More effective targeted therapy and new combination regimens are needed for Acute myeloid leukemia (AML), owing to the unsatisfactory long-term prognosis of the disease. Here, we investigated the synergistic effect and the mechanism of a histone deacetylase inhibitor, Chidamide in combination with Cladribine, a purine nucleoside antimetabolite analog in the disease.Cell counting kit-8 assays and Chou-Talalay’s combination index were used to examine the synergistic effect of Chidamide and Cladribine on AML cell lines  and primary AML cells. PI and Annexin-V/PI assays were used to detect the cell cycle effect and apoptosis effect, respectively. Global transcriptome analysis, RT-qPCR, c-MYC Knockdown, western blotting, co-immunoprecipitation, and chromatin immunoprecipitation assays were employed to explore the molecule mechanisms.Waf1/Cip1 expression and downregulated CDK2/Cyclin E2 complex, and elevated cleaved caspase-9, caspase-3, and PARP. The combination significantly suppresses the c-MYC expression in AML cells, and c-MYC knockdown significantly increased the sensitivity of U937 cells to the combination compared to single drug control. Moreover, we observed HDAC2 interacts with c-Myc in AML cells, and we further identified that c-Myc binds to the promoter region of RCC1 that also could be suppressed by the combination through c-Myc-dependent. Consistently, a positive correlation of RCC1 with c-MYC was observed in the AML patient cohort. Also, RCC1 and HDAC2 high expression are associated with poor survival in AML patients. Finally, we also observed the combination significantly suppresses cell growth and induces the apoptosis of primary cells in AML patients with AML1-ETO fusion, c-KIT mutation, MLL-AF6 fusion, FLT3-ITD mutation, and in a CMML-BP patient with complex karyotype.The combination of Chidamide with Cladribine showed a significant increase in cell proliferation arrest, the G0/G1 phase arrest, and apoptosis compared to the single drug control in AML cell lines along with upregulated p21Our results demonstrated the synergistic effect of Chidamide with Cladribine on cell growth arrest, cell cycle arrest, and apoptosis in AML and primary cells with genetic defects by targeting HDAC2/c-Myc/RCC1 signaling in AML. Our data provide experimental evidence for the undergoing clinical trial  of Chidamide plus Cladribine as a new potential regimen in AML.The online version contains supplementary material available at 10.1186/s40164-023-00383-5. Acute myeloid leukemia (AML) is a group of heterogeneous malignant hematological diseases with the increased capability of precursor cells’ self-renewal, proliferation and decreased capability of differentiation. The standard induction therapy, daunorubicin for 3 days and cytarabine (Ara-C) for 7 days (3 + 7 regimen), has been used over the past 50 years. However, more than 50% of patients who achieved a complete remission (CR) will eventually relapse . TherefoHistone deacetylase inhibitor (HDACi) is a group of epigenetic modulators, including HDAC isoform-selective inhibitors  and against all types of HDACs  that have been proven to have anti-leukemia effects in AML . ChidamiCladribine  is a purine nucleoside antimetabolite analog that is metabolized by the purine nucleoside salvage pathways. CLA triphosphate, a phosphorylated metabolite of CLA, incorporates into DNA, resulting in single-strand breaks in DNA, and triggers apoptosis, is developed as a treatment for hematological malignancies , 10. CLAIn the present study, we observed the synergistic anti-leukemia effect of CHI combined with CLA by inducing cell growth arrest, cell cycle arrest, and apoptosis in AML cells and identified the underlying mechanisms of the synergy through targeting HDAC2/c-Myc/RCC1 signaling in AML.A Total of 77 bone marrow samples from AML patients and 72 control samples from healthy volunteers were collected from October 2019 to December 2021. A total of 5 AML patients presenting with high leukocyte counts consented to undergo leukapheresis to collect primary cells. Peripheral blood mononuclear cells (PBMC) from 2 healthy donors were extracted to detect the toxicity of drugs. The mononuclear cells were separated using a lymphocytes separation medium  and erythrocytes were lysed with red blood cell lysis buffer . Total RNA was isolated from samples using Trizol as described previously , and theAML cell lines U937, THP-1, and MV4-11 were purchased from the National Collection of Authenticated Cell Cultures . U937 and THP-1 cells were cultured in RPMI 1640. MV4-11 cells were cultured in Iscove’s Modified Dulbecco’s Media . All cell culture media contained 10% FBS, and were cultured at 37 °C in a humidified atmosphere with 5% CO2.CHI (Cat. No. S8567), and CLA (Cat. No. S1199) were purchased from Selleck. CHI and CLA powders were dissolved in dimethyl sulfoxide  at concentrations of 10 mM, respectively for stocking.http://cistrome.org/db/) . There was no statistically different in other clinical characteristics or genetic abnormalities of the patients in the two groups , and relapse-free survival  compared to that with low HDAC2. The same OS results were detected in AML patients from GSE12417-GPL570, and GSE37642-GPL570 datasets  and the RCC1low  expression groups based on the mRNA intensity. The clinical characteristics of the patients in the two groups were summarized in Table high than that in the RCC1low group . A significantly lower CR rate after induction therapy was observed in RCC1high versus those in the RCC1low group . According to 2017 ELA risk stratification [high than that in the RCC1low group , and the expression of RCC1 were also much higher in the adverse cohort compared to the favorable one  and complex karyotype  in RCC1high group and the RCC1 expression was also significantly elevated in the patients with complex karyotypes . The survival curves show that in all patients cohort  and the de novo cohort , patients with high RCC1 were significantly associated with shorter OS compared to that with low RCC1. A similar but nonsignificant trend was also found for RFS . The same OS results were detected in AML patients from the TCGA-LAML, GSE12417-GPL570, and GSE37642-GPL570 datasets  to screen the potential binding motif of c-Myc in the promoter region of RCC1. Two putative binding sites  with different doses of CLA , respectively. Results showed that the combination of CHI with CLA significantly increased apoptosis of primary cells in a dose-dependent manner  on CHI combined with CLA to treat AML patients, especially who are intolerant to intensive chemotherapy.We observed the effect of the combination on the primary cells from AML patients with different genetic abnormalities, MLL-AF6 rearrangement (patient I), AML1-ETO fusion gene co-existed with c-Kit mutation (patient II), R/R AML with FLT3-ITD mutation (Patient III), AML secondary to CMML  with complex karyotype and EZH2, RUNX1and SETBP1 mutations (Patient IV). These genetic defects are an indicator of poor outcomes –23. Our CIP1 [C-Myc is the key target of HDACi in cancer . Since wCIP1 , and to CIP1 . C-Myc, CIP1 . Also, oOur study showed synergistic effects of CHI combined with CLA on cell proliferation arrest, apoptosis, and cell cycle arrest by targeting HDAC2/c-Myc/RCC1 signaling in AML cells. Our results provide the pre-clinical evidence for the ongoing clinical trial  concerning the combined therapy of CHI with CLA in AML patients.Additional file 1: Table S1. Clinical features of 5 AML patients.Additional file 2: Table S2. The ChIP-seq scores of c-Myc binding targets in hematopoietic stem and progenitor cells.Additional file 3: Table S3. The shRNA oligos and primers.Additional file 4: Table S4. The primary antibodies.Additional file 5: Table S5. The half maximal inhibitory concentration of Chidamide in AML cell lines.Additional file 6: Figure S1. The inhibition effects of Chidamide (CHI) alone in MV4-11 cells. (A) The cell proliferation assays in MV4-11 cells were treated by CHI alone with gradient concentration for 24, 48, and 72 h. The statistic histogram (B) and flow cytometry histogram (C) of cell cycle effect, The statistic histogram (D) and flow cytometry scatter plot (E) of apoptosis effect in MV4-11 cells that were treated by CHI alone with gradient concentration for 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 7: Figure S2. The inhibition effects of Chidamide (CHI) alone, Cladribine (CLA) alone, or in combination on PBMC from healthy donors and AML cell lines. The cell proliferation assays in PBMC were treated with low gradient concentration (A) or high gradient concentration (C) CHI and combined with 0.02 μM CLA for 48 h. The cell proliferation assays in PBMC were treated with low gradient concentration (B) or high gradient concentration (D) CLA. The inhibition effects of CLA alone in (E) U937, (F) THP-1, and (G) MV4-11 cells for 48 h. (H) The cell proliferation assays in MV4-11 cells were treated by CHI alone with gradient concentration or CHI combined with 0.01 μM or 0.02 μM CLA for 48 h. (I) The normalized isobologram plot of the combination of CHI and CLA in MV4-11 cells. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 8: Table S6. The combination index value of Chidamide plus Cladribine in AML cells.Additional file 9: Figure S3. The synergistic cell cycle effect of Chidamide (CHI) and Cladribine (CLA) in MV4-11 cells. The flow cytometry histogram (A) and statistic histogram (B) of cell cycle effect; (C) western blot of cyclin E2, CDK2, and p21 Waf1/Cip1 in MV4-11 cells that were treated by CHI alone, CLA alone, and two drugs combination for 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 10: Figure S4. The synergistic apoptosis effect of Chidamide (CHI) and Cladribine (CLA) in MV4-11 cells. The flow cytometry scatters plot (A) and statistic histogram (B) of apoptosis effect; (C) western blot of cleaved caspase-9, cleaved caspase-3, cleaved PARP in MV4-11 cells that were treated by CHI alone, CLA alone, and two drugs combination for 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 11: Figure S5. Enrichment analysis referred to (A) The Hallmark Gene Sets, (B) Canonical Pathways, (C) Chemical and Genetic Perturbations, and (D) Oncogenic Signatures datasets. (E) The expression of c-MYC in MV4-11 cells treated with Chidamide (CHI) alone, Cladribine (CLA) alone, and two drugs combination for 48 h was detected by RT-qPCR. (F) Western blot of c-Myc in MV4-11 cells that were treated by CHI alone, CLA alone, and two drugs combination for 48 h. (G) The expression of c-Myc in AML cell lines. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 12: Figure S6. (A) Co-Immunoprecipitation analysis was for c-Myc, HDAC1, and HDAC2 in MV4-11 cells. (B) The expression of HDAC2 in AML cell lines. The different expression levels of HDACs in AML patients versus normal donors in the GSE13159 (C) and GSE114868 (D) datasets. The survival curve of AML patients was grouped into high- or low- HDAC2 expression and was analyzed in GSE12417-GPL570 (E), and GSE37642- GPL570 (F) datasets. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 13: Table S7. Clinical characteristics and genetic abnormalities of AML patients respect to HDAC2 expression.Additional file 14: Figure S7. The significance of the RCC1 in AML. (A) the Venn plot of the co-expression differential expression genes regulated by two drugs and the potential c-MYC target genes. RCC1 was positively regulated with c-MYC in AML patients analyzed in the TCGA (B), GSE995 (C), and GSE5122 datasets (D). The elevated expression levels of RCC1 in AML patients versus normal donors in the GSE13159 (E) and GSE114868 (F) datasets. The survival curve of AML patients was grouped into high- or low- RCC1 expression and was analyzed in TCGA (G), GSE12417-GPL570 (H), and GSE37642- GPL570 (I) datasets. (J) The potential transcription factor for RCC1 was analyzed by Cistrome online tool (http://cistrome.org/db/). * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 15: Figure S8. (A) The expression of RCC1 in MV4-11 cells treated with Chidamide (CHI) alone, Cladribine (CLA) alone, and two drugs combination for 48 h was detected by RT-qPCR. (B) Western blot of RCC1 in MV4-11 cells that were treated by CHI alone, CLA alone, and two drugs combination for 48 h. (C-G) The inhibition effects of CLA alone in AML primary cells. * p < 0.05, ** p < 0.01, *** p < 0.001.Additional file 16: Figure S9. The scatters plots of apoptosis effect for AML primary cells that were treated by gradient concentration of Chidamide alone, Cladribine alone, and two drugs combination for 48 h.Additional file 17: Figure S10. The mechanism of the synergistic effect of Chidamide and Cladribine in AML.


