Response to kisspeptin and gonadotropin-releasing hormone agonist administration in Holstein-Friesian dairy heifers with positive or negative genetic merit for fertility traits.

Previous research has identified that Holstein-Friesian dairy heifers with positive (POS) genetic merit for fertility traits (FertBV) reach puberty earlier than heifers with negative (NEG) FertBV. The hypothalamus-pituitary-gonadal (HPG) axis is functional in heifers before the onset of puberty, with increased LH release evident as heifers progress toward puberty. We investigated the functionality of the HPG axis in peripubertal Holstein-Friesian dairy heifers with divergent POS or NEG FertBV, hypothesizing that the earlier puberty onset of POS heifers is associated with earlier activation of the HPG axis than in NEG heifers. In experiment 1, we tested the dose responsiveness of POS heifers to an intravenous injection of either kisspeptin [Kiss; 2, 4, or 8 µg/kg of body weight (BW); n = 3 per dose] or a GnRH agonist (buserelin; 5, 10, or 20 ng/kg of BW; n = 3 per dose). The use of these 2 agonists investigates the status of the HPG axis in both the hypothalamus (Kiss) and pituitary (buserelin) glands. Doses of 4 µg/kg BW of Kiss and 10 ng/kg BW of buserelin produced submaximal LH responses and were used in experiment 2, in which previously unused POS (n = 22) and NEG (n = 18) FertBV heifers were challenged with both agonists at 10 and 12 mo of age in a partial crossover design. Heifers were randomly allocated to treatment groups, balanced for age and BW. The LH response to buserelin was greater in POS heifers than NEG heifers at 10 mo of age, with no difference in response at 12 mo. The FSH response to buserelin and the LH and FSH responses to Kiss did not differ between the POS and NEG heifers at either age. These results indicate an association between divergent genetic merit for fertility and the LH release to buserelin at 10 mo of age, supporting the hypothesis that gonadotropin responsiveness to a GnRH agonist is more advanced in POS heifers than in NEG heifers.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Effect of gonadotropin-inhibitory hormone on luteinizing hormone secretion in humans.

BACKGROUND: Gonadotropin-inhibitory hormone (GnIH, human homologue of RFRP-3) suppresses gonadotropin secretion in animal models, but its effects have not been studied in the human. OBJECTIVE: We tested the hypotheses that exogenous GnIH inhibits LH secretion (i) in postmenopausal women and (ii) in men concurrently administered exogenous kisspeptin. DESIGN: Following in vitro and in vivo preclinical studies to functionally characterize the GnIH peptide, a dose-finding study (human GnIH: 1·5-150 µg/kg/h, iv for 3 h) was undertaken, and 50 µg/kg/h selected for further evaluation. Five postmenopausal women were administered 50 µg/kg/h iv infusion for 3 h or vehicle on two separate days. Four men were administered kisspeptin-10 (0·3 µg/kg iv bolus) with simultaneous infusion of GnIH (50 µg/kg/h, iv for 3 h) or vehicle. PARTICIPANTS: Healthy postmenopausal women (mean age 58 ± 2 years, LH: 30·8 ± 2·9 IU/l, FSH: 78·7 ± 6·4 IU/l, oestradiol: <50 pmol/l) and men (39·8 ± 2·1 years, mean total testosterone 12·1 ± 1·8 nmol/l, LH 2·2 ± 0·2 IU/l). PRIMARY OUTCOME: Change in area under curve (AUC) of LH during GnIHvs vehicle. RESULTS: During GnIH administration in postmenopausal women, LH secretion decreased (ΔAUC: -9·9 ± 1·8 IU/3 h) vs vehicle (ΔAUC: -0·5 ± 1·7 IU/3 h; P = 0·02). Kisspeptin-10-stimulated LH responses in men were not affected by GnIH co-administration (60-min AUC of LH 6·2 ± 0·8 IU/h with kisspeptin-10 alone, 6·3 ± 1·0 IU/h, kisspeptin-10 with GnIH, P = 0·72). Exogenous GnIH was well tolerated, with no adverse events reported. CONCLUSIONS: Gonadotropin-inhibitory hormone decreased LH secretion in postmenopausal women in this first-in-human study. Kisspeptin-stimulated LH secretion in men was not inhibited during concomitant administration of GnIH.

