Differential growth identified in salamander larvae half‐sib cohorts: Survival strategy?

In this study we describe the growth of several different larval cohorts (i.e. half-siblings of the same mother born on the same day) of a rare, xeric-adapted salamander Salamandra s. infraimmaculata Martens, 1885, under constant density and food conditions from birth to metamorphosis. The larvae spend the critical first phase of their lives in water, mostly in temporary ponds. Age and weight at metamorphosis were highly affected by varying food conditions. We have identified six different growth modes that these larvae use, both fast growing and slow growing. Each larval cohort was found to use 2-4 different such growth modes regardless of their initial weight. Fast growing modes (I-III) will enable larvae to survive dry years, and metamorphose bigger. Slow growing modes (IV-VI), used by 8% of the larval population, will enable survival only in rainy years. These last growth modes effect differential temporal dispersal in wet years by delaying the emergence of postmetamorphs onto land. Distribution of growth modes in the larval population is affected by food but not by density conditions. Late-born, fast-growing larvae will have an advantage in dry years being able to metamorphose and disperse, whereas the slow-growing larvae will survive only in wet years.     

Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in Escherichia coli

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.     

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.     

Exogenously applied 20-hydroxyecdysone increases the net photosynthetic rate but does not affect the photosynthetic electron transport or the content of photosynthetic pigments in Tetragonia tetragonioides L.

Phytoecdysteroids are steroid compounds present in many plant species (sometimes in rather large amounts), but their biological role is still far from being clear. We have found that the exogenous application of 20-hydroxyecdysone (20E) to leaves of Tetragonia tetragonioides L. causes stimulation of its net photosynthetic rate (P _N) but does not positively affect the photosynthetic electron transport or the content of photosynthetic pigments. The increase in P _N was observed shortly after 20E treatment and was statistically significant during the 4th and 6th hours after treatment but not later, which could be perhaps caused by a strictly short-term window of opportunity for ecdysteroids to significantly affect photosynthetic processes. To our knowledge, these results are the first to suggest a new potential biological function of phytoecdysteroids—regulation of photosynthesis.     

Amino-Acid Co-Variation in HIV-1 Gag Subtype C: HLA-Mediated Selection Pressure and Compensatory Dynamics

Background

Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C.

Methodology/Principal Findings

Individuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites.

Conclusions/Significance

Clusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints.     

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

Imaging characteristics, tissue distribution, and spread of a novel oncolytic vaccinia virus carrying the human sodium iodide symporter

Introduction

Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS.

Methods

GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide ¹³¹I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via ¹²⁴I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both ¹²⁴I-PET and 99m-technecium gamma-scintigraphy.

Results

GLV-1h153 successfully facilitated time-dependent intracellular uptake of ¹³¹I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10⁹ plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via ¹²⁴I-PET and 99m-technecium-scintigraphy.

Conclusion

GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.     

When Does Choice of Accuracy Measure Alter Imputation Accuracy Assessments?

Imputation, the process of inferring genotypes for untyped variants, is used to identify and refine genetic association findings. Inaccuracies in imputed data can distort the observed association between variants and a disease. Many statistics are used to assess accuracy; some compare imputed to genotyped data and others are calculated without reference to true genotypes. Prior work has shown that the Imputation Quality Score (IQS), which is based on Cohen’s kappa statistic and compares imputed genotype probabilities to true genotypes, appropriately adjusts for chance agreement; however, it is not commonly used. To identify differences in accuracy assessment, we compared IQS with concordance rate, squared correlation, and accuracy measures built into imputation programs. Genotypes from the 1000 Genomes reference populations (AFR N = 246 and EUR N = 379) were masked to match the typed single nucleotide polymorphism (SNP) coverage of several SNP arrays and were imputed with BEAGLE 3.3.2 and IMPUTE2 in regions associated with smoking behaviors. Additional masking and imputation was conducted for sequenced subjects from the Collaborative Genetic Study of Nicotine Dependence and the Genetic Study of Nicotine Dependence in African Americans (N = 1,481 African Americans and N = 1,480 European Americans). Our results offer further evidence that concordance rate inflates accuracy estimates, particularly for rare and low frequency variants. For common variants, squared correlation, BEAGLE R², IMPUTE2 INFO, and IQS produce similar assessments of imputation accuracy. However, for rare and low frequency variants, compared to IQS, the other statistics tend to be more liberal in their assessment of accuracy. IQS is important to consider when evaluating imputation accuracy, particularly for rare and low frequency variants.