Cytopenias are rare complications of prolonged beta-lactam use; however, incidence and associated risk factors are not well described. Patients aged 18-64 years in the 2010-2016 IBM MarketScan Commercial Database discharged from the hospital on cephalosporin, penicillin, or carbapenem outpatient Parenteral Antimicrobial Therapy (OPAT) were included. The primary endpoint was hospital admission coded for neutropenia, leukopenia, or thrombocytopenia within the first 6 weeks post index discharge and within 7 days of beta-lactam discontinuation. Patients with history of malignancy and those who are on chemotherapy were excluded. Significant factors in univariate analysis were incorporated into a multivariable logistic regression model with sequential exclusion of variables with p > 0.1.A total of 35,102 patients received beta-lactam OPAT; median age was 52 years and 53.6% were male. The primary outcome occurred in 150 (0.43%) patients at a median of 19 days (IQR 10-28 days after index discharge), which included 63 (0.18%) neutropenia, 85 (0.24%) thrombocytopenia, and 23 (0.07%) leukopenia admissions. Factors independently associated with readmission cytopenias included chronic liver disease (OR 4.61 [CI 2.93-7.25]), valvular heart disease (2.69 [1.71-4.24]), receipt of vancomycin (2.10 [1.42-3.12]), or antifungal therapy (4.42 [2.01-9.68]); lower risk was associated with carbapenem therapy (0.49 [0.32-0.75]) and diabetes (0.48 [0.31-0.74]) (Table 1).Readmissions with cytopenias during beta-lactam OPAT were rare and carbapenem use was associated with lower risk compared to other classes of beta-lactams. Combination of beta-lactam with vancomycin was associated with an increased risk of cytopenias, and those patients might benefit from closer monitoring.Table 1. Factors Associated with Cytopenias during Beta-Lactams Outpatient Parenteral Antimicrobial Therapy (OPAT)Margaret A. Olsen, PhD, MPH, Pfizer


Post COVID Syndrome (PCS) is significant morbidity following COVID-19. This study aims to identify biomarkers that predict PCS in a Gulf Coast cohort known for poor health outcomes.Since March 2020 the study Collection of Serum and Secretions for SARS CoV-2 Countermeasure Development (aka ClinSeqSer) has been enrolling subjects with confirmed acute COVID-19, with initial visit at 1 month and follow up every three months from symptom onset. At follow-up, subjects complete symptom questionnaire, physical examination, nasopharyngeal swab/saliva collection, blood draw. Subjects with >= one symptom new since COVID are PCS, remainder are Non-PCS experienced at initial one month visit and six months or longer. Univariate and bivariate analysis was carried out to study significant associations of currently available dataset (N=60).Figure 1. Post-COVID SymptomsIncluded if “new since covid”. For 60 subjects consented post-covid with completed questionnaire, results were analyzed. Most common symptoms reported were fatigue/tiredness or exhaustion (52%), muscle aches (38%), difficulty concentrating (33%) and headache (32%) as the most common symptoms during one month prior to their initial follow-up visit. The persistent symptoms experienced for six months or longer were fatigue/tiredness or exhaustion (25%), forgetfulness (22%), muscle aches (18%), and sleep difficulties (18%).Cohort is 36 (60%) female, 24 (40%) male, age group of 49 (82%) 18-64 years, 11 (18%) 65+ years, 33 (55%) African American, 27 (45%) Caucasian. Median follow-up time after symptom onset: 290 days. Study cohort reported fatigue (52%), myalgias (38%), difficulty concentrating (33%), headache (32%) as most common symptoms during first month from initial symptom onset. Persistent symptoms ( >=6 months) are fatigue (25%), forgetfulness (22%), myalgias (18%), sleep difficulties (18%). Bivariate analysis shows that gender , past stroke/transient ischemic attack (P=0.04), deep venous thrombosis (P=0.02), abnormal kidney function (P=0.01) associate with PCS. Convalescent antibodies  were measured and percentage inhibition of ACE2 spike interaction was recorded. Plasma inflammatory protein levels were measured using multiplex ELISA and Proximity Extension Assay technology during follow-up visit. Increased antibody ReSARS N IgG  response and higher convalescent IL-10 (P=0.04) was associated with PCS. Percent inhibition of ACE2: spike interaction was not associated (P=0.79) with PCS. Nasal swab/saliva SARS-COV-2 sequencing has not identified a specific SARS-CoV-2 virus mutation predictive of PCS. Table 1. Demographic and Clinical CharacteristicsThe bivariate analysis results showed that the gender , history of stroke or transient ischemic attack (P=0.0382), chest pain from narrow heart vessels (P=0.0479), deep venous thrombosis (P=0.0241) and abnormal kidney function (P=0.0142) were associated with Post-COVID syndrome.Table 2. Antibodies and ACE2 spike inhibition.The convalescent antibodies, ReSARS N IgG and S-RBD IgG were measured in U/mL and percentage inhibition of ACE2 spike interaction was recorded during follow-up visit for PCS vs Non-PCS subjects. The increased antibody ReSARS N IgG  response was associated with Post-COVID syndrome. Percent inhibition of ACE2: spike interaction was not associated (P=0.7932) with PCS.Table 3. Plasma inflammatory protein levels.Plasma inflammatory protein levels were measured using multiplex ELISA (MSD) and Proximity Extension Assay technology (Olink) recorded during follow-up visit for PCS vs Non-PCS subjects, revealing IL-10 (P=0.0379) was associated with development of PCS.This study identifies initial clinical and biomarker predictors of PCS in a cohort that is 55% African American.Figure 2. Antibody ReSARS N IgGReSARS N IgG measured in post-covid patients is significantly associated with post-COVID syndrome(P=0.0159). X axis: number of months from symptom onset to blood draw. Y axis: N IgG U/mL.Figure 3. Spike amino acid mutationsSpike amino acid mutations detected in SARS-CoV-2 from acute-phase respiratory isolates. Nasal swab/saliva samples were collected from subjects with acute COVID-19 at time of enrollment into ClinSeqSer, stored at -80°C followed by RNA isolation and SARS-CoV-2 qRT-PCR. Samples with Ct value of ≤30 were then sequenced using NextSeq (Illumina). All sequences are deposited on GISAID and under BioProject (ID PRJNA681020). X axis: subject ID, with ID number increasing chronologically. Y axis: amino acid position of each mutation moving from N- to C-terminus.Robert Garry, PhD, Zalgen Labs (Shareholder)


Ceftolozane/tazobactam (C/T), a cephalosporin–β-lactamase inhibitor combination, is approved for treatment of complicated urinary tract infections, complicated intra-abdominal infections (cIAI), and nosocomial pneumonia in adults. Safety and efficacy of C/T in pediatric participants with cIAI was assessed.This phase 2 study (NCT03217136) compared C/T + metronidazole (MTZ) with meropenem (MEM) for treatment of cIAI. Age- and weight-adjusted dosing is summarized in Table 1. The primary objective was to evaluate the safety and tolerability of C/T + MTZ compared with MEM. A key secondary endpoint was clinical cure at end of treatment (EOT) and test of cure (TOC).Table 1. Summary of Dosing and Pharmacokinetic Sampling Schedule by Age CohortA total of 94 participants were randomized 3:1; 91 were treated with C/T + MTZ (n=70) or MEM (n=21) comprising the modified intent-to-treat (MITT) population. The clinically evaluable population included 78 participants at EOT  and 77 participants at TOC . The most common diagnosis and pathogen in the MITT population were complicated appendicitis  and Escherichia coli . The mean (SD) intravenous therapy/overall treatment duration was 6.4 (2.8)/9.3 (3.6) days and 5.8 (1.8)/9.0 (3.2) days for C/T + MTZ and MEM, respectively. In total, ≥1 adverse events (AE) occurred in 80.0% and 61.9% of participants receiving C/T + MTZ and MEM, respectively (Table 2), of which 18.6% and 14.3% were considered drug related. Serious AE occurred in 11.4% (8/70) and 0% (0/21) of participants receiving C/T + MTZ and MEM, respectively; none were considered drug related. No drug-related study drug discontinuations occurred. In the MITT population, rates of clinical cure for C/T + MTZ and MEM at EOT were 80.0% and 95.2%, and at TOC were 80.0% and 100%, respectively ; 6 of the 14 failures for C/T + MTZ were indeterminate responses scored as endpoint failures per protocol. In the clinically evaluable (CE) population, rates of clinical cure for C/T + MTZ and MEM were 89.8% and 100% at EOT, and 89.7% and 100% at TOC, respectively .C/T + MTZ was well tolerated in pediatric participants with cIAI, and rates of clinical success were high with C/T treatment. C/T is a promising new treatment option for children with cIAI.Carl-Christian A. Jackson, MD, Merck & Co. Inc. (Shareholder) Julia Lonchar, MSc, Merck Sharp & Dohme Corp.  Feng-Hsiu Su, MPH, MBA, Merck Sharp & Dohme Corp.  Jennifer A. Huntington, PharmD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee) Mekki Bensaci, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee) Myra W. Popejoy, PharmD, Merck Sharp & Dohme Corp. (Employee) Matthew G. Johnson, MD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee) Carisa S. De Anda, PharmD, Merck Sharp & Dohme Corp.  Elizabeth G. Rhee, MD, Merck Sharp & Dohme Corp  Christopher Bruno, MD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee)


Sarcomas are highly heterogeneous in molecular, pathologic, and clinical features. However, a classification of sarcomas by integrating different types of pathways remains mostly unexplored.We performed hierarchical clustering analysis of sarcomas based on the enrichment scores of 14 pathways involved in immune, stromal, DNA damage repair (DDR), and oncogenic signatures in three bulk tumor transcriptome datasets.TP53 mutations, and the worst survival. We further validated the stability and reliability of our classification method by analyzing a single cell RNA-Seq (scRNA-seq) dataset. Based on the expression levels of five genes in the pathways of T cell receptor signaling, cell cycle, mismatch repair, focal adhesion, and calcium signaling, we built a linear risk scoring model (ICMScore) for sarcomas. We demonstrated that ICMScore was an adverse prognostic factor for sarcomas and many other cancers.Consistently in the three datasets, sarcomas were classified into three subtypes: Immune Class (Imm-C), Stromal Class (Str-C), and DDR Class (DDR-C). Imm-C had the strongest anti-tumor immune signatures and the lowest intratumor heterogeneity (ITH); Str-C showed the strongest stromal signatures, the highest genomic stability and global methylation levels, and the lowest proliferation potential; DDR-C had the highest DDR activity, expression of the cell cycle pathway, tumor purity, stemness scores, proliferation potential, and ITH, the most frequent Our classification method provides novel insights into tumor biology and clinical implications for sarcomas.The online version contains supplementary material available at 10.1186/s12967-022-03248-3. CDK4 and RB1-associated CNAs  prognosis among the sarcoma subtypes. Notably, DDR-C was likely to have the worst survival consistently in the three datasets  Fig. B, while 05) Fig. C.Fig. 3CU test, P < 0.001)   . These phenotypes are associated with tumor progression, immune evasion, drug resistance, and unfavorable prognosis , 29. Not01) Fig. D. Stemne01) Fig. E. In add05) Fig. F. AltogeP = 0.047; hazard ratio (HR) = 1.72 and its 95% confidence interval (CI)   survival analysis by the multivariate Cox proportional hazards model. We found that the subtype DDR-C remained a significant risk factor for DFS , while it showed no significant difference between Imm-C and DDR-C , while they were not significantly different between Imm-C and DDR-C  and/or increased CNAs . We founR-C Fig. A. HomoloR-C Fig. B. In addR-C Fig. C. The G-R-C Fig. D. OveralTP53 had a significantly higher mutation frequency in DDR-C than in Imm-C and Str-C  = 2.0) . On the other hand, TP53 mutations were less frequent in Str-C than in DDR-C and Imm-C . This result could explain why Str-C was more genomically stable than the other subtypes because p53 plays an important role in the maintenance of genomic stability [DYNC2H1 and MDN1 were more frequently mutated in DDR-C than in Imm-C and Str-C       < 0.05]. In contrast, 124 and 180 genes had significantly higher methylation levels in Str-C and DDR-C, respectively, compared to other subtypes. Notably, most of these genes showed