HR remained statistically significant even when adjusted for classic cardiovascular risk factors. Conclusions. In our observational study, we confirm that the ε4 allele is associated with higher total peripheral revascularization in patients with advanced atherosclerotic vascular disease at prolonged follow-up.Introduction. Atherosclerosis is a complex multifactorial disease and apolipoprotein E (APOE) polymorphism has been associated with cardiovascular events. The APOE gene, located on chromosome 19q13.2, has an important role in lipid metabolism, in particular on circulating cholesterol levels, implying further pleiotropic effects; from its polymorphism are derived three alleles , which induce different phenotypes, while its impact on carotid and femoral atherosclerosis is still controversial. Objectives. The aim of the study is to investigate the relationship between APOE genotypes and peripheral revascularization in a cohort of patients affected by advanced peripheral arterial disease (PAD) at a prolonged follow-up. Materials and methods. Some 332 patients  with severe PAD were enrolled in a longitudinal study, with a 90.75 ± 32.25 month follow-up, assessing major adverse cardiovascular events (MACE). Results. As compared with ε3/ε3, in ε4 patients we observed a significant higher incidence of carotid (13.2% vs. 5.6%; HR = 2.485, 95% CI 1.062–5.814;  Important manifestations of atherosclerotic disease, such as myocardial infarction and stroke, represent the leading cause of death in the world . ChronicIn recent decades, attention has increased towards the causes of atherosclerosis and its clinical consequences . In addiAtherosclerosis is the result of a low-grade inflammatory process that favors the deposition of inflammatory cells and cholesterol and the proliferation of smooth muscle cells in the intima . A postpAPOE polymorphism modulates susceptibility to many diseases, including neurodegenerative disorders (Alzheimer’s disease and cognitive decline) and atherosclerotic arterial disease . The ε4 A cohort of cardiovascular controlled case series was followed up in a longitudinal study fulfilling the Declaration of Helsinki, the guidelines for Good Clinical Practice and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) . The ethics committees on human experimentation of “Casa Sollievo della Sofferenza” Hospital  approved the study protocol.We identified 332 patients  affected by peripheral arterial disease who were enrolled into the study from 1 November 2009 to 31 October 2017, after informed consent, using the following criteria: Caucasian race and advanced atherosclerosis defined as the presence of carotid plaque producing more than 50% stenosis by Doppler analysis velocimetry and/or Fontaine stage II or III claudication . Exclusi2). We measured brachial-ankle pulse wave (PWV) using an AngE system , left ventricular ejection fraction (LVEF) and left ventricular mass with the Devereux formula by echocardiographic examination. Finally, we derived the ventricular mass index (LVMI) using left ventricular mass divided by body surface area. Data about medical treatments, cancer, smoking, being overweight, dyslipidemia, arterial hypertension and type-2 diabetes mellitus, according to criteria of the World Health Organization and ATP III , were coEchocardiographic examination and doppler ultrasonography of supra-aortic trunks, abdominal aorta and lower limb arteries were all performed, according to current guidelines , with thWe calculated PR, QRS and QTc intervals using a standard 12-lead electrocardiogram. We conducted a follow-up at 90.75 ± 32.25 months (range 1–124). During this period we recorded major adverse cardiovascular events (MACE), defined as cerebral ischemia, myocardial infarction, myocardial and/or peripheral revascularization, acute lower limb ischemia and cardiovascular death. We considered cardiovascular death as deaths occurring from heart failure, myocardial infarction, stroke, sudden death or ventricular arrhythmias. Furthermore, we recorded all cancer deaths. Blood samples (2 mL) from each patient were collected in EDTA-containing tubes. We used standard methods to extract genomic DNA from peripheral blood and we identified in blinded fashion APOE genotypes ,22. GeneWe observed the following genotype frequencies: 9.94% for ε2/ε3, 69.58% for ε3/ε3, 19.28% for ε3/ε4 and 1.20% for ε4/ε4. We did not identify the ε2/ε2 or ε2/ε4 genotypes. No differences were observed in respect to expected Hardy–Weinberg frequencies. Using these genotype frequencies found, we estimated allele frequencies: 4.97 for ε2, 84.19 for ε3 and 10.84 for ε4. Then, patients were grouped as follows: ε2 (ε2/ε3), ε3 (ε3/ε3) and ε4 (ε4/ε3 + ε4/ε4). We considered ε3/ε3 as a “wild-type”, being the most common genotype in the population, and patients with ε3/ε3 genotype as the reference group. 2 test. Hardy–Weinberg equilibrium was tested by a χ2 test. Quantitative data variables were compared using variance analysis (two-tailed unpaired t-test), after verifying normal distribution by a Kolmogorov–Smirnov test. p values < 0.05 were considered statistically significant. The Cox model was applied to estimate the cardiovascular event incidence by hazard ratio (HR) with 95% confidence interval (95% CI). HR was calculated crude and with adjustment for the common risk factors of BMI, hypertension, diabetes, dislipidemia, smoking, age, gender, LDL-cholesterol and triglycerides. Kaplan–Meier curves were used to show event-free rivascularization during follow-up. All statistical analyses were performed with SPSS 25.0 software .Continuous variables were presented as means ± standard deviation (SD); categorical variables were presented as frequency (%). In the groups, dichotomous variables were compared using the Pearson’s χWe recruited 332 patients  affected by advanced PAD . At baseline, 170 peripheral revascularization procedures had already been performed: in detail, 6 femoro-popliteal bypasses, 4 aorto-bifemoral bypasses, 1 ilaco-femoral thromboendarterectomy, 61 percutaneous transluminal angioplasties of the lower limb arteries, 47 carotid percutaneous transluminal angioplasties, 49 carotid thromboendarterectomies and 2 carotid bypasses. Moreover, 134 patients had a diagnosis of ischemic heart disease  and 124 had been revascularized through 41 surgical treatments, 72 endovascular treatments and 11 combined procedures. In addition, 52 patients had a history of previous cerebrovascular accidents or neuroradiological pictures of ischemic multifocal leukoencephalopathy.2); 53% were overweight (BMI ≥ 25 kg/m2) and 28% were obese (BMI ≥ 30 kg/m2). Finally, 53 subjects had a personal history of cancer (with a life expectancy of more than 6 months).Only 19% of collected patients were of normal weight . Compared to the wild type, despite being statistically non-significant, ε4 patients had higher fasting glucose levels .We observed a statistically significant difference of BMI: ε4 patients were less obese with respect to ε3 patients , more insulin resistant , had higher triglyceride values  and had greater arterial stiffness as seen with PWV . However, in ε2 patients, with respect to ε3, we noticed no statistically significant differences in microalbuminuria , QTc interval , PR interval  and QRS duration . Compared to ε3 carriers, in ε2 carriers we found a trend in LVMI  and no difference in LVEF . Compared to ε3, we found statistical trends: ε2 patients were more obese .We observed revascularizations of the lower limbs performed in 2 cases by femoral-popliteal bypass surgery, in 4 cases with percutaneous transluminal balloon angioplasty and in 13 cases with percutaneous transluminal angioplasty plus stent insertion. In all patients, lower limb revascularization was performed to improve walking interval and reduce claudication. In 8 cases, a second attempt at revascularization was necessary. We did not note any lower limb amputations. On the other hand, analyzing carotid revascularizations, we counted 1 carotid-subclavian by-pass surgery, 10 endovascular treatments by transluminal angioplasty and stenting, and 15 thromboendarterectomies. Five patients with carotid stenosis were symptomatic just before undergoing revascularization (one stroke and four transitory ischemic attacks). In one patient we recorded a major complication during the first 24 h after carotid stenting: a hemorrhagic stroke. During the follow-up, we observed two significant carotid restenoses , one intrastent and one at the anastomosis site of the carotid-subclavian bypass; both underwent a new revascularization treatment.p = 0.744), even if not statistically significant.Comparison analysis between ε3 and ε4 carriers showed a greater number of MACE in ε4 carriers . In particular, ε4 carriers had a significantly higher incidence of carotid artery  and lower limb  revascularizations  as resulting from eithercarotid  or lower limb  revascularization. We calculated crude hazard ratio with adjustment for classical cardiovascular risk factors  . With rep = 0.004; HR adjusted for triglycerides = 2.589, 95% CI 1.337–5.049; p = 0.005).The risk of ε4 carriers was higher for total peripheral revascularizations even when the variables LDL-cholesterol and triglycerides were considered , worsening of renal failure (three cases), exacerbation of respiratory failure (five cases), anemia, pneumonia, surgical complications, severe cognitive impairment and traumatic fracture. In any case, we did not find any significant difference based on APOE genotype.p = 0.107).In respect to ε3 carriers, we found a higher incidence, even if not statistically significant, of developing fatal events in ε2 carriers; in particular, cancer-related mortality was observed in ε2 carriers  during follow-up ; in detail, this group experienced 7.8% cardiovascular death and 10.1% cancer-related death, while the other group suffered 5.2% cardiac death and 5.2% cancer-related death.Additionally, the revascularized group at baseline had a higher incidence of all-cause death . We calculated hazard ratio adjusted for previous cardiovascular risk factors , confirming significant higher risk of peripheral revascularizations in ε4 carriers . The statistically significant difference for myocardial revascularization was lost when we adjusted HR for diabetes. Graphical representation of peripheral revascularization-free survival curves in the group of patients with baseline revascularizations was similar to that shown in p = 0.004) and not significant for ε3 vs. ε2 carriers (p = 0.469).Comparison analysis between ε2 and ε3 carriers showed no differences, irrespective of baseline revascularization status. In the group with baseline revascularization, we collected a statistically significant incidence of myocardial and peripheral revascularizations in ε4 carriers compared to ε3  and candidate gene evaluation studies . It is wDiffering results reported in the literature may be related to diverse outcomes and populations analyzed for both age and stage of disease, as hypothesized for the Secondary Manifestations of ARTerial disease (SMART) study. This study analyzed 7418 subjects, from The Netherlands, aged 56.7 ± 12.4 years , highlighting more events and peripheral reperfusions in ε2 carriers compared with ε3 . On the Even in the Study of Health in Pomerania (SHIP), conducted in Germany on 3327 participants aged between 20 and 79 years, ε2 carriers were more predisposed to cardiovascular events  and peripheral atherosclerosis assessed by measuring thickening intimal carotid. The ε4 allele had no effect on carotid intimal thickening, carotid plaque progression, myocardial infarction or stroke . We hypoConversely, when a cohort with known carotid atheroma is selected, as in a recent case–control study conducted in northwest China, the role of APOE genotypes in the progression of artery atherosclerosisis is more evident in ε4 carriers .An evaluation of APOE polymorphism and carotid atherosclerosis in Korean subjects, characterized by being over the age of 45 years, rural area origin, mostly female gender and not affected by peripheral atherosclerosis, showed that ε4 carriers had a higher risk of carotid plaque, and this result was mediated by lipids .p = 0.027) of developing critical carotid plaque requiring revascularization independent of common vascular risk factors.In the Three-City (3C) study, a longitudinal study conducted on 5856 elderly subjects recruited from three French cities, carotid plaques were more present in ε4 carriers compared to ε3 homozygous carriers . The corRecent clinical studies analyzed the association between APOE polymorphism and incidence of in-stent restenosis after vertebral and carotid artery stenting, demonstrating that ε4 polymorphism is an independent risk factor for restenosis ,39.According to the literature, we report a strong association between insulin resistance, hypertrigliceridemia and ε2 polymorphism, such as direct correlation between insulin resistance and arterial stiffness ; increasOn the basis of our results, we strongly believe that in the future APOE polymorphism needs to be included in complex genetic risk scores to assess cardiovascular risk, even in patients affected by advanced peripheral vascular disease. Introduction of new lipid lowering drugs, such as PCSK9 inhibitors, bempedoic acid and incliserin, will lead to better treatment and outcomes in patients affected by advanced atherosclerosis; however, the literature underlines the strict relationship between PCSK9 and APOE in both lipid profile and vascular disease ,44. In tAn approach to patients affected by peripheral artery disease needs to be completed, including different dimensions from classic risk factor profiles to more advanced genetic analysis, taking into account the role of APOEε4 for atherosclerosis development in different vascular beds.Our study presents several limitations, mainly represented by population size, a single center, observational approach, narrow regional origin of enrolled patients, Caucasian ethnicity, gender bias and a lack of apolipoprotein level dosage.In our observational study, we confirm that the ε4 allele is associated with a higher incidence of aggressive events of cardiovascular disease, in particular of peripheral revascularization (carotid and lower limb revascularization) in patients with advanced atherosclerotic vascular disease at prolonged follow-up, underlying the need for a strict periodical clinical reassessment in specific subgroups, even in the elderly population, in the setting of a personalized medicine approach.


Nature Medicine 10.1038/s41591-022-02102-9. Published online 15 December 2022.Correction to: In the version of this article initially published, there was an error in the abstract, where in the sentence now reading, in part, “In participants with HFimpEF… first worsening heart failure events (HR = 0.84),” the hazard ratio originally read “0.78,” while in the second paragraph of the “Outcomes by HFimpEF status” Results subsection, in the sentence now reading, in part, “The effect of dapagliflozin on HF outcomes was also similar in those with HFimpEF  and those with LVEF consistently over 40% (HR = 0.78),” the hazard ratio values were mistakenly interchanged. The values have been updated in the HTML and PDF versions of the article.


By comparing the triple-gene deletion mutant with the parental strain and the double-gene deletion mutant, we found that the safety of both the triple-gene deletion mutant and the double-gene deletion mutant could reach 100% in goats, while the safety of parental virus was only 50% after continually observing immunized animals for 14 days. A virulent field strain of ORFV from an ORF scab was used in the challenge experiment by inoculating the virus to the hairless area of the inner thigh of immunized animals. The result showed that the immune protection rate of triple-gene deletion mutant, double-gene mutant, and the parental virus was 100%, 66.7%, and 28.6%, respectively. In conclusion, the safety, immunogenicity, and immune-protectivity of the triple-gene deletion mutant were greatly improved to 100%, making it an excellent vaccine candidate.Contagious ecthyma is a zoonotic disease caused by the orf virus (ORFV). Since there is no specific therapeutic drug available, vaccine immunization is the main tool to prevent and control the disease. Previously, we have reported the construction of a double-gene deletion mutant of ORFV (rGS14ΔCBPΔGIF) and evaluated it as a vaccine candidate. Building on this previous work, the current study reports the construction of a new vaccine candidate, generated by deleting a third gene (gene 121) to generate ORFV rGS14ΔCBPΔGIFΔ121. The in vitro growth characteristics, as well as the in vivo safety, immunogenicity, and protective efficacy, were evaluated. RESULTS: There was a minor difference in viral replication and proliferation between ORFV rGS14ΔCBPΔGIFΔ121 and the other two strains. ORFV rGS14ΔCBPΔGIFΔ121 induced continuous differentiation of PBMC to CD4 Contagious ecthyma, also known as orf, is a contact, epitheliophilic, and highly contagious zoonotic disease caused by parapoxvirus . The disORF is caused by the orf virus, a DNA virus belonging to the genus Parapoxvirus, family Poxviridae, with a total length of approximately 138 kbp that encodes approximately 132 genes ,10. The ORFV has been proposed as a vaccine vector due to its limited host range, lack of virus-neutralizing antibodies, large genome, and broad immunomodulatory properties ,22. The + T cells, CD8+ T cells and CD80+CD86+ cells and mainly cause Th1-like immune responses.Based on the successful construction of the double-gene deletion mutantrGS14ΔCBPΔGIF  in our p2.ORFV-GS14 preserved in our laboratory  was used as a parent virus throughout the experiment and as a genetic background virus to generate target gene-deleted virus. Virus stocks were propagated in Primary Goat Testicular cells (PGT cells) and stored at −80 °C. The wild-type virus was prepared by sampling scabs from the goat and grinding them after the challenge experiment . The cbp7.7 TCID50/mL of rGS14ΔCBPΔGIF, and subsequently transfected with 4 mg of the transfer vector p121hm1-GFP-121hm2 at 24 h post-infection (hpi) using 5 mL of Lipofectamine 3000 reagent . The green fluorescent areas were picked and purified using the limited dilution method. Further purification was performed using flow cytometry until all the lesioned areas within the field of view fluoresced. Afterward, the entire DNA of the purified virus was extracted with the AxyPrep body fluid viral DNA/RNA miniprep kit . The 121 gene was identified using PCR amplification, and no band appeared as a successful deletion, and rGS14ΔCBPΔGIFΔ121-eGFP was obtained.We mainly used the principle of homologous recombination for the deletion of the 121 gene (GenBank: OM687301.1) of rGS14ΔCBPΔGIF . In the After that, the left homologous arm and the right homologous arm of the 121 gene were ligated using fusion PCR to construct the vector for transfection, and after sequencing without errors, they were transfected into LT cells in 6-well plates with access to rGS14ΔCBPΔGIFΔ121-eGFP length, and the areas with lesions that did not fluoresce were picked, repeatedly frozen and thawed, and then purified using the limited dilution method and flow cytometry. After purification to complete non-fluorescence in green, deletion identification was performed with primers and sent for sequencing to obtain rGS14ΔCBPΔGIFΔ121.The animals used in this study were 1-month-old ORFV-negative