Infection with the SARS-CoV-2 virus seems to contribute significantly to increased postoperative complications and mortality after emergency surgical procedures. Additionally, the fear of COVID-19 contagion delays the consultation of patients, resulting in the deterioration of their acute diseases by the time of consultation. In the specific case of urgent digestive surgery patients, both factors significantly worsen the postoperative course and prognosis. Main working hypothesis: infection by COVID-19 increases postoperative 30-day-mortality for any cause in patients submitted to emergency/urgent general or gastrointestinal surgery. Likewise, hospital collapse during the first wave of the COVID-19 pandemic increased 30-day-mortality for any cause. Hence, the main objective of this study is to estimate the cumulative incidence of mortality at 30-days-after-surgery. Secondary objectives are: to estimate the cumulative incidence of postoperative complications and to develop a specific postoperative risk propensity model for COVID-19-infected patients.A multicenter, observational retrospective cohort study (COVID-CIR-study) will be carried out in consecutive patients operated on for urgent digestive pathology. Two cohorts will be defined: the “pandemic” cohort, which will include all patients (classified as COVID-19-positive or -negative) operated on for emergency digestive pathology during the months of March to June 2020; and the “control” cohort, which will include all patients operated on for emergency digestive pathology during the months of March to June 2019. Information will be gathered on demographic characteristics, clinical and analytical parameters, scores on the usual prognostic scales for quality management in a General Surgery service , prognostic factors applicable to all patients, specific prognostic factors for patients infected with SARS-CoV-2, postoperative morbidity and mortality (at 30 and 90 postoperative days). The main objective is to estimate the cumulative incidence of mortality at 30 days after surgery. As secondary objectives, to estimate the cumulative incidence of postoperative complications and to develop a specific postoperative risk propensity model for SARS-CoV-2 infected patients.The protocol  was approved by the local Institutional Review Board . The study findings will be submitted to peer-reviewed journals and presented at relevant national and international scientific meetings.ClinicalTrials.gov Identifier: NCT04479150 . The multicentric design of the study will allow the recording of a large number of operated patients.The specific emergency digestive pathology considered and the recording of clinical conditions and prognostic scores will reduce the heterogeneity of population included in this study.The impact of SARS-CoV-2 viral status, COVID-19-related prognostic factors, the validity of surgical prognostic scores and the specific characteristics of initial pandemic period in Spain (March–June 2020) on postoperative outcomes will be assessed.The control cohort  will represent a proper comparative group.Due to the retrospective design of the study, and the very first contact of Spanish Health System with the COVID-19 at the beginning of the study period, some data may be lost.1–4 As of August 7th, 2020, the pandemic has infected more than 18.9 million people, with a devastating death toll of more than 709,500 worldwide .,5 Spain, with more than 314,300 confirmed cases and more than 28,500 deaths, constitutes one of most severely hit countries .,7 Multiple clinical guidelines, protocols and recommendations have been developed to standardize medical practice and protect both patients and professionals.–11 All health specialties have adopted forceful contingency plans and a profound reorganization of their healthcare activity.,11 Emergency digestive surgery has not been different.–15 During the period of maximum impact of the pandemic, most scheduled surgical interventions were drastically reduced or suspended, operating rooms were converted into critical care units, and highly versatile surgical staff were redistributed to medical services.,16–20 However, urgent and “non-delayable” interventions continued to be performed in patients who were occasionally infected with SARS-CoV-2.,20–23Since early 2020, the rapid spread of the pandemic due to the SARS-CoV-2 virus (coronavirus 2 of severe acute respiratory syndrome) has triggered a complex and profound global sanitary, social, and economic crisis.,24 Several teams have recently described classic emergency surgical pathologies with “atypical” forms of presentation and/or complicated postoperative evolution, differing from the usual “pre-pandemic” scenario.,13,25–28Some publications suggest that surgical procedures accelerate and exacerbate the clinical progression of COVID-19.,30 In the current context of the COVID-19 pandemic, in addition to the preoperative risk factors and the peculiarities inherent in all emergency surgery, other different elements seem to influence the worst postoperative prognosis, even in COVID-19-negative patients.,31 Several factors may be involved:Urgent and emergency digestive interventions, extremely prevalent in our setting, are always associated with a high risk of complications and mortality, compared to elective surgeries.1.patients’ fear of visiting the hospital during the pandemic (and/or home confinement) makes that clinicians face with more advanced surgical pathologies in the moment of consultation;2.“saturation” of the health system, collapse of the emergency departments, and the misinterpretation of septic symptoms in this context can easily add an “extra” delay in diagnosis;3.conservative treatment  may be forced beyond usual local standards, making patients who ultimately need a surgical rescue worse off;4.–23,32–35work overload of supporting services, such as intensive care units and interventional radiology, may increase “failure to rescue” in complicated patients, not just COVID-19-positive patients.Furthermore, we can’t exclude a synergistic negative effect of SARS-CoV-2 infection on the acute disease that prompted surgical consultation.,36 The surgical indications and operative techniques for urgent digestive surgeries performed during the “pandemic” period may differ from the “pre-pandemic” period, as well as the postoperative complication and mortality rates of COVID-19-negative patients during the pandemic period.,23,25,37The COVID-19 pandemic seems to directly or indirectly modify the clinical profile of the urgent surgical pathology treated in a General Surgery Emergency Department .Digestive surgeons ask several questions in this particular epidemiological context:1.,21,26To what extent does COVID-19 infection modify the nature, clinical profile, or postoperative evolution of urgent surgical pathology?;2.,32Does the socio-sanitary context of a pandemic modify the presentation, severity, diagnostic delay, or postoperative evolution of COVID-19-negative patients undergoing emergency surgery?;3.,39Do the usual postoperative risk prediction scales maintain their validity in this period or do they require any kind of adjustment based on other specific prognostic criteria?;4.,25,36,37Is it appropriate to perform emergency surgical interventions with the classic clinical indications or should some acute pathologies be deferred and the surgery be postponed until normal conditions prevail?;5.,36,37,40Are all the surgical techniques and approaches commonly used in emergency digestive surgery equally safe during COVID-19 pandemic period?,41 However, in order to control the confounding factors of the pandemic scenario, cohort studies should provide additional information on the pathology and clinical condition of patients at the time of urgent surgery, for example with prognostic surgical scores. The classic surgical prognostic scales validated for the estimation of postoperative risk  may not be adequate in patients undergoing emergency surgery during the pandemic period.,39,42–45 Therefore, it may be necessary to consider other prognostic factors and design a new and specific prediction model for patients infected with SARS-CoV-2.,47 Currently, we only have extrapolated prognostic scores for the set of COVID-19-positive patients, with little data on the specific subgroup of surgical patients, and even less information on the postoperative evolution after urgent digestive surgery.,22,48–51These and other questions should be answered as soon as possible. The analysis of real data on urgent gastrointestinal surgery in patients operated on during this period could provide the necessary information.It would also be advisable to compare a COVID-19-positive group of patients with a simultaneous COVID-19-negative group (both in the “pandemic period”) and with a “pre-pandemic” control group in the same hospitals. The few studies published to date address these issues superficially, with unadjusted raw data and a low degree of statistical analysis and scientific evidence.,35,52A registry of these characteristics would provide firmer evidence on their clinical profile and would make it possible to adequately estimate patient's postoperative risk.To contribute to the clarification of these doubts, we propose a multicenter, observational study (“COVID-CIR” study), in which multiple digestive surgical teams from different areas of Spain severely affected by the COVID-19 pandemic will participate. Main working hypothesis: infection by COVID-19 increases postoperative 30-day mortality for any cause in patients submitted to emergency/urgent general or gastrointestinal surgery. Likewise, hospital collapse during the first wave of the COVID-19 pandemic increased 30-day mortality for any cause in COVID-19-non-infected patients who required emergency/urgent general or gastrointestinal surgery. Hence, the main objective of this study is to estimate the cumulative incidence of mortality at 30 days after surgery. Secondary objectives are: to estimate the cumulative incidence of postoperative complications and to develop a specific postoperative risk propensity model for COVID-19 infected patients.,41,53The conclusions of this study could contribute to design a future action plan for similar epidemiological situations or subsequent outbreaks of SARS-CoV-2.2This protocol is reported in accordance with the STROBE guideline.2.1,13COVID-CIR is an observational, multicenter, cohort study of patients undergoing emergency/urgent gastrointestinal surgery during the period of maximum impact of the COVID-19 pandemic in Spain .This study was registered in the ClinicalTrials.gov Protocol Registration and Results System .2.1.1https://bellvitgehospital.cat/es/investiga-con-nosotros/ceic/composicion . The level of protection of confidentiality, in terms of protection of personal data as required by Spanish Law (Organic Law on Data Protection 3/2018), is also ensured. The study findings will be submitted to peer-reviewed journals and presented at relevant national and international scientific meetings.The study conforms to the stipulations of the Declaration of Helsinki. The protocol was approved by the local Institutional Review Board  and a waiver for written informed consent was given, as no additional procedures or information will be required from participants beyond that which would normally take place as part of clinical care. The list of local Institutional Review Board members is available at 2.2A total of 30 surgical teams from different Spanish hospitals have been proposed to participate in this study (see Supplementary data file 1), which has been coordinated by the Department of General and Digestive Surgery of the Bellvitge University Hospital . All hospitals participating are from Spain, with different surgical capabilities.2.3Patients operated on for emergency/urgent gastrointestinal surgery constitute the target population. Table Inclusion criteria: ≥18 years, both genders, and operated on for urgent/emergency gastrointestinal pathology.Exclusion criteria: patients who have undergone a scheduled surgical intervention or patients operated on by a service other than General and Digestive Surgery.Two cohorts will be defined:1.,14,17,46Cohort A: consecutive patients who required emergency/urgent gastrointestinal surgery from March 1st to June 30th, 2020 (“first wave” of the COVID-19 pandemic in Spain). This cohort will be stratified by SARS-CoV-2 infection status :COVID-19-positive patients (infected by SARS-CoV-2), andCOVID-19-negative patients (not infected by SARS-CoV-2).2.Cohort B: consecutive patients who required emergency/urgent gastrointestinal surgery from March 1st to June 30th, 2019 (control cohort of a “pre-pandemic” period).,57Figure 2.4The study period has been delimited by the period of the “COVID-19 pandemic”: March 1st to June 30th, 2020. In Spain, this period corresponds to the maximum impact of the pandemic on health services  and society at large .Schedule the study protocol was drawn up in March and April 2020. The IRB  approved the protocol on May 5, 2020 .At the moment of submitting this manuscript, participant recruitment is in progress. The inclusion of patients data does not end until August 31st, 2020. Provided the 30-day follow-up of the last included patient is completed by July– to August 2020, and that 1 month will be required for database cleaning and statistical analysis, it is expected that the first results  will be available in September to October 2020. Complete analysis  will be available in November to December 2020. Table 2.5The primary outcome is the cumulative incidence of mortality for any cause at 30 days after urgent gastrointestinal surgery.Secondary outcomes are:1.Cumulative incidence of all-causes mortality within 90 days after surgery.2.,58Cumulative incidence of any postoperative morbidity within 30 days after surgery: overall postoperative complications and/or complications specifically associated with the evolution of COVID-19 Table .3.,57,59Cumulative incidence of severe 30 day-postoperative complications, >IIIA grade according to the Clavien-Dindo score .6.,39,42–45“Physiological” and “operative severity” to calculate the estimated risks of mortality and postoperative complications according to 5 surgical prognostic scales ?-Was the diagnosis of the surgical pathology delayed?-Was urgent intervention delayed for logistical problems ?-Was a conservative/non-operative treatment “over-indicated”?8.,36,37,40 Did it change? [No/Yes]; if affirmative:Attitude of the surgeons due to the influence of the SARS-CoV-2 pandemic regarding the “surgical technique” used:-Was the surgical technique being “more conservative than usual” or “more aggressive than usual”?2.6All data will be gathered from electronic medical record. All consecutive patients operated on for urgent digestive surgery during the study period will be screened and included if they fulfil the inclusion criteria.,35Demographic characteristics, comorbidities, clinical and analytical variables, SARS-CoV-2 infectious status (pre- and post-operative periods), clinical diagnosis, intraoperative findings and postoperative course  will be recorded (Supplementary data file 2).,45All values necessary for the automatic calculation of the surgical prognostic scores shall also be included Table .,50,60–64Prognostic factors recently described in COVID-19-positive patients will also be recorded: advanced age, hypertension, smoking, diabetes, chronic obstructive pulmonary disease (COPD), cardiovascular history, lymphocyte and platelet count, alanine-aminotransferase (ALT), C-reactive protein (CRP), D-dimer, ferritin, procalcitonin (PCT), lactate-dehydrogenase (LDH), troponin, prothrombin time, “neutrophil-to-lymphocyte ratio” (NLR) and “platelet-to-lymphocyte ratio” (PLR).,35,58Among postoperative complications, those currently considered “characteristics” of COVID-19  will be analyzed in depth., has been created ad hoc for this study (Supplementary data file 3).An electronic Case Report Form (eCRF), based on REDCap platform 2.7Before closing the database for analysis, the data manager and the 2 principal investigators of the study (JO and ZM) will contact the senior local surgeon from each center in order to confirm completeness and accuracy of recorded data. Hospitals which failed to include all eligible patients of both periods will be excluded for analysis to avoid a selection bias. Hospitals failing to include at least 90% of operated patients fulfilling inclusion criteria during the study periods will be excluded for analysis in order to avoid selection bias. Moreover, patients with relevant missing information  will be also excluded.2.8Due to the exploratory nature of our aim, no formal calculation of sample size was performed. It will be defined by the total number of patients who underwent emergency gastrointestinal surgery during the 2 study periods at the participating hospitals. We expect to include more than 2000 patients.2.9Baseline characteristics will be described using standard descriptive statistics, and a descriptive and exploratory comparative analysis between both cohorts will be carried out.The cumulative incidence of mortality  and the incidence of morbidity  at 30 and 90 days will be estimated by positive/negative SARS-CoV-2 infection status (cohort A) and in the control group (cohort B).To minimize the selection bias effect of potential confounders, propensity scores will be estimated using a multinomial model to estimate the probability that subjects were SARS-CoV-2-positive, SARS-CoV-2-negative or control. Subjects will be matched using the propensity scores. After matching, to identify imbalance between groups, the standardized mean difference will be estimated and plotted. In the matched sample, the cumulative incidence of mortality  and the incidence of morbidity  at 30 and 90 days will be compared using a generalized linear model with a binomial distribution and a logarithm link function. Those variables that remain imbalanced between groups after matching would be added to the model with an adjusting purpose.,39,42–45 Their applicability on postoperative morbidity or mortality prediction in COVID-19 pandemic will be assessed.A generalized linear model with a binomial distribution and a logarithm link function will be used to estimate relative risk as a measure to indentify prognostic factors associated with 30 and 90 days mortality and morbidity incidences. Five predictive scores of postoperative morbidity and/or mortality will be calculated according with Table https://www.R-project.org/).Data analysis will be carried out using R version 4.02 or superior  this significant deterioration of the postoperative course described by several groups, especially in elderly patients and/or with associated diseases.,60,61,70 Simultaneously, during the pandemic period, acute surgical pathologies attended in the emergency departments appear in more advanced stages and associate worse postoperative course, even in patients not affected by SARS-CoV-2.–23,32,37In the particular context of urgent surgery, SARS-CoV-2 virus infection appears to contribute to a significant increase in postoperative morbidity and mortality, higher than expected. There is not information about the validity of these surgical predictive scores regarding urgent digestive surgery in COVID-19-positive or COVID-19-negative patients.,39,45 Therefore, it is necessary to establish the predictive capacity of these scales and/or to select other significant prognostic factors in the COVID-19 pandemic period.,41,46,48Risk scales (scores) for postoperative morbidity and/or mortality  are essential for the outcomes of rigorous surgical audits, quality care controls and as a comparative framework  of clinical outcomes.,16,35 At the same time, the COVID-19-negative subgroup of patients in the 2020 cohort will be contrasted with the 2019 control group (“pre-pandemic”), in order to compare mortality rates and detect differences in clinical indications, surgical techniques used, and postoperative evolution between the 2 epidemiological contexts.,33,36,37 The analysis of the data could provide us with useful information on the main predictors of postoperative complications or mortality of COVID-19-positive patients.This protocol has been designed to specifically assess the effect of the COVID-19 pandemic on patients (infected or not by SARS-CoV-2) operated on for some urgent digestive pathology .,25Through an online electronic Case Report Form (eCRF) on REDCap platform,As a secondary objective of the study, we are confident of obtaining sufficient data to develop our own model of postoperative risk prediction, specific to patients infected with SARS-CoV-2 (COVID-19-positive), as a useful short-, medium, and long-term clinical tool for clinical decision-making.This study has some limitation, including its inherent observational nature and the potential loss of perioperative analytical data , especially during the “pre-pandemic” period and the first weeks of the “pandemic period” .,25,41,53The information provided by this study could be a useful and extrapolable tool to surgical teams for urgent surgical decision-making on indications, surgical techniques, and resource management, both in the current pandemic context and in anticipation of future SARS-CoV-2 outbreaks.We thank IDIBELL and CERCA Program/Generalitat de Catalunya for institutional support.COVID-CIR Collaborative Group: The COVID-CIR Collaborative Group : Aurora Aldeano, Granollers General Hospital, Granollers; Eva Alonso, Cruces University Hospital, Bilbao; Martín Amarelo, Basurto University Hospital, Bilbao; Ainhoa Andrés, Donostia University Hospital, San Sebastian; Aitor Ariceta, Hospital Complex of Navarra, Pamplona; Lorena Arrabal, Donostia University Hospital, San Sebastian; Nares Arroyo, Granollers General Hospital, Granollers; Eva Artigau, Girona Dr. Josep Trueta University Hospital, Girona; Elisabet Baena, Bellvitge University Hospital, Barcelona; María Batllé, Granollers General Hospital, Granollers; Albert Caballero, Germans Trias i Pujol University Hospital, Badalona; Miguel Calle, Alto Deba Hospital, Mondragón, San Sebastián; Beatriz Campillo, Sant Joan de Deu Hospital Foundation, Martorell Hospital, Martorell; Andrea Campos, Parc Taulí Health Corporation, Sabadell Hospital, Sabadell; Aleidis Caro, Joan XXIII University Hospital, Tarragona; Ariadna Cidoncha, Parc Taulí Health Corporation, Sabadell Hospital, Sabadell; Arantxa Clavell, Germans Trias i Pujol University Hospital, Badalona; Pere Clos, Mataró Hospital, Maresme Health Consortium, Mataró; Pablo Collera, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Natalia Cornellá, Bellvitge University Hospital, Barcelona; David Luis Coroleu, Barcelona Hospital, Barcelona; Javier Corral, Germans Trias i Pujol University Hospital, Badalona; Rafael Gerardo Díaz-del-Gobbo, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Víctor Echenagusia, Araba University Hospital, Txagorritxu Hospital, Vitoria; Marina Esgueva, Cruces University Hospital, Bilbao; Roser Farré, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Roser Flores, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Miriam Flores, Granollers General Hospital, Granollers; Amador García-Ruiz-de-Gordejuela, Vall d’Hebron University Hospital, Barcelona; Alba García, Donostia University Hospital, San Sebastian; Eulogio Gardeazabal, Bidasoa Hospital, San Sebastián; Nico Garriga, Granollers General Hospital, Granollers; Elisenda Garsot, Germans Trias i Pujol University Hospital, Badalona; Marta Gil, Viladecans Hospital, Viladecans; Carlos Javier Gómez, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Concepción Gómez, Vall d’Hebron University Hospital, Barcelona; Eneko González, Basurto University Hospital, Bilbao; Carmen González, Basurto University Hospital, Bilbao; Claudio Antonio Guariglia, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Rosa Jorba, Joan XXIII University Hospital, Tarragona; Montserat Juvany, Granollers General Hospital, Granollers; Camilo Andrés López, Sant Joan Despí Moisès Broggi Hospital, Sant Joan Despí; Victoria Lucas, Parc Taulí Health Corporation, Sabadell Hospital, Sabadell; Eloy Maldonado, Girona Dr. Josep Trueta University Hospital, Girona; Lidia Martínez, Barcelona Hospital, Barcelona; Rodrigo Medrano, Sant Pau University Hospital, Barcelona; Robert Memba, Joan XXIII University Hospital, Tarragona; Estela Membrilla, Hospital del Mar University Hospital, Barcelona; Nuria Mestres, Arnau de Vilanova University Hospital, Lleida; Laura Millán, Dr. José Molina Orosa Hospital, Lanzarote; Joan Molins, Vic University Hospital, Vic; Alex Morera, Hospital del Mar University Hospital, Barcelona; Anna Muñoz, Parc Taulí Health Corporation, Sabadell Hospital, Sabadell; Ane Murúa, Basurto University Hospital, Bilbao; Esther Nve, Granollers General Hospital, Granollers; Carles Olona, Joan XXIII University Hospital, Tarragona; Jaume Ortega, Arnau de Vilanova University Hospital, Lleida; Alexander Leonel Osorio, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Amalia Pelegrina, Hospital del Mar University Hospital, Barcelona; Silvia Pérez, Arnau de Vilanova University Hospital, Lleida; Noelia Pérez, Mútua de Terrassa University Hospital, Terrassa; María Pintado, Basurto University Hospital, Bilbao; Arantxa Rada, Granollers General Hospital, Granollers; Esther Raga, Sant Joan de Reus University Hospital, Reus; Araceli Rodríguez, Donostia University Hospital, San Sebastian; Patricia Ruiz-de-León, Granollers General Hospital, Granollers; David Ruiz, Terrasa Health Consortium, Terrassa Hospital, Terrassa; Beatriz Sáinz, Hospital Complex of Navarra, Pamplona; David Salazar, Igualada Hospital, Anoia Health Consortium, Igualada; Sergi Sánchez, Igualada Hospital, Anoia Health Consortium, Igualada; Raquel Sánchez, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Lorena Sanchón, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Maite Santamaría, Arnau de Vilanova University Hospital, Lleida; María José Sara, Hospital Complex of Navarra, Pamplona; Aingeru Sarriugarte, Cruces University Hospital, Bilbao; Cristina Soto, Althaia University Hospital, Xarxa Assistencial Universitària de Manresa, Sant Joan de Déu Hospital, Manresa; Jon Ignacio Uriarte, Basurto University Hospital, Bilbao; Marina Vila, Mataró Hospital, Maresme Health Consortium, Mataró; Ibabe Villalabeitia, Cruces University Hospital, Bilbao.Biostatistics Unit : Natalia Pallarés, Judith Peñafiel, Cristian Tebé.Concept and design: ZM, JO, SB. Drafting of the manuscript: ZM, JO . Critical revision of the manuscript: AO, SV. All authors have read and approved the manuscript.Conceptualization: Sebastian Videla, Zoilo Madrazo, Javier Osorio, Sebastiano Biondo.Data curation: Zoilo Madrazo.Funding acquisition: Sebastian Videla, Javier Osorio.Investigation: Javier Osorio.Methodology: Sebastian Videla, Aurema Otero.Supervision: Zoilo Madrazo, Javier Osorio, Sebastiano Biondo.Writing – original draft: Sebastian Videla, Zoilo Madrazo, Javier Osorio, Aurema Otero.Writing – review & editing: Sebastian Videla, Zoilo Madrazo, Javier Osorio, Aurema Otero, Sebastiano Biondo.


APX genes in the Populus trichocarpa genome using bioinformatic methods. Phylogenetic analysis revealed that the PtrAPX proteins were classifiable into three clades and the members of each clade shared similar gene structures and motifs. The PtrAPX genes were distributed on six chromosomes and four segmental-duplicated gene pairs were identified. Promoter cis-elements analysis showed that the majority of PtrAPX genes contained a variety of phytohormone- and abiotic stress-related cis-elements. Tissue-specific expression profiles indicated that the PtrAPX genes primarily function in roots and leaves. Real-time quantitative PCR (RT-qPCR) analysis indicated that PtrAPX transcription was induced in response to drought, salinity, high ammonium concentration, and exogenous abscisic acid treatment. These results provide important information on the phylogenetic relationships and functions of the APX gene family in P. trichocarpa.Ascorbate peroxidase (APX) is a member of class I of the heme-containing peroxidase family. The enzyme plays important roles in scavenging reactive oxygen species for protection against oxidative damage and maintaining normal plant growth and development, as well as in biotic stress responses. In this study, we identified 11  Thus, APX plays a crucial role in redox homeostasis and the scavenging of ROS in plants [On account of their immobility, plants are frequently subject to diverse environmental stresses, including biotic and abiotic stresses, such as insect attack, drought, chilling, and flooding . Exposurn plants ,5.Arabidopsis thaliana, eight members of the AtAPX gene family have been identified, which encode three cytosolic , three peroxisomal , and two chloroplastic (AtsAPX and AttAPX) proteins [AtsAPX binds to both the chloroplast matrix and mitochondria [Oryza sativa), eight APX genes have been identified, including two cytosolic isozymes (OsAPX1 and -2), two peroxisomal isozymes (OsAPX3 and -4), three chloroplastic isozymes , and one mitochondrial isozyme (OsAPX6) [2O2 produced by β-oxidation of fatty acids [OsAPX2 plays important roles in chloroplast protection and H2O2 homeostasis [OsAPX4, which encodes a peroxisomal protein, results in a premature aging phenotype in rice [Arabidopsis [Chenopodium album [Ascorbate peroxidase is a protease encoded by members of a polygenic family. The family is divided into four classes based on the subcellular localization of the protein, namely the cytosol, peroxisomes, chloroplasts, and mitochondria ,6. In Arproteins ,8, of whchondria . In rice(OsAPX6) ,11,12. Tty acids . Cytoplaeostasis . Silenci in rice . The sambidopsis  and Chenum album .Arabidopsis apx1 mutant grows slowly and is sensitive to oxidative stress in comparison with the wild type (ecotype Col-0) [apx1 tapx double mutant of Arabidopsis shows substantial accumulation of anthocyanins and delayed flowering time under light stress [Arabidopsis apx6 mutant [OsAPX4 results in a premature senescence phenotype in leaves of rice [apx2 mutant exhibits semi-dwarf seedlings, yellow-green leaves, and sterile seeds [OsAPX gene in transgenic Arabidopsis elevates salt tolerance [SbpAPX) of Salicornia brachiata in tobacco (Nicotiana tabacum) [APX genes is up-regulated in response to abscisic acid (ABA) treatment in maize (Zea mays) [APX genes in herbaceous plant species, there is a paucity of research on genome-wide identification and expression pattern analysis of the APX gene family in woody plants.Ascorbate peroxidase genes play important roles in plant growth, development, and response to environmental stress. The e Col-0) ,17,18. Tt stress . The ROS6 mutant . Loss-of of rice . The ricle seeds . Expressolerance . Droughttabacum) . The expea mays) . HoweverPopulus is characterized by its ease of vegetative propagation and rapid growth rate, and considerable genomic information is publicly available [Populus trichocarpa is widely used in forest tree molecular breeding and resistance breeding research. The PpAPX gene in Populus tomentosa has been cloned and it has been found that overexpression of PpAPX increases drought and salt tolerance in transgenic tobacco [PtomtAPX and PtosAPX located in mitochondria of Populus tomentosa Carr were identified. PtosAPX is dual targeted to both chloroplast and mitochondria, while PtomtAPX is specifically targeted to mitochondria and plays an important role in maintaining the redox balance [PtrAPX genes and their functions require detailed investigation. In this study, we identified 11 APX genes in the P. trichocarpa genome. We analyzed the chromosomal location, gene structure, phylogenetic relationships, physicochemical properties, and expression patterns of members of the PtrAPX gene family in response to abiotic stress and exogenous ABA treatment. The results provide a foundation for future exploration of the molecular mechanisms of APXs in poplar exposed to environmental stresses.vailable ,27. As a tobacco ,29. Ptom balance . As an iAtAPX genes were downloaded from The Arabidopsis Information Resource database . We used the protein family database Pfam  to search for the hidden Markov model (HMM) profile of the APX gene family (protein family ID: PF06200) based on an expected value  cutoff of 1× 10-5 in HMMER 3.3  to find APX genes in the P. trichocarpa genome and then remove the redundancy with some specifications. All candidate PtrAPX genes were assessed for presence of the conserved Haem-peroxidase domain (IPR002016) using the InterProScan database  and peroxidase (PF00141) in the pfam database . The subcellular localization of the PtrAPX proteins was predicted using WoLF PSORT . Physicochemical properties of the PtrAPX amino acid sequences, such as theoretical isoelectric point, molecular weight, number of amino acids, aliphatic index, and grand average of hydropathicity (GRAVY) index, were estimated using the ProtParam tool of the Expasy  online portal. Transmembrane analysis was conducted using the TMHMM Server v2.0  online database.The amino acid sequences of the eight identified http://gsds.cbi.pku.edu.cn/, accessed on 6 September 2020). The conserved motifs of the PtrAPX proteins were identified using the MEME Suite 5.1.1  online tools.The exon–intron structure of the PtrAPX gene family members was analyzed using the Gene Structure Display Server . A schematic representation of the chromosomal locations was generated using the MapGene2Chromosome v2  online tool. For Ka/Ks calculation, the coding sequences of paralogous gene pairs performed multiple alignment using ClustalW, and then, the values of Ka and Ks were calculated using Synonymous and NonSynonymous Substitutions in DnaSP software (version 6.12.03) [−9 Mya [The chromosomal location of 6.12.03) . The div [−9 Mya .PtrAPX gene family member was downloaded from the Phytozome v12.1 database using the corresponding PtrAPX gene ID. The PlantCARE online database  was used to identify the abiotic stress- and phytohormone-related cis-elements in the PtrAPX promoters. IBS: Illustrator for Biological Sequences software was used to visualize the results.The promoter sequence (2 kb upstream of the initiation codon) of each P. trichocarpa, Arabidopsis, and rice using the neighbor-joining (NJ) method.The PtrAPX protein sequences were aligned using Clustal X 2.0  and Bioehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html, accessed on 12 September 2020) online software. The tertiary structure of PtrAPX proteins was predicted using the SWISS-MODEL database .We predicted the secondary structure of the PtrAPX proteins using the NPSA  using the gene ID [P. trichocarpa to verify the expression level of each PtrAPX gene in roots and leaves using RT-qPCR. The specific primers are listed in Visualized images of the tissue-specific expression pattern for each  gene ID . Then weP. trichocarpa  was cultured in a tissue culture laboratory under a 16/8 h (light/dark) photoperiod with light intensity of 50 µmol photons m−2 s−1 at 23–25 °C. Wild-type P. trichocarpa seedlings were cultured on a woody plant medium  for 3 weeks to use for abiotic stress and phytohormone treatment. For abiotic stress treatment, consistently growing 3-week-old P. trichocarpa were transferred to a WPM supplemented with 7% PEG6000, 150 mM NaCl, or 10 mM (NH4)2SO4 (high ammonium: NH4+). For phytohormone treatment, wild-type P. trichocarpa was transferred to a WPM supplemented with 200 µM ABA. Each abiotic stress and phytohormone treatment was applied for 0, 3, 6, 12, and 24 h, of which the 3 and 6 h time points served as an early response, whereas the 12 and 24 h time points served as a later response. Nontreated seedlings of P. trichocarpa served as the control. Fresh roots and leaves from plants in each stress and phytohormone treatment were sampled at the corresponding time points, immediately frozen in liquid nitrogen, and stored at −80 °C until use. Each sample consisted of three biological replicates.In vivo −△△CT method [UBQ7 gene as an internal reference gene [PtrAPX specific primers for RT-qPCR and UBQ7 gene primers are listed in Total RNA was extracted from different tissues using pBIOZOL Plant Total RNA Extraction Reagent  according to the protocol; then the extracted total RNA was treated with gDNA Eraser to remove genomic DNA as following the reaction system  . Next, total RNA (1 µg) was reverse transcribed using the PrimeScriptTM RT kit  to obtain cDNA as following reaction system . The quantitative real-time PCR used an UltraSYBR Mixture (Low ROX) (CWBIO) with three biological replicates, and the detailed procedure referred to the manufacturer’s instructions. The RT-qPCR reaction was performed by qTOWER 3G Cycler  and the relative expression level analysis was calculated using 2T method ,37. We snce gene ,39. All PtrAPX genes was conducted using Blast2GO v5.2 software. First, we uploaded all protein sequences to Blast2GO and blasted the sequences in the NCBI online database. After mapping and annotation, the GO results and visualized images were downloaded. All procedures were conducted using the default parameters.The gene ontology (GO) annotation of t test was performed using SPSS  software to determine significance. Significance was defined as * p < 0.05, ** p <0.01. The detailed statistical analysis is listed in Student’s APX gene family have been identified previously in the Arabidopsis genome, consisting of AtAPX1–6, AtsAPX , and AttAPX (thylakoid membrane- bound APX) [APX genes in the P. trichocarpa genome, we used the eight AtAPX protein sequences for a BLAST search to detect homologs in the P. trichocarpa genome. Eleven candidate APX gene family members (hereafter PtrAPX) were identified in the P. trichocarpa genome. The 11 PtrAPX genes all possessed the ascorbic acid peroxidase domain (protein ID: IPR002016) as predicted with the InterProScan database. The PtrAPX genes were designated PtrAPX1 to PtrAPX11 based on the chromosomal location. The protein length and molecular weight ranged from 96 to 486 aa and 10,226.73 to 53,657.88 Da, respectively. The aliphatic index ranged from 72.6 to 92.5. The GRAVY score ranged from −0.491 to 0.255. PtrAPX6 possessed the maximum aliphatic index and GRAVY score, and was the only gene to show a positive GRAVY score. Prediction of the subcellular localization of the PtrAPX proteins using WoLF PSORT revealed that PtrAPX2, -3, -6, -7, -9, -10, and -11 were predicted to be cytoplasmic, PtrAPX1 and 8 were predicted to be localized to the chloroplast stroma and chloroplast thylakoid membrane, respectively, and PtrAPX4 and -5 were predicted to be localized to the chloroplast. Details for the PrtAPX genes are presented in Eight members of the und APX) ,8,9. To APX genes, an unrooted NJ tree was constructed derived from Arabidopsis, rice, and P. trichocarpa APX protein sequences. The proteins were resolved into three clades , all of which received strong bootstrap support , which indicated that PtrAPX genes may play a pivotal role in ABA response. PtrAPX8 and 10 contained the P-box (a gibberellin-responsive element). PtrAPX1, 4, 5, 8, and 10 possessed the TCA element . Only PtrAPX11 contained an additional gibberellin-responsive element (TATC-box) and the TGA-element (an auxin-responsive element). Interestingly, eight PtrAPX genes possessed CGTCA and TGACG motifs, which are both involved in methyl jasmonate responsiveness. With regard to abiotic stress-related cis-elements, all but PtrAPX1, 3, 6, 10, and 11 contained TC-rich repeats (cis-acting elements involved in defense and stress responsiveness). These four genes each contained the LTR element (involved in low-temperature responsiveness) and MYB-binding site . Importantly, PtrAPX6 contained three MBS cis-elements in the promoter region, which indicated that PtrAPX6 may participate in the response to drought stress. It was notable that PtrAPX3, 6, and 8 contained all abiotic stress-related cis-elements, which indicated that these three genes may play an important role in abiotic stress response.Promoter reatment . We idenentified . With reProtein is the basic unit of life activities and its structure determines its function. The study of protein structure is helpful to gain a thorough understanding of its function . The secPtrAPX gene family members participated in antioxidant activity, catalytic activity, and binding. The PtrAPX genes participated in a variety of biological processes; for instance, two PtrAPX genes are involved in reproduction, multicellular organismal processes, growth, and developmental and reproductive processes. All PtrAPX genes participated in detoxification, cellular processes, and response to stimulus processes  [PtrAPX1, 4, and 9 exhibited a high relative expression level and PtrAPX6 and 9 showed a low expression level in roots  . The genin roots . Five Ptes, etc. .PtrAPX genes was detected in response to drought and salinity treatment in P. trichocarpa. Under drought stress, eight PtrAPX genes were induced by treatment with 7% PEG6000, and expression of PtrAPX4, 6, and 7 was suppressed at several time points. PtrAPX1 and 9 were significantly upregulated at all time points (p < 0.01). It was notable that PtrAPX9 was gradually upregulated in leaves at successive time points and expression peaked at 24 h, when the gene was up-regulated by approximately 40-fold compared with leaves of nontreated plants. In roots, PtrAPX1, 4, 9, and 11 were induced, whereas PtrAPX2, 6, 8, and 10 were suppressed at all time points. PtrAPX7 was rapidly downregulated at 3, 6, and 12 h, and thereafter returned to the normal expression level. The genes PtrAPX1, 9, and 11 were induced in roots and leaves in response to drought stress .The expression profiles of n leaves . PtrAPX4PtrAPX genes in response to 200 μM ABA treatment was investigated using RT-qPCR analysis. Half of the PtrAPX genes were induced, whereas PtrAPX4 and 5 were significantly suppressed at all time points in leaves , but was rapidly up-regulated by approximately 12-fold at 24 h. PtrAPX1 and 10 were significantly downregulated at 3 h and returned to the normal expression level at 6, 12, and 24 h. PtrAPX2, 6, and 7 were suppressed at all time points in roots. PtrAPX3, 8, and 9 were induced, whereas PtrAPX5, 6, and 7 were suppressed, in roots and leaves under ABA treatment.Expression of the PtrAPX genes, except PtrAPX1, 9, and 10, were suppressed in leaves under NH4+ treatment. PtrAPX9 was dramatically up-regulated at all time points. PtrAPX10 showed no significant change in expression in roots and gradual up-regulation with time in leaves, which peaked at 12 h (approximately four-fold increase) and thereafter declined. PtrAPX2, -4, -7, -8, and -11 were significantly down-regulated at all time points in leaves . PtrAPX2, -3, -4, -5, -7, and -8 were suppressed in roots and leaves  [Gossypium hirsutum) [APX gene family is available for the model woody plant, Populus trichocarpa. In the present study, we identified 11 APX gene family members in the P. trichocarpa genome, each of which contained the conserved ascorbic acid peroxidase domain. The number of PtrAPX genes is notably higher than that reported for Arabidopsis and rice (which both possess eight members). This difference may suggest that APX genes perform more complex functions in woody plants.Ascorbate peroxidase genes play an important role in the detoxification of photoproduced Hn stress . APX genbidopsis ,49, ricebidopsis ,50, tomaersicum) , and cotirsutum) . LimitedPopulus trichocarpa [, In this study, 11 PtrAPX genes have been identified encoding seven cytosolic isoforms, and four chloroplastic isoforms. Among the PtrAPXs genes located in chloroplast, PtrAPX1 and PtrAPX5 are dual-targeted to chloroplasts and/or mitochondria, consistent with the findings that PtosAPX and AtsAPX are dual-targeted to these two organelles [Previous studies have identified seven APX genes in chocarpa , In thisganelles ,30.PtrAPX members of class I contained eight introns, except for PtrAPX6, 7, and 9, which possessed two, six, and six introns, respectively  was detected in the promoter sequence. Thus, we speculate that PtrAPX9 may interact with other drought-related genes in the response to drought stress.It is generally accepted that promoter c stress . In the e points B, but no2O2, in chloroplasts and peroxisomes of photosynthesizing cells [2O2 may cause cellular injury [2O2 to H2O and O2, and scavenges ROS to protect against the toxic effects of ROS accumulation in higher plants [AtAPX1 performs an important function in the drought stress response by protecting chloroplasts from oxidative damage in Arabidopsis. OsAPX2 is induced by salinity stress and transgenic lines of Arabidopsis and Medicago sativa show enhanced salt tolerance [PtrAPX genes was induced by drought and salinity stress, which indicated that these genes play an important role in resistance to drought and salinity stress. PtrAPX6 expression was significantly up-regulated by salinity stress but was down-regulated under drought stress; in contrast, PtrAPX11 was up-regulated under drought stress but was suppressed under salinity stress. These results indicated that PtrAPX genes perform different physiological and biochemical functions in response to environmental stresses. We observed that PtrAPX10 was significantly upregulated in leaves under drought stress at 3, 6, and 12 h, at which point it was up-regulated approximately 25-fold compared with that of the control  [APX6 is down-regulated in response to drought and salinity stimuli, but respond positively to ABA treatment. In the present study, PtrAPX8, which is homologous to ScAPX6, exhibited a similar expression pattern in that expression was suppressed by drought and salinity stress, and was significantly up-regulated by exogenous ABA. The present RT-qPCR data were highly consistent with the aforementioned previous results, thus indicating that homologous genes may perform similar functions in different species.Abscisic acid plays an important role in drought stress . Droughtreatment . The PEGreatment . The precinarum) , APX6 isAPX genes are induced by salt stress [OsAPX2 and 7 [APX mRNA levels may maintain the high activity of APX in the cytosol to protect the cellular components from ROS-induced oxidative damage in response to salinity stress [PtrAPX1 located in chloroplasts and/or mitochondria was stongly induced by high salinity in leaves  and phytohormone treatment (ABA) in relation to promoter cis-elements and RT-qPCR analyses. The present findings provide an improved understanding of the evolution and functions of the APX gene family in P. trichocarpa, and present a framework for further detailed analysis of this gene family.Eleven


Through literature curation and public database integration, here we reported an updated database of phosphorylation sites (p-sites) in prokaryotes (dbPSP 2.0) that contains 19,296 experimentally identified p-sites in 8,586 proteins from 200 prokaryotic organisms, which belong to 12 phyla of two kingdoms, bacteria and archaea. To carefully annotate these phosphoproteins and p-sites, we integrated the knowledge from 88 publicly available resources that covers 9 aspects, namely, taxonomy annotation, genome annotation, function annotation, transcriptional regulation, sequence and structure information, family and domain annotation, interaction, orthologous information and biological pathway. In contrast to version 1.0 (~30 MB), dbPSP 2.0 contains ~9 GB of data, with a 300-fold increased volume. We anticipate that dbPSP 2.0 can serve as a useful data resource for further investigating phosphorylation events in prokaryotes. dbPSP 2.0 is free for all users to access at:  This reversibly dynamic process is precisely modulated by protein kinases (PKs) and protein phosphatases (PPs), which are involved in linking or removing a phosphate group at specific residues of protein substrates5. The first eukaryotic phosphoprotein was discovered in 1883 by Olof Hammarsten, a Swedish biochemist, who detected phosphorous in a secreted protein, casein, from milk6. Although later studies demonstrated that many proteins can be phosphorylated in eukaryotes, it was long debated whether protein phosphorylation also exists in prokaryotes until the discovery of isocitrate dehydrogenase in Escherichia coli, the first identified prokaryotic phosphoprotein, in 19798. In contrast with eukaryotic phosphorylation, which occurs mainly at specific serine (S), threonine (T) and tyrosine (Y) residues of proteins5, prokaryotic protein phosphorylation can occur at additional types of amino acids, such as arginine (R), aspartic acid (D), histidine (H) and cysteine (C)13. Given the importance of phosphorylation in the regulation of protein functions13, the identification of novel phosphorylation sites (p-sites) in proteins is fundamental for understanding the molecular mechanism and regulatory roles of prokaryotic phosphorylation.As one of the most well-characterized and important post-translational modifications (PTMs), protein phosphorylation plays an essential role in almost all signalling pathways and biological processes, from eukaryotes to prokaryotes14, in vitro kinase assay (IKA) to identify potential kinase-specific p-sites15, detection of p-sites in purified proteins with LTP mass spectrometry (LTP-MS)16, and N-terminal sequencing of phosphopeptides (NSP)17. The quality of p-sites identified in LTP studies is higher, because usually multiple assays were performed, and the biological functions of p-sites were also carefully analyzed. Recently, advances in the development of proteomic techniques using high-throughput MS (HTP-MS) have enabled the large-scale phosphoproteomic identification of p-sites in prokaryotic proteins21. For example, Macek et al. conducted phosphoproteomic profiling to detect 54 phosphoserine (pS), 16 phosphothreonine (pT) and 8 phosphotyrosine (pY) residues of 78 proteins in Bacillus subtilis, as well as 81 pS/pT/pY sites of 79 E. coli phosphoproteins19. For arginine phosphorylation, Elsholz et al. systematically identified 121 phosphoarginine (pR) residues in 87 B. subtilis proteins20, whereas Schmidt et al. later quantitatively characterized 134 phosphoproteins with 217 pR sites in B. subtilis21. More recently, Lai et al. detected 159 phosphohistidine (pH) and 69 phosphoaspartic acid (pD) sites of 197 phosphopeptides in nine prokaryotic organisms13. Because an increasing number of LTP and HTP p-site investigations have been reported, the collection, curation, integration and annotation of known phosphoproteins and p-sites in prokaryotes will provide invaluable information for better understanding the host-pathogen interaction and development of antimicrobial agents.Previously, experimental identification of p-sites with conventional biochemical assays was usually labour intensive, time consuming and expensive and was accomplished in a low-throughput (LTP) manner. The LTP methods mainly included site-directed mutagenesis (SDM) of candidate p-sites22. Compared with the second largest resource, the Phosphorylation Site Database, which curated approximately 1,400 prokaryotic p-sites23, dbPSP 1.0 had a > 4-fold greater data volume. At that time, few annotations were provided, except limited information on p-sites. Due to the large number of prokaryotic p-sites found in recent studies, here we created dbPSP 2.0, which contains 19,296 known p-sites in 8,586 proteins from 200 prokaryotic organisms, through literature curation and public database integration  1.0, which contained 7,391 experimentally identified p-sites, including 2,709 pS, 2,174 pT, 2,187 pY, 142 pR, 84 pD, 90 pH and 5 phosphocysteine (pC) sites, in 3,750 phosphoproteins of 96 prokaryotes23, UniProt24, dbPTM 201925, SysPTM 2.026 and PHOSIDA27, and our database contained a much higher number of known phosphoproteins and p-sites in prokaryotes  and annually causes 1.5 million deaths29. Due to the high virulence of M. tuberculosis, two related species including the slow-growing Mycobacterium bovis30 and the fast-growing Mycobacterium smegmatis30 were established as models to study mycobacterial physiology. Additionally, we analyzed the distribution of p-sites on different types of amino acid residues and found that pS, pT and pY sites appear more frequently than other types of residues and occupy proportions of 39.67%, 31.55% and 19.87%, respectively  (https://www.uniprot.org/proteomes/?query=taxonomy:330779). Then the proportion of phosphoproteins against all protein products were counted, and top 10 species with higher coverage values were shown. From the results, we found that the coverage values of the 10 prokaryotes ranged from 8.47% (Staphylococcus aureus) to 36.06%   Fig. . Previou24, whereas corresponding accession numbers were integrated from UniProt24, Ensembl33, EMBL34, KEGG35 and NCBI GenBank36. Moreover, functional descriptions, protein/nucleotide sequences, and keywords were derived from UniProt24 to provide the basic information for each phosphoprotein entry, while the primary references with PMIDs were provided for each p-site. The gene ontology (GO) annotations in the Gene Ontology resource37 were also included if available. Furthermore, the knowledge from 88 additional public resources, such as ChEMBL38, BacDive39, PDB40, IUPred2A41, InterPro42, BioGRID43, EggNOG 5.044 