As outlined in other chapters, the influenza virus, existing laboratory diagnostic abilities, and disease epidemiology have several peculiarities that impact on the timing and processes for the annual production of influenza vaccines. The chapter provides an overview on the key biological and other factors that influence vaccine production. They are the reason for an "annual circle race" beginning with global influenza surveillance during the influenza season in a given year to the eventual supply of vaccines 12 months later in time before the next seasonal outbreak and so on. As influenza vaccines are needed for the Northern and Southern Hemisphere outbreaks in fall and spring, respectively, global surveillance and vaccine production has become a year round business. Its highlights are the WHO recommendations on vaccine strains in February and September and the eventual delivery of vaccine doses in time before the coming influenza season. In between continues vaccine strain and epidemiological surveillance, preparation of new high growth reassortments, vaccine seed strain preparation and development of standardizing reagents, vaccine bulk production, fill-finishing and vaccine release, and in some regions, clinical trials for regulatory approval.

We studied how conduction delays of action potentials in an unmyelinated axon depended on the history of activity and how this dependence was changed by the neuromodulator dopamine (DA). The pyloric dilator axons of the stomatogastric nervous system in the lobster, Homarus americanus, exhibited substantial activity-dependent hyperpolarization and changes in spike shape during repetitive activation. The conduction delays varied by several milliseconds per centimeter, and, during activation with realistic burst patterns or Poisson-like patterns, changes in delay occurred over multiple timescales. The mean delay increased, whereas the resting membrane potential hyperpolarized with a time constant of several minutes. Concomitantly with the mean delay, the variability of delay also increased. The variability of delay was not a linear or monotonic function of instantaneous spike frequency or spike shape parameters, and the relationship between these parameters changed with the increase in mean delay. Hyperpolarization was counteracted by a hyperpolarization-activated inward current (I(h)), and the magnitude of I(h) critically determined the temporal fidelity of spike propagation. Pharmacological block of I(h) increased the change in delay and the variability of delay, and increasing I(h) by application of DA diminished both. Consequently, the temporal fidelity of pattern propagation was substantially improved in DA. Standard measurements of changes in excitability or delay with paired stimuli or tonic stimulation failed to capture the dynamics of spike conduction. These results indicate that spike conduction can be extremely sensitive to the history of axonal activity and to the presence of neuromodulators, with potentially important consequences for temporal coding.

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12)(dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(•)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.

Abstract Introduction: Acne has a significant negative impact on Quality of Life (QoL): lack of self-confidence, depressive symptoms and suicidal thoughts. The objective was to assess the impact of an initial and continued therapy in severe acne patients through patient-related outcomes (PRO). Methods: In two sequential double-blind randomized studies patients received either adapalene/BPO or vehicle, associated with doxycycline 100 mg for 12 weeks. Patients having obtained at least a good improvement according to investigator global assessment were re-randomized for a 24-week therapy with adapalene/BPO or vehicle. PROs were assessed using the Acne-QoL and a patient treatmentsatisfaction questionnaire. Results: QoL was improved at week 12 in all domains with a significant difference for the Acne-symptoms domain (p<0.001) in favor of the adapalene/BPO regimen. Additional 24-week adapalene/BPO treatment showed a sustained improvement, significant (p<0.001) for all domains except for Acne-symptoms. In the vehicle arm, QoL significantly worsened for all domains (p<0.03). At weeks 12 and 36, a significantly higher proportion of patients receiving adapalene/BPO vs. vehicle reported high satisfaction to 5 out of 6 treatment satisfaction items. Conclusions: The early and sustained improvement of these PROs is correlated to the fast onset of action of adapalene/BPO, the treatment effectiveness and a good safety profile.

A full characterization of the thermodynamic forces underlying ligand-associated conformational changes in proteins is essential for understanding and manipulating diverse biological processes, including transport, signaling, and enzymatic activity. Recent experiments on the maltose binding protein (MBP) have provided valuable data about the different conformational states implicated in the ligand recognition process; however, a complete picture of the accessible pathways and the associated changes in free energy remains elusive. Here we describe results from advanced accelerated molecular dynamics (aMD) simulations, coupled with adaptively biased force (ABF) and thermodynamic integration (TI) free energy methods. The combination of approaches allows us to track the ligand recognition process on the microsecond time scale and provides a detailed characterization of the protein's dynamic and the relative energy of stable states. We find that an induced-fit (IF) mechanism is most likely and that a mechanism involving both a conformational selection (CS) step and an IF step is also possible. The complete recognition process is best viewed as a "Pac Man" type action where the ligand is initially localized to one domain and naturally occurring hinge-bending vibrations in the protein are able to assist the recognition process by increasing the chances of a favorable encounter with side chains on the other domain, leading to a population shift. This interpretation is consistent with experiments and provides new insight into the complex recognition mechanism. The methods employed here are able to describe IF and CS effects and provide formally rigorous means of computing free energy changes. As such, they are superior to conventional MD and flexible docking alone and hold great promise for future development and applications to drug discovery.

Background: FlexTouch® is the only prefilled insulin pen that utilizes an easy touch button that does not extend at any dose in place of a push-button extension. Rigorous testing has shown that the new FlexTouch pen accurately and consistently delivers insulin doses. Methods: This study assessed dose accuracy of FlexTouch, KwikPen®, and SoloSTAR®. Dose accuracy for minimum, medium, and maximum doses of each pen type (1, 40, and 80 U for FlexTouch and SoloSTAR and 1, 30, and 60 U for KwikPen) was assessed. Results: FlexTouch delivered all doses consistently, as demonstrated by low standard deviations. FlexTouch showed similar accuracy to KwikPen at 1 U and to SoloSTAR at 40 and 80 U. However, FlexTouch was significantly more accurate at delivering 1 U of insulin than SoloSTAR (p <.0001). Conclusions: This study demonstrates that FlexTouch, a new prefilled pen, delivers insulin accurately and consistently at low, medium, and high doses. In addition, FlexTouch is currently the only prefilled pen that has a push button that does not extend at any dose, making FlexTouch easier to use than other pens.

