A study of the osmotic characteristics, water permeability, and cryoprotectant permeability of human vaginal immune cells.

Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3(+) T cells and CD14(+) macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types (Vb) were determined relative to the initial cell volume (V0) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water (Lp) and to four different cryoprotective agent (CPA) solutions (Ps) at room temperature were also measured. Results indicated Vb values of 0.516 V0 and 0.457 V0 for mucosal T cells and macrophages, respectively. Lp values at room temperature were 0.196 and 0.295 µm/min/atm for T cells and macrophages, respectively. Both cell types had high Ps values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10(-3) cm/min), but transport of the fourth CPA, glycerol, occurred 50-150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells.

Engineering of Bacillus thuringiensis Cry2Ab toxin for improved insecticidal activity.

Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.

Stability is essential for insecticidal activity of Vip3Aa toxin against Spodoptera exigua.

Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines (P591, P605 and P779) located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.