and Reactome45, was integrated to comprehensively annotate the prokaryotic phosphoproteins. These resources covered 9 aspects, namely, taxonomy annotation, genome annotation, function annotation, transcriptional regulation, sequence and structure information, family and domain annotation, interaction, orthologous information and biological pathway , class II (LP ≤ 0.75 and >0.5), class III (LP ≤ 0.5 and ≥0.25), and class IV (LP < 0.25), as previously described46. In most of these HTP studies, different reference databases, distinct search engines and/or diverse parameter configurations were adopted for phosphopeptide detection in different organisms. Thus, the aggregation of false positive identifications might result in a considerable higher FPR value in the cumulative dataset. A re-analysis of all raw MS datasets under a unified platform will generate phosphopeptides with much higher quality, although such an effort is not within the scope of dbPSP 2.0, which directly collected known p-sites from published literature.In phosphoproteomic studies, phosphopeptides were derived from mass spectrometry spectral datasets, usually with a false discovery rate (FDR) of 0.01 at the peptide-spectrum match (PSM), peptide and protein level for quality control. To pinpoint an exact p-site in a phosphopeptide, a localization probability (LP) score could be calculated by a variety of tools, such as MaxQuant47. For each orthologous group, all protein sequences were multi-aligned using MUSCLE48, and a conservation ratio was calculated for the sequences containing the same types of phosphorylatable residues against all sequences in the group. The distribution of the conservation ratio ranged from 0 to 1 was illustrated for all p-sites in the orthologous groups ups Fig. , and we ups Fig. . These hB. subtilis ClpP, an ATP-dependent Clp protease proteolytic subunit, as an example to introduce the usage of dbPSP 2.0. Two browse options, ‘Browse by phyla’ ’ to retrieve a list of phosphoproteins in a tabular format with ‘dbPSP ID’, ‘UniProt Accession’, ‘Gene Name’, ‘Protein Name’ and ‘Organism’  residues most frequently occur at the +1 position, with a lesser extent at the +2 position  in E. coli53. Additionally, Lin et al. utilized p-site information in dbPSP to analyze phosphoproteomic data and dissected the dynamic alteration of phosphorylation in various phosphoproteins during antibiotic treatment and resistance54. Moreover, Hasan et al. adopted pS and pT sites in dbPSP as training datasets and developed a useful tool, Microbial Phosphorylation Site predictor (MPSite), for predicting microbial p-sites55. In addition, the phosphorylation data of representative prokaryotes from dbPSP was utilized for kinase motif enrichment analysis, and the results demonstrated that most eukaryotic phosphorylation motifs could not be recovered in prokaryotes56.After the publication of dbPSP 1.0, it has been visited more than 180,000 times and has served as a highly useful resource for studying prokaryotic phosphorylationIn dbPSP 2.0, we collected and curated newly identified p-sites in prokaryotic phosphoproteins, which could present more complete information on phosphorylation in prokaryotes. Furthermore, dbPSP 2.0 has rich annotations for phosphoproteins and p-sites, which is critical for exploring the function and mechanism of phosphorylation events. In addition, the MSA results of orthologues were provided in this database and will be important for discovering conserved functional p-sites in prokaryote cells. Based on previous studies, dbPSP could work as a well-curated data resource of prokaryotic phosphoproteins to provide helpful support for phosphoproteomic analysis, tool development, and the investigation of prokaryotic phosphorylation events. We anticipate that the updated dbPSP 2.0 could be a comprehensive data resource for better understanding the importance of protein phosphorylation in prokaryotes.57. In 2015, we collected 7,391 known p-sites of 3,750 proteins in 96 prokaryotes from published literature and developed dbPSP 1.022 to contain these datasets. Due to the accumulation of phosphorylation information, here we released dbPSP 2.0 by adding 11,905 new entries to include newly discovered phosphoproteins and p-sites in prokaryotes. Furthermore, the rich annotations derived from 88 public databases were integrated. In total, dbPSP 2.0 contained 19,296 known p-sites in 8,586 phosphoproteins and occupied the size of ~9 GB, with a 300-fold increase compared to that of version 1.0.Protein phosphorylation is one of most well-studied PTMs and is reported to be involved in regulating numerous cellular processes in prokaryotic cells58. Inhibiting the function of ClpP was reported to affect the infectivity and virulence of microbial pathogens59. Moreover, the arginine phosphorylation of ClpP was essential for maintaining its function60. As shown in Fig. B. subtilis protease ClpP is annotated as a serine peptidase and participates in eliminating damaged proteins during heat shock, and its activity can be repressed by CtsR as well as by 20,697 compounds. Meanwhile, ClpP might interact with 9 partners and self-assemble in hexameric ring structures 24 24 Fig. . We alsoely Fig. . These d40 if available. A JavaScript molecular visualization library, 3Dmol.js61, was used to support the dynamic structure chart in the browser interface. In addition, the probabilities of disordered binding regions and disorder propensity values were predicted by using ANCHOR241 and IUPred241, respectively. The details are provided on the phosphoprotein page.The 3D structures of phosphoproteins for intuitive visualization were obtained from the PDBHTML, PHP and JavaScript were applied to develop the web interface as the front-end. The MySQL server was applied to manage the data as the back-end. The backlog and cache data will be cleared regularly, and the dbPSP database will be maintained and optimized continuously.Supplementary Table 1Supplementary Table 2Supplementary Table 3


REG4), a member of the calcium-dependent lectin gene superfamily, is abnormally expressed in various cancers, such as colorectal, gastric, gallbladder, pancreatic, ovarian, prostate, and lung cancer. REG4 is associated with a relatively unfavorable prognosis and clinicopathologic features in cancers, including advanced tumor and nodal stage, histological differentiation, and liver and peritoneal metastasis. Moreover, REG4-positive cancer cells show more frequent resistance to chemoradiotherapy, especially 5-FU-based chemotherapy. REG4 participates in many aspects of carcinogenesis, including cell proliferation, apoptosis, cell cycle, invasion, metastasis, and drug resistance. The underlying mechanisms are complex and involve a series of signaling mediators and multiple pathways. Thus, REG4 may be a potential diagnostic and prognostic biomarker as well as a candidate therapeutic target in cancer patients. In this review, we systematically summarize the advances about the clinical significance, biological functions, and mechanisms underlying REG4 in cancer to provide new directions for future cancer research.Regenerating islet-derived type 4 ( REG) family genes belong to the calcium-dependent lectin (C-type lectin) gene superfamily. There are five REG members expressed in humans: REG1A, REG1B, REG3A, REG3G, and REG4. All of these are located on the second chromosome, except REG4, which is located on chromosome 1  . REG4 isntestine , 6, and ntestine . REG4 mantestine –13. As antestine  and prom manners . The exp manners . The com manners . High exvia the EGFR/Akt/AP-1 pathway  in vitro  and promotes invasiveness and migration in gastric tumor cells . Kuniyasith REG4 . Antibod pathway , 60. Zhoc cancer .REG4 is overexpressed in pancreatic cancer tissues than in adjacent normal tissues at either the mRNA or protein level  (identified as the KS subgroup). REG4 promotes the progression in KRAS mutant lung adenocarcinoma cells progression and can be used as a novel biomarker in lung adenocarcinoma subtype . Anotherin vitro , but not in oral squamous cell carcinomas. The expression of REG4 could be correlated with nodal metastasis, poor prognosis, and pEGFR levels and that cell growth could be inhibited by anti-REG4 treatment in vitro .via activation of Akt  in malignant tumors are commonly identified , 64. Then of Akt .via other mechanisms. Kathryn et al. revealed that REG4 can modulate phosphorylation of multiple additional receptor tyrosine kinases (RTKs), including insulin receptor, insulin-like growth factor receptor, as well as their downstream effectors, EGFR, mitogen-activated protein kinase, and phosphatidylinositol-3-kinase pathways. Knockdown of REG4 affects the ability of insulin and EGF to phosphorylate downstream tyrosine kinase in human colon and prostate cancer cells  by increasing phosphorylation of Ser9 residue. Decreased GSK-3β activity induces an increased nuclear translocation of β-catenin by decreasing its phosphorylation at Ser33/37/Thr41 and sequentially increasing TCF-4 transcriptional activity, which promotes the expression of cyclin D1 and D3 coupled with CDK4 and CDK6. REG4 treatment accelerates G1/S and G2/M phase transition, coupled with increased mitotic index of colorectal cancer cells. The use of REG4 antagonists or Akt inhibitors decreased, while GSK-3β antagonist significantly increased mitotic index and proliferation in colorectal cancer cells  are essential for driving each cell cycle phase. Misregulated CDKs induce unscheduled proliferation and chromosomal and genomic instability . Furtherer cells . These r pathway Figure 1APC) and KRAS synergistically increase tumorigenesis and enhance the induction of colorectal stem cells  encoded protein regulates prostate cancer proliferation and invasion by interacting with a variety of cell surface proteins in prostate cancer  can provide a favorable microenvironment to promote tumor angiogenesis, progression and suppress adaptive immunity –79. Ma end CD163 . TAMs arnd CD163 . EGFR annd CD163 . Furthernd CD163 . Tumor-svia EGFR, comprising EGFR, ADAM17, G protein-coupled receptor 37 (GPR37), TGF-α, REG4, and transcription factor SP1 (REG4 in colon cancer cells can be enhanced by stimulation from transforming growth factor-α (TGF-α), epidermal growth factor (EGF), fibroblast growth factor, and hepatocyte growth factor  family, a group of evolutionarily conserved zinc finger-containing transcription factors, is essential for proliferation, differentiation and development in many organs  and is presented as a stem cell marker .REG4 in human cancers is complex and involves multiple pathways .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.