goats, and a total of 47 goats were used, all purchased from Honest Farm, Mancheng District, Hebei, China. The goats were randomly grouped. The animal procedures were approved by the Animal Care and Use Committee of China Institute of Veterinary Drug Control.50 assay, rGS14ΔCBPΔGIFΔ121, rGS14ΔCBPΔGIF and GS14 were diluted by a gradient from 10−1 to 10−10 and added into a 96-well plate seeded with PGT cells. The viruses were removed after two hours of adsorption at 37 °C and 5% CO2. After that, PBS was added and used to wash the cells and repeated twice. The medium was replaced with 4% fetal bovine serum maintenance medium, and cells were incubated at 37 °C with 5% CO2. Calculations were performed according to the Reed-Muench method after 7 days of incubation post-infection.Following the conventional TCID50 for inoculation and observed and recorded the CPE characteristics at different time points of dispersion.In order to investigate the cytopathic effect (CPE) differences among GS14-DCBP-DGIF-D121, rGS14ΔCBPΔGIF and GS14, we used the same TCID50 doses of rGS14ΔCBPΔGIFΔ121, rGS14ΔCBPΔGIF and GS14 viruses were inoculated into 6-well plates with full confluency of PGT cells. Cells were incubated at 37 °C with 5% CO2. The viral fluid was collected at 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h post-infection. The viral fluid was frozen and thawed three times. Then, the lysate was titrated for viral load at each time point by TCID50 and quantitative PCR (qPCR).Equal TCIDTo investigate the genetic stability of rGS14ΔCBPΔGIFΔ121, we performed consecutive virus passaging in PGT cells. PGT cells were infected with low MOI (~1) recombinant virus and harvested 7 h post infection. The virus stability of the 121 gene deletion on the 1st, 5th, 10th and 15th passage was evaluated by PCR.6TCID50 each via inner thigh scratch inoculation. The animals were photographed on days 5, 7 and 14 post-inoculation. Clinical signs were scored by 6 persons unrelated to this study every 2 days from the inoculation day. Clinical scores were performed according to Martins’ method [To assess the virulence of the gene-deficient recombinant virus on goats, 16 antibody-negative one-month-old goats were divided into four groups and inoculated with rGS14ΔCBPΔGIFΔ121 (s goats per group) (hereafter referred to as rGS14ΔCBPΔGIFΔ121 group), GS14 (3 goats per group) (hereafter referred to as GS14 group), PBS (3 goats per group) (hereafter referred to as PBS group)and rGS14ΔCBPΔGIF (3 goats per group) (hereafter referred to as rGS14ΔCBPΔGIF group) at 0.2 mL 10’ method . Four cl+, CD8+ T cells and CD80+CD86+ dendritic cell differentiation using flow cytometry. The anticoagulant containing EDTA was added to the whole blood and mixed. A 100 μL mixture was taken and added with the corresponding antibodies , PE, Thermo; CD8 Monoclonal Antibody (38.65), FITC, Thermo; CD86 antibody|IL-A190, Bio-rad; CD80 antibody|IL-A159, Bio-rad, Hercules, CA, USA) following recommended antibody instructions. The sample was vortexed and then incubated for 15 min without light. Then, the sample was added to 2 mL of hemolysin containing an immobilizer. After vortexing and incubation for 10 min, the sample was centrifuged at 1500 rpm for 5 min to discard the supernatant. Samples were washed again with PBS and finally resuspended with 200 μL PBS and analyzed using flow cytometry (BD FACSAria II).Blood was collected on days 3, 5, 7 and 14 post-inoculation to determine CD4The expression of cytokines, including IL-2, IL-4, IL-10 and IFN-γ, was also examined on day 3, 5, 7 and 14 post-inoculation using commercial kits  ELISA Kit, Goat Interleukin 4, IL-4 ELISA Kit, Goat Interferon γ, IFN-γ ELISA Kit, Cusabio Biotech Co., PGTD, Wuhan, China).6TCID50. After 14 days, the goats’ homology was challenged on the right side of the thigh in the inner hairless area with the wild-type strain ORFV isolated from the scabs of the infected goat. The strain was preserved in our laboratory and the main virulence genes, such as vIL-10, vir, cbp, gif and 121, were sequenced by Zhu et al. in 2022 [−2 dilution. The virus was challenged according to the minimum pathogenic dose, observed, photographed and recorded for 14 days, and then scored according to the scoring criteria. Scabs from the challenging site and ORFV-susceptible tissues from the muzzle area were taken at the end of the observation period. Ten percent suspensions of tissue samples were ground, after which the viral load of ORFV was determined using the fluorescent quantitative PCR method previously established in our laboratory.The goats were grouped according to rGS14ΔCBPΔGIFΔ121 (11 goats per group) (hereafter referred to as G1), rGS14-ΔCBP-ΔGIF (3 goats per group) (hereafter referred to as G2), GS14 (7 goats per group) (hereafter referred to as G3), and PBS group (10 goats per group) (hereafter referred to as G4) and inoculated with a scratch on the left side of the thigh in the hairless area, and the inoculation dose is 0.2 mL 10p < 0.05 or p < 0.01.Statistical analysis of cytokine and PBMC differentiation data was performed using two-way analysis of variance by GraphPad Prism version 8.0 . Statistically significant was determined when The 121 gene and the sequence of the left and right homologous arms of recombinant rGS14ΔCBPΔGIF were determined. The 121 gene was successfully replaced with the GFP gene. After four rounds of purification, all the cytopathic areas of the virus emitted green fluorescence A, which −7/0.1 mL, which was similar to that of the parental ORFV-GS14 (10−6.7/0.1 mL) and rGS14ΔCBPΔGIF (10−6.2/0.1 mL). There was no statistical difference in the CPE produced at different time points after infection among the three viruses , while that of rGS14ΔCBPΔGIF started to rise on day 5 and maintained a high level of expression after reaching a peak on day 7 (p < 0.0001). The number of the CD4+cell subpopulation of the rGS14ΔCBPΔGIFΔ121 group was consistently higher than that of the rGS14ΔCBPΔGIF group in the first 7 days after immunization (p < 0.05) (p < 005), while that of the rGS14ΔCBPΔGIF group peaked on day 7 (p < 0.05) and then decreased (+CD86+ cells on day 3. rGS14ΔCBPΔGIFΔ121 group peaked on day 5 (p < 0.0001) and then started to decline. The rGS14ΔCBPΔGIF group peaked on day 7 (p < 0.0001) and then declined. The GS14 group peaked on day 5 (p < 0.0001) and then declined  B. In terecreased C. rGS14Δdeclined E.p < 0.001), starting to rise on day 3 (p < 0.0001) and reaching a peak on day 5 (p < 0.0001). The expression level of IL-2 in rGS14ΔCBPΔGIFΔ121 group was always higher than that in the GS14 group during the first 7 days (p < 0.05). GS14 caused a high level of IL-2 expression on day 14 (p < 0.05) (p < 0.01), reached a peak on day 5 (p < 0.0001) and gradually decreased after day 7 (p < 0.001). The rGS14ΔCBPΔGIFΔ121 group consistently expressed high levels of IL-10 during the first 7 days (p < 0.001) and then decreased (p < 0.0001)  A. Detectecreased B. In ter 0.0001) C. By det 0.0001) D. Taken Protective efficacy is an important parameter of a vaccine. We investigated the immune-protectivity properties of different live vaccines by using wild-type strain obtained from scabs of infected goats after immunizing the animals. Within 14 days after challenging, all members of the control group developed pustules, papules and scabs, which proved the validity of this experiment. In contrast, the goats in G1 showed bleeding, redness and swelling on day 2 post-challenge. Clinical signs were gradually alleviated over time and almost completely subsided by day 7. Only a few goats still had mild inflammatory reactions, which disappeared by day 14. The immuno-protection rate could reach 100%. G2 and G3 groups showed pustules, papules and other signs on day 7 after the immunization. Some goats still showed scabs and other signs by day 14, so the protection rate could not reach 100% A. The clORF is prevalent worldwide as a zoonotic disease, and it has caused huge economic losses to the sheep industry . HoweverIn this study, we constructed a triple-gene-deletion recombinant strain rGS14ΔCBPΔGIFΔ121 based on the double-gene-deletion recombinant strain rGS14ΔCBPΔGIF constructed in the previous study  and expl+CD86+ double-positive cells, but rGS14ΔCBPΔGIF and rGS14ΔCBPΔGIFΔ121 induced higher and more expression with a longer-lasting effect. This might be related to the deletion of CBP and GIF, which could weaken the immune evasion of the virus [+CD86+ cells induced by rGS14ΔCBPΔGIF consistently showed a slow increase, suggesting that rGS14ΔCBPΔGIF also greatly weakened immune evasion. Among the variations in the horizontal expression of CD4+ T cells and CD8+ T cells, both rGS14ΔCBPΔGIFΔ121 and rGS14ΔCBPΔGIF induced higher levels of expression, which might be related to the intrinsic properties of ORFV. The immune responses elicited by both rGS14 and rGS14ΔCBPΔGIFΔ121 appeared earlier after weakening the immune evasion. GS14 group did not show the immune responses elicited by ORFV during the 14-day experiment due to its immune evasion and individual differences between animals. In the CD4+ T cell assay, the overall expression level of rGS14ΔCBPΔGIFΔ121 was consistently higher than that of rGS14ΔCBPΔGIF during the first 7 days and was significantly different from that of the rGS14ΔCBPΔGIF group, indicating that rGS14ΔCBPΔGIFΔ121 caused a much higher level of cellular immune. In the CD8+ T cell assay, rGS14ΔCBPΔGIF caused more significant changes than rGS14ΔCBPΔGIF in goats at day 5. rGS14ΔCBPΔGIFΔ121 consistently maintained high levels of CD8+cell expression within 14 days. We suggest that the deletion of the 121 gene greatly diminishes the virulence and immune evasion of ORFV itself and that it can induce an earlier, stronger and longer-lasting immune