Modern varieties of common wheat (Triticum aestivum L.) bred mainly for high productivity are often of low grain quality. The identification of NAM-1 alleles associated with high grain protein content in wheat relatives has enhanced the significance of distant hybridization for the nutritional value of T. aestivum L. grain. In this work we aimed to study the allelic polymorphism of the NAM-A1 and NAM-B1 genes in wheat introgression lines and their parental forms and evaluate the effects of various NAM-1 variants on the grain protein content and productivity traits in the field conditions of Belarus. We studied parental varieties of spring common wheat, the accessions of tetraploid and hexaploid species of the genus Triticum and 22 introgression lines obtained using them (2017–2021 vegetation periods). Full-length NAM-A1 nucleotide sequences of T. dicoccoides k-5199, T. dicoccum k-45926, T. kiharae, and T. spelta k-1731 accessions were established and registered with the international molecular database GenBank. Six combinations of NAM-A1/B1 alleles were identified in the accessions studied and their frequency of occurrence varied from 40 to 3 %. The cumulative contribution of NAM-A1 and NAM-B1 genes to the variability of economically important wheat traits ranged from 8–10 % (grain weight per plant and thousand kernel weight) to up to 72 % (grain protein content). For most of the traits studied, the proportion of variability determined by weather conditions was small (1.57–18.48 %). It was shown that, regardless of weather conditions, the presence of a functional NAM-B1 allele ensures a high level of grain protein content; at the same time, it does not significantly decrease thousand kernel weight. The genotypes combining the NAM- A1d haplotype and a functional NAM-B1 allele demonstrated high levels of productivity and grain protein content. The results obtained demonstrate the effective introgression of a functional NAM-В1 allele of related species increasing the nutritional value of common wheat. Common wheat (Triticum aestivum L.) is an importantagricultural crop that plays a key role in providing food topeople across the globe. One of the priority directions ofwheat breeding is to improve grain quality, which is primarilydetermined by the total protein content .Complex polygenic nature of the trait “grain protein content”,its variabilitywhen exposed to external factors, as well asthe negative correlation between the protein content and productivity,complicate the breeding process .In addition, low genetic diversity of modern varieties bytrait limits their use in breeding programs aimed at improvingthe nutritional value of wheat. Many related species ofT. aestivum are characterized by a higher grain protein contentcompared to cultivated varieties .New opportunities that make it possible to increase thetotal grain protein appeared in breeding with the identificationof the Gpc-B1 locus associated with protein content inwild emmer T. dicoccoides (AABB genome). The locus wasmapped in the short arm of chromosome 6B and upon detailedclarification of its localization site boundaries, a sequencewas found identified as the NAM-B1 gene belonging to NACfamily transcription factors . The genesof this family are involved in the regulation of various plantdevelopment programs, control of defense responses to bioticand abiotic stressors, and they play an important role in plantsenescence . In additionto the NAM-B1 gene, common wheat also has homologousNAM-A1 and NAM-D1 genes on chromosomes 6A and 6D.A functional NAM-B1 allele  was foundin wild emmer, providing high protein content in grain. Theallele includes three exons and two introns and encodes aprotein of 407 amino acid residues that has the conservedN-terminal region, or the NAC domain with five subdomainsand the highly variable C-terminal transcriptional activationregion . A functional NAM-B1 allele isnot found in most modern wheat varieties. Varieties have, asa rule, a 1 bp insertion in the first exon leading to a frameshift or a gene deletion  and, as aresult, to an inactive protein or its absence .Thus, a study of 218 wheat varieties from five main regionsof China did not reveal any single variety with a functionalallele of the NAM-B1 gene . Molecularcharacterization of the NAM-1 genes of Australian commonwheat varieties showed the presence of the wild allele of theNAM-B1 gene in only 2 out of 51 varieties .It was established that the NAM-A1 gene similar to NAM- B1consists of three exons; it possesses typical characteristics ofNAC-family genes and is involved in the regulation of thesame processes as NAM-B1. As a result of the analysis ofthe association of single nucleotide variants (SNPs) of theNAM-A1 gene with nitrogen remobilization from leaves andgrain protein accumulation, two functional single nucleotidesubstitutions were identified: at position 722 (T/C) and at position1509 (A/del). Based on the data obtained, a classificationof NAM-A1 haplotypes was proposed: NAM-A1a (722Cand 1509A), NAM-A1b (722C and 1509del), NAM-A1c (722Tand 1509A), and NAM-A1d (722T and 1509del) .The absence, in modern wheat varieties, of the functionalNAM-B1 allele, which provides for high grain protein contentin various environmental conditions, has strengthenedthe position of distant hybridization from a perspective ofincreased nutritional value of wheat grain. In order to enrichand to improve the common wheat gene pool, in the crossingwith T. aestivum L. varieties we used accessions of the speciesof the genus Triticum . An earlier study of genetic diversityof the collection of introgressive wheat lines using C-bandingand SSR analysis showed that in the genome of hybrid linesthe foreign genetic material is presented both in the form ofshort fragments and whole chromosomes .In this work, we aimed to study the allelic compositionof NAM-A1 and NAM-B1 genes in introgressive wheat linesand their parental forms and to evaluate the effect of differentvariants of NAM-1 genes on grain protein content and wheatproductivity traits in the field conditions of Belarus.The study included five varieties of spring common wheat; tetraploid accessions T. dicoccoides, T. dicoccoidesk-5199, T. dicoccum k-45926, and T. durum, andhexaploid accessions T. spelta k-1731 and T. kiharae of thespecies of the genus Triticum, as well as 22 introgressive lineswe had developed 1. The accessionsof foreign donors were obtained from the collection ofN.I. Vavilov All-Russian Institute of Plant Genetic Resources(VIR). Information about the pedigree of individual accessionsis not available as unpreserved .http://vavilov.elpub.ru/jour/manager/files/Suppl_Orlovskaya_Engl_27_3.pdf.Supplementary Materials are available in the online version of the paper:The plants were grown in the experimental fields of theInstitute of Genetics and Cytology of the National Academyof Sciences of Belarus, in 2017–2021 (Minsk), on sod-podzolicloamy sand soil. The characteristic of weather conditionsin the region of our experiment in 2017–2021 is presented inSupplementary Material 2. Data on average daily temperaturesand precipitation (http://rp5.by) were used to calculate thesum of active temperatures (SAT) and Selyaninov’s hydrothermalcoefficient (HTC) .When harvesting, the following traits were taken into account:plant height, the number of productive shoots per plant, thelength of the main spike, the number of spikelets and grainsof the main spike, grain weight per spike and plant, as wellas thousand kernel weight. To assess the traits, 15 plants ofeach genotype were randomly selected.To sequence full-length NAM-A1 gene sequences andthe first exon of NAM-B1, specific primers developed byR. Yang et al. (2018), were used. The sequencing reactionwas performed using the BigDye Terminator v. 3.1 CycleSequencing kit ; the separationof sequencing reaction products was carried out using theABI PRISM 3500 genetic analyzer (Applied Biosystems).Alignment of nucleotide sequences and homology analysiswere performed using the BLAST analyzer of the NationalCenter for Biotechnology Information, USA (http://www.ncbi.nlm.nih.gov/BLAST). The Chinese Spring variety was used asa reference sequence, which, according to the literature data,is the carrier of the NAM-A1a haplotype (722C and 1509A)and the NAM-B1 mutant allele (T insertion at position +11).The total protein content in wheat grain was determinedin accordance with GOST 10846-91 (2009) at the CentralRepublican Laboratory of the State Institution “State Inspectoratefor Testing and Protection of Plant Varieties” . The essence of the method lies in the mineralizationof organic matter with sulfuric acid in the presence of a catalystwith the formation of ammonium sulfate, the destruction ofammonium sulfate with alkali with the release of ammonia,the stripping of ammonia with water vapor into a solution ofsulfuric or boric acids followed by titration.The results of the experiment were summarized usingdescriptive statistics methods; two-way analysis of variance,regression, and correlation analyses (the Spearman’srank correlation coefficient was used). Statistical procedureswere implemented in the software packages Statistica 10.0,MS Excel, and web application SNPstats. A quantitativecontribution of individual factors of the dispersion analysiswas calculated on the basis of the relation of the absolute factorvariance to the sum of variances of that factor and otherfactors, according to the formulae given in .Allelic polymorphism of NAM-A1 and NAM-В1 genesFull-length NAM-A1 gene sequencing was carried out inthe parental varieties and accessions of species of the genusTriticum, in 22 introgressive lines developed on their basis.Among the parental forms, we detected haplotype NAM-A1ain Festivalnaya and Rassvet varieties; accessions T. durum,T. dicoccum k-45926, T. dicoccoides k-5199, T. dicoccoides,and T. kiharae; haplotype NAM-A1c in T. spelta k-1731; andhaplotype NAM-A1d in varieties Saratovskaya 29, Belorusskaya80, and Pitic S62 .A comparative analysis showed that NAM-A1a nucleotidesequences of T. dicoccoides k-5199, T. dicoccoides, andT. kiharae accessions did not have 100 % similarity with theNAM-A1a sequence of T. aestivum (MH160778) from theGenBank database. The sequences of wild emmer accessions(T. dicoccoides k-5199 and T. dicoccoides) that we studieddiffered from NAM-A1а of T. aestivum (MH160778) in twoSNPs: positions 538 bp (C/A in exon 2) and 1139 bp (G/Tin exon 3) (99.9 % identity level). The SNP 1139 bp of G/Tresults in the replacement of asparagine with tyrosine in theamino acid sequence of protein. The level of similarity ofNAM-A1а of the T. kiharae accession with NAM-A1а fromthe GenBank database (MH160778) was 99.7 % and differedfrom it in six SNPs: in the positions of 189 bp (C/A in exon 1),306 bp (A/C in intron 1), 1133 bp (G/A in exon 3), 1271 bp(G/T in exon 3), 1414 bp (C/G in exon 3), and 1491 bp (G/Cin exon 3). Three of these SNPs lead to changes in the aminoacid sequence of the protein: a G/A substitution in the positionof 1133 bp leads to the replacement of alanine with threonine;G/T in the position of 1271 bp results in the replacement ofalanine with serine; and G/C in the position of 1491 bp replacesglycine with alanine.The NAM-A1 gene sequence of T. dicoccum k-45926and T. durum accessions was completely homologous tothe NAM- A1a allele sequence (GenBank: MH160778); theNAM- A1 sequence of the T. spelta k-1731 accession correspondedto the NAM-A1c allele (GenBank: MH MH160777).Nucleotide sequences of the NAM-A1 gene of T. kiharae,T. spelta k-1731, T. dicoccoides k-5199, and T. dicoccumk-45926 