In present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium tumefaciens (LBA4404) carrying rd29A:PgeIF4A construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T0 plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T0 transgenic plants through Southern blot analysis. Predicted Mendelian ratio of segregation (3:1) was noted in transgenic plants at T1 generation. The T2 homozygous lines  expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length, and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. The quantitative real time—PCR results confirmed higher expression of PgeIF4A gene in L1-5, L8-3, and L16-2 plants imposed with drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging, and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.Eukaryotic translational initiation factor 4A belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. The  Arachis hypogea L.) is one of the important food legumes and oil seed crops grown in the semi-arid tropics of the world. It is being cultivated on over 25.2 million ha worldwide with a total production of 41.2 million tons with an average yield of 1.67 tons/ha. India is the second largest producer of groundnut accounting 8 million tons from 6 million ha  gene in groundnut leads to compartmentalization of Na+ ions in vacuoles, thereby improved tolerance against salinity and limited water conditions , and abscisic acid stress ripening (SbASR) genes when expressed in groundnut displayed salt and drought stress tolerance coupled with more chlorophyll retention and enhanced levels of relative water content under normal/stress conditions  simultaneously expressing three TFs  displayed to increase drought, salinity, and oxidative stress tolerance compared to wild type  homologous to eIF4A when expressed in tobacco and rice imparted salt stress tolerance with minimal accumulation of sodium in transgenic plants  showed high tolerance against moisture stress , PDH45 and PgHSF4 (Pennisetum glaucum heat shock factor) simultaneously expressed in groundnut conferred tolerance to simulated drought stress and enhanced yield  R. Br.], a hardy C4 plant, exhibits better adaptation to harsh arid or semi-arid climates as compared to other cereals  was isolated from Arabidopsis genomic DNA by using promoter specific primers overhang with restriction enzymes, Kpn I in sense primer and Nde I in anti-sense primer. The PgeIF4A gene fused in sense orientation at Nde I–Not I sites at downstream to the rd29A promoter (Kpn I–Nde I) and upstream to poly A terminator (Not I–Sac I) in plant expression vector, pGreen0229 (www.pgreen.ac.uk/) with bar  as a selectable marker. The recombinant rd29A:PgeIF4A:Poly A construct was then confirmed by restriction and sequence analysis.The cDNA encoding for rd29A-PgeIF4A–poly A cassette and pSoup were co-transformed into Agrobacterium tumefaciens (LBA4404) with following condition of 2.5 kV for 5 μS by multi- electroporator . Electroporated samples were immediately transferred into 1 ml YEM (yeast extract mannitol) liquid medium and incubated for 5 h at 28°C with 200 rpm. Then the cells were harvested and plated on YEM medium supplemented with 50 mg/L kanamycin, 20 mg/L rifampicin, and 50 mg/L streptomycin. The recombinant colonies were observed after 48 h of incubation at 28°C and confirmed the positive colonies by PCR.The pGreen0229 vector harboring cv JL-24) were collected from International Crops Research Institute for the Semi Arid Tropics (ICRISAT), Patancheru, India. Seeds were surface sterilized with 0.1% mercuric chloride for 8 min, followed through rinsing with sterile water and soaked for 4 h in distilled water. The seed coat was peeled off and the cotyledons were sliced vertically to prepare de-embryonated half cotyledon (DEC) aseptically which was used for Agrobacterium transformation.Seeds of groundnut (A. tumefaciens (LBA4404) suspension culture (OD600 = 0.5–0.6) by gentle shaking for 10 min and dried them on sterile Whatman filter paper under aseptic conditions. Then, the explants were co-cultivated for 48 h on MS basal medium  + 21.5 mg/L of 2, 4—Dichlorophenoxy acetic acid  + 5 mg/L phosphinothricin (PPT) and 3% sucrose at pH 5.8 solidified with 0.8% agar. Cultures were maintained at 26 ± 1°C at 16 h photoperiod with white fluorescent light intensity of 60 mE m2s−1. Resistant induced shoots from un-differentiated calli at the stage of 3–5 cm length were transformed to shoot elongation medium supplemented with BAP (2 mg/L) and allowed to grow further for another 2 weeks to attain shoot elongation up to 7–9 cm. Elongated shoots were sub cultured into rooting medium containing NAA (0.5 mg/L) + PPT (5 mg/L) incubated for about 15 days. After root formation, the plantlets were transferred to transgenic glasshouse for further hardening and grown them till attaining the maturity. Seeds were harvested from the putative transformed and control plants for further analysis.Healthy half DECs were infected with 0) was isolated using modified CTAB method  and marker gene (nos-bar) cassettes were PCR amplified using primer sets rd29A F1: PgeIF4A R1 and nos F1: bar R1 respectively (Supplementary Table Genomic DNA from un-transformed control and transformed plants (T0) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. The digested samples were size fractionated on agarose gel (0.8%) for 16 h at 30 V. The depurinated and denatured DNA fragments were subsequently transferred to positively charged Hybond N+ nylon membrane . Biotin labeled PgeIF4A gene was used as probe. Pre-Hybridization, washing and detection of transgene signals were carried out by following manufacturer's instructions . The seeds obtained from Southern positive were used for further transgene segregation analysis.Genomic DNA (20 μg) from control and transformed plants  were dipped in PPT (5 mg/L) solution and assessed the herbicide resistance after 10 days.The T2 seeds  were placed on half strength MS medium supplemented with mannitol  and NaCl , incubated for 15 days at 25 ± 2°C under 16 h light /8 h dark photo-period. Three seedlings constituted one biological replicate. The phenotypic growth parameters like shoot length, tap root length and number of lateral roots were recorded to evaluate the effect of drought and salinity stresses on transgenic lines. All experiments were repeated thrice and the data statistically analyzed.Mannitol and NaCl were used for simulating drought and salt stress conditions in transgenic plants at seedling stage. T2 plants of lines L1-5, L8-3, and L16-2) were excised using cork borer. The discs were floated on 5 ml solution of 0 (distilled water as control), 200, 300 mM mannitol and 100, 200 mM NaCl to simulate drought and salinity stress, respectively. These discs were incubated for 4 days under continuous illumination at 26°C for leaf senescence assay. Then, the leaf discs were macerated using mortar and pestle and diluted them in 80% acetone. The absorbance of the samples was recorded at A664 and A667 using spectrophotometer. The chlorophyll A, B, and total amount were calculated as described by Arnon  from fully expanded leaves of 1-month-old plants (Tby Arnon . Experim2 lines as per the manufacturer's instructions (Sigma Aldrich). The quality and quantity of total RNA was assessed by Nano spectrophotometer  and RNA gel electrophoresis. The total RNA (5 μg) was reverse transcribed to cDNA using oligo dT primer as per manufacturer's instructions (iScript cDNA synthesis kit). The quantitative-real time PCR (qRT-PCR) was performed in Realplex  real time system. The gene specific primers and reference gene primers were designed using Primer 3.0 software to amplify the product of PgeIF4A and glucose 6 phosphate-1-dehydrogenase  from the transgenic TTo determine the salinity induced oxidative stress, 1 month old control and transgenic plants  were exposed to NaCl (250 mM) treatment for 10 days and maintained them under controlled greenhouse conditions.−2. s−1) to perform the reaction. The photo reduction of NBT (blue formazan production) was measured at 560 nm using spectrophotometer.The SOD activity was assayed by monitoring the percentage of photochemical reduction of nitro bluetetrazolium (NBT) in leaf tissues according to a protocol described by Beyer and Fridovich . The rea−1 cm−1  content in leaf tissues through TBARS (thio barbutaric acid reactive substances) assay. Hundred milligrams of leaf tissue was homogenized in 2 mL of 0.1% TCA and centrifuged at 10,000 rpm for about 10 min. To 1 mL of supernatant 2 mL of 20% TCA and 2 mL of 0.5% TBA was added and incubated in water bath at 95 °C for 30 min, immediately cooled on ice and then centrifuged at 10,000 rpm for 10 min. The absorbance was measured at 532 and 600 nm. The value of absorbance at 600 nm was subtracted from absorbance at 532 nm. The MDA content was calculated using extinction coefficient of 155 mM1 Arnon, .Electrolyte leakage (EL) was determined by following the standard protocol described by Kumari et al. . BrieflyIn vitro localization of H2O2 was determined by DAB assay as described by Kumari et al. . Subsequently, two-way ANOVA was used to assess treatment (T), plant lines (L), and plant lines by-treatment interactions (L × T) in two different stress treatments (Mannitol and NaCl).The experiments were designed as triplicates  and data analyzed statistically. To perform one-way and two-way ANOVA analysis, CoStat version 6.204 statistical program package  were used. One-way ANOVA was carried out to test the difference between the transgenic lines  and control. Means were compared using Tukey-Kramer test and LSD at PgeIF4A gene was isolated and cloned from P. glaucum using homology based cloning approach. The cloned gene sequence was deposited in the NCBI Genbank (Accession no: EU856535). The PgeIF4A gene consists of 1224 bp encoding for 407 amino acids with predicted molecular mass 45.2 kDa and isoelectric pH 6.10. The PgeIF4A protein consists of important motifs like RNA helicase/DEAD box Q-motif, DEAD box domain and RNA-helicase C-terminal  carrying vector, pGreen0229: rd29A: PgeIF4A: poly A with nos-bar gene as selectable marker  plants, 24 showed amplification of expected 654 bp  and nos F1-bar R1 (nos-bar junction) primer sets (Supplementary Table nos-bar and rd29A-PgeIF PCR products from transgenic plants were further sequenced using Sanger's dideoxy method (Eurofins Genomics India) and annotated the sequences using multiple sequence alignment software . Both gene and marker sequences were integrated in groundnut genome exactly matched with vector T-DNA  was integrated into groundnut transgenic plants.The gene integration and copy number of s Figure . However1 progeny seeds collected from single copy integrated southern positive (T0) plants were raised in glass house to determine the inheritance pattern of the transgene. The T1 progeny of L1, L8, and L16 was tested with PCR to detect the transgene. These lines exhibited the predicted Mendelian ratio as 3:1  and transgenic lines  under both concentrations of NaCl (100 and 200 mM). The shoot and tap root lengths of transgenics were noted as 10.46 and 3.48 cm compared with control plants as 6.57 and 1.8 cm, respectively, under 200 mM NaCl stress condition Figures . FormatiPgeIF4A could have conferred high level of salt tolerance.In leaf senescence assay, on 2nd day of incubation, the leaf discs from control had bleaching of the chlorophyll as with increasing concentration of NaCl also. However, minimal bleaching was observed in leaf discs from transgenic plants. Total chlorophyll content of these leaf discs after 4 days of stress imposition was retained about 2.3–2.6 μg/mg FW, whereas in control it was observed 2.0 μg/mg FW Figure . As the PgeIF4A transcript was confirmed by semi quantitative PCR and qRT-PCR. The PgeIF4A transcript accumulated in homozygous T2 lines  grown under stress (mannitol and NaCl) conditions  was assessed by measuring th percentage of NBT inhibition. The percentage of NBT inhibition in control was noted as 26.9% compared to the transgenic lines such as L1-5, L8-3, and L16-2 was 46.7, 33.3, and 37.2%, respectively  compared to transgenic lines L1-5, L8-3, and L16-2  activity in control and transgeni lines  sensed the stress by way of coordinating with oxidative enzyme machinery. As a result high RO scavenging, more retention of chlorophyll in transgenic lines was noted and there by protected the plants by growing normally under stress and non-stress conditions. The improved JL-24 variety exhibited superior growth parameters under simulated stress condition. The plants showed tolerance to herbicide which could be additional feature to control the weeds. The eIF4A gene from P. glaucum played a key role in stress tolerance and these plants will be further validated under field conditions.cv JL-24 is an important high yielding and drought sensitive variety was used for improving drought and salt stress tolerant by transferring PgeIF4A gene through transgenic approach. In present study, three lines clearly showed drought, salt, and oxidative stress tolerance under simulated conditions. Among these lines, L1-5 exhibited super growth performance and scavenging free radicals under salt stress conditions. PgeIF4A expression in groundnut was strongly associated with enhancement of growth parameters and chlorophyll retention. This strong regulation could be due to firm regulated expression of PgeIF4A gene controlled by stress inducible rd29A promoter, which leads to the stress tolerance in subsequent generations.Groundnut TS and GM: Planned and executed the experiments; JV: biochemical analysis, PS and MR: provided gene construct GM, TS, JV, and PS: Prepared manuscript.Financial support received from Rashtriya Krishi Vikas Yojana (RKVY), Government of Andhra Pradesh.