Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach’s potential to engineer cell function is demonstrated in HIV infection studies.     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.     

Factors promoting efficient in vitro regeneration from de-embryonated cotyledon explants of <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.

Highly efficient (>90%) protocols were developed for in vitro regeneration from de-embryonated cotyledon explants of peanut (Arachis hypogaea L.). Phytohormone combinations and concentrations, explant source and orientation, period of incubation and the response of genotypes were examined for optimization of the regeneration efficiency. Adventitious shoot primordia could be induced from de-embryonated cotyledon explants when (1) the proximal end of the explant was kept in contact with the shoot induction medium-I supplemented with 5 mg l⁻¹ BAP + 2 mg l⁻¹ 2,4-D for 4 weeks, or (2) the distal end was kept in contact with shoot induction medium-II supplemented with only BAP at 20 mg l⁻¹ for the first 2 weeks, followed by subculture in the same medium containing 15 mg l⁻¹ BAP for the next 2 weeks. Orientation of placing the explant on the above media was critical for in vitro regeneration. The factors affecting the morphogenic responses like, repetitive organogenesis, shoot elongation, in vitro flowering and rhizogenesis were examined. Shoot bud formation was genotype independent. Histological studies showed multicellular origin of adventitious shoot primordia. The protocols gave healthy and fertile plants within 4 months.     

Soft tissue re-growth after different crown lengthening techniques among Indian patients

Patients often report complaining of fractured or decayed teeth with severe morphological deformities. However, all these clinical scenarios require the same level of care and consideration to rehabilitate form, function and esthetics. Some cases have sufficient clinical crown height while others often require an interdisciplinary approach in the form of orthodontic/surgical extrusion or surgical periodontal options. A common factor delaying treatment is soft tissue regrowth after crown lengthening which delays the impression required for final prosthesis. Therefore, it is of interest to compare the prevalence of soft tissue regrowth a week after different crown lengthening techniques including laser gingivectomy, electrocautery gingivectomy, modified Widman flap and apically repositioned. The parameters assessed included 1-week postoperative soft tissue regrowth after crown lengthening, age of patients and gender. It was observed that laser and electrocautery-assisted gingivectomy had a higher rate of soft tissue regrowth as compared to surgical techniques. It was further noted that laser and electrocautery assisted gingivectomy had a higher frequency of soft tissue rebound growth compared to surgical crown lengthening using modified widman flap and apically repositioned flap, which was statistically insignificant. Patients within the age groups of 26-60 years were found to have a higher tendency of soft tissue regrowth, which was found to be clinically and statistically significant (p<0.05).     

Sexual transmission of a plant pathogenic bacterium, Candidatus Liberibacter asiaticus, between conspecific insect vectors during mating

Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 μm in length and 0.19 to 0.39 μm in width. The spherical structures measured from 0.61 to 0.80 μm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees.