Interventions during childbirth may reduce maternal and neonatal deaths. The Guidelines for maternity care in South Africa (2015) stipulates that all observations during labour should be recorded on a partogram. The objective of this study was to assess the knowledge and attitudes of nursing personnel and to evaluate their practices of completing partograms at National District Hospital, South Africa.a two-phase, quantitative, cross-sectional, descriptive study design was used. In phase 1, the knowledge and attitudes of midwives and nurses were evaluated. Midwives and nurses completed anonymous, self-administered questionnaires that assessed their knowledge and attitudes. In Phase 2, partogram practices were measured by assessing completed partograms using a data collection tick sheet.twelve of the 17 nursing personnel completed the questionnaires. More than 90% of participants answered basic partogram knowledge questions correctly, but only two thirds knew the criteria for obstructive labour and just more than half that for foetal distress. Participants displayed a positive attitude toward the use of partograms. Of the 171 randomly selected vaginal deliveries during the study period, only 57.1% delivered with a completed partogram. Most elements of foetal monitoring and progress of labour scored above 80%, however, for maternal monitoring scored poorly in 26.4% of cases.although 71.4% of partograms scored more than 75% for completion, the critical components that influence maternal and foetal death, like the identification of foetal distress, maternal wellbeing and progress of labour, were lacking. Guidelines for maternity care in South Africa [Worldwide, 830 women die daily (302 000 yearly) , either h Africa  stipulatProfessor RH Philpott introduced the modern partogram, which is a graphical recording of the labour, including maternal and foetal observations that chart the progress of labour. The modern partogram is an improvement on the original partogram, developed by Friedman in 1955, as it includes the alert and action lines to identify abnormal labour . If abnoThe aim of this study was to assess the knowledge and attitudes of midwives and nurses and to evaluate their practices of completing partograms at the obstetric unit of the National District Hospital in Bloemfontein, South Africa. National District Hospital is part of the academic training complex of the University of the Free State and is involved in undergraduate and postgraduate training of medical, allied health and nursing students. During 2018, a total of 1 740 normal vaginal deliveries, 832 caesarean sections and 38 assisted deliveries were performed at the setting. At the same time, a further 239 babies were born before they arrived at the facility. Professional nurses take responsibility for the unit, which offers 24-hour doctor assistance and has theatre availability in the daytime.A two-phase, quantitative, cross-sectional, descriptive study design was used. In phase 1, the knowledge and attitudes of midwives and nurses were evaluated. In phase 2, partogram practices were evaluated.Study population and sample: the study population and sample consisted of all the advanced midwives and professional nurses working in the maternity unit of National District Hospital at the time of data collection.Measurement: midwives and nurses on duty during the study period completed an anonymous, self-administered questionnaire after they were informed about the study and had read the information leaflet. This was done during day and night duty over a two-week period to include most participants. The researchers developed the questionnaire from the Guidelines for maternity care in South Africa [h Africa  manual. Study population and sample: a systematic random sampling method was used to include every third vaginal delivery over three months (July-September 2018) and to assess their partograms. Names and file numbers were gathered from the delivery register and files were collected from the file store.Exclusion criteria: 1) elective caesarean sections were not included during the random sampling; and 2) files of mothers who had delivered before the partogram was started were excluded after selection.Measurement: partograms were assessed using a data collection tick sheet developed from information in the Guidelines for maternity care in South Africa [h Africa  as well Data management and analysis: all data from the questionnaires and the tick sheets were captured into a Microsoft Excel 2016 spreadsheet. Data were checked for correctness and completeness by the researchers and verified by the supervisor. Department of Biostatistics, University of the Free State, analysed the data. A score was calculated under each heading to assess for completeness. A score of ≥ 80% per category was considered good, and <50% as poor. The results were summarised by frequencies and percentages  and medians .Validity: the validity of the questionnaire and data collection sheets was established from information in the literature, discussion with experts in the field and through pre-testing of the collection tools.Ethical considerations: the project was approved by the Health Sciences Research Ethics Committee of the University of the Free State (HSREC UFS-HSD2018/0417/2602) and the Free State Department of Health. All data were managed confidentially, and no health care professional or patient is identified in any reporting of data.A total of four advanced midwives and eight professional nurses completed the questionnaires, while two were on leave and one did not report for work during the data collection period. Two chose not to complete the questionnaire. The response rate was 70.6%.Baseline data: one-third of respondents were advanced midwives and two-thirds held nursing diplomas. They had worked in a maternity ward for between 2 months and 17 years (median = 3 years), with 41.7% (n=5) working there for at least 9 years.Knowledge: most participants gave the correct answers for the basic knowledge questions e.g. the components of a partogram (91.7%), the duration of latent and active labour (91.7%) and the use of the partogram to identify prolonged labour and foetal distress (83.3%). Between 54.6% and 66.7% answered correctly regarding how often to monitor the mother and foetus, as well as how to identify obstructive labour and foetal distress. The knowledge results are displayed in Attitudes: the participants displayed positive attitudes toward the use of partograms. Two participants (16.7%) stated that the partogram is useful, nine (75.0%) indicated it is very useful, and only one (8.3%) did not find it useful at all. Most (83.3%) stated that partograms make caring for patients easier, and 91.7% indicated that partograms help them to identify possible complications. The factors mentioned that influence the completion of partograms are time constraints and personnel shortages. Although all participants knew that the partogram should be filled in during the labour process, 41.7% (n=5) found it difficult to divide their attention between the patient and the partogram. Shortages of personnel and busy units force participants to fill in partograms after the labour, or they do not complete it at all. One participant indicated that it is important that the partogram contains no mistakes and, therefore, partograms are “corrected” after the delivery. A quarter of participants (n=3) indicated that they had not received any formal training on completing partograms.Measurement of practices: a total of 91 partograms were assessed. The response rate of more than 70% for the questionnaires, as well as the demographic distribution of the participants, make the data collected representative of that of the nursing personnel at National District Hospital. This information is, however, not necessarily transferable to other settings, as this setting is a district hospital that is part of an academic training complex. More than 90% of participants answered basic knowledge items, for instance, the components on a partogram, correctly, but much lower percentages answered higher-level interpretation items correctly. Only two-thirds knew the criteria for obstructive labour, and just more than half for foetal distress. Taking into consideration that the aim of a partogram is to alert health care workers of foetal distress, maternal abnormalities and poor progress of labour , birth aIn this study, the attitudes of the health care workers were positive regarding the completion of partograms, with more than 90% believing that partograms are useful and could help them to identify problems during labour. Although a positive attitude towards the usefulness of partograms was identified, time constraints, lack of personnel and emphasis on the correct filling in of partograms sometimes meant participants did not fill in partograms before after the delivery, or to “correct” mistakes on the partogram. In an Eritrean study, 88.7% of participants had a positive attitude about the filling in of partograms , while mAll components of foetal assessment scored above 80%, except decelerations, which is the main indicator of foetal distress. Two recent African studies, one in Zambia  and one Limitations: due to the setting, these results may not be representative of other settings in South Africa. Despite an above 70% completion rate of questionnaires, the numbers of cases and participants are small. The large number of patients that delivered without a partogram or with only one recording on the partogram may have skewed the results; however, it highlighted the real-life situation of late presentation at labour wards, which is accompanied by its complications. The time of partogram completion (day versus night or weekday versus weekend) was not assessed. The difference between reported and measured practice must be taken into consideration when results are interpreted and compared.Although more than 70% of the partograms scored more than 75% for completion, it was the critical components that influence maternal and foetal death, such as the identification of foetal distress, maternal wellbeing, and progress of labour, that were lacking. Regular training and monitoring of partogram completion may be a first step to improving the practice of partogram use.The correct completion of partograms can save mothers and babies during childbirth;A partogram should be used to monitor each delivery.Partograms are not used optimally and therefore important danger signs are missed;Many deliveries take place without the filling of a partogram.


