Engineered Trehalose Permeable to Mammalian Cells

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.     

Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression

We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high‐resolution by X‐ray crystallography, low yield of soluble protein prevented X‐ray structure determination of a fifth designed material, T33‐09. Here we report the design and crystal structure of T33‐31, a variant of T33‐09 with improved soluble yield resulting from redesign efforts focused on mutating solvent‐exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic‐level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.     

HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01.     

Thermal biology and establishment potential in temperate climates of the aphid parasitoid, <Emphasis Type="Italic">Lysiphlebus testaceipes</Emphasis>

Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae, Aphidiinae) is a parasitic wasp which plays an important role in the biological control of a number of aphid species. Through assessment of its thermal biology and low temperature tolerance, this study ascertains the establishment potential of L. testaceipes in cool temperate climates typical of northern Europe. The developmental threshold of L. testaceipes was 5.8°C. Rearing of parasitoids at shorter day lengths and lower temperatures indicated no ability to enter a diapause state. The supercooling points (SCP) of non-acclimated and acclimated parasitoid life stages were between −24.6°C and −17.7°C, with LTemp₅₀ temperatures approaching these values, indicating a high level of cold tolerance in short exposures. At 5°C the LTime₅₀ of acclimated larvae within parasitized aphids was 42.8 days. Acclimated pupae continued to develop with 54% adult emergence from mummies within 60 days. Acclimated parasitoid larvae and pupae, within living and mummified aphids, continued to develop during 70 days of winter field exposure and emerging adult parasitoids were reproductively viable under field conditions. These data indicate that where suitable host species are available throughout the year, L. testaceipes would be able to establish in northern Europe.     

U.K. winter egg survival in the field and laboratory diapause of Typhlodromips montdorensis

Abstract. Typhlodromips montdorensis has potential for release as a glasshouse biological control agent in the U.K. against thrips and spider mites. This study investigates the field survival in the U.K. of T. montdorensis when released as eggs, and the diapause response when reared in a regime related to its location of origin. All acclimated and nonacclimated eggs of T. montdorensis die in the field within 7 days of exposure. It is not possible to induce diapause in T. montdorensis reared at 21 °C under a LD 11 : 13 h photoperiod. The results presented here support the view that T. montdorensis is unlikely to survive a U.K. winter outside of the glasshouse environment, and contribute to the understanding of the biology of this little known species.     

Ovarian activity and plasma sex steroid levels of Distichodus antonii in relation to environmental conditions in the upper basin of the Congo River

Distichodus antonii is an endemic fish species of the Congo River basin in which the stocks of wild populations are threatened by overfishing pressure. Knowledge of its reproductive biology would be useful in consideration of conservation and management options for the species. Therefore, this study investigated changes in ovarian activity and levels of steroid profiles in wild populations in relation to variation in temperature and rainfall. Adult females (n = 101, body weight of 3 183 ± 14.75 g, SE) were captured monthly over one year (2013–2014). Apart from evaluation of oocyte diameters and gonad developmental stages, gonado-, hepato-, lipososomatic indices (GSI, HSI, LSI) and plasma levels of sex steroids (testosterone-T, estradiol-17β-E2) were determined. The results suggested a synchronous development of oocytes with two annual reproductive seasons over the one-year study. Plasma T and E2 levels peaked during spawning periods likely reflecting active oogenesis. The highest values of morphosomatic indices were observed during the longest rainfall period in September, and were associated with high steroidogenic activity evidenced by increased E2 production. In addition, more vitellogenic oocytes (September and October) were observed during the latter season than during the short rainy season (in May).     

Walking speed adaptation ability of Myzus persicae to different temperature conditions

Walking speeds were calculated for nine clones of the peach potato aphid Myzus persicae collected from three countries along a latitudinal cline of its European distribution from Sweden to Spain (Sweden, UK and Spain), and the effects of collection origin and intra and intergenerational acclimation were investigated. Walking speeds declined with decreasing temperature, with maximum performance at temperatures closest to acclimation temperature (fastest median walking speed of 5.8 cm min(-1) was recorded for clone UK 3, collected from the UK, at 25°C after acclimating to 25°C for one generation). Following acclimation at both 20°C and 25°C, walking ceased (as indicated by median walking speeds of 0.0 cm min(-1)) at temperatures as high as 7.5°C and 12.5°C. However, acclimation at 10°C enabled mobility to occur to temperatures as low as 0°C. There was no relationship between mobility and latitude of collection, suggesting that large scale mixing of aphids may occur across Europe. However, clonal variation was suggested, with clone UK 3 outperforming the majority of other clones across all temperatures at which mobility was maintained following acclimation at 10°C for one and three generations and at 25°C for one generation. The Scandinavian clones consistently outperformed their temperate and Mediterranean counterparts at the majority of temperatures following acclimation for three generations at 25°C.     

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.     

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold‐based and scaffold‐free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS—five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long‐term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick‐freeze/deep‐etch electron microscopy. Both the scaffold‐free and the collagen‐based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell‐derived matrix in the scaffold‐based constructs was significantly lower than that produced by scaffold‐free constructs (controls). We also observed significant variability in the remodeling of RCS scaffold by PHCFCs. PHCFCs which penetrated the RCS scaffold did exert robust local control over secreted collagen but did not appear to globally reorganize the scaffold effectively in the time period of the study. Consistent with our hypothesis, the results demonstrate that the presence of the scaffold appears to interfere with the global organization of the cell‐derived matrix. The production of highly organized local matrix by fibroblasts which penetrated the scaffold suggests that there is a mechanism which operates close to the cell membrane capable of controlling fibril organization. Nonetheless, the local control of the collagen alignment produced by cells within the scaffold was not continuous and did not result in overall global organization of the construct. Using a disorganized scaffold as a guide to produce highly organized tissue has the potential to delay the production of useful matrix or prevent uniform remodeling. The results of this study may shed light on the recent attempts to use disorganized collagenous matrix as a temporary corneal replacement in vivo which led to a variable remodeling response. Biotechnol. Bioeng. 2012; 109: 2683–2698. © 2012 Wiley Periodicals, Inc.