Developing sporophytes transition from an inducible to a constitutive ecological strategy of desiccation tolerance in the moss Aloina ambigua: effects of desiccation on fitness

Background and Aims Two ecological strategies of desiccation tolerance exist in plants, constitutive and inducible. Because of difficulties in culturing sporophytes, very little is known about desiccation tolerance in this generation and how desiccation affects sexual fitness.Methods Cultured sporophytes and vegetative shoots from a single genotype of the moss Aloina ambigua raised in the laboratory were tested for their strategy of desiccation tolerance by desiccating the shoot–sporophyte complex and vegetative shoots at different intensities, and comparing outcomes with those of undried shoot–sporophyte complexes and vegetative shoots. By using a dehardened clonal line, the effects of field, age and genetic variance among plants were removed.Key Results The gametophyte and embryonic sporophyte were found to employ a predominantly inducible strategy of desiccation tolerance, while the post-embryonic sporophyte was found to employ a moderately constitutive strategy of desiccation tolerance. Further, desiccation reduced sporophyte fitness, as measured by sporophyte mass, seta length and capsule size. However, the effects of desiccation on sporophyte fitness were reduced if the stress occurred during embryonic development as opposed to postembryonic desiccation.Conclusions The effects of desiccation on dehardened sporophytes of a bryophyte are shown for the first time. The transition from one desiccation tolerance strategy to the other in a single structure or generation is shown for only the second time in plants and for the first time in bryophytes. Finding degrees of inducible strategies of desiccation tolerance in different life phases prompts the formulation of a continuum hypothesis of ecological desiccation tolerance in mosses, where desiccation tolerance is not an either/or phenomenon, but varies in degree along a gradient of ecological inducibility.     

Inhibition of human cytochrome p450 1b1 further clarifies its role in the activation of dibenzo[a,l]pyrene in cells in culture

Metabolic activation and DNA adduct formation of the carcinogenic aromatic hydrocarbon dibenzo[a,l]pyrene (DBP) was investigated in human mammary carcinoma MCF-7 cells and human cytochrome P450 (CYP) 1B1-expressing Chinese hamster V79 cells in culture. It has been shown that DBP is metabolically activated to DNA-binding diol epoxides both in vitro and in vivo. To further establish the role of human CYP1B1 in the activation of DBP, both cell lines were cotreated with DBP and a selective chemical inhibitor of CYP1B1, 2,4,3' ,5'-tetramethoxy-stilbene (TMS). Results from DBP-DNA adduct analyses revealed the complete inhibition of DNA binding when cells were cotreated with DBP and TMS in comparison to DBP alone. Inactivation of CYP1B1 by TMS was also demonstrated through a decrease in the 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes isolated from these cells. Emodin, 3-methyl-1,6,8-trihydroxyanthraquinone, an active ingredient of an herb, has been recently shown of being able to induce CYP1 gene expression. Examination of human CYP1B1 induction and EROD activity confirmed an increase in protein levels upon cotreatment with emodin and DBP. Despite increases in protein levels and enzyme activity, there was no significant change in DBP-DNA binding levels at very low substrate concentrations (17 nM). The data obtained in this study emphasize the central role of CYP1B1 in the activation of DBP in human cells in culture.     

Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors

Background

Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal) pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations).

Methods

Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.

Results

We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48) and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value <0.01), across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% (p-value < 10–15), supporting clonality. In the second pair, no common mutations of 184 variants were validated (p-value >0.99), supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.

Conclusion

We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.     

An experimental demonstration of the cost of sex and a potential resource limitation on reproduction in the moss Pterygoneurum (Pottiaceae)

The cost of sexual reproduction is incurred when the current reproductive episode contributes to a a decline in future plant performance. To test the hypotheses that a trade-off exists between current sexual reproduction and subsequent clonal regeneration and that resources limit reproduction and regeneration, plants of the widespread moss Pterygoneurum ovatum were subjected to induced sporophytic abortion, upper leaf removal, and nutrient amendment treatments. Sexually reproducing plants were slower or less likely to produce regenerative structures (protonemata or shoots) and produced fewer regenerative tissue areas or structures. The ability and the timeline to reproduce sexually and regenerate clonally were unaffected by an inorganic nutrient amendment. However, when leaves subtending the sporophyte were removed, the sporophytes were less likely to mature, tended to take a longer time to mature, and were smaller compared to sporophytes from shoots with a full complement of upper leaves. Our findings indicate that plants investing in sexual reproduction suffer a cost of decreased clonal regeneration and indicate that sporophyte maturation is resource-limited, with upper leaves contributing to the nutrition of the sporophyte. This study represents only the second explicit experimental demonstration of a trade-off between sexual and asexual reproduction in bryophytes.     