accessions, which we described for the first time,were registered with the International GenBank Database.Analysis of NAM-A1 gene sequencing data in introgressivewheat lines showed that 54.6 % of the lines had the NAM-A1dhaplotype; 36.4 % – the NAM-A1a haplotype; and 9.1 % –the NAM-A1c haplotype. It should be noted that wheat lineswith foreign genetic material inherited, as a rule, the NAM-A1gene of the original wheat variety but there were a numberof exceptions . A haplotypecorresponding to a related species was identified in the linesdeveloped using T. spelta k-1731 ;in line 226-7 T. durum × Belorusskaya 80 (NAM-A1a); and inline 20-1 T. kiharae × Saratovskaya 29 (NAM-A1a). Amongthe lines developed using T. dicoccoides, only lines 11-1 and13-3 obtained as a result of crossing with the Festivalnayavariety inherited the NAM-A1 gene from wild emmer with theSNPs only characteristic of it at positions 538 bp and 1139 bpof the nucleotide gene sequence.Analysis of the sequenograms of the first exon of theNAM-B1 gene in the studied genotypes revealed a functionalallele (F ) in all the accessions of related species, except forT. durum, for which the NAM-B1 allele was not identified, andin 5 out of 22 introgressive wheat lines . All parental varietiesand most of the wheat lines with the foreign genetic material(77.3 %) had a mutant allele (NF ).Effects of NAM-A1 and NAM-B1 geneson the productivity traits and the grain protein contentGenotyping results of common wheat introgression lines andparental forms by NAM-A1 and NAM-B1 genes were comparedwith the results of field trials and the data on grain proteincontent for 2017–2021. The effect of genotype, environmentalfactors, and their interaction were assessed using the generallinear model (GLM) of two-way analysis of variance .The combination of NAM-A1/B1 genes produces a statisticallysignificant effect on the manifestation of all nine traitsstudied, while exceeding a contribution of individual NAM-1genes to the variability of protein content, plant height, thenumber of spikelets per spike, and thousand kernel weight. NAM-B1 does notsignificantly affect the spike length of wheat, while NAM-A1or the combination of NAM-A1/B1 determine more than halfof the observed trait variationThe greatest length of the main spike relative to other haplotypesis typical for NAM-A1c plants (9.86 cm in averageover the 5-year observation period), and in the case of thec/F combination, this indicator increases up to 10.87 cm.The effect of NAM-B1 allelic variants on the variability ofthe number and weight of grains in the spike is almost threetimes higher than the effect of NAM-A1 haplotypes, and it isone-and-a-half times higher in the case of the NAM-A1 andNAM-B1combinationThe spike productivity in the group of samples with the wildNAM-B1 allele was significantly lower than in the genotypeswith a mutant allele . Thus, in thespike of the vast majority of genotypes with a functional allele(7 out of 10), the number of grains did not reach 30 pieces,while in the spike of genotypes with a mutant allele, as a rule,30–40 grains were formed. A significant variation in spikeproductivity traits can be noted in both groups. Individualgenotypes with a functional allele demonstrated high indicesby these traits (lines 19 of T. kiharae × Saratovskaya 29 (d/F )and 15-7-1 of T. dicoccoides × Festivalnaya (a/F )). In allthree variants of variance analysis, thousand kernel weightand the weight of grains per plant are the traits with a low(up to 10 %) contribution of genetic variances; at that, theweight of grains per plant is statistically independent of theNAM-A1 haplotype and thousand kernel weight is independentof the NAM-B1 allele. Variability of the protein contentin grain is 70 % associated with NAM-B1 polymorphism,and a contribution of NAM-A1 is significantly lower and itslightly increases when considering a combination of genealleles . Thus, on average,during the 4-year period, the highest grain protein content inthe groups with different NAM-A1 haplotypes was observedin the case of NAM-A1a genotypes (21.53 %); in the groupswith differentNAM-B1 variants – in the case of the functionalNAM-B1 allele (22.53 %); and the maximum amount of proteinwas noted (23.72 %) in the case of the a/F combination.The strength and direction of the relationship between thedifferent alleles of NAM-1 genes and economically valuabletraits was assessed using the Spearman’s correlation coefficient(Table 1).A medium-strength relationship was found between thegrain protein content and the allelic variants of NAM-1 genes,but only the correlation with NAM-B1 is statistically significant(see Table 1). Apart from that, significant dependencewas established between the NAM-B1 allelic variants and thetilling capacity, the number and weight of grains per spike.The NAM-A1 haplotype significantly correlated only with thespike length; association with other productivity traits wasweak. Variance analysis results also showed that NAM-B1allelic variants produce a significantly greater impact on thevariability of grain protein content, the number and weight ofgrains per spike, and productive tilling capacity than NAM-A1. It should be noted that bothvariance and correlation analyses established a low correlationof NAM-1 genes with the traits “plant height”, “the number ofspikelets per spike”, “grain weight per plant” and “thousandkernel weight” .In total, six combinations of NAM-A1/B1 alleles wereidentifiedin the accessions under study and their frequenciesvaried from 40 (d/NF) to 3 % (c/NF ). Some combinations ofalleles are represented by a small number of samples , and therefore, it does not seem possible tospeak about a significant difference of traits in plants withsuch combinations of allelesMean values of productivity traits of the genotypes carryingvarious combinations of NAM-A1/B1 genes are demonstratedin Table 2.The genotypes combining different NAM-A1 haplotypeswith the functional NAM-B1 allele were taller and of a highertilling capacity than the plants with a corresponding haplotypecombined with the mutant NAM-B1 allele (see Table 2). Themaximum values for these traits are typical for c/F accessions:93.36 cm and 3.86 pcs. An opposite trend was revealed forspike productivity traits: all three NAM-A1 haplotypes in combinationwith the functional NAM-B1 allele had a low numberand weight of grains per spike (see Table 2). The sampleswith NAM-A1a and NAM-A1c haplotypes in combinationwith the functional allele demonstrated a slight decrease inthousand kernel weight compared with the genotypes combiningNAM- A1a and NAM-A1c and the mutant allele (seeTable 2).For the plants with the NAM-A1d haplotype, an increase inthousand kernel weight in the group with the functional allelewas revealed (40.20 g for d/NF genotypes and 44.34 g for d/Fgenotypes). It can be noted that NAM-A1a accessions were theleast productive by this trait both in the group with functional(37.97 g) and non-functional (38.93 g) NAM-B1 alleles. Thepresence of the mutant allele of the NAM-B1 gene leads toa decrease in the protein content relative to the combinationwith the functional allele: for NAM-A1а haplotypes – by3.6 % on average; for NAM-A1d by 2.3 %; and for NAM-A1c,by 0.6 % . The maximum amount of protein in grain wasaccumulated by the lines with the a/F combination and theminimal amount – by the lines with the d/NF and c/NF combinations(see Table 2).The role of weather conditions and their relationshipwith NAM-1 genes regarding variabilityof productivity traits and grain protein contentRegardless of the model used , the high statistical significance of the contributionof weather conditions of the year of growth was shownfor all the traits. However, the predominant role of this factor(> 50.0 %) was found only in the variability of traits “plantheight” and “the number of spikelets per spike”. The interactionof the NAM-A1/B1 gene combination with weather conditionssignificantly affects all wheat productivity indices. Thecontribution of this interaction is especially high (more than60 %) in the variance of the traits “grain weight per spike andplant”, “thousand kernel weight” and “tilling capacity”. Theimpact of the interaction “NAM-B1 × weather conditions” onproductivity traits is lower than the contribution of other factors. The only exception is thetrait “number of spikelets per spike”, for which a contributionof genotype-environmental interactions with NAM-B1 is 3 %and with NAM-A1 it is statistically insignificant ( p = 0.23). Itshould be noted that the interaction of weather conditions andall three genetic factors did not affect grain protein content.Different genotypes may respond to changes in environmentalconditions differently. Additivity of NAM-A1/B1 effects andweather conditions on the manifestation of the traits studiedwas tested using the log-additive linear regression model ofthe SNPstats web application. For the traits “plant height”,“tilling capacity”, “the number of spikelets per spike”, “weightof grains per plant”, and “thousand kernel weight”, the mutualenhancement of the effects of two factors was found: NAMA1/B1 alleles and weather conditions. The additive interactionof the NAM-A1/B1 genotype and the environment is statisticallyinsignificant for the variability of traits “spike length”,“the number and weight of grains in the main spike”, and“protein content”.Variability of the studied traits of genotypes carrying variousNAM-A1/B1 combinations was assessed under the conditionsof different growing seasons relative to the productivityof plants with d/NF alleles in 2017 . With aview to searching for meteorological factors that determinegenotype-environmental interaction, the following was carriedout: (1) a comparative analysis of the ranking of alleliccombinations in 2017–2021 conditions; (2) a correlation analysisof productivity traits with meteorological parameters.An analysis of correlation coefficients showed that mostof the traits studied were significantly influenced by weatherconditions during the grain filling stage – HTC and SAT inJuly. The closest association was found between the HTC inJuly and the plant height, the number of spikelets per spike,the weight of grains per plant and thousand kernel weight . During the grain filling stage, there is an increasedsupply of mineral and organic substances to the wheat grain;unfavorable conditions during this period significantly worsenits quality and reduce yields. The optimal HTC in July (1.2) istypical for the 2020 season. In July 2021, there was an increasein air temperature and a lack of precipitation compared to thenorm, and the HTC was only 0.4, characterizing this periodas dry. In other years, the HTC in July significantly exceededthe norm and amounted to 2.8, 2.8 and 2.4 in 2017, 2018 and2019 respectively. It can be noted that the air temperature andprecipitation throughout the growing season of 2020 wereclose to the climatic norm, which contributed to the maximumrealization of wheat productivity .The conditions of 2021  ledto a significant decrease in the yields of the genotypes understudy .