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Midpoint S1P levels during adjuvant therapy were lower than baseline, with near return to baseline after completion, indicating a relationship between chemotherapy and circulating S1P. While stage of disease did not correlate with plasma S1P levels, they were lower among patients with Her2-enriched and triple-negative breast cancer as compared to luminal-type breast cancer. Plasma S1P levels are paradoxically suppressed in aggressive breast cancer and during adjuvant chemotherapy, which raises the possibility that postoperative plasma S1P levels do not reflect S1P secretion from resected breast cancer.Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that has been shown to serve an important regulatory function in breast cancer progression. This study analyzes plasma S1P levels in breast cancer patients undergoing adjuvant therapy as compared to healthy control volunteers. 452 plasma S1P samples among 158 breast cancer patients, along with 20 healthy control volunteers, were analyzed. Mean S1P levels did not significantly differ between cancer patients and controls. Smoking was associated with higher S1P levels in cancer patients. Baseline S1P levels had weak inverse correlation with levels of the inflammatory mediator interleukin- (IL-) 17 and CCL-2 and positive correlation with tumor necrosis factor alpha (TNF- Breast cancer is the second leading cause of cancer death among women in the United States with 40,610 deaths estimated in 2017 [Most investigations on biomarkers have centered on measuring circulating tumor cells, proteins, and nucleic acids released from the breast tumor or cancer cells. More recently, however, the roles of sphingolipid mediators in breast cancer have been increasingly investigated due to recent advances in mass spectrometry . One lipIn this prospective study among women with breast cancer, the association of circulating plasma S1P with demographic factors and during adjuvant therapy is investigated.All the patient and volunteer samples were collected with Virginia Commonwealth University and Massey Cancer Center Institutional Review Board (IRB) approval. Venipuncture plasma samples were collected from 158 women who underwent surgical resection of their breast cancer tumors. Paired samples were collected two weeks prior to adjuvant therapy (baseline), prior to the fourth cycle of chemotherapy (midpoint), and two weeks after completion of adjuvant therapy (completion). For a control sample, a one-time plasma sample was collected from 20 healthy women volunteers. The samples were snap-frozen and stored at −80°C.μL aliquot of ethanol : methanol : water (7 : 2 : 1), and analysis for S1P using LC-ESIMS/MC.Circulating S1P concentration was quantified using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MC) at the Virginia Commonwealth University Lipidomics Core as previously described , 14. Briγ), MCP-1, MIP-1, and tumor necrosis factor alpha (TNF-α).Additionally, levels of various circulating inflammatory cytokines were measured from baseline serum samples with enzyme-linked immunosorbent assays. The cytokines measured included interleukin- (IL-) 1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, interferon gamma  followed by African American (33.5%) and Hispanic (3.8%). There was no difference in plasma S1P level between the breast cancer patients at baseline and the control cohort  , or ethnicity distribution between the breast cancer cohort and the control cohort . Among t = 0.41) .2), and ethnicity of the control patients. This resulted in 40 matched breast cancer patients by age, BMI, and ethnicity to the control volunteers. Similar to the overall analysis, there was no difference in baseline S1P level between the control-matched breast cancer patients and the controls .Of the breast cancer patients, a matched cohort with the controls was subselected matching for age (within five years), BMI . There was no significant correlation between plasma S1P levels and BMI (p = 0.75) and plasma S1P and ethnicity (p = 0.41) in both the breast cancer and control cohorts. Among the breast cancer cohort, there was no significant difference in mean baseline plasma S1P levels between premenopausal patients and postmenopausal patients . Tobacco use, however, was associated with increased plasma S1P levels  among the breast cancer cohort, while alcohol use was not associated with S1P levels (p = 0.98).Among the breast cancer cohort, there was no significant correlation between age and S1P concentration (p = 0.688), subtype and tumor grade were associated with baseline S1P , suggesting an effect of chemotherapy on circulating S1P (p = 0.06). Additionally, there were no significant differences in S1P concentration among patients who underwent radiation therapy  and CCL-2 , while it had a positive correlation with TNF-α . Baseline plasma S1P did not have a significant correlation with the other measured inflammatory mediators.Among the breast cancer cohort, associations between baseline plasma S1P levels and various inflammatory mediators were analyzed using Spearman's Rho tests. Baseline S1P had a significant weak inverse correlation with IL-17 (This study revealed that there was no statistically significant difference in circulating S1P levels between healthy volunteers and patients with breast cancer after tumor excision. The only demographic characteristic associated with increased circulating S1P was a history of smoking. Patients with more aggressive pathologic subtypes of breast cancer paradoxically demonstrated decreased levels of circulating S1P. While radiation, hormonal therapy, and targeted biologic adjuvant therapy did not affect S1P levels, chemotherapy was associated with changes in circulating S1P during treatment.S1P itself results from the intracellular phosphorylation of sphingosine by two sphingosine kinases, SphK1 and SphK2 . Phosphoα. Further investigation is needed to clarify the inverse relationship noted in this study between S1P levels and IL-17 and CCL-2 levels.The role of S1P in inflammation and immunity has been well described . T cellsClinically, S1P levels have been shown to be high in patients with breast cancer , and phoWe have previously demonstrated that among patients undergoing total or partial mastectomy for tumors greater 1.5 cm, peritumoral tissues had significantly higher S1P and other sphingolipid levels as compared to distant tissues in the same sample . In the Further, higher grade and more aggressive subtypes such as Her2-enriched and triple-negative breast cancer patients demonstrated lower baseline circulating S1P levels. The lower baseline S1P levels in triple-negative breast cancer may be related to S1P's association with circulating estrogen , 26. In Our findings of lower baseline S1P in a more aggressive disease run counter to the previous published data demonstrating an increase in tissue S1P signaling in aggressive tumors , but areα signaling, NF-κB activation pathways, and chemokine expression for recruitment of mononuclear cells into sites for inflammation [α expression. The overall increased inflammatory state present in smokers likely explains the increased circulating S1P that was found in the present study. Another possibility is that smoking is known to be associated with dysfunction of the vessels, where S1P signaling plays fundamental roles. For instance, S1PR1-deficient mice die in utero from massive hemorrhage due to immature vessel development, and neutralization of extracellular S1P with anti-S1P antibody demonstrates a significant inhibition of angiogenesis, tumor growth, and metastasis [In support of that hypothesis is the finding that smoking was the only patient characteristic noted to result in increased circulating S1P. Smoking is known to result in systemic inflammation, and S1P's role in inflammation has been well described . Specifiammation , 29, 30.tastasis . TherefoAdjuvant therapies in breast cancer aim to decrease both local and distant recurrence and/or metastasis from nodal spread or microscopic dissemination. Adjuvant treatment options include various methods of irradiation, multiple chemotherapy regimens, use of hormonal therapy, and biologic modulators. While radiation therapy is focused on the breast tissue field and hormonal therapy targets specific receptors in the breast and ovaries, the effects of chemotherapy are more systemic. Perhaps, this study identified no change in S1P during radiation therapy and hormonal therapy, however, did find changes in S1P with chemotherapy. Specifically, with AC chemotherapy, circulating S1P increased from baseline and remained high, while with taxane-based chemotherapies, S1P appeared to decrease. This may be related to the fact that doxorubicin in AC regimen induces inflammation.The interaction between the S1P pathway and adjuvant treatment agents is an area of active investigation. Upregulation of the S1P pathway has been implicated in the development of chemotherapy resistance, whereby the addition of FTY720, a functional antagonist of S1PR1 that blocks S1P signaling, results in overcoming chemotherapy resistance , 32. TheSpecifically, in breast cancer, there have been a limited number of studies investigating the interaction between S1P and chemotherapy. The only study to date investigating circulating S1P, to the authors' best knowledge, details the detrimental effect of circulating S1P with weight gain during chemotherapy among 21 patients . In breaIn breast cancer cells, SphK1 activity has been linked to endocrine resistance, whereby overexpression of SphK1 resulted in resistance to tamoxifen with increased proliferation . TamoxifThe main limitations of this study include the limited patient sample, although to the authors' best knowledge, this constitutes the largest study investigating the association of circulating S1P with patient characteristics and during chemotherapy. The study also used a limited control subset of patients without breast cancer to investigate differences in baseline S1P between controls and postoperative breast cancer patients. Additionally, the baseline circulating S1P levels were measured from samples taken from the patients after the removal of the tumor, hindering our ability to draw conclusions regarding the association of circulating S1P with the characteristics of breast cancer. Due to the initial scope of the study, follow-up data on outcomes is not available to dissect whether changes in S1P during chemotherapy were associated with differential recurrence or survival outcome.In summary, this study displays a paradoxical association of low S1P with more aggressive subtypes, suggesting that circulating S1P may not be an accurate surrogate for tissue S1P in the tumor microenvironment. These data also reveal a variable association between various chemotherapy regimens and S1P, suggesting that S1P modulation could have a therapeutic impact in chemosensitization in breast cancer treatment.


