A novel, highly sensitive and rapid allele-specific loop-mediated amplification assay for the detection of the JAK2V617F mutation in chronic myeloproliferative neoplasms

Background The identification of the JAK2V617F mutation is mandatory in the diagnostic work-up of Philadelphia chromosome-negative myeloproliferative neoplasms. Several molecular techniques to detect this mutation are currently available, but each of them has some limits. DESIGN AND METHODS: We set up a novel molecular method for the identification of the JAK2V617F mutation based on an allele-specific loop-mediated amplification, not polymerase chain reaction analysis. This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency and rapidity. The method does not require either a thermal cycler or gel separation and the DNA amplification reaction is visible to the naked eye and can be monitored by turbidimetry. This method was validated on DNA from cell lines as well as from patients with myeloproliferative neoplasms. The results were compared with those obtained by conventional polymerase chain reaction methods. RESULTS: This assay detects, within 1 hour, the JAK2V617F mutation down to an allele burden of 0.1-0.01%. All samples positive by polymerase chain reaction (n=146) proved positive when tested by allele-specific loop-mediated amplification and none of the 80 negative controls gave false positive results. In addition, six patients with essential thrombocythemia previously diagnosed as being JAK2V617F negative by polymerase chain reaction analysis were found to be positive (at a low level) by allele-specific loop-mediated amplification. Furthermore, this assay discriminated the amount of JAK2V617F tumor allele within intervals of positivity, above 50%, between 50% and 10% and below 10%. Conclusions Allele-specific loop-mediated amplification is a simple, robust and easily applicable method for the molecular diagnosis and monitoring of JAK2V617F mutation in patients with chronic myeloproliferative neoplasms.     

18F‐fluoride PET as a noninvasive imaging biomarker for determining treatment efficacy of bone active agents at the hip: A prospective,randomized, controlled clinical study

The functional imaging technique of ¹⁸F‐fluoride positron emission tomography (¹⁸F‐PET) allows the noninvasive quantitative assessment of regional bone formation at any skeletal site, including the spine and hip. The aim of this study was to determine if ¹⁸F‐PET can be used as an early biomarker of treatment efficacy at the hip. Twenty‐seven treatment‐naive postmenopausal women with osteopenia were randomized to receive teriparatide and calcium and vitamin D (TPT group, n = 13) or calcium and vitamin D only (control group, n = 14). Subjects in the TPT group were treated with 20 µg/day teriparatide for 12 weeks. ¹⁸F‐PET scans of the proximal femur, pelvis, and lumbar spine were performed at baseline and 12 weeks. The plasma clearance of ¹⁸F‐fluoride to bone, K_i, a validated measurement of bone formation, was measured at four regions of the hip, lumbar spine, and pelvis. A significant increase in K_i was observed at all regions of interest (ROIs), including the total hip (+27%, p = 0.002), femoral neck (+25%, p = 0.040), hip trabecular ROI (+21%, p = 0.017), and hip cortical ROI (+51%, p = 0.001) in the TPT group. Significant increases in K_i in response to TPT were also observed at the lumbar spine (+18%, p = 0.001) and pelvis (+42%, p = 0.001). No significant changes in K_i were observed for the control group. Changes in BMD and bone turnover markers were consistent with previous trials of teriparatide. In conclusion, this is the first study to our knowledge to demonstrate that ¹⁸F‐PET can be used as an imaging biomarker for determining treatment efficacy at the hip as early as 12 weeks after initiation of therapy.     

European Species of Hebeloma Section Theobromina

This paper addresses section Theobromina within the genus Hebeloma (Agaricales). We recognise seven European species within this section, three of which are described as new: Hebeloma alboerumpens, H. griseopruinatum and H. parvicystidiatum. The first two of these species appear to be ectomycorrhizal with Cistaceae: Cistus and Helianthemum. Hebeloma parvicystidiatum is more likely to be in mycorrhizal association with Quercus spp. We also provide a key to the European species within sect. Theobromina and an updated key of known Hebeloma associates of Cistus. Molecular analyses based on multiple loci further illustrate the distinctness of the newly described taxa and provide molecular evidence, supporting the morphological evidence, for the relationship that exists among species of this section. The ITS is the only one from the sequenced loci that, alongside with morphology, distinguishes among all of the species of sect. Theobromina. The section gains most of its molecular support from the MCM7 locus, followed by RPB2.     

Multiplex analysis of genetic polymorphisms within UGT1A9, a gene involved in phase II of Δ9-THC metabolism

We present a novel multiplex assay for the simultaneous detection of 12 polymorphisms within the UGT1A9 sequence, which codes for enzymes involved in phase II biotransformation. The assay combines a multiplexed amplification step with single-base extension sequencing. The method described here is fast, cost-effective, and easy-to-use, combining the relevant features of screening methods for research and diagnostics in pharmacogenetics. To validate the assay, we tested reproducibility and sensitivity and analysed allele frequencies of 110 Caucasian individuals. Furthermore, we describe combining genetic information of individuals consuming Cannabis sativa products with respective plasma concentrations of a metabolite.

     

Baseline knee joint effusion and medial femoral bone marrow edema,in addition to MRI-based T2 relaxation time and texture measurements of knee cartilage,can help predict incident total knee arthroplasty 4–7 years later: data from the Osteoarthritis Initiative

Skeletal Radiology - To evaluate if baseline pathological knee conditions as assessed via single features of the MR-based Whole-Organ Magnetic Resonance Imaging Scoring (WORMS), standard T2, and T2...     

Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X₄) blocker 5-BDBD. Moreover, mechanical P2X₄ channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X₄ channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X₄ channels as mechanotransducers in podocytes.