Survey of Early-Diverging Lineages of Fungi Reveals Abundant and Diverse Mycoviruses.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions.IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Our Essential and Endangered Dentist-Scientist Workforce.

Future advances in dental medicine rely on a robust and stable pipeline of dentist-scientists who are dedicated to research inspired by the patients' condition. The biomedical research community faces external and internal pressures that have been building over years. This is now threatening the current and future status of basic, translational and patient-oriented research by dentist-scientists who study dental, oral and craniofacial diseases, population sciences, and prevention. The dental academic, research and practicing communities can no longer ignore the warning signs of a system that is under considerable stress. Here, the authors report findings of the Physician-Scientist Workforce Working Group, charged by the National Institutes of Health (NIH) Director, to perform quantitative and qualitative analyses on dentist-scientists by addressing the size, composition and activities of the group, relative to other health professions. From 1999 to 2012, trends in the numbers of grant applications and awards to dentist-scientists point to an overall decline. Disturbing are the low numbers of new investigators who apply for Early Career NIH Programs. While more seasoned dentist researchers enjoy greater success, the average age of first-time funded dentists is 52.7 y for females and 54.6 y for males, with a relatively low number of applications submitted and funded. These new data led the panel to stress the need to expand the capacity of the dentist-scientist workforce to leverage technologies and research opportunities that benefit the profession at-large. Suggestions were made to invest in developing clinical research faculty, including those with foreign degrees, through new training mechanisms. The creation of new alliances between national organizations like the American Association for Dental Research, the American Dental Education Association and the American Dental Association will undoubtedly lead to bold and concerted actions that must be pursued with a sense of urgency. A more supportive culture within dental schools and universities for dentist-scientists is needed, as their success is critical to the future career choices of their mentees. Knowledge Transfer Statement: Advances in dental medicine rely on a pipeline of dentist-scientists who are dedicated to research inspired by the patients' condition. Despite the recent advancement in technology and innovation, the dental community can no longer ignore the various pressures that threaten the future of the dentist-scientist profession. Here, the authors report findings of the Physician-Scientist Workforce Working Group of NIH that were published in 2014, and draw attention to the key issues threatening the NIH-funded pool of dentist-scientists.

Genetic approach identifies distinct asthma pathways in overweight vs normal weight children.

The pathogenesis of asthma in the context of excess body weight may be distinct from asthma that develops in normal weight children. The study's objective was to explore the biology of asthma in the context of obesity and normal weight status using genetic methodologies. Associations between asthma and SNPs in 49 genes were assessed, as well as, interactions between SNPs and overweight status in child participants of the Greater Cincinnati Pediatric Clinic Repository. Asthma was significantly associated with weight (OR = 1.38; P = 0.037). The number of genes and the magnitude of their associations with asthma were notably greater when considering overweight children alone vs normal weight and overweight children together. When considering weight, distinct sets of asthma-associated genes were observed, many times with opposing effects. We demonstrated that the underlying heterogeneity of asthma is likely due in part to distinct pathogenetic pathways that depend on preceding/comorbid overweight and/or allergy. It is therefore important to consider both obesity and asthma when conducting studies of asthma.

N-acetyltransferase 1 polymorphism increases cotinine levels in Caucasian children exposed to secondhand smoke: the CCAAPS birth cohort.

Cotinine is a proxy for secondhand smoke (SHS) exposure. Genetic variation along nicotine and cotinine metabolic pathways may alter the internal cotinine dose, leading to misinterpretations of exposure-health outcome associations. Caucasian children with available SHS exposure and hair cotinine data were genotyped for metabolism-related genes. SHS-exposed children had 2.4-fold higher hair cotinine (0.14±0.22 ng mg(-1)) than unexposed children (0.06±0.05 ng mg(-1), P<0.001). SHS-exposed children carrying the NAT1 minor allele had twofold higher hair cotinine (0.18 ng mg(-1) for heterozygotes and 0.17 ng mg(-1) for homozygotes) compared with major allele homozygotes (0.09 ng mg(-1), P=0.0009), even after adjustment for SHS dose. These findings support that NAT1 has a role in the metabolic pathway of nicotine/cotinine and/or their metabolites. The increased cotinine levels observed for those carrying the minor allele may lead to SHS exposure misclassification in studies utilizing cotinine as a biomarker. Additional studies are required to identify functional single-nucleotide polymorphism(s) (SNP(s)) in NAT1 and elucidate the biological consequences of the mutation(s).

Long-term changes in the fine-scale population structure of coho salmon populations (Oncorhynchus kisutch) subject to extensive supportive breeding.