Although the existence of DCM was established nearly four decades ago, the exact mechanisms underlying this disease pathophysiology is constantly evolving. Furthermore, the complex pathophysiology of DCM is linked with exosomes, which has recently shown to facilitate intercellular (cell-to-cell) communication through biomolecules such as micro RNA (miRNA), proteins, enzymes, cell surface receptors, growth factors, cytokines, and lipids. Inflammatory response and Ca2+ signaling are interrelated and DCM  has been known to adversely affect many of these signaling molecules either qualitatively and/or quantitatively. In this literature review, we have demonstrated that Ca2+ regulators are tightly controlled at different molecular and cellular levels during various biological processes in the heart. Inflammatory mediators, miRNA and exosomes are shown to interact with these regulators, however how these mediators are linked to Ca2+ handling during DCM pathogenesis remains elusive. Thus, further investigations are needed to understand the mechanisms to restore cardiac Ca2+ homeostasis and function, and to serve as potential therapeutic targets in the treatment of DCM.Diabetes mellitus is one of the prime risk factors for cardiovascular complications and is linked with high morbidity and mortality. Diabetic cardiomyopathy (DCM) often manifests as reduced cardiac contractility, myocardial fibrosis, diastolic dysfunction, and chronic heart failure. Inflammation, changes in calcium (Ca In the ventricular myocyte, Ca2+ moves around the sarcoplasmic reticulum, mitochondrial membrane and sarcolemma through different ion channels and ion transporters  and reverse sodium-calcium (Na+/Ca2+) exchanger i levels. Such dynamic regulation is necessary, as Na+/Ca2+ exchanger is one of the major Ca2+ extruder channels, which regulate cardiomyocyte contraction ability and pathologic responses. Though the direct phosphorylation status was not assessed, it was shown that enhanced PKC activity was associated with the decline in Na+/Ca2+ exchanger activity in diabetes i when compared to the Na+/Ca2+ exchanger. Takeda et al. have shown that decreased activity of sarcolemmal Ca2+ pump occurs earlier compared to sarcoplasmic reticulum Ca2+ pump activity and myofibrillar Ca2+ stimulated ATPase activity i overload contributing to detrimental cardiovascular outcomes.The plasma membrane Caactivity . The invactivity . In contal cells . It is p2+. The sarcolemma can bind to the Ca2+ and thus regulate Ca2+ exchanges during EC coupling cycles. In the heart, Ca2+ binding pool of sarcolemma is linked with the residues of sialic acid i and cardiac contractility, as it participates in Ca2+ release, reuptake, and storage during the Ca2+ transits associated with the cardiac contraction-relaxation cycles. Each of these functions are achieved by three-special class of proteins: (1) cytosolic Ca2+ spikers/releasers: sarcoplasmic reticulum Ca2+ release channels (RyR2); (2) Ca2+storage by the luminal Ca2+-binding proteins , sarcalumenin and junctate); and (3) Ca2+ uptakers: SERCA2a pumps for Ca2+ reuptake. As proper Ca2+ storage, release and reuptake are essential for normal cardiac function, any qualitative and quantitative changes in these regulators lead to impaired cardiac function. Several studies have shown that sarcoplasmic reticulum function is adversely affected during diabetes. For instance, sarcoplasmic reticulum-release and uptake activities play a crucial role in regulation of [Ca2+]i, which are decreased in the diabetic heart.The sarcoplasmic reticulum regulates [Ca2+]i has to be reset to low levels to facilitate cardiac dilatation and ventricular filling. SERCA2a, the major isoform that is present in the cardiac tissue, plays a central role in pumping >70% of the sarcoplasmic Ca2+ back to the sarcoplasmic reticulum lumen. SERCA2a is negatively regulated by a peptide known as phospholamban (PLN). The PLN association with SERCA2a is determined by the phosphorylation status, which causes its dissociation from SERCA2a leading to higher transportation rate of Ca2+ through the pump. Classically, β-adrenergic stimulation is involved in enhancing the heart function through the PKA-mediated PLN phosphorylation i overload through Ca2+ leak from RYR2 and reduced cellular efflux i depletion and enhanced PKC phosphorylation i homeostasis in adult cardiomyocytes [2+ release promote myocardial remodeling  resulting in heart failure due to pressure overload. These findings demonstrate that inflammation-triggered Ca2+ imbalance can contribute to cardiac remodeling [2+-release events in both postoperative atrial fibrillation cardiomyocytes and HL-1-cardiomyocytes [2+-sensing receptor (CASR) activates the NLRP3 inflammasome which is mediated by the increase in [Ca2+]i and decrease in cellular cyclic AMP [2+ signaling in Type II diabetes obese (db/db) mice. TNF-α upregulated transient [Ca2+]i amplitude and expedited its decay without changing the sarcoplasmic reticulum Ca2+ load or its spark frequency in mice i sensor i i .Epigenetic mechanisms such as histone modification and DNA methylation regulate gene expression and play an important role in different cellular processes.+/Ca2+ exchanger, which is responsible for Ca2+ flux and efflux in cardiomyocytes i concentration directly upregulates the expression of certain apoptotic genes. miRNA-145 could inhibit the Ca2+ overload and the overexpression of miRNA-145 has a protective effect against the ROS-induced cardiomyocyte apoptosis [2+ homeostasis [2+ signaling in target cells like cardiomyocytes in DCM. However, very little is known about such a possibility. Mayourian et al. [2+ homeostasis [2+ signaling related proteins or transcripts are enriched in exosomes under pathologic conditions. Interestingly, Ca2+ regulates multivesicular body formation and exosome release. For example, increased Munc13-4 (Ca2+-dependent Rab binding protein) is associated with increased Ca2+ uptake, multivesicular body formation and exosome release [2+ signaling could impact exosome biogenesis, release and cellular uptake needs further investigation.Tissue microenvironment, including diabetes-induced effects modulate exosome cargo and has been shown to regulate communication between various cardiac cells  and among the heart and peripheral tissues/organs such as bone marrow, lungs, vasculature, kidney, and immune cells . Our recproteins , 207. Yiproteins  molecularhythmia , 210. Wa SERCA2a . The ovemyocytes . The incpoptosis , 214. mipoptosis , miRNA-1eostasis . Macropheostasis . It is pn et al. , reporteeostasis . Also, i release . Whether2+ handling proteins in the development of hypertrophy and heart failure in DCM, we summarize here some of the novel therapeutic approaches targeting Ca2+ handling proteins in modulating this aberrant expression and activity to improve symptoms in patients with DCM.As this review clearly focusses on the importance of Ca2+ handling proteins, gene therapy targeting RyR2, SERCA2a and PLN is gaining fresh impetus recently, as one of the modalities to treat contractile dysfunction in DCM. S100A1 is a Ca2+ binding protein, which modulates RyR2 and SERCA2a activity [2+ homeostasis [2+ transients and cardiac function in animal models of heart failure [+/K+ ATPase [2+ uptake into the SR [By modulating expression of Caactivity . In animeostasis –223. Likeostasis . In a pieostasis . Followieostasis , 227. Al failure –231. Giv failure –234. The failure , 235. Wi+ ATPase . Also, bo the SR . Animal o the SR , 239. Ado the SR , 241. LaAnother interesting approach is by modifying SERCA2a and PLN using nitroxyl (HNO/NO−). BMS-986231 has undergone extensive clinical testing using HNO derivative. Early clinical trials have demonstrated BMS-986231 to be safe and efficacious in HFrEF patients  Clincal.2+ handling at multiple transports and ion channels located at sarcolemma, sarcoplasmic reticulum, and mitochondria  has been proven to be beneficial in ameliorating DCM. However, to find wide applicability, it is necessary to develop highly selective modulators to correct aberrant cardiomyocyte Ca2+ transits and Ca2+ handling proteins.DCM involves failure of Ca


Adequate mentoring and support of community service nurses (CSNs) in transitioning from the learning environment to the public health setting is pivotal. Despite this notion, the mentoring of CSNs is inconsistently implemented. It was therefore imperative that the researchers developed the guidelines that can be used by managers to mentor the CSNs.This article shares nine guidelines to ensure adequate mentoring of CSNs in public health settings.The study was conducted in public health settings designated for placement of CSNs, in South Africa.n = 27) and CSNs (n = 28). Quantitative data were analysed with Statistical Package for Social Science software version 23, ATLAS.ti 7 software was used to analyse qualitative data.This study followed a convergent parallel mixed-methods design whereby qualitative data were obtained from purposefully selected CSNs and nurse managers. Quantitative data were obtained from 224 CSNs and 174 nurse managers, with the use of mentoring questionnaires. Semi-structured interviews were used on focus groups of nurse managers  creation of a positive mentoring environment, (2) enhancement of collaboration between stakeholders, (3) attributes of CSNs and nurse managers in the mentoring relationship, (4) enhance orientation for nurse managers and CSNs, (5) facilitation of mentor–mentee matching process, (6) conducting mentoring meetings, (7) capacity development for CSNs and nurse managers, (8) monitoring and evaluation of mentoring process, and (9) reflections and feedback.This was the first CSNs’ guidelines to be developed in the public health setting. These guidelines could facilitate adequate mentoring of CSNs. From the South African perspective, nurses who have met the requirements of South African Nursing Council (SANC), Regulation 425 of 22 February 1985, are subsequently registered as a nurse  and Midwife SANC . AccordiAdequate mentoring and support of CSNs in transitioning from the learning environment to the public health setting is pivotal. Notably, the CSN as novices have to adequately adjust to new roles in public health settings characterised by a myriad of responsibilities. Additionally, the CSN as a newly qualified professional is expected to adapt to more complex and advanced responsibilities such as leadership, patient