[The advantages of 3D imagery in diagnosing and supervising children's and teenagers' scoliosis]. / Intérêt de l'imagerie 3D dans le diagnostic et la surveillance des scolioses de l'enfant et de l'adolescent.

Scoliosis is an abnormal curvature of the spine. One or several curves of more than 10 degrees in the frontal plane can be seen with the rotation of vertebrae in the axial plane, which modifies sagittal curves. In addition to esthetic harm, the morbidity of a scoliosis depends on the extent of the deformation. Treatment, whether it be orthopedic or surgical, is aggressive and never completely cures the condition. At best the deformation will be stabilized at the end of growth. Therefore, it is essential to detect any slight curve and quickly identify any potential progressive form in order to treat it. Visualization of scoliosis in 3D through spine modeling has several advantages at each stage of care. First, with slight curvatures, 3D modeling allows the medical staff to confirm the scoliosis by showing the modification in the three different planes. All curvatures will not progress. Orthopedic treatment is constraining and expensive; only progressive forms will receive it. When the curvature is slight and does not need immediate treatment, 3D modeling at each successive check-up will help detect any sign of likely progression quickly and reliably. Moreover, the medical observation of corset treatment and the preoperative work-up are improved because all 3D parameters of the deformation are accessible. The need for 3D modeling for scoliosis has been known for a long time, but no tool allowing a vertical study with a low level of radiation was available. The EOS imagery system meets these criteria through an optimal analysis of deformations caused by scoliosis.

Accuracy and reproducibility of preoperative three-dimensional planning for total hip arthroplasty using biplanar low-dose radiographs : A pilot study.

BACKGROUND: In total hip arthroplasty (THA), the acetabular cup and femoral stem must be correctly sized and positioned to avoid intraoperative and postoperative complications, achieve good functional outcomes and ensure long-term survival. Current two-dimensional (2D) techniques do not provide sufficient accuracy, while low-dose biplanar X-rays (EOS) had not been assessed in this indication. Therefore, we performed a case-control study to : (1) evaluate the prediction of stem and cup size for a new 3D planning technique (stereoradiographic imaging plus 3D modeling) in comparison to 2D templating on film radiographs and (2) evaluate the accuracy and reproducibility of this 3D technique for preoperative THA planning. HYPOTHESIS: Accuracy and reproducibility are better with the 3D vs. 2D method. PATIENTS AND METHODS: Stem and cup sizes were retrospectively determined by two senior surgeons, twice, for a total of 31 unilateral primary THA patients in this pilot study, using 3D preplanning software on low-dose biplanar X-rays and with 2D templating on conventional anteroposterior (AP) film radiographs. Patients with a modular neck or dual-mobility prosthesis were excluded. All patients but one had primary osteoarthritis; one following trauma did not have a cup implanted. The retrospectively planned sizes were compared to the sizes selected during surgery, and intraclass coefficients (ICC) calculated. RESULTS: 3D planning predicted stem size more accurately than 2D templating: stem sizes were planned within one size in 26/31 (84%) of cases in 3D versus 21/31 (68%) in 2D (P=0.04). 3D and 2D planning accuracies were not significantly different for cup size: cup sizes were planned within one size in 28/30 (92%) of cases in 3D versus 26/30 (87%) in 2D (P=0.30). ICC for stem size were 0.88 vs. 0.91 for 3D and 2D, respectively. Inter-operator ICCs for cup size were 0.84 vs. 0.71, respectively. Repetitions of the 3D planning were within one size (except one stem), with the majority predicting the same size. DISCUSSION: Increased accuracy in 3D may be due to the use of actual size (non-magnified) images, and judging fit on AP and lateral images simultaneously. Results for other implant components may differ from those presented. Size selection may improve further with planning experience, based on a feedback loop between planning and surgical execution. LEVEL OF EVIDENCE: Level III. Retrospective case-control study.

Congenital dislocation of the hip: optimal screening strategies in 2014.