significant inverse correlations of their expression levels with methylation levels . These proteins included Syk, PREX1, Lck, 14-3-3_epsilon, PI3K-p85, Caspase-7_cleavedD198, PRDX1, Annexin-1, G6PD, Bax, ATM, p38, STAT5-alpha, Annexin_VII, Claudin-7, p90RSK, TIGAR, CD31, and GATA3  Fig. . These pCD40LG, CDC25A, MSH2, FLT4, and ADCY2. The five genes were involved in five of the 14 pathways for clustering analysis, including T cell receptor signaling (CD40LG), cell cycle (CDC25A), mismatch repair (MSH2), focal adhesion (FLT4), and calcium signaling (ADCY2). ICMScore calculates risk score in a tumor as follows: ICMScore = 0.76 × exp(CD40LG) + 0.73 × exp(FLT4) − 0.20 × exp(ADCY2) + 1.97 × exp(CDC25A) + 1.50 × exp(MSH2), where exp(X) denotes the expression level of gene X in the tumor sample. To prove that ICMScore is an authentic risk factor in sarcomas, we compared ICMScores among the three sarcoma subtypes and analyzed their correlation with survival prognosis in sarcomas. In the three sarcoma datasets, As expected, ICMScores was significantly higher in DDR-C than in Imm-C and Str-C    to evaluate the prognostic risk of sarcomas based on the expression levels of five genes, including 05) Fig. A. Surviv05) Fig. B. These 05) Fig. C.Fig. 8AUsing the pathway-based clustering method, we analyzed a scRNA-seq dataset GSE131309), which involved gene expression profiles in 6951 single cells from 12 sarcoma [advanced synovial sarcoma (SyS)] patients. The 6951 single cells included 4371 tumor cells, 90 B cells, 943 macrophages, 185 mastocytes, 102 NK cells, 235 CD4 + T cells, 659 CD8 + T cells, 206 T cells, 79 endothelial cells, and 81 cancer-associated fibroblasts (CAFs). Using the t-distributed stochastic neighbor embedding (tSNE) algorithm , we clus, which iWe performed hierarchical clustering of the 6951 single cells based on the enrichment scores of the 14 pathways identified three subtypes of these cells  exhibited the consistent expression pattern: Imm-C < Str-C < DDR-C    were significantly higher in Imm-C than in Str-C and DDR-C Fig. D. The raR-C Fig. D. It ind01) Fig. E, suppor01) Fig. F. It is TP53 mutations, and the worst survival. It is interesting to observe that there was no significantly different TMB between DDR-C and Imm-C, while their immune infiltration levels were significantly different. Two possible reasons could explain this observation: (1) DDR-C had more frequent arm-level copy number amplifications and deletions than Imm-C; and (2) DDR-C had higher ITH than Imm-C, since both CNAs [In this study, we proposed a novel classification method for sarcomas based on the enrichment scores of 14 pathways, which were involved in immune, stromal, DDR, and oncogenic signatures. In three datasets for bulk tumors and a scRNA-seq dataset, we reproducibly identified three sarcoma subtypes: Imm-C, Str-C, and DDR-C. Imm-C had the strongest anti-tumor immune signatures and the lowest ITH; Str-C showed the strongest stromal signatures, the highest genomic stability and global methylation levels, and the lowest proliferation potential; DDR-C had the highest DDR activity, expression of the cell cycle pathway, tumor purity, stemness scores, proliferation potential, and ITH, the most frequent oth CNAs  and ITH oth CNAs  are negaThe TCGA Research Network analyzed six types of adult soft tissue sarcomas: DDLPS, LMS, UPS, MFS, MPNST, and SS . We founBased on the enrichment scores of 14 pathways associated with immune, stromal, and DDR signatures, we classified sarcomas into three subtypes. The three sarcoma subtypes were characterized by different immune infiltration levels, stromal signatures, DDR activity, genome features, tumor progression phenotypes, and clinical outcomes. Our new classification method for sarcomas provides novel insights into tumor biology and clinical implications for this disease.Additional file 1: Table S1. A summary of the datasets analyzed.Additional file 2: Table S2. Pathways, immune signatures, and biological processes and their marker genes.Additional file 3: Fig. S1. Comparisons of the expression levels of human leukocyte antigen (HLA) genes among the sarcoma subtypes. The one-way ANOVA test P-values are shown. * P < 0.05, ** P < 0.01, *** P < 0.001.Additional file 4: Table S3. Pearson correlations between the methylation levels and expression levels of the genes having significantly different methylation levels among the sarcoma subtypes.Additional file 5: Fig. S2. Heatmap showing top 30 genes with the most significant upregulation of methylation levels in each of the three subtypes.Additional file 6: Fig. S3. Positive correlations between the expression levels of 19 proteins significantly upregulated in Imm-C and immune signature scores in TCGA-SARC. * P < 0.05, ** P < 0.01, *** P < 0.001.Additional file 7: Fig. S4. Overlaps between our subtyping and other subtyping of sarcoma in TCGA-SARC. (A) Proportions of our subtypes in six types of adult soft tissue sarcomas: DDLPS, LMS (ULMS and STLMS), UPS, MFS, MPNST, and SS. (B) Proportions of our subtypes in the immune subtypes of sarcomas. (C) Proportions of the five subgroups (Clusters A-E) in our subtypes.


Enterobacterales are considered an urgent threat for patients in healthcare facilities, causing infections with significant morbidity and mortality. Most isolates are multidrug resistant with limited treatment options, so combination therapy is an alternative. Recently, synergy with piperacillin/tazobactam (P/T) + meropenem (MP) was demonstrated against 7/10 (70%) KPC-producing Escherichia coli and 9/10 (90%) OXA-48-producing K. pneumoniae using time-kill assay . The aim of the present study was to further evaluate the combination of P/T + MP against KPC-producing Enterobacter cloacae, in addition to OXA-producing K. pneumoniae using our rapid ETEST MIC:MIC synergy method.Carbapenem-resistant K. pneumoniae  and 7 KPC-producing E. cloacae  were obtained from the CDC and FDA Antibiotic Resistance Isolate Bank. ETEST MICs for P/T and MP and our ETEST synergy method were performed in triplicate for each isolate. The summation fractional inhibitory concentration was calculated, and the mean value was interpreted as: < 0.5 synergy; > 0.5-1 additivity; > 1-4 indifference; and > 4 antagonism.14 carbapenemase-producing isolates: 7 OXA-48-like K. pneumoniae and synergy (6) or additivity (1) against all 7 KPC-producing E. cloacae. No antagonism was detected.MICs (µg/mL) ranged: MP, 0.5 to > 32 (14% susceptible) and P/T, 96/4 to > 256/4 . The combination of P/T + MP showed synergy (3) or additivity (2) against 5/7 (71%) OXA-producing K. pneumoniae and 7/7 KPC-producing E. cloacae, similar to previously published findings showing synergy in 7/10 KPC-producing E. coli and 9/10 OXA-48-producing K. pneumoniae using time-kill assay. Our ETEST synergy method is simple to use and should be evaluated more extensively. Regardless of the method used, results may or may not correlate in an in vivo setting. In vivo studies are needed.Using our ETEST MIC:MIC method, the combination of P/T + MP demonstrated synergy or additivity in 5/7 OXA-producing All Authors: No reported disclosures


Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identif ied these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might inf luence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium inf lux inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1–3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative inf luence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the f luorophores such as red f luorescent protein (RFP) and yellow f luorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by inf iltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modif ication or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development. Annexins are of particular research interest due to their abilityto regulate various aspects of plant growth and development.Annexins belong to the evolutionarily conservedsuperfamily of proteins that are involved in Ca2+-dependentor Ca2+-independent binding with membrane phospholipids. Most annexinshave four putative annexin repeats of around 70 amino acids,with the conservative repeat GxGT-(38 residues)-D/E, whichconfers Ca2+/phospholipid-binding activity to these proteins. In addition,some plant annexins have motifs demonstrating F-actin bindingand peroxidase and ATPase/GTPase activities .Despite the general structural similarity of these proteins,the functions of annexins are diverse, and individual annexinsmay have specific activities. Annexins are involved in a widevariety of essential cellular processes, including the regulationof membrane organization, vesicle trafficking, cytoskeletaldynamics, exocytosis, cell cycle control, ion transport, andsignal transduction . Annexins as phospholipid-binding proteinsare being implicated in the fusion of membrane vesicles,as was shown for annexins from bell pepper and cotton . They are also involved inthe regulation of exocytosis, e. g., annexins in Zea mays rootcap cells . Moreover, annexins can functionas cationic channels activated by various stimuli in cells.Annexins can influence the Ca2+ influx in plant cells, as wasdemonstrated for a Capsicum annuum annexin, which hasCa2+-channel activity . The Arabidopsisthaliana annexin AtAnn1, which is expressed in root cells,exhibits pH-dependent cation-channel activity, while Z. maysannexins cause active conductivity of Ca2+ in lipid bilayersat slightly acidic pH . Since annexins can be Ca2+ sensors, these proteins arelikely to be involved in signal transduction; for example, theannexin from Triticum aestivum was suggested to be engagedin low-temperature signaling .Participation of annexins in the responses to cold, oxidative,and saline stresses is well-studied in plants . The annexinAtAnn1 from A. thaliana is involved in plant protectionagainst oxidative stress .The overexpression of AtAnn has been found to confer toleranceto drought and salt stresses and fungal attack in transgenicplants . Similarly,the overexpression of the wild tomato (Solanum pennellii)annexin SpAnn2 in cultivated tomato Solanum lycopersicumenhances drought and salt tolerance through the eliminationof reactive oxygen species (ROS) .Some annexins are also known to be activated in plantsduring interaction with plant-growth promoting bacteria and the development of mutualistic symbioses. During legume-rhizobial symbiosis,physiological changes occur, which are necessary for rhizobialinfection and nodule organogenesis, such as the stimulationof ion fluxes, membrane depolarization, ROS production,cytoplasm alkalinization, perinuclear calcium oscillations,and cytoskeletal rearrangements. In Medicago truncatula, thetranscription of MtAnn1 is activated directly by Nod factorsor inoculation with rhizobia in epidermal cells and later incortical cells . Studies using confocal microscopy showedGFP-labeled MtAnn1 to be localized in the cytoplasm, butprotein accumulation in response to inoculation occurred atthe periphery of the nucleus. MtAnn1 has been shown to beable to bind to the membrane phospholipid phosphatidylserine.Therefore, MtAnn1 is probably related to the eventsoccurring at the early stages of symbiosis, leading to bacterialinfection or nodule organogenesis .Transcriptome profiling of roots inoculated with rhizobiarevealed enhanced expression of MtAnn2, as well as MtAnn1. The expression of the MtAnn2 gene isassociated with cell division in the nodule primordium . Proteomic analysis revealed the MtAnn2protein presence in lipid rafts from root plasma membranepreparations . Another annexin MtAnn3was found to be important for root hair deformations inM. truncatula . The increased expression ofMtAnn1 and MtAnn2 is also associated with the early stagesof AM fungal symbiosis, which corresponds to the stages ofpre-infection and infection in this type of symbiosis . This may indicate the general role of these annexinsin the regulation of signaling pathways that lead to thedevelopment of two types of symbiosis.A protein homologous to MtAnn1 – PvAnn1 from Phaseolusvulgaris – is activated at the early stages of symbiosisdevelopment . The stimulation of Ca2+ ion transfer throughthe plasma membrane and ROS production caused by Nodfactors constitute an early response in the signal transductionpathway. Analysis of PvAnn1-RNAi transgenic roots inoculatedwith rhizobia showed a decrease in ROS productionand Ca2+ influx into the cells, which resulted in impairedprogression and decreased numbers of infection threads