Predicting metastasis in melanoma patients is important for disease management and could help to identify those who might benefit from adjuvant treatment. The aim of this study was to investigate whether the tumor microenvironment-derived protein S100A8/A9 qualifies as prognostic marker for melanoma patients, also in the setting of immunotherapy.S100A8/A9 gene and protein expression were analyzed on melanocytic nevi, primary melanomas and metastases using a cDNA library and three independent tissue-microarrays (TMA). Serum levels of S100A8/A9 were measured using a specific ELISA in two independent cohorts of 354 stage III and stage IV melanoma patients as well as in two independent cohorts of patients treated with the PD-1 antibody pembrolizumab.P < .0001) and in melanomas of short-term survivors compared to long-term survivors (P < .0001). Serum S100A8/A9 levels > 5.5 mg/l were associated with impaired overall survival in two independent cohorts (both P < .0001). Importantly, patients with serum elevated S100A8/A9 treated with pembrolizumab showed significantly impaired survival compared to patients with lower S100A8/A9 levels .cDNA analysis revealed an upregulation of S100A8 and S100A9 gene expression in melanoma metastases compared to primary melanomas. Significantly higher numbers of infiltrating S100A8/A9 positive cells were found in tissue samples of metastasizing primary melanomas compared to non-metastasizing melanomas (The tumor microenvironment-associated protein S100A8/A9 serves as a novel prognostic marker for metastasis and survival of metastatic melanoma patients and predicts response to immunotherapy with pembrolizumab. These data underscore the significance of tumor microenvironment-derived factors as suitable biomarkers for melanoma. Melanoma is a highly malignant cancer that was associated with a short median survival in the pre-immunotherapy era. Nevertheless, its immunogenicity allows immune checkpoint inhibitors like anti-PD-1 and anti-CTLA-4 antibodies to achieve impressive response rates and a striking improvement of survival [Certain cell types present within the tumor microenvironment (TME) such as myeloid cells  have been identified to serve as prognostic markers in melanoma , 6. We hThe aims of this study were to investigate (i) whether S100A8/A9 expression in tissue sections of primary melanomas and melanoma metastases discriminates between short-term and long-term survivors, (ii) whether elevated serum S100A8/A9 is associated with impaired survival of stage III and IV melanoma patients, and (iii) whether elevated serum S100A8/A9 is associated with survival of melanoma patients treated with the anti-PD-1 immune checkpoint inhibitor pembrolizumab.For this study, seven independent sets of tissue and serum samples from melanoma patients were analyzed. Furthermore, an additional set comprising 237 cDNA samples was utilized for a gene expression analysis of the S100 super-family. The study was in accordance with the ethical standards and was approved by the local ethics committee of the University Medical Center Mannheim (Project number 2010-318 N-MA).Immunofluorescence analysis was performed as described previously , and nucDNA collection, RNA preparation, procession via SAGE™, and cDNA gene expression analysis was done as described earlier , 18. BriThe analyzes of protein expression of S100A8/A9 in the above mentioned three independent TMAs were conducted using formalin fixed and paraffin embedded tissue according to a previous report . BrieflyThree independent tissue microarrays (TMA) were utilized for analyzes of S100A8/A9 expression in melanoma tissue. TMA 1 contained samples of benign melanocytic nevi, non-metastasizing primary melanomas and metastasizing primary melanomas. TMA 2 and TMA 3 contained samples of primary melanomas and melanoma metastases deriving from patients who have all been diagnosed with metastatic disease. These two TMAs were designed to compare long-term vs. short-term survivors. Long-term and short-term referred to the timespan between first occurrence of metastatic disease (at this time the metastatic tissue samples were obtained) and death. Less than 12 months was considered short-term survival, more than 30 months was considered long-term survival. The TMAs were scored by experienced dermato-histopathologists in a blinded fashion regarding to the outcome of the patients.Collection of serum and documentation of clinical data were performed after patients’ informed consent with institutional review board approval. Blood draw was done using gel-coated serum tubes (Sarstedt). After centrifugation, serum was immediately stored at − 80 °C. Serum concentration of S100A8/A9 was measured in duplicate using commercially available sandwich ELISA kits . S100B and LDH levels had been determined routinely during regular follow-up.S100A8/A9 serum levels were measured in two independent sets of serum samples from 114 stage III and stage IV melanoma patients treated between 1990 and 2009 at University Hospital Essen, Germany (training set), and from 240 stage III and stage IV melanoma patients treated between 2007 and 2010 at University Hospital Tübingen, Germany , respectively. All samples were collected immediately after first diagnosis of stage III or stage IV disease, respectively. None of the patients had been treated systemically within 4 weeks before the blood withdrawal. Serum samples were chosen from the biobanks of the two University Hospitals Tübingen, and Essen, respectively, based on the amount of serum available. 32 of the samples of the training set were excluded due to missing follow-up information. None of the 354 patients of both sets were treated with CTLA-4 or PD-1 antibodies, nor with BRAF or MEK inhibitors. All patients included in this study were followed-up and were staged systematically by the departments for Dermatology of the two University Hospitals Tubingen and Essen according to the German melanoma guidelines.S100A8/A9 serum levels were analyzed in two independent sets of 27 and 44 patients, respectively, treated with the anti-PD-1 antibody pembrolizumab at University Hospital Mannheim, Germany (pembrolizumab set 1) and at University Hospital Tubingen, Germany (pembrolizumab set 2). The serum samples of the pembrolizumab treated patients were collected prospectively after written informed consent was obtained.Samples were selected according to the following criteria: histologically confirmed cutaneous melanoma, complete documentation of medical history, course of the disease, and follow-up. Follow-up time started at the date of initiation of pembrolizumab treatment and ended at the date of last follow-up or death. Primary endpoint in pembrolizumab set 1 was overall survival (OS). Median OS was not reached in pembrolizumab set 2. Therefore, primary endpoint in this set was progression-free survival (PFS). Patients received at least one cycle of pembrolizumab over 30 min at a dose of 2 mg/kg body weight. Treatment was repeated every 3 weeks according to the protocol approved by the European medicines agency (EMA). Staging was performed every 3 months according to the structured staging guidelines of the University Hospitals Mannheim and Tubingen, Germany. Radiologic responses were assessed using contrast-enhanced CT/MRI/PET-CT at around week 12 after the first pembrolizumab infusion and clinical response was defined based on immune-related response criteria (irRC). The peripheral blood was taken up to 5 days before or on the day of the first infusion.P-value [U test. Estimates of cumulative survival probabilities according to Kaplan-Meier were compared using two-sided log-rank test. Multivariate Cox proportional hazard analyses were used to evaluate the independent effects of S100A8/A9 on survival. Throughout the analyses, p-values less than .05 were considered statistically significant. All analyses were carried out using R.All cut-off values in this study were determined utilizing a previously described algorithm which selects the ideal cut-off point based on minimizing the P-value . For theImmunofluorescence analysis of S100A8/A9 expression in tissue sections of primary melanomas revealed an exclusive and abundant expression of S100A8/A9 in cells of the TME, mainly granulocytes, whereas S100B expression was restricted to melanoma cells    as well as analyzing metastatic tissue   as well as between metastatic samples using the same cut-off for S100A8/A9 positive cells     in stage IIB/III patients . The proThe rationale to conduct a study with S100A8/A9 as putative biomarker completely differs from known concepts of biomarkers like LDH or S100B. The latter markers are thought to be released upon tumor cell necrosis due to high turnover or shortage of their blood supply and to reflect tumor burden , 28, 30.Therefore, serum S100A8/A9 might be a biomarker depicting the aggressiveness and the metastatic potential of the tumor. Hence, rising serum levels of S100A8/A9 are likely to precede bulky tumor growth which finally leads to increases of S100B or LDH. Our data support this assumption with S100A8/A9 being superior to LDH and S100B in predicting survival in stage III patients. Moreover, S100A8/A9 was particularly valuable for patients presenting with normal levels of LDH  have been shown to be reliable biomarkers for non-response to immune-checkpoint inhibition , 6. S100Although we showed that S100A8/A9 is a valuable prognostic marker for stage III and IV melanoma patients and predicts the response to immune-checkpoint inhibition, it is not a specific melanoma marker. S100A8/A9 has been reported to be also upregulated in many malignancies and in chronic inflammatory disorders [In summary, our findings demonstrate that high numbers of S100A8/A9 expressing cells predict metastasis and are a powerful new tissue biomarker associated with short-term survival. It should be further evaluated as additional tissue marker completing Breslow’s vertical tumor thickness and ulceration of the primary. Therefore, future prospective studies with large cohorts focusing on its tissue marker value are needed. Moreover, our data imply that serum S100A8/A9 could be a valuable prognostic marker for stage III and stage IV melanoma as well as for patients undergoing immune checkpoint inhibition with pembrolizumab. Large-scale prospective studies will be required in order to confirm these results and to prove whether S100A8/A9 can become a standard biomarker in clinical routine. Altogether, by presenting clinical data, this study strengthens the mechanistic impact of S100A8/A9 on metastasis and progression in melanoma patients –15, 32.Additional file 1: Figure S1. PIQOR microarray assay reveals strong expression of S100A8 and S100A9 in melanoma metastases. Figure S2. S100A8/A9 predicts overall survival in stage III and in stage IV melanoma patients. Figure S3. Combinatory analysis of S100B and LDH with S100A8/A9 in serum of stage III and stage IV patients of the combined training and independent validation sets. Figure S4. Survival analysis of serum S100A8/A9 in stage III patient with normal levels of LDH or S100B. Table S1. Main characteristics of the tissue microarray sets. Table S2. Main characteristics of the serum marker sets. Table S3. Multivariate analysis of serum marker proteins and overall survival in stage III patients of the combined training and independent validation sets. Table S4. Multivariate analysis of serum marker proteins and overall survival in stage IV patients of the combined training and independent validation sets. Table S5. Main characteristics of the pembrolizumab sets.


