Effect of Calcium Ions and Disulfide Bonds on Swelling of Virus Particles

Multivalent ions affect the structure and organization of virus nanoparticles. Wild-type simian virus 40 (wt SV40) is a nonenveloped virus belonging to the polyomavirus family, whose external diameter is 48.4 nm. Calcium ions and disulfide bonds are involved in the stabilization of its capsid and are playing a role in its assembly and disassembly pathways. Using solution small-angle X-ray scattering (SAXS), we found that the volume of wt SV40 swelled by about 17% when both of its calcium ions were chelated by ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid and its disulfide bonds were reduced by dithiothreitol. By applying osmotic stress, the swelling could be reversed. DNA-containing virus-like particles behaved in a similar way. The results provide insight into the structural role of calcium ions and disulfide bonds in holding the capsid proteins in compact conformation

Effect of Calcium Ions and Disulfide Bonds on Swelling of Virus Particles

Multivalent ions affect the structure and organization of virus nanoparticles. Wild-type simian virus 40 (wt SV40) is a nonenveloped virus belonging to the polyomavirus family, whose external diameter is 48.4 nm. Calcium ions and disulfide bonds are involved in the stabilization of its capsid and are playing a role in its assembly and disassembly pathways. Using solution small-angle X-ray scattering (SAXS), we found that the volume of wt SV40 swelled by about 17% when both of its calcium ions were chelated by ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid and its disulfide bonds were reduced by dithiothreitol. By applying osmotic stress, the swelling could be reversed. DNA-containing virus-like particles behaved in a similar way. The results provide insight into the structural role of calcium ions and disulfide bonds in holding the capsid proteins in compact conformation

Crystallization, Reentrant Melting, and Resolubilization of Virus Nanoparticles

Crystallization is a fundamental and ubiquitous process that is well understood in the case of atoms or small molecules, but its outcome is still hard to predict in the case of nanoparticles or macromolecular complexes. Controlling the organization of virus nanoparticles into a variety of 3D supramolecular architectures is often done by multivalent ions and is of great interest for biomedical applications such as drug or gene delivery and biosensing, as well as for bionanomaterials and catalysis. In this paper, we show that slow dialysis, over several hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution against salt solutions containing MgCl₂, with or without added NaCl, results in wt SV40 nanoparticles arranged in a body cubic center crystal structure with <i>Im</i>3<i>m</i> space group, as a thermodynamic product, in coexistence with soluble wt SV40 nanoparticles. The nanoparticle crystals formed above a critical MgCl₂ concentrations. Reentrant melting and resolubilization of the virus nanoparticles took place when the MgCl₂ concentrations passed a second threshold. Using synchrotron solution X-ray scattering we determined the structures and the mass fraction of the soluble and crystal phases as a function of MgCl₂ and NaCl concentrations. A thermodynamic model, which balances the chemical potentials of the Mg²⁺ ions in each of the possible states, explains our observations. The model reveals the mechanism of both the crystallization and the reentrant melting and resolubilization and shows that counterion entropy is the main driving force for both processes

Temporal silencing of an androgenic gland-specific insulin-like gene affecting phenotypical gender differences and spermatogenesis