Expression of regulatory neuropeptides in the hypothalamus of red deer (Cervus elaphus) reveals anomalous relationships in the seasonal control of appetite and reproduction.

Red deer are seasonal with respect to reproduction and food intake, so we tested the hypothesis that their brains would show seasonal changes in numbers of cells containing hypothalamic neuropeptides that regulate these functions. We examined the brains of male and female deer in non-breeding and breeding seasons to quantify the production of kisspeptin, gonadotropin inhibitory hormone (GnIH), neuropeptide Y (NPY) and γ-melanocyte stimulating hormone (γ-MSH - an index of pro-opiomelanocortin production), using immunohistochemistry. These neuropeptides are likely to be involved in the regulation of reproductive function and appetite. During the annual breeding season there were more cells producing kisspeptin in the arcuate nucleus of the hypothalamus than during the non-breeding season in males and females whereas there was no seasonal difference in the expression of GnIH. There were more cells producing the appetite stimulating peptide, NPY, in the arcuate/median eminence regions of the hypothalamus of females during the non-breeding season whereas the levels of an appetite suppressing peptide, γ-MSH, were highest in the breeding season. Male deer brains exhibited the converse, with NPY cell numbers highest in the breeding season and γ-MSH levels highest in the non-breeding season. These results support a role for kisspeptin as an important stimulatory regulator of seasonal breeding in deer, as in other species, but suggest a lack of involvement of GnIH in the seasonality of reproduction in deer. In the case of appetite regulation, the pattern exhibited by females for NPY and γ-MSH was as expected for the breeding and non-breeding seasons, based on previous studies of these peptides in sheep and the seasonal cycle of appetite reported for various species of deer. An inverse result in male deer most probably reflects the response of appetite regulating cells to negative energy balance during the mating season. Differences between the sexes in the seasonal changes in appetite regulating peptide cells of the hypothalamus present an interesting model for future studies.

IGFBP-2 inhibits adipogenesis and lipogenesis in human visceral, but not subcutaneous, adipocytes.

BACKGROUND/OBJECTIVE: IGF-binding protein (IGFBP)-2 is the principal IGFBP produced by white adipocytes during adipogenesis, and circulating levels are reduced in obesity. Overexpression of IGFBP-2 in transgenic mice prevents obesity, but depot-specific effects of IGFBP-2 on adipo/lipogenesis are unknown. The present study aimed to investigate whether IGFBP-2 affects adipo/lipogenesis in a depot-specific manner and explore potential mechanisms. METHODS: Following adipocyte characterisation, IGFBP-2 levels were measured from human subcutaneous and visceral preadipocytes, and IGFBP-2 dose-responses were then undertaken with exogenous IGFBP-2 in an in vitro IGF-I-free system to examine adipo/lipogenesis. Following this, both types of adipocytes were transfected with human siRNA IGFBP-2 to assess auto-/para-/intra-crine effects, with and without additional add-back IGFBP-2. To elucidate the potential mechanisms, visceral preadipocytes were treated with either wild-type or Heparin Binding Domain (HBD)-mutant IGFBP-2 (which is unable to bind to cell-surface components), and experiments were also undertaken using Echistatin (an integrin receptor blocker). Outcomes included gene expression profiles, protein levels and phosphorylation and lipid staining. RESULTS: Human visceral adipocytes produced significantly more IGFBP-2 than subcutaneous adipocytes. Subsequent dose-responses to IGFBP-2 demonstrated significant reductions in adipo/lipogenesis in visceral, but not subcutaneous, adipocytes in response to increasing IGFBP-2. Silencing IGFBP-2 resulted in exaggerated adipo/lipogenesis in visceral, but not subcutaneous, adipocytes, an effect completely inhibited by add-back IGFBP-2. These effects occurred in the absence of changes in IGF-I levels. HBD-mutant IGFBP-2 had reduced effects compared with wild-type IGFBP-2. Wild-type IGFBP-2 increased phosphorylation of focal adhesion kinase (FAK) and decreased phosphatase and tensin homolog (PTEN) levels, suggestive of integrin-mediated signalling. Blockade of this signalling, using Echistatin, completely negated the effects of IGFBP-2 on visceral adipo/lipogenesis. CONCLUSION: IGFBP-2 inhibits both adipogenesis and lipogenesis in visceral, but not subcutaneous, adipocytes. This depot-specific impairment appears to be independent of IGF-I and involves cell-surface association of IGFBP-2 and activation of integrin signalling pathways.