care, administrative and teaching roles which can be cumbersome  and in the Regulations relating to performance of Community Service, published in Government Notice No. 765 of 24 August 2005 SANC . FurtherThe study aimed to (1) describe and explore perceptions of CSNs and nurse managers in order to conceptualise the CSNs’ mentoring programme, (2) develop a mentoring programme for CSNs, and (3) develop and propose guidelines for operationalisation of the CSN mentoring programme. Components of the Kellogg’s Logic Model (KLM) were used in the study  South Africa, which is typically rural. The NWP is divided into four districts with subdivisions of 19 sub-districts, namely – Ngaka Modiri Molema made up of five sub-districts, Bojanala Platinum – five sub-districts, Dr Ruth Segomotsi Mompati – five sub-districts and Dr Kenneth Kaunda – four sub-districts. The sub-districts are also made up of the health facilities: hospitals, community health centres and local clinics. Services rendered at these public health facilities include primary, secondary and tertiary health care. Student nurses are placed at health facilities for experiential learning. On a yearly basis, newly qualified nurses from universities and nursing colleges are placed in the different public health settings to perform compulsory community service for a period of 12 months. There was a dearth of literature on mentoring of the CSNs. As a result, the CSNs were not retained in the NWP public health settings.n = 174) and CSNs (n = 224). In addition, a sample of 28 CSNs and 27 nurse managers were purposefully selected for the qualitative component.The target population of this study were CSNs and nurse managers working in public health settings designated for placement of CSNs. The criteria for CSNs were 6 months to a year in practice. Nurse managers were employed in the public health settings and working with CSNs. The nurse managers were selected as they were supposed to mentor, supervise and write a quarterly report for CSNs. During the quantitative component, simple random sampling was used to select a sample of nurse managers . Unstructured interviews were used in four focus groups for CSNs (n = 28). The private boardrooms of public health settings were used for the interviews. The researcher was responsible for the interviews which took 70 min to 115 min. In order to obtain data regarding mentoring perceptions, nurse managers were specifically asked the following key questions: (1) ‘What is your perceptions regarding mentoring of CSNs?’; (2) ‘In your view, what do you think should entail mentoring of CSNs?’. The CSNs were asked the following question: ‘Please tell me about your experiences regarding your mentoring as CSN’. In order to get in-depth information from participants, probing was adequately done to get more clarity. Interviews were captured using a tape recorder and simultaneous documentation of non-verbal cues was made. Results of both approaches were finally merged and contrasted. Notably, they both complemented each other, with the qualitative component providing more in-depth clarity to mentoring items described by quantitative aspects . Both CSNs and nurse managers’ quantitative data were analysed with Statistical Package for Social Science software version 23  and descriptive statistical methods  and inferential statistical tests  was used to organise, manage and analyse data Friese :4.Both CSNs’ and nurse managers’ quantitative and qualitative results were then merged with an in-depth literature review to develop the mentoring guidelines.Validity and reliability of quantitative data were ensured. Questionnaires were scrutinised by a mentoring expert and a statistician to ensure face, construct and content validity. Questionnaires were piloted on 10 CSNs and 10 nurse managers who did not form part of the whole study population. Internal consistency of questionnaires met acceptable standard with Cronbach’s alpha ranging between 0.74 and 0.83.Lincoln and Guba’s principles of trustworthiness, namely confirmability, credibility and dependability, were applied to enhance the rigour of qualitative data Krefting :220. To The rights of participants were observed throughout the study. Ethical clearance certificate was obtained from North-West University Ethics Committee (Reference number: NWU003-16-14-A9). Permissions were obtained from Department of Health and public health settings where the study was conducted. Informed consent was obtained, and voluntary participation was enhanced. Participants were provided with detailed information about the study and the rights to terminate their participation at any point in time. Anonymity and confidentiality were ensured throughout the study to respect and protect participants from harm. In order to ensure internal confidentiality, the researcher emphasised that participants should not divulge comments made during focus discussions.The converged qualitative and quantitative data obtained from CSNs and nurse managers emerged into three broad themes Nurse managers described public health facilities as lacking characteristics of a mentoring environment. Illustrations of such inadequacies were related to a lack of resources such as time, staff and equipment:‘Other gaps could be issue of shortage of staff … you find that one has to run the unit herself without mentoring.’ ‘A mentor must have time to mentor this mentee because some will have time ….’ n = 148; 85%). They also highlighted that they lacked time to fulfil the mentoring roles .Quantitatively, nurse managers regarded infrastructure and resources as pivotal in mentoring of CSNs ‘I feel like … the best mentors are each and every nursing category … because there are, things that professional nurse does not know, then EN, can help you with.’ Both participants mutually emphasised admirable and acceptable characteristics of CSNs and nurse managers as a mentee and mentor pairs in the mentoring process. In this regard, they unanimously emphasised that nurse managers should have ensuing attributes: knowledgeable, professionalism, role modelling, willingness to mentor and approachable. Both CSNs and nurse managers highlighted that CSNs should have a positive attitude, should be respectful and be willing to learn. All these attributes are bolstered in the following citations:These citations bolstered that nurse managers should be equipped with knowledge:‘She must be knowledgeable; she must have up to date information.’ .‘That is what I should learn from mentor, should be professionalism. The way she behaves should be that of a professional ….’ ‘Positive attitude is … is a necessity for a mentee and for a mentor.’ ‘So, they must be role models for the mentees as well. To learn a certain attitude, the way you treat your patients, the way you treat your mentees.’ The study revealed activities which should be included in the mentoring process of the CSNs.n = 219; 98.0%) and nurse managers  highlighted that orientation should form part of a mentoring process of CSNs. These findings were corroborated by citations from participants:Both CSNs they patch with com serves] [raising her voice].’ ‘That kind of orientation is not going down into books like in Ethos of Professional Practice … What is happening in this province, ba patcha ka ma com serves .’ ‘It’s just that I haven’t get that exposure to go and see how it is like to be in the workshop simple .’ ‘I reported to my supervisor on Monday that I was alone, at least I have tried to manage the ward. He said nothing … That would be better to give me, to give me a remark on that one [‘I do have my notebook I write everything down regarding the mentoring.’ Three main themes that appeared from converged results are given in Concluding reports were consequential from merged findings. These conclusions are illustrated in The aim of Guideline 1 is to ensure that there are enough resources to enhance mentoring of CSNs in public health settings. According to Foolchand and Maritz :7, a shoThe results of the current study and literature described several mechanisms that can be operationalised to enhance collaboration in mentoring of CSNs. Dowling, Powell, and Glendinning :308 defiShaikh :3 arguedThe current study revealed that both CSNs and nurse managers suggested that they need to be thoroughly orientated. Participants reiterated that they should not only be orientated about physical layout, rather they expected a well-structured orientation programme. Literature has also echoed the importance of orientation as an aspect of the mentoring process. Pertiwi and Hariyati :617 repoIt is pivotal that there should be proper mentor and mentee pairing. The objective of Guideline 5 is aimed at ensuring an effective pairing of mentor and mentee in the mentoring of CSNs. Compatibility between the mentee and mentor has been described as crucial in effective mentoring (Cellini et al. It is crucial that mentoring pairs meet regularly in order to realise the aim and objectives of the mentoring programme. Dirks :e14 posiThe objective of Guideline 7 is to provide capacity building for both CSNs and nurse managers in order to ensure adequate mentoring of CSNs. Mubeezi and Gidman :9 revealThe study revealed that the mentoring programme should be monitored and evaluated. Consistently, Diraditsile :78 and DThe aim of Guideline 9 is to ensure that both CSNs and nurse managers participate actively in the provision of feedback to improve the effectiveness of the mentoring process. Feedback is pivotal in enhancing the effectiveness and sustainability of the support strategies system (Shawa & Botma The purpose of the study is to report the nine guidelines developed to ensure adequate mentoring of CSNs in public health settings. Both CSNs and nurse managers suggested several activities that need to be considered in the mentoring of CSNs. Nurse managers and CSNs should be developed through workshops and training. Measures should be put in place to ensure feedback, monitoring and evaluation of mentoring processes.The study was a mixed-methods study which was resource-intensive as it involved two groups of participants, namely the CSNs and nurse managers. As a result, it was difficult at that point in time to implement and evaluate the mentoring programme and operationalise the guidelines because of time and financial constraints. However, future predictions entail the implementation of the mentoring programme with these guidelines.


Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.Calcium (Ca Ca2+ signaling regulates numerous eukaryotic processes, including host defenses against bacterial infections2. Abnormalities in Ca2+ cellular homeostasis may lead to human diseases3 or result from diseases, for example, bacterial infections6. The altered levels of extracellular Ca2+, as exemplified by elevated Ca2+ in nasal and lung liquids of Cystic Fibrosis (CF) patients7, may serve as host factors driving patho-evolution of invading pathogens. Therefore, studying Ca2+ signaling and regulation in human pathogens is of high importance.Calcium ions  at µM level, the organism generates transient increases in [Ca2+cyt] in response to external Ca2+17, and the discovery of Ca2+ channel CalC that is required for generating the [Ca2+cyt] transient spikes controlling Ca2+-dependent regulation of gene expression . However, the molecular mechanisms responsible for recognizing the fluctuations in Ca2+ levels and transducing Ca2+ signals towards cellular processes are not known.Our earlier studies showed that 2+22, the protein was named EfhP and predicted to bind Ca2+. We showed that EfhP plays an important role in Ca2+-regulated production of pyocyanin and alginate, resistance to oxidative stress and P. aeruginosa infectivity in plants17. EF-hand containing proteins belong to one of the largest family of proteins, some of which function as Ca2+ sensors, signal transducers, or modulators. One of the most well studied and versatile EF-hand Ca2+ sensors is calmodulin (CaM). CaM is known to bind Ca2+, which leads to conformational rearrangements that reveal its hydrophobic clefts and allow specific interactions with numerous binding partners. The altered activities of CaM protein targets define cellular responses to fluctuating Ca2+ (reviewed in23). Considering the presence of EF-hand motifs in EfhP amino acid sequence and its role in Ca2+-regulated processes in P. aeruginosa, we predicted that, similarly to CaM, EfhP binds Ca2+, which triggers its conformational changes and enables signal relaying towards the downstream responses. To test this hypothesis, we expressed the recombinant EfhP from E. coli and characterized its binding with Ca2+ and the triggered structural changes by a combination of isothermal titration calorimetry (ITC), fluorospectrophotometry, and nuclear magnetic resonance (NMR) approaches. Through site-directed mutagenesis, we identified the amino acid residues in EfhP that are key to Ca2+ binding. Finally, we studied the roles of EfhP in the Ca2+-regulated production of virulence factor pyocyanin in P. aeruginosa in vitro and virulence of the pathogen in animal model in vivo. The results of this study establish EfhP as a Ca2+ sensor and provide insights into better understanding of Ca2+ signaling in bacteria.We previously identified a putative EF-hand protein encoded by gene PA4107. Based on the presence of two EF-hand motifs known to selectively bind Capseudomonas.com) revealed two canonical EF-hand motifs forming a conserved pattern DxDxDG with identical key residues in the positions 1(+X), 3 (+Y), 5(+Y), 9(−X), and 12 (−Z) (Table 2+ (reviewed in24). Another residue shared by both EfhP EF-hands is glycine (G) at the position 6. This residue is highly-conserved among EF-hand proteins and known for enabling the loops to encompass Ca2+, which is critical for the high binding affinity25. Modeling the 3D structure of EfhP by I-TASSER using CaM as the top threading model (Z score of 1.94) supported the prediction of two canonical helix-loop-helix structures for EF-hand motifs , share up to 58% sequence identity. When searching for homologs among all sequenced genomes of Pseudomonas species using the pseudomonas genome database (www.pseudomonas.com), we found that 3040 genomes carry homologs of EfhP with above 42.6% amino acid sequence identities . Interestingly, 98.5% of these homologs were present in P. aeruginosa. We took a closer look at the remaining 1.5%, most of which belong to Pseudomonas sp., and performed the average nucleotide identity (ANI) analysis to retrieve their species identity. Based on the ANI, all the 26 P. sp. strains and three single strains of P. mendocina, P. otitidis and P. protegens were identified as P. aeruginosa  Table  known toifs Fig. . To evalosa Fig. . These dP. aeruginosa is known to reside in a variety of ecological niches, we hypothesized that conservation of efhP sequence reflects niche-specific adaptations and therefore correlates with the isolation source of the gene-carrying strains. To test this, we retrieved all the available isolation sources for strains carrying full-length efhP homologs and applied multiple sequence analyses followed by clustering with the isolation sources. The analyses revealed that 86.7% of the retrieved homologs belong to strains isolated from clinical sources  and the other 13.2%—from environmental . Among those representing clinical sources, a majority came from CF clinical samples  encode for EfhP. These include 79% of CF isolates, 59% of non-CF clinical isolates, and 62% of environmental isolates and show a greater incidence of EfhP among CF isolates. We also plotted the distribution of amino acid sequence identities between EfhP and its full-length homologs in these groups of isolates  was expressed in E. coli and purified (as described in the Methods). EfhPtr contains 123 amino acids (33 to 155) with a calculated molecular weight of 12.9 kDa  led to significant changes in the 1H-15 N heteronuclear single-quantum correlation (HSQC) spectrum of the protein, indicating structural rearrangements upon Ca2+ binding . The ITC experiments revealed that titrating EfhPtr with Ca2+ . The model of sequential binding fit poorly, whereas the models of one-set of binding sites and two-sets of binding sites resulted in similar fits with a slightly better statistics for the former (data not shown). Thus, we assumed the one-set model and calculated the dissociation constant, Kd of 13.7 µM  in comparison to that of the wild type EfhP µM in cotr Table D, S5D. Tng Table . These d2+-binding would lead to EfhP conformational changes that in turn increase its hydrophobicity. To evaluate the changes in EfhP hydrophobicity in response to binding Ca2+, we used the hydrophobic fluorophore ANS, known to yield increased fluorescence upon interacting with hydrophobic molecules32. The fluorescence of EfhPtr-ANS complex was measured upon titrating with Ca2+, Mg2+, and Sr2+. A concentration-dependent increase in the fluorescence of EfhPtr-ANS complex was detected with increasing concentrations of Ca2+ up to 70 µM , and two genetic complements with either wild type efhP (ΔefhP::efhP) or quadruple mutant efhP whose product was not able to bind Ca2+ (ΔefhP::efhP_q). Pyocyanin was extracted from the cultures grown for 24 h on BMM plates with and without 5 mM Ca2+  possesses the key features of a Ca2+ sensor: it specifically binds Ca2+, undergoes Ca2+-dependent structural rearrangements which increase its hydrophobicity, and it transduces the Ca2+ signal towards cellular processes. The latter is illustrated by the observation that EfhP mediates Ca2+ regulation of virulence factor pyocyanin production in vitro, which requires its ability to bind Ca2+. We also show that EfhP plays an important role in P. aeruginosa survival and virulence in invertebrate animal model in vivo.According to our previous studiesPseudomonas aeruginosa strains. This opens an intriguing question about the evolutionary pressure that secures this protein in the genus. To gain additional insights, we related the sequence similarity between efhP homologs with the sources of the strains’ isolation. The analyses suggested that fewer mutations occur within the homologs encoded in CF isolates, and more frequent mutations correlate with environmental isolates. Although the results may be skewed due to disproportionate numbers of sequences available for clinical and environmental isolates, the observation supports the role of the protein in P. aeruginosa virulence. The higher incidence and conservation of EfhP among CF isolates likely reflects pathoadaptation of the bacterium as an evolutionary driver of EfhP conservation. The fact that P. aeruginosa genome contains no homologs of EfhP signifies the role of this protein and possibly contributes to its evolutionary conservation.Bioinformatic analyses suggested that full-length EfhP is mostly prevalent among 2+ sensor, it must first selectively bind Ca2+ ion(s). ITC showed that EfhP binds Ca2+ with a Kd of 13.7 µM. This value lies within the Kd values of other EF hand Ca2+ sensors varying in the nM to µM range36. Despite some similarities between Ca2+ and Mg2+ ions and the ability of some EF-hand motifs to bind Mg2+, the interactions between EF-hand proteins and these ions differ and lead to different functional consequences37. Based on their interactions with Mg2+, EF-hand proteins can be grouped into Ca2+-specific and Ca2+/Mg2+ -binding. According to the ITC data, EfhP showed no binding to Mg2+ (and Sr2+), likely due to stereochemical constraints (reviewed in37), indicating it belongs to Ca2+-specific EF-hand proteins. Further, according to ITC and ANS fluorescence spectroscopy, replacing the D residue at position +X in the first EF-hand motif significantly impairs the capability of EfhP for binding Ca2+. This EF-hand residue is highly conserved among EF-hand proteins38 and its replacement was shown to inhibit Ca2+ binding in CaM39. When both D and E residues at positions +X and −Z were replaced within each or both EF-hands, no Ca2+ binding was observed. The E12 (-Z) is also highly conserved and supplies two of the seven coordinating oxygens within the EF hand Ca2+-binding loop41. Overall, our data support the importance of D1(+X) and E12(−Z) residues for Ca2+ binding of EfhP and suggest that both EF-hands are needed to enable EfhP binding the ion. This indicates cooperative Ca2+ binding. Positive cooperativity is a key functional attribute of EF-hand proteins and is particularly important for Ca2+ sensors, where it provides the ability to detect the small changes in Ca2+ concentrations constituting Ca2+ signals43.In order for a protein to serve as a Ca2+ sensor is the ability to transduce Ca2+ signals most commonly via Ca2+-triggered conformational changes which enable the selective binding of protein partners. This phenomena is well exemplified by CaM, which undergoes major conformational transitions upon binding Ca2+45 but not other ions46. These conformational changes expose hydrophobic surfaces of CaM and enable selective binding to a large number of diverse protein targets48. In contrast, EF-hand proteins that serve as Ca2+ buffers, for example, calbindin, bind Ca2+ without significant changes in their conformation50. Here we applied NMR spectroscopy and ANS-based fluorospectrophotometry which showed that the addition of Ca2+ triggers a significant change in the EfhP structure and hydrophobicity, respectively. The NMR-detected chemical shifts clearly illustrated binding Ca2+, which likely lead to not only local but also global rearrangements in the protein structure. Future research will focus on determining the residues affected by Ca2+ binding and building three-dimensional models of both the unbound and bound forms of EfhP, so that the more detailed mechanisms of binding can be determined. Overall, the observed Ca2+-dependent structural rearrangements in EfhP and the Ca2+-dependent increase in its hydrophobicity strongly support the ability of the protein to recognize and transduce Ca2+ signals.The second requirement for a functional Ca51), EfhP likely resides in the periplasm. Based on the E. coli study52, the periplasmic concentration of Ca2+ reflects or exceeds its extracellular levels. Considering the micromolar Kd for Ca2+, we predict that EfhP plays a particularly significant role in Ca2+ signaling in P. aeruginosa when the organism resides in the environments with micromolar levels of Ca2+. Examples of such environments include those within the host mucus or within P. aeruginosa biofilms. The low levels of Ca2+ in these environments are due to the presence of negatively charged polymers, such as F-actin, DNA, and alginate55, that chelate Ca2+ ions. These environments are clinically relevant and prevalent within the lungs of CF patients, where the mucus levels are particularly high56 and alginate-overproducing P. aeruginosa strains are predominant57. This prediction is supported by the previously observed more pronounced role of EfhP in the virulence of a mucoid FRD1 strain of P. aeruginosa51. Another example of low Ca2+ environment is within macrophages and neutrophils, which play an essential role in host defense against bacterial pathogens. The level of phagosomal Ca2+ decreases during phagocytosis from the low millimolar extracellular level to low micromolar level58. Here we show that EfhP plays role in PAO1 survival during phagocytosis, further supporting the idea of the protein’s importance for Ca2+ regulation at micromolar levels of the ion.According to the updated SignalP 5.0 prediction of the signal peptide (in contrast to the reported earlier transmembrane domain59) and a few have been proposed to function as Ca2+ sensors (reviewed in60). Although some of these proteins were shown to play important roles in virulence and host–pathogen interactions, their Ca2+ binding and the mechanisms of action were not studied in detail (Table M. tuberculosis shown to activate Nicotinamide Adenine Dinucleotide (NAD) kinase and phosphodiesterase (PDE)61 plays a role in M. tuberculosis growth and survival in macrophages63. Two CaM-like proteins in Bacillus subtilis include CALP, which stimulates PDE and NAD kinase in a