Mycobacterium tuberculosis secreted antigen Ag85A fused with the influenza A virus (IAV) HA2 protein epitopes, pEGFP/Ag85A-sHA2 (pAg85A-sHA2), was designed to provide protection against influenza. The antigen encoded by the DNA vaccine vector was efficiently expressed in mammalian cells, as determined by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence analyses. Mice were immunized with the vaccine vector by intramuscular injection before challenge with A/Puerto Rico/8/34 virus (PR8 virus). Sera and the splenocyte culture IFN-γ levels were significantly higher in immunized mice compared with the control mice. The novel vaccine group showed a high neutralization antibody titer in vitro. The novel vaccine vector also reduced the viral loads, increased the survival rates in mice after the PR8 virus challenge and reduced the alveolar inflammatory cell numbers. Sera IL-4 concentrations were significantly increased in mice immunized with the novel vaccine vector on Day 12 after challenge with the PR8 virus. These results demonstrated that short HA2 (sHA2) protein epitopes may provide protection against the PR8 virus and that Ag85A could strengthen the immune response to HA2 epitopes, thus, Ag85A may be developed as a new adjuvant for influenza vaccines.A novel DNA vaccine vector encoding the  The influenza A virus (IAV) pandemic of 2009 involving the H1N1 strain has raised international concern over the potential for outbreaks of increased severity and highlighted the need for improved global surveillance and vaccination strategies [Hemagglutinin (HA) is the most abundant protein on the viral coat and is highly immunogenic. Antibody responses against the viral surface protein HA are the major determinants of protection against IAV ,4. HowevMycobacterium tuberculosis (M. tuberculosis) has demonstrated marked immunomodulatory effects and enhanced antigen-specific antibody production when used in combination with influenza vaccines [M. tuberculosis secreted antigen Ag85A (Ag85A) is known to increase T helper type 1 (Th1) cytokine responses [The Bacille Calmette-Guérin (BCG) vaccine against vaccines ,10,11. Aesponses , such asesponses .Based on the hypothesis that the Ag85A may serve as an effective immune adjuvant for HA2, we constructed a novel influenza vaccine vector expressing the conserved region of the HA protein linked to Ag85A. The efficacy of this vaccine vector in preventing mortality and morbidity in mice was evaluated after challenge with IAV, and the cytokine profiles of the sera and cultured splenocytes were determined.The transfection of HEK293 cells with the DNA vaccine vectors, pHA, pAg85A-sHA2, pEGFP-C2, pAg85A and psHA2, all produced strong green fluorescent signals A. The reMice were immunized with pAg85A-sHA2, psHA2, pAg85A, PBS, pEGFP-C2 or pHA. Development of antibodies against HA was evaluated by hemagglutination inhibition (HI) assays. The HI titers were represented as group arithmetic means (log 2 dilution). As shown in P < 0.05). Furthermore, the IAV titers in the samples from the pAg85A-sHA2 vaccinated group were significantly lower than those in the other groups vaccinated with the plasmid vaccine vectors pEGFP-C2, psHA2 and pAg85A (P < 0.05). While not as effective as pAg85A-sHA2, vaccination with psHA2 decreased the challenge viral load to a significantly lower level compared with the pEGFP-C2 and pAg85A vaccinated groups (P < 0.05). However, the immunization of mice with pHA, psHA2 or pAg85A-sHA2, all showed an significant decrease in lung PR8 virus loads in mice compared with the PBS and pEGFP-C2 immunized control groups  (P < 0.0P < 0.05). The NAb titer from the psHA2 vaccinated group was significantly higher than those from the pEGFP-C2, PBS and pAg85A vaccinated groups (P < 0.05).The development of NAbs against HA was evaluated by the neutralization assay. The titers from each serum sample were expressed as the dilutions (log 2 transformed) that reduced the cytopathic effect (CPE) resulting from the viral inoculation by 50% in the Madin-Darby canine kidney (MDCK) cells. As shown in P < 0.05). The IgG NAb titer from the pAg85A-sHA2 vaccinated group was significantly higher than that from the psHA2 vaccinated group (P < 0.05).The titers of IgG against HA were evaluated by ELISA. The titers of each serum sample were expressed as optical density (OD) values. The ELISA substrates used were indicated for recombinant H1 HA proteins of A/PR/8/34 B or recoed group C . Splenocytes from mice vaccinated with pAg85A-sHA2 produced a 1.5-fold significantly higher IFN-γ response to HA than those from mice vaccinated with psHA2 (P < 0.05). Meanwhile, IFN-γ production from the splenocytes in the PBS group was significantly lower than that of other groups upon stimulation with HA (P < 0.05). HA induced significantly lower IFN-γ levels than did ConA (data not shown). The HA-induced IFN-γ production 2 or pHA  (P < 0.0P > 0.05) (P < 0.05)  B. Sera I < 0.05) B.P < 0.05) (P < 0.05). The survival rate of mice vaccinated with psHA2 was higher than that of mice vaccinated with pAg85A, pEGFP-C2 or PBS. Furthermore, no significant differences were observed between survival rates in the pAg85A-sHA2 group and the pHA group (P > 0.05)  . The sur > 0.05) . P < 0.05 vs. mice of PBS group, psHA2 group, pAg85A group and pEGFP/C2 group) (P < 0.05).Body weight loss could be used to follow the course of IAV infection in mice. Marked body weight loss was detected 48 h after infection. While body weights significantly decreased in the PBS, psHA2, pAg85A and pEGFP/C2 groups, those of the pAg85A-sHA2 and pHA groups did not change, indicating a clinically protective effect by the fusion of the DNA vaccine vector that was comparable to that of the pHA vaccine vector (2 group) . The bodP < 0.05).Lungs were harvested on Day 8 after infection with the PR8 virus to prepare hematoxylin and eosin (H&E)-stained lung slides for histopathological examination. The alveolar inflammatory cell numbers were reduced in the psHA2, pAg85A-sHA2 and pHA groups . Lung inWhile antibodies against HA are the major determinants of host protection against IAV infection, HA can also induce high frequencies of effector and memory CD4 T cells . The varIn this study, we developed a DNA vaccine vector encoding the Ag85A-sHA2 fusion protein with the aim of enhancing the protection against IAV that is afforded by the HA2 immunogen alone. Following the confirmation of successful expression in mammalian cells, the novel vaccine vector and corresponding controls were used to immunize mice via the intramuscular route before the challenge with the PR8 virus. Interestingly, while the sera from mice vaccinated with the pAg85A-sHA2 fusion vaccine vector exhibited apparently low functional antibody titers in the HI test, they were relatively highly effective in the neutralization assay. Furthermore, the viral loads of the mice vaccinated with the fusion construct (pAg85A-sHA2) were the lowest after challenge compared with those of the other groups given DNA vaccine vectors expressing either Ag85A or HA2 epitopes alone. The HI antibody titers were low in these groups for both the primary and booster responses, as might be expected based upon the absence of the HA1 antigen in the vaccine vector, which could induce antibodies against the receptor binding site or other epitopes involved in HI. This observation is consistent with a previous study describing NAbs to HA2 epitopes that could not completely prevent the hemagglutination of erythrocytes . It is sThe sHA2 construct did not include a signal peptide. The posttranslational processing of sHA2 did not involve its secretion into the extracellular space. We assumed that the high expression of the sHA2 ORF in our experiments, which was driven by the plasmid’s strong cytomegalovirus (CMV) promoter, led to the release of free sHA2, which was responsible for the induction of antibodies and cell damage.Our analysis of cytokine production showed that the sera IFN-γ levels in the psHA2 group were significantly higher than those in the pEGFP-C2 and PBS groups after PR8 virus challenge. IFN-γ is a representative Th1 cytokine associated with inhibition of IAV proliferation . The alvImportantly, vaccination with the pAg85A-sHA2 fusion construct produced significantly higher sera IFN-γ levels and resulted in higher survival rates than vaccination with pHA2. The IAV titer from the pAg85A-sHA2 group was lower compared to those from the pEGFP-C2, psHA2 and pAg85A groups. The alveolar inflammatory cell numbers were reduced in the psHA2, pAg85A-sHA2 and pHA groups. The increased survival rate after challenge in the pAg85A-sHA2 group was likely due to Ag85A-mediated immune activation, which enhanced the protective effects of sHA2 against PR8 virus challenge, thereby decreasing the viral load. As IAV can suppress the functions of immune cells ,21, the P < 0.05) on Day 12 after infection with the PR8 virus. These results demonstrated that pHA or pAg85A-sHA2 vaccine could increase the Th2 cytokines response. In addition to analyzing the IFN-γ responses above, we also investigated the IL-4 levels following vaccination with the various constructs. IL-4 is a cytokine of the Th2 response . Sera ILHA2 is masked by the membrane-distal portion of HA, which is a bulky and highly immunogenic globular head domain . The HA2et al. [The HA ABO21709.1) and Ag85A (AY207395.1) sequences were downloaded from the National Center for Biotechnology Information (NCBI). The B-cell and T-cell epitopes for HA2 and Ag85A were predicted bioinformatically using the network servers BCEPRED  and SYFP09.1 and et al. ,15.The pEGFP/Ag85A (pAg85A) plasmid was constructed by first cloning the Ag85A sequence (aa 1 to 250 of the N-terminus) ,15,16 frTM 2000 transfection reagent , 0.8 μg of each of the purified eukaryotic expression plasmids pHA, pAg85A-sHA2, pAg85A, pEGFP-C2 and psHA2 were transfected into 1 × 105 HEK293 cells (CRL-1573™) obtained from the American Type Culture Collection (ATCC) according to the manufacturer’s instructions. Each plasmid was transfected in triplicate wells, and PBS was applied as a mock transfection control. Plasmid expression in all sets of transfected cells was assessed by fluorescence microscopy at 48 h after transfection.Using the LipofectamineTM H Minus First Strand cDNA Synthesis Kit . cDNA samples were amplified by PCR using 2× TransTaq High Fidelity (HiFi) PCR SuperMix  in a 50 μL reaction mixture according to the manufacturer’s instructions. PCR primer pairs for mouse GAPDH were obtained from the RevertAidTM H Minus First Strand cDNA Synthesis Kit (400 bp). The following PCR primer pairs were used: pAg85A-sHA2, 5'-CCCTCGAGCATGCAGCTTGTTGACAG-3' and Total RNA from each set of transfected cells was isolated by using 1 mL TRIpure Reagent according to the manufacturer’s instructions . Contaminating genomic DNA was then eliminated from the RNA preparations by using RNase-free DNase I . Reverse transcription (RT) was performed with the ReverAid5'-CGCGGATCCTCATTTGGGGTAATCATAA-3'; pAg85A, 5'-CCCTCGAGCATGCAGCTTGTTGACAG-3' and 5'-CGGGATCCTTAGCCGTTGCCGCAGTACAC-3'; psHA2, 5'-CCGCTCGAGTATTGAAGGGGGATGGA-3' and 5'-CGCGGATCCTCATTTGGGGTAATCATAA-3'; and pHA, 5'-CATAAGCTTAACCAAAATGAAGGCAACC-3' and 5'-CGCGGATCCTCATATTTCTGAAATTCTAAT-3'. Control PCRs using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were performed on cDNA from the pHA, pAg85A-sHA2, pAg85A, pEGFP-C2 and psHA2 groups. Test PCRs for the pEGFP-C2 group were performed with the pHA primers, psHA2 primers, pAg85A primers and pAg85A-sHA2 primers. Test PCRs for the pHA, pAg85A-sHA2, pAg85A and psHA2 groups were performed with their respective primers. All PCRs were performed simultaneously under the same conditions as follows: five minutes at 95 °C, followed by 30 cycles consisting of 30 s at 95 °C, 30 s at 50 °C and 2 min at 72 °C. Following the amplification, 10 μL of each PCR product were subjected to electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. PCR amplification and sequencing were performed using an ABI Prism 377XL DNA Sequencer.A total of 120 female BALB/c mice, 4–6 weeks old, were purchased from the animal care center of Sichuan University and divided into six groups, with 20 mice in each group. They were bred under pathogen-free conditions. Each group of mice was injected separately with one of the following: pAg85A-sHA2, pAg85A, psHA2, pEGFP-C2, PBS or pHA. The mice were immunized by intramuscular injection in the quadriceps of both legs (50 μg plasmid in 50 μL PBS per leg) twice with a 3-week interval between injections. The pHA group was the positive control group.Seven days after the booster immunization, the mice were challenged with IAV, as described previously ,18. In bet al. [et al. [TM H Minus First Strand cDNA Synthesis Kit . Lung cDNA samples from all of the groups were stored at -80°C before use. A 0.6 mg left lung samples from each group was used for the histopathological analyses. The third serum samples for the cytokine measurements were obtained from each mouse via tail bleeding on Day 12 after the virus challenge. All of the sera and bronchoalveolar lavages samples were stored at −20 °C before use.Specimens were collected as reported by Zhang et al.  and Tamu [et al. . In brieet al. [The HA inhibition (HI) assay was performed as reported by Zhang et al. . In brie(DMEM) supplemented with 2 µg/mL trypsin  and 3% bovine serum albumin  were added to each well of the 24-well plate. Each dilution was performed in four replicate wells. The cytopathic effect (CPE) was assayed after incubation at 37 °C for 2 days. The titer from each sample, recorded as the dilution that reduced the CPE by 50%, was calculated by the Reed–Muench method.The neutralization assay was performed as previously described . In brieNinety-six-well plates (Costar) were coated with 0.1μg per well of purified recombinant HA protein in PBS. The recombinant HA proteins of the A/Anhui/1/2005  and A/PR/8/34  viruses were obtained from Sino Biological Inc., China. 500-fold dilutions of antisera were incubated on the plates, bound antibody was detected with peroxidase-link Goat Anti-Mouse IgG antibody  according to the manufacturer’s instructions. The experiments were repeated three times, and the results from one experiment are shown.Spleens were removed aseptically on Day 8 after the virus challenge and processed as follows. Cells were isolated, washed, adjusted to a concentration of 5 × 106 cells per mL, and grown in a 96-well flat-bottom plate (5 × 105 cells per well) in Roswell Park Memorial Institute medium (RPMI) 1640 medium, supplemented with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), glutamine and 10% heat-inactivated fetal calf serum (FCS). Cells were stimulated separately with 0.5 µg concanavalin A (ConA) or 1.25 µg recombinant HA of A/PR/8/34  and incubated at 37 °C in a humidified CO2 incubator. After 72 h, supernatants were harvested, pooled and stored frozen at −20 °C until assayed. The experiments were repeated three times, and the results from one experiment were shown. Sera were collected on Days 8 and 12 after infection with the PR8 virus. For the cytokine measurements, sera from each group were pooled and stored at −20 °C until assayed. IFN-γ and IL-4 from the sera and IFN-γ from the splenocyte culture supernatants were measured using specific enzyme-linked immunosorbent assays  according to the manufacturer’s instructions.et al. [IAV titering was performed as reported by Zhang et al. . In brieTM H Minus First Strand cDNA Synthesis Kit. The following IAV NS1 (GenBank: V01104.1) PCR primer pairs were used: 5'- GCTTTCAGGTAGATTGCT -3' and 5'-CCATTCTCATTACTGCTTCT-3'; PCRs for the pHA, pAg85A-sHA2, PBS, pAg85A, pEGFP-C2 and psHA2 groups were performed with GAPDH primers and NS1 primers. All of PCRs were performed simultaneously under the same conditions as follows: Five minutes at 95 °C, followed by the 25 cycles consisting of 30 s at 95 °C, 30 s at 50 °C and 2 min at 72 °C. After amplification, 10 μL of each PCR product were subjected to electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. PCR amplification and sequencing were performed using an ABI Prism 377XL DNA sequencer.Lung cDNA samples from each group were amplified by PCR using the 2× TransTaq High Fidelity (HiFi) PCR SuperMix  in a 50 μL reaction mixture according to the manufacturer’s instructions. PCR primer pairs for mouse GAPDH were obtained from the RevertAidLungs harvested at the 8th day after the virus challenge were used for histological examination. The specimens were fixed in 10% formalin, and embedded in paraffin. Four micrometer sections were stained with H&E. All of the sections were examined for histopathological changes in a blind manner, as described previously . In brieP values < 0.05 were considered to be significant. Data are presented as means ± SD. Group-group comparisons were analyzed using t-tests. Survival rates were determined using the Kaplan-Meier method. The expression of sHA2 alone could provide a certain level of protection against IAV, but the additional expression of Ag85A from a DNA fusion construct significantly enhanced the protective immune responses to the HA2 epitopes. The pAg85A-sHA2 vaccine vector improved Th1 type responses in the lungs and splenocytes of mice compared with the psHA2 vaccine vector, suggesting that the Ag85A-mediated immune activation was responsible for the increased protection against the PR8 virus challenge. Our findings support the further development of Ag85A as a novel adjuvant for influenza vaccines.


In acute myocardial infarction (AMI), reperfusion injury is often associated with hemorrhage and microvascular obstruction (MVO), which are independent predictors of poor patient outcomes. T2 and T2* cardiovascular magnetic resonance (CMR) approaches have been instrumental in detecting hemorrhage; however, the relative sensitivity of each of these measures remains to be investigated. In this patient study, our objective was to quantitatively compare the extent of hemorrhage relative to the area-at-risk (AAR) post-AMI using T2 and T2* mapping techniques.50 patients with STEMI underwent a CMR exam on a 1.5T scanner (GE Signa Excite) at 48 hrs post percutaneous-coronary-intervention. T2 was quantified using a cardiac-gated T2-prepared spiral imaging sequence (TE=2.9-184 ms). T2* was determined from a multi-echo gradient-echo acquisition (TE=1.4-12.7 ms). Infarct/MVO was assessed using early gadolinium enhancement (EGE) imaging. Using T2* maps, hemorrhage extent was quantified as the region where T2* was at least two standard deviations (SD) below that in remote myocardium. From T2 maps, AAR was determined as the region where T2 was at least two SD above that in remote myocardium. Any ‘signal voids' within this area were identified as hemorrhage and the computed AAR was accordingly corrected. T2 and T2* values were recorded in these regions. Areas were expressed as a percentage of LV myocardium. Only patients with MVO were included in the analysis since hemorrhage is typically associated with an MVO.19 patients (38%) presented with an MVO. Hemorrhage extent determined by T2 was significantly less than that computed from T2* . AAR assessed by T2 was underestimated if the hemorrhagic region was not accounted for . Hemorrhage extent, by both T2 and T2*, demonstrated linear relationship with AAR Fig. , althougT2* appears to be a more sensitive and specific indicator of hemorrhage. T2 underestimates the hemorrhage extent and AAR due to counteracting influence from edema. In patients with MVO/hemorrhage, both T2 and T2* imaging may be necessary to accurately quantify hemorrhage extent and AAR.NA.