andnodules . Taken together, thesefindings point to the involvement of PvAnn1 in the regulationof signal transduction at early stages.Previously performed proteomic analysis in pea (Pisumsativum L.) allowed us to reveal two annexins, the synthesisof which was increased in response to inoculation with Rhizobium leguminosarum bv. viciae RCAM1026 in 24 h. In this work, searching in therecently released pea genome database using available codingsequences for annexin genes from M. truncatula andP. vulgaris revealed 15 annexins in pea. Phylogenetic analysisshowed the relationship among members of the annexin superfamilyin other legumes and allowed the identification oftwo previously revealed pea annexins responsive to rhizobialinoculation as PsAnn4 and PsAnn8 based on their homologywith the M. truncatula and P. vulgaris proteins. To verify thefunction of these annexins, we performed comparative proteomicanalysis using pea mutant P56 (sym10) unable to formsymbiosis and wild type cv. Frisson. The approaches employedincluded quantitative RT-PCR, experiments with calciumchannel inhibitors, and localization of labeled proteins.Plant material and bacterial strain. Pea Pisum sativum L.seeds cv. Frisson were sterilized with sulphuric acid for5 min, washed with water 3 times, transferred on 1 % wateragar plates and germinated at room temperature in the dark.4–5 days-old seedlings were transferred into pots with vermiculitesaturated with Jensen medium , grown in a growth chamber at 21 °С at 16 h light/8 h dark cycles, 60 % humidity. For experiments with inhibitor,the Ca2+ channels blocker LaCl3, the plants were grownin pots saturated with Jensen medium with 100 μM CaCl2 ×2 H2O. The Rhizobium leguminosarum bv. viciae strainRCAM 1026 (WDCM 966) was cultivated at 28 °С on TY agar medium with 0.5 mg/ml of streptomycin.Fresh liquid bacterial culture was grown in B– medium and the optical density of the suspensionat 600 nm (OD600) was adjusted to 0.5. Pea seedlingswere inoculated with 2 ml of R. leguminosarum bv. viciaeper plant. Pea roots  were harvested 1 dayafter inoculation (dai).Nicotiana benthamiana seeds were surface sterilized with10 % hypochlorite for 10 min, washed with water 5 timesand left for imbibition on a plate with sterile filter paper at4 °С. All seeds were germinated in a large plastic box withsoil for seven days, and then transferred into individual potswith soil. Plants were grown at 23 °С with 16 h light/8 h darkcycles, 60 % humidity.http://www.clustal.org/omega/. The phylogenetic tree was generatedwith the Maximum Likelihood method using MEGA X https://www.megasoftware.net/ with 1000 bootstrap replicates. Thedomain composition of the corresponding encoded proteinswas assessed using PFAM https://www.sanger.ac.uk/science/tools/pfam.Phylogenetic analysis. Multiple sequence alignments wereperformed using ClustalΩ https://salilab.org/modeller/9.20/release.html. Visualization of the three-dimensional structurewas obtained using the PyMol programhttps://pymol.org/2/. The three-dimensional crystal structure of theGhAnn1 G. hirsutum protein  was used asa template for building the model. To refine the model, the (VTFM) and the method of molecular dynamics in vacuum.The reliability of the model was calculated by the formula Protein homology modeling was performed in Modeller9.20 Isolation of total protein from pea roots. A modified methodwas used to isolate proteins from pea roots . 100 mg of the roots were ground in liquid nitrogen,then extraction buffer (0.1 M tris-HCl (pH 8.0), 30 % sucrose,10 mM dithiothreitol (DTT), 2 % sodium dodecyl sulfate(SDS), a mixture of protease inhibitors was added to the material and extraction was performed at+4 °С. After centrifugation at 12 000 g for 15 min, the supernatantwas mixed in a 1:1 ratio with phenol (pH 8.0) , centrifuged at 12 000 g for 5 min.The upper phase was taken for precipitation of proteins. Fivevolumes of cold 100 mM ammonium acetate in methanol wereadded and incubated for 30 min at –20 °С. After centrifugationat 12 000 g for 5 min, the pellet was washed twice with100 mM ammonium acetate in methanol and twice with 80 %acetone. The precipitate was dried in air and dissolved in thebuffer for isoelectric focusing (25 mM tris-HCl (pH 8.0), 9 Murea, 4 % CHAPS, 50 mm DTT, 0.2 % ampholytes ). Protein concentration was measuredusing Bradford assay .Two-dimensional differential gel electrophoresis. Twodimensionaldifferential gel electrophoresis (DIGE) of proteinswas performed using staining of samples with variousfluorescent dyes . The samples were conjugatedfor 30 min on ice with fluorescent dyes Cyanine 2or Cyanine 5 (Cy2 or Cy5) in various combinations. The incubationsolution contained 400 pM of each dye dissolvedin dimethylformamide for 30 min on ice. The reaction wasstopped by adding 10 mM L-lysine (Sigma-Aldrich), followedby incubation on ice for 10 min. After that, the controland experimental samples were mixed, DTT and ampholyteswere added. Passive in-gel rehydration with immobilizedpH gradient (Bio-Rad Laboratories) was performed overnightat room temperature. The total amount of sample applied to7 cm gel  was up to 100 μg.Isoelectric focusing (IEF) was performed in a Protean IEFsystem (Bio-Rad Laboratories) at a temperature of 20 °С, thesamples were desalted at 250 V for 15 min, after which thevoltage was linearly increased to 4,000 V for 2 hours, thenIEF was carried out with increasing voltage up to 10 000 V.Before electrophoresis in polyacrylamide gel (PAGE), proteinrecovery was carried out in buffer with DTT  for10 min followed by alkylation in iodoacetamide buffer  for 15 min. The second direction of two-dimensionalelectrophoresis was carried out in tris-glycine bufferin 15 % polyacrylamide gel using a 4 % stacking gel. Afterseparation of proteins the gels were visualized using a laserscanner Typhoon FLA 9500 .Mass spectrometry. The proteins were rehydrated in trypsinsolution  on ice for1 h and then incubated for 1 h at 56 °С. The peptides wereextracted from the gel with 50 % acetonitrile, 0.1 % formicacid. This solution was evaporated in vacuum concentratorCentriVap (Labconco) at 4 °С and dissolved in phase A . Mass spectrometry was performedusing Agilent ESI-Q-TOF 6538 UHD (Agilent Technologies)combined with high performance liquid chromatographAgilent 1260 (Agilent Technologies). Chromatographywas performed in system water – acetonitrile in the presenceof 0.1 % formic acid  in the gradient of acetonitrile (from 5 to 60 % phase Bfor 25 min and to 100 % phase B for 5 min) on Zorbax 300SBC18column 3.5 μm, 150 mm length (Agilent Technologies)with flow rate 15 μl/min.RNA extraction and quantitative reverse transcriptionPCR (RT-PCR). RNA extraction and RT-PCR were performedas described previously . Thequantitative RT-PCR analysis was performed on a CFX-96real-time PCR detection system with C1000 thermal cycler(Bio-Rad Laboratories). All primer pairs (Table 1) were designedusing the Vector NTI program and produced by theEvrogen company (www.evrogen.com). PCR amplificationspecificity was verified using a dissociation curve (55–95 °С).mRNA levels were normalized against Ubiquitin and valueswere calculated as ratios relative to non-inoculated root expressionlevels. The data of two-three independent biologicalexperiments were analysed. Statistical analysis was conductedby Student’s test ( p < 0.05) to assess the differences betweenvariants.Mass spectrometry. The proteins were rehydrated in trypsinsolution  on ice for1 h and then incubated for 1 h at 56 °С. The peptides wereextracted from the gel with 50 % acetonitrile, 0.1 % formicacid. This solution was evaporated in vacuum concentratorCentriVap (Labconco) at 4 °С and dissolved in phase A . Mass spectrometry was performedusing Agilent ESI-Q-TOF 6538 UHD (Agilent Technologies)combined with high performance liquid chromatographAgilent 1260 (Agilent Technologies). Chromatographywas performed in system water – acetonitrile in the presenceof 0.1 % formic acid  in the gradient of acetonitrile (from 5 to 60 % phase Bfor 25 min and to 100 % phase B for 5 min) on Zorbax 300SBC18column 3.5 μm, 150 mm length (Agilent Technologies)with flow rate 15 μl/min.RNA extraction and quantitative reverse transcriptionPCR (RT-PCR). RNA extraction and RT-PCR were performedas described previously . Thequantitative RT-PCR analysis was performed on a CFX-96real-time PCR detection system with C1000 thermal cycler(Bio-Rad Laboratories). All primer pairs (Table 1) were designedusing the Vector NTI program and produced by theEvrogen company (www.evrogen.com). PCR amplificationspecificity was verified using a dissociation curve (55–95 °С).mRNA levels were normalized against Ubiquitin and valueswere calculated as ratios relative to non-inoculated root expressionlevels. The data of two-three independent biologicalexperiments were analysed. Statistical analysis was conductedby Student’s test ( p < 0.05) to assess the differences betweenvariants.Genetic constructs for plant transformation. To obtainthe pBIN19 vector for plant transformation, carrying the geneof interest, the coding sequence of PsAnn4 gene without stopcodonhas been amplified using cDNA as a template withcorresponding primers (see Table 1). Total RNA was isolatedfrom 2 dai pea roots of cv. Frisson. Amplification was doneusing Phusion Flash High-Fidelity PCR Master Mix (ThermoScientific). The amplified products were restricted with XbaIand EcoRI and subcloned in the pMON vector under 35Spromoter in the frame with the sequences encoding RFP orYFP and nopaline synthase terminator (Tnos). The inserts wereverified by sequencing. The cassette composed of the 35Spromoter, gene of interest fused with RFP or YFP and Tnoswas excised from pMON using Hind III, SmaI and cloned inthe pBIN19. All verified constructs were transferred into theAgrobacterium tumefaciens LBA4404.Transient protein expression in N. benthamiana leaves.A. tumefaciens strain LBA4404 was used for infiltration inN. benthamiana leaves. Bacterial culture was grown at 28 °Covernight, then centrifuged at 3000 g and resuspended in10 mM MES-KOH, 10 mM MgCl2 and 0.5 mM acetosyringoneup to culture density OD600 = 0.5. Bacterial cells wereinfiltrated into the leaves of 3-week-old N. benthamiana.Plants were analyzed 48–96 h after infiltration.ResultsPhylogenetic analysis of annexinsin pea and other legumesThe search of the sequences presumably coding for annexinsin legumes was performed using BlastX with 8 previouslyrevealed M. truncatula and 13 P. vulgaris nucleotidesequences encoding these proteins  as queries against differentplant sequence databases: https://phytozome.jgi.doe.gov/pz/portal.html for M. truncatula and P. vulgaris, http://www.kazusa.or.jp/lotus/ for L. japonicus, and the URGI databasev. 