response, which will greatly shorten the time for the host to react with an immune response when re-infected.For vaccines, safety and stability are the two important indicators. In this study, we minimized the risk of virulence reversion and mutation by knocking out the virulence gene. The safety of GS14, rGS14ΔCBPΔGIF and rGS14ΔCBPΔGIFΔ121 was evaluated by vaccinating 1-month-old goats. The results showed that both rGS14ΔCBPΔGIF and rGS14ΔCBPΔGIFΔ121 met the safety criteria. In the differentiation assay of PBMC, all three viruses could induce high expression of CD80he virus ,36. By cBy further exploring the immune response induced by the recombinant virus rGS14ΔCBPΔGIFΔ121, we found that it could cause high levels of IL-2, IL-10 and IFN-γ expression. Among these, the high level of IL-10 expression was related to the characteristics of ORFV itself. As the virus proliferated, host cells continued to secrete IL-10, thereby suppressing the cellular immune response . This suIn the assay to detect immune protection, we used wild-type ORFV on goats inoculated with recombinant virus GS14, rGS14ΔCBPΔGIF and rGS14ΔCBPΔGIFΔ121 separately. The results showed that the immune protection rates of triple-gene deletion mutant, double-gene mutant, and the parental virus were 100% (11/11), 66.7% 2/3), and 28.6% (2/7), respectively, which were different from the previous result , and 28.. NF-κ B In summary, the triple-gene deletion strain rGS14ΔCBPΔGIFΔ121 was successfully constructed in this study. The safety, immunogenicity and protective rates were evaluated. The result showed that the triple-gene deletion mutant strain rGS14ΔCBPΔGIFΔ121 could induce Th-1-like immune response and fully protect goats against the wild-type ORFV challenge. The prototype vaccine candidate, elicited earlier, increased and sustained immune responses.


There were errors in the original publication .The CAD system version and an inequality sign in the definition should be corrected.A correction has been made to 2. Materials and Methods, 2.4. Kinetic Analysis, 1st paragraph:The sentence “Kinetic information was retrospectively analyzed on a dedicated workstation using a CAD system .” should be replaced with “Kinetic information was retrospectively analyzed on a dedicated workstation using a CAD system .”.The sentence “The enhancement rate, defined as the signal change between the pre- and first post-contrast images, was categorized as slow (<50% increase), medium (50–100%), or rapid (≥100%).” should be replaced with “The enhancement rate, defined as the signal change between the pre- and first post-contrast images, was categorized as slow (<50% increase), medium (50–100%), or rapid (>100%).”.p-value should be corrected.A A correction has been made to 3. Results, 3.1. Patient and Lesion Characteristics, 1st paragraph:p < 0.001), heterogeneous enhancement (p = 0.012), and rim enhancement (p = 0.012).” should be replaced with “In the morphological analysis, malignant masses more frequently showed non-circumscribed margins (p < 0.001), heterogeneous enhancement (p = 0.012), and rim enhancement (p = 0.014).”.The sentence “In the morphological analysis, malignant masses more frequently showed non-circumscribed margins .“ should be replaced with “For the detection of all malignancies including in situ carcinoma, the enhancement degree, enhancement curve type, and size showed significantly better AUC values compared to morphological analysis alone .”.The sentence “For the detection of all malignancies including in situ carcinoma, the enhancement degree, enhancement curve type, and size showed significantly better AUC values compared to morphological analysis alone .” should be replaced with “For the detection of invasive cancers, enhancement degree and size showed significantly better AUC values compared to morphological analysis alone .”.The sentence “For the detection of invasive cancers, enhancement degree and size showed significantly better AUC values compared to morphological analysis alone (0.72 for each vs. 0.6; Incorrect dividing lines inside the In the fifth line of Figure S1, “circumscribed” should be corrected to “heterogeneous enhancement.” The corrected The authors state that the scientific conclusions are unaffected. This correction was approved by the academic editor. The original publication has also been updated.


The objective of this study was to investigate the bi-directional associations between experienced and witnessed gender-based harassment (GBH) on the one hand, and depressive symptoms and psychological treatment on the other, in an occupational setting. GBH are behaviors that derogate, demean, or humiliate an individual based on his or her gender.The analyses were based on data from the Swedish Longitudinal Occupational Survey of Health at 2018 (T1) and 2020 (T2), including 6,679 working participants (60.3% women) with a majority in the age range of 45–64. Using cross-lagged structural equational models, we analyzed experienced and witnessed GBH in relation to depressive symptoms and having received psychological treatment  over time.p ≤ 0.001) and having received psychological treatment  weakly predicted experiences of GBH over time. Having received psychological treatment was furthermore weakly associated with witnessed GBH .Our results showed that neither experienced nor witnessed GBH was prospectively associated with depressive symptoms or psychological treatment over two years. Both higher levels of depressive symptoms (β = 0.002, In conclusion, the hypothesized associations between exposure to GBH and mental health outcomes were not statistically significant, while a weak reverse association was noted. More research addressing bidirectional associations between GBH and mental health outcomes are needed. SLOSH is a longitudinal project that builds on participants of the Swedish Work Environment Surveys . SWES is a nationally representative sample of the Swedish working population whose participants are invited to participate in the SLOSH cohort. The survey has been conducted biennially since 2006 and consists of a self-completion questionnaire in two versions, one for those who work 30% or more full-time and one for those who work less or not at all. SLOSH focuses on connecting various work-related variables with issues about individuals' health and general wellbeing. A detailed description of the SLOSH cohort can be found in Magnusson Hanson et al. ( and 2020n = 7546). Due to most self-employed businesses (96%) having <10 employees  were excluded, reducing the sample to 6679 individuals (60.29% women and 39.71% men). To be able to separate the effect of witnessing GBH from the effect of experiencing GBH personally, we excluded individuals who reported that they had experienced GBH (n = 381) in the analyses of witnessed GBH. This resulted in a sample of n = 6298 (58.91% women and 41.09% men). Information on socio-demographic characteristics was obtained from the registered Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA) and linked to data from the SLOSH questionnaire. The study was approved by the Regional Research Ethics Board in Stockholm (No: 2019-05590).The overall response rates for the SLOSH waves 2018 (T1) and 2020 (T2) were 48% and 49%, respectively. For our study, we selected participants who worked at least 30% in 2018 and 2020  managers, (b) co-workers, or (c) others ?”. Witnessed GBH was measured with a single question; “Have you during the past 6 months, heard or seen someone else in the workplace being harassed because of their gender?”. The four response alternatives for all variables ranged from “At least once during the week” to “No”. Due to restrictions in statistical power, the three questions about experienced GBH were combined into one variable, and participants who reported any exposure were categorized as exposed, regardless of the reported frequency and who they identified as the harasser. The variable for witnessed GBH was also dichotomized in the same way.Depressive symptoms were measured with The Symptom Checklist-core depression (SCL-CD6)  shorter psychological treatment such as motivational or support interventions with a counselor, and (b) psychotherapy, i.e., longer psychotherapy with, e.g., a psychologist or psychotherapist”. The response options were “Yes” and “No”. Answering “Yes” to either of the two questions were considered as having received psychological treatment.Swedish Demand Control Questionnaire  to 4 (No). For our analysis, the alternatives were dichotomized into “No” or “Yes”, where yes indicated being exposed at least once. Job demands and decision authority were measured using the The following covariates were retrieved from LISA and linked to SLOSH survey items through the Swedish personal number. Information on participants' gender was available with the options “woman” and “man”. Age was categorized into five groups . Marital status was coded as a dichotomous variable, with 1 indicating married/cohabiting and 0 otherwise. Education was coded as “up to 9 years”, “up to 12 years”, “university education <3 years”, and “university education ≥3 years”. Country of birth was dichotomized as “Swedish” and “Other”. Origin of parents was categorized as “One or both parents born in Sweden” and “Parents born outside Sweden”. Disposable income was categorized into quartiles. Type of occupation was categorized from the Swedish Standard Classification of Occupations (SSYK), which in turn is based on the international standard ISCO-08, and encompasses all occupations on the Swedish labor market and includes nine categories: “Legislators, senior officials, managers”, “Professionals”, “Technicians”, “Clerks”, “Service workers”, “Agricultural and fishery workers”, “Craft workers”, “Machine Operators”, and finally “Elementary occupations”.We employed structural equation modeling (SEM) techniques to fit our data. All analyses were run in STATA using the Stata command SEM. The utilized two-wave autoregressive cross-lagged panel design allows for the examination of the mutual effects of two variables on one another over time with a 2-year time lag. This approach presents several advantages such as the construction of latent variables, simultaneous estimation with correlated residuals, and management of missing data  and the outcomes of depressive symptoms and psychological treatment. Model 1 was an unadjusted, reciprocal model including all autoregressive and cross-lagged paths between the two time points 2018 (T1) and 2020 (T2). This model examined the cross-lagged paths from experienced GBH at T1 to depressive symptoms at T2, the reversed paths from depressive symptoms at T1 to experienced GBH at T2, and the paths from experienced GBH and depressive symptoms at T1 to the same variables at T2. Model 2 extended Model 1 by adjusting for the socio-demographic covariates at T1 . Model 3 included the workplace covariates, job demands, and job control in addition to the demographic covariates. Model 4 additionally controlled workplace violence and bullying. These four models were fitted separately for experienced and witnessed GBH with the respective outcome variables of depressive symptoms and psychiatric treatment. The fit of the models was assessed via several fit statistics: the root mean square error of approximation (RMSEA), the comparative fit index (CFI), the standardized root mean square residual (SRMR), and Tucker–Lewis index (TLI), with cutoff values recommended for a good fit being RMSEA ≤ 0.08, CFI ≥ 0.90, SRMR ≤ 0.08 and TLI >0.90. Models 1–3 are presented in The descriptive statistics for the study variables in SLOSH T1 (2018) and T2 (2020) are presented in p = 0.909). The estimate for the small reverse association, from depressive symptoms at T1 to experienced GBH at T2, was . The auto-regressive paths between T1 and T2 for both experienced GBH  and depressive symptoms  indicate large effects and within-variable stability across time. The fit statistics were RMSEA = 0.000, CFI = 1.000, SRMR = 0.000, TLI = 0.995.The estimated paths between experienced GBH and depressive symptoms for the fully adjusted model 4  are shown in p = 0.139). For the reversed association between psychological treatment and experienced GBH, the estimate was small . The auto-regressive paths for experienced GBH  and psychological treatment  indicate stability in the variables across time. Fit statistics for experienced GBH and psychological treatment were RMSEA = 0.000, CFI = 1.000, SRMR = 0.000, and TLI = 1.000.The paths between experienced GBH and psychological treatment are illustrated in p = 0.906) or for the reversed path between depressive symptoms at T1 and witnessed GBH at T2 . Auto-regressive paths from T1 to T2 were for depressive symptoms 0.570 (p = 0.000) and for witnessed GBH 0.114 (p = 0.000). Fit statistics for the model were RMSEA = 0.000, CFI = 1.000, SRMR = 0.000, and TLI = 1.000.The associations between witnessed GBH and depressive symptoms for the fully adjusted model are shown in p = 0.408). Psychological treatment at T1 was associated with witnessed GBH at T2 . Auto-regressive paths from T1 to T2 indicated stability for both psychological treatment  and witnessed GBH . The corresponding fit statistic values were RMSEA = 0.000, CFI = 1.000, SRMR = 0.000, and TLI = 1.000.All associations for the fully adjusted model are depicted in In the current study, we tested hypothesized and reversed longitudinal associations between experienced or witnessed GBH on the one hand, and depressive symptoms and psychological treatment on the other, between the years 2018 and 2020. We found some support for weak reversed longitudinal associations between indicators of poorer mental health  and future exposure to experienced and witnessed GBH but no support for the hypothesized longitudinal associations.Our study sheds new light on previous findings, showing that having experienced or witnessed GBH  over time led to poorer mental health . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.JP: Conceptualization, Writing—original draft, Writing—review & editing. AN: Conceptualization, Funding acquisition, Methodology, Project administration, Writing—original draft, Writing—review & editing. PP: Data curation, Formal analysis, Methodology, Writing—review & editing.


There is a need to increase understanding of the effectiveness of bystander programmes targeting gender-based violence in the United Kingdom. There is also a need to utilise a robust theoretical models of decision-making while doing so. Changes were examined in bystanders’ attitudes, beliefs, motivations towards intervening, and intervention behavior in situations of gender-based violence. To achieve this, a quantitative examination of Mentors in Violence Prevention was conducted. There were 1396 participants  who were aged 11 to 14 years old  attending high school at the first time point. Participants were attending 17 schools (53% Mentors in Violence Prevention and 47% control) in Scotland. Outcome variables were assessed approximately one year apart using questionnaires. Multilevel linear regressions revealed that Mentors in Violence Prevention did not change outcomes reflecting bystanders’ attitudes, beliefs, motivations towards intervening, or intervention behavior in gender-based violence. Discrepancies between the current findings and those of other evaluations may be due to other studies including small numbers of schools that may be more motivated to implement the program. This study also identified two key issues that need to be addressed at stakeholder level before concluding that Mentors in Violence Prevention is ineffective at targeting gender-based violence. That the program has moved towards a more gender-neutral approach in the United Kingdom could explain the null results of this study. Furthermore, the current findings could be attributed to a failure to adequately address the theoretical model underpinning the program in practice. Gender-based violence is violence aimed at someone because of the gender with which they identify  for assessing the effectiveness of Mentors in Violence Prevention. They were therefore included in the confirmatory analyses in this study. In line with the preregistrationN = 1270), 2.4% identified as “Asian, Asian Scottish/ British”, 0.9% identified as “African”, 1.9% identified as “Mixed or multiple ethnic group”, 0.4% identified as “Caribbean or Black” and 2.9% identified as “Other ethnic group”. Socioeconomic status, as assessed by the percentage of students in each school who were registered for free school meals, ranged from 1.0% - 50.2% .The final sample at both T1 and T2 was 1396 (50% attending Mentors in Violence Prevention (MVP) and 50% attending non-MVP schools) students attending 17 (9 MVP and 8 non-MVP) mainstream high schools in Scotland between Autumn 2018 and Spring 2020. Participants were aged 11 to 14 years old . Overall, 689 (49.4%) reported their gender as female, 685 (49.1%) as male, 9 (0.6%) “prefer not to say”, and 13 (0.9%) did not report. Ninety one percent of the sample identified as “White Scottish or White British” , simulation analyses were conducted , and then subsequently again at T2, approximately one year later. Covariates  were measured at T1 only.A total of 2079 young people in S1-S3 attending 19  mainstream secondary schools in Scotland consented to take part at T1. However, between T1 and T2, five of the ‘MVP’ schools were unable to implement MVP due to staffing and structural issues: therefore, three subsequently became non-MVP schools and two withdrew entirely due to school closures related to the COVID-19 pandemic. In addition, and as a result of the pandemic, two more non-MVP schools had completed partial data collection before they were forced to close. Consequently, one school reported on intervention behavior only at T2 and the other school reported on all other variables only at T2. Both were included in the analyses. As described above, the final sample for T1 and T2 was 1396 students, with 50% (698) attending non-MVP schools and 50% attending MVP schools. However, participation within schools varied considerably due to whether positive or negative consent was sought as well as the availability of the target age groups at the time of fieldwork. Numbers of participants varied from 5 to 198 between schools.Ethical approval for this study was granted by the lead author’s institution. Scottish Local Education Authorities (LEAs) were contacted for permission to approach schools. Permission was obtained from 10 of 13 (77%) LEAs. All schools within the ten authorities were invited to take part. Of the schools contacted, 42 (26%) expressed interest in taking part and 19 did so. Time, resource limitations, and eligibility prevented some schools from participating. The research team aimed to recruit a balanced number of MVP and non-MVP schools. However, the fact that more MVP schools (14) opted into the study than non-MVP schools (5) potentially reflected the commitment of these schools to the MVP program or indeed their motivation to understand the effectiveness of a program that they had committed so much time and resource to. Schools could not be randomly assigned to these groups as the implementation of MVP is led by the Scottish Government and thus randomization was not within the control of the research team.Information letters and consent forms were distributed to all parents whose children were eligible. At the preference of the LEA, either negative (80% of LEAs) or positive (20%) consent was sought. Parents were