Plasmid CDS5 influences infectivity and virulence in a mouse model of Chlamydia trachomatis urogenital infection.

The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.

The Rowing Spine: A Review of Biomechanics, Injury, and Treatment.

OBJECTIVE: We aimed to describe spinal biomechanics and injury patterns in rowing. METHODS: In this systematic literature review, a Google and PubMed literature search was undertaken using keywords "rowing," "biomechanics," and "spine." RESULTS: Relevant articles were reviewed and synthesized to describe biomechanics, injury patterns, treatment options, and techniques for injury prevention. CONCLUSIONS: Rowing has increased in popularity throughout the United States. Up-to-date knowledge of rowing biomechanics and spinal injury patterns is necessary for prompt diagnosis and appropriate treatment of the injured rowing athlete.

Socioeconomic disadvantage is correlated with worse PROMIS outcomes following lumbar fusion.

BACKGROUND CONTEXT: Socioeconomic status (SES) has been associated with differential healthcare outcomes and may be proxied using the area-deprivation index (ADI). Few studies to date have investigated the role of ADI on patient-reported outcomes and clinically meaningful improvement following lumbar spine fusion surgery. PURPOSE: The purpose of this study is to investigate the role of SES on lumbar fusion outcomes using Patient-Reported Outcomes Measurement Information System (PROMIS) surveys. STUDY DESIGN/SETTING: Retrospective review of a single institution cohort. PATIENT SAMPLE: About 205 patients who underwent elective one-to-three level posterior lumbar spine fusion. OUTCOME MEASURES: Change in PROMIS scores and achievement of minimum clinically important difference (MCID). METHODS: Patients 18 years or older undergoing elective one-to-three level lumbar spine fusion secondary to spinal degeneration from January 2015 to September 2021 with minimum one year follow-up were reviewed. ADI was calculated using patient-supplied addresses and patients were grouped into quartiles. Higher ADI values represent worse deprivation. Minimum clinically important difference (MCID) thresholds were calculated using distribution-based methods. Analysis of variance testing was used to assess differences within and between the quartile cohorts. Multivariable regression was used to identify features associated with the achievement of MCID. RESULTS: About 205 patients met inclusion and exclusion criteria. The average age of our cohort was 66±12 years. The average time to final follow-up was 23±8 months (range 12-36 months). No differences were observed between preoperative baseline scores amongst the four quartiles. All ADI cohorts showed significant improvement for pain interference (PI) at final follow-up (p<.05), with patients who had the lowest socioeconomic status having the lowest absolute improvement from preoperative baseline physical function (PF) and PI (p=.01). Only those patients who were in the lowest socioeconomic quartile failed to significantly improve for PF at final follow-up (p=.19). There was a significant negative correlation between socioeconomic level and the absolute proportion of patients reaching MCID for PI (p=.04) and PF (p=.03). However, while ADI was a significant predictor of achieving MCID for PI (p=.02), it was nonsignificant for achieving MCID for PF. CONCLUSIONS: Our study investigated the influence of ADI on postoperative PROMIS scores and identified a negative correlation between ADI quartile and the proportion of patients reaching MCID. Patients in the worse ADI quartile had lower chances of reaching clinically meaningful improvement in PI. Policies focused on alleviating geographical deprivation may augment clinical outcomes following lumbar surgery.

Clinical Improvement After Lumbar Fusion: Using PROMIS to Assess Recovery Kinetics.

