Novel glycosylated human interferon alpha 2b expressed in glycoengineered Pichia pastoris and its biological activity: N-linked glycoengineering approach.

Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.

Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system.

CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1-2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1-2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1-2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.

Coping with spatial heterogeneity and temporal variability in resources and risks: adaptive movement behaviour by a large grazing herbivore.

Movement is a key mean for mobile species to cope with heterogeneous environments. While in herbivorous mammals large-scale migration has been widely investigated, fine-scale movement responses to local variations in resources and predation risk remain much less studied, especially in savannah environments. We developed a novel approach based on complementary movement metrics (residence time, frequency of visits and regularity of visits) to relate movement patterns of a savannah grazer, the blue wildebeest Connochaetes taurinus, to fine-scale variations in food availability, predation risk and water availability in the Kruger National Park, South Africa. Wildebeests spent more time in grazing lawns where the grass is of higher quality but shorter than in seep zones, where the grass is of lower quality but more abundant. Although the daily distances moved were longer during the wet season compared to the dry season, the daily net displacement was lower, and the residence time higher, indicating a more frequent occurrence of area-concentred searching. In contrast, during the late dry season the foraging sessions were more fragmented and wildebeests moved more frequently between foraging areas. Surprisingly, predation risk appeared to be the second factor, after water availability, influencing movement during the dry season, when resources are limiting and thus expected to influence movement more. Our approach, using complementary analyses of different movement metrics, provided an integrated view of changes in individual movement with varying environmental conditions and predation risk. It makes it possible to highlight the adaptive behavioral decisions made by wildebeest to cope with unpredictable environmental variations and provides insights for population conservation.

Hair characteristics of four Indian bear species.

Dorsal guard hairs of four species of bear (family: Ursidae) found in India were characterized using light microscopy by studying features including colour, hair thickness, cuticle pattern, medulla pattern, medullary index, cross-section and scale count index. The gross cuticular pattern was quite similar for the four species but a distinct difference was observed in the scale count index. Medulla type was narrow and amorphous with a very low medullary index (< 0.15) except for brown bear which showed a comparatively higher medullary index (0.38) and a vacuolated medulla. By combining together the parameters mentioned above it was possible to characterize bear species on the basis of their hair characteristics.