Thus, this inside-out signaling is critical for a variety of S1P cellular responses. Deregulation of S1P metabolism has been related to various diseases including cancer with increased S1P tissue levels. Various studies suggest the involvement of SphKs/S1P signaling in tumor progression and metastasis [Besides their structural role, sphingolipids including Sphingosine-1phosphate (S1P) have drawn attention as bioactive sphingolipid signaling molecules involved in the regulation of several processes, including cell growth, differentiation, survival, migration or immune response. It is now well established that S1P is involved in the onset or the progression of pathological conditions such as cancer, autoimmunity, cardiovascular conditions or diabetes to name a few. The S1P content in cells is low and is kept under control through a delicately regulated balance between its synthesis by Sphingosine kinases 1 & 2 (SphKs) and its dephosphorylation by S1P phosphatases or degradation by S1P lyase. Once produced S1P can be secreted via specific ATP-binding cassette transporters including Spns2 and exerts its extracellular functions through a family of five G-protein-coupled receptors, named S1PCell division or mitosis is a particularly complex and highly regulated process that allows the formation of two genetically identical daughter cells. A quality control mechanism called spindle assembly checkpoint , ensures fidelity of chromosome segregation. The SAC prevents anaphase onset until all kinetochores are stably attached to microtubules. In the presence of unattached kinetochore, the SAC is on and the metaphase-to-anaphase transition is inhibited. Dysfunction of SAC leads to chromosome mis-segregation and aneuploidy and is implicated in tumorigenesis , 4.We recently identified and explored a novel function of SphKs/S1P signaling during mitosis . Our stuS1P may act intra- or extracellularly. To test this hypothesis we used Sphingomab™, a high-affinity monoclonal anti-S1P antibody that neutralizes extracellular S1P . First, 1-5) mediate S1P extracellular functions. These receptors differ in their tissue distribution, and their biological effect, depending on the suite of S1P receptor subtypes expressed. Using pharmacological and RNA interference approaches we identified S1P5 receptor as target for S1P’s mitotic function. Accordingly, mitosis was not affected by S1P in mouse embryonic fibroblasts (MEFs) derived from S1P5 knockout mice. Among multiple signaling cascades activated downstream S1P receptors, PI3K/Akt pathway was activated by S1P in mitotic cells. It has been reported that Akt phosphorylates and activates the mitotic kinase Polo-like kinase 1 (Plk1) at Serine 99 at the level of kinetochore and promotes metaphase-to-anaphase transition [99. Therefore, abundance of S1P promotes mitotic progression by S1P5 and downstream activation of PI3K/Akt leading to Plk1 activation at kinetochore, to control metaphase-to-anaphase transition (Figure Five high-affinity G-coupled receptors (S1Pansition . Using T5 axis.Overall, these data demonstrate that extracellular S1P promotes mitotic progression leading to chromosome segregation defects supporting the concept that cellular microenvironment plays an important role in coordination of mitosis. In the future, it will be important to investigate what are the consequences of the S1P-induced chromosome mis-segregation in chromosome stability of normal and cancer cells and the therapeutic potential of S1P/S1P


Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4+ T cell responses.An important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts  at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with recombinant gp120 protein or MVA-expressed gp140 to enhance antibody responses elicited by the GOVX-B11 DNA prime-MVA boost vaccine. We found that both types of immunogen boosts enhanced potentially protective antibody responses, whereas the gp120 protein boosts also increased CD4+ T cell responses. Our data provide important information for HIV vaccine designs that aim for effective and balanced humoral and T cell responses. GOVX-B11, a subtype B DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine, elicits higher response rates and titers of antibodies to the gp41 transmembrane than the gp120 receptor binding subunit of Env , 4.The GOVX-B11 vaccine expresses the native ADA (clade B) gp160 Env, a clade B CCR-5-tropic Env from a chronic infection , 8 and pThe regimen for vaccination was based on the regimen used in RV144 and on our clinical experience with DNA priming and MVA boosting –11. The –+ T cells over the MVA-only group. Given the comparable levels of elicited antibodies, and the potential for vaccine-elicited CD4+ T cells both to protect from infection  and MN gp41 were measured using enzyme-linked immunosorbent assays (ELISAs) and longitudinal serum samples. gp120- and gp41-specific IgG responses developed 2 weeks after the first MVA boost (week 20), peaked after the second boost (week 28) declined with time, and were boosted again after the 3rd boost  and B. ANext, we measured IgG responses to the vaccine-matched B.63521 gp120 protein. The MVA+gp120 group showed significantly higher responses following the 2nd boost, postcontraction at the time of the 3rd boost, and after the 3rd boost than did both the MVA-only and MVA+MVAgp140 groups . The difP values that are not controlled for multiple comparison [raw_p] = 0.03 for each)   and 2 anor each)  and 2 thIn contrast to the gp120 and gp140 responses, levels of binding to the V1V2 panel were similar for sera from the MVA+gp120 and MVA+MVAgp140 groups after both the 2nd and 3rd boosts, with both being approximately 2-fold higher than that for the MVA-only group .The durability and boostability of binding antibody responses were evaluated as the fold decline of the antibody response over the 12 weeks following the 2nd boost (week 28/week 40) and the fold increase of the antibody response following the 3rd boost over that following the 2nd boost (week 42/week 40) . The MVAIgG from the vaccinated animals targeted linear epitopes in the C1, V2, C2, V3, C4, and C5 regions of gp120 . In gp41The contribution of each epitope to the total Env linear epitope binding after the 2nd and 3rd boosts was evaluated by determining the percentage of the binding intensity for each epitope within the total Env linear epitope binding intensity. Among the 3 groups, the MVA+gp120 group was the most focused on V3, with 81.1% and 70.8% of total linear epitope binding (group median) targeting V3 at weeks 28 and 42, respectively, compared to 34.2% to 49.9% for other groups . The MVAP < 0.01) were found after the 3rd boost for C2.2 (MVA+MVAgp140 and MVA+gp120 > MVA only) and after the 2nd boost for C5.1 (MVA+MVAgp140 > MVA only and MVA+gp120) and C5.2 (MVA+MVAgp140 > MVA only)  were observed among groups for binding to epitopes in C2, C4, and C5 following either the 2nd (week 28) or 3rd (week 42) boost . Highly VA only) . Due to Binding responses to peptides representing different clades/strains were generally similar among vaccine groups and at the two tested time points (following the 2nd and 3rd boosts) . All groThe avidity of the antibody elicited for three linear epitopes—clade B V3, clade C V3, and gp41–ID—was measured by an avidity index assay , 15. For50) titers up to 5,274  and MW965.26 (clade C) viruses, with 50% inhibitory dose  against tier 2 viruses after the 2nd boost, with positivity rates ranging from 40% to 100% for 2 clade C tier 2 viruses (25710-2.43 and Ce1176_A3) and being 20% for the clade B tier 2 virus TRO.11   . Group m50 neutralizing titers for tier 1A and tier 1B clade B viruses were associated with binding magnitudes to peptides in the C1, V3, and the gp41_ID regions, with Spearman r values of >0.6 and raw_p values of <0.05 . Interestingly, the highly conserved gp41_ID was among epitopes with the most scores above a Spearman r of 0.5 (raw_p < 0.05) for tier 1B and tier 2 viruses. The ADCC titers measured with B.63521Δ11 gp120-coated cells associated most strongly with two C1 epitopes . Further examination of scatter plots revealed that correlations between binding magnitudes and neutralization titers for most pairs with r values of >0.6 were driven by data from all three groups, with the exception of MW965, for which correlations were driven by higher neutralization titers for the MVA+gp120 group  than in lymph nodes but more similar levels of elicited CD40L-expressing cells in these two compartments   . No sign in PBMC .+ T cell responses. Below we discuss these results and how they might relate to protective vaccine responses.The current study compared the magnitude, durability, specificity, breadth, and functional activity of antibodies elicited by MVA+gp120 protein boosts, MVA+MVAgp140 boosts, or MVA-only boosts of a DNA-primed response. Our results reveal that the addition of either gp120 protein or MVA-expressed secreted gp140 to boosts elicits higher, broader, and more functional antibody responses than the MVA-only boosts. They also reveal that protein, but not MVAgp140, boosts enhanced CD4The goal for adding boosts to the DNA/MVA vaccine was to enhance antibody responses to gp120, especially those that target the V1V2 region of gp120. Both B.63521 gp120 and MVAgp140 boosts enhanced these antibody responses. Following the 3rd and last boost, mean magnitudes of binding to the gp120 antigen panel and the V1V2 panel were 4.3- and 3.0-fold higher and 2.1- and 2.5-fold higher for the MVA+gp120 and MVA+MVAgp140 groups, respectively . The gp1In addition to the magnitude of binding antibody responses, the boost immunogens affected the durability of antibody responses and the performance of repeated boosts. The MVA+MVAgp140 group showed a trend for a slower decline and a more effective 3rd boost of responses than for the MVA+gp120 group. This lower contraction and higher boost with MVAgp140 suggest that MVAgp140 may elicit more durable and replenishable responses than the gp120 protein boost. Although lower levels of preboost antibody responses likely contributed to the efficiency of the 3rd MVAgp140 boost, there is precedent for late boosts, with MVA being highly effective in both preclinical and clinical settings –18.