“Plant height” is a trait with an additive contribution ofNAM-1 genes and weather conditions. The presence of thefunctional NAM-B1 allele and the NAM-A1c haplotype isassociated with a statistically significant increase in culm.Maintained soil moisture (HTC > 1) during the “booting–flowering” period stimulates plant growth, while excessiveprecipitation in July-August, on the contrary, negatively affectsplant height. For example, the average height of wheat plantsin 2019 (cold and wet summer) decreased by 10.6–14.5 cm, and in 2020 (the year close to the climatic norm) it increasedby more than 20 cm relative to 2017 values. Plants with theNAM-A1c haplotype are the least sensitive to environmentalfactors that reduce the stem height, while the growth of theculm reaches 10–25 cm under favorable conditions.It was shown that the tilling capacity of plants is negativelyaffected by soil waterlogging and a large amount of precipitationin August, which is associated with the inhibition ofsecondary growth processes, lodging and disruption of gasexchange in the root system. The trait is positively correlatedwith the HTC of May and the SAT of July; that is, the absenceof frosts and drought at the tillering stage and the high intensityof photosynthesis at the maturation stage. Plants with thefunctional NAM-B1 allele form more stems under favorableconditions and significantly reduce tillering capacity underspring drought conditions .SNPstats models failed to explain spike length variabilityeither by the additive interaction of NAM-A1 and NAM-B1alleles or the interaction of genetic factors and growing conditions.Both the spike length and the number of spikelets perspike are influenced by the conditions observed at the stagesof booting and heading, namely, the HTC in June. Warm anddry conditions in May-June 2018 were accompanied by adecrease in the average length and the number of spikeletsper spike. However, for late-ripening genotypes with the c/Fcombination, a decrease in the number of spikelets turnedout to be statistically insignificant. The number and weightof grains in the main spike depend on the optimal moistureconditions at the stage of germination and tillering, the sum ofactive temperatures at the stage of heading and flowering. Lowyields of the main spike were noted in the conditions of 2017and 2021, which were characterized by a low hydrothermalcoefficient in May, but differed in temperature and hydrologicalregimes at the subsequent stages of the growing season. In2021, the maximum number of grains formed in the main spikewas noted and in 2017 – the minimum one .The weight of grains per plant and thousand kernel weightare the most important indicators of wheat yield. In cases of asignificant difference in the mean values of traits in the plantscarrying NAM-A1/B1 combination variants, genetic factorsdo not produce an additive effect. Regardless of the year ofcultivation, NAM-A1a and NAM-A1c haplotype combinationswith a functional NAM-B1 variant are associated with a slightdecrease in thousand kernel weight relative to combinationswith a non-functional gene variant. There were no significantdifferences found between d/F and d/NF genotypes by seedproductivity traits . Late-ripening genotypescarrying the NAM-A1с haplotype significantly increase thousandkernel weight in the case of a combination of high HTCin July and August, which was observed in 2019–2020 .For the formation of grain, wheat plants remobilize nitrogenand carbohydrates from the flag leaf, and therefore, the proteincontent in grain depends on the intensity of photosynthesisand the photosynthetic surface area . HighHTC in June may produce a negative effect on the proteincontent through a decrease in these parameters – low solarinsolation as a result of high cloudiness and an increase in thelamina damage area caused by phytopathogens under highhumidity conditions. Under unfavorable conditions for overallproductivity in 2021, plants with the functional NAM- B1 alleleaccumulated more protein than the genotypes with a mutantgene variant .The common wheat varieties we studied had the NAM-A1dor NAM-A1a haplotype and the mutant allele NAM-B1,which is consistent with the literature data. For example, inthe collection of Australian wheat varieties, accessions withhaplotypes NAM-A1a (50.1 %) and NAM-A1d (28 %) werethe most frequently occurring, while NAM-A1b (1.9 %) wasthe least common . F. Cormier et al. (2015)revealed in their studies that the NAM-A1d haplotype is typicalfor most modern European wheat varieties, while NAMA1ais more common among the varieties with high bakingproperties. Wheat samples with the NAM-A1b haplotype werenot found in the collection we studied. The works of foreignscientists provide data on the low frequency of occurrence ofthis haplotype. For example, in the collection of 795 wheat accessions,only one accession with NAM-A1b was found. Thereis an assumption that this haplotype has appeared recently asa result of recombination between NAM-A1a and NAM-A1d.The presence of functional NAM-B1 alleles only in wheatrelatives among the parental forms we studied is confirmedby the data of other researchers. Thus, in the work of C. Uauyet al. (2006b), a functional NAM-B1 allele was found in all42 analyzed accessions of T. dicoccoides and in 17 out of19 accessions of T. dicoccum (Schrank) Schuebl., while all57 studied varieties of durum wheat and 34 of common wheateither contained a 1 bp insertion or had a gene deletion. Among367 common wheat accessions of the INRA core collection(France) selected from 3942 genotypes of different geographicorigin, only 5 contained functional NAM-B1 alleles . Due to the fact that currently cultivatedvarieties are, as a rule, missing a functional NAM-B1 allele,five introgressive lines of wheat with the allele that we havedeveloped are of great interest from the point of view of theirability to enhance the quality of wheat grain.Analysis of differences in the mean values of quantitativetraits in the groups carrying different NAM-A1 haplotypesshowed that during all years of the experiment, NAM-A1aplants had a short spike, low grain weight per plant andthousand kernel weight, and high protein content in grain;NAM-A1c – the maximum plant height, tilling capacity andspike length but low grain content and seed weight per spike;NAM-A1d – the minimum plant height, number of productiveshoots, and the maximum values of spike and plant productivitytraits. A number of studies have shown that the presenceof the NAM-A1a haplotype is associated with a shorter periodof grain filling and a more intense process of nitrogen remobilization,which leads to increased protein content; but, at thesame time, to a decrease in the number of grains per spikeand thousand kernel weight, while the presence of NAM-A1cor NAM-A1d leads to an increase in the grain filling period,which results in an increase in the amount of nitrogen uptakeand wheat yield .There is an assumption that NAM-A1a is a functional variant of the NAM-A1 gene, which is rarely found in elite modernwheat varieties the breeding of which was carried out mainlyfor productivity purposesThe data we obtained on a high level of protein accumulationin grain in wheat genotypes with the wild NAM-B1 allelecoincide with the results of many foreign scientists. The studyof a series of almost isogenic lines based on common and durumwheat in different countries across the globe  allowed us to concludethat the introgression of a functional NAM-B1 allele into thegenome of cultivated wheats of both ploidy levels leads to anincrease in the content of protein and key minerals in grain,an improved harvesting nitrogen index, and increased proteinharvest .Throughout the entire observation period, the genotypeswith a functional NAM-B1 allele were characterized by higherplant height and tilling capacity, but lower indicators by spikeand plant productivity traits compared to the genotypes carryinga nonfunctional allele. However, we did not find anysignificant impact of the NAM-B1 allele state on thousandkernel weight. In the literature, there are data on both positiveand negative effects of the wild-type NAM-B1 allele onthe main components of wheat productivity . A significantincrease in the productive stems in common wheat lines witha functional NAM-B1 allele that we established was also describedin the works of other scientists .Tillering capacity is known to be determined by many environmentalfactors, including nitrogen availability . It is possible that a functional NAM-B1 allelecontributes to the formation of productive stems due to thefact that it improves nitrogen metabolism . According to the review article that summarizes thedata of 25 studies on the influence of allelic variants of theNAM-B1 gene on 50 wheat traits, 36 % of the studies did notshow any significant differences in thousand kernel weightbetween the genotypes with different allele variants of thisgene; correspondingly, 23 and 41 % of the studies revealedboth a significant decrease and a significant increase in thisindicator in the lines with a functional allele. It should benoted that the majority of studies (79 %) did not establish astatistically significant effect of NAM-B1 polymorphism onwheat yield, and only 4 % showed a decrease in the yield oflines with a functional allele . This factis explained by a positive effect of a functional allele on theformation of productive stems, since it is precisely due to hightillering capacity that no significant decrease takes place ingrain yield, even despite low spike productivity .Evaluation of the effect of six combinations of NAM-A1/B1alleles on the level of manifestation of a number of economicallyvaluable wheat traits showed that the maximum heightand tillering capacity are characteristic of c/F genotypes; d/ NFgenotypes are responsible for high productivity of spikes andplants; and the highest grain protein concentration is determinedby a/F (see Table 2). Similar results are presented in, regarding the influence of the alleliccomposition of NAM-1 genes on the efficiency of nitrogenuse, productivity and protein content in wheat grain. In thisstudy, the Mace variety, which has a non-functional NAM-B1allele and the NAM-A1d haplotype, significantly outperformedother genotypes in terms of yield but had the minimum grainprotein content. Varieties combining a functional allele withdifferent NAM-A1 haplotypes had high grain protein content,while a negative correlation was found between the mutantallele and grain protein content, regardless of the NAM-A1haplotype .The combination of NAM-A1/B1 genes had a significanteffect on the formation of all the traits of wheat under study,while the grain weight per plant was statistically independentof the NAM-A1 haplotype, and the spike length and thousandkernel weight were statistically independent of the NAM-B1gene allele. It is also important to note a small joint contributionof these two genetic factors to the variability of mostproductivity traits studied (7.59–18.75 %), but the predominanceof NAM-A1/B1 in the variability of protein content ingrain (72 %) should be mentioned at that.It is known that, in addition to the genetic control, crop andits components are significantly influenced by environmentalfactors . Ourstudy shows a high statistical significance of the contributionof weather conditions to the variability of wheat quantitativetraits. A particularly high role of this factor (more than 50 %)was revealed for the dispersion of traits with a wide reactionrate and a high variation coefficient strongly dependent on theambient temperature and the amount of precipitation – “plantheight” and “the number of spikelets per spike”. For the restof the traits studied, the share of variability determined byweather conditions was significantly lower and was in therange of 1.57–18.48 %; and the impact of the “NAM-A1/ B1 ×weather conditions” interaction was of great importance(> 60 %). According to the data we obtained, the effect of the“NAM-B1 × weather conditions” interaction on productivitytraits is lower than the contribution of other factors; and for thetraits “the length of spike”, “the