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.

Descriptive study of earthquake-related spinal cord injury in Nepal.

STUDY DESIGN: Descriptive study. OBJECTIVES: To describe the epidemiological features of spinal cord injury (SCI) following the 2015 earthquakes in Nepal. SETTING: Spinal Injury Rehabilitation Centre, Kavre, Nepal. METHODS: Data were collected from the medical records of all earthquake-related patients seen from 25 April 2015 through to 16 June 2016. Data collected included patient demographics, mechanism of injury, initial medical treatment, neurological assessment, complications, neurological/functional outcomes and length of stay. RESULTS: Data from 117 earthquake-related SCI patients were evaluated, with a female-to-male ratio of 1.3:1. In total, 108 patients (92%) sustained vertebral fracture and/or dislocation. Seventy-seven patients had undergone surgical fixation. The majority of patients (81%) presented with paraplegia, of whom most (60%) were incomplete. Thirty-eight (33%) patients had documented pressure ulcers upon admission; six (5%) patients developed new pressure ulcers during their rehabilitation stay. Urinary tract infection was seen in 34 (29%) patients. Seven (6%) patients were diagnosed with deep vein thrombosis. One patient developed clinically significant heterotopic ossification. Significant improvements were seen in patients' functional outcomes before discharge. Two deaths occurred in this patient population. CONCLUSIONS: The Nepal earthquakes resulted in a significant number of SCIs, the majority occurring in women. Incomplete paraplegia was the most common presentation. Pressure ulcer, the most frequent complication, primarily occurred before rehabilitation admission. Continued efforts focused on comprehensive planning, and preparedness for SCI-specific interdisciplinary care following earthquakes, particularly in resource-limited settings, is critical to ensuring survival, preventing complications and optimizing functional outcomes in this patient population.

Physiological levels of melatonin enhance gap junction communication in primary cultures of mouse hepatocytes.

Gap junction communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. Melatonin, a hormone secreted by the pineal gland, has putative oncostatic properties. Intercellular communication through gap junctions was assessed by microinjecting Lucifer yellow fluorescent dye into primary hepatocytes and visualizing the spread of the dye to adjacent neighboring cells using phase contrast/fluorescent microscopy. Treatment of primary hepatocyte cultures with a physiological range of melatonin concentrations for 24 h prior to microinjection resulted in significant enhancement in intercellular communication at 0.2 and 0.4 nmol/L but not at lower (0.1 nmol/L) or higher (0.8 or 1.0 nmol/L) concentrations. A time-dependent study showed that the changes in intercellular communication began 10 h after melatonin treatment and reached a maximum at 12 h of treatment. This nonlinear, functional gap junction response to melatonin occurred in the physiological concentration range detected in blood of mammals during nightly releases of the hormone by the pineal gland. These melatonin levels may affect the ability of gap junction communication to exert cell growth control in vivo. The uneven decline between individuals in nocturnal release of melatonin that occurs with age could identify potentially sensitive subpopulations susceptible to developing pathologies involving alterations in biological processes dependent on gap junction communication.

Dental prepayment: the past, present, and the future.

Insurance now pays for slightly less than 50% of America's dental bill. The explosive growth in dental insurance in the 1970s and 1980s is traced to the tax effect (coverage paid for with pre-tax dollars offsets fees up to a point) and the insurance effect (costs for care can be projected, although improvements in oral health status are leading to lower projected costs). An equilibrium point appears to have been reached. Dentists must weigh the trade-offs between discounted fees and increased number of patients, and carriers must weight the trade-offs between smaller discounts and wider participation by dentists. There are no market forces forecast that will substantially change this equilibrium in the near future.

Acquisition of virulence-associated factors by the enteric pathogens Escherichia coli and Salmonella enterica.

In this review we summarize recent genomic studies that shed light on the mechanism through which pathogenic Escherichia coli and Salmonella enterica have evolved. We show how acquisition of DNA at specific sites on the chromosome has contributed to increased genetic variation and virulence of these two genera of the Enterobacteriaceae.

Human visual function in the North Carolina clinical study on possible estuary-associated syndrome.

