Influenza virus H1N1pdm09 infections in the young and old: evidence of greater antibody diversity and affinity for the hemagglutinin globular head domain (HA1 Domain) in the elderly than in young adults and children

The H1N1 2009 influenza virus (H1N1pdm09) pandemic had several unexpected features, including low morbidity and mortality in older populations. We performed in-depth evaluation of antibody responses generated following H1N1pdm09 infection of naïve ferrets and of 130 humans ranging from the very young (0 to 9 years old) to the very old (70 to 89 years old). In addition to hemagglutination inhibition (HI) titers, we used H1N1pdm09 whole-genome-fragment phage display libraries (GFPDL) to evaluate the antibody repertoires against internal genes, hemagglutinin (HA), and neuraminidase (NA) and also measured antibody affinity for antigenic domains within HA. GFPDL analyses of H1N1pdm09-infected ferrets demonstrated gradual development of antibody repertoires with a focus on M1 and HA1 by day 21 postinfection. In humans, H1N1pdm09 infection in the elderly (>70 years old) induced antibodies with broader epitope recognition in both the internal genes and the HA1 receptor binding domain (RBD) than for the younger age groups (0 to 69 years). Importantly, post-H1N1 infection serum antibodies from the elderly demonstrated substantially higher avidity for recombinant HA1 (rHA1) (but not HA2) than those from younger subjects (50% versus <22% 7 M urea resistance, respectively) and lower antibody dissociation rates using surface plasmon resonance. This is the first study in humans that provides evidence for a qualitatively superior antibody response in the elderly following H1N1pdm09 infection, indicative of recall of long-term memory B cells or long-lived plasma cells. These findings may help explain the age-related morbidity and mortality pattern observed during the H1N1pdm09 pandemic.     

Cues used in host‐seeking behavior by frog‐biting midges (Corethrella spp. Coquillet)

We investigated the role of carbon dioxide and host temperature in host attraction in frog‐biting midges (Corethrella spp). In these midges, females are known to use frog calls to localize their host, but the role of other host‐emitted cues has yet not been investigated. We hypothesized that carbon dioxide acts as a supplemental cue to frog calls. To test this hypothesis, we determined the responses of the midges to carbon dioxide, frog calls, and both cues. A significantly lower number of midges are attracted to carbon dioxide and silent traps than to traps broadcasting frog calls. Adding carbon dioxide to the calls does not increase the attractiveness to the midges. Instead, carbon dioxide can have deterrent effects on frog‐biting midges. Temperature of calling frogs is not a cue potentially available to the midges. Contrary to our hypothesis, there was no supplemental effect of carbon dioxide when presented in conjunction to calls. Midge host‐seeking behavior strongly depends on the mating calls emitted by their anuran host. Overall, non‐acoustic cues such as host body temperature and carbon dioxide are not important in long‐distance host location by frog‐biting midges.     

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.     

Green synthesis of isoamyl acetate via silica immobilized novel thermophilic lipase from <Emphasis Type="Italic">Bacillus aerius</Emphasis>

Isoamyl acetate, a pear or banana flavor, is widely used in food, beverage, cosmetic, and pharmaceutical industries. In the present work, lipase from Bacillus aerius was immobilized on silica gel matrix in the presence of a cross-linking agent, glutaraldehyde, and its efficiency in synthesizing isoamyl acetate using esterification reaction was studied. The esterification of acetic acid and isoamyl alcohol by silica-bound lipase was studied as a function of time and temperatures. The incubation time of 10 h, temperature of 55°C, substrate molar ratio 1: 1, and the amount of lipase as 1% were found to be optimal for the esterification reaction. The bound lipase catalyzed the esterification of acetic acid by isoamyl alcohol with the yield of about 68% under the optimized reaction conditions. The product was identified as isoamyl acetate using gas-liquid chromatography, nuclear magnetic resonance, and Fourier transform IR spectroscopy analysis by the presence of an ester group at the wavenumber of 1720.5 cm^–1.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

Disruption of Annexin II /p11 Interaction Suppresses Leukemia Cell Binding,Homing and Engraftment,and Sensitizes the Leukemia Cells to Chemotherapy

The bone marrow microenvironment plays an important role in acute lymphoblastic leukemia (ALL) cell proliferation, maintenance, and resistance to chemotherapy. Annexin II (ANX2) is abundantly expressed on bone marrow cells and complexes with p11 to form ANX2/p11-hetero-tetramer (ANX2T). We present evidence that p11 is upregulated in refractory ALL cell lines and patient samples. A small molecule inhibitor that disrupts ANX2/p11 interaction (ANX2T inhibitor), an anti-ANX2 antibody, and knockdown of p11, abrogated ALL cell adhesion to osteoblasts, indicating that ANX2/p11 interaction facilitates binding and retention of ALL cells in the bone marrow. Furthermore, ANX2T inhibitor increased the sensitivity of primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine induced cell death. Finally, in an orthotopic leukemia xenograft mouse model, the number of ALL cells homing to the bone marrow was reduced by 40–50% in mice injected with anti-ANX2 antibody, anti-p11 antibody or ANX2T inhibitor compared to respective controls. In a long-term engraftment assay, the percentage of ALL cells in mouse blood, bone marrow and spleen was reduced in mice treated with agents that disrupt ANX2/p11 interaction. These data show that disruption of ANX2/p11 interaction results in reduced ALL cell adhesion to osteoblasts, increased ALL cell sensitization to chemotherapy, and suppression of ALL cell homing and engraftment.     

DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silico database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and MTUS1 function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2′-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC.     

Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng—Panax quinquefolium L

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47–49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.     

Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n = 5) or 150% (Fat; n = 6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater (P < 0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater (P < 0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected (P = 0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) β tended (P = 0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-β was greater in female fetuses than male fetuses from Control ewes (P = 0.05). Although amount of ER-β mRNA did not differ among male fetuses (P = 0.7), amounts tended to be less (P = 0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ (P ≥ 0.8) from male fetuses. Hypothalamic ER-α mRNA tended (P = 0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced (P = 0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended (P = 0.06) to be greater in male fetuses than female fetuses and tended to be less (P = 0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction (P = 0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ (P ≥ 0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-α, and decrease amount of ER-β in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.     

Cooperativity of Cdk4R24C and Ras in melanoma development

The importance of the Cdk4 protein in human cancer became evident following the identification of a germ line mutation in the Cdk4 locus that predisposes humans to melanoma. This mutation results in substitution of argininefirst with cysteine at position 24 (R24C). In an earlier study, we introduced the R24C mutation into the Cdk4 locus of mice using Cre-loxp-mediated “knock-in” technology and observed a very low incidence of spontaneous melanomas in Cdk4^R24C/R24C mice. This suggested that additional oncogenic mutations might be required for development of melanomas. Here we report an increased incidence of spontaneous cutaneous melanoma in mice expressing the oncogene HRAS(G12V) in melanocytes on a Cdk4^R24C background. Treatment of Tyr-HRas:Cdk4^R24C/R24C mice with the carcinogen, DMBA/TPA resulted in a further increase in the number of nevi and melanomas developed when compared with Tyr-HRas:Cdk4^+/+ mice. In summary, in Tyr-HRas:Cdk4^R24C/R24C mice, we observed that activated Cdk4 cooperates with the oncogenic HRAS(G12V) protein to increase the susceptibility of melanoma development in vivo.Key words: Cdk4^R24C, ras, melanoma, skin, carcinogen