–+ T cell response over those elicited by the MVA-only boost vaccine. The MVA-specific T cell response was not measured. However, we consider it unlikely that adding 1 × 108 50% tissue culture infective doses (TCID50) of MVA gp140 to the 1 × 108 TCID50 of MVA62B in the MVA gp140 group significantly enhanced CD4+ responses because of the very shallow dose-response curve for MVA boosts of a constant DNA prime . These correlations were strongest and most frequent for the two tested tier 1 viruses. The finding of correlations with the gp41_ID region, albeit modest, are of interest because these correlations, with a highly conserved region in Env, included 4 of 9 tested tier 1B and tier 2 viruses. Monoclonal antibodies (MAbs) to the gp41_IDR capture virions  blocked ADCC activity in over 90% of subjects . A32 did+ T cell responses. These data demonstrate that both recombinant gp120 protein and MVA-expressed secreted gp140 are effective strategies for enhancing potential protective nonneutralizing antibody responses to HIV.In conclusion, gp120 protein and MVAgp140 were similar in their abilities to increase levels of antibody responses for gp120 and the V1V2 region of gp120 over the MVA-only boosts in the GOVX-B11 DNA/MVA vaccine regimen but differed in their abilities to enhance CD4+ T cell response may allow identification of conditions under which protein boosts of DNA/MVA vaccines enhance both Ab and preclinical protective responses.The study has had a direct impact on the protein boost protocol being advanced for the clade B DNA/MVA vaccine GOVX-B11. This study clearly demonstrated that under the conditions tested, B.63521Δ11 only modestly enhanced V1V2 responses. Because of this finding, both higher doses of B.63521Δ11 and a gp120 that is more effective at enhancing V1V2 responses are being tested in nonhuman primates and advanced to phase 1 clinical trials. Also, protein boosts have enhanced antibody responses but not protective responses in preclinical trials for DNA/MVA vaccines (see above). Understanding the nature of the enhanced CD48 TCID50 per ml in a proprietary buffer for storage and inoculation. MVA encodes a secreted ADA gp140 Env that was inserted into deletion II of MVA by plasmid insertion vector pLAS-2. It coexpresses the same HXB2/BH10 gag-pol as MVA62B. MVAgp140 was produced in CEF at the Laboratory for Viral Diseases, NIAID, NIH, purified by sucrose pad centrifugation, and suspended at 1 × 108 TCID50 for storage and inoculation. B.63521Δ11mutC gp120 was produced by transient transfection at the Duke Human Vaccine Institute, Duke University. To prevent proteolytic clipping, mutations were introduced into the B.63521Δ11 gp120 V3 (SIR-GPGQT) region. Antigenicity analysis with a panel of MAbs showed similar affinities of binding of soluble CD4 (sCD4) and CD4 binding site broadly neutralizing antibodies (bnAbs) (VRC01 and CH103 MAbs) and for bnAbs for the V3 loop ASN332-centered oligomannose patch  as well as the V3 loop MAb 19b, for B63521Δ11 with and without the mutC mutation .The GOVX-B11 vaccine consists of the pGA2/JS7 DNA and MVA/HIV62B (MVA62B) immunogens. The DNA prime expresses VLPs displaying the ADA gp160 Env. The DNA used in this trial also coexpressed rhesus granulocyte-macrophage colony-stimulating factor (GM-CSF) , 28. The8 TCID50 of MVA62B into the upper left limb on weeks 18, 26, and 40. At the time of the MVA boosts, animals in group 2 also received 100 μg of B63521Δ11gp120 plus 500 μg of Alhydrogel  and animals in group 3 also received 1 × 108 TCID50 of MVAgp140, both delivered intramuscularly in 1 ml by needle and syringe to the upper right limb. Sera, PBMC, and rectal swabs were collected at regular intervals throughout the trial. Lymph nodes as well as an exsanguination bleed were collected at the end of the trial, 2 weeks following the last boost. The general health, weights, clinical blood counts (CBC), and clinical chemistries of animals were monitored throughout the trial.The animal study was conducted at Bioqual Inc., Rockville, MD, in accordance with all rules of the American Association for Laboratory Animal Care and under the supervision of the Institutional Animal Clinical Care and Use Committee. Three-year-old male rhesus macaques, prescreened for background for Env binding antibodies and activated T cells, were randomized into 3 study groups of 5 animals each by weight. For all groups, 3 mg of DNA was inoculated by needle and syringe into the upper left limb on weeks 0 and 8 and 1 × 10Binding antibody response was tested both in enzyme-linked-immunosorbent assays (ELISAs) and in binding antibody multiplex assays (BAMA). The ELISA uses Con6 gp120 produced in BHK cells (Duke) and gp41 produced in bacteria  as antigens for antibodies to gp120 and gp41, respectively. Assays were done using a standard curve of macaque IgG captured by a goat anti-rhesus antibody and results interpolated to estimate micrograms of specific IgG per ml as previously described . BAMA weSerum epitope mapping of heterologous strains was performed as previously described –34, with–The affinity maturation of the vaccine-elicited antibody responses to the immunodominant region of gp41 (gp41_ID) and V3 linear epitopes (V3.B and V3.C) was measured by an avidity index assay using the binding antibody multiplex assay. Avidity index was measured by inclusion of a denaturation step  . UntreatAntibody dependent cellular cytotoxic activity mediated by test animal plasma/serum was detected according to our modification of the previously described GranToxiLux cell-mediated cytotoxicity procedure . CEM.NKRhttp://www.hiv.lanl.gov/content/nab-reference-strains/html/home.htm.Neutralizing antibodies were measured as a function of reductions in luciferase (Luc) reporter gene expression after a single round of infection in TZM-bl cells , 40. TZMHIV-specific cellular immune responses were assessed by multiparameter intracellular cytokine staining (ICS) assays following stimulation with GOVX-B11 vaccine-specific HIV peptides (15-mers overlapping by 11) as previously described . Panel oP value of <0.05 were further tested using Wilcoxon rank sum exact test. Correlations between binding antibody responses and neutralization and ADCC responses were tested using the Spearman correlation test. P values from the Wilcoxon rank sum test and Spearman correlation test were corrected for false discovery rate (FDR) using the Benjamini-Hochberg (BH) method . Both raw_p and FDR-adjusted P values (FDR_p) are shown in Antibody and T cell response measurements were first screened in an omnibus test. Differences between pairs of groups in the levels of measurements that showed a ) method . Statist


The seroprevalences of H9N2 were significantly higher in live poultry market workers, large-scale poultry farmers and backyard farmers than in poultry slaughtering factory workers and wild bird habitant workers. The seroprevalences of A/Guangzhou/333/99 (G9) (3.42%) and A/quail/Hong Kong/G1/97 (G1) (1.37%) in Southern China were significantly higher than those in Northern China (p<0.001). The seroprevalence was highest in the elderly, followed by adults and then youths. Our results indicate that subclinical human infection with H9N2 avian influenza virus is widely distributed in China. Longer poultry exposure might contribute to the higher seroprevalence in the elderly group. The higher seroprevalence observed in Southern China than in Northern China might be caused by a higher poultry density.The H9N2 avian influenza virus circulates worldwide, predominantly in poultry. Its increasing infectivity and adaptation in poultry and mammals have enhanced the possibility of human infection. However, H9N2 human cases are difficult to detect due to their mild clinical symptoms. Serological study is valuable for risk assessment. A total of 15,700 serum samples were collected from occupationally exposed populations in 22 provinces of China and tested using hemagglutination inhibition (HI) and microneutralization (MN) assays. The sera positive rate of A/Guangzhou/333/99 (G9) was significantly higher than that of A/quail/Hong Kong/G1/97 (G1) ( In China, lowly pathogenic H9N2 avian influenza viruses are the most prevalent avian influenza viruses in poultry , 2. ThisSince the first human infection with H9N2 virus was reported in 1998 , concernThe clinical symptoms of H9N2 human infection are always mild , which mBased on avian influenza surveillance data from China, 2 representative H9N2 viruses (A/Guangzhou/333/99 (G9) and A/quail/Hong Kong/G1/97 (G1), a representative virus of the G1 lineage) were selected as the antigens for antibody detection. A/Guangzhou/333/99 (G9) was isolated from a human case by the Chinese National Influenza Center, and A/quail/Hong Kong/G1/97 (G1) was kindly provided by Dr. Jin-hua Liu. Ferret antisera raised against these viruses were used as positive controls for the HI and MN assays with the approval of the Animal Care Welfare Committee of the National Institute for Viral Disease Control and Prevention, Chinese Centers for Disease Control and Prevention.Since 2008, China has implemented a routine national serologic surveillance of occupationally exposed populations for the highly pathogenic avian influenza H5N1 virus. As part of the national pandemic-preparedness plan, the requirement for informed consent was waived according to the regulatory policy in China. In this study, a total of 15,700 stored serum samples that had been collected between 2009 and 2011 were used. Poultry workers from live poultry markets, large-scale poultry farms, backyard poultry farms, poultry slaughter factories and wild bird habitats were included in this study. Detailed demographic information is shown in For the HI assay, all serum samples were treated with 4 volumes of receptor-destroying enzyme  at 37°C for 18 hours, followed by inactivation at 56°C for 30 min. All serum samples were then adsorbed by packed turkey red blood cells (one volume of packed red blood cells to 10 volumes of RDE-treated sera) for 60 min at 4°C to remove nonspecific hemagglutinin. The HI assay was performed as previously described using 1% turkey red blood cells. Serum samples were titrated in 2-fold dilutions in phosphate-buffered saline and tested at an initial dilution of 1:10. Viruses were inactivated using 0.5‰ β-propiolactone. A Beckman automatic workstation  was used to dilute the sera and to add the antigen and red blood cells. Individuals with an HI titer of ≥40 were considered suspected seropositive, and the samples were then confirmed by MN assay.50 of the virus in MEM containing 1% BSA for 1 h before 100 μl of 1.5 x 105 MDCK cells/ml were added. After 18 h of incubation at 37°C, the cells were fixed by 80% acetone, and the influenza A virus NP protein in infected cells was detected by ELISA using a mixture of two nucleoprotein (NP) primary antibodies . The titer was calculated using the Reed-Muench method [MN assay was performed according to the WHO Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza . Human sh method . An MN tP<0.05 was used to indicate a significant difference.Seroprevalence was analyzed by chi-square or Fisher's exact tests using GraphPad Prism5  software. p<0.0001).In a preliminary study, a total of 8 representative H9N2 viruses (one of G1 lineage and seven of G9 lineage) that genetically belonged to different lineages or sublineages were antigenically analyzed by HI assay using ferret antisera raised against these viruses. The results showed that the G1 and G9 lineage viruses were antigenically significantly different. For G9 lineage viruses, a study published in 2012 reported that two G9 lineages, h9.4.2.5 and h9.4.2.6, were circulating in mainland China before 2012, with h9.4.2.5 being the predominate lineage and h9.4.2.6 a newly emerging lineage [The sera tested are not contemporary; thus, he manuscript cannot provide the true picture of how much H9N2 infection is currently in the Chinese population. The sera tested were from