Objective: FlexTouch (FT)(Novo Nordisk A/S, Bagsværd, Denmark) is a new prefilled insulin pen with a novel injection mechanism encompassing no push-button extension at any dose-setting. This study assessed the dose accuracy and injection force of FT compared with the established Next Generation FlexPen (NGFP)(Novo Nordisk A/S Bagsværd, Denmark). Research design and methods: Dose accuracy was measured at the minimum, medium and maximum doses (FT, 1, 40 and 80 international units (IU) and NGFP, 1, 30 and 60 IU). Injection force was measured during the injection of the maximum dose (FT, 80 IU; NGFP, 60 IU). Main outcomes: FT and NGFP delivered insulin accurately and consistently at all doses (mean ± s.d., FT at 1 IU, 0.98 ± 0.07; 40 IU, 39.86 ± 0.33; 80 IU, 79.76 ± 0.64; NGFP at 1 IU, 1.02 ± 0.08; 30 IU, 29.69 ± 0.30; 60 IU, 59.50 ± 0.51). FT had a significantly (p < 0.0001) lower injection force than NGFP. Conclusions: The study demonstrated that FT and NGFP deliver insulin accurately and consistently at low, medium and high doses. The novel torque spring injection mechanism of FT results in a significantly lower injection force than NGFP and a pen requiring less thumb-pressure to inject insulin may be welcomed by many people with diabetes.

Abstract Background: FlexTouch(®)(Novo Nordisk A/S, Bagsvaerd, Denmark) is a new prefilled insulin pen for people with diabetes, with a novel injection mechanism and no push-button extension at any dose setting. This study compared the injection force of FlexTouch with that of SoloStar(®)(Sanofi-Aventis, Paris, France) and KwikPen(®)(Eli Lilly & Co., Indianapolis, IN). Methods: Injection force was measured with the manufacturers' recommended needle attached to each pen (NovoFine(®)[Novo Nordisk] 32-gauge tip extra thin wall 6 mm needle for FlexTouch and BD [Franklin Lakes, NJ] MicroFine™ 31-gauge 5 mm needle for SoloStar and KwikPen) during injection of the maximum dose (60 IU for KwikPen and 80 IU for FlexTouch and SoloStar). Injection was performed at three different constant push-button speeds. Results: FlexTouch had a significantly (P<0.0001) lower injection force than SoloStar and KwikPen at all injection speeds. The mean±SD injection force of FlexTouch was 5.1±0.5 N. At 4, 6, and 8 mm/s push-button speeds, the injection force of SoloStar was 13.5±2.1, 19.1±1.9, and 26.9±2.4 N, respectively, and the injection force of KwikPen was 14.5±1.9, 20.9±1.4, and 28.2±1.4 N, respectively. Conclusions: The injection mechanism of FlexTouch means that insulin injection is driven by a torque spring and not the thumb pressure of the user. This results in a 62-82% lower injection force with FlexTouch than other prefilled insulin pens.

Most spiking neurons are divided into functional compartments: a dendritic input region, a soma, a site of action potential initiation, an axon trunk and its collaterals for propagation of action potentials, and distal arborizations and terminals carrying the output synapses. The axon trunk and lower order branches are probably the most neglected and are often assumed to do nothing more than faithfully conducting action potentials. Nevertheless, there are numerous reports of complex membrane properties in non-synaptic axonal regions, owing to the presence of a multitude of different ion channels. Many different types of sodium and potassium channels have been described in axons, as well as calcium transients and hyperpolarization-activated inward currents. The complex time- and voltage-dependence resulting from the properties of ion channels can lead to activity-dependent changes in spike shape and resting potential, affecting the temporal fidelity of spike conduction. Neural coding can be altered by activity-dependent changes in conduction velocity, spike failures, and ectopic spike initiation. This is true under normal physiological conditions, and relevant for a number of neuropathies that lead to abnormal excitability. In addition, a growing number of studies show that the axon trunk can express receptors to glutamate, GABA, acetylcholine or biogenic amines, changing the relative contribution of some channels to axonal excitability and therefore rendering the contribution of this compartment to neural coding conditional on the presence of neuromodulators. Long-term regulatory processes, both during development and in the context of activity-dependent plasticity may also affect axonal properties to an underappreciated extent.

Influenza A virus vaccines undergo yearly reformulations due to the antigenic variability of the virus caused by antigenic drift and shift. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. Inactivated influenza A seasonal vaccines are generated by classical reassortment. The classical method takes advantage of the ability of the influenza virus to reassort based on the segmented nature of its genome. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo. The genetic analysis was conducted using reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). The characterization was conducted to determine the parental origin of the gene segments (hy donor virus or wt virus), gene segment ratios and constellations. Fold increase in growth of reassortant viruses compared to respective parent wt viruses was determined by hemagglutination assay titers. In this study fifty-seven influenza A vaccine candidate reassortants were analyzed for the presence or absence of correlations between specific gene segment ratios, gene constellations and hy reassortant phenotype. We found two gene ratios, 6:2 and 5:3, to be the most prevalent among the hy reassortants analyzed, although other gene ratios also conferred hy in certain reassortants.