Rp1-D21 mutant. Functional analysis showed that ZmFNSI-1 and ZmFNSI-2, but not ZmS3H, suppressed HR conferred by Rp1-D21 and its signaling domain CCD21 when transiently expressed in N. benthamiana. ZmFNSI-1 and ZmFNSI-2 physically interacted with CCD21. Furthermore, ZmFNSI-1 and ZmFNSI-2 interacted with HCT, a key enzyme in lignin biosynthesis pathway, which can also suppress Rp1-D21-mediated HR. These results lay the foundation for the further functional analysis of the roles of FNSI in plant innate immunity.Nucleotide binding, leucine-rich-repeat (NLR) proteins are the major class of resistance (R) proteins used by plants to defend against pathogen infection. The recognition between NLRs and their cognate pathogen effectors usually triggers a rapid localized cell death, termed the hypersensitive response (HR). Flavone synthase I (FNSI) is one of the key enzymes in the flavone biosynthesis pathway. It also displays salicylic acid (SA) 5-hydroxylase (S5H) activity. A close homolog of FNSI/S5H displays SA 3-hydroxylase (S3H) activity. Both FNSI/S5H and S3H play important roles in plant innate immunity. However, the underlying molecular mechanisms and the relationship between S5H and S3H with the NLR-mediated HR are not known in any plant species. In this study, we identified three genes encoding ZmFNSI-1, ZmFNSI-2 and ZmS3H that are significantly upregulated in a maize line carrying an autoactive NLR  R) genes recognize the presence of specific pathogen-secreted molecules (effectors), triggering a series of defense responses which often include a localized programmed cell death at the point of pathogen challenge known as the hypersensitive response (HR) [To protect against the invasion of different pathogens, plants have evolved multiple sophisticated strategies including pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) ,2. ETI inse (HR) ,4,5. Thense (HR) ,7.R-genes encode so-called NLR proteins, which contain a central nucleotide-binding (NB) domain and a C-terminal leucine-rich repeat (LRR) domain [Most ) domain ,9. The N) domain . TNLs arRp1 locus on maize chromosome 10 carries multiple tandemly repeated CNL paralogs, one of which, Rp1-D, confers resistance to maize common rust caused by specific races of the fungus Puccini sorghi [Rp1-D21 mutant was formed by intragenic recombination between two paralogs, Rp1-D and Rp1-dp2 [Rp1-D21 confers a dominant spontaneous HR lesion phenotype on leaves and stalks in the absence of pathogen infection [The i sorghi . A high i sorghi . The chi Rp1-dp2 ,14. Rp1-nfection ,14. The nfection ,16.Rp1-D21 has been used as a tool to identify loci, genes and pathways associated with modulation of the severity of HR [Rp1-D21 compared to a near-isogenic wild type sibling [ty of HR ,17,18,19ty of HR ,21. BothFusarium verticillioides, a fungus causing ear rot and stalk rot in maize [The flavonoid biosynthesis pathway also plays important roles in plant disease resistance ,23,24. Fin maize . Flavonein maize . It beloin maize ,30, whicin maize ,32. Genein maize .Arabidopsis causes increased levels of apigenin [Arabidopsis ZmFNSI homolog, DOWNY MILDEW RESISTANT 6  displays both FNSI activity and salicylic acid (SA) 5-hydroxylase (S5H) activity, catalyzing the formation of 2,5-DHBA by hydroxylating salicylic acid (SA) [Atdmr6 mutant confers resistance to multiple pathogens, including Hyaloperonospora parasitica, H. arabidopsidis, Phytophthora capsici and Pseudomonas syringae. Expressing ZmFNSI in Atdmr6 restores the susceptibility to these pathogens [Atdmr6 mutant also accumulates higher levels of SA than wild type plants due to the S5H activity of AtDMR6 [Arabidopsis, DMR6-Like Oxygenase 1  and DLO2, with high similarity with AtDMR6, negatively regulate the defense response to downy mildew and P. syringae pv. tomato (Pst) DC3000 [Atdmr6 mutant restores the susceptibility to downy mildew, indicating their partially redundant function with AtDMR6 [Maize ZmFNSI-1 was demonstrated to be capable of converting the flavanones naringenin and eriodictyol into their corresponding flavones, apigenin and luteolin, respectively in plants . Two ricapigenin . The Aracid (SA) ,29,35,36athogens ,29,36. T) DC3000 . OverexpRp1-D21 compared to the near-isogenic wild type line from RNA-seq analysis. We examined their roles in modulating Rp1-D21-mediated HR. ZmFNSI-1 and ZmFNSI-2, but not ZmS3H, suppressed Rp1-D21-mediated HR when transiently co-expressed with Rp1-D21 in N. benthamiana, however, they had no obvious effect on cell death caused by other elicitors. ZmFNSI-1 and ZmFNSI-2 physically interacted with the Rp1 protein as determined by coimmunoprecipitation (Co-IP) and yeast two hybrid (Y2H) assays. Furthermore, we showed that ZmFNSI-1 and ZmFNSI-2 interacted with HCT, which can also suppress Rp1-D21-mediated HR. We discuss the importance of FNSI in modulating plant defense response and lay the foundation for the further functional analysis of the roles in plant innate immunity.The aim of the study was to investigate the function of ZmFNSIs and ZmS3H in NLR protein Rp1-D21-mediated HR. We identified three genes encoding ZmFNSI-1, ZmFNSI-2 and ZmS3H that were significantly upregulated in Rp1-D21 in two hybrid backgrounds, B73 × H95 and Mo17 × H95 [Rp1-D21 compared to the corresponding wild type, and all of them suppressed Rp1-D21-mediated HR when transiently expressed in N. benthamiana [We have previously performed RNA-seq analysis to compare a near-isogenic pair of lines with and without thamiana ,21.Rp1-D21 compared to their wild type counterparts and which had been otherwise implicated in plant disease resistance based on the literature for an initial screen. These genes included ZmFNSIs, ZmS3H, peptidoglycan-related genes, wall associated kinase, auxin-responsive genes, calmodulin binding proteins, etc.  and AtDLO2. Using AtDMR6 and AtS3H as queries to blast the maize genome database (MaizeGDB), we identified two ZmFNSI homologs, ZmFNSI-1 (Zm00001d029744) and ZmFNSI-2 (Zm00001d027423), and one ZmS3H homolog (Zm00001d002564) in maize  than ZmS3H (4.2–8.6 fold) (We observed that the transcript levels of .6 fold) .N. benthamiana. Rp1-D21 was fused with a 3× hemagglutinin tag at the C-terminus (Rp1-D21:HA), while ZmFNSI-1, ZmFNSI-2 and ZmS3H were fused with enhanced green fluorescent protein (EGFP) at the C-terminus (ZmS5H:EGFP and ZmS3H:EGFP). GUS:EGFP and HCT1806:EGFP were used as controls. Consistent with our previous studies [N. benthamiana, an obvious HR phenotype was observed at three days post-infiltration (dpi). No HR was observed in leaf sectors in which HCT1806:EGFP and Rp1-D21:HA were co-expressed , two CNL proteins conferring an autoactive HR when transiently expressed in N. benthamiana [To determine whether ZmFNSI-1 and ZmFNSI-2 could suppress HR mediated by other NLR proteins, we co-expressed them with barley MLA10(D502V) and thamiana ,40. The Phytophthora infestans and Bax is a death-promoting protein of the Bcl-2 family from mouse, both of which can induce cell death when transiently expressed in N. benthamiana [INF1 is a cell death elicitor from thamiana ,42. WhenArabidopsis AtS5H/AtDMR6, the histidine (H) at positions 212 and 269 is the catalytic residue for binding the ferrous iron atom and is critical for its FNSI enzyme activity [Atdmr6 to H. arabidopsisdis, indicating that the enzyme activity is required for the function of AtDMR6 [In activity . The subf AtDMR6 . Based oD21, a middle NB-ARC  domain and a C-terminal LRR domain [D21, but not NB or LRR, conferred autoactive HR when fused with EGFP and expressed transiently in N. benthamiana [D21-mediated HR, we co-expressed them and CCD21 in N. benthamiana, and found that ZmFNSI-1 and ZmFNSI-2, but not ZmS3H, suppressed CCD21-mediated HR  domain termed CCR domain . We havethamiana . To deteiated HR A. Ion leiated HR B.D21 and suppresses CCD21-mediated HR [D21, we used yeast two hybridization (Y2H) to show that ZmFNSI-1 and ZmFNSI-2 physically associated with CCD21 at 72 h after co-expression, while ZmS3H showed weak or no interaction with CCD21  to test whether ZmFNSI-1, ZmFNSI-2 and ZmS3H can form homomers. As shown in N. benthamiana lines harboring the nuclear marker H2B:RFP [To investigate where ZmFNSI-1, ZmFNSI-2 and ZmS3H are localized, we performed subcellular localization experiments by transient expression in transgenic  H2B:RFP . When fuD21 localized to both the cytoplasm and nucleus, and that nucleo-cytoplasm trafficking was important for HR induction [D21, we co-expressed ZmFNSI-1, ZmFNSI-2 and ZmS3H with CCD21:RFP in N. benthamiana. When CCD21:RFP was co-expressed with GUS:EGFP, it mainly localized in cytoplasm and nucleus, which is consistent with previous results [D21:RFP also mainly localized in the cytoplasm and nucleus  and AtDMR6(H269D), the substitution proteins in the histidine (H) residues required for the putative catalytic activity of AtDMR6, fail to restore the susceptibility of Atdmr6 to H. arabidopsidis, suggesting that the oxygenase enzymatic activity of AtDMR6 is required for its function in disease resistance [ZmFNSI is induced by multiple pathogens. ZmFNSI-1 is located in a quantitative trait loci (QTL) conferring resistance to southern leaf blight [Arabidopsis also has S5H activity [FNS1 belongs to the 2OGD superfamily members. In sistance . For thef blight , and it f blight . These ractivity ; it willD21-mediated HR, however, they did not substantially suppress the HR phenotype caused by the other two CNL autoactive mutants RPM1(D505V) and MLA10(D502V), and they have no obvious effect on Bax- and INF1-mediated cell death. These data suggested that ZmFNSI-1 and ZmFNSI-2 might not be general HR suppressors.ZmFNSI-1 and ZmFNSI-2 suppressed Rp1-D21- and CCArabidopsis, AtDMR6/AtS5H was reported to have high S5H activity and low FNSI activity [Atdmr6 is most likely caused by increased total SA levels [Pst DC3000 treatment, the transcript levels of AtDMR6/AtS5H and AtS3H are induced, and the accumulation of 2,5-DHBA and 2,3-DHBA is enhanced, implying that SA hydroxylation plays a role in the detoxification of excessive SA [AtDMR6/AtS5H is more sensitive to SA treatment and pathogen induction than AtS3H and the Ats5h mutant exhibited much stronger pathogen resistance than the Ats3h mutant [Atdmr6 mutant compared to wild type, and there is no significant different between the dlo1 mutant and wild type. However, in the dmr6dlo1 double mutant, the SA levels are approximately 20 times higher than that in the Atdmr6 mutant [AtDMR6/AtS5H and AtS3H have overlapping and distinct functions as negative regulators in innate immunity. Here, we found that the fold change of the transcript levels of ZmFNSI-1 and ZmFNSI-2 is higher than that of ZmS3H in the Rp1-D21 mutant compared to the corresponding wild type backgrounds  interacts with several kinases or pseudokinases to recognize effectors secreted from different pathogens [UMP and the activated ZAR1-RKS1-PBL2UMP complexes [Plant NLR genes are usually expressed at low levels in living cells. The over-accumulation of NLRs can lead to autoimmune responses, causing dwarfism and an autoactive HR phenotype . To reduathogens ,62,63. Romplexes ,65. A whP. sorghi [P. sorghi is still unknown. In our previous studies, we derived models to elucidate the