During May 19–September 28, 2019,The U.S. Influenza Surveillance Systemhttps://www.cdc.gov/flu/weekly/overview.htm, and a full description of U.S. influenza activity over the summer months is available in the influenza surveillance report, FluView (https://www.cdc.gov/flu/weekly/).During May 19–September 28, 2019, the weekly percentage of outpatient visits to health care providers for influenza-like illness (ILI) from the U.S. Outpatient Influenza-Like Illness Surveillance Network (ILINet) was below the national baseline, and all regions were below their region-specific baselines. One human infection with a novel influenza A virushttps://www.who.int/influenza/surveillance_monitoring/updates/en/.The timing of influenza activity and the predominant circulating virus in the Southern Hemisphere during May 19–September 28, 2019 varied by region.CDC genetically characterized 867 influenza viruses submitted by U.S. and international laboratories during May 19–September 28, 2019, including 263 influenza A(H1N1)pdm09 viruses, 427 influenza A(H3N2) viruses, and 177 influenza B viruses. All A(H1N1)pdm09 viruses belonged to genetic subclade 6B.1A. Among 25 antigenically characterized A(H1N1)pdm09 viruses, 96% were similarAll 21 of the influenza B/Yamagata lineage viruses analyzed belonged to clade Y3. All seven B/Yamagata lineage viruses antigenically characterized were similar to the cell culture–propagated 2019–20 Northern Hemisphere vaccine virus component. Multiple genetically and antigenically distinct B/Victoria lineage viruses cocirculated. Viruses with a two-amino acid deletion (162–163) in the HA protein belonged to subclade V1A.1, and viruses with a three-amino acid deletion (162–164) in the HA protein belonged to subclade V1A-3Del. Among the 156 influenza B/Victoria lineage viruses analyzed, the HA gene belonged to clade V1A , subclade V1A.1 , or subclade V1A-3Del . Among the 53 B/Victoria lineage viruses antigenically characterized, the V1A.1 viruses were similar to the cell culture–propagated 2019–20 Northern Hemisphere vaccine component. Ferret antisera raised to recent V1A.1 viruses, however, had reduced reactivity with many viruses expressing V1A and V1A-3Del HA proteins indicating some antigenic differences between viruses in the different B/Victoria lineage subclades. Nevertheless, sera from humans vaccinated with a V1A.1 virus cross reacted well with V1A-3Del viruses.https://www.cdc.gov/flu/professionals/antivirals/links.htm.CDC tested 812 influenza virus specimens collected during May 19–September 28 from the United States and worldwide for resistance to oseltamivir, peramivir, and zanamivir. All but two of the viruses tested (245 influenza A(H1N1)pdm09 viruses [161 international and 84 U.S. viruses], 406 influenza A(H3N2) viruses [284 international and 122 U.S.], and 161 influenza B viruses [71 international and 90 U.S.]) were susceptible to these influenza antiviral medications. One (0.1%) influenza A(H1N1)pdm09 virus contained the H275Y amino acid substitution in the neuraminidase and exhibited highly reduced inhibition by oseltamivir and peramivir, and one (0.1%) influenza B virus contained the amino acid substitution I221T and exhibited reduced inhibition by the same two neuraminidase inhibitors. Among 824 influenza virus specimens (253 A(H1N1)pdm09, 406 A(H3N2) and 165 type B assessed for susceptibility to baloxavir, one (0.1%) A(H3N2) virus contained amino acid substitution I38L in the polymerase acidic (PA) protein, which was previously associated with at least a threefold decreased baloxavir susceptibility. High levels of resistance to the adamantanes (amantadine and rimantadine) persisted among influenza A(H1N1)pdm09 and influenza A(H3N2) viruses, which is consistent with the current recommendation to avoid use of these medications against influenza. Influenza antiviral recommendations are available at WHO recommendations for influenza vaccine composition for the Southern Hemisphere 2020 season were made at the WHO Consultation and Information Meeting on the Composition of Influenza Virus Vaccines held September 23–27, 2019, in Geneva, Switzerland.From May to September 2019, influenza activity remained low in the United States, as is typical for that time of year. Influenza A and B viruses cocirculated throughout the summer months with influenza A(H3N2) viruses predominating overall and influenza B/Victoria, subclade V1A-3Del, viruses the most common influenza B virus reported by public health laboratories. Influenza A and B viruses also circulated widely in the Southern Hemisphere with the predominant virus varying by region and country. It is too early in the season to know which viruses will circulate in the United States later this fall and winter or how severe the season might be; however, regardless of what is circulating, the best protection against influenza is an influenza vaccination. Influenza vaccination has been shown to reduce the risk for influenza illness associated with outpatient health care visits and hospitalizations and reduces the risk for serious influenza outcomes that can result in hospitalization or death. CDC recommends that all persons aged 6 months and older who do not have contraindications get vaccinated, but vaccination is especially important for persons at high risk for serious influenza-associated complications, including persons aged ≥65 years, children aged <5 years, pregnant women, and persons with certain underlying medical conditions.In late September, WHO issued its recommendations for the 2020 Southern Hemisphere influenza vaccine. Compared with the composition of the 2019–20 Northern Hemisphere influenza vaccine formulation, these recommendations reflect changes to the A(H3N2) and B/Victoria-lineage components. The update for the B/Victoria-lineage component reflects the global spread and increase of V1A-3Del viruses, which had reduced reactivity to ferret antisera raised to V1A.1 viruses used in 2019–20 Northern Hemisphere vaccines. Apart from North and South America, the majority of A(H3N2) viruses circulating elsewhere globally belonged to subclade 3C.2a1 and were antigenically different from the Northern Hemisphere 3C.3a vaccine component, leading to a change in the A(H3N2) component to a 3C.2a1 subclade virus for the Southern Hemisphere. These recommendations were made specifically for the Southern Hemisphere using many factors, including evolutionary approaches to forecast specific subgroups likely to circulate 6 months into the future, determining which candidate vaccine viruses induce immunity that blocks the largest variety of viruses and which viruses escape population immunity from prior infection or vaccination. These factors vary among countries within the Southern Hemisphere and certainly vary between the Southern and Northern Hemispheres. For example, activity in Australia during recent seasons has not reflected influenza virus activity in the subsequent U.S. season. Changes to the Southern Hemisphere vaccine composition, therefore, might not be a good predictor of the upcoming U.S. influenza season. Although Australia experienced an early start to its 2019 season with influenza A(H1N1)pdm09 viruses circulating initially and A(H3N2) virus eventually predominating (https://www.cdc.gov/flu.Except for one influenza A(H1N1)pdm09 virus and one influenza B virus, all influenza viruses tested remained susceptible to oseltamivir, peramivir, and zanamivir, and only one virus contained a genetic mutation that has previously been associated with reduced susceptibility to baloxavir. Influenza antiviral medications are a valuable adjunct to annual influenza vaccination, and early treatment with influenza antiviral medication, especially within 48 hours of symptom onset, is recommended for patients with confirmed or suspected influenza who 1) have severe, complicated, or progressive illness; 2) require hospitalization; or 3) are at high risk for influenza-related complicationsAlthough influenza activity is typically low in the United States during the summer months, CDC collects, compiles, and analyzes data to monitor influenza activity throughout the year.In the United States, influenza activity remained low with cocirculation of influenza A and influenza B viruses. Influenza viruses circulated widely in the Southern Hemisphere, with A(H3) viruses predominating in most regions, although influenza A(H1N1)pdm09 and influenza B/Victoria viruses predominated in several countries.Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences.


S/Sp53mice displayed female-specific phenotype of open neural tube in brain  and spinal cord (spina bifida). The occurrence rate for embryonic exencephaly is 68.5% in female S/Sp53 mice, which is much more than that of in -/-p53 mice (37.1%) in the same genetic background. Further study found that p53N236S mutation increased neuronal proliferation and decreased neuronal differentiation and apoptosis. To rescue the phenotype, we inhibited cell proliferation by crossing -/-Wrnmice with S/Sp53 mice. The occurrence of NTDs in S/S Wrn-/-p53mice was 35.2%, thus suggesting that the inhibition of cell proliferation through a Wrn defect partially rescued the exencephaly phenotype in p53S/S mice. We also report that p53S decreased expression of UTX at mRNA and protein level via increasing Xist transcript, result in high female-specific H3K27me3 expression and repressed Mash1 transcription, which facilitating abnormal proliferation, differentiation, and apoptosis, result in the mis-regulation of neurodevelopment and neural tube defects (NTDs).The p53 is one of the most important tumor suppressors through surveillance of DNA damages and abnormal proliferation signals, and activation the cell cycle arrest and apoptosis in response to stress. However, the mutation of p53 is known to be oncogenic by both loss of function in inhibiting cell cycle progress and gain of function in promoting abnormal proliferation. In the present study, we have established a knock in mouse model containing an Asn-to-Ser substitution at p53 amino acid 236 by homologous recombination (p53N236S). Other than tumorigenesis phenotype, we found that  Neurulation is a defining feature of vertebrate development and is essential for proper nervous system development and embryonic viability The neural tube is formed by thickening of the dorsal surface ectoderm, which folds and joins at the midline. Neural tube closure is complex and requires tightly regulated coordination of numerous processes, including polarized apical constriction, proliferation and apoptosis There is growing evidence that the tumor suppressor p53 (Trp53), which is known to prevent tumor development by promoting growth arrest and apoptosis, also functions as a regulator of cell differentiation CHD7 gene. In that study, p5325, 26, 53, 54 mutant protein has been shown to stabilize and hyperactivate wild-type p53, which then inappropriately induces its target genes and triggers cell cycle arrest or apoptosis during developmentp53 is the most frequently mutated tumor suppressor gene involved in human tumorigenesis. Mutant p53 is thought to contribute to carcinogenesis through the acquisition of gain of function properties. It is highly likely that p53 mutations would result in impaired development. Indeed, more recently, p53 has been shown to have a gain of function mutation in CHARGE syndrome, a multiple anomaly disorder in humans that arises from mutations in the N236Smutation  has been reported as a somatic mutation in 35 tumor cases in IARC databases. Our previous study has described this p53 mutation in mouse MEFs. The mutation has been found in three independent tumorigenic mouse cell lines that use the alternative lengthening of telomeres (ALT) mechanism to maintain cell proliferationThe p53S/Sp53 knock-in mouse model. The p53S mutation led to decreased neuroepithelial differentiation and apoptosis, increased neuroepithelial proliferation during neurulation, and failure in neural tube closure. Strikingly, we found that knockout of the DNA helicase Wrn inhibiting cell proliferation