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis. (Endocrinology 150: 1278 -1286, 2009) E ver since it was first proposed as the source of a hypothetical masculinizing hormone in crustaceans, the androgenic gland (AG) has been studied thoroughly in many crustacean species. The consensus emerging from these studies is that the AG plays a unifying role in the bewilderingly varied sex differentiation mechanisms in crustaceans (1-5). The AG constitutes a feature unique to male crustaceans in that it is an organ regulating sex differentiation separated from the gametogenic organ (unlike the single organ of vertebrate species). This separation enables manipulation of sex differentiation without affecting the gonads (6). In decapod male crustaceans, there are two AGs, each attached to the ejaculatory region of a vas deferens. In research spanning several decades, the functioning of the AG was investigated in a number of crustacean species by following the morphological and physiological effects of AG removal or transplantation on primary and secondary sex characteristics. In the amphipod Orchestia gamarella, for example, bilateral AG ablation decreased spermatogenesis and prevented the development of secondary male characteristics (7). In the crayfish Procambarus clarkii, injection of AG extracts accelerated the development of external male characteristics (8). In the giant freshwater prawn, Macrobrachium rosenbergii, a degree of masculinization was recorded in AG-implanted females (9). In the same species, fully functional sex reversal from males to neo-females (10) and from females to neo-males (11) was achieved by bilateral AG ablation and transplantation, respectively. The possibility of sex reversal has economical implications for the farming of this sexually dimorphic species because males grow faster than females (12). It is currently widely accepted that the AG of decapod crustaceans secretes the hormone(s) responsible for male differentiation, with a high probability of such a hormone(s) being proteinaceous in nature (13). This premise is supported by a histological study in the shore crab Phachygrapsus crassipes Multicellular organisms express various insulin-like peptides differentially. The insulin-like peptides discovered in inverte- Abbreviations: AG, Androgenic gland; CHH, crustacean hyperglycemic hormone; dsRNA, double-stranded RNA; GFP, green fluorescent protein; hAG, hypertrophy and hyperplasia of the androgenic gland; Mr-IAG, Macrobrachium rosenbergii insulin-like androgenic gland gene; RNAi, RNA interference; T7P, T7 promoter site at the 5Ј of one primer; UTR, untranslated region; XO-SG, X-organ sinus gland complex

pH stability and disassembly mechanism of wild-type simian virus 40

Viruses are remarkable self-assembled nanobiomaterial-based machines, exposed to a wide range of pH values. Extreme pH values can induce dramatic structural changes, critical for the function of the virus nanoparticles, including assembly and genome uncoating. Tuning cargo–capsid interactions is essential for designing virus-based delivery systems. Here we show how pH controls the structure and activity of wild-type simian virus 40 (wtSV40) and the interplay between its cargo and capsid. Using cryo-TEM and solution X-ray scattering, we found that wtSV40 was stable between pH 5.5 and 9, and only slightly swelled with increasing pH. At pH 3, the particles aggregated, while capsid protein pentamers continued to coat the virus cargo but lost their positional correlations. Infectivity was only partly lost after the particles were returned to pH 7. At pH 10 or higher, the particles were unstable, lost their infectivity, and disassembled. Using time-resolved experiments we discovered that disassembly began by swelling of the particles, poking a hole in the capsid through which the genetic cargo escaped, followed by a slight shrinking of the capsids and complete disassembly. These findings provide insight into the fundamental intermolecular forces, essential for SV40 function, and for designing virus-based nanobiomaterials, including delivery systems and antiviral drugs

Standardization of Post-Vitrification Human Blastocyst Expansion as a Tool for Implantation Prediction

The increased use of vitrified blastocysts has encouraged the development of various criteria for selecting the embryo most likely to implant. Post-thaw assessment methods and timetables vary among investigators. We investigated the predictive value of well-defined measurements of human blastocyst re-expansion, following a fixed incubation period. Post-thaw measurements were taken exactly at 0 and 120 &plusmn; 15 min. Minimum and maximum cross-sectional axes were measured. Three groups were defined: Group 1: embryos that continued to shrink by 10 &micro;m or more; group 2: embryos that ranged from &minus;9 to +9 &micro;m; and group 3: re-expansion of 10 &micro;m or more. Patient and morphokinetic data were collected and integrated into the analysis. A total of 115 cases were included. The clinical pregnancy rate for group 1 was 18.9%; group 2, 27%; and group 3, 51.2% (p = 0.007). Pre-thaw morphologic grading and morphokinetic scores of the study groups did not reveal differences. p-values were 0.17 for the pre-thaw morphologic score, 0.54 for KID3, and 0.37 for KID5. The patients&rsquo; demographic and clinical data were similar. The clinical pregnancy rate correlated with the degree of thawed blastocyst re-expansion measured 2 h after incubation. This standardized measure is suggested as a tool to predict the potential of treatment success before embryo transfer