transition between the resting and activation states of Rp1 proteins [Rp1-D confers resistance to common rust caused by . sorghi . Howeverproteins ,21,38. WN. benthamiana, we believe that it can largely reflect the situation in maize for the following reasons. Firstly, this system has been widely employed for exploring the HR phenotype mediated by NLRs from diverse plant species [In summary, we have demonstrated that ZmFNSI-1 and ZmFNSI-2, two homologs of enzymes which catalyze key steps in the flavone biosynthesis and SA catabolism pathway, interact with the CC domain of Rp1-D21 to regulate Rp1-D21 -mediated HR. ZmFNSI-1 and ZmFNSI-2 also interact with HCT. To our knowledge, this is the first evidence that the homologs of enzymes in the flavonoid and lignin biosynthesis pathways may function together to regulate the NLR-mediated defense response. Though substantial data were derived from agrobacteria-mediated transient expression in  species ,68,69,70 species ,21. Thir species . With thZea mays) line B73 was grown at 24 °C with 12 h light/12 h dark conditions and used for isolating ZmFNSI-1, ZmFNSI-2 and ZmS3H. Wild type Nicotiana benthamiana and the transgenic N. benthamiana harboring histone 2B (H2B)-TaqRFP were grown at 23 °C with a cycle of 16 h light and 8 h dark.Maize  transformed with binary vector constructs was grown at 28 °C overnight in 8 mL of L-broth medium supplemented with appropriate antibiotics. The detailed procedures were performed according to our previous study [us study . Unless N. benthamiana. Leaf samples were collected at 30 hpi, and proteins were extracted by grinding 0.6 g of leaf samples in 1.8 mL extraction buffer (50 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM EDTA (pH 8.0), 0.5% Triton 100, 4 mM DTT, 40 µM MG132, and 1× plant protein protease inhibitor mixture . The kit containing anti-GFP microbeads  was used for the Co-IP assay. The detailed procedures for the Co-IP assay were performed according to our previous study [For protein expression analysis, three leaf discs (1.2 cm in diameter) infiltrated by agrobacteria were collected at 30 h post inoculation (hpi). The samples were ground in liquid nitrogen, and total protein was extracted in 150 µL extraction buffer (20 mM Tris·HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.1% SDS, 10 mM DTT, 40 µM MG132, and 1× plant protein protease inhibitor mixture ). For the Co-IP assay, Myc-, EGFP-, and 3×HA-tagged constructs were transiently co-expressed into us study  with somD21 were constructed into the pGBKT7 (BD) vector . AD- and BD-derived constructs were co-transformed into competent cells of the yeast strain Y2HGold. The Y2H interaction assay was performed according to the protocol provided by the manufacturer .ZmFNSI-1, ZmFNSI-2 and ZmS3H were constructed into the pGADT7 (AD) cloning vector  by LR reactions, and CCN. benthaminana leaves were used for observation at 48 hpi by a confocal microscope  with a PMT detector. The representative areas were photographed under a 20× objective. EGFP fluorescence was excited at 488 nm and observed between 495 and 550 nm. RFP was excited at 561 nm and observed between 580 and 675 nm.Confocal microscopy observation was performed according to our previous study  with somRp1-D21 is KF951062 and AIW65617, respectively. RNA-seq data: SRP060286.Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession numbers. Nucleotide sequence and protein sequence of


IUCrJ published from 2019 onwards reflect the breadth and outreach of this new and important subject.The articles in crystal engineering and structural chemistry in  IUCrJ during 2019 and early 2020 exemplify these trends. Crystal engineering is all about the design of molecular crystal structures and there is now greater emphasis on property design.As the subject of crystal engineering diversifies and extends its outreach into many aspects of structural science, it is but natural that the 30 or so papers published in this area in The present focus on pharmaceutical solids and the importance of crystal engineering in the synthesis of new solid forms, be they cocrystals, polymorphs or solvates, has resulted in a concomitant increase in papers dealing with such topics. Cocrystals are especially attractive from the design viewpoint: there is nascent interest in formulation technologies for cocrystals and one even comes across the term ‘cocrystal engineering’!IUCrJ since the start of 2019. For comparison, about 150 papers in the crystal engineering area have been published since the inception of the journal itself. Recent papers that have been published in IUCrJ cover a variety of frontier topics and some representative papers are listed below (Alex et al., 2019et al., 2020et al., 2020et al., 2019et al., 2019et al., 2020The 30 above-mentioned crystal engineering papers appear among the 120 or so that have been published in IUCrJ and it is important to maintain this high benchmarking. The 24th Congress of the IUCr will take place this year and the program is, as usual, strong in crystal engineering and structural chemistry; it is to be hoped that this will have its impact on the number and quality of crystal engineering papers in IUCrJ during the coming year.I would certainly like to see more papers in this section in 2020–2021, possibly more in the MOF/COF areas, and an increase in the journal impact factor. It is not easy to publish in


In the recent original research published on International Journal of Fertility and Sterility the association betweentumor necrosis factor-alpha (TNF-α) genetic polymorphisms and endometriosis in 150 Iranian patients suffered thisdisease. The authors notably found a lower frequency of TNF-α -863C/A allele A among the affected patients incomparison with healthy women, although this difference was not significant by adjusting multiple testing. We deemthat the authors should specify, if these patients had peritoneal nodules, ovarian endometrioma/deep infiltratingendometriosis (DIE) nodules or combination of them, since it has been hypothesized that these phenotypes may representthree distinct pathogenetic entities of endometriosis. Dear editor of International Journal of Fertility and Sterility,We recently read the article by Babaabasi et al.  entitledTNF-α is an inflammatory cytokine with a critical rolein activating several transcription factors involved in inflammation, such as NF-Kappa-B and c-Jun N-terminalkinases (JNK) . SeveralIn this letter, we would like to point out a methodological concern of this interesting study: in the “Materials and Methods” section, the authors indicated thatanalysis of some genotypes in the 5'-untranslated regionof TNF-α was done on DNA obtained from peripheralblood samples of 150 Iranian women with confirmedendometriosis. In particular, the polymorphisms weregenotyped by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Wedeem that the authors should specify, if these patientshad peritoneal nodules, ovarian endometrioma/deep infiltrating endometriosis (DIE) nodules or combinationof them, since these three phenotypes of endometriosismay have different pathogenesis. Moreover, it wouldbe of particular interest to know if the risk of havingendometriosis in presence of the TNF-α specific -863A allele was lower, regardless of such endometriosisphenotypes. In fact, we believe that different polymorphisms of TNF-α gene may lead to peculiar transcriptional activity and final protein function, definitivelygiving a different pathogenic contribution to each endometriotic phenotype .Other genetic polymorphisms have been correlatedwith specific phenotypes of this disease: for example,it has been reported a significant association of progesterone receptor þ331G/A polymorphism with DIE, suggesting a potential role of this variant in the hormonaldependent invasive behavior of endometrial cells .In a poOverall, it has been described that nodules of DIE tendto have higher pro-inflammatory microenvironment andoxidative stress, with a more aggressive clinical behavior and different response to conventional therapies incomparison with the other endometriosis phenotypes, 8. Hormin vitro studies havedemonstrated that TNF-α is responsible for proliferation ectopic and eutopic endometrial cells as well as thecell adherence within the endometriotic lesions (Investigations on medical therapeutic approaches fortreating endometriosis, represent one of our topics of research: in particular, we recently reviewed the role ofexperimental drugs targeting inflammation and immunesystem in this setting . Notably lesions , 11.TNF lesions , 13. Mor lesions . Until n lesions . Althoug lesions , the stuOverall, findings of the study performed by Babaabasiand colleagues  are inno


Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines.Porcine Parvovirus (PPV) is a K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice.After optimization according to the codon bias of K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make  Parvoviridae taxonomic family and are divided into two subfamilies, the Parvovirinae and the Densovirinae, that infects vertebrates and arthropods respectively [Parvovirinae subfamily, parvoviruses in the genera Protoparvovirus, Bocaparvovirus, Copiparvovirus and Teraparovirus, were also designated as porcine parvovirus types 1 through 6 [Ungulate protoparvovirus 1, was first isolated from a contaminant pig kidney culture in 1965 in Germany [Parvoviruses are small, non-enveloped DNA viruses belonging to the ectively . In the hrough 6 . Classic Germany . In the  Germany . PPV was Germany , 8. VaccPPV is a negative, single-stranded DNA virus with a genome about 5000 nucleotides, and its mature viron is an icosahedral symmetric particle with approximately 25 nm in diameter. The genome of PPV contains two large non-overlapping Open Reading Frames (ORFs), the 5′-end ORF, encoding a non-structural protein, and the 3′-end ORF, encoding three structural proteins VP1, VP2 and VP3 . VP1 andSaccharomyces cerevisiae, Pichia pastoris and Escherichia coli. In S. cerevisiae recombinant VP2 spontaneously assmebled into VLPs intracellularly, but only 8–9 mg VLPs were recovered from one liter of induced yeast culture [Pichia pastoris, another yeast that is widely used for protein expression, produced the 595.76 mg/L VP2 proteins via secretory expression, but whether VLPs were formed extracellularly remains unknown [E. coli is the most popular bacterial systems of producing recombinant protein, while most of VP2 proteins formed inclusion body after induction even if it was coexpressed with chaperones GroES-GroEL-tig [VP2 proteins can spontaneously self-assemble into virus-like particles (VLPs), mimicking the morphology of pathogenic virus and maintaining an identical hemagglutination activity . Recombi culture . Pichia  unknown . E. coliroEL-tig .PPV VLPs are safe and practical alternatives to inactivated infectious viruses as a vaccine that can provoke a strong protective immune response without the risk of diseases and offer a ready platform for facilitating recognition, uptake, and processing by the immune system . As selfKluyveromyces marxianus. This strain is an aerobic, Crabtree negative, and homothallic hemiascomycetous yeast that can generate energy from both respiratory