Infectivity of adeno-associated virus serotypes in mouse testis

Background

Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.

Results

We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.

Conclusions

In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

     

The role of stroma in hematopoiesis and dendritic cell development

Development of the immune system is depicted as a hierarchical process of differentiation from hematopoietic stem cells (HSC) to lineage-committed precursors, which further develop into mature immune cells. In the case of dendritic cell (DC) development, this linear precursor-progeny approach has led to a confused picture of relationships between various subsets of DC identifiable in vivo. A possible reconciliation of the diversity of DC precursors and DC subsets in vivo encompasses the role of the microenvironment in DC hematopoiesis. We propose here that various niches for DC hematopoiesis within lymphoid organs could account for the diversity of DC in vivo. A tridimensional space consisting of stromal cells which produce a range of membrane-bound and secreted molecules providing signals to DC progenitors would define these niches.     

Uncoupling Protein 1 and Sarcolipin Are Required to Maintain Optimal Thermogenesis,and Loss of Both Systems Compromises Survival of Mice under Cold Stress

The importance of brown adipose tissue as a site of nonshivering thermogenesis has been well documented. Emerging studies suggest that skeletal muscle is also an important site of thermogenesis especially when brown adipose tissue function is lacking. We recently showed that sarcolipin (SLN), an uncoupler of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) pump, could contribute to heat production in skeletal muscle. In this study, we sought to understand how loss of UCP1 or SLN is compensated during cold exposure and whether they are both necessary for thermogenesis. Toward this goal, we generated a UCP1;SLN double knock-out (DKO) mouse model and challenged the single and DKO mice to acute and long-term cold exposures. Results from this study show that there is up-regulation of SLN expression in UCP1-KO mice, and loss of SLN is compensated by increased expression of UCP1 and browning of white adipose tissue. We found that the DKO mice were viable when reared at thermoneutrality. When challenged to acute cold, the DKO were extremely cold-sensitive and became hypothermic. Paradoxically, the DKO mice were able to survive gradual cold challenge, but these mice lost significant weight and depleted their fat stores, despite having higher caloric intake. These studies suggest that UCP1 and SLN are required to maintain optimal thermogenesis and that loss of both systems compromises survival of mice under cold stress.     

Analysis of Fluorescence Decay by the Nonlinear Least Squares Method

Fluorescence decay deconvolution analysis to fit a multiexponential function by the nonlinear least squares method requires numerical calculation of a convolution integral. A linear approximation of the successive data of the instrument response function is proposed for the computation of the convolution integral. Deconvolution analysis of simulated fluorescence data were carried out to show that the linear approximation method is generally better when one of the lifetimes is comparable to the time interval between data.     

Characterization of a phosphoprotein phosphatase activity in ribonucleoprotein particles from HeLa cell nuclei.

Ribonucleoprotein particles containing heterogenous nuclear RNA (hnRNP) have a phosphoprotein phosphatase activity. This activity is optimal at pH 7.5, inhibited by divalent cations and by increasing ionic strength above 200 mM NaCl, stimulated by 2-mercaptoethanol and inhibited by N-ethylmaleimide. It is clearly distinct from non specific alkaline phosphatase and resembles the phosphoprotein phosphatase present in Novikoff hepatoma nucleoli (Olson et al. B.B.R.C. (1976), 70, 717–721). This enzyme may be involved in regulating the phosphorylation level of hnRNP proteins in combination with the protein kinase previously described (Blanchard et al. Eur. J. Biochem. (1977) in press).     

Relative growth before and after sexual maturity in Ocypode platytarsis (Milne Edwards) and Ocypode cordimana (Desmarest) (Crustacea: Decapoda)

The relative growth of two species of the genus Ocypode inhabiting different ecological niches was studied before and after sexual maturity. The growth coefficient of both sexes of O. cordimana, which inhabits supra-littoral zones, is higher than that of O. platytarsis found in wave-wash zones.s

Some characteristics such as eyestalk length, major chela width and obdomen width do not show any difference in the growth pattern of either species of Ocypode at the onset of sexual maturity. Other characteristics such as minor and major chela length in male O. platytarsis; carapace length, minor chela length and width in female O. platytarsis; major chela length and third walking leg length in male O. cordimana showed a difference in the growth pattern after the onset of sexual maturity.

Major chela length of males of both species is clearly a sexually dimorphic feature since it showed an increase in the growth coefficient after sexual maturity. Merus length in male O. platytarsis and minor chela length in female O. cordimana also showed such sexual dimorphism.

There are several characteristics in which the growth coefficient declines after sexual maturity. The change in growth coefficient of different characteristics may depend on the adaptive value of the characteristic in reproductive or post-reproductive activities of O. cordimana and O. platytarsis.