Ca2+-dependent manner64 and YtaF, suggested to activate Ca2+ dependent proteins required for sporulation65. Ca2+ binding was quantitatively characterized only in one of the reported bacterial EF-hand proteins, SdrD from Staphylococcus aureus67. This Serine-Aspartate-Repeat (SD repeat) domain protein contains five EF hands within the B1-B5 domains that are responsible for binding Ca2+. The binding triggers a conformational change and stabilizes the protein, which enables its interaction with host cells and promotes adhesion72. SdrD was also shown to contribute to virulence and inhibition of innate immune response73, but mostly through its function as a Sdr family adhesin. To our knowledge, here we present the first detailed report of Ca2+-binding properties in a bacterial EF-hand Ca2+ sensor, the only EF-hand Ca2+ sensor in P. aeruginosa.In bacteria, a number of EF hand-proteins have been reported 16, which contained (per liter): 9.0 mM sodium glutamate, 50 mM glycerol, 0.02 mM MgSO4, 0.15 mM NaH2PO4, 0.34 mM K2HPO4, and 145 mM NaCl, 200 µl trace metals, 1 ml vitamin solution. Trace metal solution (per liter of 0.83 M HCl): 5.0 g CuSO4.5H2O, 5.0 g ZnSO4.7H2O, 5.0 g FeSO4.7H2O, 2.0 g MnCl2.4H2O. Vitamins solution (per liter): 0.5 g thiamine, 1 mg biotin (Gold bio). The pH of the medium was adjusted to 7.0. When needed, 5 mM CaCl2.2H2O (Sigma) was added. PAO1 was used to obtain genomic DNA for cloning efhP. For DNA manipulations, E. coli and P. aeruginosa cultures were grown in Luria–Bertani (LB) broth  at 37 °C with shaking at 200 rpm. Antibiotics used for E. coli were (per ml) 50 μg kanamycin (Kan) and 100 μg ampicillin (Amp) and for P. aeruginosa, (per ml) 60 μg tetracycline (Tet), and 100 µg carbenicillin (Carb).The bacterial strains and plasmids used in this study are listed in supplementary Table No live vertebrate animals were used in this study.www.pseudomonas.com and submitted to I-TASSER for 3D structure prediction77. I-TASSER predicts 3D structure using Protein Data Bank templates and the multiple threading approach LOMETS. PyMol v.1.8.6.078 was used to visualize and highlight the structural features. To test the sequence conservation of efhP among Pseudomonas species, Basic Local Alignment Search Tools, BLASTN and BLASTP79 were used in non-redundant NCBI80 and Pseudomonas.com databases81. Multiple sequence alignments were performed by using MEGA v.7.082. To study potential correlations between sequence conservation and the isolation sources, an in-house python script was generated by Eric King83 and used to extract the isolation source data for each sequence from public databases.The amino acid sequence of EfhP (PA4107) was obtained from the efhP) excluding the 5′ 96 nucleotides encoding the signal peptide was PCR amplified from the P. aeruginosa PAO1 genomic DNA using High Fidelity Phusion polymerase (NEB) and primers efhP_om_F and efhP_R, which contain NdeI and BamHI recognition sequences . Successful transformants were verified by PCR using efhP-specific primers (Table E. coli Tuner BL21 (DE3) resulting in EcoREN strain that was used for production of the 6 × His fusion protein. EcoREN was grown at 37 °C in LB supplemented with Kan until the OD600 reached 0.6 and induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After 3 h, the cells were harvested by centrifugation at 14,650 g for 30 min and stored at − 20 °C. For lysis, the frozen pellets were thawed on ice, resuspended in 50 mL Buffer A . Lysozyme was added to the cell lysate at 0.5 mg/mL. The cells were incubated on ice for 40 min and sonicated in a Fisher ultrasonic processor XL 2010 for a total of 100 s in cycles of 20 s on and 20 s off at power setting of 5 followed by centrifugation at 14,650 g for 30 min at 4 °C. The supernatant was collected and incubated with 4 ml of HisPur Nickel Nitrilotriaceitic acid resin (Ni–NTA) , which was pre-equilibrated with buffer A in a 50 ml falcon tube on a rocker for 1 h at 4 °C. The Ni–NTA resin was subsequently transferred into a 25 ml gravity flow column and washed with 100 mL of ice-cold Buffer A. The protein was eluted by using 20 ml buffer B . The eluates were collected in fractions of 1.5 ml in microcentrifuge tubes and analyzed by SDS-PAGE to confirm the presence of the purified protein of the correct molecular weight (MW). To cleave the N-terminal 6X His-tag, the eluates containing the protein were pooled and treated with recombinant Tobacco Etch Virus (TEV) protease at 1:40 (TEV: protein sample) molar ratio. The 38 ml mixture was transferred into a dialysis membrane with 12–14 kDa MW cutoff and subjected to dialysis against 1 L buffer  at 4 °C overnight. The non-tagged protein was further purified by a second subtracting Ni–NTA column as described in86. Briefly, the dialyzed protein mixture was loaded into a pre-equilibrated Ni–NTA column, and the flow-through was collected as the non-tagged protein. Purified EfhP protein was concentrated by using a spin-column with 10 K MW cut-off (Millipore) to 5–10 mg/ml, aliquoted, and flash frozen in liquid nitrogen for storing at − 80 °C as described in87. The mutant EfhP proteins were purified following the same procedure.The PAO1 genomic DNA was isolated by using the Wizard Genomic DNA Purification Kit (Promega). A 372 bp region of PA4107  was carried out using AKTA PURE protein purification system . For purification purposes, a 120 ml HiLoad column was used, whereas a 24 ml Superdex200 column was used for analytical purposes. Prior to analyses, the columns were equilibrated with 20 mM HEPES, 100 mM NaCl, pH 7.8. The injection volumes on Superdex200 and Hiload columns were 500 µl and 5 ml, respectively. The MWs of the proteins in the eluted fractions were calculated based on the retention volumes and the calibration curve generated by using the Gel Filtration standards (Bio-Rad).2+ in the protein samples was determined by using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) at the Center of Applied Isotope Studies, University of Georgia, Atlanta. The accurate amount of EfhP in the sample was measured by using amino acid hydrolysis at the Protein Chemistry Laboratory at Texas A and M University. Then the molar ratio of protein to Ca2+ was determined. To remove the residual Ca2+, the proteins were decalcified by using a Chelex-100 resin (Sigma) column as described in88. Briefly, the protein samples were mixed with equal volume of regenerated Chelex-100 resin equilibrated with buffer solution and incubated at 4 °C for 30 min. The remaining Ca2+ concentration was measured by BAPTA absorption assay as described in88 and was below 0.8 µM.The residual level of Ca2, MgCl2, and SrCl2 were prepared in the HEPES buffer at 1 mM. Prior to titration, both the protein sample and ligand solutions were filtered through 0.2 µM nylon membrane filter (VWR) by centrifugation for 1 min at 4 0C at 15,600 g. A total of 1 ml or 300 µL of purified protein (0.1 M or 50 µM) was loaded into the sample well pre-cleaned with nanopure water using a vacuum pump. Each titration experiment consisted of a total of twenty injections of 10 µl aliquots of ligand solutions with 300 s intervals with Nano ITC III or 18 injections of 2 µl aliquots of ligand solutions with 150 s intervals with MicroCal PEAQ-ITC. After each titration, the isotherm of the decalcified HEPES buffer titrated with the corresponding ligand was subtracted from the protein sample isotherm as the blank. The resulting binding isotherms were fitted by using Bindworks software algorithms followed by modeling for independent binding sites, from which the dissociation constant (Kd) was calculated.ITC measurements were performed by using a Nano-Isothermal Titration Calorimeter III  or MicroCal PEAQ-ITC . His-tag cleaved protein samples collected from SEC were pooled, dialyzed in HEPES buffer , and adjusted to a concentration of 0.1 M. Solutions of CaCl89 using a Horiba Jobin Yvon (HJY) Fluoromax 3 spectrofluorimeter (JOBIN YVON-SPEX). The effect of Ca2+ and Mg2+ on EfhP hydrophobicity was determined by titrating protein solution with 1 M CaCl2 or MgCl2 solutions prepared in HEPES buffer. A total of ten 10 µl aliquots of the ligand solutions were added reaching a final concentration of 10–100 µM. Spectra of HEPES buffer alone and 50 µM ANS in HEPES were collected and subtracted from the EfhP/ANS spectral data, and the differences were plotted at the corresponding ion concentrations. All the measurements were performed three times.To monitor changes in protein surface hydrophobicity, 8-anilino-1-naphthalenesulfonic acid (ANS) (Molecular Probes) was used. In a 1 ml quartz cuvette, 50 µl of 10 µM protein was mixed with 950 µl of 50 µM ANS followed by acquiring an emission spectrum (excitation at 350 nm)1H/13C/15 N TXI high resolution solution NMR probe in a Bruker DMX 500 MHz spectrometer (www.bruker-biospin.com). The pulse sequence, hsqcetgpsi2 (www.bruker-biospin.com) was optimized and calibrated using the water peak chemical shift, 4.7 ppm as a reference. The 1H–15 N HSQC experiment with 4092 t2 points and 256 t1 points was run as described in90. The data were processed using nmrPipe91 and visualized in NMRFAM-Sparky . The 15 N-labelled 6xHis-tagged proteins were purified as described above and prepared by adding 60 µl of deuterium oxide to 540 µl of 297 µM protein solution. After the initial 1H–15 N heteronuclear single quantum coherence (HSQC) spectrum was acquired a titration with CaCl2 was performed. Aliquots of CaCl2 were added to yield final concentrations of 0.03, 0.15, 0.3, 0.6, and 1.5 mM.NMR experiments were performed at 25 °C using a triple resonance 2+-bound and decalcified protein samples were collected with a protein concentration of 25–100 μM. CD spectra of buffer (15 mM Phosphate or 10 mM HEPES at pH 6.5) at the corresponding Ca2+ levels (as was added to the protein sample) were collected as control. 150 mM CaCl2 stock solutions were prepared in the same buffers as in protein samples. During data acquisition, the data were averaged over 5 scans with a response time of 4 s and with a scan speed of 50 nm/min. All spectra were corrected by subtracting the blank spectra (buffer containing appropriate amount of Ca2+). CD ellipticity values were converted to normalized values (mean molar ellipticity per residue) by standard method according to the manufacturer’s manual. Every experiment was repeated for consistency using freshly purified protein samples.CD experiments were performed on a Jasco J-810 automatic recording spectropolarimeter using 0.05-cm quartz cell cuvette at room temperature. The far-UV (200–250 nm) CD data of Ca2+ binding in EfhP, point mutations were generated and the mutated efhP variants were cloned into expression and complementation vectors. The single mutant of EfhP with the D1 (position X) of the 1st EF hand replaced to N was generated by using an inverse PCR with DreamTaq Green PCR Master Mix and primers with introduced point mutation and expression vector pDAV  was cloned into XbaI-digested vector pUC19. A series of consecutive point mutations replaced the D1 (position X) and the E12 (position -Z) amino acids of each EF-hand with N and Q, respectively  or quadruple (D88N/E99Q/D115N/E126Q) mutated full-length efhP were cloned into the complementing pMF470. The resulting plasmids, pASK101, pASK102 and pASK106 were introduced into the ΔefhP mutant strain PAO1043 by electroporation. The successful transformants were selected by growth on LB agar plates with 300 µg/ml carbenicillin and verified by PCR with gene specific primers . The remaining 3.5 ml of cell suspensions were transferred into a 10 ml separatory funnel and mixed with 3.5 ml of chloroform. The mixture was shaken at 200 rpm on a platform at room temperature for 30 min. Following this, the funnels were let to stand in an upright position for 10–15 min until the clear light blue layer was formed at the bottom, which was collected into 20 ml scintillation vials. The chloroform extraction was repeated two more times and the blue extracts were combined, mixed with 3.5 ml of 0.2 N HCl, and shaken at 200 rpm for 30 min. The vials were left to stand in an upright position until a reddish upper layer of pyocyanin was formed. From this layer, 200 µl of the extracts were transferred into a clear-bottom 96-well plate, and absorbance was measured at 520 nm by Synergy Mx 2 Multi-Mode plate reader with Gen5 2.05 PC software (BioTek Instruments). The values were multiplied by an extinction coefficient of 17.193, and pyocyanin production in µg/mg of total cellular protein was calculated.To assess the role of EfhP and its ability to bind CaP. aeruginosa survival during phagocytosis, murine macrophages were infected with wild type PAO1 and ΔefhP cultures. J774.1 macrophages (ATCC) were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% v/v heat-inactivated fetal bovine serum  and 1% w/v penicillin G sodium salt and streptomycin sulfate (Life Technologies) at 37 °C with 5% CO2. For infection assays, cells were seeded at a concentration of 5 × 105 cells/well in 24 well plates in DMEM with 10% FBS. P. aeruginosa was grown overnight in lysogeny broth at 37 °C under constant shaking until reaching OD600 of 1.0. Bacteria were then washed with phosphate buffered saline (PBS), diluted 1:10 in DMEM supplemented with 10% FBS at a concentration of 5 × 108 CFU/ml, and added to the cells. Plates were centrifuged for 5 min at 1200 rpm and incubated at 37 °C with 5% CO2. Cells were incubated with bacteria for 15 min before being washed three times with PBS. To quantify initial adherence and uptake, cells were lysed with distilled water. Cell lysates were then serially diluted and plated on Pseudomonas Isolation Agar (PIA) to quantify viable bacteria. To quantify bacterial survival after initial uptake, cells were incubated with DMEM with 10% FBS and 100 µg/ml gentamycin sulfate (Fisher Scientific) for 90 min to kill extracellular bacteria before being washed three times with PBS and lysed with distilled water. Cell lysates were serially diluted and plated on PIA to quantify viable bacteria. Each experiment contained three technical replicates and was repeated three times on three different days.To evaluate the role of EfhP in P. aeruginosa virulence, Galleria mellonella infection model was used as described in94 with modifications. This invertebrate model has been established for studying bacterial pathogenesis94. Active G. mellonella larvae (Speedy Worms or American Cricket Ranch) of 2–3 cm length, stored in the dark at 4 °C without feeding for no more than 48 h were chosen for injections. P. aeruginosa, PAO1, ΔefhP, and ΔefhP::efhP strains were grown in BMM with 5 mM or no added Ca2+ for 12 h (mid log). Cultures were serially diluted to achieve two-five colony forming units (CFU) per injection. To verify the injected infection dose, the remaining cultures prepared for injection were plated on Luria–Bertani (LB) agar plates for CFU count. After the gentle rinse with 70% ethanol followed by 1 mg/mL rifampicin, ten larvae were injected with 5 μL of normalized cell suspension grown at 0 mM or 5 mM Ca2+. In addition, five larvae were injected with 5 μL of PBS with or without 5 mM CaCl2 to be used as negative controls. The larvae were incubated at 37 °C and tested for death at 24 h, which was selected to exclude the effect of cocooning that begins soon after. Death for each larva was reported when no movement was observed in response to turning over with a sterile toothpick. A total of 50 larvae were used for each experiment, which was repeated five times. The survival rates were averaged between five independent experiments and statistically analyzed by using SPSS  software, IBM, SPSS Statistics for Macintosh, version 28.0.1.0 (142), IBM Corp., Armonk, N.Y., USA.To assess the role of EfhP in Supplementary Information.