metabolism and ethanol fermentation [K. marxianus, we achieved a 2.5 g/L VLPs of the Kresse strain in a 5 L fermentor. This production is much higher than previous reports. PPV VLPs produced in K. marxianus elicited high titers of IgG antibody and hemagglutination inhibition antibody, and therefore can be used to develop anti-PPV veterinary vaccines.In the current study, we sought to achieve a high production of PPV VLPs using a nonconventional yeast entation –27. It hentation –31. UsinK. marxianus Fim-1ΔURA3 is a uracil auxotroph strain originated from K. marxianus Fim-1 deposited in China General Microbiological Culture Collection Center  was optimized and synthesized by Genewiz Biotechnology Co., Ltd . The optimized VP2 gene was deposited in the NCBI GenBank database under an accession number MT932328. The synthetic VP2 gene was amplified with Phanta® Super Fidelity DNA Polymerase  using the following oligonucleotide primers (underlining indicates the homologous sequences from pUKD-N115 vector): 5′-TTTTTTTGTT AGATCCGCGG ATGAGCGAAA ACGTGGAGC-3′) and 5′-AGCTTGCGGC CTTAACTAGT CTAGTACAAC TTTCTTGGG-3′. The amplicon was ligated with the EcoR I and Hind III linearized pUKDN115 by Gibson assembly [URA3 strain according to the Lithium acetate transformation method [According to the assembly , and then method . Transfo2HPO4, 1.8 mM KH2PO4, pH 7.4) twice, and then suspended in 500 µl lysate buffer . Approximately 400 µl of glass beads  was added to disrupt cells on a Bead-beater  at 6 m/s for 2 min. Cell lysates were centrifuged at 12,000 rpm, 4 °C for 20 min, and supernatants were used for SDS-PAGE and Western blotting assays. An anti-PPV VP2 polyclonal antibody and a goat anti-mouse IgG alkaline phosphatase-conjugate  was used as the primary and secondary antibody in western blotting respectively.Fresh clones of KM-PPV-VP2 were inoculated in 50 mL YD medium  and cultured at 30 °C, 220 rpm for 72 h. One milliliter of yeast cells harvested by centrifugation was washed with 1 ml PBS buffer , generating the pET-28a/VP2 plasmid. After transformation into E. coli BL21(DE3), VP2 protein was induced by 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and purified by Ni-NTA  affinity basically as previously described [The native VP2 gene of Kresse strain was cloned into a pET-28a (+) vector within the escribed . Six weeTEM scans of PPV VLPs were performed on a JEM-2100 Electron Microscope  according to Bucarey et al. . Briefly600 nm) and wet cell weight (WCW). For SDS-PAGE analysis of VP2 productions, cell samples were diluted 1:10 with PBS buffer before disruption using the glass bead disruption described above. VLPs quantification was performed on an Agilent Series 1100 System  using a TSKgel G4000 SWXL column (300 mm × 7.8 mm i.d.)  and a TSKgel SWXL guard column (40.0 mm × 6.0 mm i.d.) (Tosoh Bioscience) as previously described [High cell-density fermentation was conducted in a 5 L fermentor  as described recently . The KM-escribed .KM-PPV-VP2 cells were collected by centrifugation at 5000 rpm for 10 min, followed by washing with deionized water twice. Rinsed cells were then suspended with PBS buffer pH7.4 for cation exchange, or with 20 mM Tris-HCl buffer pH 8.0 for anion resins. Cell lysates were prepared by high-pressure homogenization on a JN-02C Homogenizer  under a condition of 1500 bar, 4 °C for 2 times, followed by centrifugation at 10,000 rpm, 4 °C for 30 min.Screens of IEX media were performed on the Poly-Prep®Chromatography Columns  packaged 2 ml of different cation or anion resins listed in Table To purify PPV VLPs by IEX chromatography, yeast cells were suspended in PBS buffer pH7.4 and disrupted by high-pressure homogenization. Cell lysates were subsequently adjusted to pH 4.0. After centrifugation at 10,000 rpm, 4 °C for 30 min, the supernatants of pH adjusted cell lysate were loaded onto an XK 50/30 column  packed with 400 mL of Capto S ImpAct resin. Binding VLPs were eluted with 20 mM sodium acetate buffer containing 500 mM NaCl. To elevate the recovery of VLPs, the precipitates of pH adjusted cell lysates were redissolved in an equal volume of 20 mM Tris-HCl buffer pH 8.0. Through centrifugation, the clarified supernatant were loaded onto an XK 50/30 column packed with 400 mL of Capto Q XP resins. After elution with 20 mM Tris-HCl buffer pH 8.0 plus 500 mM NaCl, fractions were diafiltrated for 10 volumes of PBS on ÄKTA flux  equipped with a 750 kDa column . Further polishing purification of PPV VLPs was performed on an AKTA Purifier 100  using a HiPrep™ 26/60 Sephacryl® S-500 HR column . About 4 ml IEX purified sample was injected and eluted with PBS at a rate of 0.5 mL/min. Protein concentration was measured by the BCA Protein Assay Kit .Purified VLPs were diluted with PBS buffer and emulsified with MONTANIDE™ Gel 01 adjuvant  at a rate of 10%, giving a final antigen concentration of 240 µg/mL. Fifteen of 6-week old female SPF Balb/c mice were randomly divided into 3 groups (n = 5). Grouped mice were subcutaneously injected with 20 µg, 40 µg PPV VLPs, and 250 µL of PBS as a control, respectively. Blood samples were collected from cheek each week till 49 dpi. Sera were separated by incubating blood at 37 °C for 1 h, followed by centrifugation at 3000 rpm for 4 min, and stored in small aliquots at − 20 °C.96-well Costar Assay Plates  were coated with the Ni-NTA purified VP2 protein according to the method described previously . ELISA dHI antibody titers of serum samples were determined in U-bottom 96-well plates according to the standard method . Briefly6 cells/mL. After adding 0.2 µg Concanavalin A , the plates were incubated at 37 °C for 48 h. Proliferative responses were detected using the Cell Titer 96® AQueous One Solution Cell Proliferation Assay Kit , and stimulation indexes (SI) were calculated as the ratio of the stimulated sample divided to the unstimulated control at OD490 nm. Cytokines secreted by the spleen cells were measured using the Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine Kit .At 42 dpi, three mice from the groups injected with 20 µg PPV VLPs and PBS were euthanized to separate spleen lymphocytes in a mouse lymphocyte separation medium . The separated spleen cells were then cultivated in 96-well plates containing 100 µL of 1640 culture medium , at a concentration of 3 × 10VP2 gene of the Kresse strain was used to express in K. marxianus, since this PPV strain displayed an increased virulence compared with other virulent strains that could kill immunocompetent fetuses [K. marxianus, and then inserted directly downstream of the K. marxianus inulinase promoter of the pUKDN115 vector  , 41, 42.600 nm) reached 560, with a dry biomass more than 120 g/L  type 2 by expressing the capsid Cap protein in K. marxianus  [K. marxianus to express large capsid protein, assembly of large capsids, and production of different VLPs, the PPV VP2, may be among the largest capsid protein of known viruses, was used to express in our K. marxianus strain. Attribute to high growth rate and facile high biomass, the recombinant K. marxianus strain produced 2.5 g/L PPV VLPs after just a 48 h fermentation. As a comparison, the production level of VLPs in Pichia pastoris, E. coli, S. cerevisiae is 595.76 mg/L [K. marxianus has a notable advantage over other strains, such as S. cerevisiae and E. coli, on high production of VLPs that there is no additional inducement during its fermentation since glucose serves as the carbon resources to support cell growth as well as an inducer for its inulinase promoter to obtain high expression of heterologous proteins [The commercially available PPV vaccines are inactivated whole-virus and live-attenuated vaccines. Irrespective of safety risks such as incomplete inactivation and reversion to virulence, albeit small probability, there are intrinsic disadvantages involved with the administration of inactivated or attenuated vaccines that it hampered to discriminate the differentiation of infected from vaccinated animals (DIVA) in seropositive animals . Intrigurxianus  . However.76 mg/L , 15 mg/L.76 mg/L , and 8–9.76 mg/L  respectiproteins .As to yeast cells, there is no additional treatment to remove endotoxins, but other downstream operations, such as disrupting the rigid wall of the yeast cells to release VLPs, clarification of cell lysates, and purification processes, are the main factors that significantly increased the production costs overall . In prevK. marxianus expression platform. Another, recombinant VLPs can be easily changed to desired genotype based on a well-established expression platform in a shorter time.Currently, most of inactivated or attenuated PPV vaccines were originated from the Cluster A strain NADL-2. The Kresse strain, isolated in the USA in 1985, is the most serious type of PPV-1 that can kill the immunocompetent fetuses . These tK. marxianus, high titers of specific IgG antibodies were induced in mice. As is known, PPV virus could agglutinate erythrocytes of chicken, guinea pig, mouse, human, monkey, rat, and cat [K. marxianus maintained the natural morphology of pathogenic virus [Compared to inactivated PPV vaccines, facile high titers of PPV VLPs elicited better cellular immune responses in gilts, and induced high neutralization antibody and high hemagglutination inhibition antibody in guinea pigs , 50. Whe and cat . In our ic virus . But theic virus , 51, 53.ic virus . These r


Cytokines are proteins secreted in the central nervous system by neurons,microglia, astrocytes and infiltrating peripheral immune cells underphysiological and pathological conditions. Over the last 20 years, agrowing number of reports have investigated the effects of thesemolecules on brain plasticity. In this review, we describe how the keycytokines interleukin 1β, interleukin 6 and tumour necrosis factor αwere found to support long-term plasticity and learning and memoryprocesses in physiological conditions. In contrast, duringinflammation where cytokines levels are elevated such as in models ofbrain injury or infection, depression or neurodegeneration, theeffects of cytokines are mostly detrimental to memory mechanisms,associated behaviours and homeostatic plasticity. N-methyl-d-aspartate receptors(NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptorsexpression was found to be induced following fear conditioningand during spatial memory tasks . No impaPeripheral IL-1β injection stimulates the release of monoamines inthe brain , which aRodent studies have shed light on the role of IL-6 in learning andmemory  in diffeThe mechanisms by which IL-6 modulates cognitive function are farfrom being elucidated . DecreasVery few studies have investigated TNF-α role under physiologicalconditions. Mice lacking TNF-α performed poorly in a novelobject recognition task and in the Barnes maze test thatevaluates spatial memory and learning effectiveness . A positTNF-α role in synaptic activity has been more extensively studied.TNF-α regulates synaptic scaling by promoting the insertion ofAMPAR lacking GluR2 subunit at the plasma membrane of synapses,hereby promoting excitatory signalling over inhibitorysignalling. TNF-α also modulates gliotransmission by elevatingastrocytic calcium levels which in turn increase by twofold thenumber of glutamatergic