Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

Effect of pre-incubation pH on the growth characteristics of Aeromonas hydrophila at 5°C, as assessed by two methods

Two strains of Aeromonas hydrophila (the type strain ATCC 7966 and a food-derived strain JAH4) were pre-incubated at 5°C in Brain Heart Infusion (BHI) broth with pH adjusted to 6.0 or 7.0, and then incubated at the same temperature in BHI broth with pH adjusted to 6.0, 6.5, 7.0 and 7.5. Growth kinetics during incubation were determined by two methods: viable count (VC) and measurement of optical density (O.D.). Pre-incubation at different pH values did not significantly affect the maximum specific growth rates of the strains during incubation, but the lag phases were shorter after pre-incubation at pH 6.0 than at pH 7.0. The VC method was more sensitive than O.D. measurements for assessing lag phase.     

Evaluation of fasciotomy and vasodilator for treatment of frostbite in the dog

Severe freezing injury was produced in the hind foot of 26 mongrel dogs. All dogs were given daily whirlpool treatment and protective bandaging for 14 days following injury. In addition, certain dogs received a vasodilator, fasciotomy, or both vasodilator and fasciotomy following injury. Deep foot temperatures, foot volumes, tissue pressures, and 14 day tissue loss-salvage scores were compared. Significant differences between fasciotomy and nonfasciotomy dogs were seen in foot temperature, volume, and tissue pressure immediately following fasciotomy. Though there was no significant difference in 14 day tissue loss, there was clinically apparent prolongation of integrity of the local vascular system for 2 to 5 days following fasciotomy, and total foot salvage in several dogs receiving fasciotomy.     

Characterization, mapping, and distribution of the two XMRV parental proviruses

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1(n) receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.     

Inter‐α‐inhibitor deficiency in the mouse is associated with alterations in anxiety‐like behavior,exploration and social approach

In recent years, several genome‐wide association studies have identified candidate regions for genetic susceptibility in major mood disorders. Most notable are regions in a locus in chromosome 3p21, encompassing the genes NEK4‐ITIH1‐ITIH3‐ITIH4. Three of these genes represent heavy chains of the composite protein inter‐α‐inhibitor (IαI). In order to further establish associations of these genes with mood disorders, we evaluated behavioral phenotypes in mice deficient in either Ambp/bikunin, which is necessary for functional ITIH1 and ITIH3 complexes, or in Itih4, the gene encoding the heavy chain Itih4. We found that loss of Itih4 had no effect on the behaviors tested, but loss of Ambp/bikunin led to increased anxiety‐like behavior in the light/dark and open field tests and reduced exploratory activity in the elevated plus maze, light/dark preference and open field tests. Ambp/bikunin knockout mice also exhibited a sex‐dependent exaggeration of acoustic startle responses, alterations in social approach during a three‐chamber choice test, and an elevated fear conditioning response. These results provide experimental support for the role of ITIH1/ITIH3 in the development of mood disorders.     

Enteric absorption and pharmacokinetics of oseltamivir in critically ill patients with pandemic (H1N1) influenza

Background

Whether the enteric absorption of the neuraminidase inhibitor oseltamivir is impaired in critically ill patients is unknown. We documented the pharmacokinetic profile of oseltamivir in patients admitted to intensive care units (ICUs) with suspected or confirmed pandemic (H1N1) influenza.

Methods

We included 41 patients 18 years of age and older with suspected or confirmed pandemic (H1N1) influenza who were admitted for ventilatory support to nine ICUs in three cities in Canada and Spain. Using tandem mass spectrometry, we assessed plasma levels of oseltamivir free base and its active metabolite carboxylate at baseline (before gastric administration of the drug) and at 2, 4, 6, 9 and 12 hours after the fourth or later dose.

Results

Among the 36 patients who did not require dialysis, the median concentration of oseltamivir free base was 10.4 (interquartile range [IQR] 4.8–14.9) μg/L; the median concentration of the carboxylate metabolite was 404 (IQR 257–900) μg/L. The volume of distribution of the carboxylate metabolite did not increase with increasing body weight (R² = 0.00, p = 0.87). The rate of elimination of oseltamivir carboxylate was modestly correlated with estimations of creatinine clearance (R² = 0.27, p < 0.001). Drug clearance in the five patients who required continuous renal replacement therapy was about one-sixth that in the 36 patients with relatively normal renal function.

Interpretation

Oseltamivir was well absorbed enterically in critically ill patients admitted to the ICU with suspected or confirmed pandemic (H1N1) influenza. The dosage of 75 mg twice daily achieved plasma levels that were comparable to those in ambulatory patients and were far in excess of concentrations required to maximally inhibit neuraminidase activity of the virus. Adjustment of the dosage in patients with renal dysfunction requiring continuous renal replacement therapy is appropriate; adjustment for obesity does not appear to be necessary.A substantial number of cases of pandemic (H1N1) influenza have involved young adults and adolescents without serious comorbidities who present with severe viral pneumonia complicated by acute respiratory distress syndrome, rhabdomyolysis, renal failure and, occasionally, shock.^1,2 Antiviral therapy in such critically ill patients typically requires oral or nasogastric administration of the neuraminidase inhibitor oseltamivir. Current guidelines from the World Health Organization for the pharmacologic management of progressive or severe pandemic (H1N1) influenza recommend the consideration of high-dose therapy (≥ 150 mg twice daily).^3,4 Critically ill patients exhibit defects in gastrointestinal absorption because of impaired gut perfusion, edema of the bowel wall and ileus as a consequence of critical illness and shock.⁵ Whether the enteric absorption of oseltamivir is impaired in such patients is unknown.We undertook this study to document the pharmacokinetic profile of oseltamivir administered orally or by nasogastric tube in patients admitted to intensive care units (ICUs) with respiratory failure due to suspected or confirmed pandemic (H1N1) influenza.     

Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.