STUDY DESIGN: Retrospective review of a single institution cohort. OBJECTIVE: The goal of this study is to identify features that predict delayed achievement of minimum clinically important difference (MCID) following elective lumbar spine fusion using Patient-Reported Outcomes Measurement Information System (PROMIS) surveys. SUMMARY OF BACKGROUND DATA: Preoperative prediction of delayed recovery following lumbar spine fusion surgery is challenging. While many studies have examined factors impacting the achievement of MCID for patient-reported outcomes in similar cohorts, few studies have assessed predictors of early functional improvement. METHODS: We retrospectively reviewed patients undergoing elective one-level posterior lumbar fusion for degenerative pathology. Patients were subdivided into two groups based on achievement of MCID for each respective PROMIS domain either before six months ("early responders") or after six months ("late responders") following surgical intervention. Multivariable logistic regression analysis was used to determine features associated with odds of achieving distribution-based MCID before or after six months follow up. RESULTS: 147 patients were included. The average age was 64.3±13.0 years. At final follow-up, 57.1% of patients attained MCID for PI and 72.8% for PF. However, 42 patients (49.4%) reached MCID for PI by six months, compared to 44 patients (41.1%) for PF. Patients with severe symptoms had the highest probability of attaining MCID for PI (OR 10.3; P =0.001) and PF (OR 10.4; P =0.001) Preoperative PROMIS symptomology did not predict early achievement of MCID for PI or PF. Patients who received concomitant iliac crest autograft during their lumbar fusion had increased odds of achieving MCID for PI (OR 8.56; P =0.001) before six months. CONCLUSION: Our study demonstrated that the majority of patients achieved MCID following elective one-level lumbar spine fusion at long-term follow-up, although less than half achieved this clinical benchmark for each PROMIS metric by six months. We also found that preoperative impairment was not associated with when patients would achieve MCID. Further prospective investigations are warranted to characterize the trajectory of clinical improvement and identify the risk factors associated with poor outcomes more accurately.

Depression State Correlates with Functional Recovery Following Elective Lumbar Spine Fusion.

OBJECTIVE: To determine how depression state impacts postoperative Patient-Reported Outcomes Measurement Information System (PROMIS) scores and achievement of minimum clinically important difference (MCID) following lumbar fusion. Depression has been shown to negatively impact outcomes following numerous orthopedic surgeries. Situational and major clinical depression can differentially affect postoperative outcomes. METHODS: Adult patients undergoing elective 1-3 level lumbar fusion were reviewed. Patients with a formal diagnosis of major depression were classified as "clinically depressed" whereas patients with at least "mild" PROMIS Depression scores in the absence of formal depression diagnosis were deemed "situationally depressed." analysis of variance testing was used to assess differences within and between groups. Multivariate regression was used to identify features associated with the achievement of MCID. RESULTS: Two hundred patients were included. The average age was 65.9 ± 12.2 years. 75 patients (37.5%) were nondepressed, 66 patients (33.0%) were clinically depressed, and 59 patients (29.5%) were situationally depressed. Situationally depressed patients had worse preoperative physical function (PF) and pain interference (PI) scores and were more likely to have severe symptoms (P = 0.001, P = 0.001). All groups improved significantly from preoperative baseline scores. All groups met MCID PF at different rates, with highest proportion of situationally depressed reaching this metric (P = 0.03). Rates of achieving MCID PI were not significantly different between groups (P = 0.47). Situational depression was predictive of achieving MCID PF (P = 0.002) but not MCID PI. CONCLUSIONS: Our study investigated the relationship between depression and postoperative PROMIS scores and identified situationally depressed patients as having the worst preoperative impairment. Despite this, the situationally depressed cohort had the highest likelihood of achieving MCID PF, suggestive of a bidirectional relationship between lumbar degenerative disease and subclinical, situational depression. These findings may help guide preoperative counseling on expectations, and patient selection.

Chlamydia trachomatis clinical isolates identified as tetracycline resistant do not exhibit resistance in vitro: whole-genome sequencing reveals a mutation in porB but no evidence for tetracycline resistance genes.

Chlamydia trachomatis is the most common bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness in developing countries. Tetracycline is commonly the drug of choice for treating C. trachomatis infections, but cases of antibiotic resistance in clinical isolates have previously been reported. Here, we used antibiotic resistance assays and whole-genome sequencing to interrogate the hypothesis that two clinical isolates (IU824 and IU888) have acquired mechanisms of antibiotic resistance. Immunofluorescence staining was used to identify C. trachomatis inclusions in cell cultures grown in the presence of tetracycline; however, only antibiotic-free control cultures yielded the strong fluorescence associated with the presence of chlamydial inclusions. Infectivity was lost upon passage of harvested cultures grown in the presence of tetracycline into antibiotic-free medium, so we conclude that these isolates were phenotypically sensitive to tetracycline. Comparisons of the genome and plasmid sequences for the two isolates with tetracycline-sensitive strains did not identify regions of low sequence identity that could accommodate horizontally acquired resistance genes, and the tetracycline binding region of the 16S rRNA gene was identical to that of the sensitive control strains. The porB gene of strain IU824, however, was found to contain a premature stop codon not previously identified, which is noteworthy but unlikely to be related to tetracycline resistance. In conclusion, we found no evidence of tetracycline resistance in the two strains investigated, and it seems most likely that the small, aberrant inclusions previously identified resulted from the high chlamydial load used in the original antibiotic resistance assays.

