Stable Stem Research Articles

The novel 172 sheep (Ovis aries) microRNAs and their targets

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about ≈22 nucleotides in length. The comparative genomics approach due to their conserved nature is a good source for the novel miRNAs discovery. In this study, total 172 novel miRNAs from 140 precursor sequences belonging to 114 families were identified in sheep (Ovis aries), the most important livestock animal. All the miRNA families (oar-mir-95, 129, 130, 186, 214, 219, 223, 324, 339, 423, 450, 499, 544, 562, 568, 584, 669, 671, 763, 935, 1281, 1282, 1306, 1552, 1584, 1587, 1603, 1607, 1706, 1711, 1718, 1732, 1738, 1761, 1771, 1777, 1778, 1780, 1807, 1843, 1895, 1930, 2127, 2139, 2182, 2284, 2287, 2295, 2296, 2310, 2311, 2314, 2315, 2316, 2320, 2359, 2378, 2381, 2382, 2395, 2400, 2404, 2410, 2412, 2423, 2426, 2435, 2470, 2477, 2482, 2487, 2881, 2883, 2885, 2888, 2889, 2896, 2901, 2904, 2917, 2964, 3063, 3064, 3074, 3080, 3432, 3529, 3533, 3613, 3649, 3654, 3658, 3661, 3662, 3940, 3960, 4273, 4426, 4447, 4459, 4468, 4493, 4507, 4647, 4680, 4785, 4788, 4800, 5102, 5105, 5109, 5115, 5125 and 5132) are found for the first time in Sheep. All 140 miRNA precursors form stable minimum free energy stem loop and the mature miRNAs reside in the stem portion of the stem loop structure. Their putative targets are involved in transcription factors (26%), signaling (19%), metabolism (18%), transportation (10%), immunity (9%), cancer and tumor related (5%), growth and development (5%), stress related (4%), and structural proteins (3%).

Compositions of equi-dimensional fold maps

According to Ando’s theorem, the oriented bordism group of fold maps of n-manifolds into n-space is isomorphic to the stable n-stem. Among such fold maps we define two geometric operations corresponding to the composition and to the Toda bracket in the stable stem through Ando’s isomorphism. By using the operations we explicitly construct several fold maps with favourable properties, including a fold map which represents the generator of the stable 6-stem.

DHHC5 Protein Palmitoylates Flotillin-2 and Is Rapidly Degraded on Induction of Neuronal Differentiation in Cultured Cells

Post-translational palmitoylation of intracellular proteins is mediated by protein palmitoyltransferases belonging to the DHHC family, which share a common catalytic Asp-His-His-Cys (DHHC) motif. Several members have been implicated in neuronal development, neurotransmission, and synaptic plasticity. We previously observed that mice homozygous for a hypomorphic allele of the ZDHHC5 gene are impaired in context-dependent learning and memory. To identify potentially relevant protein substrates of DHHC5, we performed a quantitative proteomic analysis of stable isotope-labeled neuronal stem cell cultures from forebrains of normal and DHHC5-GT (gene-trapped) mice using the bioorthogonal palmitate analog 17-octadecynoic acid. We identified ∼300 17-octadecynoic acid-modified and hydroxylamine-sensitive proteins, of which a subset was decreased in abundance in DHHC5-GT cells. Palmitoylation and oligomerization of one of these proteins (flotillin-2) was abolished in DHHC5-GT neuronal stem cells. In COS-1 cells, overexpression of DHHC5 markedly stimulated the palmitoylation of flotillin-2, strongly suggesting a direct enzyme-substrate relationship. Serendipitously, we found that down-regulation of DHHC5 was triggered within minutes following growth factor withdrawal from normal neural stem cells, a maneuver that is used to induce neural differentiation in culture. The effect was reversible for up to 4 h, and degradation was partially prevented by inhibitors of ubiquitin-mediated proteolysis. These findings suggest that protein palmitoylation can be regulated through changes in DHHC PAT levels in response to differentiation signals.