1 https://urgi.versailles.inra.fr/blast for P. sativum L.. As a result, we were able to identify 18 codingsequences (CDSs) for annexins in M. truncatula, 15 inP. sativum L., and 13 in L. japonicus (Table 2). Twenty-threegenes had been previously found to encode annexins in soybean. The coding sequences for annexinsfrom P. sativum were named based on their phylogeneticrelationships with the corresponding homologous sequencesfrom M. truncatula and P. vulgaris (see Table 2) .The phylogenetic analysis  was performed usingthe deduced amino acid sequences of annexins found andannotated for P. sativum along with those of other legumes and non-legumes , whichwere available in the Phytozome database v. 12.1 and otherdatabases.Based on our analysis, the previously found MtAnn1(Medtr8g038210) and PvAnn1 (Phvul.011g209300) clusteredin the subclade with proteins corresponding to P. sativumPsat4g147120 and Psat4g191080, named PsAnn1a andPsAnn1b (see Table 2). Revealed in M. truncatula MtAnn2(Medtr8g038220) and P. vulgaris PvAnn2 (Phvul.011g209200)clustered in the subclade with Psat4g191040, named PsAnn2.Two previously described pea annexins induced in rootsin response to rhizobial inoculation  were identified as proteins corresponding to Psat5g217440and Psat2g074960 coding sequences using a new databasehttps://urgi.versailles.inra.fr/blast for P. sativum (see Table 2). The phylogenetic analysis depicted anadditional branch in the phylogenetic group with MtAnn1/PvAnn1 and MtAnn2/PvAnn2, comprising MtAnn4(Medtr3g018780), PvAnn4 (Phvul.005g030100), and theirhomolog Psat5g217440, named PsAnn4 (identified by proteomicscreening) (see Table 2). Another previously foundpea annexin, Psat2g074960, might be closely related toMedtr5g063670 and Phvul.008G173100.1, defined as MtAnn8and PvAnn8 based on phylogenetic analysis (see Table 2).Analysis of the domain composition of pea annexinsand modeling of three-dimensional structureof PsAnn4 and PsAnn8Analysis of the domain composition of the correspondingproteinsin pea showed the presence of four typical domainsof plant annexins . This suggests that the annexin genefamily indeed comprises several members in pea. Althoughplant annexins have four putative annexin repeats, not all Ca2+-binding motifs in these repeats seem to be functional. In plantannexins, the Ca2+-binding site is highly conservative in thefirst (I) repeat but is not conservative in the second (II) andthird (III) repeats, while in the fourth (IV) repeat moderateconservatism is preserved .The crystal structure of the Gossypium hirsutum annexinGhAnn1 bound to calcium was obtained in an earlier study. Since PsAnn4 and PsAnn8 may be involvedin regulation of pea-rhizobial symbiosis, we modeled thethree-dimensional (3D) structure of these two annexins usingGhAnn1, with 50 % sequence identity for PsAnn4 and 78 %sequence identity for PsAnn8 as a template .The resulting 3D structures of PsAnn4 and PsAnn8 proteinsindicated the coordination of calcium ions in the first andfourth annexin repeats. In the first repeat of both proteins, thecalcium-binding site of the type II was coordinated by threecarbonyl oxygen atoms of the residues Phe-23, Gly-25, andGly-27, and carboxylate of Glu-67 in PsAnn4 and PsAnn8, as was shown earlier for GhAnn1.We suppose that the second calcium ion is bound in the loopof the fourth annexin repeat of PsAnn4 and PsAnn8 proteins. Itis coordinated in the binding site of type II by Ile-254, Lys- 256,and Gly-258 in pea annexins . The thirdcalcium ion (in the binding site of type III) is coordinated bytwo oxygen atoms of the residues Val-296 and Thr-299 andcarboxylate of Glu-304 in this protein   . However, in thefourth repeat of PsAnn4 protein, the Val-296 is replaced bySer and Glu-304 by Lys . This might potentially obstruct the binding of the calcium ion, as was shown in ourmodeling . Although we cannot rule out that thismight be due to low homology between PsAnn4 and GhAnn1,which was used as a template in the modeling, the results suggestthe potential difference in Ca2+ binding between PsAnn4and PsAnn8 proteins.Comparative analysis of protein patternsin wild-type and non-nodulating pea mutantTo verify whether the stimulation of synthesis of PsAnn4 andPsAnn8 proteins depends on Nod factor perception, the proteinpatterns were analyzed in wild-type pea cv. Frisson anda P56 mutant with a defective sym10 gene (which encodes aputative Nod factor receptor) .Two-dimensional differential in-gel electrophoresis-basedproteomics was used to characterize the pattern of proteindistribution . Two spots corresponding to the locationof the previously characterized annexins  were excised from the gel. Mass spectrometric analysisconfirmed their identity to annexins Psat5g217440 (PsAnn4)and Psat2g074960 (PsAnn8). Enhanced level of PsAnn4 wasfound in the inoculated roots of wild type pea plants (cv. Frisson)compared to the inoculated P56 mutant roots.The amount of PsAnn8 protein was also slightly higher inresponse to inoculation in the wild type than in the P56 mutant,but not as essential as for PsAnn4. In accordance with this,low amounts of PsAnn4 and PsAnn8 proteins were found inthe roots of the P56 mutant and didn’t change in response toinoculation. This suggests that the up-regulation of both annexinsmay depend on Nod factor recognition in pea plantsand may be connected with the functioning of these annexinduring symbiotic interaction of plants with rhizobia at earlystages. Since the increase in the amount of PsAnn4 proteinwas more significant in response to inoculation, we focusedon this annexin in our next experimentsPsAnn4 expression pattern in response to rhizobialinoculation and treatment with Ca2+ inhibitorsThe PsAnn4 expression pattern in response to rhizobial inoculationwas analyzed in our experiments . A quantitativeRT-PCR analysis revealed that Rhizobium infectionenhanced the PsAnn4 gene expression at the early stages ofnodulation, starting from 1–3 days after inoculation up to5 days after inoculation, but thereafter their transcript levelsdid not significantly change upon nodule development . In our experiments the expression of another annexingene, PsAnn1a, the closest homolog of MtAnn1 genewas also analyzed . As it was expected, thePsAnn1a gene expression was primarily enhanced at theearly stages of symbiosis development and reached the highestlevels in the nodules. Similar pattern had been previouslyfound for MtAnn1 .Therefore, up-regulation of PsAnn4 expression may be relatedto the early stages of nodulation. The upregulation of thePsAnn4 transcription level was not as significant as it wasat the protein level, which implies that the regulation of thisannexin can be mainly achieved at the post-transcriptionaland translational level.To verify the influence of calcium inhibitors on the regulationof PsAnn4 gene, its expression level was estimated after plant treatment with the Ca2+ channel blocker LaCl3 .Two previously described as symbiosis-specific genes PsNINand PsEnod5 were also used in our experiments as a controlfor effective inoculation. In pea roots, the upregulation ofPsAnn4 expression in response to inoculation was revealedin 1 dai, corresponding with experiments on the dynamics ofthis gene expression upon nodulation. The significant decreasein the expression of PsAnn4 was found in our experiments inthe presence of LaCl3. Down-regulation of symbiosis-specificgenes PsEnod5 and PsNIN was also observed, which indicatedthe importance of Ca2+ influx for their regulation. Therefore,the influx of calcium ions into the cell, which is observed atthe early stages of symbiosis development, may affect theexpression level of PsAnn4 in pea roots .Subcellular localization of pea PsAnn4 annexinTo follow the PsAnn4 protein localization in plant cells, itwas fused to the fluorophores such as red fluorescent protein(RFP) and yellow fluorescent protein (YFP) at the C-terminusand expressed under the transcriptional regulation of the 35Spromoter in N. benthamiana leaves by infiltration with A. tumefaciens. The infiltration of constructs for thesynthesis of proteins fused with RFP and YFP allowed us tovisualize the protein in leaf tissues after transformation. In thecells of N. benthamiana leaves, PsAnn4 protein was localizedin the plasma membrane or in the cell wall. In addition, we alsoestimated the presence of PsAnn4 in different cell fractionsby Western-blot hybridization using anti-YFP or anti-RFPantibodies. PsAnn4-YFP was found in insoluble fraction ofleaf tissue pelleted at 36 000 g . It suggests thatPsAnn4 may be involved in cell wall or membrane modificationas well as in ion transport.DiscussionAvailable pea genome information  allowedus to determine the composition of the annexin genefamily in this legume. Database searches revealed 15 annexingenes in P. sativum L., 18 in M. truncatula as well as 13 inboth P. vulgaris and L. japonicus. Based on the phylogeneticanalysis of these annexins, close homologs can be identifiedamong these legume species .At present, only one pea annexin, p35, has been functionallycharacterized . The localization of thisannexin in root cells involved in active secretion suggests itsfunction in exocytosis. Subsequently, the use of antibodiesagainst this protein revealed its localization in epidermalcells of the leaf and stem . However,annexins involved in nodulation have not been characterizedin P. sativum. In contrast, in M. truncatula, two annexins,MtAnn1 (Medtr8g038210) and MtAnn2 (Medtr8g038220),demonstrated a high level of expression during nodulationand were found to be involved in controlling bacterial infectionand nodule organogenesis .Another annexin, MtAnn3 (Medtr4g097180), was found to beimportant for root hair deformations in M. truncatula . At the same time, close homologs of MtAnn1 –PvAnn1 (Phvul.011g209300) and LjAnn1 (Lj0g3v0203419),which belong to the same phylogenetic group as MtAnn1, playimportant roles in the symbiotic process in P. vulgaris andL. japonicus .In our earlier work, two annexins activated at the earlystages of symbiosis development in pea were found using theproteomics approach . This approachmight be helpful for the identification of new regulators ofsignal transduction pathways at the initial stages of nodulationin pea. Our present analysis revealed that these two identifiedannexins of pea belong to different phylogenetic groups,defined as homologs of MtAnn4, PvAnn4 and MtAnn8,PvAnn8, respectively. Although PsAnn4, and MtAnn4 andPvAnn4 have high levels of homology with MtAnn1 andPvAnn1, they belong to another group of annexins basedon phylogenetic analysis. PsAnn8 belongs to a less studiedphylogenetic group. Therefore, two previously unknown annexinswere identified in our study. In addition to stimulationduring rhizobial inoculation, the dependence of PsAnn4 andPsAnn8 activation on the LysM-receptor-like kinase SYM10,encoding a putative Nod factor receptor, was revealed in thepresent study , which suggested that rhizobialsignaling molecules Nod factors may be important for theiractivation. It also suggests the participation of these twoannexins in the development of the symbiotic interaction ofplants with rhizobia.Phylogenetic analysis and prediction of the overall 3Dstructure of PsAnn4 and PsAnn8 proteins showed differencesin the Ca2+-binding motif in the fourth annexin repeat of theseproteins, and therefore, in the potential ability to bind calciumions. This can potentially influence the binding of these annexinsto phospholipids by means of a calcium bridge mechanism.It was predicted that three calcium ions were coordinatedin the first and fourth repeats, which is consistent with the dataof the canonical binding of the G. hirsutum annexin GhAnn1and animal annexins to the phospholipids of membranesusingthe mechanism of calcium bridges . Inthe predicted structures of Arabidopsis annexins , the canonicity of the Ca2+-bindingmotif in the first repeat and the presence of modified motifsin the fourth repeats of AtAnn1 and AtAnn3 were also shown,while AtAnn4 had no recognizable Ca2+ – or phospholipidbindingmotifs .Since the level of PsAnn4 synthesis in response to inoculationwas more significant in the roots of wild type pea plantscompared with mutant defective in symbiosis, we carried outthe analysis of this annexin in more detail. It was shown thatthe regulation of PsAnn4 annexin in pea could be achievedat the transcriptional level as well as post-transcriptionaland translational levels, probably. Significant activation ofMtAnn1 and MtAnn2 gene expression level was found in theroots of M. truncatula treated with Nod factors or inoculated with rhizobia . Meanwhile, theexpression of PvAnn1 in P. vulgaris was slightly upregulatedin developing nodules . However,a phosphoproteomic approach revealed that PvAnn1 wasa phosphorylated protein with enhanced levels of synthesisduring nodulation . Hence, theregulation of annexins involved in nodulation might be differentand is probably connected with different functions thatannexins fulfil in this processLocalization of annexins might differ depending on theirfunction. Some annexins show cytoplasmic and nuclear localization,while other annexins are associated with variousplant membranes, including the plasma membrane, endoplasmicreticulum, and nuclear membrane . Some annexins maybe embedded in the membrane in the form of monomers oroligomers. One of the distinctive characteristics of annexinsis their ability to change their cellular localization in responseto various stimuli. In our experiments, the localization ofannexin 4 (PsAnn4) in the cell wall or plasma membranewas shown, suggesting the participation of this annexin inprocesses associated either with membrane modification orion transport at the early stages of symbiosis establishment inpea. Similarly, the localization of the other annexin, MtAnn2,involved in nodulation in M. truncatula, was revealed to beassociated with the plasma membrane, particularly with lipidrafts from root plasma membrane preparations . In addition, the annexin PvAnn1 is essential forROS-dependent regulation of Ca2+ influx into the cells ofP. vulgaris, which strongly suggests the localization of thisprotein in the plasma membrane. Therefore, specific subcellularlocalization of annexins might be associated with theirfunction signal transduction at the early stages of symbiosis.In this study, phylogenetic analysis of the pea annexinsPsAnn4 and PsAnn8 was performed based on their homologywith annexins from other legumes. The modeling approachallowed us to estimate the structural features of these annexinsthat might influence their functional activity. To verify thefunctions of these annexins, we performed comparative proteomicanalysis, experiments with calcium influx inhibitors,and localization of labeled proteins. Essential down-regulationof PsAnn4 synthesis in a non-nodulating pea mutant P56(sym10) suggests an involvement of this annexin in the rhizobialsymbiosis. The localization of PsAnn4 in the cell wall orplasma membrane of plant cells may indicate its participationin membrane modification or ion transport.The authors declare no conflict of interest.Bateman A., Coin L., Durbin R., Finn R.D., Hollich V., Griffiths-JonesS., Khanna A., Marshall M., Moxon S., Sonnhammer E.L.L.,Studholme D.J., Yeats C., Eddy S.R. The Pfam protein families database.Nucleic Acids Res. 2004;32:D138-D141. DOI 10.1093/nar/gkh121.Bradford M.M. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of proteindyebinding. Anal. Biochem. 1976;72(1-2):248-254. DOI 10.1016/0003-2697(76)90527-3.Breakspear A., Liu C., Roy S., Stacey N., Rogers C., Trick M., MorieriG., Mysore K.S., Wen J., Oldroyd G.E.D., Downie J.A., MurrayJ.D. The root hair “Infectome” of Medicago truncatula uncoverschanges in cell cycle genes and reveals a requirement for auxin signalingin rhizobial infection. Plant Cell Online. 2014;26(12):4680-4701. DOI 10.1105/tpc.114.133496Breton G., Vazquez-Tello A., Danyluk J., Sarhan F. Two novel intrinsicannexins accumulate in wheat membranes in response to low temperature.Plant Cell Physiol. 2000;41(2):177-184. DOI 10.1093/pcp/41.2.177.Carrasco-Castilla J., Ortega-Ortega Y., Jáuregui-Zúñiga D., Juárez-VerdayesM.A., Arthikala M.K., Monroy-Morales E., Nava N., SantanaO., Sánchez-López R., Quinto C. Down-regulation of a Phaseolusvulgaris annexin impairs rhizobial infection and nodulation.Environ. Exp. Bot. 2018;153:108-119. DOI 10.1016/j.envexpbot.2018.05.016.Carroll A.D., Moyen C., Van Kesteren P., Tooke F., Battey N.H.,BrownleeC. Ca2+, annexins, and GTP modulate exocytosis frommaize root cap protoplasts. Plant Cell. 1998;10(8):1267-1276. DOI10.1105/tpc.10.8.1267.Clark G.B., Dauwalder M., Roux S.J. Purification and immunolocalizationof an annexin-like protein in pea seedlings. Planta. 1992;187(1):1-9. DOI 10.1007/BF00201617.Clark G.B., Dauwalder M., Roux S.J. Immunological and biochemicalevidence for nuclear localization of annexin in peas. Plant Physiol.Biochem. 1998;36(9):621-627. DOI 10.1016/S0981-9428(98)80010-7.Clark G.B., Morgan R.O., Fernandez M.P., Roux S.J. Evolutionaryadaptationof plant annexins has diversified their molecular structures,interactions and functional roles. New Phytol. 2012;196(3):695-712. DOI 10.1111/j.1469-8137.2012.04308.x.Clark G.B., Rafati D.S., Bolton R.J., Dauwalder M., Roux S.J. Redistributionof annexin in gravistimulated pea plumules. Plant Physiol.Biochem. 2000;38(12):937-947. DOI 10.1016/S0981-9428(00)01206-7.Clark G.B., Sessions A., Eastburn D.J., Roux S.J. Differential expressionof members of the annexin multigene family in Arabidopsis.Plant Physiol. 2001;126(3):1072-1084. DOI 10.1104/pp.126.3.1072.Dam S., Dyrlund T.F., Ussatjuk A., Jochimsen B., Nielsen K., GoffardN., Ventosa M., Lorentzen A., Gupta V., Andersen S.U., EnghildJ.J., Ronson C.W., Roepstorff P., Stougaard J. Proteome referencemaps of the Lotus japonicus nodule and root. Proteomics.2014;14(2-3):230-240. DOI 10.1002/pmic.201300353.Davies J.M. Annexin-mediated calcium signalling in plants. Plants.2014;3(1):128-140. DOI 10.3390/plants3010128De Carvalho Niebel F., Lescure N., Cullimore J.V., Gamas P. The Medicagotruncatula MtAnn1 gene encoding an annexin is induced byNod factors and during the symbiotic interaction with Rhizobiummeliloti. Mol. Plant Microbe Interact. 1998;11(6):504-513. DOI10.1094/MPMI.1998.11.6.504.De Carvalho-Niebel F., Timmers A.C.J., Chabaud M., Defaux-Petras A.,Barker D.G. The Nod factor-elicited annexin MtAnn1 is preferentiallylocalised at the nuclear periphery in symbiotically activatedroot tissues of Medicago truncatula. Plant J. 2002;32(3):343-352.DOI 10.1046/j.1365-313X.2002.01429.x.Espinoza C., Liang Y., Stacey G. Chitin receptor CERK1 links saltstress and chitin-triggered innate immunity in Arabidopsis. Plant J.2017;89(5):984-995. DOI 10.1111/tpj.13437Feng Y.M., Wei X.K., Liao W.X., Huang L.H., Zhang H., Liang S.C.,Peng H. Molecular analysis of the annexin gene family in soybean.Biol. Plant. 2013;57(4):655-662. DOI 10.1007/s10535-013-0334-0.Gerke V., Moss S.E. Annexins: from structure to function. Physiol. Rev.2002;82(2):331-371. DOI 10.1152/physrev.00030.2001.Gong Z.Y., Song X., Chen G.Y., Zhu J.B., Yu G.Q., Zou H.S. Molecularstudies of the Medicago truncatula MtAnn3 gene involved in roothair deformation. Chinese Sci. Bull. 2012;57(15):1803-1809. DOI10.1007/s11434-011-4937-6.Gorecka K.M., Konopka-Postupolska D., Hennig J., Buchet R., PikulaS. Peroxidase activity of annexin 1 from Arabidopsis thaliana.Biochem. Biophys. Res. Commun. 2005;336(3):868-875. DOI10.1016/j.bbrc.2005.08.181Hofmann A., Proust J., Dorowski A., Schantz R., Huber R. Annexin 24from Capsicum annuum. X-ray structure and biochemical characterization.J. Biol. Chem. 2000;275(11):8072-8082. DOI 10.1074/jbc.275.11.8072.Hu N.J., Yusof A.M., Winter A., Osman A., Reeve A.K., Hofmann A.The crystal structure of calcium-bound annexin Gh1 from Gossypiumhirsutum and its implications for membrane binding mechanismsof plant annexins. J. Biol. Chem. 2008;283(26):18314-18322.DOI 10.1074/jbc.M801051200.Ijaz R., Ejaz J., Gao S., Liu T., Imtiaz M., Ye Z., Wang T. Overexpressionof annexin gene AnnSp2, enhances drought and salt tolerancethrough modulation of ABA synthesis and scavenging ROSin tomato. Sci. Rep. 2017;7(1):1-14. DOI 10.1038/s41598-017-11168-2.Jáuregui-Zúñiga D., Ortega-Ortega Y., Pedraza-Escalona M., Reyes-Grajeda J.P., Ruiz M.I., Quinto C. Phosphoproteomic analysis inPhaseolus vulgaris roots treated with Rhizobium etli nodulation factors.Plant Mol. Biol. Report. 2016;34(5):961-969. DOI 10.1007/s11105-016-0978-y.Kirienko A.N., Porozov Y.B., Malkov N.V., Akhtemova G.A., Le SignorC., Thompson R., Saffray C., Dalmais M., Bendahmane A., TikhonovichI.A., Dolgikh E.A. Role of a receptor-like kinase K1 inpea Rhizobium symbiosis development. Planta. 2018;248(5):1101-1120. DOI 10.1007/s00425-018-2944-4.Kodavali P.K., Skowronek K., Koszela-Piotrowska I., Strzelecka-KiliszekA., Pawlowski K., Pikula S. Structural and functional characterizationof annexin 1 from Medicago truncatula. Plant Physiol.Biochem. 2013;73:56-62. DOI 10.1016/j.plaphy.2013.08.010.Konopka-Postupolska D., Clark G. Annexins as overlooked regulatorsof membrane trafficking in plant cells. Int. J. Mol. Sci. 2017;18(4):1-34. DOI 10.3390/ijms18040863.Konopka-Postupolska D., Clark G., Goch G., Debski J., Floras K.,Cantero A., Fijolek B., Roux S., Hennig J. The role of annexin 1in drought stress in Arabidopsis. Plant Physiol. 2009;150(3):1394-1410. DOI 10.1104/pp.109.135228.Konopka-Postupolska D., Clark G., Hofmann A. Structure, functionand membrane interactions of plant annexins: An update. Plant Sci.2011;181(3):230-241. DOI 10.1016/j.plantsci.2011.05.013.Kreplak J., Madoui M.-A., Cápal P., Novák P., Labadie K., Aubert G.,Bayer P.E., Gali K.K., Syme R.A., Main D., Klein A., Bérard A.,VrbováI., Fournier C., D’Agata L., Belser C., Berrabah W., ToegelováH., Milec Z., Vrána J., Lee H., Kougbeadjo A., Térézol M.,Huneau C., Turo C.J., Mohellibi N., Neumann P., Falque M., GallardoK., McGee R., Tar’an B., Bendahmane A., Aury J.-M., Batley J.,Le Paslier M.-C., Ellis N., Warkentin T.D., Coyne C.J., Salse J., EdwardsD., Lichtenzveig J., Macas J., Doležel J., Wincker P., BurstinJ. A reference genome for pea provides insight into legume genomeevolution. Nat. Genet. 2019;51(9):1411-1422. DOI 10.1038/s41588-019-0480-1.Kwon Y.S., Lee D.Y., Rakwal R., Baek S.B., Lee J.H., Kwak Y.S.,Seo J.S., Chung W.S., Bae D.W., Kim S.G. Proteomic analyses ofthe interaction between the plant-growth promoting rhizobacteriumPaenibacillus polymyxa E681 and Arabidopsis thaliana. Proteomics.2016;16(1):122-135. DOI 10.1002/pmic.201500196.Laohavisit A., Davies J.M. Annexins. New Phytol. 2011;189(1):40-53.DOI 10.1111/j.1469-8137.2010.03533.x.Laohavisit A., Mortimer J.C., Demidchik V., Coxon K.M., StancombeM.A., Macpherson N., Brownlee C., Hofmann A., Webb A.A.R.,Miedema H., Battey N.H., Davies J.M. Zea mays annexins modulatecytosolic free Ca2+ and generate a Ca2+-permeable conductance.Plant Cell. 2009;21(2):479-493. DOI 10.1105/tpc.108.059550.Lefebvre B., Furt F., Hartmann M.-A., Michaelson L.V., Carde J.-P.,Sargueil-Boiron F., Rossignol M., Napier J.A., Cullimore J., BessouleJ.-J., Mongrand S. Characterization of lipid rafts from Medicago truncatula root plasma membranes: A proteomic study revealsthe presence of a raft-associated redox system. Plant Physiol. 2007;144(1):402-418. DOI 10.18362/bjta.v4.i1-2.59. Leppyanen I.V., Kirienko A.N., Lobov A.A., Dolgikh E.A. Differentialproteome analysis of pea roots at the early stages of symbiosiswith nodule bacteria. Vavilovskii Zhurnal Genetiki i Selektsii =Vavilov Journal of Genetics and Breeding. 2018;22(2):196-204.DOI 10.18699/VJ18.34.7.Limpens E., Moling S., Hooiveld G., Pereira P.A., Bisseling T., BeckerJ.D., Küster H. Cell- and tissue-specific transcriptome analysesof Medicago truncatula root nodules. PLoS One. 2013;8(5):e64377.DOI 10.1371/journal.pone.0064377.Lizarbe M.A., Barrasa J.I., Olmo N., Gavilanes F., Turnay J. Annexinphospholipidinteractions. Functional implications. Int. J. Mol. Sci.2013;14:2652-2683. DOI 10.3390/ijms14022652.Madsen E.B., Madsen L.H., Radutoiu S., Olbryt M., Rakwalska M.,Szczyglowski K., Sato S., Kaneko T., Tabata S., Sandal N., StougaardJ. A receptor kinase gene of the LysM type is involved in legumeperception of rhizobial signals. Nature. 2003;425(6958):637-640. DOI 10.1038/nature02045Manthey K., Krajinski F., Hohnjec N., Firnhaber C., Pünler A., PerlickA.M., Küster H. Transcriptome profiling in root nodules andarbuscular mycorrhiza identifies a collection of novel genes inducedduring Medicago truncatula root endosymbioses. Mol. Plant-Microbe Interact. 2004;17(10):1063-1077. DOI 10.1094/MPMI.2004.17.10.1063.Mortimer J.C., Laohavisit A., Macpherson N., Webb A., Brownlee C.,Battey N.H., Davies J.M. Annexins: multifunctional componentsof growth and adaptation. J. Exp. Bot. 2008;59(3):533-544. DOI10.1093/jxb/erm344.Ordog R. PyDeT, a PyMOL plug-in for visualizing geometric conceptsaround proteins. Bioinformation. 2008;2(8):346-347. DOI 10.6026/97320630002346.Orosz L., Sváb Z., Kondorosi A., Sik T. Genetic studies on rhizobiophage16-3. I. Genes and functions on the chromosome. Mol. Gen.Genet. 1973;125(4):341-350. DOI 10.1007/BF00276589.Sievers F., Wilm A., Dineen D., Gibson T.J., Karplus K., Li W., LopezR., McWilliam H., Remmert M., Söding J., Thompson J.D., HigginsD.G. Fast, scalable generation of high-quality protein multiplesequence alignments using Clustal Omega. Mol. Syst. Biol. 2011;7(1):539. DOI 10.1038/msb.2011.75.Talukdar T., Gorecka K.M., de Carvalho-Niebel F., Downie J.A., CullimoreJ., Pikula S. Annexins – calcium- and membrane-binding proteinsin the plant kingdom: potential role in nodulation and mycorrhizationin Medicago truncatula. Acta Biochim. Pol. 2009;56(2):199-210. DOI 20091709.Van Brussel A.A.N., Planque K., Quispel A. The wall of Rhizobium leguminosarumin bacteroid and free-living forms. J. Gen. Microbiol.1977;101(1):51-56. DOI 10.1099/00221287-101-1-51.Van Brussel A.A.N., Tak T., Wetselaar A., Pees E., Wijffelman C. Smallleguminosae as test plants for nodulation of Rhizobium leguminosarumand other rhizobia and agrobacteria harbouring a leguminosarumsym plasmid. Plant Sci. Lett. 1982;27(3):317-325. DOI10.1016/0304-4211(82)90134-1.Voss T., Haberl P. Observations on the reproducibility and matchingefficiency of two-dimensional electrophoresis gels: consequencesfor comprehensive data analysis. Electrophoresis. 2000;21(16):3345-3350. DOI 10.1002/1522-2683(20001001)21:16<3345::AIDELPS3345>3.0.CO;2-Z.Webb B., Sali A. Comparative protein structure modeling using Modeller.Curr. Protoc. Bioinform. 2016;54(1):5.6.1-5.6.37. DOI 10.1002/cpbi.3.Wienkoop S., Saalbach G. Proteome analysis. Novel proteins identifiedat the peribacteroid membrane from Lotus japonicus root nodules.Plant Physiol. 2003;131(3):1080-1090. DOI 10.1104/pp.102.015362.