Distribution and sequence of gonadotropin-inhibitory hormone and its potential role as a molecular link between feeding and reproductive systems in the Pekin duck (Anas platyrhynchos domestica).

The reproductive status of adult Pekin drakes is very sensitive to nutritional status. Thus, the purpose of this study was to increase our understanding of the neurobiology underlying the depressive effect of fasting on the secretion of reproductive hormones. It was hypothesized that this effect was mediated by gonadotropin-inhibitory hormone (GnIH). Networks of GnIH fibers were present throughout the diencephalon, and cell bodies were present primarily, in the hypothalamic paraventricular nucleus (PVN). The duck GnIH gene was cloned and sequenced and found to encode GnIH and two GnIH-related peptides (GnIH-RP1, GnIH-RP2) which have a similar identity to those found in other avian species. Intracerebroventricular injection of GnIH, but not of GnIH-RP1, depressed plasma LH and stimulated feeding. Fasting for 48h depressed plasma LH and induced fos expression in about half the population of GnIH-ir neurons. These data suggest that GnIH neurons are mediators between feeding and reproductive systems in Pekin drakes.

C2 quad-screws facilitate 4-rod fixation across the cervico-thoracic junction.

BACKGROUND: Cervical spine deformity is a potentially devitalizing problem. Contemporary techniques for repair and reconstruction include fusion using rods of tapered diameter alone, or quadruple-rod constructs in which primary rods are joined to floating accessory rods by connectors. Here, we present how we utilized a quadruple-rod construct to perform five C2 to thoracic spine fusions. METHODS: Our hospital electronic medical record revealed five patients who underwent the four rod C2-thoracic spine fixation. Patients ranged in age from 14-years-old to 78-years-old. The mean operative time was 715.8 min (range 549-987 min), and average estimated blood loss was 878 cc (range 40-1800 cc). RESULTS: None of the five patients sustained any intraoperative complications, and none demonstrated progressive kyphotic deformity over the average follow-up interval of 8 months. CONCLUSION: We successfully treated five patients with degenerative or oncologic cervical pathology requiring fixation across the cervicothoracic junction utilizing a 4-rod C2-cervicothoracic fusion technique.

Cement-Augmented and Dual-Headed Posterior Screw Reconstruction After Corpectomy for Metastatic Tumor Resection.

BACKGROUND: Metastatic spinal tumors have a well-documented deleterious effect on the overall strength of the bony spine. Surgical interventions must address not only removal of the tumor itself, but the integrity of reconstructive hardware constructs as well. METHODS: We present a series of 8 patients with metastatic spine tumors who were successfully treated with tumor resection and reconstruction of residual 3-column defect with cement-augmented fenestrated pedicle screws and dual-rod posterior stabilization. RESULTS: All patients demonstrated resolution of their presenting neurologic symptoms. CONCLUSIONS: This series supports the use of the aforementioned constructs in conjunction to provide added stability and reduce hardware failure when treating a diversity of spinal tumors.

Distal Ventral Iliac Pathway for Spinopelvic Fixation: Technique Description and Case Series.

