Stable Stem Research Articles

Involvement of Cytoskeleton-associated Proteins in the Commitment of C3H10T1/2 Pluripotent Stem Cells to Adipocyte Lineage Induced by BMP2/4

The developmental pathway that gives rise to mature adipocytes involves two distinct stages: commitment and terminal differentiation. Although the important proteins/factors contributing to terminal adipocyte differentiation have been well defined, the proteins/factors in the commitment of mesenchymal stem cells to the adipocyte lineage cells have not. In this study, we applied proteomics analysis profiling to characterize differences between uncommitted C3H10T1/2 pluripotent stem cells and those that have been committed to the adipocyte lineage by BMP4 or BMP2 with the goal to identify such proteins/factors and to understand the molecular mechanisms that govern the earliest stages of adipocyte lineage commitment. Eight proteins were found to be up-regulated by BMP2, and 27 proteins were up-regulated by BMP4, whereas five unique proteins were up-regulated at least 10-fold by both BMP2/4, including three cytoskeleton-associated proteins (i.e. lysyl oxidase (LOX), translationally controlled tumor protein 1 (TPT1), and αB-crystallin). Western blotting further confirmed the induction of the expression of these cytoskeleton-associated proteins in the committed C3H10T1/2 induced by BMP2/4. Importantly, knockdown of LOX expression totally prevented the commitment, whereas knockdown of TPT1 and αB-crystallin expression partially inhibited the commitment. Several published reports suggest that cell shape can influence the differentiation of partially committed precursors of adipocytes, osteoblasts, and chondrocytes. We observed a dramatic change of cell shape during the commitment process, and we showed that knockdown of these cytoskeleton-associated proteins prevented the cell shape change and restored F-actin organization into stress fibers and inhibited the commitment to the adipocyte lineage. Our studies indicate that these differentially expressed cytoskeleton-associate proteins might determine the fate of mesenchymal stem cells to commit to the adipocyte lineage through cell shape regulation.

Controlled nonviral gene delivery and expression using stable neural stem cell line transfected with a hypoxia‐inducible gene expression system

Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration. We introduced a hypoxia-regulated plasmid-based system into mouse neural stem cells (NSCs) as an efficient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemic-regulated gene expression in the spinal cord. A synthetic hypoxia-responsive erythropoietin (Epo) enhancer, the SV40 minimal promoter and the luciferase (Luc) reporter gene were incorporated in a DsRed-expressing double-promoter plasmid for cell lipofection and Zeocin-selection to establish a hypoxia-regulated stable NSC line (NSC-Epo-SV-Luc). A nonhypoxia-regulated stable NSC line (NSC-SV-Luc) was also established as a control. Under the transcriptional regulation of the Epo enhancer, in vitro luciferase expression in NSC-Epo-SV-Luc, but not in NSC-SV-Luc, was sensitively augmented according to the strength and duration of the hypoxic stimulus and was quickly down-regulated to a low basal level after reoxygenation of the hypoxic cells. Furthermore, deoxygenation of the reoxygenated cells clearly enhanced the luciferase activity again. After transplantation into a rat spinal cord injury (SCI) model, only NSC-Epo-SV-Luc showed ischemic injury-specific luciferase expression Notably, the engineered NSC lines kept the neural differentiation potential and retained the hypoxia-regulated luciferase expression after differentiation. We propose that NSCs engineered with the Epo-SV-therapeutic gene will be valuable for developing a controllable stem cell-mediated nonviral gene therapy for SCI or other central nervous system diseases accompanied with chronic or episodic hypoxic/ischemic stresses.

Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops

Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.

Revision Total Hip Arthroplasty for a Vancouver Type B3 Periprosthetic Fracture Using an Allograft-Cemented Stem Composite by the Telescoping Technique

We describe a case of successful revision total hip arthroplasty for a Vancouver type B3 periprosthetic femoral fracture with extensive bone stock deficiency and osteoporotic diaphyseal bone. The femur was reconstructed with an allograft-cemented stem composite using a telescoping technique and a drainage hole for surplus cement. This procedure facilitated stable stem fixation to the host femur without cement interference and bony fusion between the allograft and host bone, as revealed by bone scintigraphy. This technique provides a surgical option for a severe periprosthetic femoral fracture in which the femoral diaphyseal bone is too osteoporotic to support the fixation of an allograft-cementless stem composite.

MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

Advances in Small Isometric Multicomponent ssDNA Viruses Infecting Plants.