The U.S. Environmental Protection Agency assisted the North Carolina Department of Health and Human Services in conducting a study to investigate the potential for an association between fish kills in the North Carolina estuary system and the risk for persistent health effects. Impetus for the study was recent evidence suggesting that estuarine dinoflagellates, including members of the toxic Pfiesteria complex (TPC), P. piscicida and P. schumwayae, may release a toxin(s) that kills fish and adversely affects human health. This report describes one component of the study in which visual system function was assessed. Participants working primarily in estuaries inhabited by TPC or in off-shore waters thought not to contain TPC were studied. The potentially exposed estuary (n = 22) and unexposed offshore (n = 20) workers were matched for age, gender, and education. Visual acuity did not differ significantly between the cohorts, but visual contrast sensitivity (VCS), an indicator of visual pattern-detection ability for stimuli of various sizes, was significantly reduced by about 30% in the estuary relative to the offshore cohort. A further analysis that excluded participants having a history possibly predictive of neuropsychological impairment showed a similar VCS reduction. Additional analyses indicated that differences between the cohorts in age, education, smoking, alcohol consumption, and total time spent on any water did not account for the difference in VCS. Exploratory analyses suggested a possible association between the magnitude of VCS reduction and hours spent in contact with a fish kill. The profile of VCS deficit across stimulus sizes resembled that seen in organic solvent-exposed workers, but an assessment of occupational solvent, and other neurotoxicant, exposures did not indicate differences between the cohorts. These results suggest that factor(s) associated with the North Carolina estuaries, including the possibility of exposure to TPC toxin(s), may impair visual system function.

Typhoid fever and genetic polymorphisms at the natural resistance-associated macrophage protein 1.

Control of Salmonella enterica serovar Typhimurium (S. typhimurium) infection in the mouse model of typhoid fever is critically dependent on the natural resistance-associated macrophage protein 1 (Nramp1). In this study, we examined the role of genetic polymorphisms in the human homologue, NRAMP1, in resistance to typhoid fever in southern Vietnam. Patients with blood-culture-confirmed typhoid fever and healthy control subjects were genotyped for 6 polymorphic markers within and near NRAMP1 on chromosome 2q35. Four single base-pair polymorphisms (274 C/T, 469+14 G/C, 1465-85 G/A, and D543N), a (GT)(n) repeat in the promoter region of NRAMP1 and D2S1471, and a microsatellite marker approximately 130-kb downstream of NRAMP1 were examined. The allelic and genotypic frequencies for each polymorphism were compared in case patients and control subjects. No allelic association was identified between the NRAMP1 alleles and typhoid fever susceptibility. In addition, neither homozygotes nor heterozygotes for any NRAMP1 variants were at increased risk of typhoid fever.

Serology of typhoid fever in an area of endemicity and its relevance to diagnosis.

Currently, the laboratory diagnosis of typhoid fever is dependent upon either the isolation of Salmonella enterica subsp. enterica serotype Typhi from a clinical sample or the detection of raised titers of agglutinating serum antibodies against the lipopolysaccharide (LPS) (O) or flagellum (H) antigens of serotype Typhi (the Widal test). In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated with individuals from a region of Vietnam in which typhoid is endemic, and their usefulness for the diagnosis of typhoid fever was evaluated. The antibody responses to both antigens were highly variable among individuals infected with serotype Typhi, and elevated antibody titers were also detected in a high proportion of serum samples from healthy subjects from the community. In-house enzyme-linked immunosorbent assays (ELISAs) for the detection of specific classes of anti-LPS and antiflagellum antibodies were compared with other serologically based tests for the diagnosis of typhoid fever (Widal TO and TH, anti-serotype Typhi immunoglobulin M [IgM] dipstick, and IDeaL TUBEX). At a specificity of > or =0.93, the sensitivities of the different tests were 0.75, 0.55, and 0.52 for the anti-LPS IgM, IgG, and IgA ELISAs, respectively; 0.28 for the antiflagellum IgG ELISA; 0.47 and 0.32 for the Widal TO and TH tests, respectively; and 0.77 for the anti-serotype Typhi IgM dipstick assay. The specificity of the IDeaL TUBEX was below 0.90 (sensitivity, 0.87; specificity, 0.76). The serological assays based on the detection of IgM antibodies against either serotype Typhi LPS (ELISA) or whole bacteria (dipstick) had a significantly higher sensitivity than the Widal TO test when used with a single acute-phase serum sample (P < or = 0.007). These tests could be of use for the diagnosis of typhoid fever in patients who have clinical typhoid fever but are culture negative or in regions where bacterial culturing facilities are not available.

The influence of 1.2 microT, 60 Hz magnetic fields on melatonin- and tamoxifen-induced inhibition of MCF-7 cell growth.

