in milk, daily milk yield, milk protein rate, and milk fat rate are shown in CD44 gene with somatic cell score, milk urea nitrogen, measured daily milk yield, milk protein percentage, and milk fat percentage of Holstein cattle are shown in CD44-g.2263A > G locus on milk urea nitrogen reached a very significant level (p < 0.01), and the urea nitrogen content of AA cows was significantly higher than that of AG and GG genotypes. The CD44-g.86978G > A locus had a very significant effect on the milk fat rate of dairy cows (p < 0.01), and the AA type was the highest. CD44-g.2294G > C locus had extremely significant effects on SCS and milk urea nitrogen contents (p < 0.01) and had significant effects on milk fat percentage (p < 0.05). CG-type cows had a higher milk fat percentage and milk urea nitrogen content, which were significantly higher than CC-type cows and CG-type cows; CC-type cows and CG-type cows had higher SCS. The CD44-G.86895A > G locus had significant effects on milk yield, milk fat rate, protein rate, and milk urea nitrogen content of dairy cows (p < 0.01). The milk yield, milk fat percentage, and milk urea nitrogen content of GG dairy cows were the highest, as they were significantly higher than those of other genotypes, and the protein percentage of AA and AG cows was the highest. A square visual diagram of CD44 gene difference expression is shown in The association analysis results of different genotypes of the 4 SNP loci of SPP1 gene between different genotypes and Holstein cattle somatic cell score, urea nitrogen in milk, daily milk yield, milk protein rate, and milk fat rate are shown in SPP1-g.50265G > A locus had a significant level of effect on milk yield p < 0.05) and had a highly significant effect on milk urea nitrogen content (p < 0.01). The milk yield and milk urea nitrogen content of the AG and GG cows were significantly higher than those of the AA cows. The SPP1-g.50315 C > T locus had a significant effect on the milk fat rate of dairy cows (p < 0.05), and the CT-type and TT-type were significantly higher than the CC-type. A square visual diagram of SPP1 gene difference expression is shown in The correlation analysis results of the two SNP loci of the ABCG2 gene polymorphism and milk yield and milk composition of Kalanfris cattle and found that the three genotypes of SNP on exon 14 had significant effects on the milk fat percentage of cows [ABCG2 had a significant impact on high milk fat yield, high fat, and protein percentage [ABCG2 gene, and all these polymorphisms were located in the intronic region. Existing studies have shown that introns can generate splicing signals through their own sequence properties to affect transcript splicing, thereby affecting protein sequences and individual phenotypes [ABCG2-G.57261A > G locus had a significant effect on the milk fat percentage of dairy cows, and GG type was the highest. Milk urea nitrogen content in the AA genotype of ABCG2-G.94683A > G locus was significantly higher than that in the AG and GG genotypes. Milk protein percentage, somatic cell score and urea nitrogen of cows carrying ABCG2-G.80952G > T TT genotype were significantly higher than those of the GG and GT genotypes. Milk protein percentage, milk fat percentage, somatic cell score, and urea nitrogen were significantly higher in cows carrying the AG genotype at the ABCG2-g.120017G > A locus than in the AA and GG types. The effects of ABCG2-G.80952G > T and ABCG2-G.120017G > A on production traits of dairy cows showed similar trends, probably because of the high linkage relationship between them. The results of this study show that ABCG2 polymorphisms have significant effects on dairy cows’ milk fat rate, milk yield, milk protein rate, and other production traits, which are basically consistent with previous studies. In the selection of dairy cows, the selection of ABCG2-g.57261A > G site GG type and ABCG2-g.120017G > A site AG type dairy cows should be increased in view of improving milk fat rate. In order to improve milk production and milk protein rate of dairy cows, the selection of TT-type cows at ABCG2-g.80952G > T site and AG-type cows at ABCG2-g.120017G > A site can be strengthened. However, the SCS of cows with these two genotypes are also relatively high, which may increase the burden of udder function with the increase in milk production, aggravate the shedding of mammary epithelial cells, and increase the SCS of cows. Olsen [ABCG2 gene in exon 9, resulting in a mutation of the amino acid from tyrosine to cysteine, but the differences in the five