exocytic vesicles. Released glutamateactivates neuronal NMDAR at granule cells synapses of thedentate gyrus, hereby increasing excitatory synaptic activityand potentiation of GC synapses .Beattie et al. showed, using neuronal cultures and acutehippocampal slices, that TNF-α was required for preservation ofsynaptic strength and increasing the number of AMPAR at themembrane, the number of synapses and the frequency of miniatureexcitatory post-synaptic currents . ConcomiThe role of TNF-α in other forms of plasticity is unclear. Somestudies reported that LTP induction or maintenance did notrequire TNF-α as TNF-α KO mice displayed normal LTP in the CA1pyramidal layer . HoweverCentral and peripheral inflammation in response to pathogens, injury ordisease is a defence mechanism by which microglia and astrocytesbecome activated and potentially produce chemoattractant proteins thatpromote extravasation of monocytes from the periphery into the brain. TherefoA detrimental role of IL-1β on learning and memory was described inan inflammatory environment  created Escherichia coli, IL-1β is upregulated inthe aged rat hippocampus (Legionellapneumophila before subjecting them to the Morriswater maze (MWM). Sick mice displayed impaired learning; theirtask performance was, however, restored to control levels ifthey were treated with an anti-IL-1β antibody  antibody .Lipopolantibody . Followiantibody . IP injeantibody and impaantibody ,an effeantibody .A mouseantibody . IL-1β iantibody . Interesantibody .Stressors like inescapable shocks also trigger an increase of IL-1βprotein levels in the brain, although this increase can bedifficult to highlight as it is likely to be masked by aninhibitory action of glucocorticoids .ChronicIn a model of Parkinson’s disease, elevated IL-1β production in thebrain worsened neurodegeneration course by supporting neuronaldeath, either directly or via nitric oxide production . IL-1β lSome mechanisms by which IL-1β modulates memory function have beendescribed. It has been reported that the synaptic sensitivity toIL-1β increases with age due to a ratio change of theIL-1-receptor type 1 subunits AcP (proinflammatory) and AcPb that facilitates memory impairment . There iIn illnesses such as depression, arthritis and others where chronicinflammation is settled, patients report sickness and depressivebehaviours and cognitive decline, often accompanied by elevatedlevels of IL-6 . IndividIL-6 plays a key role in activating astrocytes and microglia duringinflammatory conditions. IL-6 KO mice displayed reduced reactiveastrogliosis and microgliosis after IP injection of the toxicglutamate receptor agonist kainic acid (model of seizure; The role of TNF-α in learning and memory under inflammatoryconditions is likely to be age dependent. Indeed, studies using30-day-old mice overexpressing TNF-α did not find any learningand memory impairment in the water maze test ,whereasChronic pain is often accompanied by memory deficits and a reducedhippocampal volume .In a moAstrocytic and microglial TNF-α release under inflammatoryconditions is a feature of neurodegenerative diseases  and is aIn an inflammatory context, TNF-α levels can rise from picomolar tomillimolar levels; phosphorylation of MAPKs  have beeSurprisingly, the effects described above do not occur in thedorsolateral striatum of rodents. In this region rich ininhibitory GABAergic neurons, TNF-α promotes the internalisationof AMPAR permeable to calcium thereby reducing synapticstrength, which could be an adaptative mechanism of the brain todelay motor symptoms in diseases characterised by movementdisorders and elevated TNF-α levels . OverallCytokines are much more than basic signalling inflammatory molecules as theyhave key roles in modulating cognitive functions in physiologicalconditions. We propose that the length, strength and location in aparticular area of the brain of cytokine activity will dictate theactivation of specific signalling pathways together with the stimulation ofother inflammatory systems that will contribute to the complexity of theresponse. This renders the distinction between mechanisms that would besolely engaged either in physiological or in inflammatory conditions verydifficult. We suggest that the homeostasis equilibrium shifts whenpathological, chronic expression of cytokines sustains in time; then thecytokine actions often become detrimental and lead to neuronal death andlearning and memory impairments. In that view, a better understanding of theinteraction between the whole immune system and synaptic plasticity wouldcertainly be key to develop treatments for neuronal, cognitive and mooddisorders that present deficits in these mechanisms.


Interleukin (IL)-33 is a member of the IL-1 family, which acts as an alarmin. Several studies suggested that IL-33 inhibited osteoclastogenesis and bone resorption. Tumor necrosis factor-α (TNF-α) is considered a direct inducer of osteoclastogenesis. However, there has been no report regarding the effect of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption. The objective of this study is to investigate the role of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption. In an in vitro analysis of osteoclastogenesis, osteoclast precursors, which were derived from bone marrow cells, were treated with or without IL-33 in the presence of TNF-α. Tartrate-resistant acid phosphatase (TRAP) staining solution was used to assess osteoclast formation. In an in vivo analysis of mouse calvariae, TNF-α with or without IL-33 was subcutaneously administrated into the supracalvarial region of mice daily for 5 days. Histological sections were stained for TRAP, and osteoclast numbers were determined. Using micro-CT reconstruction images, the ratio of bone destruction area on the calvariae was evaluated. The number of TRAP-positive cells induced by TNF-α was significantly decreased with IL-33 in vitro and in vivo. Bone resorption was also reduced. IL-33 inhibited IκB phosphorylation and NF-κB nuclear translocation. These results suggest that IL-33 inhibited TNF-α-induced osteoclastogenesis and bone resorption. Osteoclasts are large multinucleated cells responsible for bone resorption, which are formed by differentiation and fusion of precursor cells derived from a monocyte/macrophage lineage . ReceptoThe cytokine interleukin (IL)-33 is a member of the IL-1 family . IL-33 iAlthough IL-33 is likely to inhibit osteoclastogenesis, the exact mechanism remains unclear. In the present study, we performed osteoclast formation using TNF-α, and investigated whether IL-33 inhibits TNF-α-induced osteoclastogenesis and bone resorption.To investigate whether IL-33 affects osteoclast precursors directly, we analyzed the effects of IL-33 on RANKL- and TNF-α-induced osteoclastogenesis of osteoclast precursors. A large number of tartrate-resistant acid phosphatase (TRAP)-positive cells were observed among osteoclast precursors treated with M-CSF and RANKL/TNF-α, whereas this was significantly lower among osteoclast precursors when additionally treated with IL-33 a,b. FurtTo determine whether IL-33 inhibits TNF-α-induced osteoclast formation in vivo, TNF-α was injected subcutaneously into the supracalvarial region of mice for 5 days with or without IL-33. When TNF-α was administered, the number of TRAP-positive cells in the suture of histological sections was significantly increased. By contrast, the number of TRAP-positive cells was decreased in the TNF-α plus IL-33 group compared with TNF-α alone group a. The peCalvariae of all mouse groups were scanned by microfocus computed tomography (micro-CT), and bone resorption was analyzed. The ratio of bone resorption area to total area in the TNF-α alone group was significantly higher than the phosphate-buffered saline (PBS) alone and IL-33 alone groups. Co-application of TNF-α and IL-33 reduced the ratio of bone resorption area compared with the TNF-α alone group a,b.We evaluated the molecular mechanism by which IL-33 inhibits TNF-α-induced osteoclast formation. TNF-α or TNF-α plus IL-33 were added for specific periods . When TNF-α was added, phosphorylation of MAPK increased transiently . IL-33 fTo evaluate the effect of IL-33 on NF-κB nuclear translocation, we performed immunofluorescence imaging of osteoclast precursors cultured with TNF-α or TNF-α plus IL-33 a. This aIn the present study, we evaluated the effect of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption in vitro and in vivo. In vivo, we focused on TNF-α-induced osteoclastogenesis. The number of TNF-α-induced TRAP-positive cells was significantly decreased by IL-33. Moreover, the ratio of the bone destruction area was also decreased by IL-33. Our results suggested that IL-33 inhibited TNF-α-induced osteoclastogenesis and bone resorption in vitro and in vivo. To the best of our knowledge, this is the first report to assess the inhibitory effect of IL-33 on TNF-α-induced osteoclastogenesis and bone resorption.RANKL gene displayed severe osteopetrosis [TNF-α plays a central role in inflammatory osteoclastogenesis. RANKL has been considered the essential factor for osteoclast differentiation since it was reported that mice with a disrupted petrosis . Howeverpetrosis . In the petrosis , few stupetrosis . In the petrosis ,19,20. Hpetrosis . We can We have previously demonstrated that osteoclasts can be induced in mouse calvariae treated with TNF-α in vivo . In the NF-κB is generally located in the cytoplasm of osteoclast precursors and is naturally inhibited by IκB. Binding of TNF-α to the receptor causes phosphorylation of IκB, which results in dissociation of IκB from NF-κB. The activated NF-κB is then translocated into the nucleus and activates transcription . We haveIL-33 could provide a potential target for therapeutic intervention in a wide range of diseases . IL-33 iEight-week-old male C57BL/6J mice were purchased from CLEA Japan . The protocols for all animal procedures were approved by the Tohoku University of Science Animal Care and Use Committee. Four mice were randomly assigned to each experimental group and were injected reagents. Recombinant mouse TNF-α was prepared in our laboratory as previously described . Recombi2. Non-adherent cells were removed with PBS and adherent cells were collected using a trypsin-ethylenediamineteraacetic acid (EDTA) solution . The obtained cells, bone marrow macrophages, were used as osteoclast precursors in the present study. Osteoclast precursors (5 × 104 cells) were seeded in 200 μL α-MEM containing 10% FBS and 1% PS in a 96-well plate and cultured in medium containing M-CSF alone (100 ng/mL), M-CSF (100 ng/mL) plus RANKL (100 ng/mL) or TNF-α (100 ng/mL), M-CSF (100 ng/mL) plus RANKL (100 ng/mL) or TNF-α (100 ng/mL) with IL-33 (100 ng/mL), or M-CSF (100 ng/mL) with IL-33 (100 ng/mL). After 5 days, the cultured cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 for 1 h at room temperature. TRAP staining solution was used to visualize osteoclasts. TRAP solution was prepared by mixing acetate buffer (pH 5.0), 50 mM sodium tartrate, naphthol AS-MX phosphate , and Fast Red Violet LB salt . TRAP-positive cells with ≥3 nuclei were considered to be osteoclasts and were counted under a light microscope [To isolate bone marrow cells from mice, femora and