We independently examined the findings of Harland and Liburdy, who reported that 1.2 microT(rms), 60 Hz magnetic fields could significantly reduce the inhibitory action of physiological levels of melatonin (10(-9) M) and of pharmacological levels of tamoxifen (10(-7) M) on the growth of MCF-7 human breast cancer cells in vitro. We used two testing protocols. In the melatonin study, the cell numbers per dish on day 7 of treatment were determined using a hemocytometer assay. In the tamoxifen study we used an expanded protocol, employing an alternative cell counting assay to characterize the cell numbers per dish on days 4, 5, 6, and 7. In both the melatonin and tamoxifen studies, cells were plated on 35 mm dishes and placed in each of two exposure chambers inside 5% CO(2) incubators. One exposure chamber was energized to produce 1.2 microT(rms), 60 Hz magnetic fields and the other chamber was not energized. Treatment was continuous until assays were performed. Cells were harvested at selected times, and enumerated without knowledge of treatment. In the melatonin study, the experiment was repeated three times, whereas in the tamoxifen study, each experiment was repeated nine times. In the melatonin study, cell numbers per dish were significantly reduced (by 16.7%) in the melatonin treated cultures after 7 days of incubation compared to control cultures, whereas in the presence of 1.2 microT(rms), 60 Hz magnetic fields, the melatonin treated cultures had the same cell populations as the control cultures. In the tamoxifen study, tamoxifen reduced the cell growth by 18.6 and 25% on days 6 and 7, respectively, in the chamber not energized, while in 1.2 microT(rms), 60 Hz fields, tamoxifen reduced the cell growth only by 8.7 and 13.1%, respectively. These results are consistent with those reported by Harland and Liburdy. A critical element of this successful replication effort was the constructive communication established and maintained with the original investigators. Bioelectromagnetics 22:122-128, 2001. Published 2001 Wiley-Liss, Inc.

Typhoid fever: pathogenesis and disease.

Typhoid fever is an infectious disease of global distribution. Although there is a wealth of data on Salmonella typhimurium infection in the mouse and the interaction of this serovar with human cell lines in vitro, there is a relatively small amount of data on S. typhi and the pathogenesis of typhoid fever. In this review we focus on three areas: adherence to and invasion of gut epithelial cells, dissemination to systemic sites, and survival and replication within host cells. In addition, we attempt to put current salmonella research into the context of typhoid fever.

Search for a parity-violating energy difference between enantiomers of a chiral iron complex

Mossbauer spectra for l and d enantiomers of the Fe(phen)3Sb2(C4H2O6)(2);8H(2)O complex are reported. Four independent experiments show a small but reproducible energy shift of the Fe-Mossbauer spectra for the two enantiomers of 0.004+/-0. 002 mm/sec ( 1.9x10(-10) eV). This exceedingly small energy difference is comparable to that predicted by the parity-violating energy difference (PVED) using a Z6.2 scaling law applied to low Z ( Z = 6) molecules. Theoretical calculations suggest that the PVED for the Fe(phen)2+3 moiety should be smaller than this estimate, however, PVED effects of the chiral antimony tartrates are not taken into account.

Adrenocorticotropin (ACTH) and corticosterone secretion by perifused pituitary and adrenal glands from rodents exposed to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD).

Although in utero maternal stress has been shown to have lasting effects on rodent offspring, fetal effects of chemically-induced alterations of the maternal hypothalamic-pituitary-adrenal axis (HPA) have not been well studied. This study examined the effects of in vivo 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on pituitary-adrenal function in the male rat, pregnant female rat and pregnant female mouse. The secretion of adrenocorticotropin (ACTH) and corticosterone (CORT) in pituitary and adrenal glands, respectively, was assessed in ex vivo perifusion cultures. Male and pregnant female (gestation day 8) Sprague-Dawley rats were gavaged once with 10 microgram/kg TCDD, pregnant female mice once with 24 microgram/kg TCDD, and euthanized 10 days later. Hemi-pituitary (rat) or whole anterior pituitaries (mice) and right adrenal glands from the same animal were quartered, perifused under baseline and stimulated conditions. In both males and pregnant females, TCDD did not affect corticotropin releasing hormone (CRH)-stimulated ACTH secretion. Neither total pituitary ACTH nor plasma ACTH was altered in either sex or species by TCDD treatment. ACTH-stimulated CORT secretion was not affected by TCDD in either sex or species, and adrenal tissue and plasma CORT levels were unchanged in males and pregnant females by TCDD. However, the plasma ACTH:CORT ratio was decreased about 46% in male rats treated with TCDD. Plasma CORT levels were 23-fold higher and plasma ACTH levels were 1.5-fold higher in pregnant females than in male rats. In male versus female rats, adrenal CORT and anterior pituitary ACTH tissue levels were about 7.5- and 1.75-fold higher and ACTH, respectively. Female mouse adrenal tissue CORT was about 4-fold greater than female rat. The reduced plasma ACTH:CORT ratio in the male rat suggests that TCDD disturbs HPA function. Exposure of male rat to a 5-fold higher dose in earlier studies clearly demonstrated effects of TCDD on male rat HPA. The present study identified substantial HPA performance differences between male and pregnant female rats. The failure to detect a response to TCDD in pregnant female rat and mouse could be a function of both TCDD dose and the high level of secretion of both ACTH and CORT in pregnant animals. For the rat or mouse, a single exposure to TCDD during pregnancy does not appear sufficient to induce maternally-mediated developmental, reproductive and behavioral toxicity via the HPA axis.