could partially rescue the phenotypes in S/Sp53 embryos, thus demonstrating that cell proliferation contributes to phenotypes resulting from p53S mutation.In the present study, we identified the role of p53S in mammalian development by producing a In this context, we propose that p53S is involved in neural tube closure through regulating H3K27me3 expression to tightly coordinate neuroepithelial cell proliferation, differentiation and apoptosis, during neurulation.S/Sp53 knock-in mice were generated by a commercial service. The details are described elsewhere S/Sp53homozygote mutant in a single PCR reaction  were S/Sp53mice  homozygous mutants, a value less than the anticipated value of 50%  S/Sp53 mutant embryos were observed from S/+p53intercrosses.To address whether S/Sp53 embryos exhibit open neural tube defects (NTDs) with exencephaly; at E13.5, some exencephalic S/Sp53embryos also exhibited spina bifida; whereas some mutant embryos have closed neural tubes  had exencephaly  in -/-p53female embryos under the same genetic background (C57BL/6 × 129SvEv)  in hindbrain revealed a significant increase in the number of cells in M phase in p53 mutant opening neural tube than that in S/Sp53 mice. We cannot properly overexpress p21 in the S/Sp53 mice, thus chose to use another strategy. We inhibited cell proliferation through knockout Wrn gene. Wrn encodes Werner syndrome protein (WRN) with both DNA helicase and exonuclease activity. Mutations in the Wrn gene are associated with a progeroid syndrome in humans , which is characterized by accelerated aging, cellular senescence, genomic instability and so on. -/-Wrnmice exhibit many of the WS phenotypes including a decreased life expectancy, but do not show any signs of NTDs  homozygous mutant (-/-p53S/SWrn) offspring from breeding -/-p53S/SWrnmales to -/-p53S/+Wrnfemale mice, 21/165(12.7%) -/-p53S/SWrn offspring from -/-p53S/+Wrnmales to -/-p53S/+Wrnfemale mice at the time of genotyping  had exencephaly, a value less than the number of exencephalic S/Sp53 mice (68.5%) marks early post-mitotic neurons, which are first seen at about E9.5. S/Sp53 embryos showed significantly decrease Tuj1+ cells compared with -/-p53 and wild-type at E10.5 ; can be erased by H3K27me3-specific demethylases, UTX . We examined UTX and EZH2 expression in S/Sp53 NTDs embryos and observed decreased UTX expression in S/Sp53 NTDs embryos relative to S/Sp53 normal embryos, while EZH2 expression no change , many developmental genes exhibit 'bivalent state' marked by bivalent trimethylation of both histone H3-lysine 27 (H3K27me3) and H3-lysine 4 (H3K4me3) Next, we observed a significantly decrease in Mash1 transcripts Fig F. Mash1,S/Sp53 NTDs embryos relative to WT or -/-p53 female embryos, which is consistent with the observation of histology . The details are described in another paper The Genotyping was performed by using a three-primer PCR assay of genomic DNA prepared from either tail clippings of pups or embryonic tissues. A 3′primer (Neo11-R primer) downstream of the neo cassette was combined with two 5′primers located downstream of the neo cassette(Neo10-F) and in the loxp site (F3) to generate different-sized PCR products from the wild type allele (458 bp) and N236S mutant . Primer sequences are as follows: Cre primers: 5'-TCCAATTTACTGACCGTACACCAA-3'; 5'-CCTGATCCTGGCAATTTCGGCTA-3'and the p53S primers: NeoF: 5'-CTGCACCCTACGAGAACTGACTT-3'; NeoR: 5'-GGGATGAAGTGATGGGAGCTAG-3'; and F3: 5'-GCATAAGCTTGGATCCGTTCTTCGGAC-3').The PCR to determine embryo sex was done with male-specific SRY primer. PCR of the male will generate a 420 bp band and female no band. β-actin provides a PCR control. Primer sequences are as follows:SRY primers: 5'-ATTTTTAGTGTTCAGCCCTAC-3'; 5'-CTACTCCAGTCTTGCCTGTAT-3'; β-actin primers: 5'-TTCTTCTGCCGTTCTCCCAT-3'; 5'-GCTTTGTCACACGAGCCATTG-3'.PCR was run for 35 cycles . PCR products were visualized on a 2% agarose gel stained with ethidium bromide.Crosses of heterozygous mice or heterozygous with homozygous mice were performed using mice that were backcrossed with C57BL/6 mice. The day on which the vaginal plug was detected was considered to be day 0.5 post-coitum (designated E0.5). Pregnant females were sacrificed at different day's post-coitum. Embryos were dissected from the uterus in PBS and examined for external abnormalities, including exencephaly, spina bifida.Embryonic samples from timed matings were fixed in 4% paraformaldehyde (PFA) at 4°C overnight, dehydrated in graded solutions of alcohol, cleared in xylene, embedded in paraffin, and cut into 3μm thick sections before hematoxylin and eosin (H&E) staining.Sections were blocked with 5% goat serum, incubated in primary antibody in blocking solution at 4ºC overnight. pH3 .For whole-mount LysoTracker evaluation of cell death, embryos were dissected in PBS, incubated in LysoTracker Red (1:5000) (Life Technology L7528) for 45 min at 37ºC. Samples were then washed three times in PBS and imaged.Brain tissue lysates were prepared at E10.5. Samples with equal concentrations of protein (25 ug/lane) were electrophoresed. The samples were then transferred to a methanol-preactivated PVDF membrane  and blocking was done for 60 minutes at room temperature using a solution of 5% w/v dried non-fat milk in TBS plus 0.1% Tween-20. Antibodies used for western blot were anti-phospho-p53 (Ser15) , anti-H3K27me3 , anti-pH3 , anti-Tuj1 , anti-EZH2 , anti-UTX (1:500 Abcam).For cartilage staining, E13.5 embryos were fixed in Bouin's solution. After washing in 70% ethanol, the embryos were equilibrated in 5% acetic acid. The embryos were stained in 0.05% Alcian blue in 5% acetic acid for 2 h, followed by two washes in 5% acetic acid and then cleared in methanol for 2 h before stored in 1:2 mixture of benzyl alcohol and benzyl benzoate.-△△Ct method. All experiments were repeated three times.Total RNA was isolated from the heads of WT and p53S/S embryos using Trizol (Invitrogen) and reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). RNA quantity was determined by agarose gel electrophoresis and by spectrophotometry. Quantitative real-time PCR was performed using SYBR  in a 10uL system on an ABI PRISM 7300 sequence detector system (Applied Biosystems). The levels of mRNA were normalized against the levels of β-actin. The Primer sequences are as follows: p21 primers: 5'-CCAGGCCAAGATGGTGTCTT-3'; 5'-TGAGAAAGGATCAGCCATTGC-3'; Mash1 primers: 5'-AAGAGCTGCTGGACTTTACCAACTG -3'; 5'-ATTTGACGTTGGCGAGA-3'; β-actin primers: 5'-AGAGGGAAATCGTGCGTGAC-3'; 5'-CAATAGTGATGACCTGGCCGT-3'. The relative expression level of each mRNA was analyzed by the 2Student′s t-tests were used to assess the significance of the differences between two groups of data (p < 0.05 is deemed significant). The Chi squared tests were used to evaluate the significance of differences between the expected and observed genotype distributions.


We report the first isolation of a reassortant clade 2.3.2.1c avian influenza A(H5N6) virus isolated from a wild bird sample in Jiangxi, China, in 2016. Sequence analyses indicated that this virus most likely evolved from Eurasia-derived H5N1 and H6N6 viruses through frequent interactions at the wild-domestic bird interface. We report the first isolation of a reassortant clade 2.3.2.1c avian influenza A(H5N6) virus isolated from a wild bird sample in Jiangxi, China, in 2016. Sequence analyses indicated that this virus most likely evolved from Eurasia-derived H5N1 and H6N6 viruses through frequent interactions at the wild-domestic bird interface. Alphainfluenzavirus genus in the family Orthomyxoviridae (Avian influenza virus (AIV) is a single-stranded segmented negative-sense RNA virus classified in the oviridae . The hemoviridae . Among toviridae , 4. It ioviridae . In contoviridae .Streptopelia decaocto and yellow-legged button quail, respectively. Virus isolation using these specimens was conducted in 9- to 11-day-old specific-pathogen-free embryonated chicken eggs. The viral RNAs were extracted from allantoic fluid of 13 samples with hemagglutination activity using an RNeasy minikit . The SuperScript III reverse transcriptase (RT) PCR kit  was used for the reverse transcription.Here, we provide a report on the first detection and nearly complete genome sequence of a novel reassortant clade 2.3.2.1c H5N6 sample isolated from a wild bird in China. A total of 488 tracheal and cloacal swab samples were collected during a routine bird survey in Suichuan County, Jiangxi Province, which were preserved in a sample solution in the fridge (4°C) and subsequently shipped to a laboratory and stored frozen at −80°C. From these samples, 89 and 85 samples were taken from The subtype of each of the 13 positive samples was first determined using PCR of a marker gene , 7. SeveThe amino acid sequence at the HA cleavage site is RERRRKR/GL, which is characteristic of high pathogenicity in poultry. Additionally, HA had Q222 and G224 (H3 numbering) at the receptor binding site associated with an adaptation to avian-like receptors . NA had A BLAST search in the GenBank database showed that all the eight genes of Jiangxi H5N6 virus had high nucleotide identities with viruses isolated from terrestrial poultry in China 2014 to 2015 . These hThis detection suggests continuous reassortment and generation of emerging variants. Further investigation and active surveillance are required to detect new AIV variants.MN165550 to MN165557.The genome sequence of Jiangxi clade 2.3.2.1c H5N6 virus has been deposited in GenBank with the accession numbers


A highly pathogenic avian influenza A(H5N6) virus of clade 2.3.4.4 was detected in a domestic duck found dead in Taiwan during February 2017. The endemic situation and continued evolution of various reassortant highly pathogenic avian influenza viruses in Taiwan warrant concern about further reassortment and a fifth wave of intercontinental spread. Since 1996, H5 A/goose/Guangdong/1/1996 (Gs/GD) lineage of highly pathogenic avian influenza viruses (HPAIVs) originating in Asia have caused outbreaks in Asia, Europe, Africa, and North America . We performed full-length genome sequencing by using reverse transcription PCR amplification and Sanger sequencing (We detected Dk/Tw/17 virus by using reverse transcription PCR and isolated the virus by using egg inoculation as described previously (quencing .We classified Dk/Tw/17 as an HPAIV on the basis of the amino acid sequence at the hemagglutinin cleavage site (PLRERRRKR/G) and its high lethality in chickens (intravenous pathogenicity index 3.0). Necropsy and histologic examination revealed virus-specific necrotic and inflammatory lesions in the pancreas, heart, and brain  Figure 2The genotype C5 comprises 17 H5N6 HPAIVs identified from wild waterfowl in China, Japan, and South Korea during November–December 2016; a virus identified from a chicken farm (A/chicken/Korea/H23/2016 [H5N6]) in South Korea in November 2016; and the Dk/Tw/17 virus. Genotype C5 is phylogenetically distinct from viruses that caused outbreaks in poultry farms in Japan and South Korea during 2016–2017. This genotype has independently evolved and been maintained in wild bird populations in the bird flyway of East Asia, highlighting how wild waterfowl play an important role in the maintenance and dissemination of this HPAIV. In addition, the median-joining phylogenetic network analysis suggests that the A/chicken/Korea/H23/2016 (H5N6) is not the direct ancestor of the Dk/Tw/17 virus, which was likely caused by separate introduction from wild birds , panel BThe site where the dead duck was collected is adjacent to a river and located near many ponds used for duck farming. After identification of Dk/Tw/17, intensified active surveillance conducted over 3 months detected additional clade 2.3.4.4C H5N6 HPAIVs from 12 farms in 4 counties  Figure 1Materials, methods, and supplementary data on reassortant H5N6 clade 2.3.4.4 of highly pathogenic avian influenza airus, Taiwan, 2017.Authors and originating and submitting laboratories of the sequences from the GISAID EpiFlu Database, on which this study is based.


