Studies on the hyperplasia ('regeneration') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity

Early Antiretroviral Therapy at High CD4 Counts Does Not Improve Arterial Elasticity: A Substudy of the Strategic Timing of AntiRetroviral Treatment (START) Trial

BACKGROUND: Both human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) may increase cardiovascular disease (CVD) risk. Vascular function assessments can be used to study CVD pathogenesis. We compared the effect of immediate versus deferred ART initiation at CD4 counts >500 cells/mm(3) on small arterial elasticity (SAE) and large artery elasticity (LAE). METHODS: Radial artery blood pressure waveforms were recorded noninvasively. Small arterial elasticity and LAE were derived from analysis of the diastolic pulse waveform. Randomized treatment groups were compared with linear models at each visit and longitudinal mixed models. RESULTS: Study visits involved 332 participants in 8 countries: mean (standard deviation [SD]) age 35 (10), 70% male, 66% nonwhite, 30% smokers, and median CD4 count 625 cells/mm(3) and 10-year Framingham risk score for CVD 1.7%. Mean (SD) SAE and LAE values at baseline were 7.3 (2.9) mL/mmHg × 100 and 16.6 (4.1) mL/mmHg × 10, respectively. Median time on ART was 47 and 12 months in the immediate and deferred ART groups, respectively. The treatment groups did not demonstrate significant within-person changes in SAE or LAE during the follow-up period, and there was no difference in mean change from baseline between treatment groups. The lack of significant differences persisted after adjustment, when restricted to early or late changes, after censoring participants in deferred group who started ART, and among subgroups defined by CVD and HIV risk factors. CONCLUSIONS: Among a diverse global population of HIV-positive persons with high CD4 counts, these randomized data suggest that ART treatment does not have a substantial influence on vascular function among younger HIV-positive individuals with preserved immunity

Delay in diagnosis of muscle disorders depends on the subspecialty of the initially consulted physician

<p>Abstract</p> <p>Background</p> <p>New therapeutic strategies in muscular dystrophies will make a difference in prognosis only if they are begun early in the course of the disease. Therefore, we investigated factors that influence the time to diagnosis in muscle dystrophy patients.</p> <p>Methods</p> <p>A sample of 101 patients (mean age 49 years; range 19-80; 44% women) with diagnosed muscle dystrophies from neurological practices and the neuromuscular specialty clinic in Berlin, Germany, was invited to participate. Time from first consultation to diagnosis, subspecialty of physician, and sociodemographic data were assessed with self-report questionnaires. The association between time to diagnosis and potential predictors (subspecialty of initially consulted physician, diagnoses, gender, and age at onset) was modeled with linear regression analysis.</p> <p>Results</p> <p>The mean time span between first health-care contact and diagnosis was 4.3 years (median 1). The diagnostic delay was significantly longer if patients were initially seen by a non-neurological specialist compared to a general practitioner (5.2 vs. 3.5 years, p = 0.047). Other factors that were independently associated with diagnostic delay were female gender and inherited muscle disease.</p> <p>Conclusion</p> <p>Action to improve clinical awareness of muscle diseases in non-neurological specialists is needed.</p

Van Gogh and Frizzled Act Redundantly in the Drosophila Sensory Organ Precursor Cell to Orient Its Asymmetric Division

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs

Targeting BRAF for patients with melanoma

The prognosis of patients with metastatic melanoma is poor and not influenced by systemic therapy with cytotoxic drugs. New targeted agents directed against the RAS-RAF-MEK-ERK pathway show promising activity in early clinical development and particular interest is focused on selective inhibitors of mutant BRAF, which is present in one half of the cases of metastatic melanoma. The majority of patients on early trials of these drugs develop secondary resistance and subsequent disease progression and it is, therefore, critical to understand the underlying escape mechanisms leading to resistance

Study of FoxA Pioneer Factor at Silent Genes Reveals Rfx-Repressed Enhancer at Cdx2 and a Potential Indicator of Esophageal Adenocarcinoma Development

Understanding how silent genes can be competent for activation provides insight into development as well as cellular reprogramming and pathogenesis. We performed genomic location analysis of the pioneer transcription factor FoxA in the adult mouse liver and found that about one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. Virtually all of the FoxA-bound silent sites are within conserved sequences, suggesting possible function. Such sites are enriched in motifs for transcriptional repressors, including for Rfx1 and type II nuclear hormone receptors. We found one such target site at a cryptic “shadow” enhancer 7 kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically induced in the early metaplastic condition of Barrett's esophagus, its expression is not necessarily present in progressive Barrett's with dysplasia or adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal epithelium becomes gradually extinguished during progression to cancer, i.e, expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We propose that this decreased expression of Rfx1 could be an indicator of progression from Barrett's esophagus to adenocarcinoma and that similar analyses of other transcription factors bound to silent genes can reveal unanticipated regulatory insights into oncogenic progression and cellular reprogramming

Intra- and Inter-Tumor Heterogeneity of BRAFV600EMutations in Primary and Metastatic Melanoma

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful

Use of imaging biomarkers to assess perfusion and glucose metabolism in the skeletal muscle of dystrophic mice

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease that affects 1 in 3500 boys. The disease is characterized by progressive muscle degeneration that results from mutations in or loss of the cytoskeletal protein, dystrophin, from the glycoprotein membrane complex, thus increasing the susceptibility of contractile muscle to injury. To date, disease progression is typically assessed using invasive techniques such as muscle biopsies, and while there are recent reports of the use of magnetic resonance, ultrasound and optical imaging technologies to address the issue of disease progression and monitoring therapeutic intervention in dystrophic mice, our study aims to validate the use of imaging biomarkers (muscle perfusion and metabolism) in a longitudinal assessment of skeletal muscle degeneration/regeneration in two murine models of muscular dystrophy.</p> <p>Methods</p> <p>Wild-type (w.t.) and dystrophic mice (weakly-affected mdx mice that are characterized by a point mutation in dystrophin; severely-affected mdx:utrn-/- (udx) mice that lack functional dystrophin and are null for utrophin) were exercised three times a week for 30 minutes. To follow the progression of DMD, accumulation of ¹⁸F-FDG, a measure of glucose metabolism, in both wild-type and affected mice was measured with a small animal PET scanner (GE eXplore Vista). To assess changes in blood flow and blood volume in the hind limb skeletal muscle, mice were injected intravenously with a CT contrast agent, and imaged with a small animal CT scanner (GE eXplore Ultra).</p> <p>Results</p> <p>In hind limb skeletal muscle of both weakly-affected mdx mice and in severely-affected udx mice, we demonstrate an early, transient increase in both ¹⁸F-FDG uptake, and in blood flow and blood volume. Histological analysis of H&E-stained tissue collected from parallel littermates demonstrates the presence of both inflammatory infiltrate and centrally-located nuclei, a classic hallmark of myofibrillar regeneration. In both groups of affected mice, the early transient response was succeeded by a progressive decline in muscle perfusion and metabolism; this was also evidenced histologically.</p> <p>Conclusions</p> <p>The present study demonstrates the utility of non-invasive imaging biomarkers in characterizing muscle degeneration/regeneration in murine models of DMD. These techniques may now provide a promising alternative for assessing both disease progression and the efficacy of new therapeutic treatments in patients.</p