Background: Coronavirus disease (COVID-19) has spread rapidly since 2019. Approximately 15% of the patients will develop severe complications such as multiple organ disease syndrome related to cytokine release syndrome (CRS). Continuous renal replacement therapy (CRRT) can remove inflammatory cytokines through filtration or adsorption. We evaluated the effectiveness of CRRT in COVID-19 patients with CRS.Methods: This retrospective, multicenter, descriptive study included 83 patients with CRS from three hospitals in Wuhan.Results: In COVID-19 patients with CRS, the fatality rate was even higher in CRRT group (P=0.005). However, inflammatory markers such as C-reactive protein, neutrophil counts, and D-dimer decreased after CRRT (P<0.05). Results of Lasso model showed that tracheotomy (β -1.31) and convalescent plasma (β -1.41) were the protective factors. In contrast, CRRT (β 1.07), respiratory failure (β 1.61), consolidation on lung CT (β 0.48), acute kidney injury (AKI) (β 0.47), and elevated neutrophil count (β 0.02) were the risk factors for death.Conclusions: Our results showed that although CRRT significantly reduced the inflammation, it did not decrease the fatality rate of patients with CRS. Therefore, the choice of CRRT indication, dialysis time and dialysis mode should be more careful and accurate in COVID-19 patients with CRS. An outbreak of coronavirus disease COVID-19) in December 2019 in Wuhan, China, has developed into a global pandemic. COVID-19 is caused by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus 9 in Dece. AlthougCytokine release syndrome (CRS) plays a vital role in the occurrence and disease progression of the COVID-19 . It has Although there are no standard diagnostic criteria for CRS, some treatments based on managing CRS have been widely applied clinically. These include antagonism at the IL-6 receptor (using the antagonist Tocilizumab), artificial-liver blood-purification therapy that can remove cytokines from the circulation, and continuous renal replacement therapy (CRRT) can remove inflammatory factors such as cytokines from the circulation via filtration and adsorption . TocilizOf the 83 cases included in our study, 67 were classified as critical, and 16 as non-critical. Of the 67 critical patients, 38 cases were treated with CRRT during hospitalization. A total of 45 patients died, and 36 were cured or improved. The outcome of two patients is unknown.The mean age of the patients was 67.3±12.6 years. Almost three-quarters (73.5%) of the patients were male; 67 were critical cases, and 13.3% of the patients had a history of smoking. The underlying medical conditions of the patients included hypertension (55.4%), diabetes (24.1%), tumors (6%), and chronic obstructive pulmonary disease (1.2%). Pulmonary CT revealed that 51.7% of the critical patients had consolidation, and 73.3 % of non-critical patients had ground-glass changes in the lungs. The mean peak creatinine value during hospitalization was 133.2 pg/mL . AKI was present in 60.2%, MODS in 36.1%, and ARDS in 42.2% of the patients. Compared to the non-critical groups, the critical group had more patients with an IL-6 value >4000 pg/mL (29.9% vs.18.8%) .Patients in the critical group had a significantly higher WBC count (P=0.001), neutrophil count (P<0.001), neutrophil percentage (P<0.001), LDH (P<0.001), peak creatinine (P=0.01), high-sensitivity troponin I (P<0.001), CRP (P<0.001), ferritin (P=0.001) and procalcitonin (PCT) (P=0.043) than those in the non-critical group. Patients in the critical group had significantly lower lymphocyte counts (P=0.018) and albumin levels (P=0.015) than patients in the non-critical group .Treatments such as CRRT (P=0.001), antibiotics (P=0.002), hormones (P=0.004), globulin therapy (P<0.001), invasive mechanical ventilation (P<0.001) and non-invasive ventilation (P<0.001), were each more commonly used in critical patients than in non-critical patients. Moreover, critical patients had a higher incidence of AKI (P<0.001), respiratory failure (P<0.001), ARDS (P<0.001), MODS (P<0.001), gastrointestinal bleeding (P=0.004), acute liver dysfunction (P<0.001) and acute myocardial injury (P<0.001) .Over half of the critical patients (38/67) were treated with CRRT. Those who received CRRT had a lower blood platelets (PLT) (P=0.023), and lower albumin (P=0.041), a higher peak creatinine (P=0.018), and a greater incidence of MODS (P<0.001) than those who did not receive CRRT. More patients receiving CRRT also underwent tracheal cannulation (P=0.003) or invasive mechanical ventilation (P=0.002). Most patients who received CRRT (64.7%) had pulmonary consolidations on CT . Unexpec9g/L vs. 0.64  109g/L] followed the same pattern, demonstrating lower values in the CRRT patients. The CRP was higher in the CRRT than in the non-CRRT group [97.7  mg/L vs. 89.16  mg/L)]. Moreover, the incidence of AKI and ARDS in critical patients treated with CRRT was 81.1% and 71.4%, respectively, which was higher than the corresponding figures of non-CRRT . The blood oxygen saturation in patients who received CRRT was lower than in the non-CRRT group [90 % vs.93 %] and the lymphocyte count , and differences between the groups were determined using a two-sample t-test or Wilcoxon rank-sum test. Lasso and accurate logistic analysis were conducted to identify the factors related to death. The explanatory variables included treatments , respiratory failure, consolidation on lung CT, AKI, elevated neutrophil percentage, cough, dyspnea, hormone treatment, invasive mechanical ventilation, acute myocardial injury, White blood cell (WBC) count (x 10The data underlying this article are available in the article and its online supplementary material.


Trichoderma are well-studied filamentous fungi generally observed in nature, which are widely marketed as biocontrol agents. The secondary metabolites produced by Trichoderma have gained extensive attention since they possess attractive chemical structures with remarkable biological activities. A large number of metabolites have been isolated from Trichoderma species in recent years. A previous review by Reino et al. summarized 186 compounds isolated from Trichoderma as well as their biological activities up to 2008. To update the relevant list of reviews of secondary metabolites produced from Trichoderma sp., we provide a comprehensive overview in regard to the newly described metabolites of Trichoderma from the beginning of 2009 to the end of 2020, with emphasis on their chemistry and various bioactivities. A total of 203 compounds with considerable bioactivities are included in this review, which is worth expecting for the discovery of new drug leads and agrochemicals in the foreseeable future. Moreover, new strategies for discovering secondary metabolites of Trichoderma in recent years are also discussed herein.Fungi play an irreplaceable role in drug discovery in the course of human history, as they possess unique abilities to synthesize diverse specialized metabolites with significant medicinal potential.  Trichoderma is a fungal genus that was first described in 1794 , and six known trichothecenes, trichodermol (9), trichodermin (10), trichoderminol (11), trichodermarin A (12) and trichodermarin B (13), and 2,4,12-trihydroxyapotrichothecene (14), were isolated from Trichoderma brevicompactum ADL-9-2, which was obtained as an endophyte of marine algae Chondria tenuissima (8), featuring a 2’-N-acetylglucosaminyl moiety, represented the first aminoglycoside-bearing trichothecene. Chemical investigations of the marine fungus Trichoderma cf. brevicompactum TPU199 fermented with NaI afforded three new trichothecenes, trichobreols A–C (15–17) , whereas compounds 16 and 17 were found only under NaI-containing culture conditions. Additionally, isolation of the same fungus yielded two new trichothecenes, trichobreols D (18) and E (19) (20) and B (21), two trichothecenes linked with octa-2,4,6-trienedioyl moiety, were isolated from the biofertilizer fungus T. brevicompactum (CGMCC19618) , 8-deoxy-trichothecin (23), and trichothecinol B (24) were yielded  . Interesd E (19) . HarzianCC19618) . From a  yielded .T. virens Y13-3, eight undescribed carotane sesquiterpenes, trichocarotins A–H (25–32), along with the known compounds CAF-603 (33), trichocarane B (34), 7-β-hydroxy CAF-603 (35), and trichocarane A (36), were discovered (From the marine-derived fungus scovered  (Shi et scovered . Carotanscovered .37), containing a previously unrecognized site of an exocyclic olefin functionality at C-10, was obtained from the marine-derived fungus T. virens Y13-3 , and three known ones, i.e., aspergilloid G (41), rhinomilisin E (42), and rhinomilisin G (43), were characterized from the marine sponge-derived fungus Trichoderma sp. SM16 , was produced by the marine fungus T. harzianum (XS-20090075), which was isolated from soft corals -isocyclonerotriol (45) and (10Z)-isocyclonerotriol (46), which were characterized as the first example with an isomerized ring in cycloneranes (47), methyl 3,7-dihydroxy-15-cycloneranate (50), and 10-cycloneren-3,5,7-triol (51), as well as two structurally related cycloneranes, 9-cycloneren-3,7,11-triol (48) and (–)-cyclonerodiol (49), were obtained from T. harzianum X-5, an endophyte of the marine alga Laminaria japonica  and 10(E)-cyclonerotriol (52), were obtained from T. longibrachiatum, an endophyte of the highly halophile Suaeda glauca , II (54), and III (55) (56), 3-oxoneomacrophorins I (57) and II (58), neomacrophorin VII (59), 5′-epimacrophorin B (60), and 5′-deoxyneomacrophorin IV (61), as well as four novel premacrophorin congeners, i.e., premacrophorin III (62), premacrophorindiol (63), premacrophorintriols I (64), and II (65), were isolated from the same fungus (56 and 57) or 2,3-epoxybenzosemiquinol substructures (58–60 and 62–64). Premacrophorins 62–65 carried acyclic isoprenoid side chains biosynthetically derived from neomacrophorins in the early stage, rather than the common drimane skeleton.Chemical exploration of an endophyte III (55)  (Hirose III (55) . They bee fungus . These mTrichoderma sp. 307 and aquatic pathogenic bacterium Acinetobacter johnsonii B2 afforded two undescribed furan-type isoeremophilane sesquiterpenes, microsphaeropsisins B (66) and C (67) , trichoacorenol (69), and trichoacorenol B (70) were isolated from marine-derived T. harzianum X-5, and they were structurally characterized as acorane sesquiterpenes (71), an unusual norsesquiterpene with a novel tricyclic-6/5/5--decane framework, was characterized to originate from T. longibrachiatum (72) and B (73), were produced by T. atroviride S361, an endophyte of Cephalotaxus fortunei  and B (75), were obtained from T. brevicompactum ADL-9-2, which was isolated from marine algae C. tenuissima , were isolated from T. asperellum residing in Panax notoginseng   and B (81)  . It is bterpenes . Trichodachiatum . Two newfortunei . Compounnuissima . Compounoginseng . The newe C (79) . Chemicad B (81) . InteresTrichoderma species (82–86), and three previously reported diterpenes, i.e., 3S-hydroxyharzianone (87), harziandione (88), and harzianol A (89), were isolated from the endophyte T. atroviride B7 of Colquhounia coccinea var. mollis (90), an undescribed harziane lactone, was isolated from an endophyte T. longibrachiatum A-WH-20-2 of marine algae Laurencia okamurai (91), was obtained from the soft coral-sourced T. harzianum (XS-20090075) by chemical epigenetic manipulation strategy , was produced by T. harzianum X-5 -dihydro-harzianone (93) and harzianelactone (94), were produced by Trichoderma sp. Xy24 from mangrove plant Xylocarpus granatum , as well as five new lactones, i.e., harzianones A–D (97–100) and harziane (101) (102), was isolated from Trichoderma erinaceum derived from Acanthaster planci  . Finallyr planci .103), a rare norditerpene, was produced by an endophyte T. citrinoviride cf-27 , was characterized from the marine algicolous fungus T. harzianum X-5 , characterized as a novel chlorinated cleistanthane diterpenoid, was isolated from the marine fungal strain T. harzianum (XS-20090075) (Trichoderma for the first time.Citrinovirin (de cf-27 . Compounanum X-5 . 