BACKGROUND: Pelvic fixation improves the stability of spinal instrumentation and can be used in high-grade degenerative disease, trauma, deformity, and destabilizing invasive pathologies, such as infection and tumor. Classic techniques for spinopelvic fixation include traditional iliac screws and S2-Alar-Iliac screws. We present a case series describing the distal ventral iliac pathway (DVIP) for spinopelvic fixation and discuss surgical indications and merits of this technique. We describe the use of the DVIP for spinopelvic fixation in the setting of degenerative and traumatic pathologies, compare this technique with existing approaches, and summarize literature to support this approach. METHODS: One hundred twenty-eight cases of DVIP screws were identified at 1 academic medical center, and 3 cases were chosen as representative examples for technique demonstration. RESULTS: Patient ages ranged from 19 to 81 (mean 62) years. Intraoperative and postoperative complications include 12 incidental durotomies, 3 suprafascial infections, and 2 compressive hematomas. There were 22 instances of hardware failure and 8 instances of pseudoarthrosis. Overall, 26 patients underwent revision surgery. Mean estimated blood loss, operative time, and time under fluoroscopy were 1959 mL, 386 minutes, and 3.19 minutes, respectively. CONCLUSIONS: The DVIP is both safe and effective as a treatment for patients with degenerative and traumatic lumbosacral pathology. Spinopelvic fixation provides improved soft tissue coverage and fewer hardware complications at minimum of 1 year follow up. This case series demonstrates a novel surgical technique for spinopelvic fixation in the setting of numerous spinal pathologies. LEVEL OF EVIDENCE: 4. CLINICAL RELEVANCE: This surgical technique is less technically challenging than current approaches, minimizes radiation exposure, and obviates the need for horizontal connector rods. In addition, in highly destabilizing pathologies, this technique also allows for multiple screw placement within the ilium, while maintaining the ability to connect to a single rod construct. This technique is safe, technically approachable, and broadly applicable to an array of spinopelvic pathologies.

Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity.

The previously determined nucleotide sequence of the porA gene, encoding the class 1 outer membrane protein of meningococcal strain MC50, has been used to clone and sequence the porA gene from two further strains with differing serosubtype specificities. Comparison of the predicted amino acid sequences of the three class 1 proteins revealed considerable structural homology with major variation confined to two discrete regions (VR1 and VR2). The high degree of structural homology between the sequences gave predicted secondary structures that were almost identical, with the variable domains located in hydrophilic regions that are likely to be surface located and hence accessible to antibody binding. The predicted amino acid sequences have been used to define the epitopes recognized by mAbs with serosubtype specificity. A series of overlapping decapeptides spanning each of the class 1 protein sequences have been synthesized on solid-phase supports and probed with mAbs. Antibodies with P1.16 and P1.15 subtype specificity reacted with sequences in the VR2 domain, while antibodies with P1.7 subtype specificity reacted with sequences in the VR1 domain. Further peptides have been constructed to define the minimum epitopes recognized by each antibody. Thus we have been able to define linear peptides on each class 1 protein molecule that are responsible for subtype specificity and that represent targets for a protective immune response.

Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor.

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Šresolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

Laminopedicular Osteotomy for En-bloc Resection of Posterolateral Thoracic Osteoblastoma: Technical Note.

BACKGROUND: Osteoblastomas are a type of primary osseous neoplasm that exhibit a proclivity for the spine, primarily the posterior elements. While generally considered benign, some variants of osteoblastoma exhibit aggressive growth with lytic osseous destruction and soft tissue invasion, with some recurring after initial treatment. Given their proximity to vital structures and potential for rapid growth, these tumors are often managed with aggressive surgery, with en-bloc resection preferred. METHODS: Here we describe our osteotomy technique for resecting en bloc a posterolateral thoracic osteoblastoma causing rapidly progressive myelopathy in a 17-year-old male. RESULTS: Successful treatment of osteoblastoma in a 17-year-old male demonstrates the efficacy of our laminopedicular osteotomy technique in treating 1 instance of a rapidly presenting spinal tumor. CONCLUSIONS: This case bolsters the growing body of literature that favors the outcomes of a more conservative approach to en-bloc resection of spinal tumors.

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide.

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.

Sequence and genome organization of a human small round-structured (Norwalk-like) virus.

Small round-structured viruses (SRSVs), also known as Norwalk or Norwalk-like viruses, are the major worldwide cause of acute, epidemic nonbacterial gastroenteritis in humans. These viruses, which contain a single-stranded RNA genome, have remained refractory to molecular characterization because of the small amounts of virus in clinical samples and the absence of an animal model and an in vitro culture system. The complete genomic nucleotide sequence of an SRSV, Southampton virus, was determined. The 7696-nucleotide RNA genome encodes three open reading frames whose sequences and organization strongly support proposals that SRVSs are members of the Caliciviridae.