Transcriptional factor E2A is crucial for the normal development and differentiation of B and T lymphocytes. Dysregulation of E2A leads to leukemia and tumorigenesis of some solid tumors. The expression and clinical significance of E2A as well as its role in colorectal cancer (CRC) are still unknown. This study aims to assess E2A expression in CRC tissues, evaluate its prognosis value, and investigate its role in colon cancer cell growth.E2A expression in CRC tissues and normal mucosa was detected by immunohistochemical staining; Kaplan-Meier survival curve and Cox regression model were used to evaluate the prognostic value of E2A. Lentivirus was used to construct E2A stably knocked-down cells. MTT assay was employed to detect cell proliferation change; cell cycle was analyzed by flow cytometry; and chromatin immunoprecipitation (ChIP) assay was used to validate the predicted binding target of E2A.Expression of E2A was lower in CRC tissues than normal mucosa; low E2A expression correlated with advanced TNM stage and larger tumor size, and predicted poor prognosis of CRC patients. E2A knockdown resulted in increased cell proliferation rate and cell cycle acceleration. ChIP assay showed miR-320a was a direct target of E2A and upregulation of miR-320a in E2A downregulated cells could reverse cell proliferation and cell cycle changes caused by E2A deficiency.E2A is an independent prognostic factor for CRC patients and targets miR-320a to regulate cell proliferation of colon cancer cells. The mammalian E2A gene is located at chromosome 19 and is non-tissue-specifically and ubiquitously expressed in a wide range of cell types. Through alternative splicing, the E2A gene encodes two isoforms, E12 and E47 (collectively referred as E2A proteins), which both have the basic-Helix-Loop-Helix (bHLH) domain and could regulate gene transcription by binding to the E-box DNA sequences, CAGGTG. Though widely expressed, E2A is not essential in some organogeneses like skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, and neurogenesis Besides its crucial role in normal B and T cell development, E2A also participates in tumorigenesis. Studies had reported the well-established role of E2A in leukemogenesis: two fusion proteins, E2A-HLF The expression of E2A in colorectal cancer (CRC) and its prognostic value have not been discussed before and it’s still unknown whether E2A promotes or represses the development of CRC. In this study, to our knowledge, for the first time we investigated the clinical significance of E2A in CRC and found its usefulness as a prognostic marker; furthermore, we found E2A suppressed tumor growth by targeting miR-320a in colon cancer cells, demonstrating its tumor-suppressive role in CRC.A total of 98 colorectal cancer patients were included; the patients were treated with surgery between June 2007 and January 2008 at Shanghai Minimally Invasive Surgery Center. Patients were excluded if they: had received neoadjuvant chemoradiotherapy; had unresectable colorectal cancers; had tumors of other organs; were unlikely to be interviewed during the follow-up. Demographic and clinicopathological data were extracted by chart review. Patients were interviewed by telephone at three months, six months, and then annually after surgery. Tumor samples were cut immediately after surgical specimens' removal during the operations and then fixed with formalin and preserved at 4°C for two weeks before next treatment; normal tissues were defined as mucosa located at least 5cm away from the tumor margin and cut, fixed, preserved and treated as above. This study was conducted with the written informed consents of all enrolled patients before surgery and under the protocol approved by the Ethical Committee of Ruijin Hospital Shanghai Jiao Tong University School of Medicine.2O2 to inactivate endogenous peroxidase. Slides were incubated with E2A primary antibody  at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Santa Cruz) for 30 minutes at room temperature. Complex visualization was done with a 2-Solution DAB Kit (Invitrogen). Negative controls were obtained by replacing the E2A primary antibody with preimmune rabbit serum.Immunohistochemistry staining was performed as protocol previously described Slides were examined by two researchers (Huang and Quan) independently. Scoring criteria used were as previously described with minor modifications. Staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining); positive cells on each section were scored as 0 (less than 10%), 1 (10%-25%), 2 (26%–50%), and 3 (>50%). The final score was a product of scores of intensity and positive cell of each slide. Slides with score 0–3 were defined as low expression and 4–9 as high expression.2.Colon cancer cell lines LOVO, HCT116, Caco-2, HT29, SW480, and SW1116 were obtained from American Type Culture Collection (ATCC) and subcultured and preserved by Shanghai Institute of Digestive Surgery; normal human colon mucosal epithelium cell NCM460 was kindly donated by Jingqing Zeng . LOVO was cultured in F-12K Nutrient Mixture Medium , HCT116 and HT29 in McCoy's 5A (Corning Cellgro®), SW480 and SW1116 in Leibovitz' L-15 (Corning Cellgro®), and NCM460 in DMEM (Corning Cellgro®). All culture medium above was supplemented with 10% fetal bovine serum (FBS) . Caco-2 was cultured in Minimum Essential Medium (Corning Cellgro®) with 20% FBS. Cells were placed in the incubator  at 37°C, in a humidified atmosphere with 5% CO562 using microplate reader . Western blot was performed according to previously reported protocol Cells were harvested at a confluence of 80% with RIPA  and the protein concentration was determined with BCA Protein Assay Kit  by measuring OD5′-CCACT TCACT GAGTC GCACAG-3′; reverse: 5′-GTCTC TCCCG AAGGA GGCATA-3′) was evaluated by qRT-PCR, using SYBR Green PCR Master Mix (Applied Biosystems); the RNA level of GAPDH  was used for normalization. The expression of miR-320a was evaluated by qRT-PCR using Taqman microRNA RT Kit , TaqMan MicroRNA Assays (Applied Biosystems), and Taqman universal PCR master mix , in accordance with the manufacturers' instructions; the U6 miRNA level was used for normalization.Total cell RNAs were extracted using Trizol reagent (Invitrogen) and total cell microRNAs (miRNAs) were extracted with mirVana™ miRNA Isolation Kit . RNA reverse transcription was performed by using PrimeScript RT Master Mix Perfect Real Time . The mRNA level of E2A (forward: E2A/shRNA-eGFP-lentivirus particles and E2A/sh-negative control (shNC)-eGFP-lentivirus particles, namely E2A/shRNA-LV and E2A/shNC-LV, were purchased from Novobio . shNC was synthesized with the same bases of shRNA but with scrambled sequence. Lentiviral transfection and cell screening were performed according to manufacturer's instruction to establish the E2A stably downregulated and the stably NC transefected SW480 clones, i.e. SW480/shE2A and SW480/shNC. Transfection efficiency was evaluated by visual examination of the percentage of eGFP-expressing cells under fluorescence microscope , western blot, and qRT-PCR.5 cells/well, 24 hours before transfection. Lipofectamine 2000  was used for transfection, in accordance with manufacturer's instruction. Efficiency of transient transfection was examined by western blot or qRT-PCR. Cells transfected with vectors were used as control.Plasmids, pEZ-M29-E12 (plasmid encoding isoform E12), pEZ-M29-E47 (plasmid encoding isoform E47), pEZ-M29-NC (plasmid with negative control sequence) and vector controls were purchased from Genecopoeia  and validated by sequencing; miRNA-320a mimics (double-strand RNA molecules mimicking miR-320a) and mimics NCs  were purchased from GenePharma . Cells of log phase were harvested and seeded in 6-well plates at a density of 4*10Cell proliferation assay was performed with the Cell Counting Kit-8 (CCK8) . Briefly, cells were digested with trypsin , washed with phosphate-buffered saline (PBS) twice, filtered with a strainer , resuspended in RIPA 1640 medium, counted, and diluted to a final concentration of 5 cells/µl. Then cells were seeded in 96-well plates, 200 µl (1000 cells) per well, in sixplicate, and placed in the incubator for 6 days. Viable cells were quantified at each 24 h interval with CCK8, in accordance with manufacturer's protocol, and the absorbance at 450 nm was measured by using microplate reader .6) were harvested 48 hours after transient transfection, as well as the corresponding controls. Then, cells were washed twice with ice-cold PBS, fixed with 75% ethanol and stored at 4°C overnight. Upon analyzing, cells were washed with PBS twice and treated with RNase at 37°C for an hour, followed by staining with Propidium Iodide for 30 minutes in dark. Cell cycle analysis was then performed with flow cytometry .Before analysis, cells  was purchased to perform the ChIP assay, according to the manufacturer's protocol. Briefly, one 15 cm cell culture plate of SW480 cells (about 80% confluence) was fixed with 1% PFA (Sigma). Then cells were lysed, sonicated to shear DNA and immunoprecipitated with anti-E2A (Santa Cruz) and control antibody (IgG). After that, protein/DNA crosslink was reversed and DNA was purified for the following PCR, using Takara Ex Taq Hot Start Version (Takara).P<0.05 was considered statistically significant.All statistical analyses were conducted by using the SPSS 15.0 software. The correlation of clinical factors with E2A expression was examined by Pearson correlation analysis. The effect of E2A on survival was estimated using the Kaplan-Meier curve and log-rank test. Univariate and multivariate Cox's proportional hazards model were used to evaluate the effects of E2A expression and clinicopathological parameters on 5-year OS and DFS, respectively. Student's t test was used to analyze differences between two groups and one-way ANOVA was employed in case of data consisted of more than two groups. A two-tailed value of P<0.01). Staining of E2A in normal mucosa presented in nucleus and cytoplasm both, but in cancer tissues, E2A staining was predominantly nuclear (P<0.01) and larger tumor size (P = 0.035), but was not related to patients age (P = 0.761), gender (P = 0.655), tumor histology (P = 0.985), or tumor site (P = 0.120). Thus E2A was likely to be involved in the development and progression of CRC.Firstly, we evaluated the expression of E2A protein in CRC tissues and normal mucosa by immunohistochemistry . Colorec nuclear . More im nuclear . We thenP = 0.000, for OS and DFS both). Next, we employed the Cox regression model to examine the prognostic value of E2A. In univariate analysis, TNM stage and E2A expression were found to be predictive for OS and DFS; while in multivariate analysis, both factors also predicted worse clinical outcomes  have been found to be involved in the pathogenesis of human diseases including cancer Then, we asked whether miR-320a was the target through which E2A regulated cell proliferation. By transfecting miR-320a mimics into SW480/shE2A cells, we found the cell cycle progression caused by E2A knockdown was arrested and cell proliferation rate was decreased . Also, cDespite great efforts having been made to improve the prevention and treatment of CRC, its morbidity and mortality still remain high: both were estimated to be the third among all cancers in USA, 2013 The role of E2A in normal B and T cell development and leukemogenesis has been well studied Previous studies have shown the self-contradictive roles of E2A in proliferation: silencing of E2A in breast As a transcriptional factor, E2A exerts its functions by regulating gene expression and studies have reported its downstream targets including p21, Id1, c-Myc, etc In summary, we present convincing evidence showing that E2A is a prognostic factor for CRC patients and plays a tumor-suppressive role in CRC cells. Through binding directly to miR-320a, E2A regulates cancer cell cycle progression and controls cell growth. Yet, the role of E2A in solid tumors has not been fully understood and our findings only partially unveil the molecular targets and mechanisms of action of E2A. Hence, future studies are required to validate our findings and thoroughly elucidate the role of E2A in CRC.Figure S10/G1 phase of SW480/WT cells and decreased the S phase; (E) E2A regulates cell cycle progression in NCM460 cells; (F) Transfection of E12 or E47 upregulated the expression of miR-320a, compared to negative control. Data is expressed as the means ± SD from 3 separate experiments. .(A) Left: E2A protein expression of wild type SW480, control SW480, and knocked-down SW480 cells. Right: Change of E2A expression in SW480 cells after transfection of shE2A, E12 or E47: shE2A reduced the expression of E2A in SW480, while E12 and E47 increased E2A expression in SW480/shE2A cells, relative to the controls; (B) Transfection of E12 or E47 inhibited SW480/WT cell growth; (C) E2A regulates cell growth in NCM460 cells; (D) Transfection of E12 or E47 increased G(DOCX)Click here for additional data file.