Multicomponent ssDNA plant viruses were discovered during 1990s. They are associated with bunchy top, yellowing and dwarfing diseases of several economic plants under family Musaceae, Leguminosae and Zingiberaceae. In the current plant virus taxonomy, these viruses are classified under the family Nanoviridae containing two genera, Nanovirus and Babuvirus. The family Nanoviridae was created with five members in 2005 and by 2010, it has expanded with four additional members. The viruses are distributed in the tropical and subtropical regions of Asia, Australia, Europe and Africa. The viruses are not sap or seed transmissible and are naturally transmitted by aphid vector in a persistent manner. The genome is consisted of several circular ssDNAs of about 1kb each. Up to 12 DNA components have been isolated from the diseased plant. The major viral proteins encoded by these components are replication initiator protein (Rep), coat protein, cell-cycle link protein, movement protein and a nuclear shuttle protein. Each ssDNA contains a single gene and a noncoding region with a stable stem and loop structure. Several Rep encoding components have been reported from each virus, only one of them designated as master Rep has ability to control replication of the other genomic components. Infectivity of the genomic DNAs was demonstrated only for two nanoviruses, Faba bean necrotic yellows virus and Faba bean necrotic stunt virus (FBNSV). A group of eight ssDNA components of FBNSV were necessary for producing disease and biologically active progeny viruses. So far, infectivity of genomic components of Babuvirus has not been demonstrated.

260. Controlled Nonviral Gene Delivery and Expression Using Stable Neural Stem Cell Line Transfected with a Hypoxia-Inducible Gene Expression System

260. Controlled Nonviral Gene Delivery and Expression Using Stable Neural Stem Cell Line Transfected with a Hypoxia-Inducible Gene Expression System

A Novel Molecule Integrating Therapeutic and Diagnostic Activities Reveals Multiple Aspects of Stem Cell-based Therapy

Stem cells are promising therapeutic delivery vehicles; however pre-clinical and clinical applications of stem cell-based therapy would benefit significantly from the ability to simultaneously determine therapeutic efficacy and pharmacokinetics of therapies delivered by engineered stem cells. In this study, we engineered and screened numerous fusion variants that contained therapeutic (TRAIL) and diagnostic (luciferase) domains designed to allow simultaneous investigation of multiple events in stem cell-based therapy in vivo. When various stem cell lines were engineered with the optimized molecule, SRL(O)L(2)TR, diagnostic imaging showed marked differences in the levels and duration of secretion between stem cell lines, while the therapeutic activity of the molecule showed the different secretion levels translated to significant variability in tumor cell killing. In vivo, simultaneous diagnostic and therapeutic monitoring revealed that stem cell-based delivery significantly improved pharmacokinetics and anti-tumor effectiveness of the therapy compared to intravenous or intratumoral delivery. As treatment for highly malignant brain tumor xenografts, tracking SRL(O)L(2)TR showed stable stem cell-mediated delivery significantly regressed peripheral and intracranial tumors. Together, the integrated diagnostic and therapeutic properties of SRL(O)L(2)TR answer critical questions necessary for successful utilization of stem cells as novel therapeutic vehicles.

Revision Total Hip Arthroplasty with a Strut Allograft and an Extensively Porous-Coated Femoral Stem

Purpose: We wanted to report on the outcomes of using a strut allograft and extensively porous-coated femoral stems in revision total hip arthroplasty that was performed due to extensive femoral bone loss. Materials and Methods: Between 1998 and 2005, we performed 167 consecutive revision total hip arthroplasties. Among them, twelve cementless femoral revision surgeries with a strut allograft and extensively porous-coated stems were retrospectively reviewed. The average follow up was 4.6 years. The average age at the time of the index revision was 55.9 years. The reasons for the revisions were periprosthetic fracture due to extensive osteolysis in 5 hips and aseptic loosening in 7 hips. Results: The Harris hip score improved from a mean of 40.8 points before revision surgery to a mean of 85.1 points at the latest follow up. Radiographic evidence of bony stable stems were present in 11 hips and a fibrous stable stem was present in 1 hip. Moderate stress-shielding was noticed in one hip. Nonunion of the allograft was observed in 1 hip due to deep infection. To date, no significant wear or osteolysis has been observed. Conclusion: Revision total hip arthroplasty with a strut allograft and an extensively porous-coated femoral stem for treating cases of extensive femoral bone loss seems to be a reasonable choice. However, the concerns related to stress shielding, the difficulties in re-revisions and the complications associated with an allograft will require longer term follow up.

