The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Effects of inbreeding on male function and self-fertility in the partially self-incompatible herb Campanula rapunculoides (Campanulaceae)

We examined the effect of inbreeding on fitness (through both male and female functions) and changes in self-fertility in the partially self-incompatible species Campanula rapunculoides. Individuals in natural populations of C. rapunculoides varied extensively in their strength of self-incompatibility (SI). We crossed 11 individuals that differed in their strength of SI to generate families with four levels of inbreeding (f = 0.0, 0.25, 0.5, and 0.75). Progeny were scored for three traits related to male fitness and for outcrossed and selfed seed production. Analyses of variance revealed significant inbreeding depression for the three male traits and seed set. Families with strong or weak SI differed in their response to inbreeding. Families with weak SI had lower levels of inbreeding depression for most traits than families with strong SI, but strong SI families had a greater increase in selfed seed set, but not self-fertility, with inbreeding. Finally, we found evidence of a significant linear response to inbreeding for all three male reproductive traits and outcrossed seed, indicating that inbreeding depression was primarily caused by partially or fully recessive deleterious alleles. Variation in genetic load was associated with variation in self-fertility, a finding that suggests an evolutionary role for partial self-fertility in natural populations of C. rapunculoides.     

Evaluation of the suchey–brooks method of age estimation in an Australian subpopulation using computed tomography of the pubic symphyseal surface

Despite the prominent use of the Suchey–Brooks (S–B) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential interpopulation error rates between geographical locations. This study assessed the accuracy of the S–B method to a contemporary adult population in Queensland, Australia and provides robust age parameters calibrated for our population. Three‐dimensional surface reconstructions were generated from computed tomography scans of the pubic symphysis of male and female Caucasian individuals aged 15–70 years (n = 195) in Amira® and Rapidform®. Error was analyzed on the basis of bias, inaccuracy and percentage correct classification for left and right symphyseal surfaces. Application of transition analysis and Chi‐square statistics demonstrated 63.9 and 69.7% correct age classification associated with the left symphyseal surface of Australian males and females, respectively, using the S–B method. Using Bayesian statistics, probability density distributions for each S–B phase were calculated, providing refined age parameters for our population. Mean inaccuracies of 6.77 (±2.76) and 8.28 (±4.41) years were reported for the left surfaces of males and females, respectively; with positive biases for younger individuals (<55 years) and negative biases in older individuals. Significant sexual dimorphism in the application of the S–B method was observed; and asymmetry in phase classification of the pubic symphysis was a frequent phenomenon. These results recommend that the S–B method should be applied with caution in medico‐legal death investigations of Queensland skeletal remains and warrant further investigation of reliable age estimation techniques. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

Evaluating Human Risk from Exposure to Alkylated PAHs in an Aquatic System

Polycyclic aromatic hydrocarbons (PAHs) are a large class of organic chemicals typically found as mixtures in the aquatic environment from natural, petrogenic, and pyrogenic sources. People can be exposed to PAHs through ingestion or dermal contact with contaminated sediments or through ingestion of finfish and shellfish exposed to contaminated sediments. Although more than 100 PAHs have been identified, human exposure and risk are commonly evaluated for 18 individual PAHs. Other PAHs, such as alkylated PAHs, likely contribute to biological activity of environmental PAH mixtures; however, insufficient toxicity data are available to quantify their potential risk. This article presents an initial evaluation of the potential for human health risk from exposure to alkylated PAHs in sediment and fish. Individual alkylated PAHs have been observed to have potentially mutagenic, tumor-promoting, or carcinogenic activity. However, except for 1-and 2-methylnaphthalene, insufficient toxicity data are available to quantify toxicity or cancer risk from exposure to individual alkylated PAHs or mixtures of alkylated PAHs. This article describes a proposed strategy to better understand the potential human health risk from exposure to alkylated PAHs. Implementation of this strategy will contribute to evaluations of human exposure to complex PAH mixtures in the environment.     

Differentiation of Serratia from Enterobacter on the Basis of Nucleoside Phosphotransferase Production

Precoated cellulose thin-layer chromatograms were used to detect the production of guanosine 5'-phosphate from guanosine and p-nitrophenylphosphate by whole-cell preparations. One-hundred per cent of Serratia (163 strains) and 84% of E. liquefaciens (15 of 18 strains) produced the nucleotide. All other Enterobacter (23 strains), Klebsiella (10 strains), and E. coli (10 strains) were negative for the production of this nucleotide. The entire test procedure could be carried out in 4 hr. It is proposed that E. liquefaciens is more closely related to Serratia than Enterobacter and that reclassification of these organisms should be investigated.     

A longitudinal study of medicaid coverage for tobacco dependence treatments in Massachusetts and associated decreases in hospitalizations for cardiovascular disease

Background

Insurance coverage of tobacco cessation medications increases their use and reduces smoking prevalence in a population. However, uncertainty about the impact of this coverage on health care utilization and costs is a barrier to the broader adoption of this policy, especially by publicly funded state Medicaid insurance programs. Whether a publicly funded tobacco cessation benefit leads to decreased medical claims for tobacco-related diseases has not been studied. We examined the experience of Massachusetts, whose Medicaid program adopted comprehensive coverage of tobacco cessation medications in July 2006. Over 75,000 Medicaid subscribers used the benefit in the first 2.5 years. On the basis of earlier secondary survey work, it was estimated that smoking prevalence declined among subscribers by 10% during this period.

Methods and Findings

Using claims data, we compared the probability of hospitalization prior to use of the tobacco cessation pharmacotherapy benefit with the probability of hospitalization after benefit use among Massachusetts Medicaid beneficiaries, adjusting for demographics, comorbidities, seasonality, influenza cases, and the implementation of the statewide smoke-free air law using generalized estimating equations. Statistically significant annualized declines of 46% (95% confidence interval 2%–70%) and 49% (95% confidence interval 6%–72%) were observed in hospital admissions for acute myocardial infarction and other acute coronary heart disease diagnoses, respectively. There were no significant decreases in hospitalizations rates for respiratory diagnoses or seven other diagnostic groups evaluated.

Conclusions

Among Massachusetts Medicaid subscribers, use of a comprehensive tobacco cessation pharmacotherapy benefit was associated with a significant decrease in claims for hospitalizations for acute myocardial infarction and acute coronary heart disease, but no significant change in hospital claims for other diagnoses. For low-income smokers, removing the barriers to the use of smoking cessation pharmacotherapy has the potential to decrease short-term utilization of hospital services. Please see later in the article for the Editors'' Summary     

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.     

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.