Azygiahwangtsiyui , a neglected parasite of predatory fishes, is little-known in terms of its molecular epidemiology, population ecology and phylogenetic study. In the present study, the complete mitochondrial genome of A.hwangtsiyui was sequenced and characterized: it is a 13,973 bp circular DNA molecule and encodes 36 genes  as well as two non-coding regions. The A+T content of the A.hwangtsiyui mitogenome is 59.6% and displays a remarkable bias in nucleotide composition with a negative AT skew (–0.437) and a positive GC skew (0.408). Phylogenetic analysis based on concatenated amino acid sequences of twelve protein-coding genes reveals that A.hwangtsiyui is placed in a separate clade, suggesting that it has no close relationship with any other trematode family. This is the first characterization of the A.hwangtsiyui mitogenome, and the first reported mitogenome of the family Azygiidae. These novel datasets of the A.hwangtsiyui mt genome represent a meaningful resource for the development of mitochondrial markers for the identification, diagnostics, taxonomy, homology and phylogenetic relationships of trematodes. Azygia Looss, 1899 is an endoparasitic helminth found in the stomach and intestine of freshwater feral carnivorous fish , which is a parasite of numerous, but especially esocid and percid, fishes in Europe. Many researchers have added to our knowledge of this cosmopolitan species. To date, species of Azygia are frequently reported from the esophagus, stomach, and intestine of a wide range of predatory fishes from Asia, Europe and North America, including China, Japan, India, Russia, Germany, and North America ) as intermediate hosts   .Azygiidae was erected more than a century ago, its situation, and that of several species of Azygia, is still controversial and uncertain. Azygia is the only genus in the family then presenting systematic confusion: Azygialonga  in North America may be a synonym of A.lucii in Europe  of Proterometramacrostoma Horsfall, 1933 (Azygiidae) is currently available in GenBank. None of the mitochondrial genome data have been sequenced for a member of the family Azygiidae. Therefore, we determined the complete mitochondrial genome sequence of A.hwangtsiyui as a basis for the future definition of strain- and species-specific markers, and for assessing mitogenomics in resolving the interrelationships of trematodes.Mitochondrial (mt) genome and nuclear ribosomal DNA sequences are effective molecular tools for taxonomic identification, phylogeny and biogeographical research ) obtained from east Dongting Lake in Yueyang, Hunan province, China . Azygiahwangtsiyui was morphologically identified according to the original and other descriptions , according to the manufacturer’s instructions.The specimens of flatworms were isolated from the stomach of their definitive host, in this case snakehead fish , 1.5 µl of each primer (0.2–1.0 μM), 1 µl EX Taq polymerase , and 2.5 µl DNA template. PCR amplification was compliant to the following amplification protocol: initial denaturation at 98 °C for 2 min, followed by 40 cycles 10 s at 98 °C, 15 s at 50 °C, 68 °C for 1 min/kb, and 10 min at 68 °C for a final extension. The amplified PCR products were purified with TIANgel Purification Kit , and sequenced bidirectionally at Sangon Biotech (Shanghai) Co., Ltd.  based on the primer walking method using several specific primers , HWF3/HWR3 (for nad4), HWF5/HWR5 (for nad1), HWF7/HWR7 (for 16S), HWF9/HWR9 (for 12S), and HWF11/HWR11 (for cox2), respectively. On the basis of these obtained nucleotide sequences, https://www.technelysium.com.au) to remove ambiguity codes and low-quality bases. Whenever the quality was sub-optimal, sequencing was repeated until the amplicon is the consensus sequence. Before manual assembly of the entire mitochondrial genomic sequence, identification of all amplicons was performed by BLASTN check  were predicted with Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/) adopting echinoderm and flatworm mitochondrial codes, and examining the nucleotide alignment against the reference mtDNA in trematode Dicrocoeliumchinense Tang et Tang, 1978 (NC_025279.1). Whole tRNAs were inferred with the detection results of ARWEN .According to sequence chromatograms, all raw fragments were quality-proofed using CHROMAS  (NC_032295.1) and Dibothriocephaluslatus  (NC_008945.1) were included as outgroup taxa representing two different families. Species information including systematic positions and GenBank accession numbers is provided in Suppl. material PCGs from the GenBank files, export fasta files with translated amino acid datasets, and align datasets in bulk using integrated applet MAFFT with normal-alignment mode. Phylogenetic analyses were performed using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. Assessment of the best-fit evolutionary model for dataset was conducted via ModelGenerator v0.8527 (BI in MrBayes version 3.2.6 (7 metropolis-coupled Monte Carlo Markov Chain (MCMC) generations. Two independent runs with four simultaneous MCMC chains (one cold and three heated chains) were conducted for 1 × 107 million generations, sampling every 10,000 generations and discarding the initial 25% generations as burn-in. ML analysis in PHYLOSUITE was performed using MtART+I+G matrix with 1000 bootstrap replicates.For phylogenetic analyses, we utilized translated and concatenated amino acid sequences of twelve protein-coding genes for 49  v0.8527 . BI in Mon 3.2.6  was carrA.hwangtsiyui mtDNA is 13,973 bp in length (GenBank accession number: MN844889) and comprised of 12 protein-coding genes , 22 tRNA genes, two rRNA genes (rrnL and rrnS), and two non-coding regions. The 12 protein-coding gene order arrangement is cox3-cytb-nad4L-nad4-atp6-nad2-nad1-nad3-cox1-cox2-nad6-nad5 , Echinostomahortense Asada, 1926, and some species of the Fasciolidae    ; the genon, 1889 , Hypoderh, 1782) , but sho8 64 bp; . The nucA.hwangtsiyui mtDNA. The full scale of 12 concatenated protein-coding genes was 10126 bp, composed of 45.2% T, 11.5% C, 14.7% A, and 28.6% G. Average A+T content of concatenated 12 protein-coding genes was 59.9%, varying from 57.7% (cox2) to 64.8% (nad4L)  > cox1 (1554 bp) > nad4 (1272 bp) > cytb (1110 bp) > nad1 (906 bp) > nad2 (861 bp) > cox3 (660 bp) > cox2 (582 bp) > atp6 (513 bp) > nad6 (444 bp) > nad3 (360 bp) > nad4L (264 bp). There are two non-coding regions (NCR1 and NCR2) in A.hwangtsiyui mitogenome, while the mt genome of Paragonimusheterotremus Chen et Hsia (1964), C.complanatum, Fascioloidesmagna , and T.szidati have a single non-coding region  forms a hairpin-like secondary structure including a whole set of stems and loops , secondly was GTG (for five PCGs), which resembles that of the most frequent extrapolated start codons for mitogenome protein-encoding genes of digenean species (PCGs). Likewise, the most-used terminal codon was TAG (for nine PCGs), followed by TAA (for two PCGs). Only one of 12 protein-coding sequence (nad5) was terminated with abbreviated T stop codon , and Postharmostomumcommutatum  , and P.westermani Indian isolate (24 tRNAs)  is located between NCR1 and NCR2  . The cod4 tRNAs) . In A.hly Table .P.heterotremus and members of the family Schistosomatidae and Fasciolidae Raillet, 1895, the gene order of the remaining digenea taxa is virtually identical with the exception of the translocation of trnE and trnG among the remaining members of selected digenea representatives in family level. Intriguingly, there is the translocation of trnE and trnG within different species of family Fasciolidae. The translocations of three tRNAs  can be discovered even between taxa of the same subgroup. Gene order of the Brachycladiumgoliath  mt genome  is nearly same as that of P.westermani  except for the relocations of trnY between trnG and cox3, and trnE to the position between trnN and trnP. The groups of Schistosomatidae show a massive gene reorganization of protein-coding genes and tRNAs compared with other sequenced digenea mitogenome, which is in accord with previous finding reported by Comparative analysis of gene arrangement among 47 selected digenean taxa, two gene blocks (cox1-trnT-rrnL-trnC-rrnS-cox2-nad6 and cytb-nad4L-nad4-trnQ) are shared by all selected taxa  for analyzing molecular-based phylogeny. In this study, the topological structure is divided into two large clades: one consists of seven members of the family Schistosomatidae; and the other clade comprises 40 members from 16 families including the family Azygiidae (A.hwangtsiyui)  is identified as the most basal lineage of the Digenea, but separated from C.complanatum , and Cyathocotyleprussica Mühling, 1896 . Phylogenetic analyses of all complete digenea mtDNAs confirmed taxonomic and previous phylogenetic assessments (Azygiidae still awaits investigation of relationships based on a much wider taxon sampling and more mitogenome datasets.To assess phylogenetic relationships among available flatworms, we utilized concatenated amino acid sequence dataset representing 12 protein-coding genes of essments . The int


