Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6–9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.     

Spatial orientation of social caterpillars is influenced by polarized light

Processionary caterpillars of Thaumetopoea pityocampa (in Europe) and Ochrogaster lunifer (in Australia) (Lepidoptera: Notodontidae) form single files of larvae crawling head-to-tail when moving to feeding and pupation sites. We investigated if the processions are guided by polarization vision. The heading orientation of processions could be manipulated with linear polarizing filters held above the leading caterpillar. Exposure to changes in the angle of polarization around the caterpillars resulted in corresponding changes in heading angles. Anatomical analysis indicated specializations for polarization vision of stemma I in both species. Stemma I has a rhabdom with orthogonal and aligned microvilli, and an opaque and rugged surface, which are optimizations for skylight polarization vision, similar to the dorsal rim of adult insects. Stemmata II-VI have a smooth and shiny surface and lobed rhabdoms with non-orthogonal and non-aligned microvilli; they are thus optimized for general vision with minimal polarization sensitivity. Behavioural and anatomical evidence reveal that polarized light cues are important for larval orientation and can be robustly detected with a simple visual system.     

Stellaria nemorum L. andS. montana Pierrat (Caryophyllaceae) in the Forest Communities of Slovenia

Morphological and coenological differences between closely related speciesStellaria nemorum L. andS. montana Pierrat are described.Stellaria montana is frequent in Slovenia and occurs mostly in the montane belt (altitude 600–1200 m). It has been recorded in thirty two forest communities (associations or lower syntaxa). Although widely distributed in EuropeS. nemorum is more rare in Slovenia. It occurs mostly in upper-montane (altimontane) and subalpine belt (altitude 1200–1600 m) in ten forest or shrubby communities. Both,S. nemorum andS. montana are valuable diagnostic species for certain associations. They characterize sites with fresh soil, rich in nitrogen. As an example we present two syntaxa of beech forests of Slovenia, in which the species of theS. nemorum group have high constancy and cover value.     

Effects of hypoxic stimulation in experimental animals and in children with bronchial asthma]

Inhalations using hypoxic mixture of 12-15% O2 were tested in 40 rats (daily for 20 min. during 1-4 months) and in 200 children with light and mid-serious forms of bronchial asthma (for 5-10 min. 2-4 times with 5 min. break, course-10 days). Stimulation effect in animals was observed for 1-2 months and then depression came. The immunological and allergological indices were observed to distinctly improve, whereas the indices of the pulmonary function remained practically unchanged. The clinical indices in most patients improved and the similarity of this method and effects of other nonmedicamental methods was noted.     

Chemical sensitivity of Limnaea stagnalis neurons and their responses to synaptic stimulation

Responses of nerve cells to puncture, to touching the surface of the mollusk leg, osmotic stimulation, and extracellular microiontophoretic injection of acetylcholine, noradrenalin, serotonin, atropine, and propranolol were recorded intracellularly in the right parietal, left pedal, and visceral ganglia of the unisolated circumpharyngeal ring ofLimnaea stagnalis. Selective sensitivity of the neurons to the biologically active substances was observed. Results indicative of the functional differences between the various ganglia and of their neurochemical organization were obtained. Selective blocking of the unit responses to puncture of the surface of the mollusk leg by atropine or propranolol suggests that different forms of excitation reaching the central neurons evoked different and specific neurochemical processes on their subsynaptic membranes which can retain the essential informativeness of the widely different afferent volleys converging on a single nerve cell.I. M. Sechenov First Moscow Medical Institute. Translated from Neirofiziologiya, Vol. 5, No. 5, pp. 510–518, September–October, 1973.     

Investigation of antitumor potential of Ni(II) complexes with tridentate PNO acylhydrazones of 2-(diphenylphosphino)benzaldehyde and monodentate pseudohalides

Square-planar azido Ni(II) complex with condensation product of 2-(diphenylphosphino)benzaldehyde and Girard’s T reagent was synthesized and its crystal structure was determined. Cytotoxic activity of the azido complex and previously synthesized isothiocyanato, cyanato and chlorido Ni(II) complexes with this ligand was examined on six tumor cell lines (HeLa, A549, K562, MDA-MB-453, MDA-MB-361 and LS-174) and two normal cell line (MRC-5 and BEAS-2B). All the investigated nickel(II) complexes were cytotoxic against all tumor cell lines. The newly synthesized azido complex showed selectivity to HeLa and A549 tumor cell lines compared to the normal cells (for A549 IC₅₀ was similar to that of cisplatin). Azido complex interferes with cell cycle phase distribution of A549 and HeLa cells and possesses nuclease activity towards supercoiled DNA. The observed selectivity of the azido complex for some tumor cell lines can be connected with its strong DNA damaging activity.     

Activators of protein kinase A and oxytocin affect rabbit reproduction

The aim of this study was to examine the effect of activators of protein kinase A ?? 3-isobutyl-1-methyl-xanthine (IBMX) and dibutyryl cyclic adenosine monophosphate (dbcAMP) ?? and of oxytocin (OT) on rabbit female reproductive function. We used equine chorionic gonadotropin (eCG) to improve follicular development and rabbit estrous synchronization and human chorionic gonadotropin (hCG) to induce ovulation. In the experimental group, the females were stimulated using gonadotropins together with either IBMX, dbcAMP or OT. In the animals kept until parturition, the conception rate, parturition rate, and numbers of stillborn and weaned pups were recorded. In the animals euthanized 18?C19 hours after insemination, the eggs were flushed from the oviducts and cultured up to the blastocyst cell stage. Numbers of corpora lutea, zygotes, morulas and blastocysts were determined. Both dbcAMP and OT, but not IBMX, decreased conception and parturition rate. Both IBMX and OT, but not dbcAMP, decreased pup mortality rate. All three tested substances increased the weaning rate. Both IBMX and dbcAMP, but not OT, increased the numbers of corpora lutea, zygotes, and embryos at morula and hatching blastocyst stages. These observations confirm the stimulatory role of the protein kinase A-dependent signaling pathway activated by IBMX and dbcAMP in rabbit reproduction. OT may decrease pups mortality.