To evaluate the efficacy of saracatinib (AZD0530), an oral Src inhibitor, in colorectal cancer (CRC) and to identify biomarkers that predict antitumor activity.
Twenty-three CRC cell lines were exposed to saracatinib, and baseline gene expression profiles of three sensitive and eight resistant cell lines in vitro and in vivo were used to predict saracatinib sensitivity in an independent group of 10 human CRC explant tumors using the gene array K-Top Scoring Pairs (K-TSP) method. In addition, fluorescence in situ hybridization (FISH) and immunoblotting determined both Src gene copy number and activation of Src, respectively.
Two of 10 explant tumors were determined to be sensitive to saracatinib. The K-TSP classifier (TOX>GLIS2, TSPAN7>BCAS4, and PARD6G>NXN) achieved 70% (7 of 10) accuracy on the test set. Evaluation of Src gene copy number by FISH showed a trend toward significance (P = 0.066) with respect to an increase in Src gene copy and resistance to saracatinib. Tumors sensitive to saracatinib showed an increase in the activation of Src and FAK when compared with resistant tumors.
Saracatinib significantly decreased tumor growth in a subset of CRC cell lines and explants. A K-TSP classifier (TOX>GLIS2, TSPAN7>BCAS4, and PARD6G>NXN) was predictive for sensitivity to saracatinib. In addition, increased activation of the Src pathway was associated with sensitivity to saracatinib. These results suggest that FISH, a K-TSP classifier, and activation of the Src pathway have potential in identifying CRC patients that would potentially benefit from treatment with saracatinib.
The predominance of circulating and unique recombinant forms (URFs) of Human Immunodeficiency Virus Type 1 (HIV-1) in Cameroon suggests that dual infection occurs frequently in this region. Despite the potential impact of these infections on the evolution of HIV diversity, relatively few have been detected. The failure to detect dual infections may be attributable to the laborious and costly sequence analysis involved in their identification. As such, there is a need for a cost-effective, more rapid method to efficiently distinguish this subset of HIV-positive individuals, particularly in regions where HIV diversity is broad. In the present study, the heteroduplex assay (HDA) was developed to detect dual HIV-1 infection. This assay was validated on sequential specimens obtained from 20 HIV+ study subjects, whose single or dual infection status was determined by standard sequence analysis. By mixing gag fragments amplified from the sequential specimens from each study subject in HDA reactions, it was shown that single and dual infection status correlated with the absence and presence, respectively, of heteroduplex bands upon gel electrophoresis. Therefore, this novel assay is capable of identifying dual infections with a sensitivity and specificity equivalent to that of sequence analysis. Given the impact of dual infection on viral recombination and diversity, this simple technique will be beneficial to understanding HIV-1 evolution within an individual, as well as at a population level, in West-Central Africa and globally.
HIV-1; Dual infection; Heteroduplex assay
Rapid 21st-century climate change may lead to large population decreases and extinction in tropical montane cloud forest species in the Andes. While prior research has focused on species migrations per se, ecotones may respond to different environmental factors than species. Even if species can migrate in response to climate change, if ecotones do not they can function as hard barriers to species migrations, making ecotone migrations central to understanding species persistence under scenarios of climate change. We examined a 42-year span of aerial photographs and high resolution satellite imagery to calculate migration rates of timberline–the grassland-forest ecotone–inside and outside of protected areas in the high Peruvian Andes. We found that timberline in protected areas was more likely to migrate upward in elevation than in areas with frequent cattle grazing and fire. However, rates in both protected (0.24 m yr−1) and unprotected (0.05 m yr−1) areas are only 0.5–2.3% of the rates needed to stay in equilibrium with projected climate by 2100. These ecotone migration rates are 12.5 to 110 times slower than the observed species migration rates within the same forest, suggesting a barrier to migration for mid- and high-elevation species. We anticipate that the ecotone will be a hard barrier to migration under future climate change, leading to drastic population and biodiversity losses in the region unless intensive management steps are taken.
Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex® Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%–100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.
HIV; immunology; microbiology; SIV
Barn Swallows (Hirundo rustica) breed almost exclusively in the Northern Hemisphere. However, since the early 1980's, a small disjunct breeding population has become established in eastern Argentina, presumably by birds previously derived from those breeding in North America. Currently, it is unknown where these individuals go following breeding and how they have adjusted to a reversal in phenology. Their austral wintering period corresponds to the breeding period of the northern ancestral population and so they can potentially return to these more traditional breeding sites or they may occupy other South American wintering regions left vacant by conspecifics returning to the Northern Hemisphere.
We used a three-isotope (δ13C, δ15N, δ2H) approach to investigate potential wintering areas in Central and South America of individuals breeding in Argentina. Feather isotope values differed from those expected and measured at local breeding sites in Argentina indicating molt after the austral breeding period and away from the breeding grounds. Potential molting origins were identified applying likelihood-based assignment methods to a δ2H isoscape for South America and dichotomous prior information on the distribution of C3 and C4 vegetation types based on modeled vegetation-δ13C values. Barn Swallows now breeding in Argentina have changed their migratory behavior but presumably use the same cues as those used by the ancestral population, molting their feathers during the austral winter, likely in north-eastern South America.
For HIV recombination to occur, the RNAs from two infecting strains within a cell must dimerize at the dimerization initiation site (DIS). We examined the sequence identity at the DIS (697–731 bp, Hxb2 numbering engine) in patients superinfected with concordant HIV-1 strains and compared them to those with discordant strains. Viral RNA in sequential plasma from four subjects superinfected with subtype-discordant and two subjects superinfected with subtype-concordant HIV-1 strains was extracted, amplified (5′ LTR-early gag: 526–1200 bp, Hxb2 numbering engine), sequenced, and analyzed to determine their compatibility for dimerization in vivo. The concordant viruses infecting the two subjects exhibited identical sequences in the 35-bp-long DIS region while sequences from the discordant viruses revealed single nucleotide changes that were located in the DIS loop (715 bp), its flanking nucleotides (710 bp and 717 bp), and the DIS stem (719 bp). Evidence from in vitro experiments demonstrates that these in vivo changes identified can abolish dimerization and reduce recombination frequency. Therefore, these results revealing differences in the DIS of discordant strains versus the similarity noted for the concordant strains may contribute to the differences in the frequency of recombination in patients superinfected with such HIV-1 variants.
Src tyrosine kinases are overexpressed in pancreatic cancers, and the oral Src inhibitor saracatinib has shown antitumor activity in preclinical models of pancreas cancer. We performed a CTEP-sponsored Phase II clinical trial of saracatinib in previously treated pancreas cancer patients, with a primary endpoint of 6-month survival. A Simon MinMax two-stage phase II design was used. Saracatinib (175 mg/day) was administered orally continuously in 28-day cycles. In the unselected portion of the study, 18 patients were evaluable. Only two (11%) patients survived for at least 6 months, and three 6-month survivors were required to move to second stage of study as originally designed. The study was amended as a biomarker-driven trial (leucine rich repeat containing protein 19 [LRRC19] > insulin-like growth factor-binding protein 2 [IGFBP2] “top scoring pairs” polymerase chain reaction [PCR] assay, and PIK3CA mutant) based on preclinical data in a human pancreas tumor explant model. In the biomarker study, archival tumor tissue or fresh tumor biopsies were tested. Biomarker-positive patients were eligible for the study. Only one patient was PIK3CA mutant in a 3′ untranslated region (UTR) portion of the gene. This patient was enrolled in the study and failed to meet the 6-month survival endpoint. As the frequency of biomarker-positive patients was very low (<3%), the study was closed. Although we were unable to conclude whether enriching for a subset of second/third line pancreatic cancer patients treated with a Src inhibitor based on a biomarker would improve 6-month survival, we demonstrate that testing pancreatic tumor samples for a biomarker-driven, multicenter study in metastatic pancreas cancer is feasible.
Biomarker; clinical trial; pancreas cancer; Src
The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.