Fusion Gene Vectors Allowing for Simultaneous Drug Selection, Cell Labeling, and Reporter Assay in Vitro and in Vivo

Vector systems allowing simultaneously for rapid drug selection, cell labeling, and reporter assay are highly desirable in biomedical research including stem cell biology. Here, we present such a vector system including pCVpf or pCVpr, plasmids that express pf or pr, a fusion protein between puromycin acetyltransferase and green or red fluorescent protein from CV, the human cytomegalovirus enhancer/promoter. Transfection with pCVpf or pCVpr produced a ∼10% efficiency of gene transfer. A 2-day pulse puromycin selection resulted in ∼13-fold enrichment for transgenic cells, and continuous puromycin selection produced stable transgenic stem cell clones with retained pluripotency. Furthermore, we developed a PAC assay protocol for quantification of transgene expression. To test the usefulness for cell labeling and PAC assay in vivo, we constructed pVASpf containing pf linked to the regulatory sequence of medaka germ gene vasa and generated transgenic fish with visible GFP expression in germ cells. PAC assay revealed the highest expression in the testis. Interestingly, PAC activity was also detectable in somatic organs including the eye, which was validated by fluorescence in situ hybridization. Therefore, the pf and pr vectors provide a useful system for simultaneous drug selection, live labeling, and reporter assay in vitro and in vivo.

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.

Salvage of Failed Trochanteric Fracture Fixation Using the Revitan Curved Cementless Modular Hip Arthroplasty

Failed internal fixation of an intertrochanteric or subtrochanteric fracture often leads to persistent pain and diminished function. This study evaluated 16 patients treated with Revitan curved cementless modular stem (Zimmer GmbH, Winterthur, Switzerland). At a mean follow-up period of 60 months, all patients demonstrated clinically significant pain relief and return to ambulation after salvage total hip arthroplasty. Mean Harris Hip Score improved from 17.8 to 87.7 points postoperatively. Radiographic follow-up demonstrated stable stem and bony ingrowth in 16 patients without evidence of subsidence. Eight patients had slight awareness of lateral trochanteric pain with no compromise of activities, and 1 patient had nonunion of the greater trochanter. Revitan curved cementless modular stem represents a useful treatment option in salvage total hip arthroplasty of failed pertrochanteric fixation.

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

Isolation and characterization of human spermatogonial stem cells

BackgroundTo isolate and characterization of human spermatogonial stem cells from stem spermatogonium.MethodsThe disassociation of spermatogonial stem cells (SSCs) were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4)-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established.ResultsThe cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells.ConclusionsThe two-step enzyme digestion (by type I collagenase and trypsin) process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

Mitochondrial genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis

We determined the complete mitochondrial (mt) genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis. The mt genomes of A. a. coreae and A. a. chejuensis are 16,260 and 16,261 base pairs in length, respectively. The general features of the 13 protein-coding genes of the two species are similar to those of other rodents. The TAG termination codon for NADH dehydrogenase subunit (ND) 3 is unique to Apodemus in the Muroidea. The L-strand replication origin has the potential to form a stable stem–loop structure. Within the control region, a termination-associated sequence and several conserved sequence blocks were observed. The diversity of the 13 protein-coding genes, 2 rRNAs, and 1 control region between the two species ranged between 0.005 (ATP8) and 0.027 (ND4L).