tibiae were immediately dissected after euthanasia. The epiphyses of these long bones were removed using scissors, and the bone marrow was flushed out with PBS. The cell suspension was filtered through a 40-μm nylon cell strainer  and centrifuged. The obtained cells were cultured in alpha-modified minimal essential medium (α-MEM)  containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin , and M-CSF for 3 days at 37 °C and under 5% COcroscope . For resPrevious reports have demonstrated that the daily injection of TNF-α (1.5 μg) into the supracalvarial region for 5 days effectively induced osteoclast formation in vivo . We follFor in vitro experiment, osteoclast precursors were cultured on 60mm dishes in α-MEM with M-CSF alone (100 ng/mL), M-CSF (100 ng/mL) plus TNF-α (100 ng/mL), M-CSF (100 ng/mL) plus TNF-α (100 ng/mL) with IL-33 (100 ng/mL), or M-CSF (100 ng/mL) with IL-33 (100 ng/mL) for 5 days. Total RNA was obtained from lysed cells using RNeasy mini kit . Calvariae from in vivo experiments were frozen in liquid nitrogen and individually homogenized using a Micro Smash MS-100R  in TRIzol reagent . Total RNA was extracted using an RNeasy mini kit  according to the manufacturer’s protocol. cDNA was synthesized from each sample using 1 μg total RNA with Superscript IV Reverse Transcriptase . To assess the gene expression levels, a Thermal Cycler Dice Real Time system  was used for real-time RT-PCR. Each reaction comprised a total volume of 25 μL, containing 2 μL cDNA and a 23 μL mixture of TB Green Premix Ex Taq II  and 50 pmol/μL primers. The PCR cycling conditions were as follows: initial denaturation stage (95 °C for 30 s), amplification stage , and final dissociation stage . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used to normalize the gene expression levels. The primers are listed in To assess the bone destruction, calvariae fixed in 4% paraformaldehyde at 4 °C were scanned by micro-CT . Three-dimensional images of the calvariae were created using TRI/3D-BON64 software . The threshold was determined by CT phantom. Bone destruction area was measured around the bregma 150 pixels in the sagittal plane and 90 pixels in the coronal plane. Shaded areas of the same color density were considered bone destruction areas. The ratio of bone resorption area to total area was measured using ImageJ  .Osteoclast precursors were cultured in 100-mm cell culture dishes  in serum-free α-MEM (serum starvation) for 3 h. TNF-α (100 ng/mL) or TNF-α plus IL-33 (100 ng/mL) were then added to the dishes for specific periods . Cytokine-treated osteoclast precursors were washed twice with PBS, then were lysed using radioimmunoprecipitation assay (RIPA) buffer  containing 1% protease and phosphatase inhibitor . Total protein concentrations were determined using Pierce BCA protein assay kit . Protein samples were treated with β-mercaptoethanol  and laemmli sample buffer  and denatured at 95 °C for 5 min as a preparation for SDS-PAGE. Equal amounts of protein were loaded into gels 4–15% Mini-PROTEAN TGX Precast Gels  and transferred to a PVDF Trans-Blot Turbo Transfer System . The membranes were blocked in Block-Ace  for 1 h at room temperature. The membranes were probed with the following antibodies: phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP Rabbit mAb, phospho-SAPK/JNK (Thr183/Tyr185) (98F2) Rabbit mAb, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Antibody, phospho-IκBα (Ser32) (14D4) Rabbit mAb , monoclonal Anti-β-Actin antibody produced in mouse  overnight at 4 °C. The membranes were washed in tris buffered saline with Triton X-100 (TBS-T) and tris buffered saline (TBS), then incubated with anti-rabbit IgG HRP-linked Antibody  or anti-mouse antibody  for 1 h at room temperature. The membranes were washed in TBS-T and TBS again, then incubated with SuperSignal West Femto Maximum Sensitivity Substrate . The signals were detected by the FUSION-FX7.EDGE Chemiluminescence Imaging System . The stained bands were quantified with Evolution Capt  and the results expressed relative to control and normalized to β-Actin.Osteoclast precursors were cultured in 96-well plates in α-MEM containing TNF-α (100 ng/mL) or TNF-α plus IL-33 (100 ng/mL) for 1 h. The cultured cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature, washed and blocked with 3% BSA in PBS for 1.5 h at room temperature. The cells were proved with NF-κB p65 (C-20) sc-372 rabbit polyclonal Antibody  in 3% BSA in PBS overnight at 4 °C. The cells were washed with PBS and incubated with Alexa Fluor 555 goat anti-rabbit IgG  in 3% BSA in PBS for 1 h at a room temperature. The cells were washed and counterstained with 4′,6-diamidino-2-phenylindole (DAPI).p < 0.05 was considered to be statistically significant.All values are presented as the mean ± standard deviation. Statistical analyses were performed using Scheffe’s test. We demonstrated that IL-33 directly inhibited TNF-α-induced osteoclastogenesis in vitro and TNF-α-induced osteoclastogenesis and bone resorption in vivo. Therefore, we have obtained evidence that IL-33 plays an important role on the inhibitory effect in TNF-α-induced osteoclastogenesis and bone resorption.


Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution. These emerging lineages have mutations at <1% of all residues in the viral spike, and at no more than 3 of the ~200 residues in the spike receptor-binding domain (RBD)—yet these handfuls of mutations often substantially erode and in some cases even ablate the polyclonal neutralizing antibody response elicited by infection16.Control of the SARS-CoV-2 pandemic will depend on widespread population immunity acquired through infection or vaccination. But a little over a year into the pandemic, a proliferating number of new viral lineages are rising in frequency21, although antibodies that target the NTD also contribute to neutralization724. Structural and binding competition studies have shown that the most potently neutralizing anti-RBD antibodies target several distinct epitopes on the RBD’s receptor-binding motif27. However, the contributions of these different classes of RBD-targeting antibodies to the overall activity of the polyclonal antibody response remains less clear. It is therefore important to systematically determine both how viral mutations impact each antibody class, and how these antibody-specific effects shape the overall effects of viral mutations in a polyclonal context.A substantial fraction of the neutralizing activity of polyclonal antibody response to SARS-CoV-2 infection is due to antibodies that target the RBD29. We make these measurements by using a deep mutational scanning approach to systematically map how all RBD amino-acid mutations affect binding to yeast-displayed RBDs30 The resulting escape maps allow us to systematically compare how RBD mutations affect binding by the monoclonal antibodies, and we find that the antibodies cluster in the space of “viral escape” in a way that largely recapitulates prior classifications based on structural analyses of the antibody epitopes. However, some of the potently neutralizing monoclonal antibodies contribute very little to the escape maps of the polyclonal plasma, even for individuals from whom the antibodies were isolated. Instead, the plasma-escape maps usually most resemble a single antibody class  that targets the face of the receptor-binding ridge that is accessible in both “up” and “down” RBD conformations. Unfortunately, a mutation that escapes this antibody class (E484K) is present in many emerging viral lineages, including B.1.351, P.1, P.2, and B.1.5266. We suggest that domination of the RBD-targeting polyclonal response by a single antibody class is a major factor in enabling a small number of viral mutations to sometimes substantially erode neutralizing antibody immunity.Here, we comprehensively map RBD mutations that reduce binding by structurally characterized representatives of three classes of neutralizing monoclonal antibodies that target the RBD's receptor binding motif, as well as polyclonal plasma from convalescent individuals from whom some of the antibodies were isolated31. Antibodies targeting the RBD have been divided into four major classes based on structural analyses of their epitopes; two with epitopes overlapping with the ACE2 binding site (class 1 and class 2), potent neutralizers that do not directly bind to the ACE2 contact surface (class 3), and antibodies that target a cryptic epitope outside of the receptor-binding motif and are generally less potent (class 4)25. We focused our studies on several antibodies representative of the first three classes of potently neutralizing receptor-binding motif-targeting antibodies. Class 1 antibodies bind the face of the receptor-binding motif that is accessible only when the RBD is in the “up” conformation  to enrich for cells expressing RBD mutants that escaped antibody binding . We did this for the seven antibodies shown in 36, including class 4 antibodies, which bind to the RBD outside the receptor-binding motif and are generally less potently neutralizing31. Antibodies with similar escape mutations are located close to one another in the multidimensional scaling projection, and antibodies with very distinct escape mutations are far apart  or proximal (4-8 Å) contact with antibody in the resolved structures . The plasmas from both cohorts cluster together in the space of binding escape, but far from some of the antibodies  had visible effects on the polyclonal plasma, but again less so than for class 2 antibody sites , then class 1 or 3 antibodies might dominate for the remaining anti-RBD antibody activity.To visualize the escape maps in terms of the RBD’s three-dimensional structure, we projected onto the surface of the RBD the total escape at each site averaged across all antibodies in a class, or all convalescent plasma in a cohort . Again, dy sites . Howeverutations . Overall7. We hypothesized that the mutations selected during viral passage would reduce antibody binding without impairing ACE2 binding affinity. Accordingly, we examined how all of the selected mutations affected both antibody binding (as measured in the current study) and ACE2 affinity . We assessed how well our escape maps predicted the actual antibody-escape mutations that arose when virus was grown in the presence of the antibodies. Prior work selected viral escape mutants by passaging chimeric VSV encoding the SARS-CoV-2 spike in the presence of several of the monoclonal antibodies we mapped in this study7. There was generally good agreement between our escape maps and these previously measured effects of mutations on viral neutralization. Nearly all mutations with a >100-fold reduction in neutralization also had large effects in our escape maps, although in a few cases mutations with more moderate effects on neutralization were not prominent in the escape maps 4748. Consistent with our work here showing that class 2 antibodies shape how mutations affect polyclonal plasma binding, mutations at the site that most strongly affects binding by this antibody class (E484) have arisen multiple times in emerging viral lineages6. Therefore, our results show the importance of thinking about antigenic evolution in the context of different