SuprMam1 (for suppressor of mammary tumours) on chromosome 7 (110-140 Mb) and SuprMam2 on chromosome 2 (120-140MB) have been identified in the BALB/c mouse strain in the Trp53+/- mouse model of spontaneous breast cancer [SuprMam loci in C57BL/6 background) in comparison to parental strains, to identify the genes within the SuprMam loci that might be responsible for higher cancer susceptibility in BALB/c mice.Two recessive mammary tumour susceptibility loci, SuprMam alleles alone do not contribute significantly to the morphological differences of the parental strains.We analysed mammary gland wholemounts for differences in their basic morphology. The average of ductal branch count per 4x field of view was, BALB/c (diestrus) =171, BALB/c (estrus) =173, C57BL/6 (diestrus) =43, C57BL/6 (estrus) =38, SM09 (diestrus) =29 and SM09 (estrus) =23. According to these results, even though there is a significant difference between the numbers of ductal branches between the two parental strains BALB/c and C57BL/6 , there is no significant difference between congenic (SM09) and control (C57BL/6) mice during either estrus (p=0.164) or diestrus (p=0.299) stages at 12 months of age. A similar pattern was observed for total epithelial area. This suggests that BALB/c SuprMam1 (1). Using semi-quantitative RT-PCR, Dmbt1 mRNA was found to be significantly lower in mammary glands of susceptible BALB/c mice when compared to C57BL/6, while SM09 congenic mice had similar levels to the control C57BL/6 mice. This indicates that the lower level of Dmbt1 expression in BALB/c mice must be due to transcriptional factor differences outside the region, which is not present in SM09 congenics.The tumour suppressor DMBT1  has previously been identified as a candidate modifier gene within Cyp2r1, a major vitamin D hydroxylase, was identified as another potential candidate gene in SuprMam1. Plasma 25(OH)D levels were measured in SM09 and control mice to detect the effect of the different Cyp2r1 alleles. The plasma levels of 25(OH)D were 20% higher in SM09 mice compared to control mice . Surprisingly, however, this is the opposite of what is expected based on the association of CYP2R1 expression and cancer susceptibility in human populations.Using Affymetrix data comparing T cell gene expression across 5 different mouse strains, SuprMam loci. In order to confirm these results, and also to identify the possible involvement of other selected candidate genes, we aim to carry out transcriptional profiling on mammary glands of SM09 congenic and control mice.With these results, we are able to rule out two important candidate genes, DMBT1 and CYP2R1, from being responsible for the higher susceptibility of


Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic. The continuous spread of highly pathogenic avian influenza Type A (HPAI) H5N1 viruses in avian species across multiple continents and frequent reports of human H5N1 infection in China and Southeast Asia highlight the threat of a potential flu pandemic in the human population. At the same time, H5N1 viruses have grown into genetically and antigentically diversified viruses. Based on phylogenetic analysis of hemagglutinin (HA) protein gene sequences, at least 10 clades of H5N1 viruses (clades 0–9) have been identified A key component in the global strategy to prepare for and control any pending influenza pandemic is the development of an effective vaccine. Several versions of inactivated as well as live attenuated H5N1 vaccines have been tested in humans and showed an overall good safety and immunogenicity profile mainly by using a clade 1 H5N1 virus (A/Vietnam/1203/04) as the vaccine strain per recommendations by the World Health Organization (WHO) One key issue that needs to be analyzed for the above strategy is the cross reactivity between DNA vaccines expressing H5 HA antigens from different clades. It is critical to first optimize the immunogenicity of H5 HA DNA vaccines and then to test how much cross protection can be achieved with optimized H5 HA DNA vaccines. In the current report, we constructed DNA vaccines to express wild type HA antigens without mutations at the HA1 and HA2 cleavage site from four key H5N1 strains that have caused major human infection: HK/156/97 (clade 0), VN/1203/04 (clade 1), Ind/5/05 (clade 2.1), and Anhui/1/05 (clade 2.3). Rabbit sera immunized with these HA antigens were examined for their protective antibody responses against either homologous or heterologous H5N1 viruses. Our results demonstrated an imperfect cross-reactivity profile for the protective antibody responses among these four viruses. A polyvalent formulation including three different H5 HA DNA vaccines was able to produce broad protective antibody responses with high titers against these key H5N1 isolates. Information learned from this study should facilitate the selection of candidate H5N1 vaccines to form polyvalent H5N1 DNA vaccines as part of the global strategy to prevent and control a potential avian flu pandemic.One of the key findings from our previous study was that HA antigens from H1 and H3 serotypes had different structure preferences in order to elicit optimal protective antibodies In order to maximize the immunogenicity of HA DNA vaccines as shown in previous studies The first set of H5 HA DNA vaccines was produced by cloning codon optimized HA genes based on the amino acid sequences of HA antigen from an H5N1 influenza isolate A/HongKong/156/97, which was responsible for the first outbreak of H5N1 avian influenza in humans in 1997, into a DNA vaccine vector New Zealand White (NZW) rabbits were used in the current study to produce large quantities of sera for both binding antibody and functional antibody analyses. Animals were immunized with one of the three H5-HK HA DNA vaccines  via gene gun. Positive antibody responses were elicited in immunized rabbit sera against the HK HA antigen and levels of such responses increased with repeated immunizations while the negative control rabbit group that received empty DNA vector did not have HA-specific antibody responses . As measHowever, functional antibody analyses with the rabbit anti-HA immune sera showed a very different picture when these sera were further analyzed by either the hemagglutination inhibition (HI) or microneutralization (MN) assays. All three forms of H5-HK HA DNA vaccines induced protective antibody responses against the autologous wild type virus A/HongKong/483/97, but levels of protective antibodies were different among sera induced by different designs of H5-HK HA DNA vaccines. The DNA vaccine with the full length HA insert under the tPA leader sequence (H5-HK.tPA) elicited consistently higher HI and MN antibody titers when compared to the other two forms of HK-HA inserts, the full length HA with a natural leader sequence (H5-HK.wt), and the transmembrane region (TM) truncated HA (H5-HK.dTM). The difference was statistically significant (p<0.05) between H5-HK.tPA and H5-HK.dTM sera based on the HI assay  and betwIn order to rule out that the above finding was not only unique to this H5N1 virus isolate from Hong Kong in 1997, similar designs of HA inserts were produced by using a codon optimized HA DNA gene from the H5N1 strain A/VietNam/1203/04, a well-studied representative isolate for the H5N1 viruses In order to ensure that the difference in protective antibody responses between sera elicited by H5-VN.wt and H5-VN.tPA was not the result of repeated immunizations, sera collected after one or three immunizations were also measured . PseudotAdditional studies were conducted to ask if glycosylation of HA has been affected with the use of a different leader sequence which may influence the immune responses. Our previous study with a hepatitis B surface antigen suggested that post-translational modifications including glycosylation may affect the immunogenicity of DNA vaccine delivered antigens Treatment with PNGaseF allowed for the removal of N-linked glycosylations, as shown by the reduction of apparent molecular weight of HA0, HA1, and HA2 species in Western blot analysis for HA proteins expressed in cells transfected by these H5-VN HA DNA vaccines . The molBased on the above results, additional HA DNA vaccines with the HA.tPA insert design were produced by using codon optimized HA genes that encode the HA proteins from H5N1 viral strains A/Anhui/1/2005 and A/Indonesia/5/2005, both have caused human infection in recent years One set of experiments was conducted to understand the cross protection between paired H5N1 HA antigens. Rabbits were immunized with individual H5-HK.tPA, H5-VN.tPA, and H5-AH.tPA DNA vaccines and rabbit immune sera were examined for HA antigen-specific antibody responses. H5 HA-specific antibody responses against H5 HA antigens from different viruses were analyzed by ELISA and the potential cross-protective antibody responses against different H5N1 viruses were evaluated by HI and MN assays.H5-HK (A/HK/156/97), a clade 0 H5N1 isolate, and H5-VN (A/VN/1203/04), a clade 1 H5N1 isolate, represent H5N1 viruses isolated from the first human outbreak in Hong Kong in 1997 and a subsequent outbreak in Vietnam in 2004, respectively. Results shown in Protective HI and MN antibody responses induced by H5-HK.tPA and H5-VN.tPA DNA vaccines were further compared against either A/HK/483/97 or A/VN/1203/04 wild type viruses . HI and Similar analysis was conducted with rabbit immune sera elicited by H5-VN and H5-AH HA DNA vaccines . H5-AH . For funThe above results indicate that while antibody responses against one H5N1 HA antigen or virus may well cross-react with another H5N1 HA antigen or virus, the levels of antibody responses against the autologous antigen or virus were always higher. Given the uncertainty regarding which H5N1 virus may ultimately cause a pandemic H5N1 outbreak, it is important to develop a vaccine strategy that can maximize the protection efficacy against a wide spectrum of H5N1 viruses from different clades (or subtypes). It is possible that a consensus HA antigen, or a structurally-optimized HA antigen design, can cover different H5N1 viruses at various levels of protective efficacy but there is no actual data showing that such HA antigen can achieve the maximum protective antibody responses against several H5N1 viral isolates from different clades.One alternative approach is to produce a polyvalent HA formulation by including multiple H5N1 HA DNA vaccines in one injection to produce an immune sera that can induce the highest antibody response against a wide range of H5N1 viral isolates. For this purpose, we have tested one such polyvalent HA DNA vaccine formulation by injecting rabbits with three H5 HA DNA vaccines: H5-VN.tPA, H5-AH.tPA, and H5-IN.tPA DNA vaccines. A H5-IN.tPA DNA vaccine was produced expressing the HA antigen from another key H5N1 viral isolate, A/Indonesia/5/2005, a clade 2.1 H5N1 isolate. Rabbits in other groups received only one H5 HA DNA vaccine . Functional antibody analysis was conducted against pseudotyped viruses expressing different individual H5 HA and N1 antigens.The pseudotyped virus system, developed in recent years and widely used in leading influenza studies, provides high sensitivity in detecting functional antibody responses against influenza HA antigens, and at the same time, eliminates the influence of other influenza viral gene products since a common viral backbone is used for different HA pseudotyped viruses According to phylogenetic analysis, H5N1 viruses can be divided into 10 clades (0–9). Since 1997, humans have mainly been infected by H5N1 viruses from clades 0, 1, and 2, although there have also been reports of infection by clade 7 virus While progress has been made in reducing the number of required immunizations during vaccination with inactivated H5N1 vaccines by incorporating various adjuvants into the vaccine formulations, a major next-step for H5N1 vaccine research is to determine to what degree the immunity elicited by one H5 avian influenza vaccine ) can cross-protect against H5N1 viruses from other clades. Unlike human Type A influenza viruses (H1 or H3 serotypes), any potential pandemic caused by an H5N1 virus will be of avian origin and, in theory, any of the current known H5N1 avian viruses may jump to the human population leading to the next pandemic. Therefore, a systemic examination on the cross-protection among HA antigens from different clades is needed for strategic planning to determine whether more than one H5N1 vaccine is needed based on the analysis of protection profiles, and if so, what particular viral strains should be selected to