number of grains per spike”,and “the grain protein content”, it is statistically insignificant.It should be emphasized that NAM-B1 has a high degree ofimpact on the level of protein accumulation (70 %), which,along with a low contribution of weather conditions and theabsence of genotype-environment interaction, indicates theeffectiveness of functional allele introgression for the improvementof the quality of wheat grain in Belarus. A number ofworks by foreign scientists have shown a significant impact ofthe “NAM-B1 × environment” interaction on wheat productivityand grain quality traits. For example, the study by , established that differences in environmentalconditions affect the expression of the NAM-B1 gene, whichlimits the use of functional allele introgression for increasingthe level of protein accumulation in spring wheat grains inthe regions with a short vegetation period. However, whenstudying the effects of NAM-B1 on the total protein content ingrain and the main yielding components of common wheat inArgentina, for most of the traits studied (including thousandkernel weight and protein content), no significant interaction“NAM-B1 × environment” and “NAM-B1 × genotype” wasshown, while the impact of “genotype” and “environment”factors was statistically significant .For the first time, full-length nucleotide NAM-A1 sequences ofaccessions T. kiharae, T. spelta k-1731, T. dicoccoides k-5199,and T. dicoccum k-45926 were identified and registered withthe international molecular database GenBank. The studiedaccessions of related common wheat species had the NAMA1ahaplotype, except for T. spelta k-1731 (NAM-A1c). BothNAM-A1d and NAM-A1a haplotypes are characteristic ofT. aestivum varieties. Among the parental forms, a functionalallele of the NAM-B1 gene was found only in the accessionsof related species. Introgressive lines inherited, as a rule, thevariants of NAM-1 genes of the original wheat variety. Outof 22 introgressive lines, the NAM-A1 haplotype of relatedspecies was identified in 6 lines, while a functional NAM-B1allele was detected in 5. Line 13-3 T. dicoccoides × Festivalnaya(a/F ) and line 7 T. spelta k-1731 × Saratovskaya 29(c/F ) had the NAM-А1 haplotype and the NAM-B1 allele ofT. dicoccoides and T. spelta, correspondingly.The genotyping results of introgressive lines of commonwheat and parental forms by NAM-A1 and NAM-B1 geneswere compared with the results of field trials in the conditionsof Belarus and with the analysis data on protein content ingrain of 2017–2021. The combination of NAM-A1/B1 geneshad a significant effect on the formation of all the traits ofwheat studied, while the grain weight per plant was statisticallyindependent of the NAM-A1 haplotype, and the spike lengthand thousand kernel weight were statistically independent ofthe NAM-B1 allele. A joint contribution of these two geneticfactors to the variability of economically valuable traits rangesfrom 8–10 % (grain weight per plant and thousand kernelweight) to 72 % (grain protein content).For most of the traits studied, the proportion of variabilitydetermined by weather conditions was small (1.57–18.48 %).The closest correlation was established between the majorityof traits studied and the HTC during the grain filling phase.The interaction “NAM-A1/B1 × weather conditions” determines65–71 % of the variability of wheat grain productivitytraits, while it is not significant for the grain protein content.The contribution of the “NAM-B1 × weather conditions” interactionto quantitative traits is lower than the contributionof other factors; and for the traits “spike length”, “the numberof grains per spike” and “the grain protein content” it isstatistically insignificant. It was found that the presence of afunctional NAM-B1 allele provides for a high level of proteinaccumulation in grain, regardless of weather conditions, andat the same time, it does not lead to a significant decrease inthousand kernel weight.Evaluation of the effect of six combinations of NAM-A1/B1alleles on the level of manifestation of a number of economicallyvaluable traits of wheat showed that high productivityof spike and plant but a low level of protein content in grainare characteristic of d/NF genotypes, while the highest proteinconcentration and low indicators by main productivity traitsare characteristic of a/F. The optimal combination of thewheat traits studied was established for d/F genotypes. Theresults obtained prove the effectiveness of the introgressionof a functional NAM-B1 allele of related species for increasednutritional value of common wheat.The authors declare no conflict of interest.Ahmed H.G.M., Sajjad M., Zeng Y., Iqbal M., Khan S.H., Ullah A.,Akhtar M.N. Genome-wide association mapping through 90KSNP array for quality and yield attributes in bread wheat againstwater-deﬁcit conditions. Agriculture. 2020;10(9):392. DOI 10.3390/agriculture10090392.Alhabbar Z., Yang R., Juhasz A., She M., Anwar M., Sultana N., DiepeveenD., Ma W., Islam S. NAM gene allelic composition and its relationto grain-filling duration and nitrogen utilization efficiency ofAustralian wheat. PLoS One. 2018;13(10):e0205448. DOI 10.1371/journal.pone.0205448.Avni R., Zhao R., Pearce S., Jun Y., Uauy C., Tabbita F., Fahima T.,Slade A., Dubcovsky J., Distelfeld A. Functional characterizationof GPC-1 genes in hexaploid wheat. Planta. 2014;239(2):313-324.DOI 10.1007/s00425-013-1977-y.Brevis J.C., Morris C.F., Manthey F., Dubcovsky J. Effect of the grainprotein content locus Gpc-B1 on bread and pasta quality. J. CerealSci. 2010;51:357-365. DOI 10.1016/j.jcs.2010.02.004.Carter A.H., Santra D.K., Kidwell K.K. Assessment of the effects ofthe Gpc-B1 allele on senescence rate, grain protein concentrationand mineral content in hard red spring wheat (Triticum aestivum L.)from the Paciﬁc Northwest Region of the USA. Plant Breed. 2012;131(1):62-68. DOI 10.1111/j.1439-0523.2011.01900.x.Chen X., Song G., Zhang S., Li Yu., Gao J., Islam S., Ma W., Li G.,Ji W. The allelic distribution and variation analysis of the NAM-B1gene in Chinese wheat cultivars. J. Integr. Agric. 2017;16(6):1294-1303. DOI 10.1016/S2095-3119(16)61459-4.Cormier F., Throude M., Ravel C., Le Gouis J., Leveugle M., LafargeS., Exbrayat F., Duranton N., Praud S. Detection of NAM-A1natural variants in bread wheat reveals differences in haplotype distributionbetween a worldwide core collection and European elitegermplasm. Agronomy. 2015;5(2):143-151. DOI 10.3390/agronomy5020143.GOST 10846-91. Interstate standard. Grain and products of its processing.Method for determination of protein. Moscow: StandartinformPubl., 2009. (in Russian)Hagenblad J., Aspland L., Balfourier F., Ravel C., Leino M.W. Strongpresence of the high grain protein content allele NAM-B1 in Fennoscandianwheat. Theor. Appl. Genet. 2012;125(8):1677-1686.DOI 10.1007/s00122-012-1943-2.Iqbal M., Moakhar N.P., Strenzke K., Haile T., Pozniak C., Hucl P.,Spaner D. Genetic improvement in grain yield and other traits ofwheat grown in Western Canada. Crop Sci. 2016;56(2):613-624.DOI 10.2135/cropsci2015.06.0348Kronenberg L., Yates S., Boer M.P., Kirchgessner N., Walter A.,Hund A. Temperature response of wheat affects final height and thetiming of stem elongation under field conditions. J. Exp. Bot. 2021;72(2):700-717. DOI 10.1093/jxb/eraa471.Kuhn J.C., Stubbs T.L., Carter A.H. Effect of the Gpc-B1 allele in hardred winter wheat in the US Pacific Northwest. Crop Sci. 2016;56(3):1009-1017. DOI 10.2135/cropsci2015.08.0470.Kumar A., Kapoor P., Chunduri V., Sharma S., Garg M. Potential ofAegilops sp. for improvement of grain processing and nutritionalquality in wheat (Triticum aestivum). Front. Plant Sci. 2019;10:308.DOI 10.3389/fpls.2019.00308Lawlor D.W., Kontturi M., Young A.T. Photosynthesis by flag leavesof wheat in relation to protein, ribulose bisphosphate carboxylaseactivity and nitrogen supply. J. Exp. Bot. 1989;40(1):43-52. DOI10.1093/jxb/40.1.43.Mamontova L.I., Khromov S.P. Meteorological Dictionary. Leningrad:Gidrometeoizdat Publ., 1974. (in Russian)Maphosa L., Collins N.C., Taylor J., Mather D.E. Post-anthesis heatand a Gpc-B1 introgression have similar but non-additive effectsin bread wheat. Funct. Plant Biol. 2014;41(9):1002-1008. DOI10.1071/FP14060Mishra V.K., Pushpendra K.G., Balasubramaniam A., Ramesh C.,Neeraj K.V., Manish K.V., Punam S., Arun K.J. Introgression of a gene for high grain protein content (Gpc-B1) into two leading cultivarsof wheat in Eastern Gangetic Plains of India through markerassisted backcross breeding. J. Plant Breed. Crop Sci. 2015;7(8):292-300. DOI 10.5897/JPBCS2015.0514.Orlovskaya O., Dubovets N., Solovey L., Leonova I. Molecular cytologicalanalysis of alien introgressions in common wheat lines derivedfrom the cross of Triticum aestivum with T. kiharae. BMC PlantBiol. 2020;20(Suppl. 1):201. DOI 10.1186/s12870-020-02407-2.Orlovskaya O.A., Leonova I.N., Adonina I.G., Salina E.A., KhotylevaL.V., Shumny V.K. Molecular сytogenetic analysis of triticaleand wheat lines with introgressions of the genetic material of Triticeaetribe species. Russ. J. Genet. Appl. Res. 2016;6(5):527-536.DOI 10.1134/S2079059716050087Peleg Z., Saranga Y., Yazici A., Fahima T., Ozturk L., Cakmak I. Grainzinc, iron and protein concentrations and zinc-efficiency in wild emmerwheat under contrasting irrigation regimes. Plant Soil. 2008;306:57-67. DOI 10.1007/s11104-007-9417-zPuranik S., Sahu P.P., Srivastava P.S., Prasad M. NAC proteins: regulationand role in stress tolerance. Trends Plant Sci. 2012;17(6):369-381. DOI 10.1016/j.tplants.2012.02.004.Rokitsky P.F. Biological Statistics. Moscow: Vysshaya Shkola Publ.,1973. (in Russian)Tabbita F., Lewis S., Vouilloz J.P., Ortega M.A., Kade M., Abbate P.E.,Barneix A.J. Effects of the Gpc-B1 locus on high grain protein contentintrogressed into Argentinean wheat germplasm. Plant Breed.2013;132(1):48-52. DOI 10.1111/pbr.12011.Tabbita F., Pearce S., Barneix А.J. Breeding for increased grain proteinand micronutrient content in wheat: ten years of the GPC-B1 gene. J.Cereal Sci. 2017;73:183-191. DOI 10.1016/jcs.2017.01.003.Uauy C., Brevis J.C., Dubcovsky J. The high grain protein content geneGpc-B1 accelerates senescence and has pleiotropic effects on proteincontent in wheat. J. Exp. Bot. 2006a;57(11):2785-2794. DOI10.1093/jxb/erl047.Uauy C., Distelfeld A., Fahima T., Blechl A., Dubkovsky J. A NACgene regulating senescence improves grain protein, zink, and ironcontent in wheat. Science. 2006b;314(5803):1298-1301. DOI10.1126/science.1133649.Vishwakarma M.K., Arun B., Mishra V.K., Yadav P.S., Kumar H.,Joshi A.K. Marker-assisted improvement of grain protein contentand grain weight in Indian bread wheat. Euphytica. 2016;208(2):313-321. DOI 10.1007/s10681-015-1598-6.Wang X., Below F.E. Cytokinins in enhanced growth and tillering ofwheat induced by mixed nitrogen source. Crop Sci. 1996;36(1):121-126. DOI 10.2135/CROPSCI1996.0011183X003600010022x.Waters B.M., Uauy C., Dubcovsky J., Grusak M.A. Wheat (Triticumaestivum) NAM proteins regulate the translocation of iron, zinc, andnitrogen compounds from vegetative tissues to grain. J. Exp. Bot.2009;60(15):4263-4274. DOI 10.1093/jxb/erp257.Yang R., Juhasz A., Zhang Y., Chen X., Zhang Y., She M., Zhang J.,Maddern R., Edwards I., Diepeveen D., Islam S., Ma W. Molecularcharacterisation of the NAM-1 genes in bread wheat in Australia.Crop Pasture Sci. 2018;69(12):1173-1181. DOI 10.1071/CP18273Zhao D., Derkx A.P., Liu D.C., Buchner P., Hawkesford M.J. Overexpressionof a NAC transcription factor delays leaf senescence andincreases grain nitrogen concentration in wheat. Plant Biol. 2015;17(4):904-913. DOI 10.1111/plb.12296.


