Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.     

Unassisted blood collection from unanesthetized rats and gerbils

Blood collection is a common procedure in animal research. This column describes humane methods of collecting blood from the rat and the gerbil that can be carried out by a single person on an awake animal.     

Comparative genomic analysis of the Hsp70s from five diverse photosynthetic eukaryotes

We have identified 24 members of the DnaK subfamily of heat shock 70 proteins (Hsp70s) in the complete genomes of 5 diverse photosynthetic eukaryotes. The Hsp70s are a ubiquitous protein family that is highly conserved across all domains of life. Eukaryotic Hsp70s are found in a number of subcellular compartments in the cell: cytoplasm, mitochondrion (MT), chloroplast (CP), and endoplasmic reticulum (ER). Although the Hsp70s have been the subject of intense study in model organisms, very little is known of the Hsp70s from early diverging photosynthetic lineages. The sequencing of the complete genomes of Thalassiosira pseudonana (a diatom), Cyanidioschyzon merolae (a red alga), and 3 green algae (Chlamydomonas reinhardtii, Ostreococcus lucimarinus, Ostreococcus tauri) allow us to conduct comparative genomics of the Hsp70s present in these diverse photosynthetic eukaryotes. We have found that the distinct lineages of Hsp70s (MT, CP, ER, and cytoplasmic) each have different evolutionary histories. In general, evolutionary patterns of the mitochondrial and endoplasmic reticulum Hsp70s are relatively stable even among very distantly related organisms. This is not true of the chloroplast Hsp70s and we discuss the distinct evolutionary patterns between "green" and "red" plastids. Finally, we find that, in contrast to the angiosperms Arabidopsis thaliana and Oryza sativa that have numerous cytoplasmic Hsp70, the 5 algal species have only 1 cytoplasmic Hsp70 each. The evolutionary and functional implications of these differences are discussed.     

Meiotic Recombination in Human Oocytes

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of “vulnerable” crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.     

Which New Health Technologies Do We Need to Achieve an End to HIV/AIDS?

In the last 15 years, antiretroviral therapy (ART) has been the most globally impactful life-saving development of medical research. Antiretrovirals (ARVs) are used with great success for both the treatment and prevention of HIV infection. Despite these remarkable advances, this epidemic grows relentlessly worldwide. Over 2.1 million new infections occur each year, two-thirds in women and 240,000 in children. The widespread elimination of HIV will require the development of new, more potent prevention tools. Such efforts are imperative on a global scale. However, it must also be recognised that true containment of the epidemic requires the development and widespread implementation of a scientific advancement that has eluded us to date—a highly effective vaccine. Striving for such medical advances is what is required to achieve the end of AIDS.In the last 15 years, antiretroviral therapy (ART) has been the most globally impactful life-saving development of medical research. Antiretrovirals (ARVs) are used with great success for both the treatment and prevention of HIV infection. In the United States, the widespread implementation of combination ARVs led to the virtual eradication of mother-to-child transmission of HIV from 1,650 cases in 1991 to 110 cases in 2011, and a turnaround in AIDS deaths from an almost 100% five-year mortality rate to a five-year survival rate of 91% in HIV-infected adults [1]. Currently, the estimated average lifespan of an HIV-infected adult in the developed world is well over 40 years post-diagnosis. Survival rates in the developing world, although lower, are improving: in sub-Saharan Africa, AIDS deaths fell by 39% between 2005 and 2013, and the biggest decline, 51%, was seen in South Africa [2].Furthermore, the association between ART, viremia, and transmission has led to the concept of “test and treat,” with the hope of reducing community viral load by testing early and initiating treatment as soon as a diagnosis of HIV is made [3]. Indeed, selected regions of the world have begun to actualize the public health value of ARVs, from gains in life expectancy to impact on onward transmission, with a potential 1% decline in new infections for every 10% increase in treatment coverage [2]. In September 2015, WHO released new guidelines removing all limitations on eligibility for ART among people living with HIV and recommending pre-exposure prophylaxis (PrEP) to population groups at significant HIV risk, paving the way for a global onslaught on HIV [4].Despite these remarkable advances, this epidemic grows relentlessly worldwide. Over 2.1 million new infections occur each year, two-thirds in women and 240,000 in children [2]. In heavily affected countries, HIV infection rates have only stabilized at best: the annualized acquisition rates in persons in their first decade of sexual activity average 3%–5% yearly in southern Africa [5–7]. These figures are hardly compatible with the international health community’s stated goal of an “AIDS-free generation” [8,9]. In highly resourced settings, microepidemics of HIV still occur, particularly among gays, bisexuals, and men who have sex with men (MSM) [10]. HIV epidemics are expanding in two geographic regions in 2015—the Middle East/North Africa and Eastern Europe/Central Asia—largely due to challenges in implementing evidence-based HIV policies and programmes [2]. Even for the past decade in the US, almost 50,000 new cases recorded annually, two-thirds among MSM, has been a stable figure for years and shows no evidence of declining [1].While treatment scale-up, medical male circumcision [11], and the implementation of strategies to prevent mother-to-child transmission [12] have received global traction, systemic or topical ARV-based biomedical advances to prevent sexual acquisition of HIV have, as yet, made limited impressions on a population basis, despite their reported efficacy. Factors such as their adherence requirements, cost, potential for drug resistance, and long-term feasibility have restricted the appetite for implementation, even though these approaches may reduce HIV incidence in select populations.Already, several trials have shown that daily oral administration of the ARV tenofovir disoproxil fumarate (TDF), taken singly or in combination with emtricitabine, as PrEP by HIV-uninfected individuals, reduces HIV acquisition among serodiscordant couples (where one partner is HIV-positive and the other is HIV-negative) [13], MSM [14], at-risk men and women [15], and people who inject drugs [16,17] by between 44% and 75%. Long-acting injectable antiretroviral agents such as rilpivirine and cabotegravir, administered every two and three months, respectively, are also being developed for PrEP. All of these PrEP approaches are dependent on repeated HIV testing and adherence to drug regimens, which may challenge effectiveness in some populations and contexts.The widespread elimination of HIV will require the development of new, more potent prevention tools. Because HIV acquisition occurs subclinically, the elimination of HIV on a population basis will require a highly effective vaccine. Alternatively, if vaccine development is delayed, supplementary strategies may include long-acting pre-exposure antiretroviral cocktails and/or the administration of neutralizing antibodies through long-lasting parenteral preparations or the development of a “genetic immunization” delivery system, as well as scaling up delivery of highly effective regimens to eliminate mother-to-child HIV transmission (Fig 1).Open in a separate windowFig 1Medical interventions required to end the epidemic of HIV.Image credit: Glenda Gray.     

The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133

Following exposure to long‐wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole‐alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 cotranscribed genes. A two‐component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator (RR), Npun_F1278, was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain Δ1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, Δ1278 was incapable of producing scytonemin. A phenotypic characterization of Δ1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid‐soluble pigment complement of Δ1278 compared to the wildtype. The mutant, however, had a slower specific growth rate under white light and produced ~2.5‐fold more phycocyanin per cell under UVA than the wildtype. Since Δ1278 does not produce scytonemin, this study demonstrates that the RR gene, Npun_F1278, is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production.     

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky’s fixative and OsO₄ and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.