The Pif1 signature motif of Pfh1 is necessary for both protein displacement and helicase unwinding activities, but is dispensable for strand-annealing activity.

Pfh1, the sole member of the Pif1 helicases in Schizosaccharomyces pombe, is multifunctional and essential for maintenance of both the nuclear and mitochondrial genomes. However, we lack mechanistic insights into the functions of Pfh1 and its different motifs. This paper is specifically concerned with the importance of the Pif1 signature motif (SM), a 23 amino acids motif unique to Pif1 helicases, because a single amino acid substitution in this motif is associated with increased risk of breast cancer in humans and inviability in S. pombe. Here we show that the nuclear isoform of Pfh1 (nPfh1) unwound RNA/DNA hybrids more efficiently than DNA/DNA, suggesting that Pfh1 resolves RNA/DNA structures like R-loops in vivo. In addition, nPfh1 displaced proteins from DNA and possessed strand-annealing activity. The unwinding and protein displacement activities were dependent on the SM because nPfh1 without a large portion of this motif (nPfh1-Δ21) or with the disease/inviability-linked mutation (nPfh1-L430P) lost these properties. Unexpectedly, both nPfh1-L430P and nPfh1-Δ21 still displayed binding to G-quadruplex DNA and demonstrated strand-annealing activity. Misregulated strand annealing and binding of nPfh1-L430P without unwinding are perhaps the reasons that cells expressing this allele are inviable.

G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase.

Certain guanine-rich sequences have an inherent propensity to form G-quadruplex (G4) structures. G4 structures are e.g. involved in telomere protection and gene regulation. However, they also constitute obstacles during replication if they remain unresolved. To overcome these threats to genome integrity, organisms harbor specialized G4 unwinding helicases. In Schizosaccharomyces pombe, one such candidate helicase is Pfh1, an evolutionarily conserved Pif1 homolog. Here, we addressed whether putative G4 sequences in S. pombe can adopt G4 structures and, if so, whether Pfh1 can resolve them. We tested two G4 sequences, derived from S. pombe ribosomal and telomeric DNA regions, and demonstrated that they form inter- and intramolecular G4 structures, respectively. Also, Pfh1 was enriched in vivo at the ribosomal G4 DNA and telomeric sites. The nuclear isoform of Pfh1 (nPfh1) unwound both types of structure, and although the G4-stabilizing compound Phen-DC3 significantly enhanced their stability, nPfh1 still resolved them efficiently. However, stable G4 structures significantly inhibited adenosine triphosphate hydrolysis by nPfh1. Because ribosomal and telomeric DNA contain putative G4 regions conserved from yeasts to humans, our studies support the important role of G4 structure formation in these regions and provide further evidence for a conserved role for Pif1 helicases in resolving G4 structures.

Hepatorenal syndrome in hospitalized patients with chronic liver disease: results from the Nationwide Inpatient Sample 2002-2012.

Hepatorenal syndrome (HRS) is one of the leading causes of hospitalizations in patients with chronic liver disease (CLD). We conducted a retrospective national database study to determine the epidemiology of HRS in hospitalized patients with CLD. Data from a Nationwide Inpatient Sample were extracted from 2002 to 2012 using ICD-9-CM codes related to CLD and HRS. The following outcomes were examined: in-hospital mortality, total charges, length of stay (LOS), patient demographics, procedures, complications, and comorbidities. Statistical analysis including regression was performed to examine factors associated with HRS. During 2002-2012, hospital discharges related to CLD increased from 407,246 to 836,475 with an increase of 37.9% for HRS as a complication in this population. Patients with CLD and HRS had worse outcomes compared with patients with CLD without HRS. This was manifested as a higher mortality rate (32.0% vs 10.3%), increased LOS (median 7 vs 5 days), and increased hospital costs (median $16,000 vs $11,000). Logistic regression demonstrated that HIV/AIDS (adjusted OR 2.9, 95% CI 2.2 to 3.9), pneumonia (aOR 2.8, 95% CI 2.3 to 3.2), and esophageal variceal bleeding (aOR 1.9, 95% CI 1.7 to 2.0) were associated with higher mortality in patients with HRS. Conversely, liver transplantation (aOR 0.1, 95% CI 0.1 to 0.1), transjugular intrahepatic portosystemic shunt (aOR 0.5, 95% CI 0.4 to 0.6), and hospitalization in the Midwest region of the USA (aOR 0.7, 95% CI 0.6 to 0.7) were associated with reduced mortality. The incidence of HRS in hospitalized patients with CLD increased during 2002-2012. HRS is associated with significant mortality and morbidity in these patients.

