Efficient cell delivery mediated by lipid‐specific endosomal escape of supercharged branched peptides

Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell‐permeable prototypes with increasing charge density. We establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. Cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. Derivatives with multiple TAT branches induce the leakage of BMP‐containing lipid bilayers, liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not cause leakage, induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in charge density of these branched structures leads to the emergence of lipid specific membrane‐disrupting and cell‐penetrating activities.      

Fire effects and ecological recovery pathways of tropical montane cloud forests along a time chronosequence

Tropical montane cloud forests (TMCFs) harbour high levels of biodiversity and large carbon stocks. Their location at high elevations make them especially sensitive to climate change, because a warming climate is enhancing upslope species migration, but human disturbance (especially fire) may in many cases be pushing the treeline downslope. TMCFs are increasingly being affected by fire, and the long‐term effects of fire are still unknown. Here, we present a 28‐year chronosequence to assess the effects of fire and recovery pathways of burned TMCFs, with a detailed analysis of carbon stocks, forest structure and diversity. We assessed rates of change of carbon (C) stock pools, forest structure and tree‐size distribution pathways and tested several hypotheses regarding metabolic scaling theory (MST), C recovery and biodiversity. We found four different C stock recovery pathways depending on the selected C pool and time since last fire, with a recovery of total C stocks but not of aboveground C stocks. In terms of forest structure, there was an increase in the number of small stems in the burned forests up to 5–9 years after fire because of regeneration patterns, but no differences on larger trees between burned and unburned plots in the long term. In support of MST, after fire, forest structure appears to approximate steady‐state size distribution in less than 30 years. However, our results also provide new evidence that the species recovery of TMCF after fire is idiosyncratic and follows multiple pathways. While fire increased species richness, it also enhanced species dissimilarity with geographical distance. This is the first study to report a long‐term chronosequence of recovery pathways to fire suggesting faster recovery rates than previously reported, but at the expense of biodiversity and aboveground C stocks.     

Do differences in plant and flower age change mating patterns and alter offspring fitness in Raphanus sativus (Brassicaceae)?

When more pollen is present on stigmas than needed to fertilize all ovules, selection among pollen grains may occur due to effects of both pollen donors and maternal plants. We asked whether increasing plant age and flower age, two changes in maternal condition, altered the pattern of seed paternity after mixed pollination. We also asked whether changes in seed paternity affected offspring success in an experimental garden. While flower age did not affect seed paternity, there was a dramatic shift in pollen donor performance as plants aged. These differences were seen in the offspring as well, where the offspring of one pollen donor, which sired more seeds on young plants, flowered earlier in the season, and the offspring of another pollen donor, which sired more seeds on old plants, flowered later in the season. Thus, change in maternal condition resulted in altered seed paternity, perhaps because the environment for pollen tube growth was different. The pattern of seed paternity and offspring performance suggests that pollen donors may show temporal specialization.     

Lapatinib,a dual HER1/HER2 tyrosine kinase inhibitor,augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2‐driven cancer cell growth

The ultimate biological and clinical meaning of shed HER2 extracellular domain (ECD) has remained largely unclear until recently. Oversecretion of soluble HER2 ECD has been shown to inhibit growth of HER2‐overexpressing cancer cells by promoting HER2 ECD dimerization with HER transmembrane receptors thus impairing their cross‐tyrosine phosphorylation and decreasing their activation status. HER2‐targeted drugs capable to enhance the occurrence of basal HER2 ECD shedding but simultaneously preventing formation of truncated cell membrane‐bound HER2 intracellular fragment, which exhibits an undesirable constitutive kinase activity, might be extremely efficient at managing HER2‐positive cancer disease. The dual HER1/HER2 Tyrosine Kinase inhibitor lapatinib, which works intracellularly and directly targets the TK domain of HER2, drastically augments basal shedding of HER2 ECD to inhibit HER2‐driven cancer cell growth. Lapatinib treatment significantly augments the concentration of the inactive (unphosphorylated) form of HER2 protein at the tumor cell membrane and promotes an exacerbated HER2 ECD shedding to the extracellular milieu of HER2‐overexpressing cancer cells. Exacerbated sensitivity of trastuzumab‐resistant cancer cells, which contain nearly undetectable levels of soluble HER2 ECD when compared with trastuzumab‐sensitive parental cells to lapatinib‐induced cell growth inhibition, takes place when lapatinib treatment fully restores high levels of basal HER2 ECD shedding. The dramatic augmentation of HER2 ECD shedding that occurs upon treatment of with lapatinib is fully suppressed in lapatinib‐refractory HER2‐positive cells. These findings, altogether, may provide crucial insights concerning clinical studies aimed to accurately describe HER2 ECD as a potential predictor of response or resistance to the HER2‐targeted drugs trastuzumab and lapatinib. J. Cell. Physiol. 226: 52–57, 2010. © 2010 Wiley‐Liss, Inc.     