Chlamydia trachomatis is an obligate intracellular pathogen that actively promotes invasion of epithelial cells. A virulence-associated type III secretion system contributes to chlamydial entry and at least four effectors have been described that are deployed during this time. Two of these invasion-related effectors, the translocated membrane-associated effectors A and B (TmeA and TmeB), are encoded in a bi-cistronic operon. TmeA directly activates host N-WASP to stimulate Arp2/3-dependent actin polymerization. According to current working models, TmeA-mediated N-WASP activation contributes to invasion. TmeB has not been functionally characterized. Unlike a tmeA null strain, loss of tmeB does not impact invasion efficiency of C. trachomatis. Using strains deficient for multiple genes, we provide evidence that TmeA is dispensable for invasion in the absence of TmeB. Our data indicate that overabundance of TmeB interferes with invasion and that this activity requires active Arp2/3 complex. We further show that TmeB is capable of interfering with Arp2/3-mediated actin polymerization. In aggregate, these data point to opposing functions for TmeA and TmeB that manifest during the invasion process. These studies raise intriguing questions regarding the dynamic interplay between TmeA, TmeB, and branched actin polymerization during chlamydial entry. Chlamydia trachomatis is a prevalent obligate intracellular bacterial pathogen that is responsible for genital and ocular diseases. Genital-specific serotypes (Serovars D-K) collectively are the most common bacterial sexually transmitted disease world-wide  and perihepatitis . C. trachomatis (serovars A-C) is also the leading cause of preventable blindness. Additional species pathogenic for humans include the respiratory pathogen C. pneumoniae and the zoonotic avian pathogen C. psittaci  was used as the parent strain in these studies. Previously described strains include C. trachomatis Δtarp (tmeB (tepp (tmeA (tmeA polar), and tmeA-lx (tmeA). C. trachomatis ΔtmeAB and ΔtmeAB-tarp were generated via FRAEM  and 695@gfpNmPR (5’- GCCCCGCCC TGCCACTCATCGTTAGATATTCCCAACCGAAGAAGGATCTTCC-3’). For ΔtmeAB, pSUmC expressing gfp and aadA flanked by 2.2 kb of chlamydial flanking DNA was generated. Sequences 5’ of tmeA were amplified from L2 genomic DNA with ctl0063-SalI-S (5’-GCAAAAGAGCTGATCCTCCGTCACTGCAGGTACCGCTCCAGCGTTGCGTATTGTTTGTGG-3’) and ctl0063-SalI-AS (5’-CGTATAATGTATGCTATACGAAGTAGG AATGCCTCCGCCGAAGCAATAACTTTTAATTCC-3’) whereas sequences downstream of tmeB were amplified with ctl0064-SbfI-S (5’-GTATAGCATACATTATACGAAGTTA TGCATTTTCTAATAGGGAAGAGGATAAATAGCGTG-3’) and ctl0064-SbfI-AS (5’-CTTTGATCTTTCTACGGGGTCTGACGCCCGACCATTTTACTTTGGAATAAG TGTGATATC-3’). Amplicons were sequentially cloned into pSUmC using Gibson Assembly of SalI-cut or SbfI-cut plasmid, respectively. The tmeA and tmeB double deletion strain was generated by transformation of C. trachomatis L2 with pSUmC-ΔtmeAB and allelic replacement using FRAEM as described  and transferred to 0.45 µm PVDF membranes (Millipore). Primary antibodies used include: MOMP , TmeA H, TmeB M, Tarp C, Tepp  and collection of the supernatant of a 90 minute, 100,000 xg, 4°C spin in a Beckman Optima MAX TL Ultracentrifuge using a TLA 55 rotor (Beckman Coulter). Approximately 30 µg of pyrene-labeled actin was mixed with 1-2µg of indicated proteins  in a volume of 500 µl for 5 min before the addition of 1/20th volume of polymerization buffer . The reaction (contained in a semi-microcuvette and holder assembly) was monitored for 30 min with a LS 55 Luminescence spectrophotometer equipped with the biokinetic accessory and directed by FL Winlab software version 4.0  with 2.5 nm bandwidth at 365 nm excitation wavelength and 2.5 nm bandwidth at 407 nm emission wavelength.Pyrene actin polymerization assays were performed as previously described . Full-leUnless otherwise noted, presented data are representative of triplicate experiments where quantitative data were generated from experiments containing triplicate biological replicates. Calculation of standard deviation of the mean and assessment via Student’s t-test with Welch’s correction. Statistical analyses were performed using GraphPad Prism 6 version 6.04 (Graphpad Software Inc).C. trachomatis. This approach  strain appeared to exhibit a more robust invasion defect than the parent polar strain. We directly tested for a potential difference by performing side-by-side invasion studies  to enable marker-less gene deletion in approach  to excismeA null  created bundance . During tmeA and tmeB to address whether the invasion defect manifested by ΔtmeA strains was due solely to the absence of TmeA. C. trachomatis L2 was transformed with pSUmC-ΔtmeAB, and FRAEM was employed to replace the entire coding region for tmeA and tmeB with a gfp-aadA cassette. Gene deletion was first confirmed in a clonal isolate via qPCR  to show that TmeA activates N-WASP to promote Arp2/3-dependent actin polymerization . We therC. trachomatis early effectors that are encoded in a bi-cistronic operon, share the secretion chaperone Slc1, and are translocated into human cells during invasion via T3S . CK-666 is a small molecule inhibitor of the Arp2/3 complex that binds in the pocket between Arp2 and Arp3 blocking the two subunits from forming a short-pitched dimer necessary to bind actin (Epithelial cell ARPC1, but not N-WASP, co-precipitated with ectopically expressed TmeB, raising the possibility of a direct interaction with the Arp2/3 complex. Although TmeB interfered with TmeA-N-WASP-mediated Arp2/3 activation in the nd actin . Our datnd actin . The Tmend actin . It is iC. trachomatis utilizes several early effectors to manipulate the host actin cytoskeleton to promote invasion of epithelial cells (tmeA-tmeB double mutant does not manifest an invasion defect implies a functional link between TmeA and TmeB activities. These data indicate that TmeA is only essential for invasion in the presence of TmeB. N-WASP is likely required since trans-complementation with TmeA deficient for N-WASP binding failed to restore WT levels of entry (tmeA-tmeB-tarp triple mutant showed decreased invasion efficiency. This would be consistent with the notion that TmeA and Tarp contribute independently to invasion (al cells . In addial cells  or indiral cells . Our obsof entry . Tarp-deinvasion  and thatinvasion . Analysiinvasion . The contmeA strain failed to manifest actin-based pedestal formation, leading them to postulate that TmeA-mediated N-WASP activation leads to protrusions associated with EBs (de novo production (C. pneumoniae Cpn0677 is not homologous to TmeA but is encoded within a syntenic locus (C. pneumoniae is currently lagging so the model has not been confirmed genetically. If C. trachomatis TmeA functions similarly, TmeB could contribute to cessation of actin polymerization around the nascent inclusion. This notion is reinforced by the fact that C. pneumoniae Cpn 0676 is homologous to C. trachomatis TmeB (Why TmeB may balance TmeA activity depends on the true role of TmeA during invasion. Faris, et al. showed that the non-polar Δwith EBs . Given twith EBs . While Soduction . Interesic locus . Cpn0677ic locus . Genetictis TmeB  and coulLegionella pneumophila kinase LegK2 is a type IV secretion system effector protein capable of inhibiting Arp2/3 through phosphorylation of the ARPC1 and Arp3 subunits (Legionella pneumophila type IV secretion system phosphotyrosine-phosphatase effector protein WipA has also been shown to disrupt Arp2/3-directed actin polymerization, through a different mechanism than LegK2. WipA reduces the tyrosine phosphorylation of N-WASP and Arp3 during infection resulting in a reduction of actin polymerization (Legionella containing vacuoles from the endocytic pathway (tmeB strain grows similarly to WT (Salmonella Typhimurium (Beyond TmeA, there are additional possibilities regarding the overarching functional consequences of Arp2/3 inhibition by TmeB. Other bacterial effectors have been shown to inhibit the Arp2/3 complex. subunits . TmeB harization . Both Le pathway . TmeB coly to WT . More brly to WT . We posthimurium . Activathimurium . SptP rehimurium  to antaghimurium . In aggrThe original contributions presented in the study are included in the article/KS and GK contributed equally to this work. All authors contributed to the article and approved the submitted version.


FKS1 genotypes of 13 clinical isolates of Candida auris that were recovered from 4 patients at a tertiary care center in Salvador, Brazil. Three isolates were categorized as echinocandin-resistant, and they harbored a novel FKS1 mutation that led to an amino acid change W691L located downstream from hot spot 1. When introduced to echinocandin-susceptible C. auris strains by CRISPR/Cas9, Fks1 W691L induced elevated MIC values to all echinocandins .We determined the echinocandin susceptibility and  In Candida spp., pharmacodynamic resistance to echinocandin class drugs occurs predominantly when a fungal strain develops mutations in two highly conserved hot spot (HS) regions of the FKS genes that encode the catalytic subunits of β-1,3-d-glucan synthase. In vitro, the presence of amino acid substitutions in Fks subunits manifests as elevated MIC values  and causes hospital outbreaks , and D1350 to L1357 (HS2), and echinocandin resistance-conferring mutations were reported at several HS positions in clinical isolates (C. aurisFKS1 mutants harboring F635Δ (HS1) and M690I (downstream from HS1) were obtained through the serial in vitro caspofungin exposure of the strain B112020  , were isolated after echinocandin treatment in a murine gastrointestinal colonization model  that were recovered from 4 patients  at a tertiary care center in Salvador, Brazil, were evaluated for their echinocandin susceptibility and FKS1 sequences. We performed antifungal susceptibility testing (AFST) with echinocandins , according to the CLSI methodology , as we described previously . The remaining 10 isolates were echinocandin-susceptible  and presented a WT FKS1 genotype , which is predicted to be embedded within the outer leaflet of the membrane and may interact with the extended aliphatic tails of echinocandins  FKS1 gene fragment in echinocandin-susceptible C. auris DPL1384  and CBS 10913  was replaced with a 465 bp FKS1 fragment (C1899 to C2364) harboring W691L (amplified from isolate B61) by using a CRISPR/Cas9 system, as described previously (CTTCTTCTTGACTTTGTCCTTGAGAGATCCT ATTAGAAACTTGTCCACC-3′), and Cau_FKS1_CRISPR_R1 (5′-GGTGCTCTCAAAGTTCTCTTGCCCTCGATTTCAGAAGGAACCTGGTGATAC-3′) primers. The crRNA target sequence was CGATTTCAGAAGGAACCTGGTGG, and the Cau_FKS1_CRISPR_R1 primer introduced a modified PAM site (without an amino acid change) into the PCR product to prevent the Cas9 digestion of the repair template. The transformants were selected on YPD plates containing 4 mg/L MCF. We previously used a similar selection approach to introduce the non-hot spot modification E655K in the Fks2 of C. glabrata   .Saccharomyces cerevisiae revealed active enzyme-membrane interactions and altered lipid arrangements around the mutated enzymes (The interactions between Fks1 enzyme variants and echinocandin drugs are not well-understood. However, a recent study presenting the cryo-electron microscopy structures of the Fks1 of  enzymes . AlthougC. auris. Our discovery emphasizes that, despite the significant progress that has been made in recent years in understanding echinocandin resistance in C. auris, the diversity of the FKS1 mutations that contribute to C. auris echinocandin resistance may be underestimated. Ultimately, improving the knowledge of the resistance mechanism will help diagnose and treat C. auris-infected patients.In conclusion, we report Fks1 W691L as a novel, echinocandin resistance-conferring, non-hot spot modification in