classes of antibodies that recognize different epitopes on the RBD.We comprehensively mapped all mutations that escape binding by three major classes of antibodies targeting the SARS-CoV-2 RBD, and compared these escape maps to those for convalescent polyclonal plasma. We find that a single antibody class (class 2) largely shapes how RBD mutations affect binding by polyclonal plasma, even for individuals from whom potent neutralizing antibodies of other classes were isolated. The dominance of class 2 antibodies in RBD-targeting portion of polyclonal plasma could be due in part to the twin facts that their epitope is exposed in both “up” and “down” conformations of the RBD, and that such antibodies are often generated from frequently observed germline genes  at the site that most potently escapes antibodies of this class. Once viruses have escaped class 2 antibodies, antibodies of other classes  will contribute most remaining RBD-targeted antibody immunity. In this respect, it is noteworthy that two of the most prominent emerging viral lineages have also acquired a mutation (K417N/T) at the site that most potently escapes binding by class 1 antibodies. Fortunately, no viral lineages currently combine these class 1 and 2 escape mutations with a class 3 escape mutation, although a moderate class 3 escape mutation (L452R) is present in the B.1.427/B.1.429 viral lineage that lacks other RBD escape mutationshttps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_RockefellerComplete computational pipeline for escape-mapping data analysis: https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/summary.mdMarkdown summaries of the escape-mapping data analysis steps: https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/supp_data/all_samples_raw_data.csv.Raw data tables of mutant escape fractions are in Raw Illumina sequencing for the escape mapping: NCBI SRA, BioProject: PRJNA639956, BioSample SAMN18148595.https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/counts/variant_counts.csvProcessed Illumina sequencing counts for the escape mapping: 21. Plasma samples profiled in this study were obtained 21-35 days post-symptom onset21. Samples were obtained upon written consent from community participants under protocols approved by the Institutional Review Board of the Rockefeller University (DRO-1006). All samples were heat-inactivated prior to use by treatment at 56 C for 60 minutes. Prior to use in each assay, plasma samples were centrifuged for 15 min at 2000 ×g to pellet platelets.Plasma samples were previously described and collected as part of a prospective longitudinal cohort study of SARS-CoV-2 convalescent individuals in New York, NY21. Briefly, the IGH, IGL, and IGK genes were sequenced using IgG-spedfic primers from single-cell sorted RBD+, CD20+ memory B cells (CD3 − CD8 − CD14 − CD16 − CD20 + Ova − RBD-PE + RBD-AF647 +). Recombinant monoclonal antibodies were produced and purified as previously described51. Antibodies were produced with a human IgG1 heavy chain and human IgK  or human IgL2  constant regions. These antibodies were previously structurally characterized in complex with SARS-CoV-2 S trimer29. A subset was functionally characterized in52. The PBD accessions for the antibody-S complex structures are: 6XCM and 6XCN for C105, 7C01 for LY-CoV016, 7K8S and 7K8T for C002, 7K8X and 7K8Y for C121, 7K90 for C144, 7K8Z for C135, and 7K8V for C11029.Antibodies were isolated from individuals in the cohort above as previously described30 with previously described duplicate yeast-displayed mutant RBD libraries32. These libraries were generated in the RBD background of the SARS-CoV-2 isolate Wuhan-Hu-1  via NNS codon tiling PCR mutagenesis, which introduced an average of 2.7 amino acid mutations per library variant. RBD variants were linked to unique 16-nucleotide barcode sequences to facilitate downstream sequencing and bottlenecked to library sizes of ~100,000 uniquely barcoded variants. The libraries contain 3,804 of the 3,819 possible amino-acid mutations, with >95% present as single mutants on at least one barcode in the libraries. We previously used these libraries to measure the effect of all RBD mutations on yeast-surface RBD expression and ACE2 affinity32. As previously described, these libraries were sorted to eliminate variants that lose ACE2 binding prior to mapping the antibody-escape variants30.Monoclonal antibody and polyclonal clonal plasma selection experiments were performed in biological duplicate using a deep mutational scanning approach30. Specifically, 9 OD aliquots of RBD libraries were thawed and grown overnight at 30°C 275 rpm in 45mL SD-CAA . Libraries were diluted to an OD of 0.67 in SG-CAA+0.1% dextrose , and incubated for 16–18 hours at room temperature with mild agitation to induce RBD surface expression. For each antibody selection, 20 OD units of induced cells were washed twice with PBS-BSA (0.2 mg/mL), and incubated in 4mL of PBS-BSA with monoclonal antibody or plasma for 1 h at room temperature with gentle agitation. Incubations were performed with 400 ng/mL for each monoclonal antibody  or with a sub-saturating dilution of polyclonal plasma such that the amount of fluorescent signal due to plasma antibody binding to RBD was approximately equal across plasma . Labeled cells were washed with ice-cold PBS-BSA followed by secondary labeling for 1 h at 4°C in 2.5 mL 1:200 PE-conjugated goat anti-human-IgG (Jackson ImmunoResearch 109-115-098) to label for bound monoclonal antibody or 1:200 Alexa-647-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064) to label for bound plasma antibodies, and 1:100 FITC-conjugated anti-Myc  to label for RBD surface expression. Labeled cells were washed twice with PBS-BSA and resuspended in 2.5mL PBS. Yeast expressing the unmutated SARS-CoV-2 RBD were prepared in parallel to library samples, labeled at the same 400 ng/mL and 100× reduced 4 ng/mL antibody concentrations for the monoclonal antibodies, and with 1× and 10× reduced plasma concentrations for the polyclonal plasma.Antibody labeling and selection was performed essentially as described in Greaney, et al. (2020)Yeast cells expressing RBD variants with substantially reduced antibody binding were selected via fluorescence-activated cell sorting FACS) on a BD FACSAria II. For monoclonal antibody selections, FACS selection gates were drawn to capture 95% of yeast expressing unmutated SARS-CoV-2 RBD labeled at 100× reduced antibody concentration relative to library samples. For polyclonal plasma selections, FACS selection gates were drawn to capture 2.8–5% of the RBD mutants with the lowest amount of plasma binding for their degree of RBD expression –C. NearlS on a BD32. Barcodes were sequenced via 50 bp single-end reads on an Illumina HiSeq 2500, targeting at least 2.5× as many sequencing reads as FACS-selected cells, and pre-sort reference populations of at least 2.5e7 reads.Plasmid samples were prepared from up to 7.5 OD units (4e7 CFUs) of overnight cultures of antibody-escaped cells, and 30 OD units (1.6e8 CFUs) of pre-selection yeast populations (Zymoprep Yeast Plasmid Miniprep II) per manufacturer instructions, with the addition of a −80°C freeze-thaw step prior to cell lysis. The 16-nucleotide barcode sequences identifying each RBD variant were amplified by PCR and prepared for Illumina sequencing exactly as described previously30, with minor modifications as noted below. Specifically, we used the dms_variants package  to process Illumina sequences into counts of each barcoded RBD variant in each pre-sort and antibody-escape population using the barcode/RBD look-up table from Starr et al. (2020)32. Markdown renderings of these steps in the computational analysis are at https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/aggregate_variant_counts.md and https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/counts_to_cells_ratio.md.Escape fractions were computed as described in Greaney et at. (2021)30. These escape fractions represent the estimated fraction of cells expressing that specific variant that falls in the antibody escape bin, so a value of 0 means the variant is always bound by antibody and a value of 1 means that it always escapes antibody binding. We then applied a computational filter to remove variants with low sequencing counts or highly deleterious mutations that might cause antibody escape simply by leading to poor expression of properly folded RBD on the yeast cell surface. Specifically, we ignored all variants with pre-selection sequencing counts that were lower than the counts for the 99th percentile of the stop-codon containing variants because stop codon variants are largely purged by the earlier sorts for RBD expressing and ACE2-binding variants and so any residual presence provides an indication of low-count “noise.” Next, we removed any variants that had poor RBD expression or ACE2 binding, or contained mutations that individually cause poor RBD expression and ACE2 binding to eliminate misfolded or non-expressing RBDs. Specifically, we removed variants that had (or contained mutations with) ACE2 binding scores < -2.35 or expression scores < -1, using the variant- and mutation-level deep mutational scanning scores32. Note that these filtering criteria are slightly more stringent than those used in Greaney et al. (2021)30 but are identical to those used in Greaney et al. (2021) and Starr et al. (2021)33. The ACE2 binding cutoff of −2.35 is used to represent the binding of RaTG13 to human ACE232, which possesses the lowest known affinity capable of mediating cell entry53. The RBD expression cutoff of −1 is used to eliminate mutations that have as large an expression deficit as mutations to core disulfide residues. 