Cryopreserved Morulae can be used to Efficiently Generate Germline-transmitting Chimeras by Blastocyst Injection

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30–75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.     

The ontogeny of the urogenital system of the spotted hyena (Crocuta crocuta Erxleben)

Studies were conducted to elucidate the importance of androgen-mediated induction of the extreme masculinization of the external genitalia in female spotted hyenas. Phallic size and shape; androgen receptor (AR) and alpha-actin expression; and sex-specific differences in phallic retractor musculature, erectile tissue, tunica albuginea, and urethra/urogenital sinus were examined in male and female fetuses from Day 30 of gestation to term. Similar outcomes were assessed in fetuses from dams treated with an AR blocker and a 5alpha-reductase inhibitor (antiandrogen treatment). Clitoral and penile development were already advanced at Day 30 of gestation and grossly indistinguishable between male and female fetuses throughout pregnancy. Sex-specific differences in internal phallic organization were evident at Gestational Day 45, coincident with AR expression and testicular differentiation. Antiandrogen treatment inhibited prostatic development in males and effectively feminized internal penile anatomy. We conclude that gross masculinization of phallic size and shape of male and female fetuses is androgen-independent, but that sexual dimorphism of internal phallic structure is dependent on fetal testicular androgens acting via AR in the relevant cells/tissues. Androgens secreted by the maternal ovaries and metabolized by the placenta do not appear to be involved in gross masculinization or in most of the sex differences in internal phallic structure.     

Direct inoculation of simian immunodeficiency virus from sooty mangabeys in black mangabeys (Lophocebus aterrimus): first evidence of AIDS in a heterologous African species and different pathologic outcomes of experimental infection

A unique opportunity for the study of the role of serial passage and cross-species transmission was offered by a series of experiments carried out at the Tulane National Primate Research Center in 1990. To develop an animal model for leprosy, three black mangabeys (BkMs) (Lophocebus aterrimus) were inoculated with lepromatous tissue that had been serially passaged in four sooty mangabeys (SMs) (Cercocebus atys). All three BkMs became infected with simian immunodeficiency virus from SMs (SIVsm) by day 30 postinoculation (p.i.) with lepromatous tissue. One (BkMG140) died 2 years p.i. from causes unrelated to SIV, one (BkMG139) survived for 10 years, whereas the third (BkMG138) was euthanized with AIDS after 5 years. Histopathology revealed a high number of giant cells in tissues from BkMG138, but no SIV-related lesions were found in the remaining two BkMs. Four-color immunofluorescence revealed high levels of SIVsm associated with both giant cells and T lymphocytes in BkMG138 and no detectable SIV in the remaining two. Serum viral load (VL) showed a significant increase (>1 log) during the late stage of the disease in BkMG138, as opposed to a continuous decline in VL in the remaining two BkMs. With the progression to AIDS, neopterin levels increased in BkMG138. This study took on new significance when phylogenetic analysis unexpectedly showed that all four serially inoculated SMs were infected with different SIVsm lineages prior to the beginning of the experiment. Furthermore, the strain infecting the BkMs originated from the last SM in the series. Therefore, the virus infecting BkMs has not been serially passaged. In conclusion, we present the first compelling evidence that direct cross-species transmission of SIV may induce AIDS in heterologous African nonhuman primate (NHP) species. The results showed that cross-species-transmitted SIVsm was well controlled in two of three BkMs for 2 and 10 years, respectively. Finally, this case of AIDS in an African monkey suggests that the dogma of SIV nonpathogenicity in African NHP hosts should be reconsidered.     

Sepsis progression and outcome: a dynamical model

Background  

Sepsis (bloodstream infection) is the leading cause of death in non-surgical intensive care units. It is diagnosed in 750,000 US patients per annum, and has high mortality. Current understanding of sepsis is predominately observational and correlational, with only a partial and incomplete understanding of the physiological dynamics underlying the syndrome. There exists a need for dynamical models of sepsis progression, based upon basic physiologic principles, which could eventually guide hourly treatment decisions.     

Identification and characterization of the water gap in physically dormant seeds of Geraniaceae, with special reference to Geranium carolinianum

Background and Aims

Physical dormancy in seeds of species of Geraniaceae is caused by a water-impermeable palisade layer in the outer integument of the seed coat and a closed chalaza. The chalazal cleft has been reported to be the water gap (i.e. location of initial water entry) in innately permeable seeds of Geraniaceae. The primary aim of this study was to re-evaluate the location of the water gap and to characterize its morphology and anatomy in physically dormant seeds of Geraniaceae, with particular reference to G. carolinianum.

Methods

Length, width, mass, anatomy and germination of two seed types (light brown and dark brown) of G. carolinianum were compared. Location, anatomy and morphology of the water gap were characterized using free-hand and microtome tissue sectioning, light microscopy, scanning electron microscopy, dye tracking, blocking and seed-burial experiments.

Key Results

Treatment with dry heat caused a colour change in the palisade cells adjacent to the micropyle. When placed in water, the ‘hinged valve’ (blister) erupted at the site of the colour change, exposing the water gap. The morphology and anatomy in the water-gap region differs from those of the rest of the seed coat. The morphology of the seed coat of the water-gap region is similar in G. carolinianum, G. columbinum, G. molle and G. pusillum and differs from that of the closely related species Erodium cicutarium.

Conclusions

Dislodgment of swollen ‘hinged valve’ palisade cells adjacent to the micropyle caused the water gap to open in physically dormant seeds of G. carolinianum, and it was clear that initial water uptake takes place through this gap and not via the chalazal opening as previously reported. This water gap (‘hinged valve gap’) differs from water gaps previously described for other families in morphology, anatomy and location in the seed coat.     

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE*

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.     

Xeric limestone prairies of eastern United States: Review and synthesis

Xeric limestone prairies (XLPs) are open, nonforested areas in which herbaceous plant communities occur on shallow, rocky soils derived from calcareous substrates. These grasslands are characterized by dominance of C₄ perennial grasses (particularlySchizachyrium scoparium) and are distributed in eastern United States from Missouri and Pennsylvania south to Arkansas and Georgia. XLPs occur in the Ozark Plateaus, Central Lowland, Interior Low Plateaus, Appalachian Plateaus, Ridge and Valley, and Coastal Plain physiographic provinces, and they are developed on Alfisols, Ultisols, Mollisols, Inceptisols, and Vertisols derived from Paleozoic limestones (also Eocene), dolomites, and calcareous shales. The C₄ perennial prairie grassS. scoparium is the characteristic dominant taxon in XLPs of eastern United States. However, C₃ perennial forbs are dominant in some sites, and C₄ annual grasses (Sporobolus spp.) may be locally dominant in shallow-soil-zone microsites. Thirteen taxa apparently are endemic, or nearly so, to this vegetation type, including eight in the Ridge and Valley in Alabama (Cahaba River valley), four in the Ozark Plateaus in Missouri and Arkansas, and one in the Ridge and Valley of West Virginia and Virginia. Various types of information are used to construct a conceptual model of the origin, maintenance, and successional dynamics of XLPs. Affinities of XLPs in eastern United States to other herbaceous vegetation types in eastern and western North America are discussed, and directions for future research are suggested.