Spatial Patterns of Channel Instability Along an Ozark River, Southwest Missouri

In the Ozark Highlands of Missouri, unstable river reaches that display rapid planform change are described as active reaches. While active reaches can be part of the natural morphodynamic regime, accelerated gravel bar deposition and bank erosion have been linked to historical and recent anthropogenic activities. Relationships between geomorphic controls and specific forms of channel instability are poorly understood in the Ozarks. The objectives of this research were to (1) develop an active reach classification scheme that provides an objective means of identifying and evaluating longitudinal patterns of instability in this and other Ozark rivers, (2) identify active reaches and different forms of instability along 80 km of the Finley River in southwest Missouri, and (3) investigate physical controls on active reaches. Historical aerial photographs and geographic information system (GIS) analysis showed that active reaches occur along 21% of the length of the main stem, preferentially in valleys with confinement ratios between 10 and 30 and near major tributary confluences. Four active reach forms were identified with proportion of length as follows: extension (8%), megabar (6%), cutoff (5%), and translation (2%). Depositional megabar-type active reaches tended to form directly downstream of erosional extension-type active reaches, probably due to excess gravel supply by reworking of historical floodplain deposits. The lack of a dominant active reach form, along with a stable main stem sinuosity over time, suggests that the Finley River is a semi-stable, self-organized system in balance with watershed inputs since at least 1955, the date of the earliest photo series.

Analysis of factors predicting success and failure of treatment after type B periprosthetic humeral fractures: a case series study.

The purpose of this study was to investigate which factors predict the failure and success of treatment of periprosthetic type B humeral fractures that have occurred traumatically. The institutional admission database and the trauma registry were retrospectively reviewed. A total of 8 patients suffering from periprosthetic humeral fractures were included. The time span was 10years (2000-2010). The average age at the time of the fracture was 77years. Surgery was performed at an average of 5.6days after injury. In three patients with a well-fixed and one with an unstable humeral component, open reduction and internal fixation with the use of a plate and screws was performed. Two patients with a Delta prosthesis had an unstable humeral component. A proximal humeral resection and an implantation of an HMRS prosthesis was performed in one patient. The other patient received a Delta revision stem prosthesis, cable and plate fixation. Two patients were treated conservatively. Early surgical treatment with angular stable implants in fractures with a stable stem and replacement with a revision long-stem component in fractures with a loose prosthesis is recommended. Special attention should be paid to bone quality and anatomical proximity to the radial nerve. Conservative treatment of type B fractures is not sufficient to achieve union, especially in short oblique or transverse fractures.

The mitochondrial genome of Apodemus peninsulae (Rodentia, Muridae)

The complete mitochondrial (mt) genome of the Korean field mouse Apodemus peninsulae was sequenced and found to be 16,266 bp in length. The mt protein-coding genes of A. peninsulae had ATG, GTG, ATC, and ATA as initiation codons and TAA, TAG, TA, and T as termination codons. Two forms each of trnL and trnS and the three tRNA clusters, IQM, WANCY, and HSL were identified, as in the typical Rodentia mt genome. Among tRNAs, abnormal cloverleaf structure of trnS(AGY) was identified in DHU arm. The l-strand replication origin has the potential to form a stable stem–loop structure and control region has several conserved sequence elements.

Isolation and characterization of spheroid cells from the HT29 colon cancer cell line

Colorectal cancer stem cells (Cr-CSCs) are involved in the growth of colon cancer, but their specific role in tumor biology, including metastasis, is still unclear. Currently, methods for sorting Cr-CSCs are based on the expression of surface markers (e.g., CD133(+), CD44(+), and aldehyde dehydrogenase 1 (ALDH1(+))); however, the specificity of these markers for Cr-CSCs is uncertain. This study aimed to develop more effective ways of isolating and purifying Cr-CSCs. Suspension culture was used for isolation of Cr-CSCs. And spheroid cells were performed by side population technology, and the putative molecular marker analysis of colorectal cancer stem cell. Migration assay and chemoresistance experiment were conducted between the adherent cells and spheroid cells. HT29 colon cancer cells grew well in suspension culture. The percentage of CD44(+) cancer cell of spheroid cells was 68 times higher than that of adherent cells (89.5% vs. 1.3%), but there was no obvious difference in the percentage of CD133(+) cells (6.25% vs. 5.6%). Moreover, it is worth noting that the percent of CD133 (+)/CD44(+) cells remarkably rose (from 0.6% to 5.4%). The expression of ALDH1 was markedly increased (7.5% vs. 20.5%) for the spheroid cells than the adherent cells. The side population within the spheroid population dramatically increased from 0.2% to 6.3%. The resistance of spheroid cells to 5-FU was higher than that of adherent cells, as was their ability to migrate in the presence of SDF-1α. Suspension culture is an effective approach for enriching Cr-CSCs and can provide an inexhaustible supply of genetically stable colon cancer stem cells for targeted Cr-CSC studies. Spheroid cell models also enable the study of colon cancer chemoresistance and metastasis and may help to elucidate the role of cancer stem cells in colon cancer.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Achieving Stable Human Stem Cell Engraftment and Survival in the CNS: Is the Future of Regenerative Medicine Immunodeficient?