occupationally exposed populations that may not be a representative sample of the Chinese population; as a result, the generalizability of the study results is limited. As it is reported, the seroprevalence of the H9N2 virus was significantly higher than that of H5N1 in populations in China . This rep = 0.0023). The seroprevalence in live poultry market workers was 3.88%, which was the highest among all occupationally exposed populations (p = 0.041). The large-scale poultry farmers (1.65%), backyard farmers (1.3%) and live poultry market workers (1.09%) were the top three groups that had positive rates over 1%  were markedly higher than those of A/quail/Hong Kong/G1/97 (G1) among different occupationally exposed populations. For A/Guangzhou/333/99 (G9), the seroprevalences among different occupationally exposed populations were significantly different  (p<0.0001), with the highest rate found in the elderly (4.45% for A/Guangzhou/333/99 (G9) and 2.31% for A/quail/Hong Kong/G1/97 (G1)), followed by adults (3.06% for A/Guangzhou/333/99 (G9) and 1.1% for A/quail/Hong Kong/G1/97 (G1)) and youths (1.38% for A/Guangzhou/333/99 (G9) and 0.39% for A/quail/Hong Kong/G1 (G1))   and A/quail/Hong Kong/G1/97 (G1)  viruses (p>0.05) . HoweverG1 (G1)) . In chilIn our study, the seroprevalence was highest in the elderly and lowest in children. This is inconsistent with our case surveillance result. Over the past few years, most of the human cases were in children. The symptoms of human infection with H9N2 virus are very mild, whereas adults with infections who have very mild symptoms may not go to sentinel hospitals, resulting in those cases being missed in the surveillance. In this circumstance, serological investigations in large populations will present a more accurate human infection rate. In mainland China, the H9N2 virus was first reported in chickens in 1994 , and thep<0.001)   and A/qup<0.001) .From a geographic perspective, the H9N2 virus has been widely distributed in poultry in China , 27, andSustained circulation of H9N2 virus in poultry has caused human infections. Most of these infections are mild or subclinical and are thus difficult to detect through influenza-like illness surveillance. The intensity of the population’s exposure to live poultry is the main factor underlying the higher seroprevalence detected in the elderly, those involved in live poultry sales and farming, and occupationally exposed groups in Southern China. The surveillance of H9N2 in poultry and occupationally exposed populations should be intensified, especially in Southern China.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Influenza virus evolves rapidly due to the accumulated genetic variations on the viral sequence. Unlike in North America and Europe, influenza season in the tropical Southeast Asia spans both the rainy and cool seasons. Thus, influenza epidemiology and viral evolution sometimes differ from other regions, which affect the ever-changing efficacy of the vaccine. To monitor the current circulating influenza viruses in this region, we determined the predominant influenza virus strains circulating in Thailand between January 2016 and June 2017 by screening 7,228 samples from patients with influenza-like illness. During this time, influenza A(H3N2) virus was the predominant influenza virus detected. We then phylogenetically compared the hemagglutinin (HA) gene from a subset of these A(H3N2) strains (n = 62) to the reference sequences and evaluated amino acid changes in the dominant antigenic epitopes on the HA protein structure. The divergence of the circulating A(H3N2) from the A/Hong Kong/4801/2014 vaccine strain formed five genetic groups (designated I to V) within the 3C.2a clade. Our results suggest a marked drift of the current circulating A(H3N2) strains in Thailand, which collectively contributed to the declining predicted vaccine effectiveness (VE) from 74% in 2016 down to 48% in 2017. Influenza A virus is an important respiratory pathogen responsible for the annual influenza outbreak and considerable socio-economic burden on the public healthcare system . The mulThe hemagglutinin (HA) surface glycoprotein of A(H3N2) possesses defined antigenic and receptor-binding sites ,5. The HTimely analysis of the genetic variations on the HA1 sequence of the circulating influenza virus strains is crucial for the prediction of VE. We therefore determined whether A(H3N2) was regionally predominant in the current influenza season and compared the genetic composition of the circulating A(H3N2) to the current vaccine strain A/Hong Kong/4801/2014.Respiratory samples from patients with influenza-like illness were analyzed in the Center of Excellence in Clinical Virology at King Chulalongkorn Memorial Hospital as part of the routine influenza surveillance program. This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine at Chulalongkorn University (IRB No. 377/57). The IRB waived the need for consent because the samples were de-identified and anonymous.5’-AGCAGAAGCA-3’) [5’-AGCAAAAGCAGG-3’) [2, 0.5 μM each of forward and reverse primers, 2 μl of cDNA template, and nuclease-free water to a final volume of 25 μl. Amplification in a thermal cycler was performed under the following conditions: initial denaturation for 3 minutes at 94°C, followed by 40 cycles of 30 seconds at 94°C for denaturation, primer annealing for 30 seconds at 55°C, 90 seconds at 72°C for extension, and 7 minutes of final extension at 72°C. Amplicons were agarose gel-purified using Expin Combo GP kit  and the HA gene sequenced. A(H3N2) nucleotide sequences were assembled using the SeqMan Pro software  and deposited in the GenBank database under the accession numbers (MF673231-MF673292)  according to the manufacturer’s instructions. We used a previously described real-time reverse-transcription polymerase chain reaction (RT-PCR) to identify influenza virus A(H1N1pdm09), A(H3N2), and influenza B virus . InfluenAGCA-3’) ,22. AmonCAGG-3’)  and the CAGG-3’) . BrieflyF673292) .http://www.ncbi.nlm.nih.gov) and GISAID (http://platform.gisaid.org) databases, were aligned using ClustalW and translated into amino acid residues using BioEdit Software version 7.0.9.1  observed on all HA sequences were analyzed using the single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), and mixed effects model of evolution (MEME) algorithms implemented in the HYPHY software [P-value of 0.1. Amino acid residue numbering was based on the HA1 of the A(H3N2) vaccine strain A/Hong Kong/4801/2014 unless otherwise indicated. Residues different from those of the A/Hong Kong/4801/2014 were placed on a three-dimensional HA protein structure  using VMD 1.9.2 [A total of 91 A(H3N2) HA nucleotide sequences, 62 obtained from this study and an additional 29 sequences identified in Thailand publicly available from the NCBI  vaccine and circulating strains. Antigenic distance defined by the Pepitope was calculated from the fraction of substituted amino acid residues in the dominant HA epitope [Pepitope is given by VE = -2.47 × Pepitope + 0.47 in which VE is 47% when Pepitope = 0.The predicted vaccine effectiveness (VE) was estimated using the  epitope . For A. The one-way ANOVA test was used to analyze VE divergence, and In all, 15.1% of the samples  tested positive for influenza viruses, of which 78.6%  were influenza A virus and 21.4%  were influenza B virus . As expeNinety-one complete coding sequences of the HA gene showed between 98.5–99.6% nucleotide sequence identity (98.1–99.8% amino acid sequence identity) to the A/Hong Kong/4801/2014 vaccine strain for A(H3N2). Among these, the vast majority significantly diverged from the A/Hong Kong/4801/2014 . Most beComparison of the deduced amino acid sequences to that of A/Hong Kong/4801/2014 showed that group I differed from the vaccine strain by residues N171K and I77V+G155E(HA2), and group II by N121K+N171K/R and I77V+G155E(HA2). Some strains in group II also possessed additional residue variants K92R+H311Q (9/20), or G150E (HA2) (6/20) . Group IA(H3N2) strains circulating in Thailand collectively showed a total of 39 residue substitutions in the antigenic epitopes. Some of the strains possessed as many as seven amino acid changes . Among 5N/dS ratio showed an average of 0.305  among the A(H3N2) strains in this study, suggesting no positively selected site. Meanwhile, the FEL method revealed that residues 142, 171, 261 and 406 (HA numbering) were under positive selection pressure. Moreover, the MEME method indicated five positively selected sites . Therefore, these data provided strong evidence of positively selected sites within epitope A , D (171) and E (261) (The receptor-binding sites of A(H3N2) are highly conserved at positions 98, 136, 153, 183, 190, 194, 195 and 228 on HA1 [ E (261) .Pepitope value was 0.049 (n = 53) for all of the A(H3N2) strains identified in 2016, suggesting a predicted VE against the virus of 74.17%. Meanwhile, the average Pepitope was 0.099 (n = 38) for strains identified in 2017, suggesting a predicted VE against the virus of 47.87%  in Thailand beginning in 2016 by determining clade designation and identifying mutations in the antigenic sites impacting the predicted VE. We categorized the strains identified in this study and those reported elsewhere between 2016 and 2017 using the HA sequences. Due to evolving genetic variations of A(H3N2) and the lagging nomenclature standard, we attempted to reconcile the most recent 3C.2a strains identified to date. Group I and II strains in this study collectively represented clade 3C.2a1 designated by the WHO . Group INew antigenic variants emerge when at least one substitution occurred in the antigenic sites ,34. SeveSoutheast Asian countries use the influenza vaccine formulated for the southern hemisphere, which for some years have different inclusion strains in the vaccine than those formulated for the northern hemisphere. A/Hong Kong/4801/2014 belonging to clade 3C.2a is a component in both northern and southern hemispheres since 2016. This vaccine strain was well-matched for the circulating A(H3N2) in Thailand that first season (predicted VE of approximately 80%) . Since tThis study had several limitations. We were unable to ascertain the vaccination status of the individuals in which the samples were derived, which would have improved the evaluation of the predicted VE. The scope of this study did not allow us to determine additional evolutionary relationships among the strains identified in different countries, which would have required additional nucleotide sequences from around the world not yet deposited in the databases and a longer study time frame. The antigenic drift and predicted VE were estimated from the accumulated mutations on the antigenic epitopes and would benefit from additional antigenic characterization such as hemagglutination inhibition assay. Finally, immunity against the neuraminidase contributing to the antigenic drift was not assessed in this study. Nevertheless, any genetic surveillance of influenza viruses will continue to be an important component in influenza prevention and vaccine improvement.S1 FigDominant epitopes (A through E) for each sequence are denoted in different colors .