lactation performances among the three genotypes were not significant. In the selection of dairy cow genotypes, it is necessary to comprehensively select the SNPs genotypes corresponding to the target traits, so we are required to use different methods to find more SNP loci that affect dairy cow traits.Previous studies analyzed the association between  of cows . Tantia rcentage . In thisenotypes . The resCD44 have focused on disease treatment. CD44 gene rs13347 polymorphism is significantly associated with elevated cancer risk in Asians [CD44 gene has been reported to reduce proinflammatory cytokines [CD44 antibodies reduced inflammation [CD44 in cattle have shown that CD44 is related to dairy production traits. Jiang [CD44 gene could affect the synthesis of triglyceride in bovine mammary epithelial cells, thereby affecting the lipid content in milk. A total of 4 SNP loci were found in this study, CD44-g.2263A > G and CD44-g.2294G > C were located in the intron region, CD44-G.86895A > G and CD44-G.86978G > A were located in the exon region, the CD44-g.86895A > G site is a nonsense mutation, and the CD44-g.86978G > A site is a missense mutation. Association analysis found that the milk urea nitrogen content of cows with CD44-g.2263A > G locus AA genotype was significantly higher than that of AG and GG genotypes. The CD44-g.86978G > A locus had a significant effect on milk fat percentage in cows, with the AA type having the highest. Cows with the CD44-G. 2294G > C CG genotype had a higher milk fat percentage and milk urea nitrogen content, which were significantly higher than those with CC and CG genotypes, while cows with CC and CG genotypes had higher SCS. Cows carrying the CD44-g.86895A > G locus GG genotype had the highest milk yield, milk fat percentage, and milk urea nitrogen content, which were significantly higher than other genotypes, while cows with AA and AG genotypes had the highest protein rate. From the results of this study, the genotypes of the three SNP loci have a significant impact on the milk fat rate of dairy cows, which proves that CD44 is related to the lipid metabolism of dairy cows. This result is consistent with previous studies. In order to improve milk fat percentage, the selection of dairy cows carrying CD44-G.86978G > A AA genotype, CD44-G.2294G > C CG genotype and CD44-G.86895A > G GG genotype should be strengthened. There are more and more studies on SPP1 gene polymorphism, which provides a powerful reference for disease treatment and improving livestock and poultry production.Most of the existing studies on n Asians . Deletioytokines , and treammation . Previous. Jiang  believedSPP1 gene polymorphism in Salda sheep and found that rs161844011 locus in exon 7 was associated with SCS. Liang [SPP1 gene had a reduced risk of developing breast cancer. Furthermore, SPP1 gene polymorphism was significantly correlated with lactation persistence of dairy cows, and G was its dominant allele [SPP1 gene polymorphism and milk yield of Girorando cows and found that 305-day cows carrying the T allele at position g.8514C > T had higher milk yield, but it did not reach a significant level [SPP1 genes obtained in this study were located in the intronic region. The milk yield and milk urea nitrogen content of the AG and GG genotypes of SPP1-G. 50265G > A were significantly higher than those of the AA genotypes. SPP1-g.50315 C > T loci CT and TT genotypes had significantly higher milk fat percentages than the CC types. The selection of cows carrying the G allele at the SPP1-G.50265G > A locus can be strengthened in future dairy breeding to improve milk yield. From the perspective of improving the milk fat rate of dairy cows, the selection of dairy cows carrying the SPP1-g.50315 C > T locus T allele can be strengthened.Maria  conducteS. Liang  found tht allele . Mello ant level . The SNPABCG2, CD44 and SPP1 genes and analyzed the association between the ABCG2, CD44, and SPP1 genes and Chinese Holstein production performance. The milk yield, milk fat rate, milk protein rate, somatic cell score, and blood urea nitrogen content of Holstein cattle with different genotypes of these 10 SNP loci were different. Among them, ABCG2-G.80952G > T locus, ABCG2-G.120017G > A locus and CD44-G.2294G > C locus had significant effects on the somatic cell score (p < 0.01). The present study elucidated that ABCG2, CD44 and SPP1 could be selected for marker-assisted selection and will benefit future precise molecular breeding.This work performed GO annotation and KEGG pathway enrichment analysis on the