104 and0090075) . TricyclT. harzianum KZ-20 afforded four new cyclodepsipeptides belonging to the destruxin family, i.e., trichodestruxins A–D (106–109), and two previously reported derivative, i.e., destruxin E2 chlorohydrin (110) and destruxin A2 (111) , trichomide B (113), and homodestruxin B (114), characterized as cyclohexadepsipeptides of the trichomide series, were produced by T. longibrachiatum (115) and halobacillin (116) were obtained from the endophyte T. asperellum (Bioassay-guided fractionation of the plant endophytic fungus A2 (111)  (Liu Z. A2 (111) . Destruxachiatum . Finallyperellum .Trichoderma sp. TPU199 is a producer of a series of diketopiperazines (117–127) (117), gliovirin (118), and trichodermamide A (119). 117 and 118 possessed an unusual epipolythiodiketopiperazine (ETP) skeleton. Then, this strain with sodium halides added to the culture medium afforded the halogenated gliovirin-type ETPs DC1149B (120), DC1149R (122), and iododithiobrevamide (123). Subsequently, chlorotrithiobrevamide (124), the first trisulfide derivative in the type of ETP, was characterized. Furthermore, a highly modified dipeptide, dithioaspergillazine A (125), was obtained after the long time cultivation. Finally, two undescribed ETPs, i.e., 5-epi-pretrichodermamide A (126) and 5-epi-trithiopretrichodermamide A (127), were characterized under NaI-containing culture conditions. Pretrichodermamide G (128) was established as a 1,2-oxazadecaline ETP, and it was identified from the endophyte T. harzianum of Zingiber officinale (129), and a biogenetically related metabolite aspergillazin A (130) were produced by the marine-sourced T. harzianum D13 (129 and 130 were novel ETP derivatives with the sulfur bridge locating at different positions. Dehydroxymethylbis(dethio)bis(methylthio)gliotoxin (131) and -6-(para-hydroxybenzyl)-1,4-dimethyl-3,6-bis(methylthio)piperazine-2,5-dione (132), which were structurally characterized as two undescribed sulfurated diketopiperazines, were produced by an algicolous isolate of T. virens Y13-3  (133) (The marine fungus 117–127)  (Yamazak117–127) . Initialficinale . A rare anum D13 . Notablyns Y13-3 . The funo) (133) .T. virens FKI-7573 generated an undescribed N-containing compound, i.e., trichothioneic acid (134) (134 contained a heptelidic acid and an L-ergothioneine substructure. Ethyl 2-bromo-4-chloroquinoline-3-carboxylate (135) was produced by the soft coral-sourced T. harzianum (XS-20090075) in Czapek’s medium  and B (137), isolated as stereoisomers originating from the PKS-NRPS mixed pathway, were isolated from T. gamsii, an endophyte of P. notoginseng (138) and B (139), were identified from Trichoderma sp. strain MF106 from the Greenland Seas (140), a nitrogen heterocyclic siderophore, was isolated from T. harzianum M10 -1-methyl-3,5-dioxopyrrolidin-2-ylmethyl]-3-methyl-butyric acid. Atrichodermone A (141) was a unique compound with a dimeric cyclopentenone framework that was discovered from the endophytic fungal strain T. atroviride  and 5′-acetoxy-deoxycyclonerin D (143), were obtained from the marine fungus T. asperellum A-YMD-9-2   (Miyano id (134) . 134 cons medium . 135 wasoginseng . Two rarand Seas . Harziananum M10 . 140 wasroviride . 141 was-YMD-9-2 .144), a natural product, methyl-trichoharzin (145), and the known trichoharzin (146) and eujavanicol A (147) were produced by the marine fungus T. harzianum XS-20090075  was identified from a fungal strain of T. harzianum F031  and B (150) were isolated from an endophytic fungus T. spirale, and characterized as new octahydronaphthalene derivatives , 1,6-di-epi-koninginin A (152), 15-hydroxykoninginin A (153), 10-deacetylkoningiopisin D (154), and koninginin T (155), along with two previously reported derivatives, koninginin L (156) and trichoketide A (157), were produced by Trichoderma koningiopsis QA-3 (151–153 were characterized as tricyclic polyketides with an octahydrochromene skeleton. Koninginins I (158), J (159) and K (160), which were structurally characterized as new koninginin-type compounds, were produced by T. neokongii 8722  and trichodermaketones A–D (162–165) (162 and 163 represented unprecedented tricyclic polyketides having a bistetrafuran skeleton. Trichoketides A (166) and B (167), two undescribed octaketides, were isolated from Trichoderma sp. TPU1237  (herein reported as a new compound) and M (168) were isolated from solid fermentation of T. koningii 8662  and M (168) was an oxygen bridge located between the C-10 and C-7 positions.Five new polyketides, sis QA-3 . Compoungii 8722 . Chemica162–165) . 162 and TPU1237 . 166 andgii 8662 . A serie169) and trichoderone B (170), together with three previously reported cytochalasans, aspochalasins D (171), J (172), and I (173), were obtained from T. gamsii from P. notoginseng  and D (175), as well as three known cytochalasans, aspochalasins D (171), M (176), and P (177), were obtained from the abovementioned strain T. gamsii , was isolated from endophytic T. atroviride (179), was produced by the marine-sourced Trichoderma sp. HPQJ-34  and B (181), were found from the marine fungus T. harzianum (XS-20090075) (180 and 181 had a C9 polyketide framework with a γ-lactone moiety. Trichoderpyrone (182), a novel cyclopentenone-pyrone mixed polyketide, was produced by T. gamsii , a new cyclopentenone, was obtained from the marine Trichoderma sp. , which was identified as a new isocoumarin derivative with a 6,8-dihydroxyisocoumarin moiety, was isolated from T. citrinoviride A-WH-20-3 (187) and 4,6-dihydroxy-5-methylphthalide (188), were produced by the fungus T. harzianum F031 (189 and 190) with a 1-hydroxyhepta/methoxyhepta-3,5-dien-2-one moiety were produced in the transformant of Trichoderma afroharzianum (191) and E (192), were obtained from dragonfly associated T. harzianum QTYC77 (193), elucidated as but-2-enoic acid 7-acetoxy-6-hydroxy-2- methyl-10-oxo-5,6,7,8,9,10-hexahydro-2H-oxecin-5-yl ester, was isolated from cultural filtrates of Trichoderma cremeum -7-hydroxy-de-O-methyllasiodiplodin (194) and (3R)-5-oxo-de-O-methyllasiodiplodin (195), were isolated from the cocultivation of Trichoderma sp. 307 and A. johnsonii B2 (196), and the known nafuredin A (197), were produced by marine fungus T. harzianum D13  . Compoun. gamsii . 182 haderma sp. . Compoun Fes1712 . Trichop-WH-20-3 . Previounum F031 . Two strarzianum . Two undm QTYC77 . A new 1 cremeum . Two undsonii B2 . An undeanum D13 .198) and B (199), were produced by a culture broth of T. polypori FKI-7382  and II (201). Notably, both of them were characterized from Trichoderma for the first time (202) and B (203), two new compounds with 4-(2-hydroxyethyl) phenol moieties, were isolated from an endophyte T. gamsii  associated with secondary metabolites are cryptic  or expressed at very low levels under general laboratory conditions . TherefoTrichoderma sp. TPU199 was found to produce a series of diketopiperazines under different conditions , 122 (Br derivative of 117), and 123 (I derivative of 177) when added with NaCl, NaBr, and NaI in culture medium, respectively. Moreover, TPU199 supplemented with DMSO yielded 124, a new trithio derivative of 120. A continuous study indicated that, with the long time cultivation, an undescribed modified dipeptide 125 was obtained. Finally, two undescribed ETPs 126 and 127 were characterized under NaI-containing culture conditions. It is undoubtedly proven that changing the culture conditions can activate cryptic metabolic pathways.The marine-derived fungus nditions  (Yamazaknditions . ChemicaTrichoderma sp. 307 and pathogenic bacterium A. johnsonii B2 yielded two undescribed sesquiterpenes (66 and 67) and de-O-methyllasiodiplodin (194 and 195) . This is the first report of cleistanthane diterpenoids isolated from Trichoderma species. This study provided solid example to show that it is efficient to activate the silent genes of Trichoderma species by chemical epigenetic manipulation.Microorganism coculture based on microbial interspecies competition is an efficient path to stimulate cryptic BGCs. Cocultivation of and 195) . HPLC anabolites . A histoT. afroharzianum, a laeA-like gene overexpression transformant was built (189 and 190). This study indicated that transcriptional control could be a considerable strategy in activating more secondary metabolites and enhancing the silent potential metabolism of Trichoderma species.The transcriptional control has also proven to be an effective approach. To activate the chemical potential of the endophytic fungus as built . Further1–203 are listed in The producing fungus, environmental source, and bioactivities of compounds 1–14 were assayed for antifungal activity against Botrytis cinerea, Cochliobolus miyabeanus, Fusarium oxysporum f. sp. cucumerium, F. oxysporum f. sp. niveum, and Phomopsis asparagi  among these trichothecenes indicated that the acetoxy and methyl functionalities (compound 10) were necessary, while the epoxide moiety and the ether linkage were other possibilities   exhibited potent effect on Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus, with EC50s of 7.7, 7.7, and 9.9 μg/mL, respectively  was active against B. subtilis , Staphylococcus epidermidis (24.28 μM), and C. albicans (25.38 μM) (148) displayed mild activity against Colletotrichum gloeosporioides, with an MIC value of 128 μg/mL  was inactive against C. albicans (MIC > 125 μg/mL). However, it was active at 125 μg/mL when treated with 0.05 μg/mL ketoconazole  . Further10 μg/mL . The nov 8 μg/mL . The newectively . It seem.4 μg/mL . Cyclopeectively . The new5.38 μM) . Trichoh28 μg/mL . Polyket64 μg/mL . Trichodconazole . New iso32 μg/mL . Polyket32 μg/mL . The newd 7.0 mm . The newa solani . Nafuredectively .Amphidinium carterae, Heterocapsa circularisquama, Heterosigma akashiwo, and Prorocentrum donghaiense) of trichothecene derivatives 1–14 was evaluated. Notably, 10 featured the strongest effect, with IC50s of 1.7, 0.82, 0.91, and 1.4 μg/mL - and (10Z)-cyclonerotriol, the isomerization of the five-membered ring greatly suppressed their antimicroalgal activities , while the new proharziane diterpene 104 potently inhibited with IC50s of 1.2–4.3 μg/mL , H. akashiwo (9.1 μg/mL), and