However, the relationship between P2X7R-mediated cell death and the progression of OA requires investigation. In this review, we correlate potential links between P2X7R, cartilage degradation, and inflammatory factor release in OA. We specifically focus on inflammation, apoptosis, pyroptosis, and autophagy. Lastly, we discuss the therapeutic potential of P2X7R as a potential drug target for OA.Osteoarthritis (OA) is the most common joint disease. With the increasing aging population, the associated socio-economic costs are also increasing. Analgesia and surgery are the primary treatment options in late-stage OA, with drug treatment only possible in early prevention to improve patients’ quality of life. The most important structural component of the joint is cartilage, consisting solely of chondrocytes. Instability in chondrocyte balance results in phenotypic changes and cell death. Therefore, cartilage degradation is a direct consequence of chondrocyte imbalance, resulting in the degradation of the extracellular matrix and the release of pro-inflammatory factors. These factors affect the occurrence and development of OA. The P2X7 receptor (P2X7R) belongs to the purinergic receptor family and is a non-selective cation channel gated by adenosine triphosphate. It mediates Na Osteoarthritis (OA), as an age-related degenerative joint disease, presents with physical pain and disability in patients. Finding effective therapies for OA remains a pertinent health problem . The occThe most important structural part of the joint is the cartilage, and its condition directly affects the occurrence and development of OA. Cartilage consists of structural proteins, such as collagen (primarily type II collagen), non-collagen proteins, proteoglycan, elastin, and aminoglycan, which form a stable network structure providing elasticity and compression resistance to joints. In OA tissues, this network structure loses its integrity with a resulting loss of tensile strength . The comThe P2X7 purinergic receptor (P2X7R) is a trimeric adenosine triphosphate (ATP)-gated cation channel, which is expressed in several eukaryotic cells, such as immune cells and bone cells. As a key inflammatory switch, the activation of P2X7R mediates several downstream reactions, including the release of inflammatory factors, cell proliferation, death, and phenotypic changes . Owing tMost studies on OA focus on the synovium-mediated development of inflammation. Synoviocytes identify the foreign fragments that fall into the joint cavity as byproducts of cartilage degradation. This results in synovial angiogenesis, an increase in the release of auto-inflammatory factors, and the stimulation of chondrocytes to synthesize and secrete MMPs. Through this mechanism, synovitis can promote cartilage degradation and aggravate OA . SynoviaAlthough our understanding of the mechanism underlining OA has improved, limited progress has been made with respect to its treatment. Currently, analgesia and joint replacement are primarily used to treat end-stage OA  which neDrug treatment often goes hand-in-hand with prevention, with commonly used drugs in clinical practice, such as paracetamol (acetaminophen) and non-steroidal anti-inflammatory drugs (NSAIDs) also being able to effectively relieve pain symptoms . FurtherP2RX7 gene) is most closely related to inflammation and immunity, and belongs to the trimeric ligand-gated cation channel. Compared with other P2X receptors, P2X7R requires a higher concentration of ATP for activation and has a higher affinity for the selective agonist BzATP, with 10–30 times more potency than ATP (Purine receptors can be divided into two categories: adenosine (ADO) activated P1 receptors and purine and pyrimidine nucleotides (ATP and ADP) activated P2 receptors. P2 receptors can further be divided into P2X ion channel receptors and P2Y metabotropic receptors. There are seven different members of the P2X family (P2X1-7), of which the P2X7 receptor  and long carboxyl- (C) termini, and two hydrophobic transmembrane fragments separated by glycosylated extracellular ATP binding domains (transmembrane domains); its topology is similar to that of other ionic P2X receptors . The funrization . When thrization .+ concentration, and mechanical stress stimulation, pannexin-1 has high ATP permeability  via ATP signaling, which can also regulate the activity of P2X7 ion channels; the regulation of caspase, cathepsin, and MMP release; and the regulation of the pro-inflammatory mediator prostaglandin E2 (PGE-2). PGE-2 is an important downstream inflammatory pathway of P2X7R, which may make P2X7R a potential anti-inflammatory target to replace cyclo-oxygenase that regulates the activation of transcription factors, such as NF-κB p65, HIF-1α, and PI3K-AKT; glutamate efflux; endocytosis; cell proliferation; and cell death. Briefly, P2X7R regulates ion flow, protease activation, and various secretory responses, which constitute the most common signaling pathways in inflammation  to serve as potential new therapeutics for these diseases.When OA occurs, the most significant cellular response is inflammation, which can be triggered by external and internal mechanisms. External factors include mechanical stress , while the mechanoreceptors (ion channels and integrins) present on the surface of joint cells convert abnormal mechanical stress into activated intracellular signals . AccordiIn daily life, physical exercise causes mechanical stress on joints that can be beneficial. Studies have shown that regular exercise can alleviate low-intensity inflammatory conditions, as are observed in OA, cancer, and cardiovascular disease, and reduce the risks associated with a high-fat diet. P2X7R is one of the factors that determine the level of IL-1β in the plasma after exercise. Furthermore, P2X7R, NF-κB, NLRP3, and caspase-1 levels have been shown to progressively increase between sedentary individuals, trained athletes, and endurance athletes . Post-ex2+ influx and K+ efflux inducing the activation and release of cytokines , hypochlorite ions (OCl–), superoxide anions (O2–), nitric oxide (NO), and hydrogen peroxide (H2O2). ROS is mainly produced through the following pathways: mitochondrial (through oxidative phosphorylation) and non-mitochondrial membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase (XO) pathways , decreased cytokine levels, and the response to intracellular damage  damages mitochondria as part of the apoptotic intrinsic pathway, destroying its DNA integrity and repair ability  exists in the cytoplasm and can recognize PAMPs  or DAMPs  . NLRP3 a+ efflux  and non-classical pathway . The latter induces cell death similar to pyroptosis . The C-t+ efflux . Hypoxia+ efflux  are invo+ efflux . P2X7R a+ efflux .Under normal circumstances, inflammasomes activate the innate immune system, producing IL-1β, IL-18, and other pro-inflammatory cytokines to protect cells from infection and reduce damage . HoweverAs stated before, cartilage degradation in OA can result from disrupted anabolic and catabolic chondrocyte balance . Once in+ efflux, ROS, mitochondrial-, and lysosome dysfunction, is an important approach in the treatment of OA , maintains the chondrocyte phenotype , and further induces hypoxia factors (HIFs)  , which ps (HIFs) . Bcl-2 ps (HIFs) , while Hs (HIFs) , activats (HIFs) , inducess (HIFs) . The ATPs (HIFs) . HIF-1α s (HIFs) . Interess (HIFs) . P2X7R rs (HIFs) , which ls (HIFs) , b. P2X7s (HIFs)  caused bs (HIFs) . P2X7R as (HIFs) .2+ influx, promoted autophagy, and aided in inducing resistence against Mycobacterium tuberculosis in macrophages . Both Nrf2 and autophagy can alleviate pyroptosis and intervertebral disc degeneration  inhibitors targeting P2X7R have been used in phase I and II clinical studies . To dateIn conclusion, there remains a need for more specific OA therapies to be developed. P2X7R of the purinoceptor family, is closely related to inflammation and a promising drug target for OA. However, research into P2X7R and its role in the pathogenesis of OA requires further investigation. For the benefit of patients and scientific progress, we believe that these expectations will be realized in the near future.ZL performed the literature review, drafted the manuscript, and prepared the figure. ZH and LB edited and revised the manuscript. All authors contributed to the article and approved the submitted version.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.