The aim of this study was to evaluate the interradicular septum bone morphometric characteristics using cone beam computed tomography (CBCT) images, as well as to establish quantitative shortcuts to allow clinicians to make a faster and more reliable plan for immediate implant placement in the maxillary molars area. This retrospective quantitative study was conducted on CBCT images obtained from 100 patients. The morphometric analysis of the maxillary molars region was based on the parameters obtained on the sagittal and axial slices. The analysis performed on sagittal slices showed that the first maxillary molars had a wider interradicular septum when compared to the second molars, but the septum height in the first molars was significantly below the height in the second maxillary molars. The axial CBCT slices analysis showed that both interradicular septum perimeter and surface area were significantly more pronounced in the first than in the second maxillary molars. The interradicular furcation angle significantly correlated with the surface area (positively) and septum height (negatively) for both molars. The results of this study may recommend CBCT image analysis as a useful tool in predefining the circumstances that can allow for substantially better planning of immediate implant placement procedures in the region of maxillary molars. The interradicular septum is the bone structure located between dental sockets that separates the tooth roots from furcation line to the apical limit of the roots . The uppIt is well known that a significant improvement in the analysis of this maxillary region was achieved by using cone beam computed tomography (CBCT). With all respect to the most commonly used radiographic diagnostic procedures in dentistry, the use of CBCT has overcome the shortcomings of other radiological methods such as the superimpositions and distortions of 2D radiography, limited access of computed tomography, poor resolution, difficulties in interpretation in dentistry, longer scanning time, high cost, disturbance of metal artifacts, and high radiation exposure of computed tomography and multi-sliced computed tomography ,7. AdditThe use of CBCT methodology has already proven remarkable characteristics for the subtle morphometric analysis of structures that may be of potential interest in planning the procedures accompanied by implant placement ,14,15.In addition to morphological examination of anatomical structures, CBCT and related software also have the possibility for the insertion of the virtual implant ,17. UsinThe literature data offer a variety of studies (sometimes contradictory) considering the interradicular septum dimensions related to the final outcome of the prime implant fixation. Thus, it has been reported that minimal bone height should be predefined at 5 mm , but alsTaken together, it is obvious that the morphometric analysis of the maxillary molars region using the advanced diagnostic methodology has potential clinical importance. Therefore, the aim of this study was to evaluate the interradicular septum bone morphometric characteristics using CBCT images, in order to estimate the previous findings, as well as to establish quantitative shortcuts that can be useful for clinicians to make a faster and more reliable plan for immediate implant placement in maxillary molars area.This retrospective quantitative study was conducted on CBCT images obtained from patients at the Department of Dentistry of the Faculty of Medical Sciences, University of Kragujevac, Serbia, during January and February 2022, and carried out in accordance with the Declaration of Helsinki and with the approval by the institutional review board of Faculty of Medical Sciences, University of Kragujevac . The inclusion criteria for involvement in this study were as follows: patients over 18 years of age, at least one maxillary molar present, and formal consent for the usage of personal clinical data for scientific purposes. The excluding criteria were predefined as low image quality and no impacted teeth or other pathology present in the area of interest. According to those criteria, the total number of participants included in this study was 100, with the total number of individual maxillary molar images of 213.The images were obtained using an Orthophos XG 3D device , and analyzed using GALAXIS software v1.9.4  according to the previously described procedure .The morphometric analysis of the maxillary molars region was based on the parameters obtained on the sagittal and axial slices. The sagittal views were used for measurement of the following parameters: the interradicular septum width at different levels A, in mm,The analysis of the obtained parameters was performed according to the following algorithm :p < 0.05 was considered to be significant. Statistical analysis was performed with the SPSS version 20.0 statistical package .All data obtained in this study were expressed as means ± SEM. Following initial submission to Levene’s test for homogeneity of variance and to the Shapiro–Wilk test of normality, the comparisons between groups were performed using the Student’s t-test. Furthermore, Pearson’s coefficient of correlation was used to analyze relationships between parameters, and simple linear regression analyses were performed. A value of p = 0.122).This study was performed on CBCT images obtained from 100 patients , with an average 42.12 ± 1.52 years of age , with no significant age difference between sexes . The analysis of sagittal CBCT slices of the second molar also confirmed no significant differences for contralateral sides of the interradicular septum for the same estimated parameters . The axial CBCT slices analysis again showed no significant side differences between the first maxillary molars’ interradicular septum perimeters at the levels A, B, C and D , as well as for the surface area . Almost the same results were obtained for the bilateral comparison of the parameters obtained on the second maxillary molars’ interradicular septum .The evaluation of the first maxillary molars’ interradicular septum characteristics using sagittal CBCT slices showed no significant bilateral differences for all estimated parameters: the interradicular septum width at the A, B, C and D level; the interradicular septum height; and the angulation of furcation and the distance between the base of interradicular septum to the sinus floor (p < 0.01). In contrast, the interradicular septum height of the upper second molars was significantly higher when compared to the first molars (p < 0.05). As shown in p < 0.05). However, the distance between the interradicular septum base to the sinus floor of the first and second molars showed no significant difference  obtained on sagittal CBCT slices is shown in fference G.p < 0.05). The same parameter was significantly higher at all  levels on the left side (p < 0.01). In contrast, the values for the interradicular septum height and angle furcation were significantly different only on the right side . Still, there was no difference between interradicular septum base to the sinus floor distances on either side . Not surprisingly, a similar observation was achieved for the interradicular septum surface areas comparison between the first and the second upper molars. Again, the surface areas values for the first molars were significantly above the second maxillary molars at all estimated levels (p < 0.01).As presented in p < 0.01). The very same observation was confirmed on the left side (p < 0.01).Interestingly, the analysis observed on axial CBCT slices, presented in p = 0.0066 and 0.0004, respectively).In order to estimate the relationship between the furcation angle and surface area, we estimated that interconnection at the level A, as the most critical (narrow) point of clinical importance. The linear regression analyses revealed a significant positive correlation for both first and second maxillary molars .Furthermore, we also estimated the relationship between the furcation angle and the height of interradicular septum. Again, we observed a significant correlation (only negative) for both the first and second upper molars  molars, neither on sagittal nor axial CBCT slices. The etiology of the observed phenomenon can be found in the study of Som and coworkers , who desHowever, the key point of our study was to estimate the differences between the interradicular septum bone morphological characteristics of the first and second maxillary molars using the CBCT technique. In order to allow for better insight into the interradicular septum properties, we performed linear measurements of its width at the four predefined levels. In that sense, the results obtained in this study showed that the first maxillary molars had a wider interradicular septum when compared to the second molars at all estimated levels. In contrast, the interradicular septum height values observed in the first molars were significantly below the height in the second maxillary molars. Unlike for the interradicular septum width, due to lack of literature data, the results considering the interradicular septum height obtained in this study have been previously described. However, this morphometric parameter was reported to be either enhanced  or lowerAs Levin and colleagues  revealedFurthermore, the distance between interradicular septum base to the maxillary sinus floor, which has not been evaluated before, also showed no significant differences according to the maxillary molar position, suggesting that this parameter may not be of significant clinical importance in different planning of immediate implant placement procedure for the first and second upper molars.We also estimated the interradicular angle furcation, as a parameter that may significantly influence the planning of immediate implant placement procedures in the maxillary molar region. Our analyses showed that this parameter showed significantly higher values in the first molars F when coUsing the axial CBCT slices, we evaluated the interradicular septum perimeter and surface area at A, B, C and D levels. The analyses strongly confirmed that both interradicular septum perimeter and surface area were significantly more pronounced in the first than in the second maxillary molars. This difference appeared congruently on either side. Although comparable only at the lower part of the interradicular septum (level A in this study), our results are in line with Agostinelli and colleagues , who repWe also estimated the perimeter and interradicular septum area of the first and second right molars using an axial cross-section. The first molar showed larger values of both (perimeter and the area) in comparison to the second molars. The same results were reported on the left side. This analysis confirms that immediate implant placement in the second molar area of both sides could require additional surgical procedures in order to achieve successful implant therapy.Finally, the estimation of the interconnection between the interradicular furcation angle and surface area  showed, as presented in The results of this study obtained using CBCT images may recommend this technique as a useful tool in predefining the circumstances that can allow for substantially better planning of immediate implant placement procedures in the region of maxillary molars. Additionally, it could be noticed that, in most cases, the interradicular septum of the first maxillary molars allowed for wider implant (in diameter) placements, while its characteristics in the second maxillary molars could be directed to the selection of the enhanced length (instead of the width) implants in order to achieve primary stability.