2,034 of the 3,819 possible RBD amino acid mutations passed these filtering steps and were included in our escape maps. All previously reported escape mapping data36 were reanalyzed in this study with the parameters listed above. A markdown rendering of the computation of the variant-level escape fractions and the variant filtering is at https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/counts_to_scores.md.For each antibody selection, we then computed the “escape fraction” for each barcoded variant using the deep sequencing counts for each variant in the original and antibody-escape populations and the total fraction of the library that escaped antibody binding via the formula provided in Greaney et al. (2021)54 implemented in the dms_variants package, as detailed at . In this fitting, we excluded variants that contained mutations that were not seen as either single mutants or in at least two multiple-mutant variants. We then computed the estimated effect of each mutation as the impact of that mutation on the “observed phenotype” scale transformation of its “latent phenotype” as computed using the global epistasis models, and applied a floor of zero and a ceiling of 1 to these escape fractions. All of the above analysis steps were performed separately for each of the duplicate mutant libraries. We then only retained mutations that passed all of the above filtering and were measured in both libraries or had at least two-single mutant variant measurements in one library. The reported scores throughout the paper are the average across the libraries; these scores are also in https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/scores_to_frac_escape.md.Because some library variants contain multiple amino acid mutations, we next deconvolved variant-level escape scores into escape fraction estimates for single mutations using global epistasis modelshttps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/call_strong_escape_sites.md.For plotting and analyses that required identifying RBD sites of “strong escape” 7 using the WebPlotDigitizer tool v4.4 https://apps.automeris.io/wpd/. We convert those previously measured IC50s to fold-change IC50 relative to wildtype RBD. The numerical IC50s found in Weisblum et al. (2020)7 are also tabulated at https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/experimental_data/data/weisblum_ic50.csv.In 38 as of March 15, 2021 were downloaded and aligned via mafft55. Sequences from non-human origins and sequences containing gap or ambiguous characters were removed, as were sequences with extremely high numbers of RBD mutations relative to other sequences, leaving 679,502 retained sequences. All RBD amino-acid mutations were enumerated compared to the reference Wuhan-Hu-1 SARS-CoV-2 RBD sequence . We acknowledge all contributors to the GISAID EpiCoV database for their sharing of sequence data .For the analysis in https://jbloomlab.github.io/dmslogo/) version 0.6.2; a markdown rendering of the code that creates these logo plots is at https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Rockefeller/blob/main/results/summary/escape_profiles.md. For each plasma, the y-axis is scaled to be the greatest of (a) the maximum site-wise escape metric observed for that plasma, (b) 20x the median site-wise escape fraction observed across all sites for that plasma, or (c) an absolute value of 1.0 .The static logo plots in the paper were created using dmslogo 56.The interactive visualizations of the escape maps and their projections on the RBD-antibody structures available at 29. Structural distances were computed using the bio3d package in R57. Surface representations of the RBD for non-antibody-bound structures utilize PDB 6M0J58.The static structural views , S5 in t25 and colored accordingly. We define the class 1 epitope as residues 403+405+406+417+420+421+453+455−460+473−476+486+487+489+504, the class 2 epitope to be residues 455+456+472+483−487+489+490+491+492+493+494, and the class 3 epitope to be residues 345+346+437−452+496+498−501. There are 5 residues that overlap between the class 1 and class 2 epitopes: 455+456+486+487+489.In many of the visualizations, the RBD sites are categorized by epitope region , defined bySupplemental Files1


Aberrant tumour necrosis factor (TNF) signalling is a hallmark of many inflammatory diseases including rheumatoid arthritis (RA), irritable bowel disease and lupus. Maladaptive TNF signalling can lead to hyper active downstream nuclear factor (NF)-κβ signalling in turn amplifying a cell's inflammatory response and exacerbating disease. Within the TNF intracellular inflammatory signalling cascade, transforming growth factor-β-activated kinase 1 (TAK1) has been shown to play a critical role in mediating signal transduction and downstream NF-κβ activation. Owing to its role in TNF inflammatory signalling, TAK1 has become a potential therapeutic target for the treatment of inflammatory diseases such as RA. This review highlights the current development of targeting the TNF-TAK1 signalling axis as a novel therapeutic strategy for the treatment of inflammatory diseases. Its role in mediating inflammatory diseases, such as autoimmune and chronic pain syndromes, has been widely documented and resulted in the development of targeted therapies aimed at inhibiting TNF . To date2.in vitro experiments using cytokine stimulations have shown that TAK1 forms a ternary complex with TAB1 and TAB2/3 and undergoes phosphorylation on residues of its activation loop  and K63-linked polyubiquitination at Lys 158 to be fully functional [Transforming growth factor-β-activated kinase 1 (TAK1) serves as a key node in the TNF inflammatory signalling pathway. It is a serine/threonine kinase and can be activated by a range of proinflammatory cytokines and ligands including TNF, interleukin-1β (IL-1β), liposaccharide (LPS) and transforming growth factor beta (TGFβ) . TAK1 isnctional . While pnctional .Figure 3.3.1.Much of the intracellular actions of TNF are thought to be mediated through TAK1 . Followiet al. showed following stimulation of THP-1 macrophages by LPS and interferon gamma (IFNγ), cells treated with the TAK1 inhibitor, takinib, showed selective inhibition of TNF (greater than ninefold reduction) compared to 110 other cytokines/chemokines profiled. Interestingly, other inflammatory cytokines stimulated by LPS and IFNγ, such as interleukin-6 (IL-6) and IL-1β, showed significantly less suppression by TAK1 inhibition, supporting the role that TAK1 signalling potently regulates TNF expression over other inflammatory cytokines [in vitro genetic knockout studies in THP-1 macrophages. Genetic knockout of TAK1 has shown that loss of TAK1 blocks LPS induced TNF pro-inflammatory responses [In addition to blocking intracellular TNF signalling through the TNF receptor, TAK1 inhibition has also been shown to downregulate TNF expression from other inflammatory ligands such as LPS . Data haytokines . Furtheresponses . Thus, i3.2.In addition to its role in TNF signalling, TAK1 regulates various upstream inflammatory signalling cascades, including the inflammatory cytokine IL-1β. Like TNF, IL-1β signalling regulates a variety of inflammatory responses following infection. Many immune cells  and epithelial cells are sensitive to IL-1β, which can induce transmigration to target tissues including cartilage, bone, blood vessels, the hypothalamus and the pancreas ,17. Foll3.3.in vitro and in vivo immune response to LPS [Toll-like receptors (TLRs) are a large receptor family and belong to the pattern recognition receptors (PRR), which are a crucial part of innate inflammation and immunity. While several endogenous and exogenous ligands for TLRs are known, TLR4 will be discussed because of the importance of its LPS ligand and downstream TAK1 signalling. Following LPS binding to TLR4, a signalling cascade similar to IL-1β is activated. MyD88 recruits IRAK proteins, leading to TAK1 activation  figure . TAK1 hae to LPS –22. In te to LPS .3.4.TGFβ is a pleiotropic cytokine that regulates angiogenesis, migration, cell proliferation, differentiation and apoptosis in a variety of tissues . TGFβ bi4.MAP3K7 (TAK1) as well as enrichment of the TAK1 activator protein TAB1 [MAP3K7 and TNFSF18 (Crohn's disease), TNFSF14 and 16NFKB1 (ulcerative colitis), TRAF3IP2 and TNFAIP2  are associated with higher risk for disease development [Inflammatory disorders, such as RA, Crohn's disease and inflammatory bowel disease (IBD), have shown increased expression of TNF, IL-1β and IL-6, which contribute to joint erosion and tissue destruction –30. Genoein TAB1 . Furtherelopment –34. AlthIn RA, TNF is overexpressed and promotes disease progression. Following initial joint injury, immune cells secrete TNF, leading to a tumour-like cell transformation of fibroblast-like synoviocytes (FLS) and increased migration, protease expression, and secretion of inflammatory mediators . While e4.1.in vivo genetic studies in mice have raised doubts to the therapeutic potential of TAK1. Early studies of TAK1 in vivo had shown that global knockout of TAK1 in mice led to early embryonic lethality owing to abnormal neural tube development [in vivo function of TAK1. Immune cell-specific knockouts such as B cell-TAK1KO demonstrated impaired B cell maturation and antigen-induced immune responses [Based on the potent regulation of TNF signalling in immune cells, TAK1 may represent a novel intracellular therapy for TNF mediated diseases. However, elopment ,48. Addielopment ,50. Thusesponses –53.in vivo studies of TAK1 difficult. Despite contradictory genetic data supporting the therapeutic potential of TAK1, early pharmacological data have strongly supported the potential of pharmacological manipulation as a therapeutic approach. Benefits of a pharmacological approach include the retention of TAK1's scaffolding function as well as ability to restore TNF signalling to normal levels rather than completely ablate it. Thus, the development of a selective TAK1 inhibitor will advance our understanding of the therapeutic potential of TAK1 in disease contexts.Loss of function TAK1 mutations in humans have not yet been identified, but recent discovery of a gain of function developmental phenotype owing to expression of a truncated form of the protein kinase has been reported . Patient4.2.50 15 nM) and suppresses IL-2 production (IC50 9 nM) [50 < 100 nM, but also showed low nM potency against HIPK2 (IC50 3 nM) and CDK9 (IC50 9 nM) [50 22 nM) with similar potencies [50 9 nM) TAK1 inhibitor [Owing to its role in mediating TNF signalling, TAK1 is a potential novel drug target for the treatment of inflammatory diseases. Until recently, pharmacological inhibitors of TAK1 lacked selectivity in the kinome, limiting their use to support the therapeutic role of TAK1 in diseases. However, discovery of the selective TAK1 inhibitor, takinib, has provided the first selective chemical entity to probe the therapeutic potential of TAK1 in the absence of off target inhibition . Previou50 9 nM) ,70,71. P50 9 nM) . In an i50 9 nM) . Anotherotencies . Drug cootencies . Recentlnhibitor . In a panhibitor ,36.FiguThe limited development of potent TAK1 inhibitors may be a result of targeting the kinase in the active/DFG-in/type I or inactive/DFG-out/type II conformation. While type I binders often form hydrogen bonds within the hinge region of the ATP binding site, type II binders extend beyond the hinge region into a hydrophobic pocket in close proximity to the ATP-binding site and are generally of higher molecular weight. A general core structure for type II binders has been developed, consisting of a hydrophobic moiety with hydrogen-bond acceptors, a linker region that often consists of amide moieties, and another hydrophobic moiety with hydrogen bond donors and acceptors ,73. It iAll previously described TAK1 inhibitors occupy the ATP-binding pocket of TAK1, and no allosteric binders have been described to this date . From th5.TAK1 represents a novel drug target for the regulation of TNF signalling in autoimmune diseases. As a key node in major disease-related cytokine signalling pathways, it activates proliferative and inflammatory gene programmes relevant to disease progression. Despite its biological importance, a lack of selective and potent TAK1 inhibitors and contradictory genetic evidence had greatly reduced our therapeutic understanding of TAK1. The most widely used TAK1 inhibitor 5ZO is a nonselective natural product, which inherently limits conclusions of studies with this compound. However, recent advent of novel TAK1 inhibitors such as takinib have revitalized the field and provided the first selective TAK1 inhibitor, allowing for enhanced understanding and evaluation of the therapeutic potential of TAK1 in inflammatory disorders.