A prospective multi-centre nationwide study of patients with congenital dislocation of the hip (CDH) diagnosed after 3 months of age was conducted with support from the French Society for Paediatric Orthopaedics (Société Française d'Orthopédie Pédiatrique [SoFOP]), French Organisation for Outpatient Paediatrics (Association Française de Pédiatrie Ambulatoire [AFPA]), and French-Speaking Society for Paediatric and Pre-Natal Imaging (Société Francophone d'Imagerie Pédiatrique et Prénatale [SFIPP]). The results showed inadequacies in clinical screening for CDH that were patent when assessed quantitatively and probably also present qualitatively. These findings indicate a need for a communication and educational campaign aimed at highlighting good clinical practice guidelines in the field of CDH screening. The usefulness of routine ultrasound screening has not been established. The findings from this study have been used by the authors and French National Health Authority (Haute Autorité de Santé [HAS]) to develop recommendations about CDH screening. There is an urgent need for a prospective randomised multi-centre nationwide study, which should involve primary-care physicians.

Child calcaneonavicular coalitions: MRI diagnostic value in a 19-case series.

INTRODUCTION: In case of hindfoot pain, diagnosis of calcaneonavicular tarsal coalition may be missed on X-ray due to the absence of any visible synostosis. All other possible etiologies (too-long anterior process (TLAP) of the calcaneum, synchondrosis, syndesmosis) must be investigated. The literature tends to recommend imaging associating standard X-ray and CT, and possibly bone scintigraphy. MRI is, however, also worth assessing, due to the many non-osseous forms calcaneonavicular pain may take. MATERIAL AND METHODS: Thirty-two cases of surgically treated calcaneonavicular tarsal coalition were studied. Nineteen cases, in 14 children, over a 10-year period, showed no visible synostosis on initial standard X-ray. In seven cases, bone scintigraphy was performed, CT in seven and MRI in 12. On the basis of the literature, our attitude was in favor of X-ray associated to CT in our early experience. Repeated diagnostic difficulties, however, led us to replace CT by MRI in case of foot pain combined to symptomatology suggestive of coalition. RESULTS: The series comprised four cartilaginous forms, four fibrous forms and eight TLAPs. In 10 of the 19 feet, radiology was strictly normal, the others showing indirect osseous signs. Only three of the seven scintigraphies showed hyperfixation. CT-scan enabled diagnosis in seven cases (two synchondroses and five rudimentary forms), and missed diagnosis in four (two cartilaginous and two fibrous forms). Second intention MRI showed two synchondroses and two syndesmoses. In the light of these 11 cases, a subsequent series of eight feet was assessed by MRI in first intention, obtaining systematic diagnosis. In all the feet of the series, the symptomatic coalition was treated by surgery, allowing peroperative findings to be compared with the imaging data. DISCUSSION: Given a rigid and painful foot syndrome suggestive of tarsal coalition, two diagnostic situations arise: (a) the clinical aspect is suggestive and standard X-ray enables diagnosis; (b) the clinical aspect is suggestive, but radiography proves non-contributive, in which case we recommend MRI with sagittal, frontal and axial slices in gadolinium-enhanced T1-weighted and fat-sat T2-weighted sequences, revealing direct (cartilaginous or fibrous coalition) or indirect signs (peripheral inflammation, osteomedullary edema, chondral lesion) unobtainable on CT scans. MRI is particularly effective in as much as most of the children concerned will not have reached bone maturity. CONCLUSION: We consider MRI to be the most effective means of precise diagnosis (causes and consequences) of tarsal coalition, especially for calcaneonavicular locations. It entails minimal invasion and irradiation, at a lower cost than CT associated to scintigraphy. LEVEL OF EVIDENCE: IV. Diagnostic study.

Pulmonary recruitment protocol for organ donors: a new strategy to improve the rate of lung utilization.

Because lung transplantation is the only effective therapy for terminal respiratory failure, the demand for donor lungs has increased steadily. However, the number of donors has remained fairly constant over the years, which results in an increasing duration of waiting for lung transplantation. To overcome the lack of organs, various strategies have been developed by transplant centers including use of marginal donors. To increase the lung utilization rate in multiorgan donors, we implemented a simple lung recruitment protocol involving a brief period of controlled sustained inflation. In 2005, the lung utilization rate in the transplant program at our institution was only 20% in multiorgan donors. With the lung recruitment protocol, the rate of lung utilization for transplantation increased to 33%, in 2006, 24% in 2007, and 24% in 2008. Following the lung recruitment protocol, the arterial oxygen tension/fraction of inspired oxygen ratio increased to greater than 15% in more than 40% of donors. We were able to improve gas exchange sufficiently that as many as two-thirds of the lungs were suitable for transplantation. During the protocol, no complications were reported, and no patient became hemodynamically unstable, precluding organ procurement. We believe that optimization of multiorgan donor management with simple interventions may improve oxygenation, reducing the number of inadequate donor lungs and increasing the overall donor pool and organ availability.

First Report of Longidorus breviannulatus Associated with Damage on Creeping Bentgrass Golf Greens in Québec, Canada.

Creeping bentgrass, Agrostis stolonifera L., is the most important turfgrass species cultivated on golf greens in Canada. The needle nematode, Longidorus breviannulatus Norton & Hoffman, has several plant hosts including Gramineae such as corn, Zea mays L. (3), and creeping bentgrass (1). This large, plant-parasitic nematode is found most frequently in sandy soils and is encouraged by irrigation (2). In 2006, irregular, yellowish/chlorotic, and dead turfgrass patches were observed for the first time on several sand-based creeping bentgrass cv. Penncross greens on a golf course in Bromont, Québec (45°19'N, 72°39'W). Furthermore, a noticeable decline of the turfgrass root system was observed. Creeping bentgrass was grown with the following management practices: mowing height 3.18 mm, fertilization 2.27 kg N/0.45 kg P2O5/3.18 kg K2O/92.9 m2/year, aeration two times per year with 9.53-mm-diameter hollow core. On 5 July 2006, soil (0.5 kg) was sampled from two damaged areas of green no. 11. Three plugs (5-cm diameter × 15 cm deep) were taken from each area with a soil probe and pooled to form two separate samples. Another set of soil samples was collected on 12 July from three golf greens (nos. 10, 11, and 16). One sample was taken from each of three damaged areas and two healthy areas of each green. Plant-parasitic nematodes were extracted from 100-ml volumes of each soil sample by the Baermann pan and funnel extraction methods. Numbers of L. breviannulatus from the soil sampled on 5 July were counted with a stereo-microscope after 4 days of extraction, while numbers of L. breviannulatus in the rest of the samples were counted after 7 days of extraction. Identification was determined by morphological examination of a small number of adult female nematodes (n = 7). Characteristics distinguishing these specimens as L. breviannulatus include: amphidial pouches (bilobed and extending to the guiding ring), length 5,115 µm (4,780 to 6,230 µm), distance of the guiding ring from the oral aperture 26 µm (24 to 30 µm), odontostyle length 83 µm (78 to 90 µm), and tail length 42 µm (37 to 50 µm). In the two soil samples collected on 5 July, 0 and 183 juveniles per kilogram of dry soil were recovered with the Baermann pan method. In samples collected on 12 July from damaged areas of three greens, averages of 16 (min 0 and max 60) and 22 (min 0 and max 80) juveniles per kilogram of dry soil were obtained with the Baermann pan and funnel methods, respectively. No L. breviannulatus was found in any sample from healthy areas. Although no damage threshold has been established for L. breviannulatus in creeping bentgrass, low numbers of needle nematodes can be damaging in other crops such as corn. In our study, the occurrence of the damage was related to populations of the needle nematode, and therefore, the nematode was the likely cause of the damage. In Canada, L. breviannulatus is reported from Ontario (4). To our knowledge, this is the first report of the occurrence of L. breviannulatus in Québec. References: (1) L. B. Forer. Plant Dis. Rep. 61:712, 1977. (2) R. B. Malek et al. Plant Dis. 64:1110, 1980. (3) D. C. Norton and J. K. Hoffmann. J. Nematol. 7:168, 1975. (4) W. Ye and R. T. Robbins. J. Nematol. 36:220, 2004.

Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA.

OBJECTIVE: To determine whether survival motor neuron (SMN) expression was stable over time. METHODS: We developed a multiplex real-time reverse transcriptase (RT)-PCR assay to quantify SMN transcripts in preclinical blood samples from 42 patients with spinal muscular atrophy (SMA) drawn for three time points per patient; most blood samples were shipped to a centralized laboratory. RESULTS: We obtained a sufficient amount (9.7 +/- 5.6 microg) of good-quality total RNA, and RNAs were stable for up to a 3-year interval. This allowed RNA samples collected during a 9- to 12-month period to be analyzed in a single run, thus minimizing interexperimental variability. SMN expression was stable over time; intersample variability for baseline measures, collected during a 17-month interval, was less than 15% for 38 of 42 SMA patients analyzed. This variability was well below the 1.95-fold increase in full-length SMN (flSMN) transcripts detected in SMA fibroblasts treated with 10 mM valproic acid. CONCLUSION: Real-time quantification of SMN messenger RNA expression may be a biomarker that is amenable to multicenter SMA clinical trials.

[Osteochondritis dissecans of the femoral condyles: report of 892 cases]. / L'ostéochondrite disséquante des condyles fémoraux.

PURPOSE OF THE STUDY: Osteochondritis rarely involves the femoral condyles. Discovery in this localization raises several questions concerning the nature of the articular cartilage, the potential for spontaneous healing, and, in the event of a free fragment, the outcome after its loss or repair. MATERIAL AND METHODS: This multicentric study included 892 pediatric and adult cases, the cutoff between two series being defined by fusion of the inferior growth plate. We excluded medical or surgical osteochondritis, cases involving the patella, osteochondral fractures, juvenile polyosteochondrosis, adult osteonecrosis, and osteochondritis beginning after the age of 50 years. RESULTS: Mean age at diagnosis was 16.5 years. Mean age at treatment onset was 22 years. Pain was the predominant symptom. 80% of cases were unilateral and 70% involved the medial condyle. The anatomic lesions were different in adults, showing more advanced degradation. At diagnosis, Bedouelle stages Ia and IIb constituted 80% of the cases observed among children while in adults, 66% were Bedouelle stages IIb to IV. Outcome was very good for the majority of children with Hughston clinical stage 4 while half of the x-rays were Hughston stage 3 and 4. There were thus a large percentage of children with abnormal xrays whose disease history was not yet terminated. In the adult series, the percentages of Hughston 3 and 4 was about the same as clinically. The x-rays were rarely perfectly normal since half of the clinical stage 3 patients were noted in stage 4. An abnormal x-ray with a very good clinical presentation was observed in a very large proportion of patients. DISCUSSION: It is difficult to interpret the plain x-ray and identify patients with a potentially unfavorable prognosis. We defined three radiographic classes: defect, nodule and empty notch. The Bedouelle classification uses information from all available explorations, particularly MRI and arthroscopy. Numerous therapeutic methods are used. Interruption of sports activities is the first intention treatment for children. Data in the literature and the findings of this symposium do not demonstrate any beneficial effect of immobilization on healing compared with simple abstention from sports activities. Transchondral perforation is a simple operation with low morbidity. In 85% of cases, it was used for lesions with an intact joint cartilage considered stable in 96% of cases. Healing was achieved in six months for 48% if the growth plate had not fused. The fragment was fixed in 43% of the cases with a loose cartilage fragment. Outcome was fair but degraded with the state of the joint cartilage and thus the stability of the fragment. Fixation must stabilize the fragment but not prevent further consolidation via osteogenesis. This is why deep perforations are drilled beyond the ossified area and additional osteochondral grafts are used. The Wagner operation gives less satisfactory results than more complicated procedures. Removal of a sequestrum is a simple, minimally invasive procedure with an uneventful postoperative period, but in the long term it favors osteoarthritic degradation, especially when performed in adults. Mosaic grafts give good mid term results. Morbidity is low especially if the grafts are harvested above the notch. The question of chondrolysis around the grafts was beyond the scope of this study. Chondrocyte grafting is difficult to accomplish and is expensive. The mid term results are good for large lesions. Osteotomy is logical only in the event of early stage osteoarthritic degradation. DECISION ALGORITHM IN CHILDREN AND ADOLESCENTS: If the plain x-ray reveals a defect (class I), simple interruption of sports activities should be proposed. Two situations can then develop. First, in a certain number of patients, the pain disappears as the defective zone ossifies progressively. Complete cure is frequent before the age of 12 years. In the second situation, the knee remains painful and the x-ray does not change or worsens to a class II nodular formation. In this case an MRI must be obtained to determine whether the joint cartilage is normal. There are two possibilities. First, the osteochondral fragment is viable and most probably will become completely re-integrated, particularly if the lesion is far from the growth plate. Necrosis is the other possibility. Transchondral perforations are needed in this case. If on the contrary the cartilage is altered, there is little hope for spontaneous cure. Arthroscopy may be needed to complete the exploration. Fragments, especially if there is a large surface area, must be fixed. Perforations to favor revascularization are certainly useful here. In the last situation (class III), the fragment wobbles on a thin attachment or has already fallen into the joint space. This is the type of problem generally observed in adults. The decision algorithm in adults is the same as in children for the rare nodular aspects (class II). There could be a discussion between transcartilage perforation and fixation. If there are a large number of fragments, fixation may not be fully successful and the lesion might be considered class III. For class III lesions, three operations can be used: removal of the sequestrum, mosaic bone-cartilage grafts, or autologous chondrocyte grafts. At the same follow-up, mosaic grafts give better results than excision of sequestra. It may be useful to remove sequestra in a limited number of situations: if there is just a small area of osteochondritis, the lesion is old and partially healed, or the zone is non weight-bearing. For other lesions, we favor mosaic grafts. We still do not have enough follow-up to assess the long-term outcome with these mosaic grafts, but simple excision clearly favors osteoarthritic degradation. Can chondrocytes grafts be compared with mosaic grafts? Chondrocyte grafts have been used for very large lesions and have given results similar to mosaic grafts. It might also be possible to combine fixation of a loose fragment and a mosaic graft. LESSONS FROM THIS STUDY: 1) The prognosis of osteochondritis is better before than after fusion of the growth plate but the lesion does not always heal in children. 2) Presence of osteochondritis requires complementary anatomic and functional exploration to determine the stability and the vitality of the fragment. 3) Attention must be taken to perform transchondral perforations early enough, particularly in children. 4) Screw fixation is not always sufficient. The trophicity of the fragment and its blood supply must be improved. 5) Mosaic grafts are preferable to excision of the fragment. 6) Chondrocyte grafts will be more widely used in the future.