provide the maximum breadth of protection.DNA vaccination is an attractive strategy to provide relatively quick and straightforward production of vaccines against an influenza pandemic when the demand for such vaccines suddenly increases. However, a key issue surrounding the use of DNA vaccines is their low immunogenicity in humans. In recent years, the success of the prime-boost strategy has greatly enhanced the utility of DNA vaccination for future human applications Results included in the current report indicated that the tPA leader sequence and the C-terminal transmembrane domain/cytoplasmic region of H5 HA both contribute to better functional antibody responses in H5.tPA DNA vaccines when compared to H5.wt and H5.dTM DNA vaccines. The above findings were different from our previous results on protective antibody responses induced by differently designed flu H1 and H3 HA DNA vaccines Studies were conducted in this report to identify the mechanism responsible for better protective antibodies in rabbit immune sera elicited by the H5.tPA HA insert design but the exact mechanism is currently unclear. First, we tested whether a higher level of HA antigen expression was produced with the tPA-leader design. As shown in Therefore, it is very likely that HA antigen expressed with the tPA leader may be more effective in eliciting conformational antibodies. This hypothesis is supported by two pieces of evidence. First, there was no difference in the levels of binding antibodies as measured by ELISA, which indicated that there was no difference in the general immunogenicity between WT and tPA leader designs; only a difference in the functional antibody was observed. Second, the HA.dTM DNA vaccine design also used the tPA leader but did not have better functional antibodies, proving that the proper folding of the HA antigen in the presence of a tPA leader is important and is dependent on the presence of an intact HA2 domain. It is possible that such an HA antigen conformation is part of a trimer structure of HA since the HA2 domain is involved in the formation of HA trimers. Only antibodies against the trimer structure are more functionally relevant to block the trimer form of HA spikes on viral particles.By using the optimal H5.tPA HA insert design, studies in this report further demonstrated that there are good levels of cross protection by one H5 HA DNA vaccine against multiple H5N1 viruses from different clades. It is well documented that cross protection among H5N1 viruses can be detected However, as shown in the current study, not all H5 HA vaccines can elicit the same levels of cross protective antibodies, and more significantly, maximum levels of protective antibodies were usually detected against the autologous viral isolates. Given the knowledge that protective anti-flu antibody responses in humans are much lower than in experimental animals, cross protection may not be very high in humans with one randomly selected H5 HA vaccine. In the current set of studies, it was encouraging to observe that the polyvalent H5 HA DNA vaccine was able to elicit high level protective antibody responses against multiple key H5N1 viruses. Such a polyvalent flu DNA vaccine can be used for stockpiling against a potential H5N1 pandemic even before information is available on which viral isolates may cause a human outbreak. At the same time, there are alternative approaches including the use of consensus HA antigen designs to achieve a broad coverage of various viral strains . It will be interesting to compare the relative efficacy between polyvalent and consensus HA DNA vaccines in their abilities to elicit protective antibody responses. One unique advantage of the polyvalent formulation is its flexibility; one alternative HA antigen can replace or be added to the earlier polyvalent formulation in the event that a new strain of virus becomes a threat while it will be necessary to re-design the whole consensus HA insert in order to allow for broader coverage.While HI titers against heterologous virus (cross-clade) were significantly reduced compared to HI titers against homologous virus in the current study, the heterologous virus titers were generally above 1∶100, and in humans, a HI titer of 1∶40 has been associated with protection No matter the design of DNA vaccines that may be used, recent studies have indicated that DNA priming immunization is effective as part of the prime –boost strategy for flu vaccine applications. In addition to DNA prime-inactivated flu vaccine boost Based on the results published in the current report and other recent similar studies, H5N1 HA DNA vaccines evaluated in the current study should be included in the design of human studies to understand whether results reported here can be reproduced in humans when they are used as part of DNA prime, either individually or as part of the polyvalent HA DNA formulation. The finding from such studies will be very useful in the identification of simple yet powerful approaches to develop vaccines against major influenza pandemics.Homo sapiens. The less optimal codons in HA genes were changed to the preferred codons in mammalian systems to promote higher expression of the HA proteins, as previously described The codon usage of HA genes from influenza A H5N1 viruses A/HongKong/156/97 (H5-HK), A/VietNam/1203/04 (H5-VN), A/Anhui/1/2005 (H5-AH) and A/Indonesia/5/2005 (H5-Ind) was analyzed with the MacVector software 7.2 against codon preference of GACCAGATCTGCATCGGCTAC) and H5-HA-opt-2 (agtcacggatcc TCAGATGCAGATCCGGCACTG). The individual H5-HK or H5-VN HA gene was cloned into the pSW3891 vector at the NheI and BamHI sites downstream of the tPA leader sequence and designated as H5-HK.tPA or H5-VN.tPA DNA vaccine constructs. For the third version, the HA natural leader sequence was replaced by a tPA leader sequence and the transmembrane (TM) and cytoplasmic region (37 aa at the C-terminus) of H5 and HA was deleted for both H5-HK and H5-VN. The truncated H5-HK or H5-VN HA genes were PCR amplified from the full length codon optimized H5-HK or H5-VN HA gene using primer pairs: H5-HA-opt-1 and H5-HA-opt-4 (agtcac ggatccTCACTGGTAGGTGCCCATGCTCTC), or H5-HA-opt-1 and H5-HA-opt-8 (agtcacggatccTCACTGGTAGATGCCGATGCTTTC), respectively. The truncated H5-HK or H5-VN gene insert was individually cloned into the pSW3891 vector at the NheI and BamHI sites downstream of the tPA leader sequence and designated as H5-HK.dTM or H5-VN.dTM.For either H5-HK or H5-VN HA DNA vaccines, three versions of codon optimized HA gene inserts were cloned into DNA vaccine vector pSW3891 Escherichia coli (HB101 strain) with a Mega purification kit  for both in vitro transfection and in vivo animal immunization studies.For the H5-AH HA DNA vaccine, the construct with the full length HA under tPA-leader sequence was made as described above. Each individual DNA vaccine plasmid was prepared from NZW rabbits (∼2 kg body weight) were purchased from Millbrook Breeding Labs  for immunogenicity studies, and housed in the Department of Animal Medicine at the University of Massachusetts Medical School in accordance with IACUC approved protocol. The rabbits (3 rabbits/group) were immunized with a Helios gene gun (Bio-Rad) at the shaved abdominal skin as previously reported This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Massachusetts Medical School's Institutional Animal Use and Care Committee (IACUC) (Protocol: A-1674). All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.6 293T cells at 50% confluence in a 60 mm dish were transfected with 10 µg of plasmid DNA, and a total of 3 ml supernatant and 100 µl of cell lysate were harvested 72 hours later. Equal amounts of each transiently expressed HA antigen (10 ng of protein in 10 µl) were loaded for the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under denatured conditions, then transferred onto PVDF membranes . After being blocked overnight at 4°C in blocking buffer , the membranes were incubated with a 1∶500 dilution of rabbit sera immunized with HA DNA vaccines for 30 min followed by washes. Then, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG at 1∶5000 dilution for 30 min. Following washes, the signals were detected using a chemiluminescence-based Western-Light Kit .Transient expression of the HA antigens from various HA DNA vaccine constructs were verified by Western blot analysis. HA DNA vaccine constructs were first transfected into the human embryonic kidney 293T cells using the calcium phosphate precipitation method. Briefly, 2×10To analyze the N-linked glycosylation of H5 HA antigens expressed by various forms of H5 HA DNA vaccines, the HA antigens expressed from 293T cells 2SO4, and the plates were read at OD 450 nm. The end titration titer was determined as the highest serum dilution that has an OD reading above twice of that from the negative control serum.ELISA was conducted to measure HA-specific antibody (IgG) responses in immunized rabbits and mice. The 96-well flat-bottom plates were coated with 100 µl of ConA (50 µg/ml) for 1 hour at room temperature, and washed 5 times with PBS containing 0.1% Triton X-100. Subsequently, the plates were incubated overnight at 4°C with 100 µl of transiently expressed HA antigen at 1 µg/ml. After being washed 5 times as above, the plates were then blocked with 200 µl/well of blocking buffer  for 1 hour. After five washes, 100 µl of serially diluted rabbit or mouse serum was added in duplicate wells and incubated for 1 hour. After another set of washes, the plates were incubated for 1 hour at 37°C with 100 µl of biotinylated anti-rabbit or anti-mouse IgG  diluted at 1∶1000 in Whey dilution buffer . Then, 100 µl of horseradish peroxidase-conjugated streptavidin (Vector Laboratories) diluted at 1∶2000 in Whey buffer was added to each well and incubated for 1 hour. After the final washing, the plates were developed with 3,3′,5,5′ Tetramethybenzidine (TMB) solution at 100 µl per well  for 3.5 minutes. The reactions were stopped by adding 25 µl of 2 M HInfluenza A viruses of the H5N1 A/HongKong/483/97 (H5N1), A/Viet Nam/1203/04 (H5N1), A/Anhui/1/2005 (H5N1), and A/Indonesia/5/2005 (H5N1) were grown in the allantoic cavity of 10-day-old embryonated hen eggs at 37°C for 26 to 40 h. Allantoic fluid pooled from multiple eggs was clarified by centrifugation and frozen in aliquots at −70°C. The 50% egg infectious dose (EID50) for each virus stock was calculated by the method of Reed and Muench following serial titration in eggs. All experiments with HPAI viruses were conducted under Biosafety Level 3 containment.HI assays were performed by standard methods 50 was incubated with equal volume of two-fold dilutions of the specific heat-inactivated serum overnight at 37°C in a 5% CO2 humidified atmosphere for 1 hr. After the incubation, 100 µl virus-serum samples were added to a 96-well plate containing Madin Darby Canine Kidney (MDCK) cell monolayer and incubated for 5 days at 37°C and 5% CO2. The microneutralization titer was defined as the highest dilution of serum that neutralized 100 TCID50 of virus in MDCK cell MN assays was performed as described previously 6 cells plated the day before) were transfected with 13.43 µg of pNL 4-3.Luc.R-E- (NIH AIDS reference and reagent program), 1.2 µg of H5-HA-wt DNA vaccine plasmid and 0.3 µg of N1-NA plasmid using 75 µg of polyetheleneimine transfection reagent. Supernatants were harvested 48 hours later, frozen at −80°C and then standardized by infectivity in 293A cells using a luciferase-based TCID50 measurement. For neutralization assays, serum samples (5 µl) were heat inactivated at 56°C for 30 minutes, then three-fold serially diluted in culture medium in flat-bottomed microtiter plates. Pseudotyped H5N1 virions were then added to the plates at 200 TCID50/well and incubated for 1 hour at 37°C. After incubation, 293A cells were trypsinized and added to each plate at a dilution of 1×104 cells per well. Following a 48-hour incubation, plates were developed using a luciferase assay system (Promega). Values averaged from triplicate wells were then used to determine IC50 based on wells that displayed 50% reduction in infection as compared to control wells containing virus plus pre-immune sera.The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described Figure S1Western blot analysis of potential N-linked glycosylations of HA protein expressed by differently designed H5-VN (A/VietNam/1204/04) DNA vaccines: H5-VN.wt, H5-VN.tPA, H5-VN.dTM or empty vector, transfected 293T cell lysate or supernatant, with (F) or without (U) PNGase F treatment.(TIF)Click here for additional data file.