Recent studies have demonstrated that both the potency and breadth of the humoral anti-HIV-1 immune response in generating neutralizing antibodies (nAbs) against heterologous viruses are significantly enhanced after superinfection by discordant HIV-1 subtypes, suggesting that repeated exposure of the immune system to highly diverse HIV-1 antigens can significantly improve anti-HIV-1 immunity. Thus, we investigated whether sequential plasma from these subjects superinfected with discordant HIV-1 subtypes, who exhibit broad nAbs against heterologous viruses, also neutralize their discordant early autologous viruses with increasing potency. Comparing the neutralization capacities of sequential plasma obtained before and after superinfection of 4 subjects to those of matched plasma obtained from 4 singly infected control subjects, no difference in the increase in neutralization capacity was observed between the two groups (p = 0.328). Overall, a higher increase in neutralization over time was detected in the singly infected patients (mean change in IC50 titer from first to last plasma sample: 183.4) compared to the superinfected study subjects (mean change in IC50 titer from first to last plasma sample: 66.5). Analysis of the Breadth-Potency Scores confirmed that there was no significant difference in the increase in superinfected and singly infected study subjects (p = 0.234). These studies suggest that while superinfection by discordant subtypes induces antibodies with enhanced neutralizing breadth and potency against heterologous viruses, the potency to neutralize their autologous viruses is not better than those seen in singly infected patients.
Defects in cilia and basal bodies function are linked to ciliopathies, which result in kidney cyst formation. Recently, cell division defects have been observed in cystic kidneys, but the underlying mechanisms of such defects remain unclear. Wtip is an LIM domain protein of the Ajuba/Zyxin family, but its role in ciliogenesis during embryonic development has not been previously described. We report Wtip is enriched in the basal body and knockdown of wtip leads to pronephric cyst formation, cloaca malformation, hydrocephalus, body curvature, and pericardial edema. We additionally show that wtip knockdown embryos display segment-specific defects in the pronephros: mitotic spindle orientation defects are observed only in the anterior and middle pronephros; cloaca malformation is accompanied by a reduced number of ciliated cells; and ciliated cells lack the striated rootlet that originates from basal bodies, which results in a lack of cilia motility. Our data suggest that loss of Wtip function phenocopies Vangl2 loss of function, a core planar cell polarity (PCP) protein located in the basal body protein. Furthermore, we demonstrate that wtip and vangl2 interact genetically. Taken together, our results indicate that in zebrafish, Wtip is required for mitotic spindle orientation in the anterior and middle of the pronephros, cloaca morphogenesis, and PCP, which may underlie the molecular etiology of ciliopathies.
Ciliopathies; Mitotic spindle; Basal body; Zebrafish
Little is known regarding the likelihood of recombination between any given pair of nonidentical HIV-1 viruses in vivo. The present study analyzes the HIV-1 quasispecies in the C1C2 region of env, the vif-vpr-vpu accessory gene region, and the reverse transcriptase region of pol. These sequences were amplified from samples obtained sequentially over a 12- to 33-month period from five dually HIV-1-infected subjects. Analysis of an average of 248 clones amplified from each subject revealed no recombinants within the three loci studied of the subtype-discordant infecting strains, whose genetic diversity was >11% in env. In contrast, two subjects who were initially coinfected by two subtype-concordant variants with genetic diversity of 7.4% in env were found to harbor 10 unique recombinants of these strains, as exhibited by analysis of the env gene. The frequent recombination observed among the subtype-concordant strains studied herein correlates with prior sequence analyses that have commonly found higher rates of recombination at loci bearing the most conserved sequences, demonstrating an important role for sequence identity in HIV-1 recombination. Viral load analysis revealed that the samples studied contained an average of 8125 virus copies/ml (range, 882–31,626 copies/ml), signifying that the amount of viral RNA in the samples was not limiting for studying virus diversity. These data reveal that recombination between genetically distant strains may not be an immediate or common outcome to dual infection in vivo and suggest critical roles for viral and host factors such as viral fitness, virus diversity, and host immune responses that may contribute to limiting the frequency of intersubtype recombination during in vivo dual infection.