First Report of the Barley Root-Knot Nematode Meloidogyne naasi Infecting Annual Bluegrass on a Golf Course in Quebec, Canada.

Annual bluegrass, Poa annua L., is an important component of the vegetation on golf course greens and fairways throughout Canada and is widely distributed across the world (2). The barley root-knot nematode, Meloidogyne naasi Franklin, infects and damages cereals such as barley (Hordeum vulgare), sorghum (Sorghum vulgare), and wheat (Triticum durum and T. vulgare), mainly in Europe. Of the root-knot species, M. naasi in particular, prefers grasses over other hosts, and infestations of this nematode can reduce the growth and vigor of turfgrasses (1). In July 2005, annual bluegrass at Beaconsfield Golf Club in Pointe Claire (45°26'N, 73°50'W), Quebec showed signs of an unidentified disease. Irregular yellowish/chlorotic patches were observed on the collar of one golf green. Lower turfgrass density in the turf areas with symptoms was noted. When roots were examined under a dissecting microscope, galling was observed. Upon dissection of washed galls, root-knot nematode egg masses and mature females inside the roots were observed. Nematode extractions of the soil from two samples of 50 g from the green and around the collar and perimeter were also done by the modified Baermann pan method. No juveniles were recovered from soil samples from the healthy area and an average of 400 and 1,500 juveniles/kg dry soil were counted in collar and perimeter samples of the patches. Morphological identification of mature females and juveniles were performed and confirmed to be M. naasi. Currently, there are no regulations to control the introduction of this nematode into Canada. To our knowledge, this is the first report of the occurrence of M. naasi in Canada where it is causing noticeable damage to turfgrass in Quebec. A soil survey is currently underway to examine the distribution of this nematode on golf course turfgrasses in Ontario and Quebec. References: (1) R. Cook and G. W. Yeates. Nematode pests of grassland and forage crops. Pages 305-350 in: Plant Parasitic Nematodes in Temperate Agriculture. K. Evans et al., eds. CAB International, Wallingford, UK, 1993. (2) S. I. Warwick. Can. J. Plant Sci. 59:1053, 1979.

First results of the search for neutrinoless double-beta decay with the NEMO 3 detector.