Supplemental vitamin D modulates inflammatory cytokines and skeletal muscle function, but results are inconsistent. It is unknown if these inconsistencies are dependent on the supplemental dose of vitamin D. Therefore, the purpose of this study was to identify the influence of different doses of supplemental vitamin D on inflammatory cytokines and muscular strength in young adults.n = 15) and women (n = 15) received a daily placebo or vitamin D supplement (200 or 4000 IU) for 28-d during the winter. Serum 25-hydroxyvitamin D (25(OH)D), cytokine concentrations and muscular (leg) strength measurements were performed prior to and during supplementation. Statistical significance of data were assessed with a two-way  analysis of variance (ANOVA) with repeated measures, followed by a Tukey's Honestly Significant Difference to test multiple pairwise comparisons.Men (P < 0.05) across time in the placebo group. Supplemental vitamin D at 200 IU maintained serum 25(OH)D concentrations and increased IL-5 (P < 0.05). Supplemental vitamin D at 4000 IU increased (P < 0.05) serum 25(OH)D without altering IL-5 concentrations. Although serum 25(OH)D concentrations correlated (P < 0.05) with muscle strength, muscle strength was not changed by supplemental vitamin D.Upon enrollment, 63% of the subjects were vitamin D sufficient (serum 25(OH)D ≥ 30 ng/ml). Serum 25(OH)D and interleukin (IL)-5 decreased D and IL-5 concentration decreases, and that muscular strength does not parallel the 25(OH)D increase induced by a high-daily dose of supplemental vitamin D. Considering that IL-5 protects against viruses and bacterial infections, these findings could have a broad physiological importance regarding the ability of vitamin D sufficiency to mediate the immune systems protection against infection. In kidney and immune cells, 1α-hydroxylase (1α-OHase) converts 25(OH)D to 1,25(OH)D. This enzymatic conversion is limited by substrate availability since 1α-OHase operates below its Km. Thus, an adequate circulating 25(OH)D concentration is necessary to maintain substrate availability for the enzymatic conversion of vitamin D to its active form.Vitamin D status is influenced by a variety of factors, including but not limited to geographical latitude, season and supplement use. Living in extreme northern and southern climates and the winter season result in low vitamin D levels ,2, whileLow serum 25(OH)D concentrations result in skeletal muscle weakness ,8, and iH1)-type cells and pathways [H2-type cell pathways [in vitro, evidence illustrating concurrent alterations in serum 25(OH)D and cytokine concentrations in humans is more ambiguous. For example, in healthy-adults during the winter, low serum 25(OH)D concentrations corresponded with increased IFN-γ and IL-10 production [Vitamin D regulates inflammatory cytokines, which orchestrate host defenses by the immune system. Interferon (IFN)-γ is a cytokine that regulates T helper 1 D simultaneously with circulating cytokines in healthy adults during the winter, and the range of supplemental vitamin D  doses were narrow  . AdditioThe Institutional Review Board at Intermountain Healthcare  approved this study. Subjects were informed of the experimental protocol and procedures and provided both written and verbal consent prior to participation. Recreationally active (minimum of 30 minutes of continuous exercise at least 3 time per week for 1 year prior to enrollment), non-smoking males and females between the ages of 18 and 45 years of age were recruited to participate in this study. Subjects were excluded from participation if they experienced a lower-extremity injury or injuries during the year prior to enrollment. Subjects were also excluded from participation if they were taking a daily dietary-supplement during the previous year, planning on increasing or decreasing the amount of time spent in the sun or tanning bed, or traveling south of 37°N latitude during enrollment. Subjects were asked to refrain from using any other supplements during study participation. Data was collected during two successive winters. First, between December 1, 2009 and April 26, 2010, and second, between December 1, 2010 and March 15, 2011 in Salt Lake City, UT USA (40°N latitude). We chose to collect data during the winter because that is when vitamin D levels are at their nadir. This occurs because the solar angle during the winter is inadequate to stimulate cutaneous vitamin D synthesis from sun (ultraviolet B radiation) exposure.This study consisted of a randomized, double-blind, placebo-controlled experimental design. Subjects were randomly assigned to one of three groups: vitamin D  supplementation at: (1) 200 or (2) 4000 IU, or (3) a matching-placebo. Supplements were taken daily for 28-d. Supplements were permutated into random blocks of six. Subjects were asked to refrain from using aspirin, ibuprofen, naproxen sodium, and acetaminophen throughout the duration of the study.It should be noted, that upon study initiation, 200 IU/d of vitamin D was the recommended-adequate intake and 4000 IU/d was double the upper limit for the targeted population. Currently, 200 IU/d is lower than the current recommended dietary allowance and 4000 IU is the upper limit for the studied population. These recommendations are provided by the Institute of Medicine for the United States and Canada . Althougg for 6 min within 20 min of sample collection. Following separation, plasma samples were sent to clinical laboratories for analytical chemistries (see below). Serum was separated by centrifugation  at 1100 g for 20 min within 20 min of sample collection and after coagulation. Serum samples were immediately stored at -80°C  until the day of 25(OH)D and cytokine concentration analyses (see below).Each subject provided five fasting blood draws: (1) prior to (Pre), and (2) 7-, (3) 14-, (4) 21- and (5) 28-d after supplementation. Subjects were asked to refrain from physical activity 72-h prior to each blood draw to ensure that prior activity did not confound serum 25(OH)D and cytokine concentration results. Fasting blood draw samples were obtained from the antecubital vein into one 4.0 ml purple-top Becton Dickinson (BD) Vacuatainer tube (K2 EDTA 7.2 mg), one 4.5 ml light green-top BD Vacutainer tube , and one 6.0 ml red-top serum BD Vacutainer tube. Plasma was separated by centrifugation  at 2400 3 as an internal standard  in a 2 mL centrifuge tube. Samples were immediately vortexed and allowed to sit on ice for 10 min. Samples were then spun at 15,000 g for 5 min and the supernatant transferred to a new 2 mL centrifuge tube containing 500 μL chloroform. Samples were vortexed and allowed to stand for 5 min. To achieve phase separation, 750 μL of ddH2O was added, vortexed, and centrifuged for 2 min at 15,000 g. The aqueous phase (top), and any debris between the two phases, was removed and discarded. The remaining organic phase was dried down to completeness in a centrifugal vacuum concentrator for 18 min at 45°C under negative pressure. The pellet was then resuspended in 100 μL of methanol and added to high performance-liquid chromatography (HPLC) vials.Serum 25(OH)D concentrations (ng/ml) were measured at USANA Health Sciences, Inc.  using a modified Bligh-Dyer technique for extraction  and a sc2 and 25(OH)D3 were detected on an Agilent tandem mass spectrometer  using atmospheric pressure chemical ionization (APCI) detection . The 25(OH)D3, dueterated 25(OH)D3 and 25(OH)D2 precursor ions were 383.3, 386.3 and 395.4, respectively. The 25(OH)D3, dueterated 25(OH)D3 and 25(OH)D2 productions were 365.3, 368.3 and 208.9, respectively. Serum 25(OH)D2 and 25(OH)D3 concentrations were determined relative to authentic standards and corrected for recovery of the 25(OH)D3 internal standard. The detection limit was determined to be < 1 ng/ml for all analytes. The sum of 25(OH)D2 and 25(OH)D3 concentrations was used as the 25(OH)D total concentration. However, since serum 25(OH)D2 was not detected in any subjects, serum 25(OH)D total concentrations are referred to as serum 25(OH)D concentrations hereafter.Analytes were separated by injecting 10 μL into an Agilent HPLC  and a Phenomenex Inertsil 3 micron, 150 × 4.60 mm column. Method conditions were: 0-8 min, 90% MeOH/10% (0.03% formic acid in water); 8-15 min, 30% MeOH/70% 2-propanol; 15-20 min, 90% MeOH/10% (0.03% formic acid in water). 25(OH)DA multiplex microsphere-bead array was used to measure a number of circulating inflammatory cytokines (pg/ml) as described previously ,43. BrieWe have reported the procedural precision of this multiplex-cytokine bead assay previously ,43. In bPlasma parathyroid hormone  and calcium (mg/dl) concentration measures were performed at ARUP Laboratories . Plasma creatinine (mg/dl) concentrations were measured at the Central Laboratory with Intermountain Healthcare .2) was calculated by the following equation:To assess renal function prior to and following supplementation, the estimated glomerular filtration rate  were excluded from participation. Leg strength from a single randomly selected leg was performed prior to (Pre) and 28-d after (Post) placebo or vitamin D supplementation. Single-leg strength testing was performed on a horizontal plyo-press . Plyo-press output data were measured from output signals obtained from a mounted force plate  and displacement transducer . All data were sampled at 200 Hz with a low-pass filter at 10 Hz using DartPower software . Before every testing session, the mounted force plate was zeroed and load calibrated.For the single-leg peak isometric force measurements, the plyo-press sled was adjusted for each subject to align the knee and hip joint flexion angles to 90° with the abdominal, lower back region secured and stabilized to the plyo-press sled with a harness. The plyo-press sled position was documented and reproduced to achieve the desired knee and hip joint flexion angles at the follow-up visits. Leg selection was randomized. Subjects were verbally instructed and strongly encouraged to exert maximal force against the mounted force platform. Each subject performed three maximal single-leg isometric contractions. Each isometric contraction was 3 sec in duration and separated by approximately 1 min. Peak isometric force (N/kg) was defined as the highest resultant force applied during the 3 sec test for each trial. To calculate average isometric force, peak isometric forces from the 3 trials at each visit were averaged.Single-leg peak power output measures followed the single-leg isometric contractions and were measured on the same horizontal plyo-press with the same securing procedures described above. Starting from an extended position (full extension = 0°), subjects performed repetitive single-leg jumps  as fast as possible. Subjects were instructed to jump as high as possible. Each test was 20 sec in duration with the weight-stack resistance set at 75% of body mass. The time-aligned product of the resultant forces (N) acquired from the force platform and weight-stack velocities (m/s) data obtained from the displacement transducer were used to calculate power output and expressed relative to body mass (W/kg). Peak power output was defined as the highest power output produced during the 20 sec test for each leg. The peak power output for each jump during the 20 sec test were averaged to provide an average power output for each testing session in each leg.P < 0.05. Data presented as mean (SD) unless otherwise noted.To achieve normality, rank transformations were performed on eGFR, serum 25(OH)D changes from Pre, and serum cytokine changes from Pre. Transformations were checked for normality with a Kolmogorov-Smirnov test. Statistical significance of data were assessed with a two-way  analysis of variance (ANOVA) with repeated measures, followed by a Tukey's Honestly Significant Difference to test multiple pairwise comparisons. One-way ANOVA tests were performed on subject characteristics. Relationships between variables were examined with a Pearson Product Moment Correlation. All statistical analyses were performed with SysStat software . Statistical significance was set at a Subject characteristics were similar between groups Table . For exan = 4), 23.3 (n = 7) and 63.3% (n = 19) of the subjects were vitamin D deficient (serum 25(OH)D ≤ 20 ng/ml), insufficient (serum 25(OH)D 21-29 ng/ml) or sufficient (serum 25(OH)D ≥ 30 ng/ml), respectively.Upon enrollment, serum 25(OH)D concentrations were 32.2 (10.0) ng/ml. Prior to randomization, 13.3  increased in 4000 IU group and compared to those in the placebo and 200 IU groups D concentrations. Serum 25(OH)D concentrations progressively and significantly  to be higher in the 200 IU group upon study enrollment  increased in the 4000 IU group and compared to those in placebo and 200 IU groups  different between the placebo and 200 IU groups D concentrations.Due the anticipated seasonal decrease (which could change vitamin D status from sufficient to insufficient) and the efficacy of supplemental vitamin D at 4000 IU to increase serum 25(OH)D concentrations, PTH and calcium were measured in each blood draw sample. PTH and calcium concentrations were not significantly different within or between groups (Table P < 0.05) IL-5 decrease in the placebo group  increased in the 200 IU group and compared to that in the placebo group at 28-d  and average  isometric forces correlated with serum 25(OH)D concentrations.Although supplemental vitamin D did not significantly change isometric forces, peak D increase induced by a high-daily dose of supplemental vitamin D in young, reportedly active and healthy adults.Vitamin D regulates inflammatory cytokines and skeletal muscle function, especially in isolated immune cells and various pathophysiological conditions in humans. In the present investigation, we extend those previous reports by providing several original and impactful results obtained from healthy adults during the winter. First, we provide new data identifying serum 25(OH)D and IL-5 concentration decreases. Secondly, supplemental vitamin D at 200 IU/d prevented serum 25(OH)D and IL-5 concentration decreases. Third, supplemental vitamin D at 4000 IU/d induced an immediate increase in serum 25(OH)D concentrations without modulating circulating IL-5. Finally, despite serum 25(OH)D concentrations correlating with muscle strength, supplemental vitamin D did not moderate muscular strength alterations. These unique data indicate that subtle fluctuations in serum 25(OH)D concentrations modulate a TAs expected, serum 25 (OH) D concentrations responded disparately to different doses of supplemental vitamin D Figures  and 2. C+ cells obtained from mice when incubated in 1,25(OH)D and stimulated with IL-2, phorbol myristate acetate (PMA) and ionomycin [H0-cells and remained unchanged in TH1- and TH2-cells [A novelty of the present investigation was the IL-5 results obtained with and without supplemental vitamin D Figure . In the onomycin . These ronomycin . FurtherH2-cells . RegardiH2-cells  demonstrin vitro, which makes it is difficult to equate or interpret results relative to supplemental D or serum 25(OH)D concentration studies in humans that do not report 1,25(OH)D. Recently, Zhang et al. [in vitro. In contrast, 30 ng/ml of serum 25(OH)D significantly inhibited cytokine production and was comparable to that shown at 0.04 ng/ml of 1,25(OH)D [Regarding the form of vitamin D, most studies have investigated the influence of 1,25(OH)D on inflammatory cytokines g et al.  demonstr,25(OH)D . These p,25(OH)D .H2-type cytokines, such as IL-4 and IL-13 [H2-cells [+ cells derived from mice, GATA-3 message expression increased when incubated with 1,25(OH)D [Vitamin D receptors are located on T-cells and mast cells ,51, and nd IL-13 ,54. GATAH2-cells ,55. In C,25(OH)D . In an e,25(OH)D . Thus, wEvidence indicates that several types of hyper-eosinophilic syndromes are mediated by IL-5 . In patiIn contrast to our hypothesis, supplemental vitamin D did not increase muscular strength Figure  and 4B oAnother probable explanation for the inconsistencies in the literature is that there are few prospective, randomized studies investigating the influence of supplemental vitamin D on serum 25(OH)D concentrations and muscular strength. The majority of the evidence identifying the beneficial influence of vitamin D on muscular strength or physical performance is correlative ,12,14,69n = 10/group). Future studies investigating the influence of different doses of supplemental vitamin D on inflammatory cytokines and muscle strength are encouraged to include a larger sample size. Fourth, our data collection was limited to weekly blood draws and emerging evidence is suggesting that the cytokine modulating property of vitamin D could be time sensitive [Study limitations include: first, a rather short-intervention phase. Future studies are encouraged to conduct longer durations of supplemental interventions when examining the influence of vitamin D on inflammatory cytokines and muscular strength. Second, the level of physical activity was not stringently examined. Recording activities and activity intensities and volumes performed during the preceding seasons both indoor and outdoor is recommended. Third, this study consisted of thirty subjects total D and IL-5 decreases in vitamin D sufficient adults during the winter. These impactful findings defy current belief by suggesting that a low dose of supplemental vitamin D (plus dietary intake) that is easily attainable through dietary sources maintains serum 25(OH)D and IL-5 concentrations.H2-type cytokine decrease, which could be influential in protecting against viral and bacterial infections; and apparently in reportedly healthy and vitamin D sufficient adults, that a further increase in serum 25(OH)D mediated by a high dose of supplemental vitamin D does not improve muscular strength.Additionally, despite concentrations correlating with muscle strength, our shocking data reveal that muscular strength does not parallel the increase in serum 25(OH)D concentrations induced by supplemental vitamin D at 4000 IU/d. We conclude that maintaining serum 25(OH)D concentration during the winter prevents a TH1: T helper 1; TH2: T helper 2.1α-OHase: 1α-hydroxylase; 1,25(OH)D: 1,25-dihydroxyvitamin D; 25(OH)D: 25-hydroxyvitamin D; eGFR: Estimated glomerular filtration rate; IL: Interleukin; IFN: Interferon; PTH: Parathyroid hormone; TThe authors declare that they have no competing interests.TB contributed to the study conception and experimental design, acquisition of data, analysis and interpretation of data, and first draft of the manuscript. TBM carried out the cytokine analysis, BMD, EDS and AD carried out the vitamin D metabolite measurements, VTH performed the single-leg strength analyses. HRH, CRK, BMD and LKW made significant contributions to the critical revision and intellectual content of this manuscript. All authors approved the final manuscript version.


Brevibacillus latersporus, which was shown via genomic analysis to contain a large number of gene clusters presumably involved in secondary metabolite biosynthesis. A scale-up culture of B. latersporus PE36 yielded three bioactive compounds that inhibited the growth of methicillin-resistant Staphylococcus aureus (basiliskamides A and B and 12-methyltetradecanoic acid). Further studies of the isolate's secondary metabolome provided both new (auripyrazine) and previously-described pyrazine-containing compounds. In addition, a new peptidic natural product (auriporcine) was purified that was determined to be composed of a polyketide unit, two L-proline residues, two D-leucine residues, one L-leucine residue, and a reduced L-phenylalanine . An examination of the genome revealed two gene clusters that are likely responsible for generating the basiliskamides and auriporcine. These combined genomic and chemical studies confirm that new and unusual secondary metabolites can be obtained from the bacterial associates of wild mammals.Bacteria associated with mammals are a rich source of microbial biodiversity; however, little is known concerning the abilities of these microbes to generate secondary metabolites. This report focuses on a bacterium isolated from the ear of a feral hog from southwestern Oklahoma, USA. The bacterium was identified as a new strain (PE36) of  Nature has served as a valuable source of bioactive compounds with many natural products (secondary metabolites) having entered into clinical use The microbial associates of animals, especially those from vertebrate hosts, represent a virtually untapped source of bacterial and archaeal diversity Brevibacillus laterosporus was obtained from a feral hog originating in southwestern Oklahoma, USA. The natural product biosynthetic potential of this isolate was revealed using a combination of LC-MS, bioassays, and genomic data. These efforts provided several compounds including a new and unusual peptidic metabolite, auriporcine (6); a new pyrazine, auripyrzine (5); and the previously described antifungal metabolites basiliskamides A and B . This research highlights how the integrated application of genomics and metabolomics presents an opportunity for mining new natural products from bacteria associated with wild mammals.This report describes the use of an opportunistic sampling approach Staphylococcus aureus were applied over the surfaces of the plates. A number of isolates exhibited antibiosis toward S. aureus as demonstrated by zones of inhibition devoid of visible growth of the pathogen in the overlay layer . The genome of Brevibacillus phR was included due to its close and unresolved phylogenetic relationship among strains of B. laterosporus. This analysis warranted the designation of PE36 as a separate strain of B. laterosporus . A total of 63 scaffolds with a genome size of 5.14 Mbp were obtained upon assembly. These scaffolds had an N50 of 155.81 Kbp and a maximal scaffold length of 457.91 Kbp producing a near-complete genome assembly (489-fold coverage). After annotation, 4,791 coding sequences were split between 430 subsystems along with 100 RNAs. The complete 16S small subunit rRNA sequence was used to identify isolate PE36 as a member of the family Paenibacillaceae and the genus bacillus . A sequerosporus  and its B. laterosporus PE36 was prepared for the purification and structure characterization of its natural products. Silica flash chromatography, C18 vacuum liquid chromatography (VLC), and preparative and semi-preparative C18 reversed-phase high performance liquid chromatography (RP-HPLC) were used to purify the metabolites (1) and B (2) 1H NMR) and two-dimensional (2D) heteronuclear single quantum coherence spectroscopy (1H-13C HSQC) data. Compounds 1 and 2 exhibit potent antifungal activities and modest antibacterial properties Brevibacillus associated with a marine tubeworm collected in Papua New Guinea 1H NMR as 12-methyltetradecanoic acid (3) A scale-up culture of abolites  responsiB. laterosporus PE36 metabolome, two additional fractions that lacked antibacterial activities were selected for chemical analysis. Liquid-chromatography-mass-spectrometry (LC-MS) profiling of the first inactive fraction revealed the presence of two compounds possessing similar UV absorbance spectra (λmax ∼220). One of the compounds was identified as 2,5-bis(3-indolylmethyl)pyrazine (4), whereas the second compound (5) is reported here for the first time as a natural product. High resolution ESI-MS analysis of compound 5 yielded a molecular ion at m/z 222.1039 [M-H]-, which substantiated a molecular formula of C14H13N3. The 1H and 13C NMR data for compound 5 were very similar to those obtained for compound 4 , a singlet integrating for two hydrogens at δH 4.17, and a methyl singlet at δH 2.45. These features could be accounted for if one of the two indoles attached to the pyrazine in compound 4 was replaced by a methyl group in compound 5. The proposed structural change was subsequently confirmed by 2D NMR to establish the structure of auripyrazine (5).To further probe the chemical diversity of the mpound 4 . Analysi6 provided a molecular ion with m/z 799.5330 [M+H]+, which indicated that the compound had a molecular formula of C43H70N6O8. An initial inspection of the 1H NMR data revealed that compound 6 was likely peptidic in nature with signals characteristic of amide doublet protons, as well as amino-acid α-hydrogen spins  or upon changes to the NMR probe temperature. Therefore, we conjectured that compound 6 might exist in two dominant conformational states , 2D gradient heteronuclear multiple bond correlation spectroscopy (1H-13C gHMBC), and 2D nuclear Overhauser effect spectroscopy (1H-1H NOESY) experiments provided evidence for several discrete spin systems attributable to two proline and three leucine residues that were linked to form a peptidic Pro-Leu-Pro-Leu-Leu fragment. Another spin set was probed in greater detail with 1H-13C gHMBC leading to the generation of a six carbon fragment in which two of the carbon atoms were attached to oxygen atoms  (1H-13C gHMBC data helped link this fragment to the peptide portion of the new metabolite (N-terminus of the peptide was further supported by a one-bond proton-nitrogen heteronuclear correlation (1H-15N HSQC) experiment from which we found no evidence for primary amide hydrogens. The remaining carbon atoms in compound 6 were determined to comprise a phenylalaninol residue (Pho) at the C-terminus of the peptide  . Examinatabolite . The pla peptide . The