(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The present study aimed to investigate the expression of STIM1 and its downstream molecules Orai1/TRPC1 in the context of myocardial ischemia/reperfusion injury (MIRI) and the effect of STIM1 inhibition on Ca2+ accumulation and apoptosis in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Objective: Previous studies have demonstrated Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ concentration was detected by laser scanning confocal microscopy using Fluo-3/AM probe. Furthermore, the opening of mitochondrial permeability transition pore (mPTP) was assessed by coloading with calcein AM and CoCl2, while ROS generation was evaluated using the dye DCFH-DA in H9C2 cardiomyocytes.Methods: Expression of STIM1/Orai1/TRPC1 was determined by RT-PCR and Western blot in mice subjected to MIRI and H9C2 cardiomyocytes subjected to H/R. To knock-down STIM1, H9C2 cardiomyocytes was transfected with Stealth SiRNA. Apoptosis was analyzed by both flow cytometry and TUNEL assay. Cell viability was measured by MTT assay. Intracellular Cain vivo and H/R in vitro. In H9C2 cardiomyocytes subjected to H/R, intracellular Ca2+ accumulation significantly increased compared with control group, along with enhanced mPTP opening and elevated ROS generation. However, suppression of STIM1 by SiRNA significantly decreased apoptosis and intracellular Ca2+ accumulation induced by H/R in H9C2 cardiomyocytes, accompanied by attenuated mPTP opening and decreased ROS generation. In addition, suppression of STIM1 increased the Bcl-2/Bax ratio, decreased Orai1/TRPC1, and cleaved caspase-3 expression.Results: Expression of STIM1/Orai1/TRPC1 significantly increased in transcript and translation level after MIRI Conclusion: Suppression of STIM1 reduced intracellular calcium level and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cardiomyocytes. Our findings provide a new perspective in understanding STIM1-mediated calcium overload in the setting of MIRI. Actually, abundant evidence suggested that intracellular Ca2+ overload might participate in the pathogenesis and progress of the I/R injury i could reduce or delayed myocardial death. The molecular mechanisms underlying [Ca2+]i accumulation has not been fully elucidated. Both the Na+–H+ exchanger (NHE) and the voltage gated L-type calcium channel have been demonstrated to be the primary mechanisms leading to lethal Ca2+ overload during the reperfusion stage. Previous study revealed that activation of STIM1 in cardiomyocytes led to enhanced Ca2+ entry i accumulation in H9C2 cardiomyocytes induced by H/R injury was significantly attenuated after suppression of STIM1. These results suggest that Ca2+ accumulation mediated by STIM1 may contribute to Ca2+ overload following H/R injury.Abundant evidence from previous studies have demonstrated that a rise in intracellular Ca2+ entry . This inertrophy ,24. In a+ influx ,21. In a2+ influx has been shown to increase mitochondrial ROS production in many different cells [Increase in ROS is also a key mediator of myocardial I/R injury. Indeed, STIM1-mediated Cant cells –27. Simi2+ accumulation may involve in ROS production and then induce the opening of mPTP, finally lead to cell death and apoptosis in H9C2 cardiomyocytes subjected to H/R injury. Our findings provide a novel cellular mechanism involved in the pathophysiology of myocardial I/R injury.In summary, our results show that both STIM1 and its target molecules Orai1/TRPC1 were up-regulated in the context of myocardial I/R injury. Furthermore, STIM1-induced intracellular Cain vitro H/R model. However, the extent to which H9C2 cells can accurately mimic the H/R responses of primary cardiac myocytes has not yet been fully established. In addition, the present study on H9c2 cells was designed to uncover the potential signaling mechanisms impacting on Ca2+ accumulation after I/R. Due to the complexity of the in vivo environment, especially after I/R, results from H/R study are not sufficient to explain the exact function of STIM1/Orai1/TRPC1 in Ca2+ overload post I/R. But this study suggested at least a feasibility of carrying such study on mammalian myocardial cells, and provided a theoretical and experimental basis to put these findings into further understanding of the mechanisms underlying the accumulation of calcium via STIM1/Orai1 signaling pathway in the myocardial systems under both physiological and pathological conditions.The present study has some limitations. First, in the present study, we used the H9C2 cardiomyocytes to establish the


Toll-like receptor (TLR) agonists are frequently studied for their role as vaccine adjuvants, largely because of their ability to enhance initiation of immune responses to antigens by activating dendritic cells. However, TLRs are also expressed on B cells and may contribute to effective B cell activation and promote differentiation into antigen-specific antibody producing plasma cells in vivo. We sought to discover an adjuvant system that could be used to augment antibody responses to influenza and anthrax vaccines. We first characterized an adjuvant system in vitro which consisted of two TLR ligands, poly I:C (TLR3) and Pam3CSK4 (TLR2), by evaluating its effects on B cell activation. Each agonist enhanced B cell activation through increased expression of surface receptors, cytokine secretion and proliferation. However, when B cells were stimulated with poly I:C and Pam3CSK4 in combination, further enhancement to cell activation was observed. Using B cells isolated from knockout mice we confirmed that poly I:C and Pam3CSK4 were signaling through TLR3 and TLR2, respectively. B cells activated with Poly I:C and Pam3CSK4 displayed enhanced capacity to stimulate allogeneic CD4+ T cell activation and differentiate into antibody-producing plasma cells in vitro. Mice vaccinated with influenza or anthrax antigens formulated with poly I:C and Pam3CSK4 in DepoVax™ vaccine platform developed a rapid and strong antigen-specific serum antibody titer that persisted for at least 12 weeks after a single immunization. These results demonstrate that combinations of TLR adjuvants promote more effective B cell activation in vitro and can be used to augment antibody responses to vaccines in vivo.Vaccines that can rapidly induce strong and robust antibody-mediated immunity could improve protection from certain infectious diseases for which current vaccine formulations are inefficient. For indications such as anthrax and influenza, antibody production  Pre-existing antibodies offer the best protection against infection, and many vaccines are available that can effectively mitigate risk of serious infection through prophylactic immunizations. However, for some indications current vaccine formulations do not provide adequate protection. Hence, there is an urgent need to develop novel vaccine technologies to meet these needs. Influenza and anthrax are two examples of indications for which improved and/ or new vaccines are required. Although these indications are quite different in terms of pathogenesis and antigen type, antibody production is the correlate of a protective immune response for both. Therefore, an optimal vaccine for these infectious diseases would induce a rapid and long lasting antibody mediated response with minimal immunizations , 2.™ (DPX) is a unique water-free formulation that generates strong, long lasting immune responses. DPX is a lipid-in-oil formulation that can incorporate a variety of different peptide or protein antigens. Upon injection, DPX creates a depot that facilitates active uptake by antigen-presenting cells -TdR; MP Biomedical, Irvine, CA) for the last 18 hours of culture. Cells were harvested onto fiberglass filter mats  with a Titertek Cell Harvester (Skatron Instruments). [3H]-TdR uptake was assessed using a Beckman LS6000IC liquid scintillation counter  and quantified as counts per minute (CPM). The values of each experiment were taken from the average of triplicate wells.To measure proliferation, B cells were incubated for 3 days and pulsed with 0.5 μCi of tritiated thymidine (-TdR uptake (n = 2–7). Data shown as average ± SEM of 2–7 individual B cell preparations pooled from at least 2 separated experiments. Statics by 1-way ANOVA with Dunnett’s post-test comparing each dose to unstimulated, *p<0.05, **p<0.01, ***p<0.001.B cells were isolated from the spleens of naïve C57BL/6 mice (n = 3) and stimulated with various concentrations of poly I:C and Pam3SK4. Expression of CD40 (A), CD80 (B) and MHC class II (C) was determined by flow cytometry after 24 hour stimulation. Dashed line indicates level of unstimulated B cells. Data are shown as average ± SEM of 3 individual B cell preparations as indicated and was collected in a single experiment(D) Proliferation of B cells was measured after 3 days incubation by [(TIF)Click here for additional data file.S4 FigB cells were isolated from the spleens of naïve C57BL/6 mice (n = 3) and stimulated with various concentrations of poly I:C and Pam3SK4 alone and in combination for 24 hours. Expression of CD80 (A), CD40 (B), MHC class II (C) was detected by flow cytometry. Secretion of IL-6 (D) was detected by ELISA, BLD: below limit of detection. Results are shown as the average of 3 individual B cell preparations and was collected in a single experiment, the selected combination of poly I:C (25 μg/mL) and Pam3CSK4 (1 μg/mL) is bolded.(PDF)Click here for additional data file.S5 Fig+ B cells were stimulated with poly I:C (25 ug/mL), Pam3CSK4 (1 ug/mL) or the combination of both adjuvants for 24 hours. B cells were then analysed by flow cytometry for expression of CD86, CD80, CD25, MHC class II (IA/IE), CD69 and CD40. Results from multiple experiments are summarized in Purified C57BL/6 CD19(PDF)Click here for additional data file.S6 Fig+ B cells were purified from TLR2-/- (n = 4) or C57BL/6 wild type (n = 4) mice and stimulated with poly I:C (25 ug/mL), Pam3CSK4 (1 ug/mL) or the combination of both adjuvants for 24 hours in (A) T-cell-independent and (B) T-cell-dependent conditions. B cells were analysed by flow cytometry for expression of CD40, CD86, MHC class II, CD25 and CD80. (C) Supernatants were analysed by ELISA for CXCL10.CD19(TIF)Click here for additional data file.S7 Fig+ B cells were purified from TLR3-/- (n = 5) or B6;129SF2/J wild type (n = 4) mice and stimulated with poly I:C (25 ug/mL), Pam3CSK4 (1 ug/mL) or the combination of both adjuvants for 24 hours in (A) T-cell-independent and (B) T-cell-dependent conditions. B cells were analysed by flow cytometry for expression of CD40, CD86, MHC class II, CD25 and CD80. (C) Supernatants were analysed by ELISA for IL-6. (D) Supernatants were analysed by ELISA for CXCL10.CD19(TIF)Click here for additional data file.S8 FigCD-1 mice were vaccinated with rPA antigen (2 ug) formulated with (A) poly I:C or (B) Pam3CSK4, at indicated doses, in DPX. Antigen-specific antibodies were detected in serum at 4 and 8 weeks post immunization.(TIF)Click here for additional data file.