High-risk cohorts in East Africa and the United States show rates of dual HIV-1 infection—the concomitant or sequential infection by two HIV-1 strains—of 50% to 100% of those of primary infection, and our normal-risk HIV-positive cohort in Cameroon exhibits a rate of dual infection of 11% per year, signifying that these infections are not exceptional. Little is known regarding the effect of dual infections on host immunity, despite the fact that they provide unique opportunities to investigate how the immune response is affected when challenged with diverse HIV-1 antigens. Using heterologous primary isolates, we have shown here that dual HIV-1 infection by genetically distant strains correlates with significantly increased potency and breadth of the anti-HIV-1 neutralizing antibody response. When the neutralization capacities of sequential plasma obtained before and after the dual infection of 4 subjects were compared to those of matched plasma obtained from 23 singly infected control subjects, a significant increase in the neutralization capacity of the sequential sample was found for 16/28 dually infected plasma/virus pairs, while only 4/159 such combinations for the control subjects exhibited a significant increase (P < 0.0001). Similarly, there was a significant increase in the plasma dilution capable of neutralizing 50% of virus (IC50) for 18/24 dually infected plasma/virus pairs, while 0/36 controls exhibited such an increase (P < 0.0001). These results demonstrate that dual HIV-1 infection broadens and strengthens the anti-HIV-1 immune response, suggesting that vaccination schemes that include polyvalent, genetically divergent immunogens may generate highly protective immunity against any HIV-1 challenge strain.
West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (~40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.
HIV-1 Diversity; Rural Cameroon; phylogenetics
The most common first-line, highly active anti-retroviral therapy (HAART) received by individuals infected with HIV-1 in Cameroon is the combination therapy Triomune, comprised of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-NRTI (NNRTI). To examine the efficacy of these drugs in Cameroon, where diverse non-B HIV-1 subtypes and recombinant viruses predominate, the reverse transcriptase (RT) viral sequences in patient plasma were analyzed for the presence of mutations that confer drug resistance. Forty-nine HIV-1-positive individuals were randomly selected from those receiving care in HIV/AIDS outpatient clinics in the South-West and North-West Regions of Cameroon. Among the 28 patients receiving HAART, 39% (11/28) had resistance to NRTIs, and 46% (13/28) to NNRTIs after a median of 12 months from the start of therapy. Among those with drug-resistance mutations, there was a median of 14 months from the start of HAART, versus 9 months for those without; no difference was observed in the average viral load (10,997 copies/ml vs. 8,056 copies/ml). In contrast, drug-naïve individuals had a significantly higher average viral load (27,929 copies/ml) than those receiving HAART (9,527 copies/ml). Strikingly, among the 21 drug-naïve individuals, 24% harbored viruses with drug-resistance mutations, suggesting that HIV-1 drug-resistant variants are being transmitted in Cameroon. Given the high frequency of resistance mutations among those on first-line HAART, coupled with the high prevalence of HIV-1 variants with drug-resistance mutations among drug-naïve individuals, this study emphasizes the need for extensive monitoring of resistance mutations and the introduction of a second-line HAART strategy in Cameroon.
drug-resistance mutations; HIV-1; NRTI; NNRTI; HAART; drug naïve
West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (∼40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.
HIV-1 Diversity; Rural Cameroon; phylogenetics
BAK1 and BKK1 are two functionally redundant leucine-rich repeat receptor-like protein kinases (LRR-RLKs) involved in brassinosteroid signal transduction by their direct interactions with the BR receptor, BRI1. Recent studies from our group and others indicated that the two RLKs also play critical roles in regulating pathogen-related and pathogen-unrelated cell-death controls. Genetic data suggest that the two kinases are essential for plant survival because the double mutants show spontaneous cell-death and seedling lethality phenotypes. Physiological analyses further suggest that the cell-death of the double mutant is triggered by the light, as dark-grown seedlings do not show any cell-death symptoms. These observations indicate that BAK1 and BKK1 regulate a novel signaling pathway to detoxify or to limit the production of a yet unknown toxin/toxins produced by plants under light conditions.
receptor-like kinases; cell-death; light; reactive oxygen species