In this study, a novel polymeric nanomaterial was synthesized and characterized, and it its potential usability in hypertension treatment was demonstrated. For these purposes, a poly-based polymeric nanomaterial (p(HEMPA)) was synthesized using a mini-emulsion polymerization technique. The nanomaterials were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and zeta size analysis. The synthesized p(HEMPA) nanomaterial had a diameter of about 113 nm. Amlodipine-binding studies were optimized by changing the reaction conditions. Under optimum conditions, amlodipine’s maximum adsorption value (Qmax) of the p(HEMPA) nanopolymer was found to be 145.8 mg/g. In vitro controlled drug release rates of amlodipine, bound to the nanopolymer at the optimum conditions, were studied with the dialysis method in a simulated gastrointestinal system with pH values of 1.2, 6.8 and 7.4. It was found that 99.5% of amlodipine loaded on the nanomaterial was released at pH 7.4 and 72 h. Even after 72 h, no difference was observed in the release of AML. It can be said that the synthesized nanomaterial is suitable for oral amlodipine release. In conclusion, the synthesized nanomaterial was studied for the first time in the literature as a drug delivery system for use in the treatment of hypertension. In addition, AML–p(HEMPA) nanomaterials may enable less frequent drug uptake, have higher bioavailability, and allow for prolonged release with minimal side effects. For this reason, the entropy increased with temperature. The increased binding capacity with increasing temperature indicated the binding between hydrophobic phenylalanine and AML. Furthermore, since physical adsorption is an exothermic process, it is expected to be higher at lower temperatures but to decrease with increasing temperature.It is thought that the dominant forces between the MPA used as the functional monomer and AML were hydrophobic interactions and hydrogen bridge bonds that formed between the amlodipine molecule’s -CH monomer . A decre2/h), and higher entrapment efficiency (88.11%) [As shown in (88.11%) . In anot(88.11%) .The release of AML was evaluated with AML-loaded-nanopolymers in SGF without enzymes with the dialysis method. 3 groups of AML. These results also indicate that the AML-loaded nanopolymer remained stable in a gastric fluid environment, whereas at pH 6.8, the AML–p(HEMPA) drug conjugate showed 45.6%, 55.8%, 86.8%, and 89.9% release rates of AML in 12 h, 24 h, 48 h, and 72 h, respectively. The reason for the rapid release of AML at pH 6.8 is thought to be the decrease in the interaction between the nanopolymer and drug as a result of AML deprotonation. The release amount of AML at an acidic pH was lower than the release amount at a neutral pH; this indicates that the release of AML from the AML–p(HEMPA) nanopolymer will be suppressed in the gastrointestinal tract and that AML release can be promoted when the nanopolymers enter the small intestine. At the same time, the AML–p(HEMPA) drug conjugate at pH 7.4 showed 86.4%, 97.3%, 99.02%, and 99.5% release rates of AML at 12 h, 24 h, 48 h, and 72 h, respectively. Similar to our results, Mei et al. reported that in the highly acidic pH environment of the stomach, a network formed due to the electrostatic interactions of the oppositely charged polymers and resulted in stronger retention of the drug in the polymer [The interactions between AML and p(HEMPA) nanopolymers are thought to comprise hydrophobic interactions and hydrogen bridge bonds. In SGF (pH 1.2), the AML–p(HEMPA) drug conjugate showed AML release rates of 21.5%, 22.3%, 22.4%, and 22.5% in 12 h, 24 h, 48 h, and 72 h, respectively. Even after 72 h, no difference was observed in the release of AML. At pH 1.2, the release of AML was slow and stable as a result of hydrogen bridge bonds between the protonated form of the –NH group in the pyridine ring of the AML and the –OH group of the p(HEMPA) nanopolymer. In addition, there were hydrophobic interactions between functional MPA monomer and the nitrobenzene and CH polymer . Another polymer .2 = 0.9947)  nanopolymer. The value of the correlation coefficient for release data at pH 7.4 was given by the Higuchi model, which was fitted best according to the value of the correlation coefficient (R 0.9947) . This mo2) value was the best-fitting model. As can be seen from the results, the value of R2 was the highest for the Peppas–Sahlin equation in almost all pH environments and the Korsmeyer–Peppas equation at pHs of 7 and 7.4; therefore, these two models were best fit for APB release. The first order and zero-order equations did not follow the drug release. The relative contribution from the diffusion and relaxation process was evaluated with the Peppas–Sahlin equation. Data analysis showed that the Higuchi and Hixon-Crowell models did not follow release kinetics.Similarly, Kapoor et al. also found that the best fit model was the Higuchi model (0.981) for an amlodipine nano lipid carrier. The Peppas model showed an n value of 0.344, indicating that drug release was regulated through non-Fickian diffusion . In anot2 = 0.9947). As a result, AML–p(HEMPA) nanomaterials may enable less frequent drug uptake, have higher bioavailability, and allow for prolonged release with minimal side effects. Evidently, it can be concluded that AML–p(HEMPA) nanopolymers may increase the oral bioavailability of hydrophobic agents, increase plasma half-life, and minimize the side effects associated with anti-hypertensive drugs by reducing their dosage requirements and intake frequency.In conclusion, the current study was undertaken to design a new polymeric nanomaterial and to evaluate the controlled-release oral administration of AML. For this purpose, poly-based polymeric nanomaterials (p(HEMPA)) were synthesized using a mini-emulsion polymerization technique. The synthesized p(HEMPA) nanomaterial is about 113 nm in diameter. Amlodipine-binding studies were optimized by changing the reaction conditions. Under optimum conditions, amlodipine’s maximum adsorption value (Qmax) of the p(HEMPA) nanopolymer was found to be 145.8 mg/g. In vitro controlled drug release studies of amlodipine, bound to the nanopolymer at the optimum conditions, were conducted in a simulated gastrointestinal system with pH values of 1.2, 6.8 and 7.4. It was found that 99.5% of amlodipine loaded on the nanomaterial was released at pH 7.4 and 72 h. Even after 72 h, no difference was observed in the release of AML. The drug release data were fitted to several kinetic models of AML release from the p(HEMPA) nanopolymer. The value of the correlation coefficient for release data in pH 7.4 was given by the Higuchi model, which was fitted best according to the value of the correlation coefficient (R


Lagovirus europaeus GI.1 belongs to Lagovirus in the Caliciviridae family. GI.1 causes an acute, septic, and highly lethal disease in rabbits. Lagovirus europaeus GI.2, a new variant of GI.1, has caused explosive mortality in rabbits of all ages in Sichuan Province, China. To explore the differences in pathogenicity of rabbits infected with GI.1/GI.2, we investigated the virulence and disease progression of a naturally occurring GI.1/GI.2 in 4-week-old, 13-week-old, and 25-week-old New Zealand White laboratory rabbits after GI.1/GI.2 infection. Objective measures of disease progression were recorded using continuous body-temperature monitoring. We observed the kittens were infected with GI.2 during the most urgent course of the disease, and GI.1 was not lethal to kittens. We found that the target organ of both GI.1 and GI.2 was the liver, but the disease course of the two viruses was differed. Our study enriches the research on the pathogenicity of GI.1 and GI.2 under the same conditions. Consequent upon the 2017 attempt to standardise the Lagovirus nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus, RHDV belongs to the GI genogroup, which is further divided into the GI.1(former G1–G6), GI.2 (RHDV2/b), GI.3  and GI.4  genogroups2. GI.1 was first reported in China in 19843. South Korea was the following country to report RHD outbreaks due to the rabbit fur imported from China4. The disease then emerged in Europe and first reported in Italy in 19865, from where it spread to other parts of Europe and became endemic in several countries. In Australia and New Zealand, rabbits are considered an important agricultural pest and a threat to animals and plants 6, and GI.1 has been used to control rabbit populations 7.Rabbit hemorrhagic disease (RHD) is also known as rabbit fever or hemorrhagic pneumonia. It is caused by two lagoviruses of the Caliciviridae family9. GI.1 and GI.2 are different genotypes of the lagovirus causing RHD. GI.2 can infect both rabbits and hares, and rabbits are more susceptible, especially kitten and subadult rabbits. Additionally, GI.2 causes disease in several hares (Lepus) species and can overcome GI.1 vaccine protection 12.In 2010, the novel lagovirus GI.2, which is genetically and antigenically distinct from GI.1, was reported in wild and domestic rabbits in France and Spain 13. In a previous study, we isolated a GI.2 strain SCCN04 from these dead rabbits at a rabbit farm in Jintang 14.On April 1, 2020, an acute death case of rabbits occurred on a rabbit farm in Jintang, Sichuan Province, China. The clinical symptoms and pathological changes in the dead rabbits were very similar to those of RHD, and deaths occurred in rabbits of all ages. Rabbits were injected with GI.1, and Pasteurellosis multocida combined inactivated vaccine before and five days after the onset of rabbit farm, but the effect was not obvious15, their results indicate that recombination contributed to the emergence, persistence and dissemination of GI.2 as a pathogenic form and that all described GI.2 strains so far are the product of recombination. In addition, with pathogenic (variants GI.1a and GI.1b) and benign (genotype GI.4) strains that served as donors for the non-structural part while GI.2 composed the structural part. In our previous study14, we performed genetic characterization of SCCN04, where the whole genome phylogenetic tree, the non-structural phylogenetic genes tree and the structural genes phylogenetic tree were constructed, and recombination events were analysed. The results showed that SCCN04 and Chinese isolate SC2020/0401(MT586027.1) formed a small branch and were the most closely related among all the genome phylogenetic trees, and SCCN04 was closely related to European GI.2 isolates on a large branch. In addition, we confirmed SCCN04 recombination by RDP5 and identified 2020 isolate MT586027.1 (SC2020/0401) as the main parental virus. This indicates that GI.2 may have a new restructuring