given at least one week to return consent. Each young person also consented before participation. Participants completed anonymous questionnaires within a classroom or assembly hall context. At both T1 and T2, data collection occurred in two phases: all measures (see section below) except intervention behavior were completed in the first phase and reported intervention behavior was assessed in a second phase approximately one month later. The questionnaire completed in phase 1 took one school period (45–55 min) to complete, and 5–10 min to complete in phase 2. Teachers and members of the research team were present during data collection. Participants were subsequently debriefed. Students who were not participating were usually given another classroom task to complete or attended a non-participating classroom that was undertaking their work as normal.Scale items were drawn from the literature . The participants were asked to respond to each item using a 9-point unipolar scale . Similarly, six items were used to measure negative attitudes towards intervening . The participants were asked to respond to each item using a 9-point unipolar scale . For both the positive and negative scales, each of the six items were divided into “a” and “b” components, essentially making each of the scales consist of 12 indices. Factor scores were created where a higher score indicated higher positive or higher negative attitudes. Internal reliability was high at T1  and T2 .Both positive and negative attitudes were measured separately ”, from 1 = none of them to 9 = all of them. For this scale, each of the three items were divided into “a” and “b” components, essentially making the scale consist of six items. Factor scores were created, and a higher score indicated higher levels of perceived social pressure to intervene. Internal reliability was high at T1 (α = 0.82) and T2 (α = 0.79).A three-item scale was adapted ”, from 1 = not at all confident to 9 = very confident. For this scale, each of the three items were divided into “a” and “b” components, essentially making the scale consist of six items. Factor scores were generated where a higher score indicated higher self-efficacy to intervene. Internal reliability was high at T1 (α = 0.75) and T2 (α = 0.73).A three-item scale was adapted ”, from 1 = definitely no to 9 = definitely yes. For this scale, each of the four items were divided into “a” and “b” components, essentially making the scale consist of eight items. Factor scores were created, and higher scores indicated that participants regarded themselves as being more like the perceived prototypical student who intervenes regularly. Internal reliability was high at T1 (α = 0.91) and T2 (α = 0.90).A four-item scale was adapted ”, from 1 = strongly agree to 7 = strongly disagree. As described at the start of this section, three of the items included examples of verbal/emotional violence and the other three included examples of physical/sexual violence. Factor scores were created, and items were reverse scored so that a higher score indicated higher moral disengagement. Internal reliability was high at T1 (α = 0.91) and T2 (α = 0.90).A six-item scale was adapted …over the next month and no-one else there was doing anything about it/ none of your friends were intervening/ no-one else was around. How willing would you be to do something?” . For this scale, each of the three items were divided into “a” and “b” components, essentially making the scale consist of six items. The factor analytic approach revealed that this measure constituted “less serious” and “more serious” gender-based violence components, where “a” items loaded onto one factor and “b” items loaded onto another. These were therefore included as separate measures. Internal reliability was high for T1 , and T2 .A three-item scale was adapted , that which they intervened positively in more serious situations (positive interventionMoreSerious), that which they intervened negatively in less serious situations (negative interventionLessSerious) and that which they intervened negatively in more serious situations (negative interventionMoreSerious).Mentees reported if they had witnessed eight gender-based violence situations in the previous month  and could likely reflect student absences as well as refusals to complete the questionnaires on the date that this fieldwork was completed, which was around one month after the longer questionnaire, as described in the earlier design and procedure. Table boy and 1 = girl for analyses.Gender was reported as “a boy”, “a girl”, or “prefer not to say”. Those who responded “prefer not to say” or with missing responses were omitted due to small numbers . Gender was coded as 0 = Ages ranged from 11–15. No treatment of this variable occurred for analyses.White Scottish or White British, and 1 = other ethnic group for analyses.Participants reported their ethnicity as “White Scottish or White British”, “Asian, Asian Scottish/ British”, “African”, “Mixed or multiple ethnic group”, “Caribbean or Black”, and “Other ethnic group”. Due to small numbers in each of these categories of response aside from “White Scottish or White British” (see participants section), all other ethnic groups were collapsed into one group “other ethnic group”. Ethnicity was coded as 0 = never true to 4 = always true. A mean score was created where a higher score indicated higher affective empathy. Internal reliability was satisfactory (α = 0.77).A six-item scale was used ”, from 1 = no control at all to 9 = complete control. For this scale, each of the two items were divided into “a” and “b” components, essentially making the scale consist of four items. Factor scores were created, and a higher score indicated higher perceived control over intervening. Internal reliability was high at T1 (α = 0.88) and T2 (α = 0.90).A two-item scale was adapted ”, from 1 = very unlikely to 5 = very likely. As described at the start of this section, four of the items included examples of verbal/emotional violence and the other four included examples of physical/sexual violence. Factor scores were generated where a higher score indicated higher intentions to intervene. Internal reliability for this scale was high at T1 (α = 0.95) and T2 (α = 0.93).An eight-item scale was adapted , and the covariates were gender, age, ethnicity, and affective empathy. The multilevel approach allowed within school effects to be estimated using intraclass correlations. Three exploratory multilevel linear regressions were also planned. Due to the large number of analyses, alpha for exploratory analyses was adjusted to p < 0.01.A series of confirmatory multilevel linear regressions were planned for the anticipated outcomes. A multiple indicator factor-analytic approach was preregistered to be used to compute latent change scores representing outcome variables. However, the number of parameter estimates (116) outnumbered the number of clusters (16 schools) in the study, and so a more parsimonious model was required. An observed change score was thus computed  to 1223 participants (12.4% missingness). For the self-reported intervention behavior outcome variables, missing data was determined by whether or not participants had the opportunity to intervene in more serious or less serious gender-based violence in the preceding month (see measures section). This was true for both T1 and T2, so some participants who had the opportunity to intervene at T1 may not have had the opportunity at T2 and vice-versa. For the more serious intervention behavior outcome variables, the sample size was 288 (79.4% missingness) who had the opportunity to intervene at both T1 and T2. For the less serious intervention behavior outcome variables, the sample size was 618 (55.5% missingness) who had the opportunity to intervene at both T1 and T2. For the covariates, a Full Information Maximum Likelihood (FIML) approach was implemented in MPlus to address any missing data.Table p = 0.002, d = −0.12, and moral disengagement by justifying gender-based violence, t (df = 548) = 2.55, p = 0.006, d = −0.11, improved from T1 to T2. However, positive interventionMoreSerious (positive intervention in more serious gender-based violence) deteriorated from T1 to T2, t (df = 135) = 1.85, p = 0.033, d = −0.16.The differences between T1 and T2 scores across all outcomes were not statistically significant for non-MVP schools. For MVP schools, negative attitudes towards intervening, t (df = 565) = 2.91, MoreSerious) had higher similarity among those within the same schools than others , and that non-MVP schools generally had more outcomes showing higher similarity between those in the same school than MVP schools.ICC scores ranged from 0.00 to 0.09 (Median = 0.01) in non-MVP schools and 0.00–0.08 (Median = 0.08) in MVP schools, indicating that some outcomes , and positive interventionLessSerious (positive intervention in less serious violence). Specifically, as age increased the change scores in subjective norms concerning beliefs about other bystander’ intervention behaviors deteriorated, and the change scores in positive and negative interventionLessSerious improved. Empathy also predicted self-efficacy, so as empathy increased, self-efficacy concerning confidence in one’s own ability to intervene improved.As can be seen in Tables p < 0.001, d = 0.15, and non-MVP, t (df = 596) = −3.46, p < 0.001, d = 0.14, schools. ICC scores were 0.01 for MVP schools and 0.00 for non-MVP schools. MVP exposure did not have an effect on this variable’s change score , suggesting that those in MVP schools did not change significantly in their levels of perceived behavioral control over intervening compared to those in non-MVP schools. There was also no effect for the covariates.Perceived behavioral