This approach provides a practical research direction for the design of MOF-derived carbon materials in the application of high-performance Li–S batteries.Nanocomposites that combine porous materials and a continuous conductive skeleton as a sulfur host can improve the performance of lithium–sulfur (Li-S) batteries. Herein, carbon nanotubes (CNTs) anchoring small-size (~40 nm) N-doped porous carbon polyhedrons (S-NCPs/CNTs) are designed and synthesized via annealing the precursor of zeolitic imidazolate framework-8 grown in situ on CNTs (ZIF-8/CNTs). In the nanocomposite, the S-NCPs serve as an efficient host for immobilizing polysulfides through physical adsorption and chemical bonding, while the interleaved CNT networks offer an efficient charge transport environment. Moreover, the S-NCP/CNT composite with great features of a large specific surface area, high pore volume, and short electronic/ion diffusion depth not only demonstrates a high trapping capacity for soluble lithium polysulfides but also offers an efficient charge/mass transport environment, and an effective buffering of volume changes during charge and discharge. As a result, the Li-S batteries based on a S/S-NCP/CNT cathode deliver a high initial capacity of 1213.8 mAh g For Li-S batteries, the S8 ring molecules first react with Li+ to form long-chain Li2S6. Then, Li2S6 transforms into short-chain Li2S4 through successive cleavage of S-S bonds [−30 S cm−1) and Li2S (10−13 S cm−1) leads to the low utilization of active materials and poor rate capability [2Sx 4 ≤ x ≤ 8) form during cycling. These polysulfide intermediates migrate from the cathode to the anode, under a gradient concentration, where they react with metallic lithium to form shorter polysulfides, and vice versa, activating the so-called “shuttle effect” which leads to low Coulombic efficiency and rapid capacity fade [Compared with the currently used Li-ion battery intercalation electrode materials, the S element has overwhelming advantages with highly crustal abundance, low production cost, and environmental benignity ,2,3, esppability ; (2) aboity fade .Over the past few decades, great efforts to improve the electrical conductivity and buffer the volume changes for the S cathode as well as suppress the shuttle effect of soluble polysulfides during cycling, have been focused on the S-based composite cathode with these advanced features of high conductivity, efficient ion pathway, and sufficient void space to accommodate the volume changes . Carbon-+ transport pathways, leading to improved rate performance. Thus, MOFs have prospective applications in Li-S batteries [2S2 onto their porous structures. The high surface area and porosity of MOFs provide sites where Li2S2 molecules can physically adsorb or even be encapsulated within the pores. This interaction can help mitigate the shuttling effect of intermediate sulfur species, reducing their migration within the battery and improving overall battery performance [Metal–organic framework (MOF) materials are a new type of porous organic–inorganic hybrid materials that have many advantages. First, MOFs are characterized by a high specific surface area which allows for significant sulfur loading, increasing the overall energy density of the Li-S batteries. Second, MOFs are of well-defined porosity which can also accommodate the diffusion of sulfur, leading to improved electrochemical performance. Third, MOFs can be designed with tunable pore sizes which enable the optimization of Liatteries . Moreoveformance . Howeverformance ,26,27. T−1 at a current rate of 1 C and a substantial capacity of 913.8 mAh g−1 after 500 cycles, corresponding to a low capacity decay of only 0.02% per cycle.Herein, we present the in situ growth of small-size zeolitic imidazolate framework-8 (ZIF-8) nanoparticles on CNTs, resulting in a composite of CNTs anchoring small-size (~40 nm) N-doped porous carbon polyhedrons (S-NCPs/CNTs) after thermal treatment. In the composite, the S-NCPs serve as an efficient host for trapping polysulfides through physical adsorption and chemical bonding, while the interleaved CNT networks offer an efficient charge transport environment. Moreover, the S-NCP/CNT nanocomposite with great features of a large specific surface area, high pore volume as well a short electronic/ion diffusion path serves as an efficient host for the S cathode (S/S-NCPs/CNTs), which not only demonstrates high trapping capacity for soluble lithium polysulfides but also offers an efficient charge/mass transport environment, and an effective buffering of volume changes during charge and discharge. The Li-S batteries based on the S/S-NCP/CNT cathode deliver an initial capacity of 1017.1 mAh g3SO3, 0.5 M LiNO3 in 1,2-dimethoxyethane (DME): 1,3-dioxolane (DOL) =1:1 vol%) was purchased from DodoChem Co., Ltd., Zhuhai, China.Polyvinylpyrrolidone , 2-methylimidazole , zinc nitrate hexahydrate , carbon nanotubes , polyvinylidene , fluoride methanol , N-methyl-pyrrolidone , and concentrated nitric acid  were used as received without further purification. The lithium–sulfur electrolyte (0.5 M LiCF3)2·6H2O was added to the above methanol solution and stirred for 15 min and then the resulting mixture was transferred into a Teflon-line autoclave and heated to 90 °C for 6 h. After the solvothermal reaction, the obtained precipitates, S-ZIF-8/CNT composites, were collected by centrifugation, washed three times with methanol and deionized water, and then dried in vacuum at 60 °C overnight. Finally, annealing the as-obtained S-ZIF-8/CNTs precursors under an Ar atmosphere in a tube furnace at 920 °C for 3 h with a ramp rate of 2 °C min−1 generated S-NCP/CNT nanocomposites.The S-NCP/CNT nanocomposites were constructed using the CNTs anchoring small-size (~10 nm) N-doped porous carbon polyhedrons via annealing the precursor of small-size zeolitic imidazolate framework-8 grown in situ on CNTs (S-ZIF-8/CNTs). First, the S-ZIF-8/CNT precursors were synthesized by the solvothermal method. Typically, 150 mg of acidified CNTs and 3.0 mmol of 2-MeIm were successively added into 200 mL of methanol containing 100 mg of PVP under vigorous stirring to form homogeneous ink. Subsequently, 1.5 mmol of Zn(NO2 solution (25 mg/mL) for 72 h, in which the weight ratio of S-NCPs/CNTs and sulfur was controlled at 2 to 8. Then, the mixture was dried at 40 °C. Next, the obtained sulfur-containing nanocomposites were heated at 155 °C in a sealed autoclave for 12 h, generating the S/S-NCP/CNT nanocomposite.A simple method of solution infiltration and heat treatment was applied to controllably load sulfur into S-NCP/CNT nanocomposite, generating sulfur-containing nanocomposite S/S-NCPs/CNTs. Before loading sulfur, the S-NCP/CNT nanocomposite was heated at 120 °C maintained for 3 h for activation treatment. In a typical fabrication of S/S-NCPs/CNTs, the as-activated S-NCP/CNT nanocomposite was immersed in sulfur/CSX-ray diffraction (XRD) patterns were recorded in an X-ray diffractometer  with Cu-Ka radiation (λ = 0.1540 nm) at 40 kV, 40 mA and scanning rate of 10°/min in a 2θ range of 10–80°. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) experiments were performed on a Phenom ProX Scanning Electron Mircoscope and a JEM1200EX high-resolution transmission electron microscope, respectively. Thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) were conducted with a TGA thermogravimetric analyzer in argon at a scan rate of 10 °C/min from room temperature to 800 °C. The Brunauer–Emmett–Teller (BET) surface area was determined through nitrogen adsorption isotherms using an Autosorb iQ3 autosorb automated gas sorption system.3SO3, 0.5 M LiNO3 in DME: DOL = 1:1 vol%). The sulfur cathodes were prepared by mixing NC/CNT/S composites, carbon black, and PVDF with a weight ratio of 8:1:1, dispersed in NMP to form the equal slurry, and coated on an Al foil. The S mass loading is about 1.27 mg cm−2. Thus, the ratio of sulfur/electrolyte is 0.025 mg µL−1. The cyclic voltammetry (CV) measurements were carried out using a CHI760e electrochemical workstation at a scan rate of 0.1 mV s−1 with a voltage range of 1.7–2.8 V. Electrochemical impedance spectroscopy (EIS) was performed on a PARSTAT 2273 in a frequency range from 10−1–105 Hz. The galvanostatic charge/discharge tests were conducted on LAND CT2001A in a voltage window between 1.7 and 2.8 V at 0.1–5 C rates (1 C = 1675 mA g−1).Electrochemical measurements were carried out in CR2025 coin-type cells using sulfur cathodes, Li foil anodes, commercial PP, separators, and 40 μL of electrolyte (0.5 M LiCFThe fabrication procedure of S/S-NCPs/CNTs is illustrated in The morphologies of the samples were characterized using SEM and TEM techniques. 0 < 0.1), suggesting the presence of a large volume of micropores. The micropore size distribution ranges from 1 to 2 nm. The surface area and the pore volume of the S-NCPs/CNTs were measured to be 1135.2 m2 g−1 and 0.47 cm3 g−1, respectively. The large surface area and pore volume are not only beneficial to accommodate volume change of the active material S caused by full lithiation and delithiation during discharge and charge processes but also can provide the trapping capability of carbon-based materials to sulfur species; hence, it is expected that the S-NCPs/CNTs can possess excellent capabilities of immobilizing sulfur and trapping polysulfides for effectively reducing the sulfur loss and inhibiting the polysulfide migration. The XPS technique was applied in the identification of the chemical composition and electronic states of the S-NCPs/CNTs. As shown in The crystal structure of the samples before and after annealing was characterized using the XRD technique. There is only the appearance of characteristic peaks of ZIF-8 crystalline (JCPDS No. 62-1030) [2 atmosphere with temperatures ranging from 35 to 600 °C, as demonstrated in 2 g−1 of the S-NCPs/CNTs to 80.4 m2 g−1 of the S/S-NCPs/CNTs, according to the BET test. Additionally, the micropore volume also reduces dramatically from 0.47 cm3 g−1 of the S-NCPs/CNTs to 0.02 cm3 g−1 of the S/S-NCPs/CNTs, based on the change in pore size distribution before and after sulfur loading  are presented in 2S4–8), and further reduction from Li2S4–8 to insoluble products Li2S2/Li2S, respectively. In the charge process, a single long charge plateau from 2.2 to 2.4 V indicates the conversion of Li2S2/Li2S into elemental sulfur and lithium [To evaluate the practical applicability of the S/S-NCPs/CNTs as an effective cathode for Li-S batteries, a series of electrochemical assessments were carried out through assembling coin cells with metallic lithium as the anode. The capacities of all Li-S cells were obtained on the basis of the mass of sulfur. The galvanostatic charge/discharge profiles of the S/S-NCPs/CNTs recorded at a current rate of 0.2 C , a constant phase element (CPE) representing the double-layer capacitance, and a Warburg element (W) accounting for diffusion processes [The electrode kinetics were studied using EIS technology. rocesses ,40. The rocesses . This furocesses . We furt−1 at a current rate of 0.2 C and a substantial capacity of 1114.2 mAh g−1 after 100 cycles, corresponding to a high capacity retention of 91.7%. Moreover, the Li-S battery based on the S/S-NCP/CNT cathode possesses a long lifespan (500 cycles) at 1.0 C which is close to that of high-energy-density LIBs, showing great potential in the energy storage market. The strategy demonstrated in this work provides an effective approach for the rational design of high-performance sulfur cathodes and can be extended to other fields.In summary, we demonstrated an advanced S/S-NCP/CNT cathode in the form of CNTs anchoring small-size NCPs as efficient sulfur hosts. In the S/S-NCP/CNT cathode, the S-NCPs with great features of a large specific surface area and pore volume as well as short electronic/ion diffusion paths not only demonstrate a high trapping capacity for soluble lithium polysulfides but also effectively buffer the volume changes during the charge and discharge, while the interleaved CNT networks offer an efficient charge transport environment. As a result, the Li-S battery based on the S/S-NCP/CNT cathode delivers a high initial capacity of 1213.8 mAh g


