Binding Parameters and Thermodynamics of the Interaction of the Human Cytomegalovirus DNA Polymerase Accessory Protein, UL44, with DNA: Implications for the Processivity Mechanism

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12 bp, with apparent dissociation constants in the nanomolar range for DNAs > 18 bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, $4 \pm 1$ monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity

25-hydroxyvitamin D levels and bone histomorphometry in hemodialysis renal osteodystrophy

25-hydroxyvitamin D levels and bone histomorphometry in hemodialysis renal osteodystrophy.BackgroundThe importance of 25-hydroxyvitamin D (25-OHD) serum levels in hemodialysis chronic renal failure has not been so far histologically evaluated. Information still lacking relate to the effect of 25-OHD deficiency on serum parathyroid hormone (PTH) levels and on bone and its relationship with calcitriol levels.MethodsThis retrospective study has been performed on a cohort of 104 patients on hemodialysis from more than 12 months, subjected to transiliac bone biopsy for histologic, histomorphometric, and histodynamic evaluation. The patients, 61 males and 43 females, mean age 52.9 ± 11.7 years, hemodialysis length 97.4 ± 61.4 months, were treated with standard hemodialysis and did not receive any vitamin D supplementation. Treatment with calcitriol was not underway at the time of the biopsy. Transiliac bone biopsies were performed after double tetracycline labels. In addition, serum intact PTH (iPTH), alkaline phosphatase, and 25-OHD were measured. Calcitriol serum levels was also measured in a subset of patients (N = 53). The patients were divided according to serum 25-OHD levels in three groups: (1) 0 to 15 (15 patients), (2) 15 to 30 (38 patients), and (3) >30 ng/mL (51 patients).ResultsThere was no significant difference in average age, hemodialysis age, serum PTH [490 ± 494, 670 ± 627, and 489 ± 436 pg/mL, respectively (mean ± SD)], alkaline phosphatase, and calcitriol between the three groups. The parameters double-labeled surface, trabecular mineralizing surface, and bone formation rate were significantly lower in group 1 than in the other groups (P < 0.03, < 0.03, and < 0.02, respectively). Osteoblast surface and adjusted apposition rate were borderline significantly lower in group 1 (P < 0.06 and < 0.10). There was no statistical difference in the biochemical and bone parameters between groups 2 and 3. A positive significant correlation was found between several bone static and dynamic parameters and 25-OHD levels in the range 0 to 30 ng/mL, showing a vitamin D dependence of bone turnover at these serum levels. However, actual evidence of an effect on bone of 25-OHD deficiency was found at serum levels below 20 ng/mL. With increasing 25-OHD levels beyond 40 ng/mL, a downslope of parameters of bone turnover was also observed.ConclusionSince PTH serum levels are equally elevated in low and high 25-OHD patients, while calcitriol levels are constantly low, an effect of 25-OHD deficiency (group 1) on bone, consisting of a mineralization and bone formation defect, can be hypothesized. The effect of vitamin D deficiency or bone turnover is found below 20 ng/mL. The optimal level of 25-OHD appears to be in the order of 20 to 40 ng/mL. Levels of the D metabolite higher than 40 ng/mL are accompanied by a reduction of bone turnover

Atherosclerotic renal artery stenosis: one year outcome of total and separate kidney function following stenting

BACKGROUND: Renal artery stenosis (RAS) is a known cause of hypertension and ischemic nephropathy. Stenting of the artery is a valid approach, in spite of cases of unexpected adverse evolution of renal function. METHODS: In this study, 27 patients with unilateral RAS were subjected to stenting and followed for a period of one year, while 19 patients were observed while on medical treatment only. The group of 27 patients, 67.33 ± 6.8 years of age, creatinine of 2.15 ± 0.9 mg/dl, following stenting, were followed at intervals with biochemical tests, renal scintigraphy and doppler ultrasonography. The control group (70.0 ± 6.1 years, creatinine 1.99 ± 0.7 mg/dl) was also followed for one year. RESULT: One year after stenting mean creatinine clearance (Ccr) increased from 36.07 ± 17.2 to 40.4 ± 21.6 ml/min (NS). Arterial BP, decreased after 1,3,6, and 12 months (p < 0.05). The number of antihypertensive drugs also decreased (p < 0.05). A significant increase in proteinuria was also observed. In the control group both Ccr, BP and proteinuria did not show significant changes. Based on renal scintigraphy and Ccr at subsequent times, it was possibile to evaluate the timecourse of renal function in both kidneys of the stented patients. In the stented kidneys Ccr increased significantly. On the controlateral kidney a decrease of renal function (p < 0.05) was observed. Resistance index appeared to be a risk factor of the functional outcome. CONCLUSIONS: Stenting of RAS due to atherosclerosis is followed by stabilization or improvement of Ccr, mainly at the stented kidney, while contralateral renal function showed a decrease

Immunohistochemical localization and mRNA expression of matrix Gla protein and fetuin-A in bone biopsies of hemodialysis patients

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Atherosclerotic ischemic renal disease. Diagnosis and prevalence in an hypertensive and/or uremic elderly population

BACKGROUND: Atherosclerotic ischemic renal disease is a frequent cause of end-stage renal failure leading to dialysis among the elderly; Its prevalence is inferred from autopsy or retrospective arteriographic studies. This study has been conducted on 269 subjects over 50 with hypertension and/or CRF, unrelated to other known causes of renal disease. METHODS: All 269 patients were studied either by color-flow duplex sonography (n = 238) or by renal scintigraphy (n = 224), and 199 of the 269 patients were evaluated using both of these techniques. 40 patients, found to have renal artery stenosis (RAS), were subjected to 3D-contrast enhancement Magnetic Resonance Angiography (MRA) and/or Selective Angiography (SA). An additional 23 cases, negative both to scintigraphy and to ultrasound study, underwent renal angiography (MRA and/or SA). RESULTS: Color-duplex sonography, carried out in 238 patients, revealed 49 cases of RAS. MR or SA was carried out in 35 of these 49 patients, and confirmed the diagnosis in 33. Color-duplex sonography showed a PPV value of 94.3% and NPV of 87.0% while renal scintigraphy, carried out in 224 patients, had a PPV of 72.2% and a NPV of 29.4%. Patients with RAS showed a higher degree of renal insufficiency compared to non stenotic patients while there were no differences in proteinuria. RAS, based on color-duplex sonography studies, was present in 11% of patients in the age group 50–59, 18% in the 60–69 and 23% at age 70 and above. CONCLUSIONS: A relatively large percentage of the elderly population with renal insufficiency and/or hypertension is affected by RAS and is at risk of developing end-stage renal failure. Color-duplex ultrasonography is a valid routine method of investigation of population at risk for renal artery stenosis

Academy of Emergency Medicine and Care-Society of Clinical Biochemistry and Clinical Molecular Biology consensus recommendations for clinical use of sepsis biomarkers in the emergency department.

Increasing evidence is emerging that the measurement of circulating biomarkers may be clinically useful for diagnosing and monitoring sepsis. Eight members of AcEMC (Academy of Emergency Medicine and Care) and eight members of SIBioC (Italian Society of Clinical Biochemistry and Laboratory Medicine) were identified by the two scientific societies for producing a consensus document aimed to define practical recommendations about the use of biomarkers for diagnosing of sepsis and managing antibiotic therapy in the emergency department (ED). The cumulative opinions allowed defining three grade A recommendations (i.e., highly recommended indications), entailing ordering modality (biomarkers always available on prescription), practical use (results should be interpreted according to clinical information) and test ordering defined according to biomarker kinetics. Additional grade B recommendations (i.e., potentially valuable indications) entailed general agreement that biomarkers assessment may be of clinical value in the diagnostic approach of ED patients with suspected sepsis, suggestion for combined assessment of procalcitonin (PCT) and Creactive protein (CRP), free availability of the selected biomarker(s) on prescription, adoption of diagnostic threshold prioritizing high negative predictive value, preference for more analytically sensitive techniques, along with potential clinical usefulness of measuring PCT for monitoring antibiotic treatment, with serial testing defined according to biomarker kinetics. PCT and CRP were the two biomarkers that received the largest consensus as sepsis biomarkers (grade B recommendation), and a grade B recommendation was also reached for routine assessment of blood lactate. The assessment of biomarkers other than PCT and CRP was discouraged, with exception of presepsin for which substantial uncertainty in favor or against remained

Academy of Emergency Medicine and Care-Society of Clinical Biochemistry and Clinical Molecular Biology consensus recommendations for clinical use of sepsis biomarkers in the emergency department.

Increasing evidence is emerging that the measurement of circulating biomarkers may be clinically useful for diagnosing and monitoring sepsis. Eight members of AcEMC (Academy of Emergency Medicine and Care) and eight members of SIBioC (Italian Society of Clinical Biochemistry and Laboratory Medicine) were identified by the two scientific societies for producing a consensus document aimed to define practical recommendations about the use of biomarkers for diagnosing of sepsis and managing antibiotic therapy in the emergency department (ED). The cumulative opinions allowed defining three grade A recommendations (i.e., highly recommended indications), entailing ordering modality (biomarkers always available on prescription), practical use (results should be interpreted according to clinical information) and test ordering defined according to biomarker kinetics. Additional grade B recommendations (i.e., potentially valuable indications) entailed general agreement that biomarkers assessment may be of clinical value in the diagnostic approach of ED patients with suspected sepsis, suggestion for combined assessment of procalcitonin (PCT) and Creactive protein (CRP), free availability of the selected biomarker(s) on prescription, adoption of diagnostic threshold prioritizing high negative predictive value, preference for more analytically sensitive techniques, along with potential clinical usefulness of measuring PCT for monitoring antibiotic treatment, with serial testing defined according to biomarker kinetics. PCT and CRP were the two biomarkers that received the largest consensus as sepsis biomarkers (grade B recommendation), and a grade B recommendation was also reached for routine assessment of blood lactate. The assessment of biomarkers other than PCT and CRP was discouraged, with exception of presepsin for which substantial uncertainty in favor or against remained

Risk factors of one year increment of coronary calcifications and survival in hemodialysis patients

<p>Abstract</p> <p>Background</p> <p>Heart and coronary calcifications in hemodialysis patients are of very common occurrence and linked to cardiovascular events and mortality. Several studies have been published with similar results. Most of them were mainly cross-sectional and some of the prospective protocols were aimed to evaluate the results of the control of altered biochemical parameters of mineral disturbances with special regard to serum calcium, phosphate and CaxP with the use of calcium containing and calcium free phosphate chelating agents. The aim of the present study was to evaluate in hemodialysis patients classic and some non classic risk factors as predictors of calcification changes after one year and to evaluate the impact of progression on survival.</p> <p>Methods</p> <p>81 patients on hemodialysis were studied, with a wide age range and HD vintage. Several classic parameters and some less classic risk factors were studied like fetuin-A, CRP, 25-OHD and leptin. Calcifications, as Agatston scores, were evaluated with Multislice CT basally and after 12-18 months.</p> <p>Results</p> <p>Coronary artery calcifications were observed in 71 of 81 patients. Non parametric correlations between Agatston scores and Age, HD Age, PTH and CRP were significant. Delta increments of Agatston scores correlated also with serum calcium, CaxP, Fetuin-A, triglycerides and serum albumin. Logistic regression analysis showed Age, PTH and serum calcium as important predictors of Delta Agatston scores. LN transformation of the not normally distributed variables restricted the significant correlations to Age, BMI and CRP. Considering the Delta Agatston scores as dependent, significant predictors were Age, PTH and HDL. A strong association was found between basal calcification scores and Delta increment at one year. By logistic analysis, the one year increments in Agatston scores were found to be predictors of mortality. Diabetic and hypertensive patients have significantly higher Delta scores.</p> <p>Conclusions</p> <p>Progression of calcification is of common occurrence, with special regard to elevated basal scores, and is predictive of survival. Higher predictive value of survival is linked to the one year increment of calcification scores. Some classic and non classic risk factors play an important role in progression. Some of them could be controlled with appropriate management with possible improvement of mortality.</p