This study suggests that HA A150V, PA A343T, and PB2 E627K are crucial in the adaption and increased pathogenicity of H5N6 in mammalian hosts.H5N6 avian influenza viruses (AIVs) can cause severe pneumonia and death in humans. However, the molecular determinants of H5N6 influenza virus mammalian adaption are still unclear. Three amino acid substitutions  are observed in H5N6 virus A/duck/Zhejiang/6D2/2013 (6D2) in lung-to-lung passage in mice. These substitutions are crucial to the pathogenicity of mouse-adapted virus. In this study, we investigated the contribution of each amino acid substitution in the virus by reverse genetics. The results demonstrate that HA A150V greatly altered the receptor binding preference of 6D2. Virus bearing this substitution acquired increased mortality than mice infected with wild-type 6D2. The PA A343T substitution mildly enhanced viral polymerase activity but the reduced survival rate in mice indicates this substitution may change the immunoreaction of the host. The well-known PB2 E627K substitution increased eight folds the relative polymerase activity compared to PA A343T and resulted in 100% death rate in mice. In addition, we show that PA A343T dramatically exacerbates the effect of PB2 E627K on viral polymerase activity; when combined, these two substitutions work synergistically. However, HA A150V and PA A343T seemed to attenuate PB2 E627K  H5 subtype avian influenza viruses (AIVs) can transmit directly to humans from wild or domestic birds and causes high mortality, raising concerns of potential pandemic threats. Since 2014, sporadic human cases of H5N6 have been documented. As of September 2017, WHO reported 16 laboratory-confirmed cases of human infection with H5N6 AIVs in China; 11 of the 16 cases were fatal, bringing the case-fatality rate to 69% . Recent Avian influenza viruses frequently infect across species and cause pandemics in mammals . There aHemagglutinins in the influenza virus binds receptors on host cells, closely related to host range  and are The transcriptional activity of avian influenza viral polymerase complex in human cells is restricted , consequtrans-species adaptation, including transmission and replication. However, the molecular mechanism underlying the highly virulent H5N6 in humans is still unclear. Recently, we reported that a mouse-adapted (MA) H5N6 influenza virus A/duck/Zhejiang/6D2/2013 (6D2) carried several amino acid substitutions, including HA A150V, PA A343T, and PB2 E627K in the process of mouse lung-to-lung passage . All experiments using H5N6 AIVs were performed in a biosafety level 3 laboratory.Human embryonic kidney (293T) cells, Madin-Darby canine kidney (MDCK) cells, and adenocarcinomic human alveolar basal epithelial (A549) cells were propagated and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , non-essential amino acids , and antibiotics . Cells were used for virus propagation, plasmid transfection, and tissue culture infectious doses (TCID50) determination.TM RT reagent Kit , then amplified by PCR using 6D2 gene specific primers and PrimeSTAR Max DNA Polymerase . This was then cloned into pHW2000 plasmid  to determine receptor binding specificity using a previously described method . BrieflyThe minigenome assay was performed using a system described previously , with mi5 cells per well) were infected with different viruses at a multiplicity of infection (MOI) of 0.01 and incubated at 37°C for 1 h. Cells were then washed with PBS and further incubated with culture medium . Supernatants were collected at 24, 48, and 72 h and subjected to virus tittering by TCID50. Virus titer was determined by end point titration in MDCK in 96-well plates  were infected with 106 TCID50 virus in 50 μL PBS and observed for weight loss and mortality for 14 days post infection. Lungs from infected mice were fixed in 10% neutral buffered formalin for 3 days, embedded in paraffin, cut to 4 μm-sections, then subjected to hematoxylin–eosin staining (H&E staining).us study , with a P < 0.05 is considered as statically significant.A one-way ANOVA was used to determine statistical significant between experimental groups. Figure 1).Three amino acid substitutions  discovered in a previous study were examined . A reverFigures 2A,B). The r6D2-MA (HA/PA/PB2) and r6D2-MA (PB2) group suffered the most drastic weight loss . The weight of the r6D2-MA (HA) group declined sharply (average weight loss was 12.7% at day 5), indicating that this substitution plays a role in the increase of virulence. All the mice in group r6D2-MA (PB2), r6D2-MA (PA/PB2), and r6D2-MA (HA/PA/PB2) died prior to day 9 post infection. The 100% death rate demonstrates the pivotal role of PB2 E627K in H5N6. HA A150V alone caused a death rate of 60% . PA A343T alone decreased the survival rate of mice compared with wild-type 6D2 . However, HA A150V or PA A343T seems to attenuate PB2 E627K in vivo .Six groups of BALB/c mice were infected and the weight change and survival rate of mice was recorded (P > 0.05), implying that the high death rate caused by these two substitutions may not due to high virus titer. All virus containing PB2 E627K grew faster, yielding more than 1,000 folds virus titer than the wild-type virus (Table 1).At day 3 post infection, the average lung virus titer of r6D2-MA (HA) and r6D2-MA (PA) is lower than r6D2-WT . r6D2-MA (HA) and r6D2-MA (PA)-inoculated mice showed thickening of the alveolar wall . The mice infected with recombinant r6D2-MA (PB2) and r6D2-MA (PA/PB2) displayed severe inflammatory cell infiltration, hemorrhage, and bronchopneumonitis . The pathological changes of r6D2-MA (HA/PA/PB2) infected mice are the most severe .The H&E staining of mouse lungs infected with r6D2-WT is morphologically normal . In MDCK cells, the titer of virus containing PA A343T is ten times the wild-type virus (P > 0.05), but not in A549. This suggests that PA A343T alone is not sufficient to induce strong in vitro replication. The virus titer of r6D2-MA (PB2) and r6D2-MA (HA/PA/PB2) grew similarly  in A549 at 48 h post infection, 50 times higher than r6D2-WT (P < 0.01), indicating that PB2 E627K increased replication efficiency markedly. r6D2-MA (HA) grew more efficiently than r6D2-WT in A549 cells at 48 h post infection , demonstrating that HA A150V is responsible for the enhanced growth in human cells.To explore the contribution of mouse adapted substitutions to growth ability in mammalian cells, we used six reassortant viruses to infect A549 and MDCK cells  using the Swiss-model . To test whether the substitution found in mouse adaption is also relevant in human strains, we examined all the sequences of H5 influenza A virus in the Influenza Virus Database of NCBI (Table 3).The hemagglutination assay suggests that HA A150V completely abolishes the binding of HA with SAα2,3 receptors, but enhanced binding with SAα2,6 receptors . 293T cells were transfected with reconstructed plasmid expressing wild-type and mutated NP, PA, PB1, and PB2. The polymerase activity of the RNP containing PA 343T is 22 folds higher than wild-type (P > 0.05). Therefore, PA A343T alone is not sufficient to enhance replication ability substantially. Notably, the polymerase activity of the RNP containing PB2 627K alone or combined PA 343T and PB2 627K are 182 and 273 folds higher than wild-type (P < 0.001), respectively. Combining the two substitutions has a synergistic effect on polymerase activity.The PA and PB2 proteins are components of viral RNA polymerase. We hypothesized that PA A343T and PB2 E627K affect the polymerase activity of reconstituted ribonucleoprotein (RNP) complexes  indicates that HA 150 localizes near the 130 loop of the receptor binding site. We speculate this substitution is related with enhancement of receptor binding affinity. Crystallographic studies show that when compared with 150A (position 134 in their study), 150V increased the distance of 130 loop and 220 loop in the receptor-binding site , our results suggest that PA A343T mildly increases polymerase activity when compared to the wild-type influenza polymerase . This is not sufficient to explain the increased pathogenicity in vivo . These results imply that pathogenicity is not only determined by virus growth but also by the immunoreaction to infection, including cytokine storm . RNA-dependent RNA polymerase in influenza lacks proof-reading mechanism, in natural conditions, the form of existence can be a cloud of diverse variants called quasispecies in vivo (in vivo. Mixed viral populations grow better than single population (All viruses containing single substitution has a lower mice survival rate than r6D2-WT, however, the results of cell infection and  in vivo . The sub in vivo , therefopulation , homogenpulation .This study revealed the significance of HA A150V, PA A343T, and PB2 E627K in the mammalian adaption process of an H5N6 avian influenza virus. The adaption of 6D2 consists of a change in receptor affinity from avian to human type, and the enhancement of polymerase activity. Our findings elucidate the mechanism of these substitutions in mammalian adaption and provides information on pathogenicity markers in the surveillance of avian influenza.XmP and FL performed the research, analyzed the data, and drafted the paper. HW and NW designed the research and revised the manuscript. XrP, YX, LW, BC, TS, FY, and SJ performed the research. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Unlike canonical pre-mRNAs, animal replication-dependent histone pre-mRNAs lack introns and are processed at the 3’-end by a mechanism distinct from cleavage and polyadenylation. They have a 3’ stem loop and histone downstream element (HDE) that are recognized by stem-loop binding protein (SLBP) and U7 snRNP, respectively. The N-terminal domain (NTD) of Lsm11, a component of U7 snRNP, interacts with FLASH NTD and these two proteins recruit the histone cleavage complex containing the CPSF-73 endonuclease for the cleavage reaction. Here, we determined crystal structures of FLASH NTD and found that it forms a coiled-coil dimer. Using solution light scattering, we characterized the stoichiometry of the FLASH NTD-Lsm11 NTD complex and found that it is a 2:1 heterotrimer, which is supported by observations from analytical ultracentrifugation and crosslinking. In eukaryotic cells, histones play important roles in genomic DNA packaging as well as epigenetic regulation of gene expression. The levels of histone mRNAs are carefully controlled throughout the cell cycle and they dramatically increase during S phase to meet the growing demand for packaging the newly replicated DNA from the replicating genome . In metaBiochemical studies of the 3’-end processing machinery that cleaves replication-dependent histone pre-mRNAs have shown that it is comprised of the stem-loop binding protein (SLBP), U7 small nuclear ribonucleoprotein (U7 snRNP), FLASH, and the histone pre-mRNA cleavage complex (HCC) , 3–6. SLLsm11 has an extended N-terminal domain  that is Biochemical studies revealed that the interacting N-terminal regions of Lsm11 and FLASH form a docking platform that recruits the HCC to the U7 snRNP . The HCCThe molecular details of how FLASH acts as a mediator between Lsm11 and HCC are still unclear. To shed some light on the essential role of FLASH in 3’-end processing of replication-dependent histone pre-mRNA processing [Drosophila to mammals for residues 55–137 in the N-terminal segment.We determined a structure of the wild-type human FLASH NTD at 2.6 Å resolution using X-ray crystallography . The ini2 , and one of them appears to adopt a straighter conformation . For eacram PISA ). The maWe also observed that Cys83 is situated in the dimer interface with the thiol side chains from the two protomers positioned near one another . While tTo rule out the possibility that the observed FLASH NTD dimer is a crystallographic artifact caused by oxidized cysteine residues, we determined the structures of the FLASH NTD C54S/C83A double mutant in two different crystal forms at 2.1 and 2.6 Å resolution, respectively . In addiInterestingly, residues 53–70 from one of the protomers in the structure at 2.6 Å resolution are stabilized by crystal packing, showing strong electron density corresponding to an α-helix . This prOverall, our structural data suggest that FLASH NTD alone forms a stable coiled-coil dimer from residues 71–137 while residues 53–70 can form another helix. It appears that the helix for residues 53–70 does not dimerize, and it may also be