P. donghaiense (5.9 μg/mL)  .38–43 were evaluated against NCIH-460, NCI-H929, and SW620 cell lines  showed cytotoxicity against human adenocarcinoma cells (COLO 201) with an IC50 of 46 μg/mL  was observed to exhibit moderate cytotoxicity against NCI-H1975 , HepG2 (60.88 μM), and MCF-7 (53.92 μM) cell lines  , HepG2 (30.8 μM), and HeLa (33.9 μM)  , c. The 33.9 μM) . Cyclopeell line . The sel20) and B (21) decreased the shoot and root lengths of the dicot species Brassica chinensis and induced inhibitory effect of seed germination at 2 μg/mL . The results indicated that 20 and 21 possess potent herbicidal potential for dicotyledon and/or monocotyledon weeds. All of the isolated harzianes 95–101 exhibited potent phytotoxicity at 200 ppm  lethal rates of 38.2 and 42.7% at 200 μg/mL  showed OH radical-scavenging and singlet oxygen-quenching ability in a dose-dependent manner, which was equivalent to those of positive controls (140) as a plant growth promoter was evaluated   .Trichoderma from 2009–2020. Their chemical structures were classified into terpenoids , cyclopeptides (104–116), diketopiperazines (117–133), alkaloids and other nitrogen-containing compounds (134–143), polyketides , and other compounds (198–203) according to their putative biogenetic sources. As shown in Trichoderma are considerable producing strains of novel terpenoids. It should be pointed out that some terpenoids, such as harzianes, are isolated exclusively from Trichoderma species. This review described 21 harziane diterpenes produced by Trichoderma. Considering their intriguing structures and bioactivities, much more attention should be devoted to this type of terpenoid in subsequent chemical studies.A total of 203 natural products were reported from Trichoderma comprises more than 340 species. Some of them are used as biocontrol agents, while some of them are promising producers of enzymes for industrial purposes. On the other hand, some Trichoderma species possess the unique capacity to synthesize various secondary metabolites with potent biological activities. In this review, a total of 17 identified species, including T. harzianum, T. brevicompactum, T. virens, T. gamsii, T. atroviride, T. longibrachiatum, T. asperellum, T. koningiopsis, T. koningii, T. citrinoviride, T. neokongii, T. spirale, T. afroharzianum, T. polypore, T. polyalthiae, T. erinaceum, and T. cremeum, are reported as the producing strains of the described metabolites. Among them, T. harzianum and T. brevicompactum were the most prolific strains, with 48 (23.76%) and 33 (16.34%) metabolites identified, respectively  have been characterized from T. brevicompactum.The genus ectively . The funTrichoderma is widely distributed and has been isolated in soils, decaying wood, and endophytes in the inner tissue of host plants. Previous studies have mainly focused on terrestrial species of Trichoderma. However, Trichoderma from the marine environment are unexploited. It would be useful to examine marine-derived Trichoderma species since they may be induced to produce specific metabolites in hyperhaline environments. Accordingly, in recent years, increasing attention has been devoted to marine Trichoderma. As shown in Trichoderma. From the above analysis, it can be concluded that marine environment and endophytes are more abundant sources of those productive strains.The genus 22 and 23 showed higher antifungal effect on C. lagrnarium  than the synthetic fungicide carbendazim  , anticancer (20.61%), and antimicroalgal (17.98%) activities were dominant in assessing the pharmacological potential of these metabolites . It shou2 μg/mL) . Cyclopeell line . The sel effects . These iTrichoderma from the beginning of 2009 to the end of 2020. As a result, a total of 203 metabolites are described herein, including their structural diversity and biological activities. Moreover, new strategies for discovering secondary metabolites of Trichoderma in recent years have also been discussed. Trichoderma has proven to be a treasure house of interesting secondary metabolites with medicinal importance. The biochemical studies of Trichoderma are untapped. Although a mass of metabolites have been isolated from Trichoderma species, the further excavation of those metabolites is worth expecting. By using new approaches to activate their silent gene clusters, including cultivation-based approaches, metabolomic profiling, and genome mining-based molecular approaches, an ever-increasing number of bioactive compounds will be obtained, which will be beneficial for the new drug discovery in the near future.In the present review, we offer a detailed summary of recently isolated metabolites from J-LZ and W-LT wrote this manuscript. Q-RH, Y-ZL, M-LW, L-LJ, CL, XY, H-WZ, and G-ZC collected and reorganized the literature data. X-XZ supervised the research work and revised the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Uropathogen resistance, Fluoroquinolone-resistance (FQR) and Extended spectrum beta-lactamase (ESBL), has been observed to be emerging worldwide with prevalences above recommended thresholds for routine empirical treatment. We sought to determine recent resistance prevalence from a geographically diverse sample of US Emergency Departments (ED).Escherichia coli. We conducted a multi-center, observational cohort study utilizing a network of 15 geographically diverse US EDs. Patients ≥ 18 years of age with the primary international classification of diseases (ICD-10) diagnosis code of cystitis, pyelonephritis, or urinary tract infection (UTI) and were discharged home from the ED from 2018-2020 were included. We calculated descriptive statistics for uropathogens and susceptibilities. Logistic regression analysis was used to identify antimicrobial resistance risk factors associated with fluoroquinolone (FQ)-resistant E. coli was the most common pathogen (62.9%), followed by Klebsiella pneumoniae (13%) and Enterococcus species (5.8%). Across all sites, overall E. coli FQ-resistance prevalence was 22.1%, ranging from 10.5 to 29.7% by site. The prevalence of ESBL-producing uropathogen was 4.4%, ranging from 2.3% to 8.6% by site. Previous IV or oral antimicrobial use in the last 90-days and complicated vs. uncomplicated UTI were associated with FQ-resistant E. coli . Of the most prescribed oral antibiotics upon patients discharged from the ED, E. coli resistance to nitrofurantoin and cephalexin was 1.8% and 0.9%, respectively.Among 3,779 patients who met inclusion criteria, median age was 62.9 years (IQR: 41-77.6) and 76.3% were female. The most common diagnoses were complicated (40.9%) and uncomplicated cystitis (39.4%). Six hundred and forty-five (17%) patients reported receiving antimicrobials in the previous 90-days. E. coli is widely prevalent and ESBL-mediated resistance appears to be emerging across US sites highlighting the need for ongoing monitoring of antimicrobial resistance and, at some locations, modification of empirical treatments. FQ-resistant Brett Faine, PharmD, Spero Therapeutics (Research Grant or Support) Megan A. Rech, PharmD, MS, BCCCP, FCCM, Spero (Research Grant or Support) David A. Talan, MD, AbbVie (Consultant)GSK (Consultant)SPERO Therapeutics (Grant/Research Support)


Athelia (Fibularhizoctonia) sp. strain TMB , which forms termite-egg-mimicking sclerotia for which termites care. We further compare its repertoire of psilocybin gene homologs to homologs previously reported for Fibularhizoctonia psychrophila.Atheliales is a diverse order of crust-forming Basidiomycota fungi. Here, we report the draft genome of the “cuckoo fungus,”  Athelia (Fibularhizoctonia) sp. TMB strain TB5 , which forms termite-egg-mimicking sclerotia that termites tend. We further compare its repertoire of psilocybin gene homologs to homologs previously reported for Fibularhizoctonia psychrophila.Atheliales is a diverse order of crust-forming Basidiomycota fungi. Here, we report the draft genome of the “cuckoo fungus,”  Fibularhizoctonia psychrophila CBS 109695, associated with carrot spoilage  psilocybin gene cluster  is an ecologically diverse –4 order –spoilage , 5, and Athelia (Fibularhizoctonia) sp. TMB  produces sclerotia  that chemically and structurally mimic Reticulitermes sp. eggs and receive care in over 70% of colonies of some Reticulitermes species . Sclerotia were cultured in potato dextrose broth at room temperature for 14 days with shaking. Tissue was filtered through Miracloth, flash-frozen with liquid nitrogen, and pulverized with a mortar and pestle. Genomic DNA was immediately extracted using the DNeasy plant minikit (Qiagen). Short-read DNA libraries were prepared using the NEBNext Ultra DNA library preparation kit and were sequenced with a 150-bp paired-end format on a NovaSeq 6000 system (Illumina). Long reads were generated by fragmenting genomic DNA with a Covaris g-TUBE, preparing libraries using the SQK-LSK108 ligation sequencing kit, and sequencing the libraries on a MinION system (Oxford Nanopore Technologies) using an R9.4 flow cell. Sequencing generated 62,281,267 Illumina reads with an average coverage of 230.98× and 228,721 MinION reads with a mean length of 3,983.38 bp, a median length of 1,834 bp, and a mean coverage of 10.94×. Coverage was calculated by mapping reads to the assembly with Bowtie 2 v2.4.1-2  data set (F. psychrophila expressed sequence tag (EST) and transcript data , Coniophora olivacea MUCL 20566 (https://mycocosm.jgi.doe.gov/Conol1), Coniophora puteana (https://mycocosm.jgi.doe.gov/Conpu1), Paxillus involutus ATCC 200175 (https://mycocosm.jgi.doe.gov/Paxin1), Pisolithus microcarpus 441 (https://mycocosm.jgi.doe.gov/Pismi1), Pisolithus tinctorius Marx 270 (https://mycocosm.jgi.doe.gov/Pisti1), Rhizopogon vesiculosus Smith (https://mycocosm.jgi.doe.gov/Rhives1), Scleroderma citrinum Foug A (https://mycocosm.jgi.doe.gov/Sclci1), Serpula himantioides MUCL 38935 (https://mycocosm.jgi.doe.gov/Serla_varsha1), and Suillus brevipes Sb2 v2.0 (https://mycocosm.jgi.doe.gov/Suibr2). Gene prediction was finalized using OrthoFiller v1.1.1 , C. olivacea MUCL 20566, F. psychrophila CBS 109695, Galerina marginata , Laccaria bicolor v2.0 (https://mycocosm.jgi.doe.gov/Lacbi2), P. croceum F 1598, Plicaturopsis crispa (https://mycocosm.jgi.doe.gov/Plicr1/Plicr1.home.html), Psilocybe serbica (https://mycocosm.jgi.doe.gov/Psiser1), and S. brevipes Sb2 v2.0.Illumina reads were trimmed using Trimmomatic v0.36  with the v1.0.11  and the  v1.0.11 . Genes w v1.0.11 , AUGUSTU v1.0.11 , Glimmer v1.0.11 , and Gen v1.0.11  via the  v1.0.11 . We refedata set , F. psycipt data , and pror v1.1.1  with defF. psychrophila and Athelia sp. TMB TB5 was calculated from the BLASTp . Protein domains were aligned (MAFFT v7.467 [Average amino acid identity (AAI) between e BLASTp  identitye BLASTp  with P. equences  were obtequences  domains T v7.467  with --aT v7.467 , -automaT v7.467 ). Close Athelia sp. TMB TB5 has a mean AAI of 82.58%  with F. psychrophila across 1,727 single-copy orthologs, consistent with substantial divergence time. Multiple copies of psilocybin decarboxylase, hydroxylase, and kinase in Athelia sp. TMB TB5 , and SRR12880627 (Illumina read library).The assembly and annotation of