The as-obtained sample was characterized by XRD, FT-IR, SEM, TEM, and XPS measurements. In addition, the electrochemical properties of VO2(B)/GN were tested. Due to its great electrochemical performance and mechanical properties, graphene could increase the electrochemical performance and strengthen the structural stability of the material at the same time. With increasing loading amount of GN, the specific capacitance of VO2(B)/GN increased correspondingly. With 20% GN loading, the initial discharge specific capacity could reach 197 F g−1 at 0.5 A g−1, and 160 F g−1 at 1 A g−1 in 0.5 M Na2SO4 electrolyte, which is better than that of pure rod-like VO2(B). The capacitance of the VO2(B)/GN (20%) composite electrode retains 95.49% after 1000 cycles, which is higher than that of a pure VO2(B) electrode (85.43%), indicating that the VO2(B)/GN composite possesses better cycling stability. Moreover, a symmetrical solid-state supercapacitor (SCs) using VO2(B)/GN(20%) as the anode was assembled. Four printed SCs were connected in series to light up a 1.5 V red LED. This demonstrates its potential application in intelligent packaging to trace food safety.More multipurpose and convenient demand driven by Radio Frequency Identification (RFID) and intelligent packaging require flexible power sources. A VO Food indicators applied in intelligent packaging have aroused worldwide attention. Furthermore, the use of power-driven indicators has been an emerging trend to set up the interaction between customers and products by displaying product information and freshness. Among them, flexible supercapacitors become a promising candidate. The rapid growth of flexible and portable electronics has stimulated enormous efforts to develop multi-purpose, high-power supercapacitors ,3,4,5,6.Metallic vanadium (V) is a single crystal polyvalent metal . It reac2, NaxVO2, and other lithium or sodium intercalated complexes. In addition, inorganic metal oxides are often used as electrode materials. However, some irreversible changes in volume often take place when ions insert and extract, and the structures are, therefore, damaged, resulting in poor cyclic stability of the electrode materials. Vanadium dioxides/GN composites could be a great candidate to solve this problem during the application of electrode materials, which would not only improve the cyclic stability of the materials, but also help each component to play a synergistic role in supercapacitors. Deng et al. [2 composites materials by a one-step hydrothermal reaction. The RG/VO2 electrode material with RG content of 26 wt% can reach 225 F g−1 at a current density of 0.25 A g−1 in 0.5 mol L−1 K2SO4 solution. A coaxial-structured VO2(B)/MWCNT  hybrid material was prepared through the sol-gel method by Liang et al. [−1 in 1M Na2SO4 electrolyte at 0.5 A g−1. Pan et al. [2O3/VOx core–shell nanomaterials, and the assembled electrochemical supercapacitors (ECs) possesses excellent power density of 1000 kW kg−1 at an energy density of 10 Wh kg−1. Xiao et al. [2(B)/grapheme through a hydrothermal reaction. The initial specific capacitance of the composites reached 290.4 F g−1 at 0.2 A g−1 in a voltage window from −0.4 to 0.6 V. However, the supercapacitor was not further fabricated to test its electrochemical performance.Vanadium oxides can be intercalated by lithium (sodium) ions under electrochemical action to form layered LixVOg et al.  preparedn et al.  preparedo et al.  prepared2(B)/GN core-shell composites by a one-pot hydrothermal reaction through mesh printing, which could meet the requirements of power supply for packaging. The electrochemical properties of the VO2(B)/GN composite can be optimized by adjusting the mass ratio of GN. Subsequently, its morphology, crystalline structure, chemical bonds, and chemical components were investigated. The whole procedure of preparing VO2(B)/GN composites and fabrication of symmetrical supercapacitors are demonstrated in The as-prepared flexible SCs in this paper can be employed as power source supplying for electronic devices in intelligent packaging, such as LEDs, RFID, electronic sensors, intelligent voice devices, and so on, to trace food information and indicate food freshness. Normally, the power source applied in packaging does not require high voltage and specific capacitance, and studies focus on the properties of flexibility, low-cost, and large-scale production. Herein, in this work, we prepared rod-like VON-methyl pyrrolidone (NMP), sulfuric acid, phosphoric acid, hydrochloric acid, potassium permanganate, oxalic acid dehydrate, and absolute ethyl alcohol were purchased from Sinopharm Chemical Reagent Co, Ltd. , polyvinylidene fluoride (PVDF) was purchased from Arkema Innovative Chemistry Co. Ltd. , vanadium pentoxide was purchased from Sun Chemical Technology Co. Ltd. . Acetylene black was purchased from Tianjin Ebory Chemical Co., Ltd. . Graphite was purchased from Sigma-Aldrich Co. Ltd. . Lithium chloride (LiCl) was purchased Tianjin Fuchen Chemical Reagents Factory . The deionized water used in the experiments was lab-made. All chemical reagents were analytical grade and used without further purification.Polyvinyl alcohol, 2SO4/H3PO4 (volume ratio was 9:1), and 13.5 g of KMnO4 was slowly stirred into the mixed solution. Then the mixed solution was heated to 55 °C for 12 h. When the mixture was cooled to room temperature, it was slowly poured into an ice bath containing 2.5 mL 30% H2O2 until the solution turned to golden yellow and no bubbles were produced. Next, the mixture solution was filtered through 0.45 μm PTFE membrane and washed with 5% HCl and deionized water until it is neutral and without residual SO42− (detected with BaCl2). The graphite oxide (GO) was prepared by drying in vacuum at 60 °C for 48 h.The synthetic procedure of graphite oxide is based on the modified Hummers method reported by Marcanos et al. . Firstly2O5 powder and 1.26 g of oxalate dihydrate were dissolved in 80 mL deionized water by ultrasonic for 20 min. The mixture was then transferred to stainless steel autoclave and heated at 210 °C for 24 h. The obtained black products was collected and washed by distilled water and absolute ethanol for several times, respectively. The final product was dried in vacuum at 80 °C for 10 h.To simplify, 0.91 g of V2O5 powder and 1.26 g of oxalate dihydrate were dissolved in 30 mL deionized water by ultrasonic for 20 min, and a certain amount of GO was dispersed in 30 mL deionized water by ultrasonic. The two above mixtures were then transferred to a stainless steel autoclave and heated at 210 °C for 24 h. The subsequent procedures were the same as those described above and the VO2(B)/GN composite was obtained.Similarly, 0.91 g of V2(B)/GN composites screen printing ink was prepared containing 80 wt% VO2(B)/GN composites, 10 wt% acetylene black and 10 wt% polyvinylidene fluoride (PVDF). PVDF was dissolved completely in N-Methyl pyrrolidone (NMP) ultrasonically in advance. Then VO2(B)/GN composites and acetylene black were added under continuous stirring until a homogenous ink was formed.Briefly, the VO2(B)/GN ink, followed by vacuum drying at 60 °C. Screen printing was operated at room temperature using a mesh (100 mesh-counts). Then, the solid-state supercapacitor was assembled from two identical electrodes, where LiCl/polyvinyl alcohol (PVA) gel was used as the electrolyte. For preparing the LiCl/PVA gel, 2 g of PVA and 2 g of LiCl were added simultaneously into deionized water and stirred at 85 °C until a clear gel electrolyte was formed, followed by immersing in the as-prepared electrolyte for 5 min before fabrication. Next, the electrodes were dried at room temperature for 3 h to remove the excessive water. Finally the electrodes were pressed together under a pressure of 1 MPa for 10 min and sealed with plastic wrap to assemble the symmetric supercapacitor. The average mass loading of electrode materials in one supercapacitor was about 6.0 mg.Firstly, electrodes were printed by screen printing on Ni foams using as-prepared VO2(B)/GN composites. The X-ray diffraction  from PANalytical X’Pert Pro with Cu Ka radiation and X-ray photoelectron spectroscopy  from Thermo Scientific ESCALAB 250Xi system with Al-Ka as the radiation source to characterize the structural information and phase purity. Raman spectroscopy was characterized on a Confocal Raman Microspectroscopy  at room temperature with Ar+ laser of 514.5 nm excitation. The chemical bonds information was measured with a Nicolet 5700 Fourier transformed infrared spectrometer . The electrochemical activity of the electrodes and SC devices were investigated by using a CS 350 electrochemical workstation in a conventional three-electrode system .Scanning electron microscope  images, energy dispersive X-ray spectroscopy  data, and transmission electron microscope  images were performed to investigate the morphology of VO2(B)/GN composites screen printed on Ni foams (0.5 mm thick and purity >99.9%) can be directly used as working electrode where Ni foams acted as the current collector. The Ni foams were sonicated in acetone for 10 min, and washed copiously with 0.1 M HCl, ethanol and deionized water. Then 80 wt% active materials, 10 wt% acetylene black, 10 wt% PVDF, and a few drops of NMP were mixed to form a syrup. It was screen printed on Ni foam and pressed under 10 MPa for 1 min to form the electrode (the thickness is about 0.15 mm), and dried at 60 °C in vacuum overnight. The mass of the active material on each electrode is 3~5 mg. A platinum electrode, a saturated calomel electrode (SCE) and 0.5 M Na2SO4 solution were used as counter electrode, reference electrode and electrolyte, respectively.The VO2(B) exist of nanorods. Besides, the as-prepared nanorods are several micrometers long with widths of 40~60 nm. 2(B)/GN composites with different mass ratios, at 10%, 20% and 30%, respectively. With increasing mass ratio of GO, the surface of VO2(B) nanorods become more irregular, indicating that adding a certain amount of GO may influence the morphology of VO2(B).The morphology and particle size of the prepared nanocomposite were characterized by SEM and TEM. As depicted in 2(B)/GN (20%) composites are shown in 2 as nets. In the