Femoral bone density changes after total hip arthroplasty with uncemented taper-design stem: a five year follow-up study.

We measured bone density (BD) changes to assess adaptive bone remodelling five years after uncemented total hip arthroplasty with taper-design femoral component using quantitative computed-tomography-assisted osteodensitometry (qCT). Nineteen consecutive patients (21 hips) with degenerative joint disease were enrolled in the study. A press-fit cup and a tapered uncemented stem ceramic-ceramic pairing were used in all patients. Serial clinical, radiological and qCT osteodensitometry assessments were performed after the index operation and at the one, two and five year follow-ups. At the latest follow-up, the clinical outcome was rated satisfactory in all hips. The radiological assessment showed signs of osteointegration with stable fixation of all cups and stems. Overall, there was evidence of a BD loss at year five (p = 0.004). We estimate that BD loss was between 2.2% and 12.1% in comparison with baseline postoperative values. Progressive loss of BD in the metaphyseal region was observed in all hips. We found unremarkable BD changes of diaphyseal cortical BD throughout the five year follow-up period. qCT osteodensitometry technology allows differentiation of cortical and cancellous BD changes over time. Periprosthetic BD changes at the five year follow-up are suggestive of stable stem osteointegration with proximal femoral diaphysis load transfer and metaphyseal stress shielding.

In Vivo Lentiviral Marking Demonstrates Long-Term Myeloid and Lymphoid Lineage Contribution of Individual Hematopoietic Stem Cell Clones to Murine Steady-State Hematopoiesis.

Abstract 813Most of the knowledge to date on the in vivo blood forming activity of individual hematopoietic stem and progenitor cells was gained in transplantation experiments of defined cell populations into syngeneic or xenogeneic murine hosts. Consequently, stem and progenitor cells are solely defined by their role in post-transplant reconstitution and very little is known on their clonal activity in steady-state hematopoiesis. To gain new insights into the clonal activity of stem and progenitor cells under steady-state conditions we used a genetic in vivo lentiviral marking strategy and subsequently monitored the clonal activity of marked hematopoietic cells for up to one year by highly sensitive integration site amplification using LAM-PCR. Highly concentrated GFP-expressing lentiviral vectors (LV) were injected intravenously (IV, n=10) or intrafemorally (IF, n=15) into GFP-tolerant B6.Cg-Tg (Krt1-15-EGFP) 2Cot/J (Krt15) mice to directly mark hematopoietic stem and progenitor cells. 5 mice from each of the two cohorts were treated with 5-Fluorouracil (5-FU, 150 mg/kg) to mobilize hematopoietic stem cells prior to LV-marking. The clonality of the transduced myelopoiesis and lymphopoiesis was analyzed by LAM-PCR. A small proportion of all peripheral blood cells in LV-injected mice consistently expressed GFP for up to one year (5-100 GFP+ cells per 20000 PB cells analyzed). Pre-treatment with 5-FU did not affect the percentage or lineage distribution of marked blood cells even when the vector was injected intravenously. Even though the initial percentage of marked cells was similar after IV and IF vector injection (p>0.05) the marking kinetics were different. Whereas the percentage of GFP expressing cells in PB of IF-marked mice remained stable over the whole observation period for up to 1 year, a 2-fold decline of the percentage of marked cells was detected two weeks after IV-marking indicating that predominantly short-lived more mature cells were transduced after IV vector injection. LAM-PCR analyses of sorted cell lineages showed that multiple clones contributed to the marked myeloid and lymphoid long-term hematopoiesis after IF-injection. In summary, our data demonstrate stable marking of steady-state hematopoiesis for up to one year. Our results demonstrate that remarkably stable stem cell clones with myeloid and lymphoid differentiation potential contribute to murine steady-state long-term hematopoiesis. In vivo marking will further allow to directly address the response of individual stem cell clones to hematopoietic stress including chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Principles of Treatment for Periprosthetic Femoral Shaft Fractures Around Well-fixed Total Hip Arthroplasty

Postoperative periprosthetic femoral fractures around the stem of a total hip arthroplasty are increasing in frequency. To obtain optimal results, full appreciation of the clinical evaluation, classification, and modern management principles and techniques is required. Although periprosthetic femoral fracture associated with a loose stem requires complex revision arthroplasty, fractures associated with a stable femoral stem can be managed effectively with osteosynthesis principles familiar to most orthopaedic surgeons. Femoral fracture around a stable femoral stem is classified as a Vancouver type B1 fracture. The preferred treatment consists of internal fixation, following open or indirect reduction. Emerging techniques, such as percutaneous plating and the use of locking plates, have been used with increasing frequency. Preliminary results of these techniques are promising; however, further prospective comparative studies are required.