event in China.According to the results published by Abrantes et al. 19, and pointing out some differences in the pathogenicity of the two viruses, but these studies were not all conducted under the same conditions, so the results are not directly comparable. Our study reports a systematic comparison, control the relevant parameters of GI.1/GI.2 challenge experiment uniformly . To compare the difference in pathogenicity between GI.2 and GI.1, GI.2 (SCCN04) and GI.1 (SCH04) were used to infect rabbits of various ages, respectively. Changes in the virus copies in blood, feces, and saliva over time after infection with GI.1/GI.2 were detected by RT-qPCR established according to the VP60 gene, because the VP60 gene of the same genotype isolates (GI.1or GI.2) was highly conserved. The β-actin gene was used as a reference gene, and the GI.1 and GI.2 RT-qPCR methods established in our laboratory20,21 were used to establish a relative quantitative assay to detect the GI.1 and GI.2 load distribution in rabbits on different days. SCCN04 (Accession: MW178244.1) and SCH04 (Accession: KX844830.1) were isolated from dead rabbits in Chengdu, Sichuan Province, China, preserved, and provided by Sichuan Agricultural University.There had been many previous studies dealing with the pathogenicity of GI.1 or GI.250, and 3 rabbits of each group were selected for the control group.GI.1 and GI.2-infected animals trials included three identical groups , respectively. Group S included 4-week-old kitten . All animal experiments were performed in accordance with the Laboratory Animals Welfare and Ethics guidelines published by the General Administration of Quality Supervision, Inspection, and Quarantine of the People’s Republic of China.50) of the virus stock. For titration, groups of six adult rabbits were inoculated intramuscularly with10-fold serial dilutions of the concentrated virus stock, and the Reed-Muench method was used to determine the 50% endpoint22. The RID50 measurement results are shown in Table 50 of GI.2 and GI.1 are 106.5 and 105.5 respectively. Rabbits were infected orally with a dose (1000 RID50) of the virus, in a final volume of 1 mL.The virus stocks were prepared by the Key Laboratory of Animal Diseases and Human Health of Sichuan Province. Briefly, the virus was amplified in New Zealand White rabbits, liver homogenates were semi-purified, and freeze-dried virus stocks were prepared and titrated to derive the 50% infectious rabbit dose  and ketamine 30 mg/kg administered intramuscularly.Blood, saliva, and feces were collected before infection. Samples of blood, saliva, and feces were collected every 3 h during the first 12 h of infection, and every 6 h thereafter. Specifically, 2 mL of blood was collected through the ear vein, saliva was dipped into the rabbit's mouth with cotton swabs, and fecal particles were collected. Due to the rapid course of RHDV infection, animals died peracute between monitoring timepoints. Therefore, when we found more than 80% of the rabbits appeared to have terminal disease at monitoring timepoints in a group, all rabbits in this group were killed humanely. At the same time, the rabbits in the control group were sacrificed and dissected. Samples of the liver, spleen, lung, heart, and kidney were collected sequentially from each dead rabbit. RNA was extracted from each tissue sample and reverse-transcribed into cDNA, and GI.1/GI.2 RT-qPCR was performed. The viral RNA extraction kit and reverse transcription kit were purchased from TaKaRa . Genious2 × SYBR Green Fast qPCR Mix (No ROX) was purchased from ABclonal .23. The GI.1 and GI.2 RT-qPCR methods established in our laboratory were used to detect the CT values of the target genes. Ct values for all genes were recorded, and the relative viral load was calculated using the 2−ΔΔCT method24. All primers used are listed in Table β-Actin gene (GenBank accession number: NM_001101683.1) was stably expressed in all organs of rabbits, and the Ct value of the β-actin gene was detected using the RT-qPCR method established by Chen RuiAll Statistical analysis was performed with GraphPad Prism 8.0.2 , and all data were analyzed by one-way ANOVA.In animal trials, all rabbits' (control and infected groups) hearts, livers, spleens, lungs, and kidneys were soaked in 4% paraformaldehyde and sent to Sevicebio Company for section preparation and pathological scoring. Pathological changes were scored on a five-point scale, with no or very few lesions scored as 0; mild or few lesions were scored as 1; moderate lesions or moderate lesions were scored as 2; severe or multiple lesions were scored as 3; extremely serious or massive lesions were scored as 4.https://arriveguidelines.org).All animal experiments were approved by the institutional animal care and use committee of Sichuan Agricultural University of China . All animal experiments were performed under the Laboratory Animals Welfare and Ethics published by the General Administration of Quality Supervision, Inspection, and Quarantine of the People’s Republic of China. And the study is reported in accordance with ARRIVE guidelines . The other infected rabbits died within 3 days after inoculation. The death time of rabbits in the GI.2-S group was the shortest, with all kittens dying within 18 h. All the dead rabbits showed the typical clinical symptoms of RHD. Specific manifestations included: depression, loss of appetite, sudden death, violent struggle before death, angular arch retraction, and oral bleeding in some rabbits.As shown in Fig. As shown in Fig. The kinetics of viremia after GI.1 infection are shown in Fig. The viral copies in the feces of kittens and subadult rabbits continued to increase within 9 hpi, and then tended to stabilize. The viral copies in the feces of adult rabbits increased within 3 hpi, then tended to stabilize, and then increased again after 12 hpi.The viral copies in the saliva of kittens increased within 9 hpi, then plateaued, and began to decrease after 48 hpi. At 72 hpi, GI.1 was not detected. The viral copies in the saliva of subadult rabbits and adult rabbits first increased and then tended to be stabilize.The kinetics of viremia after GI.2 infection are shown in Fig. After 6 hpi, the viral copies in the saliva of all rabbits increased to a stable level. The virus was detected in the saliva of kittens within 3 hpi, whereas it was detected in the saliva of subadult and adult rabbits after 3 hpi.The HE (Hematoxylin and Eosin) staining results of the rabbit viscera are shown in Supplementary Figures As shown in Fig. As shown in Fig. At present, lagoviruses still cannot passage continuously in the cell culture system. In most of the previous challenge studies, which used hemagglutination (HA) units to quantify the amount of virus in inoculants, we assumed that HA was only an indirect indication of infection dose. Titration of inoculants is necessary in order to more accurately compare the pathogenicity of two lagoviruses under the same conditions.19. In contrast to several previous studies, GI.1 and GI.2 Chinese strains were used to infect rabbits of three different ages. In our experiments, we found that GI.2 had a mortality rate of 100% in rabbits of all ages, but kittens were more susceptible to GI.2 infection, with a short death time and a rapid course of the disease. The mortality rate of GI.1 was 100% (12/12) in rabbits over 90 days of age but 0 (0/6) in kittens.Pathological changes and viral load after GI.2 infection have been widely reported elsewhere25. This finding is consistent with the results of our study.The liver is the main organ invaded by GI.1/GI.2. Liver failure caused by degeneration, necrosis, or apoptosis of hepatocytes, is the main cause of acute death in rabbits. GI.1/GI.2 first appeared in hepatocytes, Kupfern cells, and sinus wall endothelial cells and then spread to other cells. During the course of disease development, hepatocytes and vascular endothelial cells had been found to be preferentially infected and damagedWe found that disease progression was influenced by both age and presence of the virus. The average death time of rabbits infected with GI.2 was shorter than that of rabbits infected with GI.1. Adult rabbits infected with GI.1 had the shortest and most acute course of the disease. The kittens were infected with GI.2 during the most urgent period of the disease. This trend was also reflected in the time to the onset of pyrexia and in the progression of viremia.26 showed that kittens (4 weeks of age) are naturally resistant to GI.1, which is associated with a rapid and effective inflammatory response in the liver after GI.1 infection in kittens, with almost no hepatocytes infected by GI.1.We observed that GI.1 was not lethal to the kittens. Virus copies in the blood and saliva of kittens infected with GI.1 first increased and then decreased. A study by MarquesIn our study, we found that the target organs of GI.1 and GI.2 were the liver, but the organs colonized by the two viruses differed. Notably, GI.1 did not colonize the heart, whereas GI.2 did. Meanwhile, GI.2 did not appear to colonise the kidney of infected rabbits in terms of viral load, unlike those infected by GI.1.Comparing viral load in the spleen, we found that GI.1 in the spleen of kittens had the lowest viral load, while GI.2 in the spleen of kittens was higher than that of subadult rabbits, and loads of GI.1 and GI.2 in the spleen were higher in adult rabbits than in rabbits of other ages. The viral loads of GI.1 and GI.2 in the lungs were proportional to the age of rabbits. The older the rabbit was, the higher the viral load.Previous studies focused on disease progression and pathological damage to the liver, spleen, and lungs following GI.1/GI.2 infection. In this study, we conducted animal trials under the same conditions , which enriched the research on the pathogenicity of GI.1 and GI.2. We found that liver and spleen were the main organs of viral colonization during the acute progression of RHD. At the same time, the age of rabbits also has an effect on the viral colonization of lungs and spleen. Future studies can continue to explore why the spleen and lungs of adults are more likely to become the organs of GI.1/GI.2 colonization than those of subadults and kittens. In addition, possibly due to the high dose of infection, we found that the heart and kidney were the most unaffected organs in the rapid course of disease in rabbits of any age infected with either GI.1 or GI.2, respectively. We believe that it will be a future research to explore why GI.1 and GI.2 have less damage to the kidney and heart, and whether the longer disease course has new effects on the internal organs of rabbits.Supplementary Information.