Outcomes of Clostridium difficile infection in pediatric solid organ transplant recipients.

BACKGROUND: The incidence of Clostridium difficile infection (CDI) is increasing in the pediatric population. Pediatric recipients of solid organ transplantation (SOT) may be at a higher risk for CDI in part because of chemotherapy and prolonged hospitalization. METHODS: We utilized data from the Healthcare Cost and Utilization Project Kids' Inpatient Database to study the incidence and outcomes related to CDI as a complicating factor in pediatric recipients of SOT. RESULTS: Our results demonstrate that hospitalized children with SOT have increased rates of infection, with the greatest risk for younger children with additional comorbidities and severe illness. The type of transplanted organ affects the risk for CDI, with the lowest incidence observed in renal transplant patients. CONCLUSION: The occurrence of CDI in the pediatric SOT population contributes to a greater length of stay and higher hospital charges. However, CDI is not an independent predictor of increased in- hospital mortality in these patients.

Ageing and vascular ageing.

There is an age related decline in various physiological processes. Vascular ageing is associated with changes in the mechanical and the structural properties of the vascular wall, which leads to the loss of arterial elasticity and reduced arterial compliance. Arterial compliance can be measured by different parameters like pulse wave velocity, augmentation index, and systemic arterial compliance. There is evidence that arterial compliance is reduced in disease states such as hypertension, diabetes, and end stage renal failure. Changes in arterial compliance can be present before the clinical manifestation of cardiovascular disease. Pharmacological and non-pharmacological measures have been shown to improve arterial compliance. Arterial compliance may constitute an early cardiovascular risk marker and may be useful in assessing the effects of drugs on the cardiovascular system. Pharmacogenetics and genetics of arterial compliance in the future will improve our knowledge and understanding about vascular ageing.

An unusual case of fungal keratitis: Metarrhizium anisopliae.

PURPOSE: To report a case of fungal keratitis caused by Metarrhizium anisopliae, which to our knowledge is the first reported case in the United States. METHOD: Case report. RESULTS: A 36-year-old female librarian who wore extended-wear soft contact lenses was seen by an ophthalmologist on September 11 for an irritated right eye, and a corneal ulcer was diagnosed. Symptoms increased by September 27, and the patient was referred to another ophthalmologist who cultured the ulcer and had scrapings examined, which were Gram-negative for microorganisms. The patient was referred to one of the authors (W.J.R.). Her exam on October 1 showed vision corrected to 20/25 OD, a 5-mm epithelial defect with a 2.5-mm anterior stromal grayish-type infiltrate, and a quiet anterior chamber; the eye did not appear to be inflamed. The patient was reexamined on October 4 and was noted to have worsening vision. Because the initial cultures remained negative, the patient underwent a corneal biopsy, Gram stain, and cultures on October 6. Scrapings at the time of the biopsy revealed septate hyphal elements, as did the biopsy specimen, and on October 7, the patient was started on a treatment of bacitracin ointment once a day and natamycin 5% every hour. The eye gradually quieted down. A mold growing from the biopsy culture, which had been sent to a reference laboratory in San Antonio, Texas, was identified as M. anisopliae var. anisopliae. The patient was subsequently fitted with a rigid gas permeable lens, which resulted in a best-corrected visual acuity of 20/20, although glare remained a major problem. CONCLUSION: Although not previously reported in the United States, M. anisopliae can cause a keratomycosis, and one must consider this common insect pathogen in the differential diagnosis of fungal keratitis.

Post-axial limb defects with maternal sodium valproate exposure.

A male child is described with bilateral talipes equinovarus, absent fibulae with bowing and shortening of the tibiae and facial dysmorphic features. His mother was on a daily dose of 2000 mg sodium valproate for convulsions. This case raises the possibility that postaxial limb defects might be caused by intrauterine valproate exposure.

Efficiency and accuracy of two computerized topography software systems for fitting rigid gas permeable contact lenses.

PURPOSE: The world of computerized videokeratoscopy is continually evolving. Computerized videokeratoscope contact lens programs boast the capability to accurately design final rigid gas permeable (RGP) lens parameters and simulate contact lens fluorescein patterns. We evaluated the EyeSys System 2000 Pro-Fit software (v. 4.0) and Humphrey Atlas MasterVue Contact Lens Module (v. A6) for efficiency and accuracy in fitting RGP contact lenses on normal subjects. METHODS: Balanced manifest refractions, slit lamp examinations, and computerized topographical analysis with RGP module lens fitting were performed and compared for all eyes. Lens parameters were chosen after an optimal simulated fluorescein pattern was achieved by either accepting the initially recommended default lens or a modification. Final lens parameters were based on clinical performance. RESULTS: With the Humphrey Atlas software, which uses a default topographic tear clearance fitting protocol set by the manufacturer, 24/50 (48%) eyes required modifications to the default fitting parameters based on the simulated fluorescein patterns. Nine of 50 eyes (18%) required further modifications once the RGP lens was dispensed. The EyeSys Pro-Fit software, which allows a custom tear clearance fitting protocol set by the user as default, required modifications to 20/35 eyes (57.1%) from default fittings provided by the system. Six of 35 eyes (17.1%) required subsequent modifications once the RGP lens was dispensed. Comparison of the EyeSys to Humphrey modules revealed no significant difference in efficiency (P=0.51), defined as any changes required to the default parameters; however, the user made more modifications to the EyeSys module from the default setting. There was no significant difference in final success rates between EyeSys (82.9%) and Humphrey (82%) systems (P=1.00). CONCLUSION: Although both topography guided contact lens modules offered accuracy in RGP fitting, a user programmed default (EyeSys) protocol did not improve efficiency in RGP lens parameter selection compared to a manufacturer programmed default module (Humphrey). The need for a knowledgeable contact lens fitter remains to guide the systems in achieving the optimal fit.

One-year outcomes of panretinal photocoagulation in proliferative diabetic retinopathy.

PURPOSE: To describe the clinical features and complications of diabetic retinopathy, visual acuity, and number of repeat treatments after panretinal photocoagulation for proliferative diabetic retinopathy in a tertiary care center. METHODS: A cohort study was conducted with data collection from medical records of patients undergoing panretinal photocoagulation between 1985 and 1995 at the Scheie Eye Institute; 297 eyes of 186 patients were eligible for study. RESULTS: The presence of neovascularization of the disk at baseline, an earlier onset of diabetes, and a shorter duration of disease before panretinal photocoagulation were the strongest risk factors for needing an additional panretinal photocoagulation treatment. Sixty-two percent of eyes with poor visual acuity (< or =20/200) at baseline still had poor visual acuity at 1 year, and 76% with good visual acuity (> or =20/40) at baseline maintained good visual acuity at 1 year. Poor vision at baseline was the only risk factor for having poor vision at 1 year. Vitreous hemorrhage was present in 44% of eyes at baseline. New vitreous hemorrhage developed in 37% of eyes during the first year after panretinal photocoagulation. A traction retinal detachment was present in 4% of eyes at baseline and newly developed in 6% of eyes during follow-up. A repeat panretinal photocoagulation treatment was performed in 39% of eyes after initial treatment. A vitrectomy was performed in 10% of eyes from baseline through the 1-year follow-up visit. CONCLUSIONS: The data from this study are useful for counseling patients with respect to likely visual outcome, possibility of major complications from proliferative diabetic retinopathy, and the chance of undergoing additional laser treatment after panretinal photocoagulation.

The prevalence and implications of ocular hypertension and glaucoma in thyroid-associated orbitopathy.

PURPOSE: The authors determined the prevalence of ocular hypertension and its association with progression to glaucomatous damage in patients with thyroid-associated orbitopathy (TAO). METHODS: The charts of 500 consecutive patients with TAO seen at the Allegheny General Hospital (Pittsburgh, PA) between 1985 and 1995 were analyzed. The amount of proptosis, degree and duration of myopathy, exposure to corticosteroids, prior glaucoma treatment, and family history of glaucoma were evaluated. RESULTS: One hundred twenty (24%) patients with TAO were noted to have an intraocular pressure (IOP) greater than 22 mmHg but less than 30 mmHg. This ocular hypertensive group was composed of 34 men and 86 women with a mean age of 55 years and mean follow-up of 4 years. Seven patients were defined as glaucoma suspects, based on increased but nonprogressive cup-to-disc ratios or nonprogressive, atypical visual field changes in the presence of increased IOP. Two patients demonstrated progressive visual field abnormalities and cupping. Of the factors evaluated, only the duration of active orbital involvement was statistically associated with progression to glaucomatous damage. The mean duration of TAO was 3, 8, and 12 years for ocular hypertensives, glaucoma suspects, and glaucomatous damage, respectively. CONCLUSIONS: Only a prolonged duration of active TAO in association with ocular hypertension correlated with progression to glaucomatous damage. These patients with chronic TAO deserve special attention and close follow-up to prevent optic nerve damage.