Lead absorption and psychological function in Zagreb (Croatia) school children.

A cross-sectional study was performed on 275 pupils from the third and fourth grade of three elementary schools (three urban areas with different traffic conditions) in Zagreb. Lead exposure was environmental, mostly through leaded gasoline. The difference in traffic density around the schools was consistent with biological indicators of lead absorption. The aim of the study was to clarify the relationship between characteristic biological indicators of lead absorption including indicators of hematological status with some psychological functions. Lead absorption in pupils was relatively low (mean blood lead: 70.8 +/- 17.88 microgram/L). Pupils' socio-economic status was evaluated by parents' education. The results obtained indicate that gender and school were associated with both biological and psychological variables. After adjusting for age, parental education, and gender, lead appears to have no association with cognitive or psycho-motor measures. The nonstandardized regression coefficients for blood lead-as a measure of the size of lead effect on VIQ, NIQ, and IQ-were -0.016, -0.031, and -0.025, respectively, all nonsignificant.

Age-dependent effects of Aroclor 1254R on calcium uptake by subcellular organelles in selected brain regions of rats.

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transduction mechanisms in brain, including Ca2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC) translocation in mature neurons and adult brain homogenate preparations. Present studies were designed to investigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca2+-uptake by two subcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercial PCB mixture, Aroclor 1254R, on 45Ca2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex and hippocampus of postnatal day (PND) 7, 21, and 90-120 (adult) male and female Long-Evans (LE)-rats. In general, microsomal and mitochondrial 45Ca2+-uptake in selected brain regions increased with age; PND 7

Monogenic diabetes mellitus in youth. The MODY syndromes.

Maturity onset diabetes of the young is characterized by early onset diabetes inherited in an autosomal dominant pattern. Classic MODY occurs predominantly in Caucasians and presents before age 25, is nonketotic, and is generally not insulin-requiring. Less than 5% of cases of childhood diabetes in Caucasians are caused by MODY. ADM is a subtype of MODY that occurs in approximately 10% of African-Americans with youth onset diabetes. In contrast to MODY in Caucasians, ADM presents clinically as acute onset diabetes often associated with weight loss, ketosis, and even diabetic ketoacidosis. Approximately 50% of patients with ADM are obese. Therefore, based strictly on clinical grounds, at onset, ADM cannot be distinguished from type 1 diabetes. Months to years following diagnosis, a non-insulin-dependent clinical course develops in patients with ADM that is clearly different from type 1 diabetes. Mutations in five genes can cause MODY. These genes encode hepatocyte nuclear factor-4 alpha (HNF-4 alpha, MODY1), glucokinase (MODY2), hepatocyte nuclear factor-1 alpha (HNF-1 alpha, MODY3), insulin promoter factor-1 (IPF-1, MODY4), and hepatocyte nuclear factor-1 beta (HNF-1 beta, MODY5). These monogenic forms of MODY have been used as model systems to investigate the inheritance and pathophysiology of type 2 diabetes. Clinicians, should be able to diagnose MODY. Type 1 diabetes, the most common form of diabetes in Caucasians, is always insulin-requiring for control and survival, whereas patients with MODY do not usually require long-term insulin for survival. Diagnostic confusion can lead to inappropriate management and patient expectations. Primary care physicians must be alert to avoid therapeutic confusion when patients with ADM enter into the non-insulin-dependent stage. An approach to the diagnosis of childhood diabetes is offered in Table 4. The majority of youth onset diabetes remains type 1; however, the frequency of type 2 diabetes is rising in obese children and adolescents and especially in obese minority youth. The diagnosis of MODY can be made through a careful review of the patient's clinical course, severity of hyperglycemia, and family history. The identification of islet autoantibodies is confirmatory evidence of autoimmune (type 1) diabetes. Because testing for MODY mutations is expensive and is performed at a select number of research laboratories only, routine molecular genetic studies to search for the various MODY mutations should be limited to research investigations. In the future, the availability of gene chip technology may allow rapid screening of mitochondrial and MODY mutations.