control over intervening increased significantly from T1 to T2 in both MVP, t (df = 553) = −3.63, p < 0.001, d = 0.20, and non-MVP, t (df = 611) = −2.89, p = 0.002, d = 0.12, schools. It was not possible to compute ICC scores  for MVP schools though this was 0.01 for non-MVP schools. MVP exposure did not have an effect on this variable’s change score , however gender  and empathy  did. This suggests that those in MVP schools did not change significantly in intentions compared to those in non-MVP schools, that intentions to intervene improved more for boys and that, as empathy increased, intentions to intervene improved more.Behavioral intentions to intervene increased significantly from T1 to T2 in both MVP, t (df = 547) = −4.56, MoreSerious to intervene did not change significantly from T1 to T2 in MVP , but increased in non-MVP schools, t (df = 572) = −1.79, p = 0.033, d = 0.10, schools. ICC scores were 0.03 in MVP schools and 0.02 in non-MVP schools. MVP exposure did not have an effect on the change score for this variable , suggesting that those in MVP schools did not change significantly in their levels of willingnessMoreSerious compared to students in non-MVP schools. There were also no effects for the covariates.WillingnessThere are few peer-reviewed studies that contain pre- and post-intervention and control methods to examine the effectiveness of programmes aimed at tackling gender-based violence by using a bystander intervention approach in the United Kingdom. Furthermore, existing studies, including those that are conducted outside of the United Kingdom, tend to provide a partial examination of bystander decision-making factors, rather than a full set of factors that comprise a robust theoretical model of decision-making. This study aimed to address these issues by providing insight into changes in bystander attitudes, beliefs, motivations, and behaviors when violence is witnessed among Scottish high school students. These outcomes were identified using a robust theoretical model of decision-making  as determined in the preregistered simulation analyses (OSF: 10.17605/OSF.IO/ZCJS6). Nonetheless, the design was sufficiently powered for all other anticipated outcomes, and the researchers endeavored to maintain consistency in the analyses used. Publication of null findings is an important step in building a comprehensive evidence base for this, or any, intervention. They inform future refinements in the application of interventions (Miller-Halegoua, Little is known about the effectiveness of bystander programmes targeting gender-based violence in the United Kingdom. Furthermore, studies tend to include a partial examination of bystander outcomes that do not fully comprise robust theoretical models of decision-making. The current study aimed to address these limitations by conducting a rigorous evaluation of the Mentors in Violence Prevention Katz,  program


Recently, Roy et al. proposed a physically unclonable function (PUF)-based authentication and key exchange protocol for Internet of Things (IoT) devices. The PUF protocol is efficient, because it integrates both the Node-to-Node (N2N) authentication and the Node-to-Server (N2S) authentication into a standalone protocol. In this paper, we therefore examine the security of the PUF protocol under the assumption of an insider attack. Our cryptanalysis findings are the following. (1) A legitimate but malicious IoT node can monitor the secure communication among the server and any other IoT nodes in both N2N authentication and N2S authentication. (2) A legitimate but malicious IoT node is able to impersonate a target IoT node to cheat the server and any other IoT nodes in N2N authentication and the server in N2S authentication, respectively. (3) A legitimate but malicious IoT node can masquerade as the server to cheat any other target IoT nodes in both N2N authentication and N2S authentication. To the best of our knowledge, our work gives the first non-trivial concrete security analysis for the PUF protocol. In addition, we employ the automatic verification tool of security protocols, i.e., Scyther, to confirm the weaknesses found in the PUF protocol. We finally consider how to prevent weaknesses in the PUF protocol. In recent years, the Internet of Things (IoT) has been growing rapidly and all kinds of IoT devices are often present in our personal lives in the form of various applications, such as smart homes, smart transportation, and smart cities. However, designing authentication and key exchange protocols is an intractable task for IoT applications. Firstly, there is a huge number of IoT devices, but their computing and storage resources are usually limited. Therefore, lightweight protocols are urgently needed to realize efficient interaction between devices without overloading their performance. Secondly, IoT devices are often deployed in complex, heterogeneous network environments and can be connected to and accessed by multiple unknown and untrusted devices, leading to extreme security challenges. Robust authentication and key exchange protocols should satisfy all strict requirements from IoT devices and their applications.The physically unclonable function (PUF)  can helpOn the one hand, PUF has advantages in efficiency and security compared with cryptographic algorithms. Hence, it is expected to design more practical authentication and key exchange protocols for IoT. On the other hand, PUF-based authentication and key exchange protocols are relatively new, compared with the protocols using purely cryptographic algorithms and/or smart cards. And there are still many security issues in its designs, which should be addressed. Hence, in this paper, we investigate the PUF-based authentication and key exchange protocol for IoT devices.The PUF-based authentication and key exchange protocols using public key algorithms. The public key algorithms have high key management flexibility. This is because the communicating parties have two different keys, one of which is kept as a secret and the other is made public. There is no need to share the secret key as in symmetric key algorithms. The PUF-based protocols using public key algorithms inherit this feature. Yilmaz et al. [z et al.  applied z et al.  developez et al.  designedz et al.  proposedz et al.  proposedz et al.  proposedz et al.  proposedz et al.  introducThe PUF-based authentication and key exchange protocols using symmetric key algorithms. The advantage of symmetric key algorithms is low computational cost. These algorithms are therefore well suited for authentication and key exchange protocols for resource-constrained IoT devices and help to reduce the computational burden of these IoT devices. In the PUF-based protocols using symmetric key algorithms, shared keys are generated by PUFs and hash functions are commonly used to authenticate messages. The researchers [earchers ,14 propoearchers  presenteearchers  proposedearchers  proposedearchers  proposedearchers  leverageearchers  designedearchers  proposedearchers  further earchers  introducearchers  employedearchers  introducearchers ,27,28 prCryptographic algorithms are widely used in security protocols. According to the type of cryptographic algorithm, we divide PUF-based authentication and key exchange protocols into two categories as follows:IEEE Internet Things J. 2023, 10, 8547–8559, Roy et al. [In y et al.  proposed(1)A legitimate but malicious IoT node can monitor the secure communication among the server and any other IoT nodes, because the malicious IoT node can reveal their secret session key. The only requirement of the malicious IoT node is to observe the transmitted messages over public channel during the authentication and key exchange phase of Roy et al.’s protocol.(2)A legitimate but malicious IoT node is able to impersonate a target IoT node to cheat the server and any other IoT nodes during the authentication and key exchange phase of Roy et al.’s protocol. Finally, the malicious IoT node can establish a secret session key for the subsequent secure communication.(3)A legitimate but malicious IoT node can masquerade as the server to cheat any other target IoT nodes during the authentication and key exchange phase of Roy et al.’s protocol. Here, the malicious IoT node is able to generate the valid session key for the target IoT nodes.Due to their inherent vulnerability, IoT devices (nodes) are easily compromised by insider attackers. Therefore, in this paper, we carefully study the security of Roy et al.’s protocol under the assumption of an inside attack. Our results are summarized as follows:n-bit response iR to an m-bit challenge iC for any i∈iC∈{0,1}m and iR∈{0,1}n.PUFs make use of the intrinsic random variability in the physical microstructure of integrated circuits (ICs) to produce a unique iC and jC, with i, j∈APUFf and BPUFf, the uniqueness expression is as follows.Uniqueness: PUFs cannot output the same response for different input challenges, while different PUFs output different responses for the same input challenge. For two different t1 and t2 is:Reliability: This feature measures the reproducibility of a PUF response under different operating conditions for a given challenge. The reliability expression of the PUF instance for different time periods In fact, the PUF response is sensitive to various noise elements, such as temperature, voltage, and other environmental changes. Under the same challenge, the noise can cause the PUF to give an erroneous response, which is different from the original one. To eliminate the effects of such noise, Roy et al.  considerPUF has the following safety features:The one-time enrollment phase is responsible for enrolling the new IoT nodes in the server. The authentication and key exchange phase realizes