FGF4 independent derivation of trophoblast stem cells from the common vole.

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells.

Seventeen-year survival of the cementless CLS Spotorno stem

For primary uncemented hip arthroplasty, various stem designs are available. The cementless CLS((R)) Spotorno stem has been used for more than 20 years. We re-evaluated a group of patients previously examined for a 10-year follow-up to assess the clinical and radiological stem performance in the long run. Between 1987 and 1988, a consecutive series of 107 uncemented CLS((R))-Spotorno stems (Zimmer Ltd., Germany) were implanted in 94 patients. The patients' mean age at the time of surgery was 51 years (range 20-77 years); 80 hips out of 107 (86%) were available for a clinical examination [Harris Hip Score (HHS)] after a mean of 17 years (range 15-18 years). In addition, radiographs were available from 74 out of 80 examined hips (92%) and analyzed for radiolucency, stress shielding, stem migration and heterotopic ossification. Stable stem fixation was present in 64 hips (98.5%). With "non-traumatic loosening" as an endpoint, stem survival was 100% after 17 years. Two stems (3%) showed mild subsidence already in the 10-year follow-up with no progression after 17 years. The HHS described excellent results in 47 hips (59%), good results in 16 hips (20%) and fair or poor results in 7 hips (9%) and 10 hips (13%), respectively. Radiolucency and grades II and III stress shielding were progressive at 17 years compared with the 10-year results. Grade IV stress shielding associated with osteolysis was seen in 9 hips (14%). Thigh pain was present in 20 hips (25%). The CLS((R)) Spotorno stem allows excellent long-term results in cementless hip arthroplasty, leaving only minimal options for substantial improvements. Our findings on progressive stress shielding point towards a more diaphyseal load transfer of the CLS stem.

Isolated locked compression plating for Vancouver Type B1 periprosthetic femoral fractures

Objective Report treatment results of periprosthetic femoral fractures adjacent or at the tip of a stable femoral stem (Vancouver Type B1) using a locked compression plate as the sole method of fracture stabilisation. Design Retrospective case series. Setting Academic Level I Trauma Centre. Patients Patients operatively treated at our institution with locked compression plating for Vancouver Type B1 periprosthetic fractures between 2002 and 2006 with at least 12 weeks of clinical follow-up were included. Patient demographics, hip arthroplasty implant characteristics, and AO/OTA fracture type were recorded. Intervention Open reduction internal fixation using a locked-plate spanning a majority of the femur through a lateral soft-tissue sparing approach. No cortical onlay allografts or cerclage devices (wires or cables) were used. Main outcome measurements Clinical union was defined at a minimum of 12 weeks as ability to walk, with or without the use of a walking aide, without pain at or around the fracture site. Radiographic union was defined by bridging bone spanning two or more cortices on orthogonal radiographs of the femur. Results Ten subjects met the inclusion criteria and were followed for a mean of 27 weeks (range 14–97 weeks). All achieved fracture union at a mean of 17 weeks (range 12–27 weeks). There were no hardware failures or changes in fracture alignment from operative radiographs. There were no major complications that necessitated reoperation. Conclusions Open reduction internal fixation of Vancouver Type B1 periprosthetic femoral fractures using a lateral locked-plate that spans the full extent of the femur as the sole method of stabilisation is a successful treatment method that minimises soft-tissue dissection and provides adequate fixation strength to maintain fracture alignment to fracture union.

Modulation of alternative splicing by long-range RNA structures in Drosophila

Accurate and efficient recognition of splice sites during pre-mRNA splicing is essential for proper transcriptome expression. Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances. Using phlyogenetic comparisons of intronic sequences among 12 Drosophila genomes, we elucidated a group of 202 highly conserved pairs of sequences, each at least nine nucleotides long, capable of forming stable stem structures. This set was highly enriched in alternatively spliced introns and introns with weak acceptor sites and long introns, and most occurred over long distances (>150 nucleotides). Experimentally, we analyzed the splicing of several of these introns using mini-genes in Drosophila S2 cells. Wild-type splicing patterns were changed by mutations that opened the stem structure, and restored by compensatory mutations that re-established the base-pairing potential, demonstrating that these secondary structures were indeed implicated in the splice site choice. Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns. Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