structurally independent of residues 71–137, even though a single long helix (residues 53–137) is observed in one crystal form. The LDLY motif (residues 55–58) essential for binding the HCC is situated in the N-terminal helix . WhetherThe region of FLASH NTD that interacts with Lsm11 has been previously mapped to residues 100–137 . BecauseWe next investigated the oligomeric states of the mutants Y73A/L76A/Y80A and N101A/L104A/N108A, each containing substitutions of three consecutive residues in the dimer interface . The firE. coli and monitored whether FLASH NTD could be co-purified by the nickel-NTA agarose beads. The results showed that the Y73A/L76A/Y80A and K88A/K92A/K95A mutants still interacted with Lsm11 NTD, while the L118A/I119A mutant could no longer interact with Lsm11 NTD  in proteins . Extensiproteins , S3 Fig.In the low-salt buffer, the positions of the peaks for both the FLASH NTD-Lsm11 NTD complex and FLASH NTD C54S/C83A mutant were slightly shifted to the left, suggesting a more extended structure for both. A small amount of higher order structures was present for the FLASH NTD-Lsm11 NTD complex suggesting the formation of aggregates in low-salt buffer condition. As in the high-salt buffer, the FLASH NTD-Lsm11 NTD complex peak had a trailing edge. The weight-averaged molar masses of the samples eluting in the peaks are 34.3 kDa for FLASH NTD-Lsm11 NTD complex (with a Stokes radius of 4.3 nm) and 21.8 kDa for FLASH NTD C54S/C83A (with a Stokes radius of 3.5 nm). Due to the presence of higher order structures, the molar mass of the FLASH NTD-Lsm11 NTD complex decreased gradually from 55 kDa and higher (leading edge of peak) to 26 kDa (trailing edge of peak). The FLASH NTD C54S/C83A molar mass decreased from 23.5 kDa (leading edge) to 19 kDa (trailing edge). Overall, the polydispersity of the FLASH NTD-Lsm11 NTD complex was slightly higher in low-salt buffer.Based on the calculated molecular weights for Lsm11 NTD and FLASH NTD, the results from SEC-MALS showed that the FLASH NTD-Lsm11 NTD complex is a heterotrimer consisting of 2 molecules (a dimer) of FLASH and 1 molecule of Lsm11, while FLASH NTD C54S/C83A is a dimer .Kd values for the wild-type and C54S/C83A NTD dimers are 0.18 and 0.05 μM, respectively  sedimentation velocity and sedimentation equilibrium experiments on the FLASH NTD-Lsm11 NTD complex, as well as on wild-type FLASH NTD, FLASH NTD C54S/C83A double mutant, and Lsm11 NTD alone as controls in the low-salt condition , S4 Fig.ectively . Wild-tyKd value of 2.4 μM for the complex , this region underwent slower H/D exchange in the presence of Lsm11 and the reduced rate of exchange extended to amino acid 130 in FLASH . Since HDrosophila HLB, but abolishes processing in vivo [Additional studies are required to determine the structure of the FLASH-Lsm11 heterotrimer and identify potential mechanisms that may regulate the binding of a FLASH dimer to Lsm11 to form the FLASH-Lsm11 heterotrimer. In animal cells, components of the transcription and 3’-end processing machinery are localized in Histone Locus Bodies (HLBs), nuclear domains that assemble at histone gene loci and are present throughout the cell cycle. Strikingly, histone gene expression is repressed during G1 phase and becomes activated only with the onset of S phase and DNA replication in response to cell cycle signals, including cyclin E/CDK2-mediated phosphorylation of NPAT, a universal coactivator of histone gene expression –39. A gr in vivo , 41.Our findings suggest that N-terminus of FLASH may be present as a homodimer throughout the cell cycle. In recent studies , we haveEscherichia coli BL21 Star (DE3) strains (Novagen). The cells were induced using 0.4 mM isopropyl β-D-1-thiogalactopyranoside and grown for 18 h at 20°C. The cells were harvested by centrifugation and the pellets were re-suspended in lysis buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 17.8 μg/mL phenylmethane sulfonyl fluoride (PMSF) and 10 mM β-mercaptoethanol) and lysed by sonication. Cell lysates were then centrifuged at 25,000 x g for 40 min at 4°C. The supernatant was incubated with nickel beads for 1 h before being loaded onto a gravity flow column (Bio-Rad). The nickel beads were washed with buffer containing 20 mM Tris (pH 7.5), 500 mM NaCl, 40 mM imidazole, and 10 mM β-mercaptoethanol. The proteins were eluted with 20 mM Tris (pH 7.5), 500 mM NaCl, 500 mM imidazole and 10 mM β-mercaptoethanol. The eluted proteins were further purified using size-exclusion chromatography  with a buffer containing 20 mM Tris (pH 8.5), 250 mM NaCl, and 5 mM dithiothreitol (DTT). Relevant fractions from size-exclusion chromatography were pooled and the proteins were concentrated to 9.4 mg/mL (wild-type) and 11 mg/mL (mutant), and stored at -80°C.C-terminally hexahistidine-tagged FLASH NTD (residues 51–137) and FLASH NTD C54S/C93A mutant constructs were cloned into pET26b vector and over-expressed in Escherichia coli B834 methionine-auxotroph strain grown in the LeMaster media supplemented with selenomethionine [The selenomethionyl FLASH NTD protein was prepared using the thionine . The proSelenomethionyl FLASH NTD and native FLASH NTD C54S/C83A mutant were crystallized in a sitting drop by vapor diffusion. The sitting drops were set up by mixing 1 μL of 4 mg/mL selenomethionyl FLASH NTD or 5 mg/mL FLASH NTD C54S/C83A mutant protein with 1 μL of well solution. The well solution for selenomethionyl FLASH NTD crystals contained 100 mM Tris (pH 8.0) and 18% (w/v) PEG 4000; for FLASH NTD C54S/C83A crystal form 1, 4% (w/v) tacsimate pH 7.0, 11% (w/v) PEG 3350; and for FLASH NTD C54S/C83A crystal form 2, 100 mM sodium formate, 15% (w/v) PEG 3350, 3% (v/v) 1,6-hexanediol. The crystals were harvested and soaked in mother liquor supplemented with 15% (v/v) (selenomethionyl FLASH NTD) or 20% (v/v) (FLASH NTD C54S/C83A) ethylene glycol as cryoprotectant before being flash frozen in liquid nitrogen.Initial X-ray diffraction data for selenomethionyl FLASH NTD were collected at APS beamline 24 ID-C with wavelength 0.9792 Å. Three datasets from three different crystals were processed using XDS and merged with XSCALE , 44. TheE. coli BL21 (DE3) Star and the genes were co-expressed. The proteins were purified using the same protocol as for the FLASH NTD proteins with the exception of using 20 mM Tris (pH 7.5), 500 mM NaCl, and 5 mM DTT as the size exclusion chromatography buffer. Relevant fractions corresponding to Lsm11 NTD-FLASH NTD C54S/C83A complex and excess Lsm11 NTD alone were pooled separately and concentrated to 5.3 mg/mL and 2.4 mg/mL respectively.N-terminally hexahistidine-tagged Lsm11 NTD (residues 23–130) construct was cloned into pET28a vector. FLASH NTD C54S/C83A mutant construct (without tag) was cloned into MCS2 of pCDF Duet vector. Both plasmids were co-transformed into E. coli DH5α. Mutant constructs were confirmed by DNA sequencing.Mutant FLASH NTD constructs were generated using site-directed mutagenesis PCR. Primers see  designedE. coli BL21 Star (DE3) cells. The mutant proteins were purified by nickel affinity column as detailed in the earlier section. The eluted protein from nickel affinity purification was then injected into Superose 12 analytical gel filtration column, pre-equilibrated with 20 mM Tris (pH 7.5), 250 mM NaCl, and 5 mM DTT. Analytical gel filtration was performed with a flow rate of 0.5 mL/min using 20 mM Tris (pH 7.5), 250 mM NaCl and 5 mM DTT as buffer.The C-terminally hexahistidine-tagged FLASH NTD mutant constructs were cloned into pET26b vector and expressed in E. coli BL21 Star (DE3) and the genes were co-expressed in 5 mL LB media. The expressed proteins were co-purified with 15 μL of Ni-NTA agarose beads using the same buffers that were used for large-scale purification of FLASH NTD-Lsm11 NTD complex, and analyzed using SDS-PAGE.N-terminally hexahistidine-tagged Lsm11 NTD (residues 23–130) construct was cloned into pET28a vector. FLASH NTD mutant constructs (without tag) were cloned into MCS2 of pCDFDuet vector. Both plasmids were co-transformed into epon charcoal-filled centerpiece at 20°C with a rotor speed of 42,000 rpm. Protein solutions of 0.05 to 1 mg/ml (330 μl) in a buffer containing 20 mM Tris (pH 8.5), 250 mM NaCl, and 5 mM DTT  and reference (370 μl) solutions were loaded into the centerpiece, respectively. The absorbance at 280 nm was monitored in a continuous model with a time interval of 300 s and a step size of 0.003 cm. Multiple scans at different time intervals were then fitted to a continuous c(s) distribution model using the SEDFIT program [Kd) [The AUC experiments were performed on a XL-A analytical ultracentrifuge (Beckman Coulter) using an An-50 Ti rotor . The sed program . Additioram [Kd) .epon charcoal-filled centerpieces, and 0.11–0.13 ml buffers were loaded into the reference channels. The cells were then loaded into the rotor and run at speed of 10,000, 15,000, and 25,000 rpm each for 12 h at 20°C. Ten A280 nm scans with time interval of 8–10 min were measured for every different rotor speed to check the status of sedimentation equilibrium. Global analyses of combined sedimentation equilibrium and sedimentation velocity data were conducted with SEDPHAT using species analysis model [To determine the precise molecular weight of the protein, the sedimentation equilibrium experiment was performed . Three dis model .FLASH NTD C54S/C83A-Lsm11 NTD complex and FLASH NTD C54S/C83A were loaded sequentially onto a Superdex 200 size exclusion column (24 mL) pre-equilibrated with 20 mM Tris pH 7.5, 500 mM NaCl, 5 mM DTT  or 20 mM Tris pH 7.5, 250 mM NaCl, and 5 mM DTT . The eluted samples first passed through a Wyatt multi-angle light scattering system (DAWN HELEOS-II) and then a Wyatt Trex refractometer. The data were analyzed using ASTRA version 6 software . The monomer peak of 3 mg/ml BSA was used for normalization, delay time determination, and band broadening correction using ASTRA.® 500 centrifugal concentrators with a molecular weight cut off of 10 kDa and finally analyzed by SDS-PAGE.Cross-linking reactions were carried out in 20 mM HEPES (pH 7.5), 500 mM NaCl. A final concentration of 0.1% (w/v) of glutaraldehyde was added to 0.1 mg/mL  and 0.01 mg/mL  of FLASH NTD C54S/C83A-Lsm11 NTD complex, FLASH NTD C54S/C83A, and FLASH NTD wildtype. Controls with protein concentrations of 0.1 mg/mL without glutaraldehyde were set up for each sample type. All samples were incubated at 37°C for 3 min then chilled on ice. A final concentration of about 100 mM of Tris pH 8.0 was added into each sample to quench the cross-linking reaction. All samples were concentrated to a volume ~20 μL using Sartorius VivaspinS1 FigProtomers were superimposed using residues 71–100 from wildtype FLASH NTD protomer 1 as reference coordinates.(TIF)Click here for additional data file.S2 FigA) Sephacryl-300 gel filtration profile shows two peaks: peak 1 corresponds to the Lsm11 NTD/FLASH NTD C54S/C83A complex, peak 2 corresponds to excess Lsm11 NTD. Lsm11 contains the N-terminal hexa-histidine tag. B) SDS-PAGE analysis of nickel affinity eluate (labeled Ni/E), and corresponding fractions from S-300 chromatography. A molecular weight marker is situated on the far-left lane.(TIF)Click here for additional data file.S3 FigLight scattering (solid), refractive index (dotted), and MW information for (A) FLASH NTD in high salt buffer; (B) FLASH NTD in low salt buffer; (C) FLASH NTD-Lsm11 NTD in high salt buffer; and (D) FLAST NTD-Lsm11 NTD in low salt buffer.(TIF)Click here for additional data file.S4 Figr from the best fit results are shown in The speed of centrifugation for sedimentation equilibrium experiment (A) was 10,000 rpm (squares), 15,000 rpm (circles), and 25,000 rpm (triangles) at 20°C each for 14 h. The velocity experiment (B) was 42,000 rpm (circles) at 20°C for 6 h. The solid lines in two panels are the best fit results from global analysis of the two discrete species models by SEDPHAT (57). The residuals of each fit are shown below the panels. The calculated sedimentation coefficients and M(TIF)Click here for additional data file.S5 FigIn the FLASH NTD-Lsm11 NTD complex, FLASH NTD is the lower band , and Lsm11 NTD is the upper band. While the FLASH NTD band disappeared in the presence of glutaraldehyde, the Lsm11 NTD band mostly stayed the same. Therefore, probably very small amount of Lsm11 NTD (if any) got crosslinked in the reaction, consistent with the fact that it has only 1 Lys residue.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.