rederiments  were per6 was determined by X-ray diffraction experiment on a single crystal prepared in a vapor diffusion chamber (acetone and ether)  . Compouned state . We obseanalysis . The Maranalysis . Thus, tB. laterosporus PE36 was reexamined to provide a link between the secondary metabolites made by this isolate and their respective biosynthetic genes. Of the 11 NRPS gene clusters identified in PE36  phenylalanine incorporation followed by termination and post-production reductive tailoring; 2) tyrosine incorporation followed by termination, dehydroxylation, and reduction; or 3) full incorporation of all the predicted amino acids followed proteolysis of a tyrosine/phenylalanine → leucine bond and reduction. Based on the order of amino acid residues predicted by antiSMASH analysis and presence of appropriate modifying features , putative biosynthetic gene cluster 23 is most likely responsible for the production of 6; however, further experiments will be needed to confirm this hypothesis.The genome of  in PE36 , cluster in PE36  exhibite1 and 2). Focusing on the only two gene clusters predicted to be predominatly composed of PKS-related domains  domain. Instead the modules receive carbon building blocks from a shared AT, which in this case is a fused tandem AT domain 1 and 2. The starter unit is loaded by a predicted acetyl-loading AT of the GCN5-related superfamily (GNAT) E or Z configurations The antiSMASH data were also probed to determine the probable gene cluster responsible for the production of the basiliskamides . Our group has initiated studies to investigate this and related questions about the bacteria recovered from the microbiomes of wild mammals. Nevertheless, our past and current screening efforts involving other wild mammals and their associated bacteria suggest that these microbes have tremendous potential for accessing new dimensions of microbial biodiversity and chemodiversity.The results of these combined chemical and genomic studies suggest that bacteria associated with wild mammals are a potential source of unique and bioactive secondary metabolites. Despite the promising opportunities revealed through this work, we can only speculate about the nature of the biological relationship between 18 column, 100 Å, 75×3.0 mm). The HPLC system utilized SCL-10A VP pumps and system controller with a Gemini 5 µm C18 column . X-ray data were collected using a diffractometer with a Bruker APEX ccd area detector and graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). All solvents were HPLC grade or better.UV data were collected on a Hewlett Packard 8452A diode array spectrophotometer. Optical rotation data were determined on a Rudolph Research Analytical Autopol III automatic polarimeter. UV–CD spectra were measured on an AVIV circular dichroism spectrometer model 202-01. NMR data were obtained on a Varian VNMR spectrometer (500 MHz). Accurate mass data were collected on an Agilent 6538 HRESI QTOF MS coupled with an Agilent 1290 HPLC. LC-MS analyses were performed on a Shimadzu LC-MS 2020 system (ESI quadrupole) coupled to a photodiode array detector. The samples were separated using a Phenomenex Kintex column  in southwestern Oklahoma, were swabbed with sterile cotton swabs. Since our opportunistic sampling strategy does not involve the handling of live animals, only sampling carcasses, we have been assigned an internal case-tracking number (R11-021) by the University of Oklahoma IACUC. A State-of-Oklahoma-issued Scientific Collector Permit (permit #5250) was obtained for the purpose of sampling carcasses. Permission was granted to sample the head of the hog carcass; the research team did not take ownership of the carcass at any time. The tip of each swab was removed and placed into a sterile 15 mL Falcon tube containing a 0.9% NaCl (w/v) solution. The tubes were vortexed and diluted 1∶10 with 0.9% NaCl. Aliquots of 50 µL were spread on half-strength tryptic soy agar (TSA) plates. The plates were incubated at 37°C under a 5% COS. aureus was applied to the surface of each plate. The plates were incubated at 37°C under a 5% CO2 atmosphere for five days and periodically examined for zones of S. aureus inhibition.Bacterial isolates were arrayed onto half strength TSA plates and incubated for up to one week. A layer of half strength TSA seeded with methicillin-resistant de novo assembly algorithm . Contigs smaller than 800 bp were discarded. After assembly, scaffolds were initially submitted to the RAST server for total-genome annotation Genomic DNA was extracted using the PowerBiofilm DNA Isolation kit  by spinning down 2.0 mL of turbid culture and following the manufacturer instructions. The purity of gDNA was confirmed by spectrophotometery (Implen) and submitted to the Oklahoma Medical Research Foundation genomics core facility for sequencing on an Illumina MiSeq using TruSeq LT 2×150 bp chemistry . Reads were assembled using the CLC Genomics Workbench suite Starter cultures of the bacterium were prepared in tryptic soy broth (TSB) and shaken on a rotary shaker at 130 rpm at room temperature for 12 h. For scale-up preparation, 1 L Erlenmeyer flasks containing 300 mL of sterile TSB were inoculated with 1 mL of starter culture. The flasks were shaken at 130 rpm at room temperature for 1 week and the cultures were pooled prior to partitioning. The pooled culture broth with cells was partitioned three times against EtOAc (1∶1 vol/vol) and the solvent removed from the organic layer under reduced pressure on a rotary evaporator.2Cl2-MeOH gradient on an Isolera System . The purification of each compound was achieved as follows:The crude extract (∼4 g from 30 L culture) was absorbed onto silica gel and subjected to flash chromatography fractionation using a hexane-CH18 RP-HPLC. A MeOH-H2O gradient  resulted in a single active fraction. This fraction was purified by semi-preparative RP-HPLC  to provide 3 (10 mg). The structure of the metabolite was determined by 1H NMR and GC-MS analysis.Silica flash column chromatography was performed (hexane-DCM-MeOH) and the fraction that eluted with 100% DCM was further separated using C18 VLC  was performed and the fraction that eluted with 100% MeOH was further separated by C18 RP-HPLC. A MeOH-H2O gradient  resulted in a single active fraction. This fraction was further processed by semi-preparative C18 RP-HPLC  to yield 1 and 2 . The compounds were identified based on comparisons of their experimental and published HR-ESIMS, UV, and NMR data C1 and 2 was collected and separated by C18 RP-HPLC  to provide 4 (7 mg). The structure of 4 was determined by comparing its HR-ESIMS and 1H NMR data to published values An HPLC fraction that eluted after 1 and 2 was collected and fractionated by preparative C18 RP-HPLC . Purification by semi-preparative C18 RP-HPLC  provided 5 (5 mg).A fraction obtained prior to the elution of 18 RP-HPLC gradient that yielded 1 and 2 was pursued for further analysis. Semi-preparative C18 RP-HPLC with acetonitrile and H2O treated with 0.1% trifluoroacetic acid  resulted in the purification of 6 (2 mg).A late eluting fraction from the same C6. Briefly, approximately 1 mg of 6 was dissolved in 6 M HCl and heated at 100°C for approximately 18 h. After heating, the sample was dried under reduced pressure, redissolved in 50 µL of water and transferred to a 1.5 mL centrifuge tube. Aliquots consisting of 50 µL of 50 mM of the D and L enantiomers of each amino acid were independently treated in 1.5 mL centrifuge tubes. To each standard and sample, 100 µL of 1% FDAA and 20 µL of 1 M NaHCO3 were added. Tubes were capped and heated in a 40°C water bath with periodic mixing for 1 h. After cooling to room temperature, 10 µL of 2 M HCl was added to each tube. The mixtures were dried under reduced pressure and redissolved in 200 µL of 9∶1 MeOH-H2O. Samples were diluted further to one tenth of their original concentration with 9∶1 MeOH-H2O prior to LC-MS analysis.Marfey's analysis 6 with dimensions 0.44×0.16×0.12 mm was selected for structure analysis. The sample was cooled to 100 K. Cell parameters were determined from a non-linear least squares fit of 6,210 peaks in the range 2.41<θ<26.04°. A total of 40,661 data points were measured in the range 1.554<θ<28.344° using φ and ω oscillation frames. The data were corrected for absorption by the empirical method giving minimum and maximum transmission factors of 0.964 and 0.990. The data were merged to form a set of 10,800 independent data with R(int)  = 0.0586 and a coverage of 99.9%. The orthorhombic space group P212121 was determined by systematic absences and statistical tests and verified by subsequent refinement. The structure was solved by direct methods and refined by full-matrix least-squares methods on F2. The positions of hydrogens bonded to carbons were initially determined by geometry and were refined using a riding model. Hydrogens bonded to nitrogens and oxygens were located on a difference map, and their positions were refined independently. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atom displacement parameters were set to 1.2 (1.5 for methyl) times the isotropic equivalent displacement parameters of the bonded atoms. A total of 573 parameters were refined against 118 restraints and 10,800 data to give wR(F2)  = 0.1925 and S = 1.011 for weights of w = 1/[σ2 (F2) + (0.0900 P)2 + 5.0000 P], where P = [Fo2 + 2Fc2]/3. The final R(F) was 0.0707 for the 9,029 observed, [F>4σ (F)], data. The largest shift/s.u. was 0.012 in the final refinement cycle. The final difference map had maxima and minima of 0.315 and -0.374 e/Å3, respectively. The absolute structure could not be determined by refinement of the Flack parameter. Further documentation of these data can be obtained in A colorless prism-shaped crystal of Auripyrazine (5): yellow solid; UV (MeOH) λmax 222 (log ε 3.49); 1H and 13C NMR data refer to -m/z 222.1039 . See Auriporcine (6): white solid; UV (MeOH) λmax 206 (log ε 4.19), [α]D (c 0.065) -27.7; 1H and 13C NMR data refer to +m/z 799.5330 . See 6.Figure S12D NMR correlations used to determine the planar structure of auriprocine, 6. Important 2D NMR correlations used in the structure elucidation of 6 are shown: 1H-1H TOCSY (shown as rust colored bonds), 1H-1H NOESY (illustrated as orange double-headed arrows), and 1H-13C gHMBC (shown as blue single-headed arrows).(TIF)Click here for additional data file.Figure S218 LC-MS results of the Marfey's analysis.Overlaid selective ion trace chromatograms showing the C Single ion trace detection of the derivatized amino acids: derivatized proline (blue), leucine (red), and phenylalaninol (green).(TIF)Click here for additional data file.Figure S31H  spectrum of compound 5. Signals resulting from impurities are marked with red dots.(TIF)Click here for additional data file.Figure S41H-13C HSQC  spectrum of compound 5. CH groups are shown in blue, CH2 groups are in red.(TIF)Click here for additional data file.Figure S51H-13C gHMBC  spectrum of compound 5.(TIF)Click here for additional data file.Figure S6HRESI-MS (negative mode) data for compound 5.(TIF)Click here for additional data file.Figure S7UV data for compound 5.(TIF)Click here for additional data file.Figure S81H NMR  spectrum of compound 6.(TIF)Click here for additional data file.Figure S91H-13C HSQC  spectrum of compound 6. CH groups are shown in blue, CH2 groups are in red.(TIF)Click here for additional data file.Figure S101H-13C gHMBC  spectrum of compound 6.(TIF)Click here for additional data file.Figure S111H-13C TOCSY  spectrum of compound 6.(TIF)Click here for additional data file.Figure S121H-1H gCOSY  spectrum of compound 6.(TIF)Click here for additional data file.Figure S131H-1H NOESY  spectrum of compound 6.(TIF)Click here for additional data file.Figure S141H-14N HSCQ  spectra of compound 6. Overlay of two NMR experiments. Data from the first experiment, optimized to show NH signals only, are illustrated in red. Data from the second experiment, optimized to show both NH and NH2, are illustrated in blue. Data from both experiments show complete overlap indicating there are no secondary amines.(TIF)Click here for additional data file.Figure S15Thermal ellipsoid plot of compound 6.(TIF)Click here for additional data file.Figure S16HRESI-MS (positive mode) of compound 6.(TIF)Click here for additional data file.Figure S17UV data for compound 6.(TIF)Click here for additional data file.Figure S18CD data for compound 6.(TIF)Click here for additional data file.Table S1Crystal data and structure refinement data for compound 6.(DOCX)Click here for additional data file.CIF File S1X-ray coordinate data for compound 6.(CIF)Click here for additional data file.Movie S1Rotating PyMOL representation of compound 6.(MP4)Click here for additional data file.