The long-term viability of a metapopulation depends partly on the gene flow among sub-populations. Management approaches such as translocations and supportive breeding between closely related populations may affect gene flow and overall structure, and therefore viability. Here, we examined temporal changes in the fine-scale population structure of coho salmon (Oncorhynchus kisutch) by comparing archived (1938) and modern (2001-2005) populations in six rivers within a single conservation unit (Puget Sound, Washington) sampled before and after an extended period of between-river transfers and releases of millions of cultured salmon. Genotype frequencies at eight microsatellite loci showed that current populations descended from historical Puget Sound populations, but populations in different rivers that exchanged fish for hatchery propagation share more of their ancestry recently than they did historically. Historically, populations in different rivers were isolated by geographic distance, but that relationship is no longer significant. Allelic richness among all populations declined significantly, suggesting that genetic drift has increased because of a population bottleneck. Populations in different rivers and within the same river have become more diverged, providing further evidence for a widespread bottleneck. Previously, we observed that genetic distance significantly decreased with the number of fish exchanged; however, some populations apparently resisted introgression. Altered gene flow and lost diversity may affect the complexity, and therefore resiliency of sub-populations within a conservation unit. Plans for artificial culture need to maintain existing genetic diversity and avoid disrupting the fine-scale structure by using local populations for parents whenever possible.

A genetically informative developmental study of the relationship between conduct disorder and peer deviance in males.

BACKGROUND: Conduct disorder (CD) and peer deviance (PD) both powerfully predict future externalizing behaviors. Although levels of CD and PD are strongly correlated, the causal relationship between them has remained controversial and has not been examined by a genetically informative study. METHOD: Levels of CD and PD were assessed in 746 adult male-male twin pairs at personal interview for ages 8-11, 12-14 and 15-17 years using a life history calendar. Model fitting was performed using the Mx program. RESULTS: The best-fit model indicated an active developmental relationship between CD and PD including forward transmission of both traits over time and strong causal relationships between CD and PD within time periods. The best-fit model indicated that the causal relationship for genetic risk factors was from CD to PD and was constant over time. For common environmental factors, the causal pathways ran from PD to CD and were stronger in earlier than later age periods. CONCLUSION: A genetically informative model revealed causal pathways difficult to elucidate by other methods. Genes influence risk for CD, which, through social selection, impacts on the deviance of peers. Shared environment, through family and community processes, encourages or discourages adolescent deviant behavior, which, via social influence, alters risk for CD. Social influence is more important than social selection in childhood, but by late adolescence social selection becomes predominant. These findings have implications for prevention efforts for CD and associated externalizing disorders.

TEM immunocytochemistry of a 48 kDa MW organic matrix phosphoprotein produced in the mantle epithelial cells of the Eastern oyster (Crassostrea virginica).

Immunohistochemical TEM of Eastern oyster (Crassostrea virginica) mantle epithelial cells using a polyclonal antibody to a gel purified 48 kDa MW oyster shell phosphoprotein revealed that it is phosphorylated in the Golgi, packaged into secretory vesicles and subsequently exocytosed across the apical membrane of specialized cells. These phosphoprotein producing cells are concentrated along the mantle side facing the shell, in the region of the outer mantle lobe. A layer of calcium enriched immuno-reactive mucous is associated with the apical microvilli of these cells. The 48 kDa phosphoprotein forms a component of the fibrous organic matrix and appears to be involved in calcium supply thus enabling crystal growth at the mineralization front.

Genomewide linkage study in the Irish affected sib pair study of alcohol dependence: evidence for a susceptibility region for symptoms of alcohol dependence on chromosome 4.

Alcoholism is a relatively common, chronic, disabling and often treatment-resistant disorder. Evidence from twin and adoption studies indicates a substantial genetic influence, with heritability estimates of 50-60%. We conducted a genome scan in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD). Most probands were ascertained through alcoholism treatment settings and were severely affected. Probands, affected siblings and parents were evaluated by structured interview. A 4 cM genome scan was conducted using 474 families of which most (96%) were comprised by affected sib pairs. Nonparametric and quantitative linkage analyses were conducted using DSM-IV alcohol dependence (AD) and number of DSM-IV AD symptoms (ADSX). Quantitative results indicate strong linkage for number of AD criteria to a broad region of chromosome 4, ranging from 4q22 to 4q32 (peak multipoint LOD=4.59, P=2.1 x 10(-6), at D4S1611). Follow-up analyses suggest that the linkage may be due to variation in the symptoms of tolerance and out of control drinking. There was evidence of weak linkage (LODs of 1.0-2.0) to several other regions, including 1q44, 13q31, and 22q11 for AD along with 2q37, 9q21, 9q34 and 18p11 for ADSX. The location of the chromosome 4 peak is consistent with results from prior linkage studies and includes the alcohol dehydrogenase gene cluster. The results of this study suggest the importance of genetic variation in chromosome 4 in the etiology and severity of alcoholism in Caucasian populations.