Histoplasmosis—A Review of Three Cases Studied in San Diego County

Three cases of histoplasmosis, a disease seldom reported in California, were diagnosed clinically by the authors in San Diego County. It is probable that there is a higher incidence of this disease in California than is at present recognized.Travel history, histoplasmin skin testing and serologic studies for mycotic infection are important in the diagnosis. Cultures of secretions and biopsy material are of great value if positive; but negative cultures (at least in non-endemic areas) do not rule out the disease. Travel and migration to and from endemic areas present opportunities for this disease to constitute a diagnostic problem far from the geographic area in which the disease was acquired.Although usually benign, histoplasmosis may be severe in the acute state, may disseminate or may be chronically active and progressive. Amphotericin B is the only effective chemotherapeutic agent and it is usually reserved for these forms of the disease.     

17α-Methyl testosterone is a competitive inhibitor of aromatase activity in Jar choriocarcinoma cells and macrophage-like THP-1 cells in culture

17α-Methyl testosterone is a synthetic androgen with affinity for the androgen receptor. 17α-Methyl testosterone is used widely as a component of hormone replacement therapy. Previous reports have indicated that contrary to testosterone, 17α-methyl testosterone is not aromatized. However, 17α-methyl testosterone still could affect local estrogen formation by regulating aromatase expression or by inhibiting aromatase action. Both possibilities have important clinical implications. To evaluate the effect of 17α-methyl testosterone on the expression and activity of aromatase, we tested the choriocarcinoma Jar cell line, a cell line that express high levels of P₄₅₀ aromatase, and the macrophage-like THP-1 cells, which express aromatase only after undergoing differentiation. We found that in both cell lines, 17α-methyl testosterone inhibits aromatase activity in a dose-related manner. The curve of inhibition parallels that of letrozole and gives complete inhibition at 10⁻⁴ M 17α-methyl testosterone, determined by the tritium release assay. 17α-Methyl testosterone does not have detectable effects on aromatase RNA and protein expression by Jar cells. Undifferentiated THP-1 cells had no aromatase activity and showed no effect of 17α-methyl testosterone, but differentiated THP-1 (macrophage-like) cells had a similar inhibition of aromatase activity by 17α-methyl testosterone to that seen in Jar cells. The Lineweaver–Burke plot shows 17α-methyl testosterone to be a competitive aromatase inhibitor. Our results show for the first time that 17α-methyl testosterone acts as an aromatase inhibitor. These findings are relevant for understanding the effects of 17α-methyl testosterone as a component of hormone replacement therapy. 17α-Methyl testosterone may, as a functional androgen and orally active steroidal inhibitor of endogenous estrogen production, also offer special possibilities for the prevention/treatment of hormone-sensitive cancers.     

A graph spectral analysis of the structural similarity network of protein chains

We present a simple method for the analysis of large networks based on their graph spectral properties. One of the advantages of this method is that it uses a single numerical computation to identify subclusters in a connected graph, which can significantly simplify the complexity involved in analyzing large graphs. This is illustrated using a network of protein chains constructed on the basis of their structural similarities. The large-scale network properties and the cluster and subcluster organization of the protein chain network are presented. We summarize the results of structural and functional analyses of the nodes present in these clusters and elucidate the implications of structural similarity in the protein chain universe.     

The gene CSTF2T,encoding the human variant CstF-64 polyadenylation protein tauCstF-64, lacks introns and may be associated with male sterility

Messenger RNA polyadenylation in male germ cells does not seem to require the AAUAAA polyadenylation signal required in all other cell types. To account for this difference, we found a variant form of the polyadenylation protein, the 64,000 Mr protein of the cleavage stimulation factor (CstF-64), in mouse meiotic and postmeiotic germ cells. This protein is a candidate to alter polyadenylation in those cells. More recently, we reported the cloning from mouse pachytene spermatocytes of mouse tauCstF-64 (gene symbol Cstf2t), which is a homolog of CstF-64 fitting the criteria we expected for the variant CstF-64 protein. Here we report the cloning and mapping of the human ortholog of mouse tauCstF-64. The human tauCstF-64 cDNA (gene symbol CSTF2T) is 2324 bp in length and encodes a protein of 616 amino acids (64,442.90 Da). Although most highly related to mouse tauCstF-64 (89.8% identity), human tauCstF-64 is also related to the human and mouse somatic CstF-64 (74.9% and 73.4% identity, respectively). Alignment of human tauCstF-64 with human genome sequence from chromosome 10 shows that CSTF2T lacks introns. Radiation hybrid mapping places the human tauCstF-64 gene at 10q22-q23, which is the site of a translocation that has been associated with human neurological problems and male infertility.     

Dyneins, autophagy, aggregation and neurodegeneration

We recently showed that the dynein motor machinery plays a role in the delivery of autophagosome contents to lysosomes, in the process of autophagosome-lysosome fusion. This may explain a number of important previous observations, including why intracellular aggregates form in mice with dynein mutations that have motor neuron-like disease. These studies highlight the importance of dyneins and autophagy in the clearance of aggregate-prone proteins in general, and also in the specific case of Huntington's disease. Since many common neurodegenerative diseases are associated with intracellular aggregate formation but the causative variants are unknown, it may be worth considering the possibility of genetic lesions affecting autophagy as contributing factors in such disorders. The importance of dyneins in autophagosome-lysosome fusion provides new insights for the microtubule dependency of autophagy. In this Addendum, we review our findings in the contexts of autophagy and neurodegeneration and consider some of the questions raised.