CD40 Gene Silencing Reduces the Progression of Experimental Lupus Nephritis Modulating Local Milieu and Systemic Mechanisms

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders.     

Protein Array Profiling of Tic Patient Sera Reveals a Broad Range and Enhanced Immune Response against Group A Streptococcus Antigens

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.     

Epidemiology of invasive pneumococcal disease in older people in Spain (2007-2009): implications for future vaccination strategies

Background

Recently, the 13-valent pneumococcal conjugate vaccine (PCV13) has been recommended for adults. We analyzed the epidemiology of invasive pneumococcal disease (IPD) in older adults in Spain before PCV13 introduction.

Methodology/Principal Findings

IPD episodes, defined as clinical findings together with an invasive pneumococcal isolate, were prospectively collected from patients aged over 65 years in three hospitals in Spain from 2007 to 2009. A total of 335 IPD episodes were collected. Pneumonia was the main clinical syndrome, while chronic obstructive pulmonary disease, diabetes mellitus and cancer were the main underlying diseases. Pneumococcal isolates were serotyped and the molecular typing was performed by PFGE/MLST. PCV13 serotypes accounted for 59.3% of isolates, the most prevalent being serotypes 19A (15.1%), 3 (9.6%), 7F (7.5%), 14 (6.9%) and 1 (5.4%). The most frequent non-PCV13 serotypes were serotypes 16F (4.5%), 22F (3.6%), 24F (3.3%) and 6C (2.1%). The most common genotypes were CC230 (8.5%, serotypes 19A and 24F), CC156 (8.2%, serotypes 9V and 14), ST191 (7.9%, serotype 7F), CC260 (6.6%, serotype 3), ST306 (5.2%, serotype 1), CC30 (4.6%, serotype 16F) and ST433 (3.6%, serotype 22F). Comparing the 335 IPD isolates to 174 invasive pneumococci collected at the same hospitals in 1999–2000, PCV7 serotypes decreased (45.4% vs 18.4%,p<0.001), non-PCV7 serotypes included in PCV13 increased (26.4% vs 41.0%,p = 0.001) and two non-PCV13 serotypes increased (serotype 6C 0% vs 2.1%, p = 0.05; serotype 24F 0.6% vs 3.3%, p = 0.04,).

Conclusion

In our older adult population two serotypes (19A and 3) included in PCV13 accounted for about a quarter of IPD episodes in people ≥65 years. Non-PCV13 emerging serotypes should be carefully monitored in future surveillance studies.     

Modeling distributions of disjunct populations of the Sierra Madre Sparrow

ABSTRACT Sierra Madre Sparrows (Xenospiza baileyi) are among the least known of all bird species in Mexico. Recent surveys have discovered previously unknown populations and the current known distribution of Sierra Madre Sparrows consists of two populations separated by >800 km. We used available distributional information to develop ecological niche models that (1) predict much of the distribution potential of the species, (2) establish that the broad disjunction separating the two populations has ecological correlates that appear to be important to the distributional of these sparrows, and (3) illustrate the extremely restricted ecological distribution of the species. We used two sets of climatic and topographic variables, with one including all 22 variables available and the second with only six variables that were positively related to quality of distributional models. Although indications of differences between the two sets of populations were found based on the full 22‐dimensional environmental dataset, such a highly dimensional analysis is vulnerable to over‐fitting; models based on the reduced dataset indicated that the two populations occur in areas with similar ecological conditions. Our models also suggest that southern population of Sierra Madre Sparrows covers most of their potential range in that region. The potential range of the northern population, however, extends beyond known points of occurrence. To clarify the distribution of Sierra Madre Sparrows and evaluate their status and conservation opportunities, detailed searches for additional populations in areas identified by the model are needed.