The outer membrane cytochromes of Shewanella oneidensis MR-1 are lipoproteins.

AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 are lipoproteins, and to assess cell surface exposure of the cytochromes by radioiodination. METHODS AND RESULTS: In anaerobic MR-1 cells grown with (3)H-palmitoleic acid, both OmcA and OmcB were radiolabelled. The identities of these bands were confirmed by the absence of each radiolabelled band in the respective mutants lacking individual OM cytochromes. Radioiodination of cell surface proteins in anaerobic cells resulted in (125)I-labelled OmcA. The identity of this band was confirmed by its absence in an OmcA-minus mutant. A ubiquitous radioiodinated band that migrates similarly to OmcB precluded the ability to determine the potential cell surface exposure of OmcB by this method. CONCLUSIONS: Both OmcA and OmcB are lipoproteins, and OmcA is cell surface exposed. SIGNIFICANCE: The lipoprotein modification of these OM cytochromes could be important for their localization or incorporation into the OM. The cell surface exposure of OmcA could allow it to directly transfer electrons to extracellular electron acceptors (e.g. manganese oxides) and is consistent with its in vivo role.

Cell surface exposure of the outer membrane cytochromes of Shewanella oneidensis MR-1.

AIM: To determine if the outer membrane (OM) cytochromes of the metal-reducing bacterium Shewanella oneidensis MR-1 are exposed on the cell surface. METHODS AND RESULTS: MR-1 cells were incubated with proteinase K or buffer and the resulting degradation of the OM cytochromes was examined by Western blotting. The periplasmic fumarate reductase (control) was not degraded. The OM cytochromes OmcA and OmcB were significantly degraded by proteinase K (71 and 31%, respectively). Immunofluorescence confirmed a prominent cell surface exposure of OmcA and a partial exposure of OmcB and the noncytochrome OM protein MtrB. CONCLUSIONS: The cytochromes OmcA and OmcB are exposed on the outer face of the OM. SIGNIFICANCE AND IMPACT OF THE STUDY: The cell surface exposure of these cytochromes could allow them to directly contact extracellular insoluble electron acceptors (e.g. manganese oxides) and is consistent with their in vivo role.

Overlapping role of the outer membrane cytochromes of Shewanella oneidensis MR-1 in the reduction of manganese(IV) oxide.

AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 have distinct or overlapping roles in the reduction of insoluble manganese(IV) oxide. METHODS AND RESULTS: The gene replacement mutant (OMCA1) which lacks OmcA was partially deficient in Mn(IV) reduction. Complementation of OMCA1 with a vector (pVK21) that contains omcB but not omcA restored Mn(IV) reduction to levels that were even greater than those of wild-type. Examination of the OM of OMCA1/pVK21 revealed greater than wild-type levels of OmcB protein and specific haem content. CONCLUSIONS: Overexpression of OmcB can compensate for the absence of OmcA in the reduction of insoluble Mn(IV) oxides. Therefore, there is at least a partial overlap in the roles of these OM cytochromes in the reduction of insoluble Mn(IV) oxide. SIGNIFICANCE: The overlapping roles of these two cytochromes has important implications for understanding the mechanism by which MR-1 reduces insoluble metal oxides. There is no obligatory sequential electron transfer from one cytochrome to the other. They could both potentially serve as terminal reductases for extracellular electron acceptors.

Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide.

Shewanella putrefaciens MR-1 can use a wide variety of terminal electron acceptors for anaerobic respiration, including certain insoluble manganese and iron oxides. To examine whether the outer membrane (OM) cytochromes of MR-1 play a role in Mn(IV) and Fe(III) reduction, mutants lacking the OM cytochrome OmcA or OmcB were isolated by gene replacement. Southern blotting and PCR confirmed replacement of the omcA and omcB genes, respectively, and reverse transcription-PCR analysis demonstrated loss of the respective mRNAs, whereas mRNAs for upstream and downstream genes were retained. The omcA mutant (OMCA1) resembled MR-1 in its growth on trimethylamine N-oxide (TMAO), dimethyl sulfoxide, nitrate, fumarate, thiosulfate, and tetrathionate and its reduction of nitrate, nitrite, ferric citrate, FeOOH, and anthraquinone-2,6-disulfonic acid. Similarly, the omcB mutant (OMCB1) grew on fumarate, nitrate, TMAO, and thiosulfate and reduced ferric citrate and FeOOH. However, OMCA1 and OMCB1 were 45 and 75% slower than MR-1, respectively, at reducing MnO(2). OMCA1 lacked only OmcA. While OMCB1 lacked OmcB, other OM cytochromes were also missing or markedly depressed. The total cytochrome content of the OM of OMCB1 was less than 15% of that of MR-1. Western blots demonstrated that OMCB1 still synthesized OmcA, but most of it was localized in the cytoplasmic membrane and soluble fractions rather than in the OM. OMCB1 had therefore lost the ability to properly localize multiple OM cytochromes to the OM. Together, the results suggest that the OM cytochromes of MR-1 participate in the reduction of Mn(IV) but are not required for the reduction of Fe(III) or other electron acceptors.