The NEMO 3 detector, which has been operating in the Fréjus underground laboratory since February 2003, is devoted to the search for neutrinoless double-beta decay (beta beta 0v). The half-lives of the two neutrino double-beta decay (beta beta 2v) have been measured for 100Mo and 82Se. After 389 effective days of data collection from February 2003 until September 2004 (phase I), no evidence for neutrinoless double-beta decay was found from approximately 7 kg of 100Mo and approximately 1 kg of 82Se. The corresponding limits are T1/2(beta beta0v) > 4.6 x 10(23) yr for 100Mo and T1/2(beta beta 0v) > 1.0 x 10(23) yr for 82Se (90% C.L.). Depending on the nuclear matrix element calculation, the limits for the effective Majorana neutrino mass are < 0.7-2.8 e/v for 100Mo and < 1.7-4.9 eV for 82Se.

The Delta>15 Kb deletion French Canadian founder mutation in familial hypercholesterolemia: rapid polymerase chain reaction-based diagnostic assay and prevalence in Quebec.

Approximately one in 500 individuals in Western population has autosomal dominant familial hypercholesterolemia due to mutations in the low-density lipoprotein receptor (LDLR) gene. Screening for these mutations is hampered by their large number, except in founder populations. We identified the breakpoint of the >15 kb deletion involving the LDLR gene promoter and exon 1, responsible for more than 60% of French Canadian hypercholesterolemia cases, as well as the breakpoint of the 5 kb deletion of exons 2 and 3 that accounts for an additional 5% of cases. Both deletions appear to be because of homologous recombination by unequal crossing-over between the left arms of Alu repeats. Using RepeatMasker, we determined that 55% of the LDLR gene is composed of Alu elements; thus, it is not surprising that most LDLR rearrangements involve at least one Alu. Furthermore, we developed a rapid polymerase chain reaction-based assay for the French Canadian-1 (>15 kb) and French Canadian-5 (5 kb) hypercholesterolemia alleles. Screening a representative population sample of 943 French Canadian youths whose LDL cholesterol levels were above the 50th percentile allowed us to estimate the prevalence of the >15 kb allele as 0.11% (95% confidence interval, 0.03-0.38).

[Sonocystography: a new method for the diagnosis and follow-up of vesico-ureteric reflux in children]. / L'écho-cystographie: une nouvelle méthode de détection et de suivi du reflux vésico-rénal chez l'enfant.

Vesicoureteral reflux (VUR) is a common finding in children presenting with acute urinary infection, with a frequency ranging from 20 to 50%. If radiological retrograde cystography still remains the standard technique, sonocystography now appears as a valuable alternative method, due to the properties of recent ultrasound contrast agents and the wide use of harmonic imaging. The analysis of the literature and the experience acquired by the authors during a clinical trial allow summarizing the current data on this new technique. Because of its accuracy, it may replace radiological cystography in the detection of VUR in girls, the follow-up in both boys and kids, and the management of recurrent infection in children presenting with normal radiological cystography.

The SMN genes are subject to transcriptional regulation during cellular differentiation.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of alpha-motor neurons and muscular atrophy. The causal survival motor neuron (SMN) gene maps to a complex region of chromosome 5q13 harbouring an inverted duplication. Thus, there are two SMN genes, SMN1 and SMN2, but SMN1-deficiency alone causes SMA. In this study we demonstrate, for the first time, down-regulation of SMN promoter activity during cellular differentiation. Specifically, the minimal SMN promoter is four times more active in undifferentiated embryonal carcinoma P19 cells compared to cells treated with retinoic acid (RA) to initiate neuronal differentiation. This effect is mediated by sequences contained within the minimal core promoter that we have confined to the 257 nucleotides upstream of exon 1. We have identified seven regions that are highly conserved between the mouse and human SMN core promoters and this region contains the consensus sequence for a number of transcription factors. Most notably, AhR, HNF-3 and N-Oct3 have already been shown to respond to RA treatment of EC cells, while E47, HNF-3, MAZ, N-Oct3 and Pit-1a have been implicated in embryonic, muscle or neural development. In addition, we have mapped two strong transcription initiation sites upstream of SMN exon 1. The novel -79 site identified in this study is preferentially utilized during human foetal development. Furthermore, analysis of RNA from SMA patients with deletions of the entire SMN1 gene or chimpanzees that lack SMN2 suggests that the level of transcription initiation at these sites may be different for the SMN1 and SMN2 genes. Taken together, this work provides the first demonstration of transcriptional regulation of these genes during cellular differentiation and development. Deciphering the underlying mechanisms responsible for regulating SMN transcription may provide important clues towards enhancing SMN2 gene expression, one target for the treatment of SMA.

