Humans possess two mitochondrial ferredoxins,Fdx1 and Fdx2, with distinct roles in steroidogenesis,heme, and Fe/S cluster biosynthesis

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.     

A standardized framework for the validation and verification of clinical molecular genetic tests

The validation and verification of laboratory methods and procedures before their use in clinical testing is essential for providing a safe and useful service to clinicians and patients. This paper outlines the principles of validation and verification in the context of clinical human molecular genetic testing. We describe implementation processes, types of tests and their key validation components, and suggest some relevant statistical approaches that can be used by individual laboratories to ensure that tests are conducted to defined standards.     

Environments for healthy ageing: A critical review

Population health outcomes are shaped by complex interactions between individuals and the environments in which they live, work and play. Environments encompass streets and buildings (physical environment), attitudes, supports and relationships with others (social environment), as well as social and political systems and policies. The impact of environments on the physical, mental health and functioning of individuals has emerged as a growing body of research in population health and health disparities. Yet, the majority of studies in this area do not focus on older adults even though older adults are particularly susceptible to the characteristics of their local environments. In this paper we review the current state of the health literature on physical environments for healthy ageing, using the International Classification of Functioning Disability and Health as a framework. Collectively, the literature emphasizes the role of supportive, barrier-free environments particularly for older adults who are at greater risk for disability and poor health. As part of our review we identify conceptual as well as methodological limitations in the current literature, including (i) a theoretical and empirical neglect of the underlying mechanisms behind the person–environment relationship; (ii) a lack of studies using nationally representative samples; (iii) over-reliance on cross-sectional data; and (iv) a need for better definition and measurement of person-centered environments. We conclude by offering some suggestions and directions for future research in this area.     

Inflammation of unknown origin versus fever of unknown origin: Two of a kind

ObjectivesA vast literature exists on fever of unknown origin (FUO), characterized by prolonged and perplexing fevers > 38.3 °C. In contrast, no studies are available to guide the approach to inflammation of unknown origin (IUO), defined as prolonged and perplexing inflammation with temperatures < 38.3 °C. We aimed to determine the diagnostic yield, the case-mix, and the outcome of patients with IUO, relative to patients with FUO.MethodsWe matched 57 patients with IUO to 57 patients with FUO of the same gender (54% male) and a similar age (median: 67 years).ResultsA diagnosis was established in 35 patients with IUO (61%) and in 33 patients with FUO (58%) (p = .70). The case-mix did not differ significantly (p = .43). Non-infectious inflammatory disorders were the dominant diagnostic category in the IUO group (16 patients), while in the FUO group, similar numbers of malignancies [10], infections [9], and non-infectious inflammatory diseases [9] were diagnosed. ¹⁸F-fluorodeoxyglucose-positron emission tomography (FDG-PET) scan contributed comparably to the diagnosis in both groups (in 18 of 50, 36%, patients with IUO and in 13 of 40, 33%, patients with FUO) (p = .83). In both groups, 7 patients (12%) died during an average follow-up of 1 year.ConclusionDiagnostic yield, case-mix, contribution of FDG-PET scan and vital outcome were similar in patients with IUO and FUO. These data suggest that the 38.3 °C boundary may be arbitrary and that the diagnostic approaches used in FUO can be applied to IUO.     

Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response

PURPOSE: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. EXPERIMENTAL DESIGN: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. RESULTS: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. CONCLUSION: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.     

Acute and Subacute Polyhydramnios in Singleton Pregnancies

Over a 10-year period when 51,022 singleton infants were delivered, 19 pregnancies (1 in 2,685) were complicated by acute polyhydramnios 17 (1 in 3,000) by subacute polyhydramnios and 501 (1 in 102) by chronic polyhydramnios. The incidence of major congenital malformations in singleton pregnancies associated with acute polyhydramnios was 63% and the perinatal mortality rate was 74%. When subacute polyhydramnios occurred in singleton pregnancies, the incidence of major congenital malformations was 65%, similar to acute polyhydramnios, but the perinatal mortality rate was only 35%. The comparable figures for chronic polyhydramnios in singleton pregnancies were a major malformation incidence of 14% and perinatal mortality rate of 10%. The type of onset of polyhydramnios, acute, subacute or chronic is therefore the most important indicator of prognosis. In patients with gross polyhydramnios, acute renal failure must be specifically excluded.     