formed core-shell structure, the core is VO2 and the shell is GN. The similar structure was reported by Zhang [2(B)/GN composites. The distance between the lattice planes is about 0.332 nm, which is consistent with the (110) lattice plane of VO2(B) [2(B)/GN composites (This speculation was confirmed by TEM testing. The TEM image of VO2(B)/GN % composiby Zhang ,27. Figuf VO2(B) . Furthermposites f. The re2(B) and VO2(B)/GN core-shell composite was also characterized by XRD. In VO2(B) pattern, the distinguish diffraction peaks can be attributed as monoclinic VO2 (space group C2/m) with lattice parameters a = 12.09, b = 3.7021, c = 6.433 Å, which is in line with the VO2 (B)  [2(B). The strong peak situated at 2θ = 25.00° is correspond to diffraction peak of (110) planes, it is the result of preferred orientation growth of VO2(B) [2(B)/GN core-shell composites, revealing the successful combination of VO2(B) and GN.As shown in 6.433 Å) ,29 witho2(B)/GN (20%) is depicted in −1 (D band) and 1604 cm−1 (G band). The G band corresponds to the vibration in all sp2-bonded carbon atoms while the D band can be attributed to the defects of the sample [D/IG ratio of 0.86, which is lower than the report for rGO (ID/IG > 1) [D/IG for the graphitized graphene (ID/IG = 0.83), indicating the existence of some structural defects [The Raman spectrum of VOe sample . The sam/IG > 1) . However defects . Moreove2(B), GO and VO2(B)/GN composite. The broad absorption bands at about 3400 cm−1 are assigned to O-H stretching while medium bands at 1620 cm−1 are ascribed to bending vibration of H2O molecule, which can be observed in all samples. The characteristic bands of VO2(B) [−1, related to V–O–V stretching mode of vibration, which are also displayed in VO2(B)/GN composite [−1 can be assigned to the C=O stretching vibration of carboxyl group (COOH), which disappears in the VO2(B)/GN composite, indicating that COOH is reduced by oxalic acid during the hydrothermal reaction. The possible mechanism of VO2(B)/GN composite preparation is illustrated in 2O5 particles are reduced and then grow into VO2(B) nanorods at relatively high temperature and pressure. The dispersed GO nanosheets with abundant carboxyl groups move over and get reduced on the surface of VO2 at the same time. Subsequently, the core-shell structure of VO2(B)/GN nanorods are formed.f VO2(B) ,33, arouomposite ,34. In G2(B)/GN composite preparation are supported by XPS analysis. The survey peak with V, C and O elements is found in 4+, while the others at 517.2 and 524.8 eV corresponds to V5+ [5+ may be due to that the unreduced V2O5 is absorbed on the surface of VO2, which is similar to the assumption reported by Wu and Lian [2(B)/GN, most of COOH groups are reduced by oxalic acid so that it is incapable of being detected by FT-IR. As shown in x), O=C/V–O (H) and O–C peaks at 529.8, 530.4, 531.9 eV, respectively [2(B) and GN are likely to be combined by C–O–V chemical bond.The proposed mechanism of VOs to V5+ ,36. The and Lian . Figure and Lian ,38. In Vectively ,39. Base2(B)/GN electrode between 0 and 0.6 V versus SCE at different scanning rate from 2 mV s−1 to 100 mV s−1; 2(B) and VO2(B)/GN (20%) at a scanning rate of 50 mV s−1. It is40 tested to demonstrate the capacitive performance of the VO2(B)/GN composite electrode. In 2(B)/GN (20%) electrode material shows a rectangular shape in this voltage range. However, there is still a relatively large deformation compared with the standard rectangle, indicating that the electrode material has lower internal resistance. Additionally, with an increasing of scan rate, the peak current of cathode and anode increases, and the potential changes along the positive direction, indicating that Na+ has been embedded and detached in the material to maintain electrical neutrality. The positions of redox peaks at different scanning rates remained unchanged, indicating that VO2(B) has rather satisfactory cyclic reversibility. Additional, the near-rectangular shapes of the CV curves still can be found at higher scanning rates.2(B) nanobelts in these three electrodes system is shown below [The reaction process of VOwn below :VO2 + xN2(B)/GN (20%) can be observed form a larger area of CV curve (2(B)/GN (20%) composite electrode displays a more regular shape, demonstrating the typical pseudocapacity. Furthermore, there are no obvious redox peaks in curves of VO2(B) and VO2(B)/GN (20%), which can be rationalized by the mild redox behavior for VO2(B) and GN [Better capacitive behavior of VOCV curve b. The CV) and GN ,40.2(B) and VO2(B)/GN (20%) composite electrodes. 2(B) and VO2(B)/GN (20%) composite electrodes at 0.5 A g−1 and 1 A g−1, respectively. Excellent capacitive behavior can be observed from their linear curves, indicating that their capacitance mainly comes from double layer capacitance. The specific capacitance (C) of VO2(B) and VO2(B)/GN (20%) composite electrodes are calculated by the following formula:I is the applied current (A), t is the discharge time (s), m is the mass of active material (g), and V is the potential window (V). The discharge time of VO2(B)/GN (20%) indicates higher specific capacitance of 160 F g−1 at 1A g−1, in comparison to the lower specific capacitance of 64, 105, 92 F g−1 for VO2(B), VO2(B)/GN (10%) and VO2(B)/GN (30%), respectively. Additionally, the specific capacitance of the VO2(B)/GN (20%) composite electrode is about 197 F g−1 at 0.5 A g−1, which performs better than 99, 153, and 134 F g−1 of VO2(B), VO2(B)/GN (10%), and VO2(B)/GN (30%) electrode materials. The specific capacitance of VO2(B)/GN (20%) composite electrode is about 160 F g−1 at 1 A g−1, which is much higher than 64 F g−1 of VO2(B) electrode. Furthermore, the specific capacitance of the VO2(B)/GN (20%) composite electrode is about 197 F g−1 at 0.5 A g−1, which performs better than the 99 F g−1 of VO2(B) electrode. 2(B) and VO2(B)/GN (20%) composite electrodes at 15 A g−1 for 1000 charge/discharge cycles. The specific capacitance of VO2(B)/GN (20%) composite electrode retains 95.49% of the original after 1000 cycles, which is higher than that of pure VO2(B) electrode (85.43%) and displays better cycling stability.Galvanostatic charge/discharge (GCD) is performed to investigate the rate capability and capacitive performance of VO2(B) and VO2(B)/GN (20%) composites electrodes to further evaluate their electrochemical performance. The electrochemical impedance spectroscopy (EIS) curves of VO2(B) and VO2(B)/GN composites consist of a semicircular high frequency region and a straight line low frequency region. The semicircle in the high frequency region mainly comes from the charge transfer reaction at the interface between the electrolyte and active material, which corresponds to the process of electron transfer. The point of contact between the high frequency region and the real axis is considered as the internal resistance of the electrode material. Additionally, the linear type low frequency region is mainly caused by Warburg impedance produced by the diffusion of Na+ at the interface of the working electrode, which corresponds to the process of diffusion process [ process , indicat2(B)/GN composites is obviously lower than that of VO2(B), showing that the introduction of GN improves the conductivity of the composites. In addition, with increasing mass ratio of GN, the reaction resistance decreases, proving that the addition of GN is beneficial to reduce the reaction resistance of electrode materials. Moreover, the linear slope of the composite is greater than that of VO2(B) in the low frequency region, which also demonstrates that the incorporation of GN into the composite can improve the diffusion rate of Na+ and is also consistent with the CV curves. The specific morphology and synergistic effect of VO2(B)/GN composites facilitate the capacitive performance.The high frequency semicircle is produced by charge transfer reaction at the electrolyte/oxide electrode interface, corresponding to charge transfer (Rct) . EIS cur2(B)/GN (20%) composite is fabricated via screen printing. −1. As shown in −1 are shown. Superb capacitive behavior can be observed from their linear curves. The capacitance (C) can be calculated with Equation (2):I is the discharge current (A), t is the discharge time (s), m is the total mass of one electrode (g), and V is the discharge voltage window (V). Obviously, with the augment of current densities from 0.2 to 1.0 A g−1, the corresponding areal specific capacitance of symmetrical SCs are 287.5, 268.9, 229.3, 209.6, and 196.4 F g−1. It exhibits great rate capability.To develop flexible power sources, the solid-state flexible SCs based on VOMoreover, after connecting four symmetrical SCs in series, they can light up a 1.5 V red LED as shown in 2(B)/GN composites were successfully prepared by one-pot hydrothermal reaction. When the mass ratio of GN reaches 20%, optimal electrochemical properties can be obtained. The VO2(B)/GN composites displays higher specific capacitance and better stability compared to pure VO2(B). The VO2(B)/GN (20%) composite specific capacitance of 160 and 197 F g−1 at 1 and 0.5 A g−1, respectively. It is much higher than that of pure VO2(B) (64 and 99 F g−1). Additionally, the capacitance value of VO2(B)/GN (20%) composite electrode retains 95.49% after 1000 cycles, which is significantly higher than that of pure VO2(B) (85.43%) along with a better cycle stability. To develop flexible power sources, the solid-state flexible SCs based on VO2(B)/GN (20%) composite is fabricated by screen printing with excellent performance. The assembled symmetric SCs can light up a red LED of 1.5V when four of them are connected in series. More supercapacitor behaviors and its application in intelligent packaging will be investigated in our future work.In summary, a series of VO


G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we assessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression. in vivo reporter assay. Therefore, this study demonstrates that G4 activity relies on the promoter context, providing a new insight into understanding gene regulation by G4 structures. This study also provides evidence that G4 plays a regulatory role in gene expression during HCMV infection.Although a number of G4-forming sequences are predicted in the herpesviral genomes and some G4s are characterized individually, why so many G4s are present in the genome and whether they are all functional are not yet clear. Our genome-wide bioinformatic sequence analyses and biophysical stability and structure assays of G4s present in the HCMV genome revealed that most G4s present in the HCMV gene regulatory regions form a stable G4 structure. However, it was found that only a few HCMV G4s suppress viral gene expression when the effect of G4 on gene expression was examined by  Repetitive guanosine-rich (G-rich) sequences connected by short stretches of nucleotides in the genome of an organism can fold into a distinct type of tertiary structure known as a G-quadruplex (G4). Four guanine bases connected with each other through Hoogsteen hydrogen bonding form a square planar structure known as a guanine tetrad or G-tetrad. Multiple G-tetrads can stack on top of each other in a G4 structure, which can be further stabilized in the presence of monovalent or divalent cations –3.Since the presence of G4s in the human genome was first observed in the telomere region and their structure was proposed , many stEscherichia coli are 6,754  were used as the positive and negative controls of G4 spectra, respectively  by NMM, but not by TMPyP2 ; S2 Fig.tro Figs  and G4 atro Figs . These rThe G4 formation of GQ8 (in UL35) and GQ18 (in UL75/76) was further analyzed using mutagenesis. GQ8  was chosen since its high propensity for gene regulation during virus infection is expected in both Tm and luciferase reporter analyses. Despite having low stability, we chose to study GQ18  located between the UL75 and UL76 genes, because it exerted its high suppressive effect on UL76 in cell-based reporter assays. Furthermore, the UL35 and UL76 genes are required for efficient viral growth , 58. BasWe introduced the same mutations of GQ8 and GQ18 into luciferase reporter plasmids containing the UL35, UL75, and UL76 promoters. HF cells were transfected with wild-type or mutant reporter plasmids, followed by HCMV infection in the absence or presence of NMM and TMPyP2. Since the basal activities of these viral promoters were very low in uninfected cells, mutation or NMM effect was only counted in virus-infected cells. The mutations within GQ8 led to only a weak increase of the UL35 promoter activity in control (DMSO-treated) cells, but they substantially mitigated the NMM-mediated suppression of UL35 expression . The GQ1We next evaluated the G4-mediated gene regulation during virus infection. HF cells were infected with HCMV (Toledo) for 24 h , 32 h (for early genes) and 48 h (for late genes). Virus-infected cells were untreated or treated with NMM and TMPyP2 for 24 h prior to cell harvest. The roles of GQ11, GQ8, and GQ18 in the transcription of UL37, UL35 and UL75/UL76, respectively, were assessed by measuring their mRNA levels using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that NMM treatment suppressed the UL37 mRNA level but not the US3 mRNA level (control), while the effect of TMPyP2 on these viral genes was not considerable , left. SWe also compared the levels of viral DNA with or without ligands from the infected cells and from the culture supernatant. The results of qPCR indicated that NMM treatment reduced both intracellular and extracellular viral DNA levels to 75 and 65%, respectively, of those in dimethyl sulfoxide (DMSO)-treated cells, while TMPyP2 did not significantly affect the viral DNA levels . When weFinally, the suppressive role of GQ8 in UL35 expression was investigated by producing a recombinant virus containing G4-disrupting mutations. The mutations introduced did not affect the coding potential of the overlapping UL34 gene . The HCMG-rich sequences capable of forming G4 structures are present in the genome of diverse organisms and have emerged as a therapeutic target in recent years . Structuin vitro Tm analysis, we found that the stability as represented by high Tm value was generally higher in conventional G4s than in bulged or long-loop G4s  and GQ18 (in UL76) indeed suppressed the reporter gene expression. Consistently, the mRNA levels of UL35 and UL76 were reduced in HCMV-infected cells by NMM treatment. Our study further addressed the role of G4 formation (from GQ8) in the UL35 promoter in the context of virus infection by producing a recombinant virus with mutant GQ8. The GQ8 is located adjacent to the A/T-rich sequence that appears to act as a TATA-box for the UL35 gene . Our stul genome  than in l genome . This suoriLyt region and the repeated regions of HCMV genome. Therefore, NMM-meditated regulation of G4s may also affect DNA replication. The detailed molecular mechanism other than gene suppression, by which NMM inhibits HCMV growth, awaits further investigation.An intriguing question arising from the present study is why HCMV has several G4-forming sequences in its promoters, if they suppress viral gene expression. Given that the G4 formation in the HBV pre-S2/S promoter region has a positive effect on gene transcription , we thinOur study provides a platform for assessing G4 functions for gene expression in viral genomes. Through computational search, different types of G4-forming sequences such as conventional, long-loop, and bulged can be predicted from the genome sequences. The formation of parallel and antiparallel G4s and their stability can be determined by CD spectroscopy and Tm analyses. However, the role of a G4 in gene expression should be addressed using cell-based assays in the context of viral genome sequence, as the G4 formation as well as its effect on promoter activity can be influenced by G4-neighboring sequences and associated cellular factors. Overall, our results point to a relevant physiological role of G4s in controlling HCMV viral gene expression and provide a new insight into understanding gene regulation by G4 structures. We believe that further genome-wide analyses of G4s in different viruses or more organisms containing more complex genome structures will help us to establish a general link between G4s in virus genomes and their involvement in the virus life cycle.NMM and TMPyP2 were purchased from Frontier Scientific. TMPyP4 tosylate was bought from Abcam. BRACO19 and Pyridostatin (PDS) were purchased from Sigma-Aldrich.http://www.softberry.com/berry.phtml). Three different types of G4-forming sequences were considered for the prediction: (i) conventional G4s , (ii) long-loop G4s , (iii) bulge-containing G4s . All the loop regions of long-loop G4s were analyzed using the Mfold secondary structure prediction server  to calculate secondary structure-forming ΔG values, and only the long-loop G4s that held considerably low ΔG values were selected for further analysis. Finally, we selected those G4s which were conserved in the genome of the HCMV Merlin strain. After filtering through all the selection criteria, we selected 36 possible G4-forming sequences in the putative regulatory regions from the HCMV genome for further studies.The HCMV Toledo strain (GenBank: GU937742.1) was used in the genome-wide prediction of putative G4-forming sequences within −500 to +100 regions relative to the translation initiation site of the genes. The TATA boxes have been studied for a limited number of the genes. Therefore, for most genes the possible locations of TATA boxes were predicted using the SoftBerry promoter/functional motifs prediction server  from the HCMV (Towne strain) bacmid DNA. These amplified regions contained the minimal putative regulatory regions. If G4s were identified upstream of the predicted TATA boxes, the amplified regions included upstream G4-TATA box-ATG (the translation initiation site). If G4s were predicted between the putative TATA boxes and ATG, or in the regions without any putative TATA box-like sequences, at least 300-bp regions including TATA box-G4-ATG or G4-ATG were amplified. The primer sets used for PCR are given in 2 humidified incubator at 37°C. The HCMV Towne strain and recombinant HCMV (Toledo strain) that was prepared from the HCMV (Toledo) bacmid were previously described . The annealed oligonucleotides were recombined into the Toledo-bacmid DNAs containing the rpsL-neo cassette, and the E. coli cells containing the UL35 (mGQ8) Toledo-bacmid were selected on LB plates containing streptomycin. The mutated regions were amplified by PCR and sequenced to verify the desired mutations. To generate the revertant Toledo-bacmid from the mutant, the wild-type G4 fragments were also generated by annealing using the following oligonucleotide sets: 5′-CGGGTCGCCGCGACCCCCTCACCTTCAGTCACCCCAGCCC TTACCCCCGTCACGTCCCCCATAACCCCGTTGTGTATTTAACGTCACTGGAGAACAATAA-3′ and 5′-TTATTGTTCTCCAGTGACGTTAAATACACAACGGGGTTATGGGGGA CGTGACGGGGGTAAGGGCTGGGGTGACTGAAGGTGAGGGGGTCGCGGCGACCCG-3′. These fragments were inserted into the mutant Toledo-bacmid by homologous recombination as described above.The Toledo-bacmid encoding the UL35 (mGQ8) genes were generated by using a bacterial artificial chromosome (BAC) modification kit (Gene Bridges). Briefly, the rpsL-neo cassettes flanked by homology arms with 100 nucleotides of the region upstream and downstream of the target site (UL35 GQ8) were amplified using the following primer sets: 5′-CGGGTCGCCGCGACCCCCTCACCTTCAGTCACCCCAGCCCTTACCCCCGTGGCCTGGTGATGATGGCGGGATCG-3′ and 5′-TTATTGTTCTCCAGTGACGTTAAATACACAA CGGGGTTATGGGGGACGTGTCAGAAGAACTCGTCAAGAAGGCG-3′. The amplified rpsL-neo fragments were purified and introduced into t-test and indicated by p-values < 0.05 (*), < 0.01 (**), and < 0.001 (***).Statistical significances are determined using the Student’s S1 Appendix(XLSX)Click here for additional data file.S1 FigA) or cells infected with HCMV(Towne) at an MOI of 1 (B) were treated with DW, DMSO, or G4-binding ligands  and 100 mM NaCl or KCl buffer. The CD spectrum in NaCl buffer (black) is compared with that obtained in KCl buffer (red).(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 TableThe G4-forming sequences for the analyzed 36 GQs are indicated in blue.(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S5 TableMutation sites are indicated by red characters.(TIF)Click here for additional data file.