Regulation of murine survival motor neuron (Smn) protein levels by modifying Smn exon 7 splicing.

Proximal spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron gene (SMN1). In humans, two nearly identical copies of SMN exist and differ only by a single non-polymorphic C-->T nucleotide transition in exon 7. SMN1 contains a 'C' nucleotide at the +6 position of exon 7 and produces primarily full-length SMN transcripts, whereas SMN2 contains a 'T' nucleotide and produces high levels of a transcript that lacks exon 7 and a low level of full-length SMN transcripts. All SMA patients lack a functional SMN1 gene but retain at least one copy of SMN2, suggesting that the low level of full-length protein produced from SMN2 is sufficient for all cell types except motor neurons. The murine Smn gene is not duplicated or alternatively spliced. It resembles SMN1 in that the critical exon 7 +6 'C' nucleotide is conserved. We have generated Smn minigenes containing either wild-type Smn exon 7 or an altered exon 7 containing the C-->T nucleotide transition to mimic SMN2. When expressed in cultured cells or transgenic mice, the wild-type minigene produced only full-length transcripts whereas the modified minigene alternatively spliced exon 7. Furthermore, Smn exon 7 contains a critical AG-rich exonic splice enhancer sequence (ESE) analogous to the human ESE within SMN exon 7, and subtle mutations within the mESE caused a variation in Smn transcript levels. In summary, we show for the first time that the murine Smn locus can be induced to alternatively splice exon 7. These results demonstrate that SMN protein levels can be varied in the mouse by the introduction of specific mutations at the endogenous Smn locus and thereby lay the foundation for developing animals that closely 'resemble' SMA patients.

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens.

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.

Susceptibility of the European chafer (coleoptera: scarabaeidae) to entomopathogenic nematodes (rhabditida: steinernematidae, heterorhabditidae).

The European chafer Rhizotrogus majalis (Razoumowsky) feeds on turfgrass roots, causing major damage in the urban areas of northeastern North America. A laboratory study was conducted to determine the susceptibility of third-instar chafer larvae to four species of entomopathogenic nematodes (Steinernema glaseri, S. feltiae, S. carpocapsae, and Heterorhabditis bacteriophora). Only S. glaseri was virulent, and only at high numbers (LD(50) = 294 nematodes/larva). Prolonged contact with nematodes increased host mortality. Optimal conditions for S. glaseri with respect to soil type and soil moisture were determined. Maximum larval mortality was obtained with 7% moisture and a 50/50 mixture of chernozem and sand. These results suggest that these nematodes currently have little potential for the biological control of the European chafer in turfgrass.

Intragenic complementation at the argininosuccinate lyase locus: reconstruction of the active site.

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus and the ASL alleles in the ASL-deficient cell strains of two complementation phenotypes have been identified. The frequent complementers, strains that participate in the majority of the complementation events, were found to be either homozygous or heterozygous for the Q286R allele, while the high-activity complementers, those strains in which complementation is associated with a high restoration of activity, were found to be either homozygous or heterozygous for the D87G allele. Direct proof of the intragenic complementation observed at the ASL locus has been obtained with the co-expression of the D87G and Q286R alleles in COS cells. A significant increase in the ASL activity was observed when the two alleles were co-expressed relative to the expression of each mutant allele alone. The increase in activity was comparable to that observed previously in the fibroblast complementation studies. The structure determinations of ASL and the homologous eye lens protein, duck delta II crystallin, have revealed that the active site of ASL is made up of residues from three different monomers. The structural mapping of the Q286 and D87 residues shows that both are located near the active site but that, in any one active site, each is contributed by a different monomer. The molecular symmetry of the ASL protein is such that when mutant monomers combine randomly, one active site will contain both mutations and at least one active site will contain no mutations at all. It is these 'native' active sites in the hybrid Q286R/D87G proteins that give rise to the partial recovery of enzymatic activity observed during intragenic complementation.