Clinical features of schizophrenia and linkage to chromosomes 5q, 6p, 8p, and 10p in the Irish Study of High-Density Schizophrenia Families.

OBJECTIVE: Schizophrenia is clinically heterogeneous. Recent linkage studies suggest that multiple genes are important in the etiology of schizophrenia. The authors examined the hypothesis of whether the clinical variability in schizophrenia is due to genetic heterogeneity. METHOD: Using data from the Irish Study of High-Density Schizophrenia Families (N=265 pedigrees; N=1,408 individuals), the authors attempted to predict, from major symptoms and signs of psychosis, evidence for linkage within families for schizophrenia-related disorders to chromosomal regions 5q21-5q31, 6p24-6p22, 8p22-8p21, and 10p15-10p11. RESULTS: No substantial evidence was found for associations between clinical features of schizophrenia and linkage to chromosomes 5q, 6p, or 10p. However, affected individuals from families with evidence for linkage to 8p had significantly more affective deterioration, poorer outcome, more thought disorder, and fewer depressive symptoms than affected individuals from the other families in the study. CONCLUSIONS: These results raise the possibility that the putative susceptibility gene for schizophrenia localized in the 8p22-8p21 region may predispose individuals to the core dementia-praecox syndrome described by Kraepelin more than 100 years ago.

Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1.

Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas L-lactate or NADPH did not support any activity. The addition of 10 micromol l(-1) FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p-chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone-N-oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step.

A multidimensional twin study of mental health in women.

OBJECTIVE: While researchers have increasing insight into the role of genetic and environmental factors in the etiology of psychiatric and substance use disorders, they know much less about how such factors influence the dimensions of healthy psychological functioning. METHOD: In a population-based sample of 794 female-female twin pairs, the authors examined, by using multivariate structural equation modeling, six dimensions of mental health: perceived physical health, nonconflictual interpersonal relationships, anxious-depressive symptoms, substance use, social support, and self-esteem. RESULTS: The best-fit model was complex and constituted five common factors (two genetic, one family environmental, and two unique environmental); variable-specific genetic effects for physical health, substance use, and social support; and variable-specific family environmental effects for interpersonal relationships and substance use. Genetic effects were seen for all six dimensions; total heritabilities ranged from 16% to 49%. Family environment was an important influence on interpersonal relationships, substance use, and social support. CONCLUSIONS: Mental health is a complex phenotype that is influenced by a diverse array of genetic and environmental factors. While genetic factors appear to be of moderate etiologic importance in all major dimensions of mental health, the family environment is an important influence on only interpersonal relations, social support, and substance use.

Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone.

Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.

Modeling the effect of an external electric field on the velocity of spike propagation in a nerve fiber.

The effect of an externally generated electric field on the propagation of action potentials is modeled, assuming the Hodgkin-Huxley equation for the voltage-dependent conductance of the membrane of a nerve fiber. With some simplifying assumptions, this conductance together with Maxwell's equations leads to the Hodgkin-Huxley differential equations for propagation, modified by a term proportional to the gradient of the externally generated electric field component along the nerve fiber. Computer solution of these equations shows the influence of an electric field gradient on propagation velocity. When the electric field oscillates, voltage spikes starting later along a given axon advance or lag relative to earlier spikes, so the time between spikes at the receiving end differs from the time between spike originations. The amount that a low-frequency electric field modulates pulse timing at the end of a fiber relative to that at the beginning is estimated under several conditions.

Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time.

A long-term regeneration system for garlic (Allium sativum L.) clones of diverse origin was developed. Callus was initiated on a modified Gamborg's B-5 medium supplemented with 4.5 µM 2,4-D and maintained on the same basal medium with 4.7 µM picloram+0.49 µM 2iP. Regeneration potential of callus after 5, 12 and 16 months on maintenance medium was measured using several plant growth regulator treatments. The 1.4 µM picloram+13.3 µM BA treatment stimulated the highest rate of shoot production. Regeneration rate decreased as callus age increased, but healthy plantlets from callus cultures up to 16-months-old were produced for all clones. Regeneration of long-term garlic callus cultures could be useful for clonal propagation and transformation.