Experimental determination of hydrogen bandwidth for the ion parametric resonance model.

The ion parametric resonance (IPR) model predicts that distinct patterns of field-induced biological responses will occur at particular magnetic field combinations which establish ion resonances. An important characteristic of resonance is the bandwidth response of the system, in part because it determines the required tolerances of the test system. Initial development of the IPR model used literature data to estimate the bandwidth for any ion resonance to be -/+10% of its exact resonance. Because the charge-to-mass ratio of hydrogen is much larger than any other biologically significant ion, hydrogen resonance provides a unique test case by which a single ionic bandwidth can be clearly measured. Of particular relevance is work by Trillo et al. that demonstrated a hydrogen-only, resonance-based IPR response of neurite outgrowth in PC-12 cells. The work reported here considers the response of nerve-growth-factor-stimulated PC-12 cells exposed to magnetic fields tuned at or near hydrogen resonance. This work was designed to test directly the IPR model hypothesis of a -/+10% ionic bandwidth. Consistent with the work of Trillo et al., resonance conditions were established using a 2.97 microT static magnetic field, and the AC frequency and field strength were varied to prove different aspects of the resonance. With this static field 45 Hz was the resonance frequency identified for hydrogen, 42.5 and 47.5 Hz were near-resonance frequencies, and 40 and 50 Hz bounded the assumed -/+10% hydrogen resonance bandwidth. We repeated each test three times. The cell responses at 45 Hz exposures agreed with the IPR model predictions and replicated results obtained by Trillo et al. Cells exposed to 42.5 and 47.5 Hz (near resonance) magnetic fields responded in the same general pattern as those exposed to 45 Hz fields, but neurite outgrowth was less than that observed at resonance. Measured results for cells exposed to either 40 Hz or 50 Hz fields were indistinguishable from off-resonance responses, consistent with the hypothesized bandwidth. These results confirm that the response bandwidth for the hydrogen ion is no greater than -/+10%, and give further support to the resonance-based predictions of the IPR model.

Hepatocarcinogenicity in the male B6C3F1 mouse following a lifetime exposure to dichloroacetic acid in the drinking water: dose-response determination and modes of action.

Male B6C3F, mice were exposed to dichloroacetic acid (DCA) in the drinking water in order to establish a dose response for the induction of hepatocellular cancer and to examine several modes of action for the carcinogenic process. Groups of animals were exposed to control, 0.05, 0.5, 1, 2, or 3.5 g/L DCA in the drinking water for 90-100 wk. Mean daily doses (MDD) of 8, 84, 168, 315, and 429 mg/kg/d of DCA were calculated. The prevalence (percent of animals) with hepatocellular carcinoma (HC) was significantly increased in the 1-g/L (71%), 2-g/L (95%), and 3.5-g/L (100%) treatment groups when compared to the control (26%). HC multiplicity (tumors/animal) was significantly increased by all DCA treatments-0.05 g/L (0.58), 0.5 g/L (0.68), 1 g/L (1.29), 2 g/L (2.47), and 3.5 g/L (2.90)-compared to the control group (0.28). Based upon HC multiplicity, a no-observed-effect level (NOEL) for hepatocarcinogenicity could not be determined. Hepatic peroxisome proliferation was significantly increased only for 3.5 g/L DCA treatment at 26 wk. and did not correlate with the liver tumor response. The severity of hepatotoxicity increased with DCA concentration. Below 1 g/L, hepatotoxicity was mild and transient as demonstrated by the severity indices and serum lactate dehydrogenase activity. An analysis of generalized hepatocyte proliferation reflected the mild hepatotoxicity and demonstrated no significant treatment effects on the labeling index of hepatocytes outside proliferative lesions. Consequently, the induction of liver cancer by DCA does not appear to be conditional upon peroxisome induction or chemically sustained cell proliferation. Hepatotoxicity, especially at the higher doses, may exert an important influence on the carcinogenic process.