The findings from this preclinical study suggest that an LAIV-LAIV vaccination regimen would be more efficient in providing broadly protective immunity against influenza virus infection as compared to other approaches tested here.Due to continuous antigenic drift and occasional antigenic shift, influenza viruses escape from human adaptive immunity resulting in significant morbidity and mortality in humans. Therefore, to avoid the need for annual reformulation and readministration of seasonal influenza virus vaccines, we are developing a novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccine, which is comprised of sequential immunization with antigens containing a conserved stalk domain derived from a circulating pandemic H1N1 strain in combination with “exotic” head domains. Here, we show that this prime-boost sequential immunization strategy redirects antibody responses toward the conserved stalk region. We compared the vaccine efficacy elicited by distinct vaccination approaches in the preclinical ferret model of influenza. All ferrets immunized with cHA-based vaccines developed stalk-specific and broadly cross-reactive antibody responses. Two consecutive vaccinations with live-attenuated influenza viruses (LAIV-LAIV) conferred superior protection against pH1N1 and H6N1 challenge infection. Sequential immunization with LAIV followed by inactivated influenza vaccine (LAIV-IIV regimen) also induced robust antibody responses. Importantly, the LAIV-LAIV immunization regimen also induced HA stalk-specific CD4 Type A influenza viruses (both H1N1 and H3N2 subtypes) and type B influenza viruses continue to co-circulate globally in humans causing seasonal epidemics that result in morbidity and mortality worldwide . To matcClinicalTrials.gov # NCT03300050).To mitigate these concerns, there is a strong public health interest to develop a broadly protective or universal influenza virus vaccine to induce durable and broadly cross-reactive antibody responses, which would confer cross-protection against all influenza viruses and would alleviate potential disease burden once a pandemic influenza virus strain emerges , 7, 8. DNasal spray delivery of LAIV vaccines was reported to elicit stronger and longer-lasting immunity (both robust B-cell and T-cell responses) than IIV vaccination in children , 29. In The objective of this preclinical study was to assess the protective immunity induced by immunization with group 1 hemagglutinin-specific influenza virus vaccines based on chimeric hemagglutinins (cHAs) in a ferret model of influenza. The experimental designs and immunization strategies are summarized in Four-month-old neutered male ferrets were initially primed with a replication competent influenza B virus expressing cH9/1 (B-cH9/1 live virus), followed by booster vaccination(s) with cH8/1 LAIV, cH8/1 LAIV followed by cH11/1 LAIV (LAIV-LAIV), or cH8/1 LAIV followed by AS03-adjuvanted cH5/1 IIV (LAIV-IIV). Since human adults have a primed HA stalk-specific B-cell repertoire , 30, fer50 value of 566 and 310, respectively . B-cH9/1 virus-primed ferrets induced anti-H1 stalk IgG titers of 1:1100 to 1:1600 at day 21 (pre-boost #1). At day 49 (pre-boost #2), anti-stalk antibodies were boosted in animals that received one dose of LAIV vaccines. The B-cH9/1 virus primed groups generally had 3-fold higher stalk-specific antibody titers than the unprimed groups . Notablyectively .p = 0.02). Notably, LAIV-LAIV immunization also induced higher H2-specific IgG antibody titers than the LAIV group , and H3 (group 2) HAs were measured on day 91 by ELISA . The PriIV group . A similIV group . Taken t6 PFU of a pH1N1 influenza virus isolate  on day 91 post-prime immunization. Nasal wash and oropharyngeal swab samples were harvested on days 1 and 3 post-challenge to examine the kinetics of virus replication in the upper respiratory tract. All animals were then humanely euthanized on day 4 post-challenge infection for collection of respiratory tract specimens and analysis of viral titers  suggested that an LAIV component induced robust HA stalk-specific antibody responses and conferred the greatest level of protection against infection by pH1N1 influenza virus. To confirm this observation, the LAIV-LAIV (group 1) and LAIV-IIV (group 2) vaccine regimens were selected for a follow-up vaccination study. Since our previous study demonstrated that IIV-IIV vaccination also elicited high stalk-reactive antibody titers , here we6 PFU of an H6N1 (tk/97) avian influenza virus. Nasal wash and oropharyngeal swab samples were harvested on days 1 and 3 post-challenge infection, and animals were euthanized on day 4 post-challenge for collection of tissue specimens  strain  were analyzed by flow cytometry after peptide stimulation. An HIV gp120 peptide was included as a control. The gating strategy is diagrammed in +IFN-γ+ and CD8+IFN-γ+) responses among PBMCs as compared to the naive immunized group. No apparent differences for NP-specific CD4+IFN-γ+ and CD8+IFN-γ+ T cells were observed. Low T cell responses for the naïve group were observed upon stimulation with irrelevant gp 120 peptide  than the naïve immunized animals , which supports the concept that the LAIV backbone induces cell-mediated immunity  than the Prime-LAIV-IIV, H3N2 LAIV and mock immunized groups  at day 4 post-viral challenge to assess the recall of cellular responses that may have contributed to the observed protection against pH1N1 or H6N1 influenza virus infection. To analyze influenza virus-specific T cell responses to the H1 stalk domain, we designed a panel of peptides based on A/California/04/09  sequences, which potentially correspond to predicted CD4) strain . Nucleop peptide . From th animals . The Priimmunity . In addid groups .Targeting the highly conserved HA stalk region , 19, 24 50) per gram of tissue. Of note, the A/teal/HK/W312/97 (tk/97) H6N1 influenza virus used for the second vaccination/challenge experiment encodes a glutamate residue at position 627 of the PB2 which is a host determinant for influenza virus replication in avian species  , has been reported to coordinate GC reactions to stimulate the expansion of plasmablast and memory B cells, thus inducing high quality influenza virus-specific antibody responses   appear cesponses , 46. To esponses  which coesponses .The Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control and Prevention (CDC) recently announced the re-inclusion of LAIV vaccines into the list of recommended vaccines for the 2018–2019 influenza season . This chMadin-Darby Canine Kidney (MDCK) cells used for plaque assays were maintained in Minimum Essential Media supplemented with 10% fetal bovine serum  and 100 U/mL penicillin plus 100 μg/mL streptomycin (1x P/S). Virus stocks were grown in 8-day-old specific pathogen-free embryonated chicken eggs (Charles River Laboratories) at 37°C for 2 days or at 33°C for 3 days, and were then chilled down to 4°C overnight. Allantoic fluid was harvested, centrifuged at 290 × g for 5 min, and titrated by plaque assays as previous study described . PlaquesCal09N1Cal09 (cH5/1) IIV is composed of an HA head domain of A/Vietnam/1203/04 (H5N1), an H1 stalk domain and N1 NA from the Cal/09 strain while the internal genes were derived from the H1N1 A/Puerto Rico/8/34 (PR8) virus. The cH8/1Cal09N1Cal09 (cH8/1) IIV consists of an HA head domain from A/mallard/Sweden/24/02 (H8N4) while the remaining genes as well as the stalk domain are the same as for the cH5/1 IIV. All IIV vaccines used in this preclinical study were adjuvanted with Adjuvant System 03 (AS03) in the same formulation as for our previous study  LAIVs are based on the Leningrad vaccine strain , which has been licensed and successfully used in humans . The cH5/1us study , 32, andus study . The bacn humans . The H3Nn humans . cH8/1 Ln humans  and prodn = 4 or 8) included in each experimental group is indicated in the figure legends. 6 PFU of B-cH9/1 virus as in our previous approach  for a more stringent evaluation of the protective immune responses elicited by these cHA-based vaccines. A “standard of care” group of ferrets which received two human doses of QIV  and a single dose of H3N2 LAIV vaccination group were also included. The challenge infection dose of either Cal/09 pH1N1 virus or A/teal/HK/W312/97 (tk/97) H6N1 virus was 106 PFU/per ferret in 1 mL of PBS. For evaluation of vaccine effectiveness from both challenge studies, nasal wash and oropharyngeal swab samples were collected from anesthetized ferrets at day 1 and 3 post-challenge infection. At day 4 post-challenge infection, anesthetized ferrets were euthanized by exsanguination followed by intracardiac injection of Sleepaway euthanasia solution . Tissue specimens  were collected from each individual ferret to quantify viral titers by plaque assays.Outbred 4 to 5-month-old castrated male Fitch ferrets were confirmed to be seronegative for circulating H1N1, H3N2, and B influenza viruses prior to purchase from Marshall BioResources . All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the ISMMS. Ferrets were randomly assigned to the different treatment groups. The number of ferrets  (100 μl at 5 μg/ml) were added to the plates and held for another 2 h incubation at RT. The supernatants were aspirated, washed three times by PBST and developed by 3, 3′, 5, 5′ tetramethyl benzidine (TMB) substrate (Biolegend). After 15 min, the reaction was stopped by addition of 3 M phosphoric acid and plates were read at a wavelength of 410 nm in a plate reader (Biotek). 105 PFU of virus  was used for subsequent NI assays. For NI assay, ELISA plates were coated and blocked in a same manner used for the NA assay. While plates were blocking, ferret serum samples  were 2-fold serially diluted in a separate 96-well plates using blocking buffer, starting with a 1:50 dilution, at a final volume of 125 μl. One hundred and twenty five microliter of virus stock (at a concentration of 105 PFU) was added on top of the diluted serum and briefly tapped for mixture, and incubated at 37°C for 1 h. Two hundred microliter of the virus/serum mixture were transferred in parallel to the fetuin-coated plates, after which the plates were incubated at 37°C for 2 h. The rest of the NI assay protocol was identical to that of the NA assay. The readouts obtained from the plate reader were plotted in Prism 7.0 software, normalized by the average of the negative control (defined as 0%) and the average of positive control . Percent inhibition was calculated by subtracting the NA activity from 100. The corresponding 50% inhibitory concentrations (IC50s) were defined as the reciprocal serum dilutions inhibiting 50% of viral NA enzymatic activity.The inhibition of N1NA enzymatic activity in serum was measured by ELLA. To determine the optimal concentration of virus used for neuraminidase inhibition (NI) assay, the NA activity induced by PR8 x H7N1 virus  and N1 NA from Cal/09 strain) was first examined as our previous study described , 54. Brireceptor-destroying enzyme (RDE) (Denka Seiken) for 16–18 h at 37°C, followed by addition of 2.5% sodium citrate for 30 min at 56°C to block non-specific sialic acid binding. PBS was then added to each sample which resulted in an assay starting dilution of 1:10. Working stocks for each influenza virus strain were prepared by diluting the virus stock to a final HA titer of 8 HA units/50 μL in FTA Hemagglutination Buffer . Twenty five microliter of pre-diluted RDE-treated sera were then mixed with 25 μL of the working stock of each influenza virus strain at 96 V-well microtiter plates and incubated for 30 min at RT to allow any HA-specific antibodies present in the serum to neutralize the influenza virus. Next, 50 μL of a 0.5% suspension of turkey red blood cells was added and incubated for 45 min at 4°C. The HI titer was defined as the reciprocal of the highest dilution of serum that inhibited red blood cell hemagglutination by influenza virus.The HAI assays were performed as described in our previous study , 55. In g for 30 min at RT with the lowest