MARIS: Method for Analyzing RNA following Intracellular Sorting

Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.Howard Hughes Medical InstituteHarvard Stem Cell InstituteNational Institutes of Health (U.S.) (grant 2U01DK07247307)National Institutes of Health (U.S.) (grant RL1DK081184)National Institutes of Health (U.S.) (grant 1U01HL10040804

Resolving the ancestry of Austronesian-speaking populations

There are two very different interpretations of the prehistory of Island Southeast Asia (ISEA), with genetic evidence invoked in support of both. The “out-of-Taiwan” model proposes a major Late Holocene expansion of Neolithic Austronesian speakers from Taiwan. An alternative, proposing that Late Glacial/postglacial sea-level rises triggered largely autochthonous dispersals, accounts for some otherwise enigmatic genetic patterns, but fails to explain the Austronesian language dispersal. Combining mitochondrial DNA (mtDNA), Y-chromosome and genome-wide data, we performed the most comprehensive analysis of the region to date, obtaining highly consistent results across all three systems and allowing us to reconcile the models. We infer a primarily common ancestry for Taiwan/ISEA populations established before the Neolithic, but also detected clear signals of two minor Late Holocene migrations, probably representing Neolithic input from both Mainland Southeast Asia and South China, via Taiwan. This latter may therefore have mediated the Austronesian language dispersal, implying small-scale migration and language shift rather than large-scale expansion

Clinical risk factor assessment had better discriminative ability than bone mineral density in identifying subjects with vertebral fracture

Summary: This study evaluated the characteristics of patients with vertebral fractures and examined the discriminative ability of clinical risk factors. The findings provide further insights into possible development of a simple, cost-effective scheme for fracture risk assessment using clinical risk factors to identify high-risk patients for further evaluation. Introduction: Vertebral fractures are the most common complication of osteoporosis. The aim of this study was to evaluate the characteristics of patients with vertebral fractures and to determine the discriminative ability of bone mineral density (BMD) and other clinical risk factors. Methods: Postmenopausal Southern Chinese women (2,178) enrolled in the Hong Kong Osteoporosis Study since 1995 were prospectively followed up for fracture outcome. Subjects (1,372) with lateral spine radiographs were included in this study. Baseline demographic, BMD, and clinical risk factor information were obtained from a structured questionnaire. Results: Subjects (299; 22%) had prevalent vertebral fractures. The prevalence of vertebral fractures increased with increasing age, number of clinical risk factors, and decreasing BMD. The odds of having a prevalent vertebral fracture per SD reduction in BMD after adjustment for age in Hong Kong Southern Chinese postmenopausal women was 1.5 for the lumbar spine and femoral neck. Analysis of the receiver operating characteristic curve revealed that bone mineral apparent density did not enhance fracture risk prediction. Subjects with ≥4 clinical risk factors had 2.3-fold higher odds of having a prevalent vertebral fracture while subjects with ≥4 clinical risk factors plus a low BMD (i.e., femoral neck T-score <-2.5) had 2.6-fold. Addition of BMD to clinical risk factors did not enhance the discriminative ability to identify subjects with vertebral fracture. Conclusions: Based on these findings, we recommend that screening efforts should focus on older postmenopausal women with multiple risk factors to identify women who are likely to have a prevalent vertebral fracture. © 2010 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

Selected MicroRNAs Define Cell Fate Determination of Murine Central Memory CD8 T Cells

During an immune response T cells enter memory fate determination, a program that divides them into two main populations: effector memory and central memory T cells. Since in many systems protection appears to be preferentially mediated by T cells of the central memory it is important to understand when and how fate determination takes place. To date, cell intrinsic molecular events that determine their differentiation remains unclear. MicroRNAs are a class of small, evolutionarily conserved RNA molecules that negatively regulate gene expression, causing translational repression and/or messenger RNA degradation. Here, using an in vitro system where activated CD8 T cells driven by IL-2 or IL-15 become either effector memory or central memory cells, we assessed the role of microRNAs in memory T cell fate determination. We found that fate determination to central memory T cells is under the balancing effects of a discrete number of microRNAs including miR-150, miR-155 and the let-7 family. Based on miR-150 a new target, KChIP.1 (K + channel interacting protein 1), was uncovered, which is specifically upregulated in developing central memory CD8 T cells. Our studies indicate that cell fate determination such as surface phenotype and self-renewal may be decided at the pre-effector stage on the basis of the balancing effects of a discrete number of microRNAs. These results may have implications for the development of T cell vaccines and T cell-based adoptive therapies

Vertebroplasty and kyphoplasty: a comparative review of efficacy and adverse events

Vertebroplasty and kyphoplasty have become common surgical techniques for the treatment of vertebral compression fractures. Vertebroplasty involves the percutaneous injection of bone cement into the cancellous bone of a vertebral body with the goals of pain alleviation and preventing further loss of vertebral body height. Kyphoplasty utilizes an inflatable balloon to create a cavity for the cement with the additional potential goals of restoring height and reducing kyphosis. Vertebroplasty and kyphoplasty are effective treatment options for the reduction of pain associated with vertebral body compression fractures. Biomechanical studies demonstrate that kyphoplasty is initially superior for increasing vertebral body height and reducing kyphosis, but these gains are lost with repetitive loading. Complications secondary to extravasation of cement include compression of neural elements and venous embolism. These complications are rare but more common with vertebroplasty. Vertebroplasty and kyphoplasty are both safe and effective procedures for the treatment of vertebral body compression fractures

Balloon kyphoplasty in malignant spinal fractures: a systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Spinal fractures are a common source of morbidity in cancer patients. Balloon Kyphoplasty (BKP) is a minimally invasive procedure designed to stabilize fractures and correct vertebral deformities. We performed a meta-analysis to determine the efficacy and safety of BKP for spinal fractures in cancer patients.</p> <p>Methods</p> <p>We searched several electronic databases up to September 2008 and the reference lists of relevant publications for studies reporting on BKP in patients with spinal fractures secondary to osteolytic metastasis and multiple myeloma. Outcomes sought included pain relief, functional capacity, quality of life, vertebral height, kyphotic angle and adverse events. Studies were assessed for methodological bias, and estimates of effect were calculated using a random-effects model. Potential reasons for heterogeneity were explored.</p> <p>Results</p> <p>The literature search revealed seven relevant studies published from 2003 to 2008, none of which were randomized trials. Analysis of those studies indicated that BKP resulted in less pain and better functional outcomes, and that these effects were maintained up to 2 years post-procedure. While BKP also improved early vertebral height loss and spinal deformity, these effects were not long-term. No serious procedure-related complications were described. Clinically asymptomatic cement leakage occurred in 6% of all treated levels, and new vertebral fractures in 10% of patients. While there is a lack of studies comparing BKP to other interventions, some data suggested that BKP provided similar pain relief as vertebroplasty and a lower cement leakage rate.</p> <p>Conclusion</p> <p>It appears that there is level III evidence showing BKP is a well-tolerated, relatively safe and effective technique that provides early pain relief and improved functional outcomes in patients with painful neoplastic spinal fractures. BKP also provided long-term benefits in terms of pain and disability. However, the methodological quality of the original studies prevents definitive conclusions being drawn. Further investigation into the use of BKP for spinal fractures in cancer patients is warranted.</p

Mystery Solved: The Identification of the Two Missing Romanov Children Using DNA Analysis

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters – doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters

A Critical Analysis of Atoh7 (Math5) mRNA Splicing in the Developing Mouse Retina

The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3′RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although ∼10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments