Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

     

Peptide nanotube aligning side chains onto one side

A novel pseudo cyclic penta‐β‐peptide composed of a β‐naphthylalanine, two β‐alanines, and a sequence of ethylenediamine‐succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the maximum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES forms rod‐ or needle‐shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer emission, which are different from a reference cyclic tri‐β‐peptide composed of a β‐naphthylalanine and two β‐alanines. Insertion of a sequence of ethylenediamine‐succinic acid into β‐amino acids in the cyclic skeleton is therefore suggested to be effective to make the side chains aligning on one side of the PNT. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of photosynthetically active radiation and temperature on the photosynthesis of two Vietnamese species of Sargassum,S. mcclurei and S. oligocystum,based on the field and laboratory measurements

SUMMARY The effects of photosynthetically active radiation (PAR) and temperature on the photosynthesis of two Vietnamese brown algae, Sargassum mcclurei and S. oligocystum (Fucales), were determined by field and laboratory measurements. Dissolved oxygen sensors and pulse‐amplitude modulated (PAM) fluorometry were used for the measurements of photosynthetic efficiency. A Diving‐PAM revealed that underwater measurements of the effective quantum yield (Φ _PSII ) of both species declined with increasing incident PAR, with minimum Φ _PSII occurring during noon to early afternoon. Φ _PSII recovered in the evening, indicating photo‐adaptation to excessive PAR. In laboratory experiments, Φ _PSII also decreased under continuous exposure to 1000 μmol photons m^?2 s^?1; and full recovery occurred after 12 h of dark acclimatization. The net photosynthesis – PAR experiments of S. mcclurei and S. oligocystum conducted at 28°C revealed that the net photosynthetic rate quickly increased at PAR below the saturation irradiance of 361 and 301 μmol photons m^?2 s^?1 and nearly saturated to maximum net photosynthetic rates of 385 and 292 μg O₂ g_ww ^{? 1} min^?1 without photoinhibition, respectively. Gross photosynthesis and dark respiration experiments determined over a range of temperatures (12–40°C), revealed that the maximum gross photosynthetic rates of 201 and 147 μg O₂ g_ww ^{? 1} min^?1 occurred at 32.9 and 30.7°C for S. mcclurei and S. oligocystum, respectively. The dark respiration rates increased exponentially over the temperature ranges examined. The estimated maximum value of the maximum quantum yield occurred at 19.3 and 20.0°C and was 0.76 and 0.74, respectively. Similar to the natural habitat of the study site, these two species tolerated the relatively high temperatures and broad range of PAR. The ability of these species to recover from exposure to high PAR is one of the mechanisms that allow them to flourish in the shallow water environment.     

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P(Zn)), the accessory Zn-BChl a (B(Zn)), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P(Zn)(*), measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B(Zn)(*) to P(Zn), and the latter, the electron transfer from P(Zn) to H. The angle between the transition dipoles of B(Zn) and P(Zn) was estimated to be 36 degrees based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P(Zn).     

Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.     

Primary pulmonary malignant peripheral nerve sheath tumor: a case report

BACKGROUND: Although the histologic features of malignant peripheral nerve sheath tumor (MPNST) have been described, the cytologic features of primary pulmonary MPNST have not been reported in the literature. CASE: We report a case of primary pulmonary MPNST in a 78-year-old man. Follow-up computed tomography of colon cancer, renal cancer, penile cancer and gingival cancer revealed a nodular lesion, 12 mm in diameter, in the right upper lobe of the lung. In frozen section, a diagnosis of malignant neoplasm, not otherwise specified, was rendered for the imprinting specimen and histologic specimen. Imprinting specimens were composed of small cellular aggregates and discohesive neoplastic cells with obvious malignant features. Histologically, spindle cells with pleomorphic nuclei arranged infascicular patterns and multinucleated tumor giant cells were also observed. More than 25 mitotic figures were observed per 10 high-power fields. Tumor cells were positive only for vimentin and S-100, and the Ki-67 labeling index was 10%. Clinical and imaging investigation failed to identify an alternative primary site. We histologically diagnosed this case as primary pulmonary MPNST. CONCLUSION: MPNST has a varied cytomorphology with frank nuclear atypia showing no definite differentiation. Multinucleated neoplastic giant cells with immunopositivity for S-100 may permit more accurate diagnosis of MPNST.