accelerating and brake off for descending. The buffy coat containing the peripheral blood mononuclear cells (PBMC) was collected and washed with enriched RPMI (eRPMI) media (RPMI1640 media containing 5% FBS and 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)]. Contaminating red blood cells (RBC) present in the single-cell suspensions were lysed with RBC lysis buffer (Affymetrix eBioscience). Mediastinal lymph nodes (MLNs) were excised, dissociated with digestion media [eRPMI media containing 30 μg/mL of DNase I  and 1 mg/mL of Type IV collagenase (Thermo Fisher)] for 30 min at 37°C. Next, the enzyme digested-lymph nodes were mechanically rinsed and passed through 70 μm nylon cell strainers . Contaminating RBCs were lysed and washed out prior to cryopreservation. Purified single-cell suspensions of PBMC and MLN leukocytes were immediately cryopreserved following isolation from tissues for flow cytometric analyses.Peripheral whole blood samples were obtained from anesthetized ferrets by intracardiac puncture and collected in Vacutainer EDTA (K2) tubes on the last day of the virus challenge experiment. Whole blood was gently layered onto HistoPaque-1077 (Sigma-Aldrich) in a 1:1 ratio and immediately centrifuged at 400x http://www.iedb.org/) and examined in the cellular immune responses of mice or humans to influenza virus (GenScript peptide services). Each peptide was composed of 15 to 17-mers that overlapped by 10 amino acids (aa) to cover the predicted region as shown in 283−297)  was also included as a negative control. The amino acid sequences for NP encoded by Cal/09 and tk/97 were retrieved from GenBank .Predicted HA stalk-specific peptides were designed based on potential T cell epitopes which have been posted on IEDB database . Cells were, respectively stimulated with 0.1 μg of pooled NP peptides, irrelevant gp120 peptide, or 0.1 μg for each HA stalk peptide overnight. The next day, the cells were washed twice using flow cytometry staining buffer containing 2% FBS and 0.01% sodium azide in PBS) and centrifugation at 450x g at 4°C. Cells were first stained with a viability dye, LIVE/DEAD Aqua Fixable dead-cell stain kit (Thermo Fisher), according to the manufacturer's instruction, followed by surface staining with specific T cell markers [CD4  and CD8 ] for 30 min at 4°C. Then, cells were washed, fixed by intracellular Fixation buffer for 20 min at 4°C, and permeabilized using permeabilization buffer (Affymetrix eBioscience). Next, the cells were intracellular stained with anti-CD3  and anti-biotin conjugated IFN-γ  antibodies for 30 min at RT. Antibodies to CD4, CD8, and CD3 were directly conjugated to distinct fluorochromes to distinguish T cell subsets. The anti-biotin IFNγ antibody was detected by a secondary antibody . After the T cell staining, the cells were washed twice and resuspended in flow cytometry staining buffer for data acquisition using a BD 13-colors LSR-II instrument with 488-, 633-, and 405-nm lasers and FACS Diva software. All data files were analyzed using FCS Express software, version 6.04.0034.We phenotyped the T cell responses following the procedure described in our previous report , 61. In *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Serum analysis and viral titer quantification were analyzed in an un-blinded manner.One-way or two-way ANOVA were, respectively used for comparisons of more than two groups with single or multiple time points, followed by a Tukey's or Dunnett's post-test to adjust for multiple comparisons. All statistical analyses were performed in GraphPad Prism 7.0. The asterisks shown in the figures refer to the level of significance, All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai (ISMMS).W-CL, RN, DS, FK, and RA: performed experiments; W-CL, AS, RN, FB-S, AG-S, PP, FK, and RA: designed and coordinated experiments; W-CL and RA: analyzed data and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.AG-S, FK, and PP are inventors in patent applications filed and owned by the Icahn School of Medicine at Mount Sinai regarding influenza virus vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmonella enterica Serotype 4,[5],12:i:-, a monophasic variant of S. Typhimurium, with high virulence and multidrug resistance is distributed globally causing pathogenicity to both humans and domesticated animals. BOX-A1R-based repetitive extragenic palindromic-PCR (BOX)-PCR proved to be superior to three other repetitive element-based PCR typing methods, namely, enterobacterial repetitive intergenic consensus (ERIC)-, poly-trinucleotide (GTG)5-, and repetitive extragenic palindromic (REP)-PCR (carried out under a single optimized amplification condition), in differentiating genetic relatedness among S. 4,[5],12:i:- isolates from feces of hospitalized patients (n=12) and isolates from minced pork samples of S. 4,[5],12:i:- (n=6), S. Typhimurium (n=6), and Salmonella Serogroup B (n=4) collected from different regions of northern Thailand. Construction of phylogenetic trees from amplicon size patterns allowed allocation of Salmonella isolates into clusters of similar genetic relatedness, with BOX-PCR generating more unique clusters for each serotype than the other three typing methods. BOX-, (GTG)5-, and REP-PCR indicated significant genetic relatedness between S. 4,[5],12:i:- isolates 1 and 9 from hospitalized patients and S. 4,[5],12:i:- isolate en 29 from minced pork, suggesting a possible route of transmission. Thus, BOX-PCR provides a suitable molecular typing method for discriminating genetic relatedness among Salmonella spp. of the same and different serotypes and should be suitable for application in typing and tracking route of transmission in Salmonella outbreaks. Salmonella (NTS) is a cause of gastroenteritis, particularly in young children, the infection arising from consumption of contaminated food or unhygienic practices ,12:i:- is an emerging serotype with distribution worldwide and a significant infection rate of humans and domestic animals ,12:i:- expresses O 4, 5, 12 antigens but not fljB (encoding phase 2 flagellum) due to defective phase switching ,12:i:- isolates from two different sources by comparing four different repetitive element-based PCR methods, namely, BOX-, ERIC-, (GTG)5-, and REP-PCR. Clustering power and discriminatory index of each technique were evaluated using the S. 4,[5],12:i:- isolates, together with S. Typhimurium and S. Serogroup B isolates. In addition, phylogenetic trees were constructed to determine relationship of clusters with other data sets, such as antibiogram profile and carriage of antibiotic-resistant genes.The study sought to simplify identification of genetic relatedness with high discrimination between Salmonella isolates consisted of S. 4,[5],12:i:- originally isolated from hospitalized patients (n=12) at Phayao Ram Hospital, Phayao province, during 2015–2017 ,12:i:- (n=6), S. Typhimurium (n=6), S. Serogroup B , and one unknown Salmonella serotype from minced pork samples collected from retail markets in five different provinces of northern Thailand ,12:i:- isolates as dots in a cubic coordinate system and also as 3D spheres to enable visualization of 3D clustering in a realistic perspective.3D visualization of similarity to dataset of BOX-PCR clustering based on multidimensional scaling (MDS) was performed using a Metric algorithm (Applied Maths), and the coordinate space window was calculated based on the similarity matrix. Coordinate space window displayed each Salmonella isolates, a discriminatory index (D) was calculated at different levels of similarity index according to the formula ,12:i:- (n=18) as well as Salmonella Serogroup B (n=3) including S. Schwarzengrund, S. Agona, and S. Saintpaul and one unknown was either from the feces of hospitalized patients or minced pork collected from 5 different provinces of the northern Thailand , were applied to eighteen S. 4,[5],12:i:- isolates from hospitalized patients and from minced pork samples collected in northern Thailand, generating 9–28 amplicons of different sizes   using a curve-based algorithm (Pearson correlation) to create a similarity scale. A phylogenetic tree was constructed from each of the four PCR amplicon profiles ,500 bp) , with BOERIC-PCR . In ordeprofiles , which smilarity . Both thSalmonella spp. of the same serotype from same or different sources. At 50% similarity, BOX-PCR and GTG5-PCR were capable of differentiating S. Typhimurium and S. 4,[5],12:i:- isolates from minced pork into 2–4 clusters, while ERIC-PCR and REP-PCR placed Salmonella isolates of same serotype into one cluster each with D value = 0  with D value = 0.6–0.7 (S. Typhimurium isolates) and 0.8–0.9   with D value = 0.9091 and S. 4,[5],12:i:- isolates from minced pork (n=6) with D value = 0.8667. In addition, BOX-PCR effectively placed the four Salmonella Serogroup B isolates into their own cluster  . BOX-PCR cluster . BOX-PCRS. 4,[5],12:i:- isolates from feces of hospitalized patients and minced pork samples was assessed by comparing the four constructed phylogeny trees  to S. 4,[5],12:i:- isolates 1 and 9 from the patients, REP-PCR that the closest genetic relatedness (95.7%) was between S. 4,[5],12:i:- isolates 1 and en 29; and BOX-PCR that the genetic relatedness of S. 4,[5],12:i:- isolates 1 to also 9 to en 20 and en 29 was 82.4 and 92.9%, respectively. ERIC-PCR showed S. 4,[5],12:i:- isolates 56 had the closest genetic relatedness (81.6%) to all isolates from minced pork. The genetic relatedness between S. 4,[5],12:i:- isolates from two different sources could be clearly shown by the 3D coordinate space window, which demonstrated two S. 4,[5],12:i:- isolates of patients (ID 1 and 9) were in the cluster of S. 4,[5],12:i:- isolates from minced pork  and minced pork samples (n=6), S. Typhimurium isolates from minced pork samples (n=6), and other Salmonella Serogroup B isolates from minced pork samples (n=4) showed 50% similarity with three clusters of S. 4,[5],12:i:- isolates, one of S. Typhimurium isolates, and 3 of S. Serotype B isolates  with optional sulphamethox/trimethoprim (SXT), while S. 4,[5],12:i:- mainly to ampicillin, streptomycin, and tetracycline (AMP/TE/S). All ESBL-producing S. 4,[5],12:i:- isolates from patients were clustered together and apart from ESBL-producing isolates of minced pork, but all with the same antibiogram (AMP/TE/S/C/CTX). There was complete linkage between chloramphenicol resistance and ESBL-producing S. 4,[5],12:i:- isolates, blaCTX-M group 1 being the predominant determinant. From cluster analysis . One ESBL-producing S. 4,[5],12:i:- resistant to meristin through acquisition of mcr-3 gene was also observed  with high genetic relatedness to isolates from minced pork, suggesting the possibility that (some) Salmonella isolates causing human infection could have come from contaminated food (minced pork) as traditional food of northern Thai food often contains raw meat, such as raw spicy minced pork. Many studies have shown contaminated raw meat and poultry are causes of Salmonella transmission if there is a lapse in food safety practices, leading to increased risks in salmonellosis outbreaks ,12:i:- isolates from feces of hospitalize patients highly shared genetic relatedness and formed a unique cluster, with their antibiograms indicating acquisition of blaCTX group 1 as reported in many countries ,12:i:- isolates resistant to meristin and harboring mcr-3 gene is the first observed in northern Thailand, which poses the risk of traveler's diarrhea to those returning after travelling in this region of the country ,12:i:- isolates from different sources and geography.Virulent ESBL-producing ountries , 40. To  country . In addiS. 4,[5],12:i:- isolates from feces of hospitalized patients in Phayao province, northern Thailand, and those from minced pork samples obtained at different locations in the same region of the country and should be adopted in tracking transmission during Salmonella outbreaks.The simple BOX-PCR typing method is effective in differentiating genetic relatedness of