New types of embryonic stem cells generated by stabilizing pluripotency

Molecules that replace, sustain pluripotency factors make “non-permissive” mouse strains yield stable embryonic stem cells

A Single EBV-Based Vector for Stable Episomal Maintenance and Expression of Gfp in Human Embryonic Stem Cells

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

High hip center bipolar hemiarthroplasty for non-reconstructable pelvic discontinuity

A 79-year-old woman with bilateral lower extremity weakness due to cervical myelopathy presented at our department in 2002 after multiple reconstructive procedures in both hips for developmental dysplasia of the hip. In 1993, a bulk allograft in combination with an acetabular cage and a cemented cup were used to treat the left massive acetabular bone loss. The defect was type IVb by the classification of the American Academy of Orthopaedic Surgeons (D’Antonio et al. 1989) and Berry et al. (1999). In 2000, the acetabular construct failed mechani-cally while the existing cemented femoral stem remained well fixed (Figure 1A). Removal of the acetabular hardware was followed by implantation of a whole acetabular allograft. The allograft was stabilized with plates and screws, and a new cemented cup was inserted. 2 years later, allograft fracture and acetabular failure occurred again.In 2002, a high hip center bipolar hemiarthroplasty was per-formed via a standard posterior hip approach. Failed acetabu-lar component and hardware were removed but the femoral stem was left in situ as it was found to be stable. Capsular and periacetabular scar tissues were preserved as much as possible to create a soft tissue cavity to seat the bipolar head into. A 60-mm bipolar femoral head was inserted onto the femoral component to articulate with the periacetabular soft tissues in a high hip center mode. Its position was further augmented with capsular repair around the neck of the prosthesis (capsu-lar noose). A femoral condyle allograft was fixed to the ilium to serve as posterior superior acetabular wall. Postoperatively, the patient was advised to gradually increase her weight bearing using a walker or crutches. No casts or braces were applied. Within 6 months, the bipolar component migrated out of the acetabulum and articulated with the iliac soft tissues (Figure 1B). Although the patient had limb shortening, she had no pain and declined further surgery.The right hip required 7 reconstructive procedures, which led to pelvic discontinuity (type IVb) and resection arthroplasty in 1999 (Figure 1A). In 2000, a re-implantation was performed by using a reinforcement ring with a cemented polyethylene cup and a long cemented femoral prosthesis. 4 years later the was inserted onto the previously implanted and stable femo-ral stem and articulated with the soft tissues adjacent to the lateral ilium (Figure 1B). After surgery, the patient was able to transfer independently and ambulate short distances in her home with a walker.At 3 years postoperatively (right hip) and 5 years postop-eratively (left hip) the patient had no pain, relatively equal leg lengths, and could sit comfortably. Due to complete loss of lower extremity motor function associated with failed spine surgery and cervical myelopathy, the patient was non-ambu-latory. However, the Harris hip score (HHS) of the patient’s left hip had improved from 39 preoperatively to 58 postopera-tively. Similarly, the HHS of the patient’s right hip increased from 14 preoperatively to 58 postoperatively.

Intergenic regions of Borrelia plasmids contain phylogenetically conserved RNA secondary structure motifs

BackgroundBorrelia species are unusual in that they contain a large number of linear and circular plasmids. Many of these plasmids have long intergenic regions. These regions have many fragmented genes, repeated sequences and appear to be in a state of flux, but they may serve as reservoirs for evolutionary change and/or maintain stable motifs such as small RNA genes.ResultsIn an in silico study, intergenic regions of Borrelia plasmids were scanned for phylogenetically conserved stem loop structures that may represent functional units at the RNA level. Five repeat sequences were found that could fold into stable RNA-type stem loop structures, three of which are closely linked to protein genes, one of which is a member of the Borrelia lipoprotein_1 super family genes and another is the complement regulator-acquiring surface protein_1 (CRASP-1) family. Modeled secondary structures of repeat sequences display numerous base-pair compensatory changes in stem regions, including C-G→A-U transversions when orthologous sequences are compared. Base-pair compensatory changes constitute strong evidence for phylogenetic conservation of secondary structure.ConclusionIntergenic regions of Borrelia species carry evolutionarily stable RNA secondary structure motifs. Of major interest is that some motifs are associated with protein genes that show large sequence variability. The cell may conserve these RNA motifs whereas allow a large flux in amino acid sequence, possibly to create new virulence factors but with associated RNA motifs intact.