Targeting of doxorubicin to the urinary bladder of the rat shows increased cytotoxicity in the bladder urine combined with an absence of renal toxicity

Targeting of anti-tumor drugs to the urinary bladder for the treatment of bladder carcinoma may be useful, since these agents generally have a low degree of urinary excretion and are highly toxic elsewhere in the body. The anti-tumor drug doxorubicin was coupled to the low-molecular weight protein lysozyme via the acid-sensitive cis-aconityl linker. All free amino groups of the lysozyme were used for drug attachment to achieve intact excretion of the doxorubicin-aconityl-lysozyme conjugate into the bladder. In the bladder, the cytotoxic drug should be regenerated through acidification of the urine. First, the doxorubicin-aconityl-lysozyme conjugate was tested in rats for its target specificity and general toxicity. Wistar rats were injected intravenously with 2 mg/kg free doxorubicin or 10 mg/kg lysozyme-conjugated doxorubicin. Total urinary excretion of doxorubicin was about 10 times higher if the drug was coupled to lysozyme (39 +/- 3% versus 4.4 +/- 0.4%). Free doxorubicin had no detectable toxic effects on heart, liver and lung but caused severe renal damage (proteinuria, N-acetylglucosaminidase excretion and glomerulosclerosis). None of the rats injected with doxorubicin-lysozyme conjugate showed such renal toxicity. Second, we tested whether doxorubicin could be released from the conjugate in the bladder through acidification of the urine and if the released doxorubicin could still exert a cytotoxic effect. Doxorubicin-aconityl-lysozyme (2 mg/kg conjugated doxorubicin, i.v.) was administered in rats with acidified urine (pH 6.1 +/- 0.1) and in rats with a high urinary pH (8.2 +/- 0.4). Ten times more doxorubicin was released from the conjugate in the group with acidified urine (15 +/- 7% versus 1.7 +/- 0.1%). In agreement with this, cytotoxicity was also higher in the low pH group (IC50 of 255 +/- 47 nM versus 684 +/- 84 nM doxorubicin). In conclusion, a specific delivery of doxorubicin to the urinary bladder combined with a reduced toxicity of doxorubicin in the kidneys can be achieved by coupling this anti-tumor drug to the low-molecular weight protein lysozyme via an acid-labile linker. A release of cytotoxic doxorubicin in the urinary bladder can be achieved by acidification of the urine. This technology, after further optimization, may provide an interesting tool for the treatment of bladder carcinoma.     

Prognosis of haemorrhagic stroke in childhood: a long-term follow-up study

Little is known about long-term physical sequelae, cognitive functioning, and quality of life of children who have had a haemorrhagic stroke. Fifty-six patients (29 females, 27 males) under 16 years of age at time of the bleeding were studied. Mean age at time of bleeding was 7.7 years (range 1 month to 15.9 years). The primary site and cause of the bleeding at baseline were determined. Occurrences of death, re-bleedings, and seizures during follow-up were recorded. Patients who survived were invited for a follow-up examination including physical check-up, general screening of cognition, and an inventory of subjective health perception. Thirteen children died directly as a result of the haemorrhage; nine experienced a recurrent bleeding, which was fatal in three; six children developed epileptic seizures. At follow-up 36 of 56 patients were still alive. Mean follow-up time was 10.3 years (range 1.3 to 19.9 years) and mean age was 18.6 years (range 1.8 to 34.1 years). There was no patient lost to follow-up. Five patients declined to visit the hospital. In 15 out of 31 patients who could be examined, no physical impairment was observed, 11 had a hemiparesis of varying severity, and three had symptoms of cerebellar ataxia. One child had persisting tetraparesis and one persisting paraparesis. Signs of cognitive deficits were found in 15 patients. Of the children who survive haemorrhagic stroke, the physical and functional prognosis is relatively good, as almost all children were independent at follow-up. However, only a quarter of the surviving children had no physical or cognitive deficit after a mean follow-up period of 10 years. The majority had low self-esteem as well as emotional, behavioural, and health problems.