mutual authentication between the server and the node(s). To maintain consistency, we employ the same notions of  and writA wants to enroll in the server, the server randomly generates an input AiC  for A’s PUF and collects its output AiR , where AiR = APUFf(AiC). Then, the server stores A’s ID-CRP {Aid, AiC, AiR} in its secure database. The server only maintains one ID-CRP record for each IoT device.This phase is run in a secure environment without the attacker. After completing this phase, the IoT nodes can be deployed in the IoT network. When any This phase has two communication models, i.e., Node-to-Node (N2N) communication and Node-to-Server (N2S) communication. N2N communication aims to provide the mutual authentication and key establishment of any two IoT nodes. Meanwhile, N2S communication realizes the mutual authentication and key establishment between any one IoT node and the server.A and B nodes want to authenticate each other and establish their session key. As shown in A, B, and the server perform the following steps.Assume that two nearby A sends a connection request {Aid} to B. Upon receiving this request, B sends the N2N connection establishment request {Aid, Bid} to the server.Step 1. Aid, AiC, AiR} and {Bid, BiC, BiR} from its secure database and generates two random numbers RN and ABT. The server computes:AiC, AM, BM, AH, BH, ABT’} to A and the message {BiC, AM, BM, AH, BH, ABT’} to B.Step 2. The server fetches corresponding ID-CRPs {AiC, AM, BM, AH, BH, ABT’}, A first computes:A then checks whether AH is equal to AH* and BH is equal to BH*. If any verification fails, A terminates the session. Otherwise, A further calculates:A sends {BT} to B.Step 3. Upon receiving the message {BiC, AM, BM, AH, BH, ABT’} and {BT}, B computes:B checks AH and BH using AH* and BH* just like A. If any verification fails, B also terminates the session. Moreover, B evaluates:B checks whether BiR is equal to BiR*. If the verification is incorrect, B terminates the session; otherwise, B computes:B sends {AT} to A and {SBM, SBH} to the server.Step 4. Upon receiving the messages {AT}, A computes:Step 5. Upon receiving the message {A checks whether AiR is equal to AiR*. If the verification is incorrect, A terminates the session; otherwise, A computes:A then sends {SAM, SAH} to the server.SAM, SAH} and {SBM, SBH}, the server computes:SAH is equal to SAH* and SBH is equal to SBH*. If any verification is incorrect, the server terminates the session; otherwise, the server computes:Aid, AiC, AiR} to {Aid, AC*i+1)(} in its secure database.Step 4. Upon receiving the message {B and the server share BRi+1)A eavesdrops on B’s {Aid, Bid} during Step 1.(2)A eavesdrops on the server’s {BiC, AM, BM, AH, BH, ABT’} during Step 2.(3)A eavesdrops on B’s {SBM, SBH}.In Step 4, A can compute B’s BCi+1)) during Step 3. And A can correctly recover B’s BRi+1)) during Step 3. Finally, we conclude that A obtains B’s {Bid, BiC, BiR} used in the next session, i.e., {Bid, BCi+1)A eavesdrops on C’s {cid}.In Step 1, (2)A eavesdrops on the server’s {BiC, CM, BM, CH, BH, CBT’}.In Step 2, A can obtain the RN by computing BiR⊕BM, where BiR is collected in A’s previous session with B. A then recovers CBT by computing CBT’⊕RN. Moreover, if A wants to monitor B’s next session, it can further perform as follows.Now, A eavesdrops on B’s {SBM, SBH}.In Step 4, A can compute B’s BCi+1)A eavesdrops on the server’s {CiC, CM, BM, CH, BH, CBT’}.In Step 2, (2)A eavesdrops on C’s {SCM, SCH}.In Step 5, A computes CiR by using CM⊕RN and CCi+1) by running a session with the target IoT node. Afterwards, the insider attacker can expand their surveillance to other IoT nodes , which run the sessions with the target IoT node. Meanwhile, the insider attacker no longer needs to participate in those session runs and still obtains those secrets. This effectively hides the insider attacker and reduces the probability of the insider attacker being identified. Moreover, the insider attacker can establish a surveillance network of the selected IoT nodes based on our proposed attack.A is able to monitor the session run of the N2S communication model between B and the server. As shown in A can perform the following operations to reveal the secret session key BRi+1)( shared by B and the server.(1)A eavesdrops on the server’s {BiC, BM, BH}.In Step 2, (2)A eavesdrops on B’s {SBM, SBH}.In Step 3, A knows B’s BiR, A can obtain the RN by computing BiR⊕BM. Now, A further recovers the secret session key BRi+1)( by computing SBM⊕RN. A can therefore observe the following secure communication using BRi+1)C’s request {Cid} in Step 1, A sends the N2N connection establishment request {Cid, Bid} to the server.Upon receiving (2)BiC, CM, BM, CH, BH, CBT’} and {BT} in Step 4, A computes RN←BiR⊕BM, CiR←CM⊕RN, CBT←CBT’⊕RN, CT←CBT⊕CiR, and BCi+1)(⊕RN and SBH←hash(BRi+1)(‖SBM). Finally, A sends {CT} to C and {SBM, SBH} to the server.Upon receiving the messages {A randomly generates BRi+1)( instead of B’s BRi+1)) and send it to B in Step 2. However, in Step 4, B retrieves its local BiR by invoking BPUFf(BiC) (see Equation (17)). Clearly, the server’s BiR and B’s BiR are always not equal because of the PUF’s safety feature. Hence, B should recover an incorrect RN using Equation (18) and fail the subsequent authentication and key exchange procedure with the server and the counterpart IoT node. Therefore, B suffers from a denial of service attack due to A’s impersonation.A can impersonate B to cheat the server, when A obtains B’s {Bid, BiC, BiR}. As shown in A’s operations as follows.In N2S communication, (1)A sends the request {Bid} to the server in Step 1.(2)BiC, BM, BH} in Step 3, A computes RN←BiR⊕BM. Then, A generates BRi+1)(⊕RN and SBH←hash(BRi+1)(‖SBM). Finally, A sends {SBM, SBH} to the server.Upon receiving the message {A’s SBH in Step 4, and therefore we omit this operation in A applies BRi+1)A randomly generates RN and CBT. A further computes CM←CiR⊕RN, BM←BiR⊕RN, CBT’←CBT⊕RN, CH←hash(CiR‖CM), and BH←hash(BiR‖BM). Then, A sends the message {CiC, CM, BM, C HBH, CBT’} to C and the message {BiC, CM, BM, CH, BH, CBT’} to B.In Step 2, (2)SCM, SCH} and {SBM, SBH} in Step 6, A omits them.Upon receiving the messages {A finally shares the session key CBT with both C and B. Moreover, A still can reuse C’s {Cid, CiC, CiR} and B’s {Bid, BiC, BiR} to impersonate the server in the subsequent session, because C and B do not verify the freshness of CiC and BiC in each session run. Certainly, in Step 6, A can also compute CR*i+1)A generates the random number RN. A computes BM←BiR⊕RN and BH←hash(BiR‖BM). Then, A sends the message {BiC, BM, BH} to B.In Step 2, (2)SBM, SBH} in Step 4, A computes BR*i+1)(←RN⊕SBM and BC*i+1)(}.Upon receiving the message {A and B establish the secret session key BR*i+1). Scyther can analyze protocols that contain multiple subjects, infinite session interaction, and the use of random numbers. We therefore use Scyther to confirm our insider attacks on Roy et al.’s protocol. We implement experimental verification on the 64-bit Windows 10 operating system, Graphviz v2.50, and Python v2.7 using the Compile-0.9.2 version of the Scyther tool.When using the Scyther tool to verify Roy et al.’s protocol, we write secret PUF responses, XOR-encrypted messages, etc., into the SPDL script to model the protocol. The details of the Scyther tool parameter settings are shown in ABT in the N2N communication and the secret session key BRi+1)) to verify B (see Equation (14)) in the N2N communication of Roy et al.’s protocol. Similarly, B needs to recovering A’s AiR (see Equation (19)) to verify A (see Equation (20)). However, we know that both AiR and BiR are the secrets of A and B, respectively. It means that A and B obtain each other secrets after Step 4 of the N2N communication, which incurs the secure breach. In fact, these verifications conducted by A and B are unnecessary because both A and B believe the server. Therefore, one suggestion is to cancel the verifications, that is, the server does not send BM and BH to A and AM and AH to B during Step 2 of the N2N communication.We know that A and B share a random number RN in the N2N communication. RN is used to encrypt their all secrets. Because of sharing the RN, the malicious IoT node can decrypt other IoT node’s secrets. Hence, another suggestion is that the server must randomly select two random numbers instead of just one RN, that is, one random number for A and the other one for B.Moreover, we find that both The above two suggestions may defend against our proposed insider attacks. However, accurate security results for improvements require formal security models, security assumptions, security definitions, and reductions. We keep these for future work.ABT or BRi+1)(, he can always impersonate not only the IoT node to cheat other IoT nodes or the server but also the server to cheat the IoT nodes in the subsequent sessions. The secure communications between the server and the corresponding IoT nodes are also possibly under the attacker’s surveillance. This means that Roy et al.’s protocol fails to provide the known key security. Our security analysis results indicate that designing PUF-based authentication and key exchange protocols for IoT remains a challenging task.In the N2N communication of Roy et al.’s protocol, the IoT node requires the counterpart IoT node’s secret output of the PUF to verify it. More importantly, the malicious IoT node can derive the counterpart IoT node’s next output of the PUF if it eavesdrops on the message transmitted to the server. These defects have led to our insider attacks on Roy et al.’s protocol. Moreover, in the cases of the N2N communication and the N2S communication, we must point out that once the attacker stole the session keys, i.e.,