There is potential for a variety of stem cell populations to mediate repair in the diseased or injured CNS; in some cases, this theoretical possibility has already transitioned to clinical safety testing. However, careful consideration of preclinical animal models is essential to provide an appropriate assessment of stem cell safety and efficacy, as well as the basic biological mechanisms of stem cell action. This article examines the lessons learned from early tissue, organ and hematopoietic grafting, the early assumptions of the stem cell and CNS fields with regard to immunoprivilege, and the history of success in stem cell transplantation into the CNS. Finally, we discuss strategies in the selection of animal models to maximize the predictive validity of preclinical safety and efficacy studies.

Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells

BackgroundThe specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear.ResultsTo identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs.ConclusionsA regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.

Regenerative medicine spoilt for choice

Summary Embryonic stem cells, induced pluripotent cells, transdifferentiated cells – there is now a wide range of options for medical researchers trying to regenerate disease-affected tissues and organs. But which approach will win the day, and can science policy keep up with the bioethical implications of rapid progress? Michael Gross investigates.

Outcome after operative treatment of Vancouver type B1 and C periprosthetic femoral fractures: open reduction and internal fixation versus revision arthroplasty

The rate of periprosthetic femoral fractures after hip arthroplasty is rising and the estimated current lifetime incidence is 0.4-2.1%. While most authors recommend revision arthroplasty in patients with loose femoral shaft components, treatment options for patients with stable stem are not fully elucidated. Against this background we performed a retrospective chart analysis with clinical follow-up examination of 32 cases that sustained a Vancouver type B1 or C periprosthetic fracture (stable stem). Overall 16 cases were treated by open reduction and internal fixation (ORIF) by plate osteosynthesis and 16 cases by revision arthroplasty (RA). Both groups were comparable regarding age, gender, follow-up time interval, time interval from primary hip arthroplasty to fracture and rate of cemented femoral components, but more type C fractures were treated by ORIF. Functional outcome expressed by the median timed "Up and Go" test did not differ significantly (30 s ORIF vs. 24 s RA, P = 0.19). However, by comparable systemic complications surgery-related complications were significantly more frequent in plate osteosynthesis (ORIF n = 10 vs. RA n = 3, P = 0.03). Based on our results, further studies, preferable via a multicenter approach, should focus on identifying patients that benefit from ORIF in periprosthetic fractures. A misinterpretation of type B2 fractures with loose implant as type B1 fractures may cause implant failure in case of ORIF. The use of angular stable implants, additional cable wires or bone enhancing means is recommended.

Identification of microRNAs in ecological model plant <i>Mimulus</i>

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 20 to 24 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. This study resulted in 34 miRNAs belonging to 21 families in ecological model plant Mimulus. All the miRNA families (mir 156, 157, 159, 160, 164, 172, 319, 393, 395, 397, 399, 400, 403, 417, 419, 472, 782, 854, 858, 867 and 2112) are found for the first time in Mimulus. The MIR-399 is found as precursor miRNA cluster with 4 mature sequences. All 34 miRNA precursors form stable minimum free energy stem loop structure as their orthologues form and the mature miRNAs reside in the stem portion of the stem loop structure. Twenty eight are from Mimulus guttatus and six miRNAs belong to Mimulus lewisii. Their targets consist of dihy-droflavonol-4-reductase, cycloidea-like protein, DNA-directed RNA polymerase II, maturase (matR) and transcription factors like; squamosa-promoter binding, MYB, palmate-like pentafoliata 1.