Pholedrine: a substitute for hydroxyamphetamine as a diagnostic eyedrop test in Horner's syndrome.

Mydriatic responses to eyedrops containing the indirect acting sympathomimetic amines tyramine, hydroxyamphetamine, and pholedrine have been compared in 10 healthy subjects. Pholedrine, the n-methyl derivative of hydroxyamphetamine, at a concentration of 1% had effects similar to those produced by 0.5% hydroxyamphetamine itself. Pretreatment with topical guanethidine attenuated its responses and in 13 patients with unilateral Horner's syndrome it distinguished clearly those five patients who had preganglionic from the eight with postganglionic lesions. It is concluded that 1% pholedrine may be substituted for 0.5% hydroxyamphetamine, which is no longer available, as a diagnostic agent for use in Horner's syndrome.

Peripheral limb gangrene following rectal biopsy. Treatment with prostacyclin and exchange transfusion.

After a rectal biopsy, a 3-week-old boy developed centripetally progressive distal limb gangrene. This was accompanied by transient hypertension and high levels of circulating immune complexes. The ischemia was reversed by prostacyclin infusion, oral corticosteroids and multiple exchange transfusions.

Yersinia enterocolitis in iron overload.

Yersinia enterocolitis with peritonitis and septicaemia developed in a 4-year-old child with thalassemia intermedia and iron overload. It is likely that the illness was exacerbated by continued administration of desferrioxamine, a bacterial siderophore, which facilitated systemic spread of Yersinia. It is recommended that Yersinia enterocolitis be considered in children with iron overload and that desferrioxamine be discontinued and prophylactic antibiotics be administered while Yersinia is sought.

Screening for intrauterine growth retardation using ratio of mid-arm circumference to occipitofrontal circumference.

Uncritical application of standard weight percentile charts, derived from white infants, to infants from different ethnic groups may result in an overestimate of the incidence of intrauterine growth retardation in those groups. The ratio of mid-arm circumference to occipitofrontal circumference was studied in 194 babies (49 Asian, 58 black, and 87 white). In contrast with birth weight the ratio did not vary among the ethnic groups; it was also independent of sex. In a prospective study of 64 neonates whose weight was below the 10th percentile on standard charts the ratio of mid-arm circumference to occipitofrontal circumference was a more accurate predictor than weight of those infants who would develop symptoms associated with intrauterine growth retardation. The ratio of mid-arm circumference to occipitofrontal circumference therefore provides a simple, accurate, and cheap way of assessing intrauterine growth retardation in areas with a large multiethnic population, where birth weight varies greatly.

Effects of locally administered anticholinesterase agents on the secretory response of human eccrine sweat glands to acetylcholine and carbachol.

The effects of locally administered physostigmine and di-isopropylphosphorofluoridate (DFP) were compared on the secretory response of sweat glands to intradermally injected acetylcholine and carbachol in healthy male volunteers (physostigmine: six subjects; DFP: one subject). The response to acetylcholine reached its peak within 10 s of injection and then rapidly declined, whereas the response to carbachol increased steadily reaching a peak between 5 and 7 min after injection. The response to acetylcholine was potentiated in the presence of both physostigmine and DFP, whilst the response to carbachol was not significantly affected by either of these drugs. The difference in the time-course of responses to acetylcholine and carbachol may be attributed to differences in the susceptibility of the two drugs to metabolism by acetylcholinesterase; carbachol, unlike acetylcholine, being virtually immune to metabolism by this enzyme. It is concluded that the response to carbachol is mediated by a direct stimulatory action on post-synaptic muscarinic receptors rather than by activation of pre-synaptic nicotinic receptors leading to the release of endogenous acetylcholine.