The tumor-associated antigen mesothelin is expressed at high levels on the cell surface of many human cancers, while its expression in normal tissues is limited. The binding of mesothelin to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. Immunotherapeutic strategies targeting mesothelin are being intensively investigated. Here, we report the crystal structures of mesothelin that reveal a compact, right-handed solenoid consisting of 24 short helices and connecting loops. These helices form a nine-layered spiral coil that resembles ARM/HEAT family proteins. Glycan attachments have been identified in the structure for all three predicted N-glycosylation sites and confirmed with samples from cell culture and patient ascites. The structures of full-length mesothelin and its complex with the Fab of MORAb-009 reveal the interaction of the antibody with the complete epitope, which has not been reported previously. The N-terminal half of mesothelin is conformationally rigid, suitable for eliciting specific antibodies, whereas its C-terminal portion is more flexible. The structure of the C-terminal shedding-resistant fragment of mesothelin complexed with a mAb 15B6 displays an extended linear epitope and helps explain the protection afforded by the antibody for the shedding sites.The structures of full-length mesothelin and its complexes with antibodies reported here are the first to be determined experimentally, providing atomic models for structural organization of this protein and its interactions with antibodies. It offers insights into the function of mesothelin and guidance for further development of therapeutic antibodies. Mesothelin (MSLN) was discovered as a cell surface protein normally expressed by mesothelial cells lining the pleura, pericardium, and peritoneum . It is aThe gene that encodes human mesothelin (MSLN gene) is located on chromosome 16 (16p13.3) and produces a 68-kDa precursor protein that is subsequently processed by the endoprotease furin in the trans Golgi apparatus to yield a 40 kDa glycosylphosphatidylinositol (GPI)-anchored mature MSLN  and a 31MSLN shares sequence homology with stereocilin and otoancorin; these proteins are also GPI anchored to the membrane of the inner ear sensory and nonsensory epithelial cells and are associated with deafness in people. It was predicted to have a superhelical structure with armadillo (ARM)-type repeats . ExperimRecent studies have also shown that the efficacy of antibodies against MSLN is modulated by its shedding from cells due to the action of membrane-bound sheddases that cleave MSLN at various sites close to the cell membrane . SheddinDespite all the progress made, information on the structure of full-length MSLN (fl-MSLN) and its conformational variability are lacking. Here, we report the crystal structures of a 207-residue MSLN fragment, of fl-MSLN alone and in complex with a therapeutic mAb MORAb-009, as well as the structure of its C-terminal shed-resistant peptide complexed with mAb 15B6. We also analyzed glycan structures of MSLN from recombinant proteins, shed from cultured cells, and from patient ascites. Our findings revealed the structural similarity of MSLN to ARM/HEAT family proteins, which suggested its potential cellular functions, and shed new light on the conformational variability of MSLN. The complete structural information of MSLN could also guide the design and development of future MSLN-targeting cancer treatments.Full-length cDNA of MSLN was inserted into the baculovirus transfer vector pAcGP67A (RRID:Addgene_41812) of BD BaculoGold (BD Biosciences) in frame with the hexahistidine tag at the C-terminus . All mutEscherichia coli (E. coli) strain Rosetta (DE3) harboring the plasmid pET24a-MSLN-nn . The cell lysate was centrifuged at 38,729 × g for 20 minutes. For MSLN-132 that was found soluble in the supernatant, the supernatant was then mixed with Ni-NTA resin (Qiagen) preequilibrated with washing buffer A  and incubated for 2 hours at 4°C. The resin was then transferred to a column. After washing the column with the washing buffer A, the bound protein was eluted by the same buffer supplemented with 300 mmol/L imidazole. The eluant was concentrated and further purified by SEC on a Superdex S-200 column equilibrated with a SEC running buffer . The fractions containing the MSLN fragment were pooled and stored at 4°C for later use.The g for 20 minutes. This washing step was repeated two more times. The purified inclusion bodies were dissolved in 80 mL solubilization buffer . The solution was stirred for 1 hour at 4°C and centrifuged at 38,729 × g for 20 minutes to remove any insoluble material. The supernatant was applied to an 8-mL Ni-NTA column preequilibrated with an equilibration buffer , which was washed with 2.5 column volume of the equilibration buffer. The bound protein was eluted with 20 mL elution buffer . The eluate was concentrated to 2–4 mg/mL using an Amicon Ultra concentrator MWCO 10 kDa (Millipore) and further purified by SEC on a Superdex S-200 column equilibrated with the SEC running buffer . The fractions containing refolded protein were pooled, concentrated to 10 mg/mL and stored at −80°C for later use.For MSLN-168, MSLN-207, and MSLN-263, which were found to be present in the form of inclusion bodies in the pellet, the pellet was washed in 80 mL of washing buffer B ), sonicated for 5 minutes and then centrifuged at 38,729 × The Fab fragment of the MSLN-specific mAb MORAb-009 (Morphotek Inc), Fab(MORAb), was prepared following the instructions provided by the Fab preparation kit (Thermo Fisher Scientific) as described previously . BrieflyThe mAb 15B6 targeting the C-terminal peptide of MSLN was obtained from GenScript. The Fab fragment of 15B6, Fab(15B6), was prepared similarly as for MORAb-009. Briefly, mAb 15B6 at 5.6 mg/mL was buffer exchanged into the supplied digestion buffer using a Zeba Spin Desalting Column. The digestion was carried out by mixing 15B6 with preequilibrated immobilized papain and incubating at 37°C with constant mixing. After 4 hours digestion, the reaction was stopped by separating the digest from the immobilized papain using centrifugation. The digested Fab/Fc mixture was passed through a NAb Protein A Plus spin column to remove Fc and any undigested IgG, and the Fab fragment in the flow through was washed out of the column with TBS buffer containing 20 mmol/L Tris and 100 mmol/L NaCl at pH 7.5. Purified Fab was buffer exchanged into TBS and concentrated to 9.4 mg/mL using an Amicon Ultra 30 kDa centrifugation filter (Millipore). The Fab digestion and purification process were monitored by SDS-PAGE.The purified fl-MSLN in 25 mmol/L Tris, pH 7.5, 200 mmol/L NaCl was mixed with MORAb-009 Fab and incubated at room temperature for 30 minutes to allow formation of the complex. The complex was then subjected to blue-native PAGE (BN-PAGE) analysis following the procedure described previously .To prepare the complex of fl-MSLN with Fab(MORAb), purified Fab(MORAb) (8 mg/mL or 0.16 mmol/L) in PBS was mixed with an excess amount of MSLN in a 1:1.1 molar ratio in a buffer consisting of 20 mmol/L Tris, pH 8.0, 100 mmol/L NaCl, 1 mmol/L β-ME, and 2% glycerol and the resulting mixture was incubated at 4°C overnight. Excess fl-MSLN and impurities were removed using a Superdex S-200 column with a running buffer consisting of 20 mmol/L Tris, pH 8.0, 100 mmol/L NaCl, 2% glycerol. Each eluted fraction was checked with SDS-PAGE and those containing the complex were pooled and concentrated to 6 mg/mL for crystallization using an Amicon Ultra MWCO 30 kDa centrifugation filter (Millipore).Crystallization of the fl-MSLN was performed robotically using a Mosquito liquid dispenser (TTP LabTech) in a 96-well hanging-drop format (Greiner 96-well U-shape plate ref: 650101). For initial screening, 85 μL of reservoir solutions from a commercial screen kit Index HT (Hampton Research) was used, and the drops were set up with 240 nL of protein at 10 mg/mL in 50 mmol/L Tris (pH 7.5) and 200 mmol/L NaCl with 240 nL of the reservoir solution. The crystallization hit condition (B6) obtained from the screen was refined. The final conditions for crystallization were mix of the protein in a 1:1 ratio with a well solution containing 0.9–1.1 mol/L Na/K phosphate, pH 8.2.2SO4. Protein and well solution were mixed in a 1:1 ratio (0.3 μL each) and incubated at 16°C. Crystals appeared after several months. Before diffraction data collection, crystals were treated with a cryoprotectant solution made of 100% glycerol and the well solution in a 1:2 ratio.Crystallization of MSLN-207 was done similarly. The protein had a concentration of 4.0 mg/mL in a solution containing 20 mmol/L Tris, pH 8, 100 mmol/L NaCl, and 1 mmol/L β-ME. The well solution had a volume of 60 μL containing 0.1 mol/L HEPES pH 7.5, 100 mmol/L KCl, 12.5% PEG3350, 100 mmol/L Li2SO4. Crystals appeared within one week to several months. Crystals were treated with a cryoprotectant solution made of 100% glycerol and reservoir solution in a 1:2 ratio.The crystallization of fl-MSLN/Fab of MORAb-009 complex was also performed robotically by the hanging-drop method. An aliquot of 0.3 μL protein of fl-MSLN/Fab(MORAb) complex at 6 mg/mL concentration in a buffer containing 25 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 2% glycerol was mixed with 0.3 μL reservoir solution (60 μL) containing 0.1 M Tris, pH 7, 150 mmol/L Na-citrate, 16.4% PEG3350, and 100 mmol/L LiThe first step in crystallizing the C-terminal peptide (residues 582–598)/Fab of Mab 15B6 complex was to dissolve the synthetic peptide powder (GenScript) in the same TBS buffer  as the Fab(15B6). The peptide solution at 4.4 mg/mL was then mixed with concentrated Fab(15B6)  at a molar ratio of 3:1 and left on a horizontal orbital shaker at 4°C overnight before setting up the crystallization trays. Crystallization was carried out robotically in a 96-well format. The reservoir solution (85 μL) contained 0.1 mol/L Na acetate, pH 4.5, 26% PEG 550 MME, 6% 2-propanol. Hanging drops were formed with 240 nL reservoir and 240 nL protein and incubated at 4°C. Small crystals appeared after one day and matured within 1 week through an Ostwald-ripening process. Crystals were flash cooled in liquid nitrogen prior to data collection.All X-ray diffraction datasets for MSLN-207, fl-MSLN/Fab(MORAb), and C-term/Fab(15B6) were collected at 100 K using beamline 22 (SER-CAT) or beamline 23 (GM/CA) at the Advanced Photon Source (APS), Argonne National Laboratory (ANL). Diffraction data images were processed using HKL2000 . The C-tThe isolation of shed MSLN from either cultured A431/H9 cell culture media or patient ascites was carried out according to the procedure published previously . Brieflywww.pdb.org) as follows: PDB ID code 7U9J for the MSLN-207, 7UED for the fl-MSLN/Fab of MORAb-009, 8CX3 for the fl-MSLN, and 7U8C for the C-term/Fab of Mab 15B6. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034397.The atomic coordinates have been deposited in the Protein Data Bank  in E. coli all produced inclusion bodies  are associated side by side in a head to tail manner, following noncrystallographic 2-fold symmetry  of 5 nmol/L  complex per AU. The structure was determined by MR using the MSLN-64/Fab(MORAb) (PDB:4F3F) as a template for phasing  complex. Crystals of the complex diffracted X-rays using synchrotron radiation, leading to a dataset at 3.0 Å resolution . The cry phasing . The finA and MSLN-207B (The fl-MSLN/Fab(MORAb) complex is L-shaped with the Fab bound to the side of the fl-MSLN spiral on Domain A . The modSLN-207B . RemovinSLN-207B . An elecSLN-207B .Kd value of 5 nmol/L toward fl-MSLN, which is one-fifth of that toward the MSLN-64 fragment  complex (The previously determined structure of MSLN-64/Fab(MORAb) showed that the complex is formed with MSLN-64 making contacts with both light and heavy chains of Fab , though fragment  and suggfragment  and foun complex , whereasSuperposition of the structures of fl-MSLN/Fab(MORAb) and MSLN-64/Fab(MORAb) showed overall good alignment of the two but revealed several important adjustments made by residues in the Fab to accommodate additional interactions . For exaR32. MR with the fl-MSLN removed from the fl-MSLN/Fab(MORAb) complex structure as a search template revealed one molecule of MSLN in the crystallographic AU. However, subsequent structure refinement failed to converge, with severe collision with symmetry-related molecules toward the C-terminal part or Domain D of MSLN. The diffraction data set was reprocessed with the symmetry of space group C2  rekindled our effort to determine the structure of fl-MSLN. The crystal lattice of fl-MSLN was initially assigned to have the symmetry of the rhombohedral space group group C2 . An MR sgroup C2 . The sixgroup C2 . Two of group C2  that wasSuperpositions of the six independently determined fl-MSLN-245 models to the fl-MSLN gave rise to an average rms deviation of 1.3 Å, whereas limiting the superposition to residues from K299 to K427, that is, from Domains A to C, reduced rms deviation significantly to about 0.55 Å . ConsistMSLN and its precursor were predicted to have an ARM-repeat solenoid . ARM-repThese ARM and HEAT repeats of MSLN have different lengths ranging from 27 to 37 residues. Sequence and structural alignment between pairs of these repeats showed little detectable homologies. Common to all repeats is the concentration of hydrophobic residues such as Leu, Ile, Val, and Phe forming hydrophobic cores . HydrophRwork and Rfree of 20.4% and 23.8%, respectively , into which a model of the 15-residue peptide beginning with N584 and ending with S598 of the MSLN sequence could be placed , followed by manual model adjustments based on the sequence of 15B6. The final model of the Fab(15B6) contains a 213-residue light chain and a 214-residue heavy chain  and 3D. e placed . The boue placed . The twoe placed . The cone placed .Isolated full-length recombinant MSLN from insect cell expression system is glycosylated. The elucidation of MSLN structure is not complete without its glycan structures. In the context of developing more effective anti-MSLN antibodies, understanding the glycan structures associated with MSLN is necessary, as the binding of CA-125 to MSLN, which facilitates metastasis of ovarian tumors, appears to be dependent on glycosylation states of MSLN . FurtherHuman MSLN contains three predicted N-glycosylation sites: N388, N488, and N515. Among them, N388 and N515 are conserved in a sequence alignment with mouse and rat genes . BecauseConsidering our fl-MSLN was produced in insect cells, whose glycobiological pathway is simpler than that of mammalian cells , we wantIn this work, we expressed and successfully purified a MSLN fragment  and fl-MSLN (E296-T600). Our structure determination effort yielded the structural models of MSLN-207 (K299-G501), the fl-MSLN-245 (K299-H546), and the fl-MSLN (K299-G585)/Fab (Mab MORAb-009), missing the C-terminal 15 residues in the full-length construct. We also determined the structure of the C-terminal peptide (N584-S598) in complex with Fab of Mab 15B6. These structures allowed us to make a full description of the MSLN structure from K299 to S598 . From stThe structure elucidation of MSLN was previously attempted by computational modeling approaches. We compared our fl-MSLN structure with an early version of a predicted structure  and withARM/HEAT proteins should not be considered as being composed of a series of independent folding units aligned like beads on a string, because they have a single rodlike domain with a continuous hydrophobic core formed by interactions between adjacent ARM/HEAT repeats. Well-known ARM/HEAT repeat proteins are importin-α, also known as karyopherin, which recognizes nuclear localization signals  and the On the basis of the definition of the ARM/HEAT repeat, the fl-MSLN contains seven ARM/HEAT repeats sandwiched between the N- and C-terminal caps . Each reTargeting MSLN for immunotherapy is based on observations of increased MSLN-specific antibodies detected in sera of about 40% of patients with mesothelioma and 42% of patients with ovarian cancer, indicating that antibody responses to MSLN was correlated with higher expression levels of MSLN on the surface of cancer cells . PromisiInterestingly, elevated serum MSLN was found in most patients with mesothelioma (71%) and ovarian cancer (67%; ref. Table S1-S7Table S1: Plasmids and primers used for protein expression.Table S2: Characterization of MSLN expression constructs.Table S3: Structural alignments of MSLN models.Table S4: Lists of interactions.Table S5: Buried surface area for three MSLN-Fab complexes.Table S6: Observed saccharide attachments at predicted glycosylation sites.Table S7: Dali search result.Click here for additional data file.Figure S1Arrangement of MSLN molecules in the crystal.Click here for additional data file.Figure S2Comparison of experimental MSLN structure with those of computational models.Click here for additional data file.Figure S3Mapping epitopes of monoclonal antibodies to the structure of MSLN.Click here for additional data file.


Introduction: The unexpected surge of respiratory syncytial virus (RSV) cases following pandemic phase of COVID-19 has drawn much public attention. Drawing on the latest antiviral research, revisiting this heightened annual outbreak of respiratory disease could lead to new treatments. The ability of sulfated polysaccharides to compete for a variety of viruses binding to cell surface heparan sulfate, suggests several drugs that might have therapeutic potential for targeting RSV–glycosaminoglycan interactions.Methods: In the current study, the binding affinity and kinetics of two RSV glycoproteins (RSV-G protein and RSV-F protein) to heparin were investigated by surface plasmon resonance. Furthermore, solution competition studies using heparin oligosaccharides of different lengths indicated that the binding of RSV-G protein to heparin is size-dependent, whereas RSV-F protein did not show any chain length preference.Results and discussion: The two RSV glycoproteins have slightly different preferences for heparin sulfation patterns, but the N-sulfo group in heparin was most critical for the binding of heparin to both RSV-G protein and RSV-F protein. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for their inhibition of the RSV-G protein and RSV-F protein–heparin interaction, and both highly negative compounds showed strong inhibition. The data from the United States Centers for Disease Control and Prevention (CDC) shows there is still an increasing number of COVID-19 cases with the emergence of new SARS-CoV-2 variants, and at the same time cases of seasonal flu, and RSV have increased dramatically since the summer of 2022 in the United States. All the three viruses, causing respiratory illnesses, share some common symptoms ranging from mild , attachment (G) and small hydrophobic (SH) proteins. RSV-G protein and RSV-F protein are the major glycoproteins on the surface of the RSV virion and play critical roles in viral entry . The RSVThe study of GAG–protein interactions at molecular level, an important theme in glycobiology, could result many therapeutic implications. In this work, we analyzed the binding of the two major glycoproteins: RSV-G protein and RSV-F protein with heparin, heparin oligosaccharides of different lengths, and chemically modified heparins using surface plasmon resonance (SPR) to elucidate binding kinetics and structure features  of heparin/HS required for this interaction. Additionally, two highly negative drugs, pentosan polysulfate (PPS) and mucopolysaccharide polysulfate (MPS) were evaluated for their inhibition of the RSV-G protein and RSV-F protein–heparin interaction.N-desulfated heparin (14 kDa), 2-O-desulfated IdoA heparin (13 kDa), 6-O-desulfated heparin (13 kDa) were from Iduron . Mucopolysaccharide polysulfate  was from Luitpold Pharma . Pentosan polysulfate  was from Bene Pharma . Sensor streptavidin (SA) chips were from Cytiva . SPR experiments were performed using a BIAcore 3000  with Biaevaluation software (version 4.0.1).RSV-G protein (Cat: 11070-V08H2), RSV-F protein (Cat: 40628-V08B) were purchased from Sino Biological Inc. The proteins were constructed as follows: 1) a DNA sequence encoding the glycoprotein G extracellular domain (Asn66-Arg297) of human respiratory syncytial virus A (93% homologous with strain rsb1734) (P27022-1) was expressed, with a C-terminal polyhistidine tag; and 2) a DNA sequence encoding the human respiratory syncytial virus fusion (AFX60213.1) (Met1-Ile525) was expressed with a polyhistidine tag at the C-terminus. Unfractionated heparin (15 kDa) was from Celsus Laboratories . Heparin oligosaccharides from tetrasaccharide (dp4) to octadecasaccharide (dp18) were from Iduron . Desulfated heparins including 2O (200 µL) added with 10 mg NaCNBH3, and reacted at 70°C for 24 h. Then additional NaCNBH3 (10 mg) was added and reacted for another 24 h. The biotinylated heparin was desalted and immobilized onto SA chips based on the manufacturer’s protocol. The successful immobilization of heparin was confirmed by the observation of a 200-resonance unit (RU) increase on the sensor chip.Biotinylated heparin was prepared as previously described . HeparinRSV-G protein was diluted in HBS-EP+ buffer (0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% surfactant P20, pH7.4) at concentrations of 500, 250, 125, 62.5, and 31.3 nM, respectively. RSV-F protein was diluted in HBS-EP+ buffer at concentrations of 80, 40, 20, 10, and 5 nM, respectively. Diluted protein samples were injected at a flow rate of 30 μL/min for 3 min at 25°C, followed by dissociation with HBS-EP+ buffer for 3 min. The sensor surface was regenerated by injecting with 30 μL of 0.25% sodium dodecyl sulfonate (SDS) after dissociation time. No protein aggregates were observed in the samples with concentrations ranging from 32 to 500 nM for RSV-G, 5–80 nM for RSV-F before the injection.Solution competition studies between surface-immobilized heparin and heparin analogs (heparin oligosaccharides and desulfated heparins) in solution were performed as previously described . RSV-G pSolution competition studies between surface-immobilized heparin and soluble glycan drugs, PPS and MPS, were performed using SPR to measure their inhibition activity D. . In brieDuring pathogen infection, GAGs play an important role in the initial attachment of pathogens to host cells . The intka; dissociation rate constant, kd) and affinity  by globally fitting the sensorgrams using 1: 1 Langmuir binding model  . The varN-sulfation but not 6-O-sulfation or 2-O-sulfation is important for RSV infection and dp10 is the minimum size that can neutralize RSV infectivity using an improved recombinant green fluorescent protein-expressing RSV to assay infection. Here, solution competition experiments were performed to examine the effect of the saccharide chain length and sulfation pattern of heparin on the interaction of heparin and RSV-G protein. Different heparin oligosaccharides (dp4-dp18) or chemically desulfated heparins at 1,000 nM concentration were applied in the competition analysis  is currently the only United States Food and Drug Administration (FDA)-approved oral therapy for the relief of bladder pain or discomfort associated with interstitial cystitis. The safety and efficacy of PPS have been demonstrated in multiple open-label and comparative clinical trials in different populations  of RSV-G protein and RSV-F protein were all at nanomolar concentrations, and the affinity of RSV-F protein was even stronger. Solution competition studies indicated that these two RSV glycoproteins exhibit different preferences for heparin chain lengths and sulfation patterns. Competition assays demonstrated that efficient binding of RSV-G protein requires longer saccharide chain, which is not necessary for RSV-F protein. We also found that 6-O-desulfated heparin and N-desulfated heparin could barely inhibit the binding of RSV-G protein to surface-immobilized heparin, whereas all chemically modified heparin derivatives showed reduced resonance signal in competition analysis of RSV-F protein–heparin interaction with the inhibition efficacy lower than that of heparin. Thus, all the sulfation sites are important for interaction between the RSV-G protein or RSV-F protein and heparin, although N-sulfo and 6-O-sulfo groups are much more critical for the binding to RSV-G protein. Most importantly, highly negatively charged PPS and MPS show promise as therapeutic and/or preventative antiviral drugs against RSV. It is promising to develop PPS or MPS related drugs targeting the interaction between RSV and HS on the cell surface to inhibit the invasion and infection of RSV.Investigating the molecular properties of RSV glycoprotein–heparin interaction, we can develop drugs that target the interaction and reduce RSV invasion. SPR analysis revealed that the binding affinity (